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Current problems and possible solutions in the treatment of nasopharyngeal carcinoma inIndonesia

Wildeman, M.A.M.

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Citation for published version (APA):Wildeman, M. A. M. (2013). Current problems and possible solutions in the treatment of nasopharyngealcarcinoma in Indonesia.

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Current problems and possible solutions in the treatment of Nasopharyngeal Carcinoma in Indonesia

Maarten Wildeman

Current problems and possible solutions in the

treatment of N

asopharyngeal Carcinoma in Indonesia

Maarten W

ildeman

Current problems and possible solutions in the treatment of Nasopharyngeal

Carcinoma in Indonesia

Maarten Wildeman

This study was performed at the Netherlands Cancer Institute - Antoni van Leeuwenhoek Hospital, Amsterdam, the Netherlands; Rumah Sakit Sardjito - Gadjah Mada University, Yogyakarta, Indonesia, Rumah Sakit Cipto Mangunkusomo - Universitas Indonesia, Jakarta, Indonesia and the VU University Medical Center, Amsterdam, the Netherlands. The Dutch Cancer Society, ZonMw and the Achmea Foundation funded this study.

Printed and lay-out by Nauka

Cover: Dede Eri Supria

ISBN: 9789491688003

© M. Wildeman 2012

Current problems and possible solutions in the treatment of Nasopharyngeal

Carcinoma in Indonesia

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctor

aan de Universiteit van Amsterdam

op gezag van de Rector Magnificus

prof. dr. D.C. van den Boom

ten overstaan van een door het college voor promoties

ingestelde commissie,

in het openbaar te verdedigen in de Aula der Universiteit

op 8 februari 2013, te 13:00

door

Melchert Adrianus Martinus Wildeman

geboren te Dordrecht

Promotiecommissie

Promotores: Prof. dr. I.B. Tan

Prof. dr. A.J.M. Balm

Copromotores: Prof. dr. J.M. Middeldorp

Dr. B. Hariwiyanto

Dr. J.P. de Boer

Overige leden: Prof. dr. S. van der Baan

Prof. dr. G.C. de Gast

Prof. dr. F.J.M. Hilgers

Prof. dr. C.R.N. Rasch

Prof. dr. D.L. Willems

Faculteit der Geneeskunde

Voor mijn moeder

Content

Chapter 1: Introduction and outline thesis ....................................................................................................9

Chapter 2: Can an Online Clinical Data Management service help in improving data collection and data quality in a developing country setting? ............................. 27

Chapter 3: Primary treatment results of Nasopharyngeal Carcinoma in Yogyakarta, Indonesia ............................................................................................................................................ 39

Chapter 4: Knowledge of general practitioners about nasopharyngeal cancer at the Puskesmas in Yogyakarta, Indonesia ................................................................................ 51

Chapter 5: Short-term effect of different teaching methods on Nasopharyngeal Carcinoma for general practitioners in Jakarta Indonesia ............................................... 61

Chapter 6: Photodynamic therapy in the therapy for recurrent/persistent nasopharyngeal cancer................................................................................................................. 75

Chapter 7: Temoporfin mediated photodynamic therapy in patients with local persistent and recurrent Nasopharyngeal Carcinoma after Curative Radiotherapy: a feasibility study .............................................................................................. 87

Chapter 8: Cytolytic virus activation therapy for Epstein-Barr virus driven tumors ...................101

Chapter 9: Summary and Discussion ...........................................................................................................119

Chapter 10: Dutch summary / Nederlandse samenvatting ...................................................................127

List of publications / Publicatielijst .........................................................................................135

Curriculum Vitae ............................................................................................................................137

Acknowledgements / Dankwoord ..........................................................................................138

Sponsor page / Sponsorpagina ...............................................................................................142

Chapter 1

Introduction

Chapter 1

10

Scope

Cancer has become a leading cause of death and disability in many low and middle-income countries. In Southeast Asia one of the most frequently encountered malignancies is Nasopharyngeal Carcinoma (NPC). Fortunately, NPC is quite sensitive to radiotherapy, making treatment highly effective when given in a timely manner and according to proper protocols. Unfortunately, in contrast to high-income countries, the infrastructure for radiotherapy in these regions is often not optimal, whereby such effective treatment cannot always be delivered. The current literature in Indonesia addressing NPC has focused primarily on the incidence of the disease, without careful consideration toward additional clinical perspectives, such as public awareness, knowledge among health care providers, treatment results and survival rates. Advances in these specific clinical areas would allow better insight into the magnitude of the NPC epidemic in Indonesia and provide a more solid basis for the development of new diagnostic and treatment strategies. This thesis is intended to contribute to these areas.

Focus of this thesis

Since 1999, the Netherlands Cancer Institute’s department of Head and Neck Surgery and Oncology has been involved in a KWF (Dutch Cancer Society) sponsored program with the Gadjah Mada University in Yogyakarta, Indonesia. The goal of this collaboration was to increase the capacity of the Head and Neck Surgery and Oncology department within the Dr Sardjito University Hospital in Yogyakarta to an international standard. After a few years into the exchange program, it became clear that essential requirements regarding pathology, radiology and radiotherapy were not sufficiently developed to achieve this.

The most striking obstacle in meeting these requirements was the lack of data management, making it impossible to evaluate treatment protocols and treatment results. Doctors, therefore, also lacked the essential feedback, resulting in the absence of a learning curve. This continued lack of data management made it further impossible to gain ground into the actual problems regarding general head and neck cancer care. Therefore, the decision was made to reduce the scope of generalized head and neck cancer to focus on the most prevalent disease encountered: NPC. By focusing on the most common head and neck malignancy in Indonesia, ranking number 4 in all cancers among males, a multi-disciplinary team of head and neck surgeons, radiation oncologists and medical oncologists could achieve optimal treatment outcomes. This choice for NPC has the added advantage that, if it turns out to be possible to develop optimal strategies for this cancer, this would provide a blueprint for the future development of similar strategies for other (head and neck) malignancies.

With these precedents in mind, this thesis will address the current pitfalls concerning care for patients with primary NPC in Indonesia, seek potential solutions, and create treatment options suitable for the specific local infrastructure in Indonesia.

Nasopharyngeal Carcinoma

NPC arises in the epithelial lining of the nasopharynx and is frequently found at the pharyngeal recess (Rosenmüller’s fossa) posteromedial to the medial crura of the Eustachian tube opening in the nasopharynx[1]. The annual incidence of NPC is 1 per 100.000 with uneven distribution throughout the world. In western countries NPC is an orphan disease. In the Netherlands, for example, the incidence of NPC is 40-80 new cases a year [2,3]. In contrast, high incidences of NPC are found in South-East Asia and Northern Africa. In Indonesia,

Introduction

11

NPC is the most frequent cancer of the head and neck; and, as mentioned above, is the fourth most common tumour occurring in males. Based on the incidence numbers of neighbouring countries, such as Malaysia, the incidence of NPC in Indonesia is estimated 6 per 100000[4]. However, this is likely to be an underestimation.

Pathology

The NPC cells are of squamous origin [5]. Histopathologically, NPC is divided into three types based on degree of differentiation, i.e. keratinizing squamous cell carcinoma (WHO type I) highly differentiated and characterized by epithelial growth patterns and keratin filaments, non-keratinizing squamous cell carcinoma (WHO type II) with retaining epithelial cell shape and growth pattern, and undifferentiated carcinoma (WHO type III), which does not produce keratin and lacks a distinctive growth pattern[6]. In endemic areas, undifferentiated type comprises the vast majority of NPC, while keratinized squamous cell carcinoma is less common [7].

Epstein-Barr virus (EBV) is consistently detected in patients with NPC from regions of high and low incidence. In nearly all tumour cells an EBV-encoded RNA signal has been demonstrated by in-situ hybridisation, whereas EBV-encoded RNA is absent from the adjacent normal tissue, except perhaps in a few scattered lymphoid cells[8]. The relation between the EBV and NPC is explained in more detail below.

Risk factors for NPC

Although genetic defects and epigenetic changes regulating the expression of various tumour suppressor genes are implicated in the pathogenesis of NPC, the most consistent risk factor in the tumour development of NPC EBV [9,10]. The undifferentiated carcinoma (WHO type III) is always associated with EBV [6]. High levels of volatile nitrosamines in preserved foods and butyrate in animal fat or dried meat, which are frequently consumed in high incidence regions and can trigger chronic EBV reactivation, have been shown to be putative co-carcinogen(s) for the development of NPC. Non-environmental risk factors include: gender, ethnicity and the family history [11-16].

Presentation

Patients with NPC present symptoms of the following categories: (1) presence of tumour mass in the nasopharynx leading to epistaxis, nasal obstruction and nasal discharge; (2) dysfunction of the Eustachian tube leading to (unilateral) otitis media with effusion, tinnitus and hearing loss; (3) skull base erosion and palsy of the 5th and 6th nerve leading to headache, diplopia, facial pain and numbness; and (4) neck mass; painless enlargement of the upper cervical lymph node.

Evaluation of 4,768 NPC subjects by Lee et al, summarized the symptoms at presentation as neck mass (76%), which related to cervical nodal metastases, nasal dysfunction (62%), headache (35%), diplopia (11%), facial numbness (8%), weight loss (7%) due to metastatic spread, and trismus (3%). Symptoms were found to be similar for both young and adult NPC subjects [17].

The early symptoms of NPC such as epistaxis, nasal obstruction, and hearing loss are not specific for NPC, which makes NPC difficult to diagnose at an early stage [18]. Accordingly, most newly diagnosed patients (75-90%) have loco-regionally advanced disease at presentation in the hospital and thus present with late stage disease[19,20].

Chapter 1

12

Staging and diagnosis

NPC staging is based on two TNM classifications, by Ho and the AJCC/UICC classification respectively (American Joints Committee/ International Union Against Cancer). Ho’s classification is predominantly used in Asia as the highest incidence region, while AJCC and UICC are utilized more in USA and Europe [21]. Main differences are the nodal classification of the Ho’s classification, which has incorporated prognostic significance, and the deviation of the t-stage in five sectors (see table 1).

Table 1 | NPC staging according to Ho and AJCC

The American Joint Committee on Cancer Staging Ho Staging

Tumour in the Nadopharynx (T) Primary tumour (T)

T1 Tumour confined to the nasopgharynx T1 Tumour confined to nasopharynx (space behind choanal orifices and nasal septum and aboveposterior margin of soft palate in resting position)

T2 Tumour extends to soft tissues of oropharynx and/or nasal fossa

T2 Tumour extended to nasal fossa, oropharynx, or adjacent muscles or nerves below base of skull

T2a without parapharyngeal extension

T2b with parapharyngeal extension

T3 Tumour invades bony structures and/or paranasal sinuses

T3 Tumour extended beyond T2 limits and subclassified as follows:

T3a Bone involvement below base of skull (floor of sphenoid sinus is included in this category)

T3b Involvement of base of skull

T3c Involvement of cranial nerve(s)

T3d Involvement of orbits, laryngopharynx (hypopharynx), or infratemporal fossa

T4 Tumour with intracranial extension and/or involvement of cranial nerves, infratemporal fossa, hypopharynx, or orbit

Regional lymph nodes (N) Regional lymph nodes (N) Regional lymph nodes (N) Regional lymph nodes (N)

NX Regional lymph nodes cannot be assessed

N0 No regional lymph node metastasis N0 Node palpable or thought to be benign

N1 Unilateral metastasis in lymph node(s), 6 cm or less in greatest dimension, above the supraclavicular fossa

N1 Node(s) wholly in upper cervical level, bounded below by the skin crease extending laterally and backward from or just below thyroid notch (laryngeal eminence)

N2 Bilateral metastasis in lymph node(s), 6 cm or less in greatest dimension, above the supraclavicular fossa

N2 Node(s) palpable between crease and supraclavicular fossa, the upper limit being a line joining the upper margin of the sternal end of the clavicle and the angle formed by the lateral surface of the neck and the superior margin of the trapezius

N3 Metastasis in a lymph node(s) N3 Node(s) palpable in the supraclavicular fossa and/or skin involvement in the form of carcinoma en cuirasse or satellite nodules above the clavicles

N3a greater than 6 cm in dimension

N3b extension to the supraclavicular fossa

Distant metastasis (M) Metastases (M)

MX Distant metastasis cannot be assessed

M0 No distant metastasis M0 No haematogenous metastases

M1 Distant metastasis M1 Haematogenous metastases present, and/or lymph nodal metastases below the clavicle

Introduction

13

Stage grouping Stage grouping

Stage 0 T1s N0 M0

Stage I T1 N0 M0 Stage I T1 N0

Stage IIA T2a N0 M0 Stage II T2 and/or N1

Stage IIB T1 N1 M0

T2 N1 M0

T2a N1 M0

T2b N0 M0

T2b N0 M0

Stage III T1 N2 M0 Stage III T3 and/or N2

T2a N2 M0

T2b N2 M0

T3 N0 M0

T3 N1 M0

Stage IVA T4 N0 M0 Stage IV N3 (any T)

T4 N1 M0

T4 N2 M0

Stage IVB Any T N3 M0

Stage IVC Any T Any N M1

Stage IV M1

Clinical examination, with endoscopic examination, can provide valuable information about mucosal involvement and tumour extension into the nasal fossae and oropharynx. Imaging with CT scan and MRI scan give a better insight in tumour extension, skull-base erosion, or intracranial spread. Furthermore, combined (cross-sectional) imaging with MRI and CT in the treatment planning of NPC, improves the effectiveness of the radiotherapy (see below). Radiological imaging has identified para-nasopharyngeal extension as one of the most common modes of extension of NPC and has shown perineural spread through the oval foramen to be an important route of intracranial extension [22,23].Compared with CT imaging, MRI is better for displaying both superficial and deep nasopharyngeal soft tissue invasion and for differentiating tumour from normal soft tissue. MRI is also more sensitive for assessment of retropharyngeal and deep cervical nodal metastases. Screening for distant metastasis is best achieved with positron emission tomography (PET). Accordingly, the combination of PET/CT and head-and neck MRI are suggested for the initial staging of NPC patients[24]. Unfortunately, this combination is not utilized or not available in Indonesia, where only CT-imaging is used for initial loco-regional staging in combination with ultra sound and bone scans for distant metastasis screening.

Primary treatment of Nasopharyngeal Carcinoma

The optimal treatment modality for primary NPC is radiotherapy. Although NPC is quite sensitive to ionizing radiation, location of the tumour at the base of skull, close to the brain stem and spinal cord, requires radiation dose limitations in order to prevent radiation-induced long-term side effects.

For locally advanced NPC, the standard care is concurrent chemo-radiotherapy with cisplatin-based regimens, resulting in three-years disease-free and overall survival of 70% and 80%, respectively [25,26]. A recent meta analysis confirmed the clinical benefit of concurrent chemoradiotherapy compared with radiation alone (RT) in the treatment of NPC in endemic areas [27].

With the introduction of intensity-modulated radiotherapy (IMRT), local and regional failure rates have declined and the biggest remaining challenge is the effective treatment of distant metastasis with occurrence

Chapter 1

14

rates of 15-19% [28-31]. Besides new radiotherapy techniques, research of the Hong-Kong group also revealed improved outcomes if the staging and treatment planning is performed with MRI compared to CT-scan[29].

Treatment of locally recurrent and persistent NPC

Options for treatment of locally recurrent NPC are brachytherapy, external re-irradiation, stereotactic radiosurgery and (minimally invasive) nasopharyngectomy. These treatment modalities can be used either alone or in combination [32-36]. Re-irradiation is the most commonly used therapy for local recurrent NPC. Responses and new developments in technology of re-irradiation are promising, but the ‘price to pay’ is a high incidence of major late complications, such as brain tissue necrosis, cranial nerve palsies, and catastrophic haemorrhages [37-41]. Presently, locally recurrent disease re-irradiation and surgery are not feasible in Indonesia due to lack of trained personnel, proper equipment, and the absence of re-treatment time due to long waiting times for treatment of primary tumours. In persistent disease, there is limited role for re-irradiation. Locally persistent tumours after radiotherapy or chemo-radiation can sometimes be addressed surgically, either through a minimal invasive approach, or with a mandibular swing procedure [42-44]. For both surgical techniques, a well-trained surgical team and high-tech operating theatre facilities are needed. Unfortunately, both are not yet available in Indonesia.

Photodynamic Therapy (PDT)

PDT is an established non-invasive treatment modality for incurable head and neck cancer[45,46]. A photosensitizer is administered to the patient followed by illumination of the tumour with a specific wavelength, of 652 nM, which causes tumour destruction. Several clinical trials with first generation haematoporphorin-derived photosensitizers (HpD or Photofrin) have shown that PDT is effective in destroying NPC, with good local tumour control and complete responses in the majority of patients with limited recurrent or persistent disease, while achieving long-term palliation in cases with extensive recurrence[47-51]. Although these results were encouraging, PDT for NPC has not yet been considered as a break through in this field. The two major drawbacks of these studies were (1) the light delivery and (2) the selection of photosensitizer. First, light delivery in the nasopharynx is extremely difficult. It is almost impossible to illuminate the whole tumour area with a lens fiber, guided with endoscopes or trans-orally. This problem of proper illumination of the nasopharyngeal cavity has now been solved by the development of a special applicator, which allows one-stage illumination of the entire nasopharynx (Figure 2)[52]. The second drawback is the use of HpD/Photophrin (First generation photsensitizer). This photosensitizer has a limited depth penetration of < 5 mm and a prolonged light hypersensitivity of several months. Temoporfin (Foscan®), a second-generation photosensitizer and approved in Europe for treatment of incurable head and neck cancer, has a depth penetration of 1 cm and light hypersensitivity of only a few weeks. Research in NPC cell lines by Yow et al. confirmed that Temoporfin showed much better PDT efficiency than HpD[53]. These authors also observed significant photo destruction of the mitochondria, especially in Foscan® mediated PDT. Mitochondria are an important sub-cellular target and may play a role in cell death [54]. Temoporfin, in combination with the special designed nasopharyngeal applicator for proper illumination, has the potential to effectively treat NPC, also in the current Indonesian medical system [55].

Introduction

15

Figure 1 | A: Nasopharynx applicator, B: schematic view of positioning and illumination. 1. Cylindrical diffuser in shielding tube. 2. Target area. 3. Soft palate is shielded. This figure has been previously used and kindly provided by Nyst et al. [52].

Treatment of metastatic NPC

For the treatment of metastatic NPC several chemotherapeutic schedules have been tested, showing that NPC has high level of response and enduring responses[56]. The first-line treatment consists of platin-based regimens; after progression under platins, the most frequently tested second-line drugs are gemcitabine, capecitabine or taxanes, all with acceptable results.

A recent review by Bensouda et al. showed that gemcitabine (GCb) is one of the most effective single agent therapies in NPC [57]. The largest study with 32 patients treated with GCb, after platinum-based chemotherapy failed, showed 43.8% partial response and 28.1 % stable disease with minimal side effects [58].

Epstein Barr Virus (EBV)

EBV is a ubiquitous human gamma herpes virus with a persistent infection ranging from 80 to 99 % of the worldwide population. Infection is via direct salivary contact. In developing countries, primary infection occurs in early infancy and is asymptomatic in most cases. In western countries primary infection is more often delayed until adolescence and then causes infectious mononucleosis in up to 25% of the cases[59].

In 1997 WHO recognized EBV as “Class 1 human carcinogenic virus.”[60]. EBV is also associated with a variety of lymphoid (Burkitt’s lymphoma, Hodgkin’s Lymphoma, Lymphoepthelioma-like carcinoma, extranodal NK/T-cell lymphoma) and gastric cancer[61].

Epstein Barr Virus (EBV) and NPC

In case of undifferentiated NPC there is a causative link with EBV. The relation between EBV infection and NPC is complex and, based on the observation that the presence of EBV in the tumour cells is associated with faster cell growth, less differentiation, and a higher metastasis rate of NPC, in comparison to tumours which do not contain EBV DNA [62].

Chapter 1

16

EBV-markers for screening, diagnostic and monitoring

Prior studies have shown that EBV-related markers can be used for early detection (screening) and prognostic monitoring. These markers include EBV (IgA) serology and EBV-DNA load since NPC patients have characteristic elevated IgG and IgA antibody titres to several EBV encoded antigens as well as increased EBV-DNA derived from shed (apoptotic) fragments from the tumour into the circulation. Increased IgA antibody levels are found against early antigen (EA), viral capsid antigen (VCA) and the latent Epstein-Barr nuclear antigen 1 (EBNA1) as well as inhibitory antibodies to the EBV specific DNase[63,64]. These antibody responses against defined viral antigens are the basis of a proposed screening test for NPC in high risk populations[65-67]. Recent insight in the molecular basis and diversity of anti-EBV IgA and IgG responses allowed the development of more defined serological tools[68-72]. EBV DNA load in the circulation and in nasopharyngeal brushings can be used in addition as independent NPC-related EBV markers, since both have been detected in a higher proportion of NPC patients than controls[73-77]. EBV IgA serology testing especially appears to fulfil criteria as a possible screening tool in the future, since the cost is relatively low and easy to use when combined with finger-prick blood sampling [71,78]. Patients with elevated EBV-IgA serology plus defined chronic symptoms suggestive of NPC may be selected for more costly EBV-DNA testing in the nasopharyngeal brushing to make early-stage NPC diagnosis possible [79].

EBV genes

The genome of EBV is a linear double stranded DNA molecule of 172,000 base pairs and encodes for 80 proteins. In most cases of EBV malignancies the viral genome is not integrated into the cellular DNA but forms circular episomes that replicate aside the host chromosomes. The integrated form of EBV DNA is occasionally found in B cell lymphoma and especially in high grade B cell lymphoma [80].

EBV infection can be latent or lytic (figure 2). In latent infected cells only a few proteins are transcribed and the genes coding for these proteins are therefore referred as ‘latent genes’[81].These latent genes of EBV are EBNA1, EBNA2, LMP1, LMP2, EBNA3, and LP. Only EBNA1 is expressed in all latent infected cells[82]. Another group of genes, the lytic genes, are involved in viral DNA replication and formation of new virions. These genes are classified in thee different types: immediate-early genes (IE genes), early genes (E genes) and late genes (L genes).

Immediate-early genes

The lytic cascade starts with activating the expression of IE genes like in all other herpes viruses. These genes are BZLF1 and BRLF1 and encode the transactivators ZEBRA (Zta) and Rta, which are responsible for activation of the other lytic phase genes. The BZLF1 gene encodes a sequence specific DNA-binding protein ZEBRA. ZEBRA can bind many ZEBRA-responsive elements (ZRE’s), which are present in the BZLF1 promoter (positive regulation) and in promoters of EBV early genes[83].The promoter of the BZLF1 gene is called Zp (ZEBRA promoter), and this promoter is thus very important in regulating the switch from latent to lytic phase. The BZLF1 gene can also be activated by the Rta, but the main expression of the BZLF1 gene is due to activation via Zp[84]. It has been demonstrated that in some of EBV-associated malignancies, sporadic tumour cells show detectable ZEBRA expression. Since true late gene transcription in these tumour cells is mostly absent, these levels of ZEBRA expression probably reflect partial (abortive) activation of the lytic phase[85]. The other IE gene, BRLF1, encodes for the protein Rta, which acts together with ZEBRA to activate the E genes.

Introduction

17

Early genes

The E genes are also called replication factors. These genes encode enzymes that are involved in DNA synthesis and nucleotide metabolism or interfere with the apoptosis pathway to ensure prolonged cell survival. Therefore, these proteins/enzymes encoded by these genes (like EBV-PK) can be potential targets for anti-viral drugs and are therefore important for the development of new anti-tumour therapies[86].

Late genes

After activation of both IE and E genes, the L genes are activated. These L genes are mainly structural proteins and the expression of these genes relies on new linear genomic templates. These genes do not only encode structural proteins of the virion, but also a few non-structural genes. Among the structural proteins are VCA-p18, a small capsid protein, and VCA-p40 and Gp125, nuclear membrane proteins. These proteins are strongly immunogenic in humans and serve as targets for sero-diagnosis, because almost all EBV carriers develop antibodies to these proteins. A non-structural late protein is vIL-10, which has been shown to be correlated with a poor prognosis.

Figure 2 | Epstein-Barr virus infection can be latent or lytic. Both states are associated with their own profile of gene expression. The lytic state consists of 3 phases, the immediate early (IE), the early (E), and the late (L) phase.

Indonesia1

Indonesia is a democratic republic, proclaimed by President Soekarno on the Declaration of Independence of Indonesia on August 17th 1945. The country is one of the largest archipelagos in the world consisting of 17,508 islands totalling 248 million inhabitants. Islam is the major religion (85.2% of the population), designating

1 Sources: http://www.en.indonesia.nl/content/view/145/60/;

http://en.wikipedia.org/wiki/Indonesia

http://www.who.int/countryfocus/cooperation_strategy/ccs_idn_en.pdf;

http://www.ino.searo.who.int/EN/Section3_256.htm;

https://www.cia.gov/library/publications/the-world-factbook/geos/id.htm

Chapter 1

18

Indonesia as the largest Muslim country in the world. The remaining population consists of Protestants (8.9%); Catholics (3%); Hindus (1.8%); Buddhists (0.8%); and other religions (0.3%).

Economy

Indonesia has abundant natural resources including crude oil, natural gas, tin, copper and gold. Despite being the second largest exporter of natural gas and an abundance of crude oil, Indonesia has recently needed to import crude oil for increased consumption. The agriculture products of Indonesia include rice, tea, coffee, spices and rubber. The major trade partners of Indonesia are Japan, the United States of America and neighbouring countries Malaysia, Singapore and Australia. Despite its wealth in natural resources, the country is still facing crucial issues of poverty because of the uneven distribution of the national income.

Since the 1990s, Indonesia had experienced an improvement in socioeconomic indicators. The proportion of population living in poverty dropped. There was a setback in mid-1997 due to the Southeast Asian economic crisis, which affected the Indonesian economy the hardest in comparison with other countries in the region. The economic crisis resulted in the departure of president Soeharto, who had been in power for 31 years. Most recently the proportion of people living in poverty has further declined. In 2006, an estimated 17.8% of the population lives below the poverty line, 49.0% of the population lives on less than US$2 per day, and the unemployment rate was 9.75%. As of 2010, an estimated 13.3% of the population was living below the poverty line, and the unemployment rate was 7.1%.

Health care system

Health expenditure in Indonesia is 5.5 % of Gross Domestic Product (GDP). This is low compared to other countries in the region, with neighbouring Malaysia at 8% of GDP. It is comparatively worse when factored against western countries such as the Netherlands, which is currently at 10,4% of GDP. The primary health care level is generally regarded as having relatively adequate levels of provision, with an average of one public health centre for every 30 000 people. However, the quality of the health workforce, deficiencies in human resources and reportedly low productivity still remain a problem. For many decades the main health problem was related to communicable disease, whereas currently an epidemiological shift towards non-communicable diseases, cancer among them, is ranking high. According to the WHO, this will become is an increasing a major problem and an additional challenge for Indonesia. Cancer is becoming a burden in terms of cost, suffering and human lives. Unfortunately these diseases are affecting not only wealthy people but also poor people, which will reduce their ability to generate adequate income and again lead to further impoverishment. Even in cases where insurance is available for the poor, it rarely covers all expenses and frequently the entire community spends all their savings for one patient. Unfortunately, in most cases, this spending is wasted due to poor treatment results, insufficient counselling, patient’s delay, doctor’s delay and long waiting periods.

Introduction

19

Brief outline Thesis

To address the major questions regarding the improvement of treatment and diagnosis of NPC in Indonesia, we performed a prospective study and a survey among GPs. The results are the fundaments of the described current problems on NPC-care in Indonesia (chapter 3 and 4). The other chapters describe possible solutions: implementation of data management (chapter 2), training of General Practitioners for better awareness and knowledge of NPC, and thus for earlier recognition of the disease (chapters 5), and the introduction of new treatment modalities for NPC to eventually improve treatment outcome (chapters 6, 7, and 8).

Chapter 2: Can an online clinical data management service help in improving data collection and data quality in a developing country setting? (Wildeman MA, Zandbergen J (contributed equally), Vincent A, Herdini C, Middeldorp JM, Fles R, Dalesio O, van der Donk E, Tan IB. Trials. 2011 Aug 8;12:190.)

Data collection by Electronic Medical Record (EMR) systems has been proven to be helpful in data collection for scientific research and in improving healthcare. This study shows that the introduction of a Clinical Trial Data Management service (CTDMS) composed of electronic Case Report Forms (eCRF) results in effective data collection and treatment monitoring. The digital nature of the CTDMS, as well as the online availability of that data, gives fast and easy insight in adherence to treatment protocols. As such, the CTDMS can serve as a tool to train and educate medical doctors and can improve treatment protocols.

Chapter 3: Primary treatment results of Nasopharyngeal Carcinoma (NPC) in Yogyakarta Indonesia (M.A. Wildeman, R. Fles, C. Herdini, S.R. Indrasari, A.D. Vincent, M. Tjokronagoro, S. Stoker, J. Kurnianda, B. Karakullukcu, K.W. Taroeno-Hariadi, O. Hamming-Vrieze, J.M. Middeldorp, B. Hariwiyanto, S.M. Haryana, I.B. Tan, submitted)

Treatment of primary Nasopharyngeal Carcinoma (NPC) with radiotherapy and/or concurrent chemo-radiation shows good response rates, as reported in many series in the literature. We present a prospective cohort of patients with curable NPC who have been treated in 2009 till 2011. In contrast to the literature, the treatment results found in this series are very disappointing. The identification of the causes for the high rate of treatment failures will hopefully lead to a series of interventions, which will eventually lead to improvement of these results.

Chapter 4: Knowledge of general practitioners about nasopharyngeal cancer at the Puskesmas in Yogyakarta, Indonesia (Fles R, Wildeman MA (contributed equally), Sulistiono B, Haryana SM, Tan IB. BMC Medical Education 2010, 10:81.)

This study reveals that general practitioners (GPs) in the Primary Health Care Centres (PHCC) in Yogyakarta lack knowledge on all aspects of Nasopharyngeal Carcinoma (NPC). This is an important finding as NPC is endemic in Indonesia and the PHCC are the institutions that provide primary medical health care in the country.

Chapter 5: Short-term effect of different teaching methods on Nasopharyngeal Carcinoma for general practitioners in Jakarta Indonesia (Wildeman MA, Fles R, Adham M, Mayangsari ID, Luirink I, Sandberg M, Vincent AD, Fardizza F, Musa Z, Armiyanto, Middeldorp JM, Gerritsen G, Suwanto R, Tan IB. PLoS One. 2012;7(3):e32756.)

This study confirms our findings from Yogyakarta regarding GPs insufficient knowledge of NPC and proves that lectures in the Primary Health Care Centre by a team of two GPs and a symposium by local head and neck surgeons have both been proven to be effective training tools in the education of GPs on NPC.

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Chapter 6: Photodynamic therapy in the therapy for recurrent/persistent nasopharyngeal cancer (Wildeman MA, Nyst HJ, Karakullukcu B, Tan BI. Head & Neck Oncology 2009, 1:40)

A review of five studies conducted in the past with Photodynamic therapy for recurrent/persistent NPC; these studies were all with first generation photo sensitizer and without a local application device tailored for optimal delivery of the laser light to the nasopharynx. Despite this, the results of these studies are very promising.

Chapter 7: Temoporfin mediated photodynamic therapy in patients with local recurrent nasopharyngeal carcinoma (Nyst HJ, Wildeman MA, Indrasari SR, Karakullukcu B, van Veen RL, Adham M, Stewart FA, Levendag PC, Sterenborg HJ, Tan IB. Photodiagnosis Photodyn Ther. 2012 Sep;9(3):274-81.)

A feasibility study with Temoporfin mediated PDT in 22 patients with persistent (n=21) and recurrent (n=1) Nasopharyngeal Carcinoma. This novel treatment with a special designed nasopharyngeal applicator and second-generation photo sensitizer showed no severe side effects and a good clinical response.

Chapter 8: Cytolytic virus activation therapy for Epstein-Barr virus driven tumours (Wildeman MA, Novalic Z, Verkuijlen SA, Juwana H, Huitema AD, Tan IB, Middeldorp JM, de Boer JP, Greijer AE. Clin Cancer Res. 2012 Sep 15;18(18):5061-70.)

Since all NPC tumour cells carry EBV, the virus itself is a potential target for therapy. In these tumour cells EBV “hides” in a latent state and expresses only few non-immunogenic viral proteins essential for EBV maintenance and contributing to tumour growth. We developed a cytolytic virus activation (CLVA) therapy, which activates virus gene expression in tumour cells by epigenetic modulation, triggering immune recognition and induces susceptibility to antiviral therapy.

Our results in the first CLVA treated patients indicate that induction therapy had a biological effect and was well tolerated. Virus specific tumour therapy will open a generic approach for treatment of multiple EBV-associated malignancies both in developed and developing countries worldwide.

Chapter 9: Summary and future perspectives

Chapter 10: Summary in Dutch

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Introduction

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67. de Vathaire F, Sancho-Garnier H, de The H, Pieddeloup C, Schwaab G, Ho JH, Ellouz R, Micheau C, Cammoun M, Cachin Y et al.: Prognostic value of EBV markers in the clinical management of nasopharyngeal carcinoma (NPC): a multicenter follow-up study. Int J Cancer 1988, 42:176-181.

68. Fachiroh J, Schouten T, Hariwiyanto B, Paramita DK, Harijadi A, Haryana SM, Ng MH, Middeldorp JM: Molecular diversity of Epstein-Barr virus IgG and IgA antibody responses in nasopharyngeal carcinoma: a comparison of Indonesian, Chinese, and European subjects. J Infect Dis 2004, 190:53-62.

69. Fachiroh J, Paramita DK, Hariwiyanto B, Harijadi A, Dahlia HL, Indrasari SR, Kusumo H, Zeng YS, Schouten T, Mubarika S et al.: Single-assay combination of Epstein-Barr Virus (EBV) E. J Clin Microbiol 2006, 44:1459-1467.

70. Paramita DK, Fachiroh J, Artama WT, van Benthem E, Haryana SM, Middeldorp JM: Native early antigen of Epstein-Barr virus, a promising antigen for diagnosis of nasopharyngeal carcinoma. J Med Virol 2007, 79:1710-1721.

71. Fachiroh J, Prasetyanti PR, Paramita DK, Prasetyawati AT, Anggrahini DW, Haryana SM, Middeldorp JM: Dried-blood sampling for epstein-barr virus immunoglobulin G (IgG) and IgA serology in nasopharyngeal carcinoma screening. J Clin Microbiol 2008, 46:1374-1380.

72. Paramita DK, Fachiroh J, Haryana SM, Middeldorp JM: Two-step Epstein-Barr virus immunoglobulin A enzyme-linked immunosorbent assay system for serological screening and confirmation of nasopharyngeal carcinoma. Clin Vaccine Immunol 2009, 16:706-711.

73. Lin JC, Wang WY, Chen KY, Wei YH, Liang WM, Jan JS, Jiang RS: Quantification of plasma Epstein-Barr virus DNA in patients with advanced nasopharyngeal carcinoma. N Engl J Med 2004, 350:2461-2470.

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75. Stevens SJ, Verkuijlen SA, Hariwiyanto B, Harijadi, Paramita DK, Fachiroh J, Adham M, Tan IB, Haryana SM, Middeldorp JM: Noninvasive diagnosis of nasopharyngeal carcinoma: nasopharyngeal brushings reveal high Epstein-Barr virus DNA load and carcinoma-specific viral BARF1 mRNA. Int J Cancer 2006, 119:608-614.

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carcinoma cells: coexpression of EBNA1, LMP1, and LMP2 transcripts. J Virol 1992, 66:2689-2697. 83. Heather J, Flower K, Isaac S, Sinclair AJ: The Epstein-Barr virus lytic cycle activator Zta interacts with methylated

ZRE in the promoter of host target gene egr1. J Gen Virol 2009, 90:1450-1454. 84. Manet E, Gruffat H, Trescol-Biemont MC, Moreno N, Chambard P, Giot JF, Sergeant A: Epstein-Barr virus bicistronic

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Chapter 2

Can an Online Clinical Data Management service help in improving data collection and data quality in a

developing country setting?

Maarten A. Wildeman*,

Jeroen Zandbergen*,

Andrew Vincent,

Camelia Herdini,

Jaap M. Middeldorp,

Renske Fles,

Otilia Dalesio,

Emile van der Donk,

I. Bing Tan

* Both authors contributed equally

Trials. 2011 Aug 8;12:190.

Chapter 2

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Background

Data collection by Electronic Medical Record (EMR) systems have been proven to be helpful in data collection for scientific research and in improving healthcare. For a multi-centre trial in Indonesia and the Netherlands a web based system was selected to enable all participating centres to easily access data. This study assesses whether the introduction of a Clinical Trial Data Management service (CTDMS) composed of electronic Case Report Forms (eCRF) can result in effective data collection and treatment monitoring.

Methods

Data items entered were checked for inconsistencies automatically when submitted online. The data were divided into primary and secondary data items. We analysed both the total number of errors and the change in error rate, for both Primary and Secondary items, over the first five month of the trial.

Results

In the first five months 51 patients were entered. The Primary data error rate was 1.6%, whilst that for Secondary data was 2.7 % against acceptable error rates for analysis of 1% and 2.5% respectively.

Conclusion

The presented analysis shows that after five months since the introduction of the CTDMS the Primary and Secondary data error rates reflect acceptable levels of data quality. Furthermore, these error rates were decreasing over time. The digital nature of the CTDMS, as well as the online availability of that data, gives fast and easy insight in adherence to treatment protocols. As such, the CTDMS can serve as a tool to train and educate medical doctors and can improve treatment protocols.

Can an Online Clinical Data Management service help in improving data collection and data quality in a developing country setting

29

Background

Data collection concerning medical needs is required to assess the effectiveness of interventions and current health care practices [1]. Furthermore, data collection by Electronic Medical Record (EMR) systems has been proven to be helpful in data collection for scientific research and can be helpful in improving healthcare. These EMR systems allow for the early identification of missing data and the patients possibly loss-to-follow-up, which is essential for the conduct of proper scientific research [2-6].

A Clinical Trial Data Management service (CTDMS) has been introduced for running a multicenter clinical trial in Indonesia and in the Netherlands. The same system has also been introduced for monitoring treatment results of Nasopharyngeal Carcinoma (NPC) in Indonesia.

In most countries NPC is an orphan disease, but overall has a worldwide incidence of 80.0000 new cases per year, being endemic in Northern Africa, Southern China and Hong Kong, and the South-East Asian peninsula, including Malaysia, Vietnam, Thailand, Singapore and Indonesia.

In Indonesia NPC is the most frequent cancer in the head and neck area and ranks as the 4th most common tumour found in males. The incidence is estimated 6 per 100.000, leading to 12.000 new cases per year [7] [8]. Little is known about treatment results of NPC in Indonesia.

The CTDMS system was selected because of the web-based nature which makes the data approachable for all participating parties. This online accessible data system has made it easier for the principal investigator to check the data for inconsistencies. The senior physician can easily see if treatment is according protocol.

This study assesses whether the introduction of CTDMS composed of online Case Report Forms (eCRF) can result in improved patient outcomes. The assessment focuses on data quality and the identification of possible bottle necks within the patient care process.

This study investigates if a web based CTDMS can be helpful in proper data collection by analysing errors in data items. Bottle necks in patient care are analysed by comparison of treatment plan and actual treatment.

Methods

The CTDMS is constructed for the NPC Clinical Trial: Early detection of primary and recurrent NPC using (anti-)EBV based tumour markers and evaluation of primary treatment for NPC (funding KWF NKI-2008-4233). A technical description of the CTDMS is provided in Appendix 1. The database is comprised of 10 online electronic Case Report Forms (eCRF’s). In order to prevent errors from being entered, data validation rules were implemented into the eCRF’s prior to commencement of the NPC Clinical trial. These data validation rules assess whether certain pre-specified conditions are valid and can therefore pin-point omissions or erroneous data. Online warning messages notify the data-manager (entering data) when errors are detected. Commonly used checks are, for instance, range checks that verify whether values are within the boundaries dictated by the study protocol, and mandatory field checks (i.e. ‘This field cannot be blank’).

Of the 10 eCRF’s, 9 were required to be completed multiple times per patient during the study and only 1 was to be completed and submitted once per patient. Each of these submissions is a unique realization of the form. For example, for one patient a laboratory form is completed during baseline measurements, just before the start of the treatment. Once this form is submitted through the CTDMS, there is one realization of the laboratory form stored in the data base for this patient. After the patient received treatment, a laboratory

Chapter 2

30

form is completed and submitted again. The data base then contains two realizations of the laboratory form, for that patient. Each realization may be submitted multiple times if it contained errors. We note that it is impossible to claim that an entered form for which no warning messages were displayed is clean, as new errors may be found later.

The data-manager completing an eCRF has the option to ignore (override) a warning message, however in such cases, he/she is required to provide an explanation which is recorded in an Audit Trail entry field (error log). Warning messages and error logs are also created when an incorrect value or data-type is entered, an omission is detected, or when a previously entered value is changed. Changed values are considered to be (previously undetected) errors that have now been rectified (except when the changed value also triggers a check to fire, in which case the data is considered unclean).

The eCRF’s contain differing quantities of data. Each field to be entered is considered a data item, which were designated either as primary or secondary. Primary data items are data that were considered essential for the assessment of the NPC Clinical trial primary endpoint, and so for assessment of treatment protocol. Secondary data items are data required to assess the clinical trial’s secondary endpoints.

As acceptable levels of data quality, an 1% error rate for primary and an 2.5 % error rate for secondary data points were adopted [9]. We present the change in error rate over the course of the trial, the number of errors per submission, and the change in data quality per form per submission.

Results

Between November 2009 and March 2010 a total of 4860 data items pertaining to 51 patients were entered. This is the first five months of an estimated 3 year long accrual period. In total 433 eCRF’s were submitted, of which 329 were unique realizations. Each CRF has been submitted between 1 to 4 times. Table 1 presents an overview of the submitted eCRF’s and data items.

Of the 433 submitted eCRF realizations, 287 were submitted for the first time without primary data errors (Table 2), while 253 forms (realizations) were submitted for the first time without secondary data errors (Table 3). No form had more than two errors in the primary data. One form contained 10 secondary data errors when it was submitted for the first time. This was baseline patient registration data for which the wrong patient was entered. In general subsequent submissions contained fewer errors (Figure 1).

Table 1 | Number of times each form has been entered and the number of data items per form.2

Patients Forms* Data Items Primary Secondary Total Items

Patient information/Physic 51 75 18 7 11 1350

Radiology diagnostics 49 92 13 0 13 1196

New Complaint 3 4 2 0 2 8

PDT Reaction/Adverse event 1 2 3 0 3 6

General patient information 23 34 8 4 4 272

PDT Therapy form 2 2 6 6 0 12

Endoscopy/NPC Diagnostics 40 68 6 6 0 408

* Number of forms submitted, some unique realizations have been submitted multiple times.


31

Primary Tumor & NPC Diagn. 2 2 14 5 9 28

Laboratory form 48 64 5 3 2 320

Pathology, Staging & Given 48 90 14 9 5 1260

Totals: 433 89 40 49 4860

For example, the ‘Pathology, Staging & Given treatment’ eCRF contained a total of 89 unique data items, with the number of data items per eCRF ranging from 2 to 18 (Table 1). Of these 89 items, 40 were classified as primary data, with the remaining 49 being classified as secondary. The error rate at first submission was 3.3% for primary data and was 8.4% for secondary data.

Table 2 | The number of forms submitted with erroneous primary data items at each submission. For example 287 forms were entered error free at the first submission, while four were entered for the fourth time, 3 error free and one with one error.

Number of errors

0 1 2

Subm

issi

on

Num

ber

1 287 31 11

2 78 9 2

3 8 3 0

4 3 1 0

To assess the change in data quality over time, the proportion of unsolved errors in primary and secondary data were plotted against time (in months). Figure 2(A) presents the cumulative number of unique data items submitted and the number of unresolved errors over the first five months of the study. Figure 2(B) presents the change in the percentage of unresolved errors of primary and secondary data items. Although the absolute number of unsolved errors is increasing with time (due to the accrual of patients), the fraction of erroneous data is declining. Five months after the start of study the error rate for the primary data items was 1.6% and for the secondary data items the error rate was 2.7%. Although not quite at the levels appropriate for final analysis, the standard of data quality is high, very early into the study.

Table 3 | The number of forms submitted with erroneous secondary data items at each submission.

Number of errors

0 1 2 3 4 10

Subm

issi

on

Num

ber

1 253 19 39 15 2 1

2 76 2 8 3 0 0

3 9 0 2 0 0 0

4 4 0 0 0 0 0

Chapter 2

32Figure 1 | Errors per submission for the six forms of the nine forms for which there have been more than one submission (per realization).

Figure 2 | (A) The cumulative number of unique data items entered (black), the number of primary data items entered (blue solid) and errors unresolved (blue dashed), the number of secondary data items entered (red solid) and errors outstanding (dashed lines). (B) The percentage of Primary (blue) and Secondary (red) errors unresolved over time. The horizontal lines are the targets for a final analysis.


33

Discussion

For this study we found an error rate of 1.6 % for primary data items, while in earlier studies in the same setting data could not be analysed because of the massive data loss and poor data quality. With this real time data monitoring and inbuilt checks we have realized acceptable levels of data quality. The CTDMS prevents us from missing data or ending up with poor quality data at the end of the study, which often at that point cannot be resolved anymore.

The presented analysis shows that after five months since the introduction of the CTDMS the error rates for both Primary and Secondary data items reflect acceptable levels of data quality. Furthermore these error rates were decreasing over time. The drop in errors per form with each form submission indicates that, while being prompted by the CTDMS, the data manager and responsible doctors are actively solving the errors. Online warning messages notify the data-manager (entering data) when errors are detected, allowing them to immediately correct the data, rather than the usual delay associated with paper based CRFs.

Clearly, the CTDMS encourages local data managers to verify the entered data and, if necessary, ask the doctor whether the information is correct. It is also likely that the reason that data managers have to specify arguments before submitting the form in case the CTDMS detects erroneous data motivates them to verify whether the available data is actually correct. This may explain why our results show a significant increase of clean data and a self-learning curve of the data manager is to be expected. Moreover, the error logs provide valuable information about the bottlenecks in the treatment of the NPC patients.

In the past authors have pointed out that existing data collections in developing countries are often deficient [10] [11]. Eiseman and Fossum (2005) emphasize that existing data collections are insufficiently comprehensive, sometimes inaccurate, and often out of date by the time the data can be acted upon. All point out that without these data the required empirical knowledge to address the health problems in developing countries is insufficient. Especially on strategic planning, priority setting, monitoring and evaluation, advocacy, and general policymaking [12-14].

These comments supported us on introducing an online medical record system which could play an important role in improving data collection and data quality. Accordingly, during analysis we have also seen that treatment procedures are often unsatisfactory. The first analysis regarding the treatment of NPC has been presented and discussed with all members of the disciplinary team. The main concern was the duration of the radiotherapy. According to the protocol the duration for administering the 66 gray radiotherapy should take to the utmost 42 days, yet analysis showed that the treatment time takes in average 66 days, which will lead to inadequate treatment [15,16]. Future analysis has to show if intervention by CTDMS system-based education of the doctors will eventually lead to better treatment outcome. The digital nature of the CTDMS, as well as the online availability of data, gives fast and easy insight in adherence to treatment protocols. As such, the CTDMS can serve as a tool to directly train and educate medical doctors. Therefore, a potential even bigger advantage of an online medical record system is the ability to monitor the data from the teaching hospitals especially in developing countries. Via this way the teachers can communicate directly or visit the participating hospitals with a custom fit teaching program, which will make such visits more effective.

Conclusion

We show that an online clinical data management service can improve data quality in a developing country setting. In the future we expect to see both less loss-to-follow-up and better treatment programmes with help

Chapter 2

34

of this CTDMS. For better and more efficient medical care programs and studies in developing countries we believe an online data system is essential.

The digital nature of the CTDMS, as well as the online availability of that data, gives fast and easy insight in adherence to treatment protocols. As such, the CTDMS can serve as a tool to train and educate medical doctors and can improve treatment protocols. Since the introduction of this system training the doctors has become much more efficient.

Reference List 1. Pollak VE, Lorch JA: Effect of electronic patient record use on mortality in End Stage Renal Disease, a model

chronic disease: retrospective analysis of 9 years of prospectively collected data. BMC Med Inform Decis Mak 2007, 7: 38.

2. Fraser HS, Jazayeri D, Nevil P, Karacaoglu Y, Farmer PE, Lyon E et al.: An information system and medical record to support HIV treatment in rural Haiti. BMJ 2004, 329: 1142-1146.

3. Simbini T: Computerised information management systems in HIV/AIDS care and outcomes research. Cent Afr J Med 2006, 52: 65-67.

4. Clifford GD, Blaya JA, Hall-Clifford R, Fraser HS: Medical information systems: a foundation for healthcare technologies in developing countries. Biomed Eng Online 2008, 7: 18.

5. Choi SS, Jazayeri DG, Mitnick CD, Chalco K, Bayona J, Fraser HS: Implementation and initial evaluation of a Web-based nurse order entry system for multidrug-resistant tuberculosis patients in Peru. Stud Health Technol Inform 2004, 107: 202-206.

6. Fraser H, Choi SS, Galipot M, Jazayeri D, Mangubat N: Successful transfer of a Web-based TB medical record from Peru to the Philippines. AMIA Annu Symp Proc 2006, 924.

7. Devi BC, Tang TS, Corbex M: Reducing by half the percentage of late-stage presentation for breast and cervix cancer over 4 years: a pilot study of clinical downstaging in Sarawak, Malaysia. Ann Oncol 2007, 18: 1172-1176.

8. Curado M.P., Edwards B., Shin H.R.. Cancer Incidence in Five Continents, Vol. IX. 2007. IARC Scientific Publications No. 160, Lyon, IARC. 2007. Ref Type: Data File

9. Sullivan EMGMASRCCG: A Statistically-based process for Auditing Clinical data listings. Drug Information Journal 1997, 31: 647-653.

10. Eiseman E, Fossum D: The Challenges of Creating a Global Health Resource Tracking System. RAND Corperation 2005.

11. Heathfield H, Pitty D, Hanka R: Evaluating information technology in health care: barriers and challenges. BMJ 1998, 316: 1959-1961.

12. Clifford GD, Blaya JA, Hall-Clifford R, Fraser HS: Medical information systems: a foundation for healthcare technologies in developing countries. Biomed Eng Online 2008, 7: 18.

13. Mantas J, Ammenwerth E, Demiris G, Hasman A, Haux R, Hersh W et al.: Recommendations of the International Medical Informatics Association (IMIA) on Education in Biomedical and Health Informatics. First Revision. Methods Inf Med 2010, 49: 105-120.

14. McKee M: A decade of experience in eastern Europe. In Leadership and management for improving global health. Edited by Foege W, Black R, Daulaire N. 2005:167-186.

15. Fu KK, Pajak TF, Trotti A, Jones CU, Spencer SA, Phillips TL et al.: A Radiation Therapy Oncology Group (RTOG) phase III randomized study to compare hyperfractionation and two variants of accelerated fractionation to standard fractionation radiotherapy for head and neck squamous cell carcinomas: first report of RTOG 9003. Int J Radiat Oncol Biol Phys 2000, 48: 7-16.

16. Thames HD, Kuban D, Levy LB, Horwitz EM, Kupelian P, Martinez A et al.: The role of overall treatment time in the outcome of radiotherapy of prostate cancer: an analysis of biochemical failure in 4839 men treated between 1987 and 1995. Radiother Oncol 2010, 96: 6-12.


35

Appendix 1

Technical description CTDMS

The selected CTDMS is ALEA™ 3.0, which uses Microsoft Infopath 2007 for eCRF form template design and Sharepoint Enterprise 2007 for rendering the eCRF’s in browsers. SQL server 2008 is used for data storage. The CTDMS provides a comprehensive electronic case report forms (eCRF) system. It uses standard browsers running on any computer connected to the internet. The system has been validated, and has been certified by registered auditors, as being in compliance with relevant regulations, such as the FDA’s CFR 21 Part 11.

The CTDMS eCRF design module is based on an industry grade enterprise electronic forms system: Microsoft Infopath 2007 for form design and Microsoft Forms Server 2007 for data entry. The components make use of a common standard representation of data and metadata: the Operational Data Model of CDISC. Within the CTDMS, the components share a database for storing and retrieving information about the trial, and a separate database for storing and retrieving patient data.

The online Data Management Module of the CTDMS is a web browser application that supports online completion of eCRF for healthcare studies. It requires initial login with a username and password, and provides a navigation menu for all trials to which the account has been granted access, and the selected investigators for which the account has been granted permissions to access. Transmission of data is SSL encrypted using RSA 1024 bit Public Key encryption.

Data validation rules were implemented into the eCRF’s using the tools Microsoft Office InfoPath provides, as well as some Xpath expressions. With data validation rules implemented, the eCRF automatically checks the data as soon as it is entered. If a value does not match the specified condition, an error alert provides the user with immediate feedback. Moreover, after completion of an eCRF, the user is prompted to provide an explanation of all data items which raised validation errors. This enables users to submit data with validation errors, while providing a comprehensive audit trail in compliance with requirements from regulatory authorities. Examples of data validation rules, which trigger error flags is provided in Table 4.

Table 4 | List of filters/rules which trigger error flags. Examples of data validation rules triggering error flags for a subset of the data items on two of the eCRF’s.

Name eCRF Data item Data validation rule

Patient information/ Physical examination form

Date of First Visit 1. The field “Date of First Visit” is mandatory and cannot be blank.2. The Date of First Visit cannot be after the Date of today

Date of Birth 1. The patient Date of Birth cannot be blank2. The patient Date of Birth cannot be after the current date.3. The Date of birth you entered is before 1-1-1900, the patient is

too old to participate in this study

Date of Signed informed consent

1. The field “Date of Signed informed consent” is mandatory and cannot be left blank

2. The Date of First Visit cannot be after the Date of today

1. The field “heart rate” cannot be blank.2. The field “heart rate” cannot be more than 220 p/m3. The field “heart rate” cannot be less than 40 p/m

Temperature 1. Patient Temperature should be between 35 and 41.5 degrees

Chapter 2

36

WHO performance rate 1. The field “WHO performance rate” is mandatory and cannot be left blank

2. During First Visit, the “WHO performance rate” cannot be either 3 or 4.

Radiology Diagnostics form CT/ MRI- scan Date Sample Taken

1. The field CT/ MRI- scan Date Sample Taken is mandatory and cannot be blank.

2. The Date of Sample Taken, CT/MRI-scan, cannot be before the Date of Visit, as specified on the General Patient Information form (belonging to this Event/Visit).

3. The Date Sample Taken CT/ MRI- scan cannot be before the Date of First Visit.

4. The Date Sample Taken CT/MRI- scan during PDT Assessment should be about 12 weeks after the Date of Foscan Administration and no less than 10 weeks after that date.

5. The Date of CT/MRI- scan cannot be later than today.

T-stage 1. The T-stage (0,1,2A and 2B,3,4) in the field “CT-MRI T-stage” has to be the same as the T (0,1,2A and 2B,3,4) in the field “T-stage” on the Pathology form.

2. The T- stage cannot be “0” if patient has been included in this study because of a recurrent or persistent disease

field CT/MRI Lesion Size 1. The field “CT/MRI Lesion Size (length)” cannot be blank when the field T-stage has been entered.

2. The field “CT/MRI Lesion Size (length)” cannot be more than 200 mm

CT/MRI site 1. The field “CT/MRI site” is mandatory and cannot be left blank2. The tumour site CT/MRI- scan selected here differs from the

Suspicious site as specified on the Endoscopy form during this Visit. .Are you sure this is correct? If yes, please provide explanation in Audit Trail entry after Validation

3. The tumour site CT/MRI- scan selected here differs from the Tumour site as specified on the Endoscopy form during this Visit. Are you sure this is correct? If yes, please provide explanation in Audit Trail entry after Validation

endoscopy 1. If Nose endoscopy has been performed, the field “Date of Endoscopy” cannot be blank

2. During (visit) Positive Test, the Date of Endoscopy should be after the Date of Endoscopy of the PDT Assessment.

3. The Date of Endoscopy should be after the Date of Endoscopy of the last PDT Follow Up

4. The Date of Endoscopy should be after the Date of Endoscopy of the PDT Visit(s)

5. The Date of Endoscopy should be after the Date of Endoscopy of the previous Follow Up visit.

6. The Date of Endoscopy should be after the Date of Endoscopy of the First Visit.

7. The Date of Endoscopy should be 3 months after the Date of Endoscopy of the Therapy Assessment

Chapter 3

Primary treatment results of Nasopharyngeal Carcinoma (NPC) in Yogyakarta, Indonesia

M.A. Wildeman,

R. Fles,

C. Herdini,

S.R. Indrasari,

A.D. Vincent,

M. Tjokronagoro,

S. Stoker,

J. Kurnianda,

K.W. Taroeno-Hariadi,

O. Hamming-Vrieze,

J.M. Middeldorp,

B. Hariwiyanto,

S.M. Haryana,

I.B. Tan

Submitted

Chapter 3

40

Abstract

Introduction

Nasopharyngeal Carcinoma (NPC) is a major health problem in southern and eastern Asia. In Indonesia NPC is the most frequent cancer in the head and neck area. NPC is very sensitive to radiotherapy resulting in 3 years disease-free and overall survival of approximately 70% and 80%, respectively. Here we present routine treatment results in a prospective study on NPC in Indonesia.

Methods

All NPC patients presenting from September 2008 till January 2011 at the ear, nose and throat (ENT) department of the Dr. Sardjito General Hospital, Universitas Gadjah Mada, Yogyakarta, Indonesia, were possible candidates. Patients were included if the biopsy was a histological proven NPC without distant metastasis and were assessed during counselling sessions prior to treatment, as being able to complete the entire treatment.

Results

In total 78 patients were included for treatment analysis. The median time between diagnosis and start of radiotherapy is 120 days. Forty-eight (61,5%) patients eventually finished all fractions of radiotherapy. The median duration of the radiotherapy is 62 days for 66 Gy. Median overall survival is 21 months (95% CI: 18-35) from day of diagnosis.

Conclusion

The results presented here reveal that currently the treatment of NPC at an Indonesian hospital is not sufficient and cannot be compared to the treatment results in literature. Main reasons for these poor treatment results are (1) a long waiting time prior to the start of radiotherapy, (2) the extended overall duration of radiotherapy and (3) the advanced stage of disease at presentation.


41

Introduction

Cancer has become a leading cause of death and morbidity in low and middle-income countries. While this has been recognized for at least twenty years, healthcare systems in these countries often cannot deliver the prevention and care needed to overcome this challenge(1-4). Most publications that address this issue are based on cancer incidence and barely report on treatment results and survival. The main reason for this is the lack of appropriate data collection in developing countries. There is a clear need for improved cancer control systems in developing country settings(5). Recently we have introduced a Clinical Trial Data Management service (CTDMS) to monitor treatment results of Nasopharyngeal Carcinoma (NPC) in a university hospital setting in Yogyakarta, Indonesia(6).

In southern and eastern Asia NPC is a major health problem. In Indonesia NPC is the most frequent cancer in the head and neck area and ranks as the 4th most common tumour found in males. The undifferentiated histological subtype, NPC WHO III, is the most prevalent NPC type in South-East Asia and Indonesia. This type of cancer is causally associated with the Epstein-Barr virus (EBV) (7). The incidence is estimated 6 per 100.000, leading to 12.000 new cases per year (8-10). Due to the insufficient national cancer registration system, the actual number is most probably much higher. NPC is very sensitive to radiotherapy at early stage (stage I-II), but this type of treatment is not generally available for many patients in developing countries and unfortunately can induce complications after treatment if not properly administered. Since the location of the tumour has a close contact with the base of skull, the brain stem and spinal cord, radiation is hindered by dose limitations on these organs at risk. For advanced NPC (stage III-IV), standard-care is concurrent chemo radiation therapy with high-dose radiation combined with cisplatin-based regimens. This treatment approach may result in 3 years disease-free and overall survival of approximately 70% and 80%, respectively (11;12). A recent meta-analysis confirmed the clinical benefit of concurrent chemo radiation therapy (CCRT) compared with radiation alone (RT) in the treatment of stage III and IV NPC in endemic areas(13). Nearly all treatment results on NPC presented in the literature derive from top-end hospitals and clinical trial settings. Here we present a prospective study on routine treatment results of NPC at a university hospital in Indonesia.

Patients and Methods

Patients Eligibility

All NPC patients presenting from September 2008 till January 2011 at the ear, nose and throat (ENT) department of the Dr Sardjito General Hospital, Universitas Gadjah Mada, Yogyakarta, Indonesia, were analysed. Patients were examined by CT-scan, chest X-ray, ultrasound of the abdomen and bone survey. Patients were included in the study if the biopsy was a histological proven NPC without distant metastasis and were assessed during counselling sessions prior to treatment, as being able to complete the entire treatment. Tissue analysis was performed at the pathology department of the Dr Sardjito hospital; WHO-histological classification was done according to the UICC 2002 criteria and EBV-encoded small RNA (EBER) staining was done with commercial reagents according to the manufacturer’s instructions (Dako, PNA-kit). Other inclusion criteria were measurable disease and a curative intent treatment plan. Patients were not eligible if they had NPC as a second malignancy.

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42

Treatment

Due to different health insurances of the patients, different treatment regimens have been administered. All patients received 66-70 Gray (Gy) (2 Gy per fraction for 5 days) external beam radiotherapy and in all cases treatment planning was 2 dimensional. The type of chemotherapy and the number of courses, and whether applied concurrent or as neo-adjuvant, was adjusted for the type of insurance and waiting time for radiotherapy at that time. Options of chemotherapy include 3-4 cycles cisplatinum-based induction chemotherapy and chemo-concurrent radiotherapy (CCRT). In induction setting either cisplatin and 5 fluorouracil (5 FU) (PF regimen) or combination of docetaxel, cisplatin and 5 FU (TPF regimen) are administered. Radiotherapy is carried out 1 month after induction chemotherapy. During the course of radiotherapy some patients also received a 6-7 cycles of weekly carboplatin AUC 2. In the setting of CCRT, a weekly 6-7 cycles of low-dose platinum (40 mg/m2) cooperated with radiotherapy.

Therapy assessment

Eight weeks after treatment the response was monitored by CT-scan, chest X-ray, ultrasound of the abdomen and bone survey. In case of histological proven local persistent or recurrent disease patients were offered to be treated with photodynamic therapy (PDT). In case of persistent neck lymph nodes patient were treated with a modified radical neck dissection.

Follow Up

All patients had a regular follow-up schedule consisting of 3 monthly visits during the first two years after radiotherapy. In case of suspicion for tumour recurrence radiological examinations were performed. In case of a local or regional recurrence patients were treated with PDT with separate treatment of the neck.

Photodynamic therapy (PDT)

Drug administration of Foscan®, light administration and treatment procedures of the PDT are completely similar to and described previously in Nyst et al 2012 (14).

Patients received the dose level and the drug light interval recommended for the treatment of patients with squamous-cell carcinoma of the head and neck. These parameters are drug dose, 0.15 mg/kg Foscan®; drug-light interval: 48 or 96 hours; light dose: 20 J/cm2.

Results

In total 188 patients presented with NPC during September 2008 till January 2011. Seventy-eight patients were included for treatment analysis, as they were considered able to complete treatment protocol based on intake counselling. The median age of the patients was 49 years (range 17-78), 50 patients were male and 28 were female patients. Only 2 patients had early stage disease at entry being stage I or IIA, and 76 patients presented with loco-regional advanced stage IIB and higher. All 78 patients had an EBER-positive, histological proven NPC and were WHO type III. Reasons for exclusion were insufficient funding to afford a curative treatment (n=27; 25%), no sufficient funding to afford staging procedures (n=24, 22%), no pathology available (n=24, 22%), decided to go for traditional treatments (n=16, 15%), eleven patients presented with distant metastasis at diagnosis (n=11, 10%), six patients due to logistic reasons (7%) and two refused the protocol (2%) . Patient characteristics are listed in table 1. Types of chemotherapy regimen administered are listed in table 2.


43

Table 1 | Patient characteristics and type of treatment. *The 8 patients who haven’t received any treatment died before the start of treatment.

None*Concurrent

+ RTNeo Adjuvant + Concurrent

+ RTNeo Adjuvant

+ RT RT Total

N = 8 N = 18 N = 9 N = 41 N = 2 N = 78

AGE

Median 48 49 45 51 38 49

(Range) (41 - 72) (17 - 73) (27 - 54) (23 - 78) (29 - 47) (17 - 78)

GENDER

F 1 ( 12%) 8 ( 44%) 2 ( 22%) 16 ( 39%) 1 ( 50%) 28 ( 36%)

M 7 ( 88%) 10 ( 56%) 7 ( 78%) 25 ( 61%) 1 ( 50%) 50 ( 64%)

T.STAGE

T1 2 ( 25%) 2 ( 11%) 1 ( 11%) 10 ( 24%) 1 ( 50%) 16 ( 21%)

T2A 1 ( 12%) 2 ( 11%) 0 ( 0%) 5 ( 12%) 0 ( 0%) 8 ( 10%)

T2B 0 ( 0%) 4 ( 22%) 1 ( 11%) 5 ( 12%) 1 ( 50%) 11 ( 14%)

T3 1 ( 12%) 7 ( 39%) 7 ( 78%) 17 ( 41%) 0 ( 0%) 32 ( 41%)

T4 4 ( 50%) 3 ( 17%) 0 ( 0%) 4 ( 10%) 0 ( 0%) 11 ( 14%)

N.STAGE

N0 0 ( 0%) 1 ( 6%) 2 ( 22%) 5 ( 12%) 1 ( 50%) 9 ( 12%)

N1 3 ( 38%) 6 ( 33%) 1 ( 11%) 4 ( 10%) 1 ( 50%) 15 ( 19%)

N2 0 ( 0%) 8 ( 44%) 2 ( 22%) 12 ( 29%) 0 ( 0%) 22 ( 28%)

N3A 2 ( 25%) 3 ( 17%) 3 ( 33%) 15 ( 37%) 0 ( 0%) 23 ( 29%)

N3B 3 ( 38%) 0 ( 0%) 1 ( 11%) 5 ( 12%) 0 ( 0%) 9 ( 12%)

AJCC.STAGE

I 0 ( 0%) 0 ( 0%) 0 ( 0%) 0 ( 0%) 1 ( 50%) 1 ( 1%)

IIA 0 ( 0%) 0 ( 0%) 0 ( 0%) 1 ( 2%) 0 ( 0%) 1 ( 1%)

IIB 0 ( 0%) 3 ( 17%) 1 ( 11%) 1 ( 2%) 1 ( 50%) 6 ( 8%)

III 0 ( 0%) 9 ( 50%) 4 ( 44%) 15 ( 37%) 0 ( 0%) 28 ( 36%)

IVA 3 ( 38%) 3 ( 17%) 0 ( 0%) 3 ( 7%) 0 ( 0%) 9 ( 12%)

IVB 5 ( 62%) 3 ( 17%) 4 ( 44%) 21 ( 51%) 0 ( 0%) 33 ( 42%)

TYPE.OF.INSURANCE

Government Insurance 0 ( 0%) 1 ( 6%) 3 ( 33%) 1 ( 2%) 1 ( 50%) 6 ( 8%)

Poor Insurance 7 ( 88%) 17 ( 94%) 3 ( 33%) 39 ( 95%) 0 ( 0%) 66 ( 85%)

Self Finance 1 ( 12%) 0 ( 0%) 3 ( 33%) 1 ( 2%) 1 ( 50%) 6 ( 8%)

Table 2 | Type of chemotherapy administered.

Concurrent + RT Neo Adjuvant + Concurrent + RT Neo Adjuvant + RT Total

N = 18 N = 9 N = 40 N = 78

TYPES OF CHEMOTHERAPY REGIMENT DURING NEOADJUVANT

Carboplatin, 5 FU 2 ( 5%) 2 ( 3%)

Cisplatin, 5 FU 36 ( 88%) 36 ( 46%)

Docetaxel, Cisplatin, 5 FU 9 ( 100%) 1 ( 2%) 10 ( 13%)

Paclitaxel, Cisplatin 1 ( 2%) 1 ( 1%)

TYPES OF REGIMENTS OF CONCURRENT CT

Carboplatin 1 ( 6%) 7 ( 78%) 8 ( 10%)

Cisplatin 17 ( 94%) 1 ( 11%) 18 ( 23%)

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44

Radiotherapy

The median time between diagnosis and start of radiotherapy is 120 days (range 13-500). Twelve (15.4%) patients died before the start of radiotherapy due to disease progression or in two cases due to side effects of the neo-adjuvant chemotherapy treatment. Four patients did not return for radiotherapy due to concern about the side effects after the neo-adjuvant chemotherapy and two patients had, despite counselling, insufficient funding to be treated with radiotherapy.

Forty-eight (61,5%) patients eventually finished all fractions of radiotherapy. The median duration of the radiotherapy is 62 days (range 46-140 days) for 66 Gy.

Response to treatment

Twenty three (29%) patients died of disease progression before treatment response could be assessed. Five patients dropped out due to insufficient funding, 5 stopped treatment early due to side effects and 6 stopped treatment due to concern for side effects. Eventually 39 (50%) patients had a treatment response measurement 8 weeks after treatment. Twenty-three (29 %) achieved a complete response. Ten patients experienced local persistent disease, in two patients regional persistent disease was found and two patients developed distant metastasis. Two patients had local and regional persistent disease, one of them also developed distant metastasis. Multiple testing for prognostic factors, like stage of disease, didn’t show any significant factor.

Survival

Median overall survival is 21 months (95% CI: 18-35) from day of diagnosis. The median disease free survival from day of diagnosis is 20 months (95% CI: 18-24). In figure 1A and figure 1B Kaplan Meijer curves present the survival probability for overall survival and disease free survival respectively.

Photodynamic therapy

Six patients with local persistent disease, which was discovered during therapy assessment after fulfilment of the regular treatment, and one patient with a local recurrence, discovered during follow up, has been treated with PDT. All patients had a complete response 12 weeks after illumination, three patients developed regional recurrences and one patient died due to the regional recurrence.

Figure 1A and figure 1B | Kaplan Meijer curves present the survival probability for overall survival and disease free survival respectively.


45

Discussion

The results presented here reveal that currently the treatment of NPC at an Indonesian academic hospital is not sufficient and cannot be compared to the treatment results in literature (11;12). Of the patients evaluable for response only 29% had a complete response directly after treatment, with the median overall survival being 21 months after diagnosis. Main reasons for these poor treatment results are (1) a long waiting time prior to the start of radiotherapy, (2) the extended overall duration of radiotherapy and (3) the advanced stage of disease at presentation.

Cancer is becoming a growing problem in low and middle-income countries. Since it is ill-defined how many of the patients actually make it to the hospital and receive a biopsy that is registered, the actual NPC incidence remains unclear. NPC is the commonest head and neck cancer in Indonesia with most patients presenting with an advanced stage of disease which poses a heavy burden on the population. A major problem is that patients cannot afford treatment or are afraid of treatment toxicity, causing them to refrain from treatment or seek alternative options. This resulted in nearly 61% of the excluded NPC patients being non-compliant with the inclusion criteria for this study. However, the NPC treatment results presented here reveal an additional problem, this being the limited capacity for proper treatment. In 2008 there were 18 linear accelerators and 17 Cobalt-60 teletherapy machines available in Indonesia with a population of 229 million, nevertheless 6 of them were under commission. Resulting in 0.13 accelerator per million inhabitants(15). This in comparison to Europe were 5.5 accelerator is available per million inhabitants in the high, 3.5 per million in the medium and 2 per million in the low resource countries(16). The recommended number of treatment units per population differs widely, in Europe guidelines of 25 low to high income countries recommended on average 5.9 per million(17).

Although Indonesia is a rising economy, meeting these criteria will not be feasible on short term. Expanding radiotherapy facilities is a time and money-consuming project. For building a new radiotherapy facility and have it installed for treatment the average time is approximately 5 years. Expansion of staffing (radiation oncologists, physicists, technologists) takes even longer, since proper education and training is necessary (16).

When patients with poor insurance in Yogyakarta finally complete diagnosis and imaging to stage the disease, they have to wait on average 4 months to start radiotherapy treatment. At this time 13% have already succumbed to disease progression. Despite optimal counselling in this study cohort, five patients who started treatment did not finish the treatment properly because they could not afford the costs of the entire treatment. Importantly, we noted that most patients who eventually started treatment received insufficient radiotherapy treatment. Optimally, a total dose of 66 to 70 Grey should be given in 33 to 35 fractions and the best therapy response is achieved when the total dose is administered in 45 to 47 days. Every day the radiotherapy is postponed a loss of the effective dose occurs. This will influence the treatment success to a great extent (18;19).

The median duration of radiotherapy treatment in this study was 62 days. This prolonged treatment time together with the 2-dimensional radiotherapy technique based on CT imaging only (i.e. not using MRI or FDG-PET to assess disease extent) are the main causes of the poor response rate (20;21). In part this prolonged treatment duration is often caused by the radiotherapy facility being intermittently operational, due to poor maintenance of the equipment. Other causes for this delay may be due to poor efficiency in administration and communication at and between hospital departments and poor patient and doctor compliance to protocols and timelines. Further research is required to reveal these causes and can eventually contribute to improve overall treatment time.

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46

The late stage of NPC at presentation in the hospital is another unfavourable prognostic factor. One possible reason for the high percentage of patients with advanced NPC could be due to poor diagnosis by general practitioners (GP) and thereby a delay in referral. In our previous study we assessed the knowledge on NPC of the GPs working in the Primary Health Care Centres in the Yogyakarta region (22). Our results indicate that the knowledge of GPs is insufficient, with many of them not being aware of the high incidence of NPC in their region.

In Malaysia prior studies have proved that the lack of awareness and knowledge of primary health care workers is one of the main reasons for delayed diagnosis(23). Given that presenting stage is the most important prognostic factor, appropriate training of GPs is critical. The relevance of adequate referral by GPs for head and neck carcinomas has been shown by Alho et al.(24), who found that 20% of 221 patients, subsequently to being diagnosed with head and neck carcinoma, were initially sent home without referral. The risk of death in this group was significantly higher when compared with the patients who were immediately referred or received a follow up appointment.

The same group from Finland, has also shown that the time between GP referral and final diagnosis is a significant factor in patient outcome in other head and neck cancers (25). Long delay in primary care resulted in a significantly worse prognosis in patients with laryngeal carcinoma (26).

All patients analysed in the presented study had WHO type III positive NPC. Prior studies have shown that EBV-related markers can be used for screening and prognostic monitoring. These markers include EBV (IgA) serology and EBV-DNA load in nasopharyngeal brushings or blood. NPC patients have characteristic aberrant IgG and IgA antibody reactivity to several EBV encoded antigens as well as increased EBV-DNA in blood plasma, derived from shed (apoptotic) tumour fragments into the circulation. Increased IgA antibody levels are found against early antigen (EA), viral capsid antigen (VCA) and the latent Epstein-Barr nuclear antigen 1 (EBNA1) as well as inhibitory antibodies to the EBV specific DNAse (27;28). These antibody responses against defined viral antigens are the basis of a proposed screening test for NPC in high-risk populations. (29-31) Recent insight in the molecular basis and diversity of anti EBV IgA and IgG responses allowed the development of more defined serological tools(32-36). Importantly, such initial NPC-risk analysis can be done in the regional hospital setting with small volumes of blood, collected by finger prick sampling on filter paper, thus providing a cheap approach (35;37) . The fingerpick sample might also allow EBV-DNA load measurement, since DNA is a rather stable molecule in dried blood. Furthermore, the nasopharyngeal brushing with EBV marker assessment, may provide a promising method for detecting tumour presence in situ, by measuring EBV-DNA and RNA in parallel. Such brushings can be taken with simple tools under nasal-endoscope guidance(38-42).

Future education programs should include referencing to the available improved EBV-based diagnostic procedures for NPC-risk screening and early detection. Improved education combined with a screening method could be a cheap and sensitive screening method for NPC in Indonesia and other high incidence countries. Application of these methods in patients with chronic complaints in the head and neck, not responding to traditional antibiotic and anti-allergic medication has already yielded successful detection of early stage NPC cases in the Sardjito hospital patient population (43).

Currently we enrolled NPC awareness programme in Jakarta, Yogyakarta and Surabaya. Short time efficacy of the training programmes have been published recently(44). Hopefully in the future this programme can be expanded to include more regions of Indonesia.

The patients treated with PDT participate in an ongoing phase II trial, the results on PDT treatment for persistent and recurrent disease will be presented in more detail when the study has been completed. Still


47

worth mentioning is that out of the seven patients who were treated with PDT, six of them are still alive. This is 17 per cent of all included NPC patients who are still alive. Based on our previous and recent experiences it seems logical to assume that Temoporfin mediated PDT, requiring relatively simple equipment and facilities, may be used in the future as primary treatment modality for superficially growing NPC, with still the availability of all other treatments. In case of complete response it will beneficial influence the overall waiting time for radiotherapy. Besides treatment of superficial growing tumours PDT could perhaps play a role in tumour reduction to overcome the waiting time for radiotherapy.

The easiest ways of addressing current problems would be establishing sufficient radiotherapy facilities in Indonesia, however this will take decades. In the mean while our goal has to be to assure that patients who will be treated have an effective treatment and the right candidates are selected. Early detection of NPC, new treatment regimens to overcome the waiting time, a counselling system that supports only those patients who have a real opportunity to complete the treatment and improved maintenance for the radiotherapy facility are feasible short-term solutions to improve treatment outcome in the near future.

Conflicts of interest

We declare that we have no conflicts of interest.

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(14) Nyst HJ, Wildeman MA, Indrasari SR, Karakullukcu B, van Veen RL, Adham M et al. Temoporfin mediated photodynamic therapy in patients with local persistent and recurrent nasopharyngeal carcinoma after curative radiotherapy: A feasibility study. Photodiagnosis Photodyn Ther 2012 September;9(3):274-81.

(15) Gondhowiardjo S, Prajogi G, Sekarutami S. History and growth of radiation oncology in Indonesia. Biomed Imaging Interv J 2008 July;4(3):e42.

(16) Slotman BJ, Cottier B, Bentzen SM, Heeren G, Lievens Y, van den Bogaert W. Overview of national guidelines for infrastructure and staffing of radiotherapy. ESTRO-QUARTS: work package 1. Radiother Oncol 2005 June;75(3):349-54.

(17) Bentzen SM, Heeren G, Cottier B, Slotman B, Glimelius B, Lievens Y et al. Towards evidence-based guidelines for radiotherapy infrastructure and staffing needs in Europe: the ESTRO QUARTS project. Radiother Oncol 2005 June;75(3):355-65.

(18) Levendag PC, Lagerwaard FJ, Noever I, dePan C, vanNimwegen A, Wijers O et al. Role of endocavitary brachytherapy with or without chemotherapy in cancer of the nasopharynx. Int J Radiat Oncol Biol Phys 2002 March 1;52(3):755-68.

(19) Akimoto T, Mitsuhashi N, Hayakawa K, Sakurai H, Murata O, Ishizeki K et al. Split-course accelerated hyperfractionation radiotherapy for advanced head and neck cancer: influence of split time and overall treatment time on local control. Jpn J Clin Oncol 1997 August;27(4):240-3.

(20) Lee N, Puri DR, Blanco AI, Chao KS. Intensity-modulated radiation therapy in head and neck cancers: an update. Head Neck 2007 April;29(4):387-400.

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effect of delayed diagnosis of cancer cases. CMAJ 2006 March 14;174(6):779-84. (25) Koivunen P, Rantala N, Hyrynkangas K, Jokinen K, Alho OP. The impact of patient and professional diagnostic delays

on survival in pharyngeal cancer. Cancer 2001 December 1;92(11):2885-91. (26) Teppo H, Koivunen P, Hyrynkangas K, Alho OP. Diagnostic delays in laryngeal carcinoma: professional diagnostic

delay is a strong independent predictor of survival. Head Neck 2003 May;25(5):389-94. (27) Cevenini R, Donati M, Caliceti U, Moroni A, Tamba I, Rumpianesi F. Evaluation of antibodies to Epstein-Barr virus in

Italian patients with nasopharyngeal carcinoma. J Infect 1986 March;12(2):127-31. (28) Chen JY, Chen CJ, Liu MY, Cho SM, Hsu MM, Lynn TC et al. Antibodies to Epstein-Barr virus-specific DNase in patients

with nasopharyngeal carcinoma and control groups. J Med Virol 1987 September;23(1):11-21. (29) Ji MF, Wang DK, Yu YL, Guo YQ, Liang JS, Cheng WM et al. Sustained elevation of Epstein-Barr virus antibody levels

preceding clinical onset of nasopharyngeal carcinoma. Br J Cancer 2007 February 26;96(4):623-30. (30) Tamada A, Makimoto K, Yamabe H, Imai J, Hinuma Y, Oyagi A et al. Titers of Epstein-Barr virus-related antibodies in

nasopharyngeal carcinoma in Japan. Cancer 1984 February 1;53(3):430-40. (31) de Vathaire F, Sancho-Garnier H, de The H, Pieddeloup C, Schwaab G, Ho JH et al. Prognostic value of EBV markers

in the clinical management of nasopharyngeal carcinoma (NPC): a multicenter follow-up study. Int J Cancer 1988 August 15;42(2):176-81.

(32) Fachiroh J, Schouten T, Hariwiyanto B, Paramita DK, Harijadi A, Haryana SM et al. Molecular diversity of Epstein-Barr virus IgG and IgA antibody responses in nasopharyngeal carcinoma: a comparison of Indonesian, Chinese, and European subjects. J Infect Dis 2004 July 1;190(1):53-62.

(33) Fachiroh J, Paramita DK, Hariwiyanto B, Harijadi A, Dahlia HL, Indrasari SR et al. Single-assay combination of Epstein-Barr Virus (EBV) E. J Clin Microbiol 2006 April;44(4):1459-67.

(34) Paramita DK, Fachiroh J, Artama WT, van Benthem E, Haryana SM, Middeldorp JM. Native early antigen of Epstein-Barr virus, a promising antigen for diagnosis of nasopharyngeal carcinoma. J Med Virol 2007 November;79(11):1710-21.

(35) Fachiroh J, Prasetyanti PR, Paramita DK, Prasetyawati AT, Anggrahini DW, Haryana SM et al. Dried-blood sampling for epstein-barr virus immunoglobulin G (IgG) and IgA serology in nasopharyngeal carcinoma screening. J Clin Microbiol 2008 April;46(4):1374-80.

(36) Paramita DK, Fachiroh J, Haryana SM, Middeldorp JM. Two-step Epstein-Barr virus immunoglobulin A enzyme-linked immunosorbent assay system for serological screening and confirmation of nasopharyngeal carcinoma. Clin Vaccine Immunol 2009 May;16(5):706-11.


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(37) Ji MF, Yu YL, Cheng WM, Zong YS, Ng PS, Chua DT et al. Detection of Stage I nasopharyngeal carcinoma by serologic screening and clinical examination. Chin J Cancer 2011 February;30(2):120-3.

(38) Lin JC, Wang WY, Chen KY, Wei YH, Liang WM, Jan JS et al. Quantification of plasma Epstein-Barr virus DNA in patients with advanced nasopharyngeal carcinoma. N Engl J Med 2004 June 10;350(24):2461-70.

(39) Stevens SJ, Verkuijlen SA, Hariwiyanto B, Harijadi, Fachiroh J, Paramita DK et al. Diagnostic value of measuring Epstein-Barr virus (EBV) DNA load and carcinoma-specific viral mRNA in relation to anti-EBV immunoglobulin A (IgA) and IgG antibody levels in blood of nasopharyngeal carcinoma patients from Indonesia. J Clin Microbiol 2005 July;43(7):3066-73.

(40) Stevens SJ, Verkuijlen SA, Hariwiyanto B, Harijadi, Paramita DK, Fachiroh J et al. Noninvasive diagnosis of nasopharyngeal carcinoma: nasopharyngeal brushings reveal high Epstein-Barr virus DNA load and carcinoma-specific viral BARF1 mRNA. Int J Cancer 2006 August 1;119(3):608-14.

(41) Tune CE, Liavaag PG, Freeman JL, van den Brekel MW, Shpitzer T, Kerrebijn JD et al. Nasopharyngeal brush biopsies and detection of nasopharyngeal cancer in a high-risk population. J Natl Cancer Inst 1999 May 5;91(9):796-800.

(42) Tong JH, Tsang RK, Lo KW, Woo JK, Kwong J, Chan MW et al. Quantitative Epstein-Barr virus DNA analysis and detection of gene promoter hypermethylation in nasopharyngeal (NP) brushing samples from patients with NP carcinoma. Clin Cancer Res 2002 August;8(8):2612-9.

(43) Hutajulu SH. Clinical, Virological and Host Epigenetic Markers for Early Identification of Nasopharyngeal Carcinoma in High Risk Populations in Indonesia 2012.

(44) Wildeman MA, Fles R, Adham M, Mayangsari ID, Luirink I, Sandberg M et al. Short-term effect of different teaching methods on nasopharyngeal carcinoma for general practitioners in Jakarta, Indonesia. PLoS One 2012;7(3):e32756.

Chapter 4

Knowledge of general practitioners about nasopharyngeal cancer at the Puskesmas in

Yogyakarta, Indonesia

Renske Fles*,

Maarten A. Wildeman*,

Beni Sulistiono,

Sofi a Mubarika Haryana,

I. Bing Tan

* Both authors contributed equally.

BMC Med Educ. 2010 Nov 18;10:81.

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Abstract

Background

Nasopharyngeal carcinoma (NPC) is one of the leading causes of cancer death in Indonesia. At initial diagnosis, 80% of the patients present with advanced stage disease. In Indonesia, primary medical care is generally provided by the health care centres; named Puskesmas. The lack of knowledge of various aspects of NPC of the General practitioners (GPs) working in these centers might contribute to the diagnostic delay. The aim of this study was to assess the knowledge of these GPs on different aspects of NPC including symptoms, risk factors and incidence.

Methods

One hundred six GPs in the Puskesmas in the Yogyakarta province were subjected to a questionnaire on different aspects of NPC based on literature and interviews with Head and Neck Surgeons.

Results

All GPs approached participated and in total 106 questionnaires were filled in. All participants were aware of NPC as a disease and 89% confirmed that it is a serious problem in Indonesia. However, 50% of the participants believed NPC has a low incidence in their region. The question on early symptoms gave a mean 4.2 answers of which 50% were incorrect.

The GPs provided a total of 318 answers when asked for the risk factors of NPC, 75% of which were incorrect. Fifty seven GPs (54%) stated that they did not receive sufficient education on NPC at the university and insufficient knowledge was gained during daily practice. Ninety-two percent of the GPs were interested in additional education, preferably in form of lectures, meetings or folders.

Conclusion

This study revealed that GPs in the Puskesmas in Yogyakarta lack knowledge on all aspects of NPC. This is an important finding as NPC is endemic in Indonesia and the Puskesmas are the institutions which provide primary medical health care in the country. Further education of the GPs in these endemic areas could be a first step to increase the rate of early detection. Therefore, we suggest 1) to conduct a medical awareness campaign for GPs on the most important subjects concerning NPC, and 2) as soon as NPC awareness among GPs has risen, provide further education on the risk factors, the early symptoms and the incidence, education to the community. We propose to extend this study to other areas in Indonesia (i.e. Jakarta, Surabaya, Central Java), using models that have been developed in Yogyakarta.

Knowledge of general practitioners about nasopharyngeal cancer at the Puskesmas in Yogyakarta, Indonesia

53

Background

NPC is a rare malignancy in most parts of the world. In year 2000 there were approximately 65,000 new cases and 38,000 deaths worldwide [1]. However, NPC is endemic in a few of well-defined populations, for example in China, Southeast Asia, Arctic, Middle East/North Africa and North America [2]. The highest incidence has been observed in Hong Kong where approximately 1 out of 40 men developed a NPC before the age of 75 [3].

NPC in Indonesia has a relatively high incidence of at least 5.7 among men and 1.9 among women per 100.000 (www.iarc.fr) compared to the worldwide incidence 1.9 among men and 0.8 among women. It should be noted that the actual incidence of NPC in Indonesia is unknown due to poor cancer registries. For examples, the Ear, Nose and Throat (ENT) department of the Dr. Sardjito Hospital in Yogyakarta already has 200 new cases of NPC a year and is 1 of the 4 big hospitals in Yogyakarta (data of unpublished study).

One of the most important factors playing a role in the tumour development of NPC is the presence of the Epstein-Barr virus (EBV). The undifferentiated carcinoma (WHO type III) is always associated with EBV [4]. In addition, high levels of volatile nitrosamines in preserved food can be found in the high incidence regions, and has shown to be a putative carcinogen for the development of NPC [5]. Also, the consumption of salty-preserved fish has been strongly associated with an increased risk of developing NPC [6-12]. The non-environmental risk factors include: gender, ethnicity and the family history.

The non-specific nature of the early symptoms makes NPC a disease challenging to diagnose at early stage. The most common presenting symptom is a painless mass in the neck. Early symptoms like nasal dysfunction, aural dysfunction and headache are non-specific for NPC but should be a warning sign in endemic areas. Given these non-specific of NPC, 80% of the patients present themselves with a stage 3 or 4 disease.

The choice of treatment for primary NPC is radiotherapy, with chemotherapy for advanced stage disease. Despite the radio responsiveness of nasopharyngeal tumours, good long-term survival is only achieved for patients with early disease and with minimal neck involvement [13-15].

Given that prognosis is better when treatment is started at an earlier stage of the disease, it is of major importance to determine the various factors that may possibly contribute to the diagnostic delay of NPC in Indonesia.

We hypothesized that one of the possible mechanisms leading to late NPC presentation observed in Indonesia is a lack of knowledge and awareness among primary health care workers; the GPs working in the primary health care centres; Puskesmas.

In this study we aimed to assess the current knowledge amongst GPs in the Puskesmas in Yogyakarta about NPC regarding risk factors, incidence and symptoms. The findings from this study will be useful to complement knowledge and awareness on this important public health issue in Indonesia.

Methods

Interview and questionnaire development

A questionnaire based on literature was designed appointing the most important aspects of NPC; including the risk factors, early symptoms, as well as the education possibilities and the interest in education. This first draft was discussed during a semi constructed interview with four Head and Neck surgeons from Universitas

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Indonesia, Jakarta, Universitas Gadjah Mada, Yogyakarta and The Netherlands Cancer Institute, Amsterdam. The questionnaire was adapted according to consensus opinions.

The second draft was filled in by 10 Indonesian ENT-specialist who gave oral and written comment. These ENT specialists listed the key aspects about NPC a GP should be familiar with, which include the awareness of early NPC symptoms and how to pursue if a patient shows these symptoms. GPs are often unaware to whom and when they should refer a patient. If they refer, patients are often referred to a general surgeon rather than to an ENT specialist.

Questionnaire

The questionnaire consists of four sections, assessing (1) general information about the GPs such as number of years of experience and the number of patients seen in one year; (2) knowledge on NPC such as early symptoms and risk factors (3) incidence, experience in daily practice regarding the extent to which the GPs had been confronted with NPC in their Puskesmas and how they dealt with patients suspected of NPC; and (4) ambition and wishes regarding future education on NPC.

The questionnaire contained open and closed questions. Possible responses were grouped together for the ease of presentation and understanding as described in the results. The open-ended questions featured the questions where multiple answers were possible, for instance when asked about risk factors or presenting symptoms.

Study population

The study population consisted of GPs from three different districts in the Yogyakarta province. In total there are 68 Puskesmas in these regions. With two or three GPs working in one Puskesmas, approximately 170 GPs are practicing in a Puskesmas in this area. One hundred and six GPs were approached in their Puskesmas to participate. They were asked to fill in the questionnaire directly in the presence of the researcher (RF).

Results

All 106 GPs approached participated in this study and completed their questionnaires (100%). All participants know NPC as a disease and 89% confirmed it is a serious problem. Fifty-nine questionnaires were completed immediately during the visit of the interviewer. The other forty-seven were completed at a later time point. Patients waiting for medical attention, was the reason for completion questionnaire without the presence of the researcher. Thirty questionnaires were collected in the centre of Yogyakarta (Kota), the other 76 questionnaires were collected in the rural areas, Sleman and Bantul. The number years of work experience of the GPs varied from less than one year to 29 years (median= 5 years). There was no correlation between working experience and knowledge on NPC. Participant details are outlined in Table 1.

Knowledge about the early symptoms and risk factors

When asked to list early symptoms on NPC, the participating GPs gave a mean of 4.2 answers. However, 51% of these answers were incorrect (Table 2). The most frequent given answer provided was swelling in the neck (n=78; 35%). Nasal obstructions and epitaxis, caused by presence of tumour mass in the nasopharynx, were mentioned 70 times (32%). Tinnitus and hearing loss due to dysfunction of the Eustachian tube, associated with the lateroposterior extension of the tumour to the paranasopharyngeal space was stated 33 times (15%). Headache, diplopia, facial pain and numbness was pointed out 25 (11%) times, these symptoms are caused


55

by skull-base erosion and palsy of the fifth and sixth cranial nerves, associated with the superior extension of the tumour.

Of the 106 participants only 20 participants provided four or more correct symptoms of NPC. The majority of the GPs (66%) could give two correct answers. The most frequent incorrect NPC symptoms stated answers were hoarseness and dysphagia. .

The risk factor questions yielded a total of 328 answers, of which only 36% were indeed risk factors for NPC (Table 2). Surprisingly, EBV was only mentioned 12 times, while smoking, which is not a risk factor for NPC in Indonesia, was answered 80 times. Seven out of the eight participants who provided only one answer gave an incorrect answer.

Knowledge on incidence and referral

Fifty percent of the participating GPs falsely believed that NPC has a low incidence in their region. Furthermore, only 45% was aware of the fact NPC can affect both children and adolescents (table 3). Seventy four percent of the GPs stated that they had never seen a patient with NPC in their Puskesmas. Enlarged lymph nodes is the most common presenting symptom for NPC. The differential diagnosis for these lymph node enlargements is limited and in most cases related to tumours in the ENT region. Strikingly, 80% of the GPs have seen patients with enlarged lymph nodes in the daily practice. In an open question on referral of a suspected NPC case, only 18% percent would refer the patient to an ENT specialist. Further details are shown in table 4.

Table 1 | Descriptive divided by the three district of the province Yogyakarta: Overview of the descriptive variables over the three districts.

Questions Kota (n=30) Sleman n=42) Bantul (n=34)

How long have you worked as a doctor? (in years) 8.2 8.1 6.7

Have you always worked in this area?

yes 26 42 32

no 3 0 2

missing 1 0 0

From which university did you get you medical degree?UGM 18 26 21

other 12 16 12

How many patients do you treat a year? 10364 7944 6236

How many GP’s are practicing in your Puskesmas? 2.9 2.8 2.9

When GPs were asked if they would be interested in additional education on NPC; 92% of the participants responded positive, 7% were neutral and only one participant strongly disagreed. Although the differences between the preferences for the way to educate were small, 96% stated a lecture from an ENT-specialist is the best form of further education on NPC. A good alternative or addition could be a folder (88%). Eighty two percent believes that receiving credits and a certification for participation is important. Finally, 40% of the participants recommended a refreshing course on NPC in the open comment box.

Table 2 | Number of correct and incorrect answers: Overview of the total numbers of correct and incorrect answers when asked about the symptoms and the risk factors of NPC.

correct incorrect total

symptoms 227 (51%) 215 (49%) 442

risk factors 113 (36%) 203 (64%) 316

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Table 3 | General knowledge of the General Practitioners about NPC in Indonesia: General questions about NPC concerning the incidence, severity, age and relation to gender.

Have you ever heard of nasopharyngeal carcinoma?

yes no missing

106 (100%) 0 0

NPC is a serious problem in Indonesia/Javastronglyagree

agree neutral disagreeStronglydisagree

missing

19 (18%) 73(69%) 9 (8%) 2 (2%) 0 3 (3%)

The number of new people suffering NPC on Indonesia per year.

10-99 100-249 250-499 500-999 >1000 missing

6 (6%) 17 (16%) 21 (20%) 9 (8%) 25 (24%) 28 (26%)

From what age people can develop NPC? 0-19 20-29 30-39 40-49 >50 missing

8 (8%) 39 (37%) 37 (35%) 15 (14%) 0 7 (7%)

Which age category has the highest incidence?

0-19 20-29 30-39 40-49 >50 missing

1 (1%) 3 (3%) 35 (33%) 49 (46%) 10 (9%) 8 (8%)

Do you know what the difference in incidence is between men and women?

1:1 1:2 2:1 1:3 3:1 missing

3 (3%) 7 (7%) 51 (48%) 4 (4%) 30 (28%) 11 (10%)

Table 4 | Question related to the situation in the puskesmas: Questions related to the frequency of patients with NPC recognized by the GP, treatment and the referring system of the GP

Did you ever treat a patient with NPC? yes no missing

16 (15%) 89 (84%) 1 (1%)

Have you ever seen a patient with NPC in your clinic?

never<2 times per year

2-5 times per year

5-10 times per year

> 10 times missing

78 (74%) 22 (20%) 4 (4%) 0 (0%) 1 (1%) 1 (1%)

The number of new people suffering NPC in your region per year?

<100 100-250 250-500 500-1000 >1000 missing

80 (75%) 0 (0%) 2 (2%) 0 (0%) 1 (1%) 23 (22%)

Have you seen a shift in the number of people suffering NPC in year clinic the last year?

yes, more patients

yes, less patients

no missing

7 (7%) 2 (2%) 91 (86%) 6 (6%)

How often do you, in average a year, treat patients with enlarged neck lymph nodes?

never<2 times per year

2-5 times per year

5-10 times per year

> 10 times missing

21 (20%) 32 (30%) 30 (28%) 13 (12%) 6 (6%) 4 (4%)

How did you treat most of the patients? No treatment medicationlocal surgery

refer to hospital

medication and referring to a hospital

missing

4 (4%) 15 (14%) 1 (1%) 62 (58%) 13 (12%) 10 (9%)

What do you do when you diagnose a patient with NPC?

refer to hospitalrefer to academic hospital

refer to ENT specialist

refer to specialist

refer if no improvement of treatment

missing

58 (55%) 3 (3%) 13 (12%) 1 (1%) 1 (1%) 30 (28%)


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Discussion

Our study revealed that knowledge on essential aspects of NPC among GPs in the Puskesmas in Yogyakarta, Indonesia is poor. This finding is of importance as NPC is endemic in Indonesia and the Puskesmas are institutions, which provided primary medical care in the country. This lack of basic knowledge of NPC might be a fundamental cause for late stage discovery of the disease by GPs.

The majority of the doctors believed that the incidence of NPC in their region is at least a tenfold lower than the estimated incidence. Together with the lack of knowledge on early NPC symptoms and risk factors we can conclude that the knowledge about NPC of these doctors is not sufficient.

Enlarged lymph nodes in the neck is the most common symptom of NPC at presentation. The participants of this study regularly see patients with enlarged neck lymph nodes, but stated they see very few cases of NPC. Combining the overall answers given regarding early symptoms and incidence, we think GPs often do not consider NPC when they see a patient with enlarged neck lymph nodes or other symptoms suspicious for NPC.

A possible solution could be education combined with a screening method. Fachiroh et al has established a cheap and sensitive screening method for NPC which could be easily adopted in the Puskasmas [16]. This method is based on the detection of the Epstein-Barr virus which is present in all NPCs in Indonesia. The combination of more knowledge and an easy way to screen might lead to early detection of NPC.

Following the answers provided by the participants, the number of NPC patients seen by the GPs is much lower compared to the incidence in this region. The results of our study imply that this may be due to the fact that GPs are unable to identify patients with NPC. It should be noted that the majority of the patients, who were treated at the ENT-departments either went directly to a hospital without referral by a GP, or were administered to a hospital as a medical emergency caused by the tumour, for example dyspnoea to massive neck metastasis. In an ongoing study we look into more detail at the referral system.

Study Limitations

Not all questionnaires were completed in the presence of the researcher. This was mostly due to lack of time on the GPs side as patients were waiting for their appointments. Despite the statement in the questionnaire not to seek help from third parties, GPs may have searched or asked colleagues for answers in our absence. The results of these 47 participants, who filled in the questionnaires in the absence of the interviewer, were notable better but overall remained poor. For example, 9 of the 12 GPs who knew that EBV was related to NPC completed the questionnaire without supervision. This could imply that even if GPs would have searched for information elsewhere the correct information was not available.

In addition, some of the questions despite our testing may not have been understood completely by the participating GPs. For instance, there was a big variance between the answers provided for the number of patients seen a year, even if these doctors worked in the same Puskesmas. For example, in one Puskesmas two GPs stated that they see 18.000 patients a year, whilst the other GP only sees 4000 patients a year.

Conclusion

Based on the results of this survey it is clear that the knowledge of the GPs about NPC in the Puskesmas in Indonesia is not sufficient to deal with this important health issue. The problem concerning the identification

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of patients suffering from NPC is most likely bigger than expected. The number of patients with NPC registered in Sardjito Hospital in the period of 2004 and 2008 might only represent the tip of the iceberg since these are the patients who actually made it to the hospital.

A medical education program should be started to educate the GPs working in the Puskesmas in Indonesia about NPC. The next step could be a campaign to increase public awareness on NPC. Similar campaigns have been enrolled for cervix carcinoma and breast carcinomas [17]. The public needs to be informed to visit the Puskesmas when they have complaints or early NPC symptoms.

References 1. Ferlay J,Bray F,Pisani P,Parkin DM: Globocan 2000: cancer incidence, mortality and prevalence worldwide. 2009 2. Chang ET, Adami HO. The enigmatic epidemiology of nasopharyngeal carcinoma. Cancer Epidemiol.Biomarkers

Prev. 2006;15:1765-1777. 3. Parkin DM, Whelan SL Ferlay J Teppo L Thomas DB: Cancer. Cancer incidence in five continents, vol. VIII. IARC

scientific publications No. 155. 2002. 4. Raab-Traub N. Epstein-Barr virus in the pathogenesis of NPC. Semin.Cancer Biol. 2002;12:431-441. 5. Poirier S, Ohshima H, de The G et al. Volatile nitrosamine levels in common foods from Tunisia, south China and

Greenland, high-risk areas for nasopharyngeal carcinoma (NPC). Int J.Cancer 1987;39:293-296. 6. Guo X, Johnson RC, Deng H et al. Evaluation of nonviral risk factors for nasopharyngeal carcinoma in a high-risk

population of Southern China. Int.J.Cancer 2009;124:2942-2947. 7. Zou J, Sun Q, Akiba S et al. A case-control study of nasopharyngeal carcinoma in the high background radiation

areas of Yangjiang, China. J.Radiat.Res.(Tokyo) 2000;41 Suppl:53-62. 8. Henderson BE, Louie E. Discussion of risk factors for nasopharyngeal carcinoma. IARC Sci.Publ. 1978 251-260. 9. Yu MC, Ho JH, Lai SH, Henderson BE. Cantonese-style salted fish as a cause of nasopharyngeal carcinoma: report

of a case-control study in Hong Kong. Cancer Res. 1986;46:956-961. 10. Yu MC, Huang TB, Henderson BE. Diet and nasopharyngeal carcinoma: a case-control study in Guangzhou,

China. Int.J.Cancer 1989;43:1077-1082. 11. Armstrong RW, Imrey PB, Lye MS et al. Nasopharyngeal carcinoma in Malaysian Chinese: salted fish and other

dietary exposures. Int J.Cancer 1998;77:228-235. 12. Yuan JM, Wang XL, Xiang YB et al. Preserved foods in relation to risk of nasopharyngeal carcinoma in Shanghai,

China. Int.J.Cancer 2000;85:358-363. 13. Chen CL, Hsu MM. Second primary epithelial malignancy of nasopharynx and nasal cavity after successful

curative radiation therapy of nasopharyngeal carcinoma. Hum.Pathol. 2000;31:227-232. 14. Wei WI, Sham JS. Nasopharyngeal carcinoma. Lancet 2005;365:2041-2054. 15. Al Sarraf M, Reddy MS. Nasopharyngeal carcinoma. Curr.Treat.Options Oncol. 2002;3:21-32. 16. Fachiroh J, Prasetyanti PR, Paramita DK et al. Dried-blood sampling for epstein-barr virus immunoglobulin G

(IgG) and IgA serology in nasopharyngeal carcinoma screening. J.Clin.Microbiol. 2008;46:1374-1380. 17. Ali TS, Baig S. Evaluation of a cancer awareness campaign: experience with a selected population in Karachi.

Asian Pac.J.Cancer Prev. 2006;7:391-395.

Chapter 5

Short-term eff ect of diff erent teaching methods on Nasopharyngeal Carcinoma for general practitioners

in Jakarta Indonesia

M. A. Wildeman,

R. Fles,

M. Adham,

I.D. Mayangsari,

I. Luirink,

M. Sandberg,

A. D. Vincent,

F. Fardizza,

Z. Musa,

Armiyanto,

J. M. Middeldorp,

G. Gerritsen,

R. Soewento,

I. B. Tan

PLoS One. 2012;7(3):e32756.

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Abstract

In Indonesia, Nasopharyngeal Carcinoma (NPC) is the most frequent cancer of the head and neck region. At first presentation in the hospital most patients already have advanced NPC. Our previous study showed that general practitioners (GPs) working in Yogyakarta, Indonesia lack the knowledge necessary for early detection of NPC. By providing training on early symptoms of NPC we hope that the diagnosis and referral will occur at an earlier stage. Here we assess the current NPC knowledge levels of GPs in Jakarta, evaluate improvement after training, compare the effectiveness of two training formats, and estimate the loss of recall over a two week period.

Methods

Two Indonesian GPs visited 31 Primary Health Care Centres (PHCCs) and provided a lecture on NPC. The alternative format consisted of a symposium at the Universitas Indonesia, Jakarta, presented by local head and neck surgeons, with all GPs in the region being invited. To evaluate the effect of both formats a questionnaire was conducted before and after.

Results

The lecture in the PHCCs was attended by 130 GPs. Sixty-six GPs attended the training in the university hospital and 40 GPs attended both. Pre training the NPC knowledge level was poor with an average of 1.6 symptoms being correctly identified out of a potential maximum of 12, this was increased to 4.9 post training (p < 0.0001). GPs attending the PHCC course recorded a greater increase in correct symptoms than those attending the symposium (3.8 vs. 2.8; p=0.01). After a two week period the knowledge levels had declined slightly from 5.5 correctly identified symptoms to 4.2 (p=0.25).

Conclusion

These results confirm our findings regarding GPs insufficient knowledge of NPC. Lectures in the PHCC and a symposium have both been proven to be effective training tools in the education of GPs.

Short-term effect of different teaching methods on Nasopharyngeal Carcinoma for general practitioners in Jakarta Indonesia

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Introduction

The Indonesian health care sector represents a mix of public and private providers. The government provides primary health care centres (PHCCs). There are more than 7600 of these centres in Indonesia. At the primary health care level, Indonesia has relatively adequate levels of provision, for every 30 000 people one public health centre on average. [1] ,[2]. The PHCCs are the first recourse for Indonesians seeking medical attention, with referral to a hospital occurring when deemed necessary.

Cancer is increasingly recognized as leading cause of death in Indonesia, and nasopharyngeal carcinoma (NPC) is the most frequent cancer in the head and neck area and is the fourth most common tumour occurring in males. The incidence is estimated 6 per 100000, leading to at least 14000 new cases per year [3]. However this may be an underestimation due to poor cancer registration. In most countries NPC is an orphan disease with a worldwide incidence of 80000 new cases per year. However, in Southern China and most of South-East Asia NPC is endemic with a yearly incidence reaching as high as 20-50 cases per 100000 annually [4],[5].

NPC arises in the epithelial lining of the nasopharynx. This neoplasm is frequently seen at the pharyngeal recess (Rosenmüller’s fossa) posteromedial to the medial crura of the eustachian tube opening in the nasopharynx [6].

NPC patients present themselves with symptoms from the following categories: (1) presence of tumour mass in the nasopharynx (epistaxis, nasal obstruction, nasal discharge); (2) dysfunction of the Eustachian tube (tinnitus, hearingloss); (3) skull base erosion and palsy of the 5th and 6th nerve (headache, diplopia, facial pain and numbness); and (4) neck mass (painless enlargement of the upper cervical lymph node). The early symptoms such as epistaxis and tinnitus are not specific for NPC, which makes it difficult to diagnose at an early stage [6].

The Epstein-Barr virus is known as the first tumour virus and was associated with NPC in 1970 [7]. Other risk factors of NPC are environmental co-carcinogens, i.e. high levels of volatile nitrosamines and butyrate derivatives in preserved food, especially in salty-preserved fish and dried meat, alcohol and smoking [8-11]. Non-environmental risk factors are gender, ethnicity and family history [12,13].

A majority of patients present with a loco-regional disease most often with an advanced lymph node metastasis in the neck [14,15]. Accordingly the most common symptom at presentation is a painless mass in the neck [15]. Presently, at intake in the hospital Cipto Mangunkusumo hospital/ University of Indonesia, Jakarta 88% of new patients already have advanced NPC. (Adham et al; Chin J Cancer, submitted). The standard treatment for primary NPC is radiotherapy to which NPC is sensitive, however in advanced cases additional chemotherapy is needed. A recent meta analysis proved the clinical benefit of concurrent chemoradiation therapy compared with radiotherapy alone in the treatment of advanced NPC in endemic areas [16]. The most important prognostic factor is presenting stage[6,17]. Patients with early stage disease (T1, T2 and N0-1 without distant metastasis) can achieve a five years overall survival of 85% compared to 66 % in patients with late stage disease (T3, T4 and N2, N3 without distant metastasis)[17]. The 10-year disease free survival for early stage NPC is 67-71% while for late stage disease this is 29-54% [18].

One possible reason for the high percentage of patients with advanced NPC could be a delay in referral due to poor diagnosis. In our previous study we assessed the knowledge on NPC of the GPs working in the PHCC in the Yogyakarta region [19]. Our results indicated that the knowledge of GPs is insufficient with many not being aware of the high incidence of NPC in their region.

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In this study we (1) assess the current knowledge concerning NPC of GPs working in the Jakarta region, (2) evaluate the improvement provided by additional training, (3) compare the effectiveness of two different training formats, and (4) estimate the loss of recall over a two week period. By providing additional training about NPC and its early symptoms we hope to increase the diagnosis and referral of patients with early stage NPC. An early detection program for breast cancer, cervical cancer and NPC was proven to be effective for down staging breast cancer and cervical cancer, however the training was not sufficient to result in a downstaging of NPC [20]. We anticipate our early detection training program to be more effective since we only focus on NPC.

Methods

Study population

For this study we invited GPs from two of the five districts of the province Jakarta.

The study population consists of three groups: (1) GPs who only attended the lecture at their own PHCC; (2) those who only attended the symposium at the hospital Cipto Mangunkusumo (Universitas Indonesia, Jakarta); and (3) those who attended the lecture at the PHCC followed by the symposium at the hospital Cipto Mangunkusumo.

Approval for both the visit at the PHCCs as well as for the symposium was given by the head of public health department for the province Jakarta, and a letter of approval was presented at each PHCC visited. For study design see figure 1.

Questionnaire

Data were collected utilizing a revised questionnaire, based on the one described in a previous study[19], which consisted of four sections: (1) general information questions concerning the GP, such as the number of years experience, and the number of patients seen per year; (2) questions concerning NPC, such as early symptoms and risk factors; (3) questions concerning the experience in daily practice regarding the extent to which a GP was confronted with NPC and his/her response for suspected NPC; and (4) questions regarding the ambition and wishes regarding future education on NPC.

The questions regarding early symptoms and risk factors allowed for the GP to list as many as he/she thought appropriate, while the questions concerning prevalence of NPC were multiple choice questions. The questionnaire was completed both prior to and after the NPC training session. All questionnaires were finished within 20 minutes, and were made in anonymity.

Primary health care centres (PHCCs)

PHCCs in the Jakarta region were visited by two physicians. The GPs working at these centres were invited to participate in the study. GPs were not informed forehand as to the purpose of the visit. The training session consisted of a lecture designed by a team of Head and Neck surgeons from Rumah Sakit CiptoMangunkusumo/ University of Indonesia, Jakarta, Indonesia and the Netherlands Cancer Institute/ Antoni van Leeuwenhoek hospital. This lecture focused on all aspects of NPC, especially on early symptoms and best referral strategy. Two Indonesian physicians who have been trained and examined by the same Head and Neck surgeons presented the lecture. The lecture was given in the Indonesian language. The lecture lasted for 35 minutes and afterwards there was time for discussion.


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Figure 1 | Study design

Figure 2 | Percentage of correct answers about the symptoms given by the GPs. a) Percentage of correct symptoms from the four different catagories given by GPs with less than 20 years of work experience. b) Percentage of correct symptoms from the four different catagories given by GPs with more than 20 years of work experience. (1) presence of tumour mass in the nasopharynx; (2) dysfunction of the eustachian tube, associated with the lateroposterior extension of the tumour to the paranasopharyngeal space; (3) skull-base erosion and palsy of the fifth and sixth cranial nerves, associated with the superior extension of the tumour; (4) neck masses.

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Symposium

All GPs visited by the two physicians in the PHCCs were invited to a subsequent symposium on NPC (one to four weeks thereafter) at the Cipto Mangunkusumo University Hospital in Jakarta, Indonesia. The symposium consisted of a lecture on the risk factors, symptoms, incidence, referral system concerning NPC patient, in addition a physical examination training of the head and neck was provided. The symposium was accredited by the Indonesian Medical Association. The training and lectures lasted for four hours.

Statistical Methods

Analyzed were the questions concerning NPC symptoms, risk factors and age at presentation. The listed NPC symptoms and risk factors were designated as being correct or incorrect, and in the case of the former being one of 12 possible correct symptoms. As a result the number of correctly listed symptoms was binomial (listed or not listed) for the 12 cases. Similarly for the questions concerning youngest and peak age at presentation the outcome was taken as correct or incorrect. While for the number of incorrectly listed symptoms, the number of correctly listed risk factors and the number of incorrectly listed risk factors, we assume the outcome to be Poisson distributed. These outcomes were modelled using generalized linear mixed effects models (GLMMs), either logistic or Poisson mixed effects models. In all models doctor ID is the random intercept with an unstructured covariance matrix. Fixed effect covariates included teaching format (PHCC vs. symposium), time (pre- or post-training), work experience (0-10, 11-20, 20+ years) and the pair-wise interactions of these. The 12 correct symptoms were divided into four categories (for details see figure 2); this covariate and its interaction with other covariates were also included as fixed effects in the model for correct symptoms. The only covariate missing data was the number of work experience years, which was imputed using the median. In these analyses only data from the doctors attending a training session for the first time was employed.

The symposium data from the doctors who had previously attending the PHCC trainings was used to assess the loss in recall over the 2 week period between PHCC and symposium training sessions. In these analyzes the GLMMs included a four level factor representing the four assessments for these individuals (PHCC pre-training, PHCC post-training, symposium pre-training, symposium post-training) as a fixed effect. The interest being in the change in knowledge between PHCC post-training session and the symposium pre-training session.

In all models, fixed effects were removed if their significance level was greater than 0.10 in a stepwise backwards procedure using log-likelihood ratio tests. No adjustments for multiple testing were performed. A Wilcox-Mann-Whitney test was performed to assess difference in work-experience years between the two groups. For all tests the level of significance set at 0.05.

Results

In total, training sessions were provided at 31 PHCCs involving 130 GPs. All GPs have voluntary participated. Two GPs could not participate in the assessment process as they were attending patients; the remaining present 128 GPs completed the questionnaire both pre and post training. The average number of GPs who participated at a PHCC was 4.3 (median: 3; range 1-12). In total 106 GPs attended the symposium, of which 76 completed both the pre- and post- training questionnaire. Fifty-four of these had not attended the trainings in the PHCCs, while 22 attended both. The overall study population is presented in figure 1.

The clinicians attending the PHCC session had more years work-experience than those attending the symposium (p=0.007). The median amount of years of work experience of clinicians attending the PHCC


67

session was 9 years, with a minimum of one year and a maximum of thirty-three years. Participants at the symposium ranged in work experience from 0 to 27 years (median 9 years). Of the 182 participating clinicians 35 (19%) had more than 20 years experience. Of these, 29 attended the PHCC sessions, while only 6 attended the symposium session.

Symptoms

The overall gain over both trainings was an increase from an average of 1.6 to 4.9 symptoms correctly identified (p < 0.0001). However, in comparison with the PHCC group, the symposium group had a smaller overall improvement (p=0.01). Prior to the training sessions an average of 1.4 symptoms were correctly listed at the PHCCs versus 1.7 at the symposium (p=0.30). After training the average number of correct symptoms listed was 5.3 in the PHCC versus 4.5 at the symposium (Table 1).

Table 1 | The mean number of accurately listed symptoms both pre and post training, by GPs attending the PHCC and the symposium sessions.

Pre-training Post-training

Symptoms correct 95% CI Symptoms correct 95% CI

PHCC 1.5 (1.3-1.7) 5.3 (5.0-5.6)

Symposium 1.7 (1.4-2.0) 4.5 (4.1-5.0)

There were no difference between GPs with less work experience (0-10 years) and those with moderate work experience (11-20 years) (p=0.46). However compared with these GPs, GPs with longer work experience (20+years) on average listed more symptoms correctly prior to training (1.9 vs. 1.5; p = 0.04), but gained less from the training sessions (4.3 vs. 5.0; p < 0.0001). In the PHCC pre-training questionnaire 71% (129/182) of the GPs correctly identified neck mass as one of the correct symptoms of NPC, however only a few GPs could describe symptoms from one of the other 3 categories in the pre-training assessment (figure 2). Post training there was an increase of correct symptoms for the non-neck mass categories.

Training decreased the number of incorrect symptoms listed by GPs from 2.0 pre-training to 0.9 post-training (p < 0.0001). As with the correctly identified symptoms, this improvement was higher for GPs attending the PHCC training than those attending the symposium (see Table 2; p<0.0001). However GPs attending the symposium recorded fewer incorrect symptoms pre-training than those attending the PHCC sessions (see Table 2; p=0.002). There was no association between the number of incorrect symptoms and number of years of work experience (p=0.85).

Table 2 | The mean number of incorrect symptoms listed in the pre- and post-training assessments by GPs attending the PHCC and symposium training sessions.


Symptoms incorrect 95% CI Symptoms incorrect 95% CI

PHCC 2.4 (2.1-2.8) 0.5 (0.4-0.6)

Symposium 1.6 (1.2-2.0) 1.2 (0.9-1.6)

Risk Factors

The number of correctly listed risk factors increased after the training sessions from 1.5 pre-training to 4.1 post-training (p < 0.0001). This improvement was not affected by training format: PHCC vs. symposium (p=0.76), nor was the number of years worked related to the number of correct risk factors listed by GPs (p=0.37). GPs

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attending the symposium knew more risk factors prior to training (and therefore after training) than those attending the PHCC only (p=0.001) (Table 3).

Table 3 | The mean number of correctly identified risk factors listed in the pre- and post-training by GPs attending the PHCC and symposium sessions.


Risk factors correct 95% CI Risk factors correct 95% CI

PHCC 1.3 (1.1-1.5) 3.8 (3.5-4.1)

Symposium 1.6 (1.4-1.9) 4.7 (4.2-5.3)

Likewise, the number of incorrect risk factors decreased after training (see Table 4; p = 0.004); this improvement was not different between those GPs attending the PHCC or symposium sessions (p=0.16); there was no association between the number of incorrect risk factors and number of years worked (p=0.13); and GPs attending the symposium listed fewer incorrect risk factors pre-training than those attending the PHCC training (p=0.02).

Table 4 | The mean number of incorrect risk factors listed in the pre- and post-training assessments by GPs attending the PHCC and the symposium sessions.


Risk factors incorrect 95% CI Risk factors incorrect 95% CI

PHCC 0.8 (0.6-1.0) 0.6 (0.4-0.7)

Symposium 0.5 (0.4-0.7) 0.4 (0.3-0.5)

Age of NPC presentation and peak incidence of age group.

The questionnaire also contained questions about the youngest age of presentation and the peak age of incidence. GPs more often answered incorrectly the youngest age at presentation when compared with the peak age at presentation question (p=0.03; see Table 5). The training sessions resulted in an improvement in knowledge (p < 0.0001), with the PHCC training resulting in a slightly higher improvement than the symposium training (p = 0.04). There was no difference between GPs who had worked 0-10 years and those who had worked 11-20 years (p=0.88), while GPs with the longest working experience (20+ years) more often got the youngest age of presentation question wrong (p=0.03).

Table 5 | The probability (%) of correctly answering questions concerning youngest age of presentation and peak age of incidence.


Work Exp. % Correct 95% CI % Correct 95% CI

Youngest Age PHCC 0-20 yrs 22 (16-30) 71 (63-78)

20+ yrs 13 ( 8-22) 57 (43-71)

Symposium 0-20 yrs 25 (17-37) 59 (46-70)

20+ yrs 15 ( 8-29) 43 (27-61)

Peak Age PHCC 0-20 yrs 32 (25-40) 81 (74-86)

20+ yrs 41 (28-55) 86 (77-92)

Symposium 0-20 yrs 20 (13-31) 52 (40-63)

20+ yrs 27 (15-45) 61 (43-76)


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Assessment of loss of recall: GPs attended who attended both training sessions.

GPs who attended both the PHCC and the symposium training sessions had a small reduction in the number of symptoms correctly identified between the end of the PHCC session and the start of the symposium session (5.5 vs. 4.2; p=0.25), however this was still a gain when compared to their pre-training knowledge (4.2 vs. 1.3; p < 0.0001). Strikingly, GPs who came back for a second training session recorded twice the number of incorrect symptoms prior to the symposium training (1.1 vs. 0.5; p=0.05), however this was still a large reduction on the number of errors made prior to the PHCC training (1.1 vs. 2.8; p < 0.0001). GPs who came back for a second training session recorded slightly fewer correct risk factors pre-symposium training as compared to PHCC post-training (4.1 vs. 3.2; p=0.11), but a significant increase when compared to their knowledge prior to their first training session (1.6 vs. 3.2; p=0.0008). Similarly there was a decrease in the probability of correctly identifying the youngest and peak ages at presentation (Youngest: 84% vs. 59%; Peak: 90% vs. 72%; p=0.39), however GPs still knew more than what they knew prior to the PHCC session (Youngest: 13% vs. 59%; Peak: 22% vs. 72%; p < 0.0001).

Discussion

NPC has a high incidence and mortality in Indonesia with late diagnosis being one of the reasons for numerous advanced disease and high mortality. GPs working in a PHCC are the first line of care for patients in need of medical attention. For a correct and early diagnosis of NPC the knowledge of these GPs, especially concerning early-stage symptoms is crucial. In a prior study in Yogyakarta, Central-Java, we have shown that the knowledge on NPC and related symptoms among GP’s working in PHCCs is insufficient for the recognition of NPC and to initiate appropriate referral. Our ongoing studies in the Yogyakarta province and the study from the Jakarta region presented here confirm these results; the GPs working in the Jakarta region have, similar to the Yogyakarta region, insufficient knowledge to refer NPC suspects to the hospital. Besides confirmation of the lack of knowledge, we investigated if improvement of knowledge is possible by introducing a focussed education program. This is the first study that examines the effect of different teaching methods to educate Indonesian GPs on NPC. Early diagnosis will influence the type of treatment patient require; only in advanced stage of disease additional chemotherapy is required, while in early stages radiotherapy alone is sufficient. The addition of chemotherapy to the treatment leads to more serious side effects and a general weakening of the patient. Furthermore, early diagnosis of NPC should lead to fewer patients presenting with distant metastasis who currently cannot be treated with curative intent.

In Malaysia prior studies have proved that the lack of awareness and knowledge of primary health care workers is one of the main reasons for delayed diagnosis. Given that presenting stage is the most important prognostic factor, the appropriate training of GPs is critical [21]. The relevance of adequate referral by GPs for head and neck carcinomas has been showen by Alho et al.[22], who found that in 20% of the 221 patients, subsequently to be diagnosed with head and neck carcinoma, were initially send home without referral. The risk of death in this group was significantly higher when compared with the patients who were immediately referred or received a follow up appointment. Although not statistically significant, patients who were initially sent home had higher cancer stage at diagnosis.

The same research team has also showen that time between GP referral and final diagnosis is a significant factor in patient outcome in other head and neck cancers [23]. Long delay in primary care resulted in a significant worsened prognosis especially by patients with laryngeal carcinoma [24].

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Educational sessions at PHCCs by a team of two GPs and a symposium by local head and neck surgeons have both shown to be effective. In general, the pre-test knowledge at the symposium was higher than in the PHCC. The reason for this could be that visit in the PHCC was unannounced, while for the symposium the GPs received an invitation so they had some time to prepare for the meeting, or were being pre-informed about the NPC topic by colleagues who had previously attended the PHCC training sessions.

Comparing the two different training formats we see a greater gain in knowledge at the PHCCs. One explanation for this could be that the PHCC sessions provided a more focused approach and direct contact/confrontation with participants. Another reason could be that the lecture at the symposium was more extensive and perhaps did not delve too deeply into the most important aspects, but rather covered all aspects of NPC to broadly.

On the other hand, during the symposium GPs also received practical education for performing physical examination of the head and neck region and a testimonial of a NPC patient. We expect this additional training aspect to be important for the recognition of NPC patients. Another remarkable result is that the more experienced GPs knew more prior to the training but learned less in both interventions. Perhaps in future the education of longer serving doctors should be adjusted.

For the four different categories of symptoms, as described by Wei et al, we see at the pre-test the most often and only given correct answer is neck mass, which only occurs in advanced NPC. At the post test the GPs are also aware of symptoms in the other categories (see figure 2), most importantly, including symptoms of early stage NPC.

The doctors who attended both trainings showed a small decline in knowledge prior to the symposium when compared with their results directly after the session in the PHCC. However they still knew far more than what they knew prior to the PHCC session. Unfortunately we are only able to assess recall over an average of 8 days. It would be of great interest to assess changes in NPC awareness over a much longer duration.

Visiting the PHCC was a time consuming exercise as the PHCC are scattered throughout Jakarta. The symposium was more time-effective with all the general practitioners present at the same venue and date. Accreditation points are only accrued after the symposium and not the PHCC sessions, thus making the symposium session more attractive for the GPs. However, every GP visited in the PHCC was willing to participate, perhaps stimulated or convinced by the approval letter from the head of public health department of the Jakarta province.

Although this study demonstrates the effectiveness of education GPs about NPC, the time between the pre- and post-training assessments is short. The goal of achieving a down-staging of NPC at presentation is still to be proven. We believe the recent introduction of an online data management service will aid in the confirmation of earlier stage presentation of patients with NPC[25].

The NPC WHO III histological subtype is the most prevalent type in SE-Asia and Indonesia. This type is causally associated with the Epstein-Barr virus (EBV). Prior studies have shown that EBV-related markers can be used for early detection (screening) and prognostic monitoring. These markers include EBV (IgA) serology and EBV-DNA load since NPC patients have characteristic elevated IgG and IgA antibody titres to several EBV encoded antigens as well as increased EBV-DNA derived from shed (apoptotic) fragments from the tumour into the circulation. Increased IgA antibody levels are found against early antigen (EA), viral capsid antigen (VCA) and the latent Epstein-Barr nuclear antigen 1 (EBNA1) as well as inhibitory antibodies to the EBV specific DNase [26,27]. These antibody responses against defined viral antigens are the basis of a proposed screening test for NPC in high-risk populations [28-30]. Recent insight in the molecular basis and diversity of anti EBV IgA and IgG responses allowed the development of more defined serological tools[31-35]. A possible assay in


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the future could be EBV DNA load in the circulation and in nasopharyngeal brushings since both have been detected in a higher proportion of NPC patients than controls[36-40]. Especially EBV IgA serology testing appears to fulfil criteria as a screening tool in the future, since the price is relative low and easy to use when combined with finger-prick blood sampling [34,41]. Future education programs should include referencing to the availability of improved diagnostic procedures for screening and early detection. Improved education combined with a screening method could be a cheap and sensitive screening method for NPC in Indonesia and other high incidence countries. Importantly, the decision for serological or EBV-DNA based analysis has to be based on complaints and duration of complaints registered and interpreted by the GP. Our ongoing research is focussed on finding the best decision tree to accomplish effective early stage diagnosis in NPC high-risk groups (Hutajulu et al; manuscript submitted).

Currently the NPC awareness programme takes place in Jakarta, Yogyakarta and Surabaya. Hopefully in the future this program can be expanded to include all of Indonesia. We also hope to raise public awareness on importance of early-stage cancer and how and when to consult a GP in all layers of Indonesian society. However, as a first step we aim to raise the level of relevant knowledge in primary health care workers. Similar campaigns to educate society on breast and cervical cancer have proven to be highly effective [42,43]. Education of the GPs and society, and combined with improved diagnostic testing will ideally result in earlier detection of NPC, better treatment outcomes, and increased overall prognosis.

Conclusion:

The current level of knowledge regarding NPC diagnosis is poor, potentially contributing to an increased rate of late stage diagnosis. Additional training sessions increased the knowledge of key symptoms, in particular early-stage symptoms. This increase was observed after conducting both types of training format: a centralized symposium and lectures in local PHCC. With improved knowledge of NPC patients should be referred to hospital at an earlier stage of NPC and as such should have an improved chance of survival.

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13. Parkin DM, Bray F, Ferlay J, Pisani P: Global cancer statistics, 2002. CA Cancer J Clin 2005, 55: 74-108. 14. Ozyar E, Atahan IL, Akyol FH, Gurkaynak M, Zorlu AF: Cranial nerve involvement in nasopharyngeal carcinoma: its

prognostic role and response to radiotherapy. Radiation medicine 1994, 12: 65-68. 15. Lee AW, Foo W, Law SC, Poon YF, Sze WM, SK O et al.: Nasopharyngeal carcinoma: presenting symptoms and

duration before diagnosis. Hong Kong Med J 1997, 3: 355-361. 16. Zhang L, Zhao C, Ghimire B, Hong MH, Liu Q, Zhang Y et al.: The role of concurrent chemoradiotherapy in the

treatment of locoregionally advanced nasopharyngeal carcinoma among endemic population: a meta-analysis of the phase III randomized trials. BMC Cancer 2010, 10: 558.

17. Lee AW, Sze WM, Au JS, Leung SF, Leung TW, Chua DT et al.: Treatment results for nasopharyngeal carcinoma in the modern era: the Hong Kong experience. Int J Radiat Oncol Biol Phys 2005, 61: 1107-1116.

18. Lee AW, Poon YF, Foo W, Law SC, Cheung FK, Chan DK et al.: Retrospective analysis of 5037 patients with nasopharyngeal carcinoma treated during 1976-1985: overall survival and patterns of failure. Int J Radiat Oncol Biol Phys 1992, 23: 261-270.

19. Fles R, Wildeman MA, Sulistiono B, Haryana SM, Tan IB: Knowledge of general practitioners about nasopharyngeal cancer at the Puskesmas in Yogyakarta, Indonesia. BMC Med Educ 2010, 10: 81.

20. Devi BC, Tang TS, Corbex M: Reducing by half the percentage of late-stage presentation for breast and cervix cancer over 4 years: a pilot study of clinical downstaging in Sarawak, Malaysia. Ann Oncol 2007, 18: 1172-1176.

21. Prasad U, Pua KC: Nasopharyngeal carcinoma: a delay in diagnosis. Med J Malaysia 2000, 55: 230-235. 22. Alho OP, Teppo H, Mantyselka P, Kantola S: Head and neck cancer in primary care: presenting symptoms and the

effect of delayed diagnosis of cancer cases. CMAJ 2006, 174: 779-784. 23. Koivunen P, Rantala N, Hyrynkangas K, Jokinen K, Alho OP: The impact of patient and professional diagnostic

delays on survival in pharyngeal cancer. Cancer 2001, 92: 2885-2891. 24. Teppo H, Koivunen P, Hyrynkangas K, Alho OP: Diagnostic delays in laryngeal carcinoma: professional diagnostic

delay is a strong independent predictor of survival. Head Neck 2003, 25: 389-394. 25. Wildeman MA, Zandbergen J, Vincent A, Herdini C, Middeldorp JM, Fles R et al.: Can an online clinical data

management service help in improving data collection and data quality in a developing country setting? Trials 2011, 12: 190.

26. Cevenini R, Donati M, Caliceti U, Moroni A, Tamba I, Rumpianesi F: Evaluation of antibodies to Epstein-Barr virus in Italian patients with nasopharyngeal carcinoma. J Infect 1986, 12: 127-131.

27. Chen JY, Chen CJ, Liu MY, Cho SM, Hsu MM, Lynn TC et al.: Antibodies to Epstein-Barr virus-specific DNase in patients with nasopharyngeal carcinoma and control groups. J Med Virol 1987, 23: 11-21.

28. Ji MF, Wang DK, Yu YL, Guo YQ, Liang JS, Cheng WM et al.: Sustained elevation of Epstein-Barr virus antibody levels preceding clinical onset of nasopharyngeal carcinoma. Br J Cancer 2007, 96: 623-630.

29. Tamada A, Makimoto K, Yamabe H, Imai J, Hinuma Y, Oyagi A et al.: Titers of Epstein-Barr virus-related antibodies in nasopharyngeal carcinoma in Japan. Cancer 1984, 53: 430-440.

30. de Vathaire F, Sancho-Garnier H, de The H, Pieddeloup C, Schwaab G, Ho JH et al.: Prognostic value of EBV markers in the clinical management of nasopharyngeal carcinoma (NPC): a multicenter follow-up study. Int J Cancer 1988, 42: 176-181.

31. Fachiroh J, Schouten T, Hariwiyanto B, Paramita DK, Harijadi A, Haryana SM et al.: Molecular diversity of Epstein-Barr virus IgG and IgA antibody responses in nasopharyngeal carcinoma: a comparison of Indonesian, Chinese, and European subjects. J Infect Dis 2004, 190: 53-62.

32. Fachiroh J, Paramita DK, Hariwiyanto B, Harijadi A, Dahlia HL, Indrasari SR et al.: Single-assay combination of Epstein-Barr Virus (EBV) E. J Clin Microbiol 2006, 44: 1459-1467.

33. Paramita DK, Fachiroh J, Artama WT, van Benthem E, Haryana SM, Middeldorp JM: Native early antigen of Epstein-Barr virus, a promising antigen for diagnosis of nasopharyngeal carcinoma. J Med Virol 2007, 79: 1710-1721.

34. Fachiroh J, Prasetyanti PR, Paramita DK, Prasetyawati AT, Anggrahini DW, Haryana SM et al.: Dried-blood sampling for epstein-barr virus immunoglobulin G (IgG) and IgA serology in nasopharyngeal carcinoma screening. J Clin Microbiol 2008, 46: 1374-1380.

35. Paramita DK, Fachiroh J, Haryana SM, Middeldorp JM: Two-step Epstein-Barr virus immunoglobulin A enzyme-linked immunosorbent assay system for serological screening and confirmation of nasopharyngeal carcinoma. Clin Vaccine Immunol 2009, 16: 706-711.

36. Lin JC, Wang WY, Chen KY, Wei YH, Liang WM, Jan JS et al.: Quantification of plasma Epstein-Barr virus DNA in patients with advanced nasopharyngeal carcinoma. N Engl J Med 2004, 350: 2461-2470.


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37. Stevens SJ, Verkuijlen SA, Hariwiyanto B, Harijadi, Fachiroh J, Paramita DK et al.: Diagnostic value of measuring Epstein-Barr virus (EBV) DNA load and carcinoma-specific viral mRNA in relation to anti-EBV immunoglobulin A (IgA) and IgG antibody levels in blood of nasopharyngeal carcinoma patients from Indonesia. J Clin Microbiol 2005, 43: 3066-3073.

38. Stevens SJ, Verkuijlen SA, Hariwiyanto B, Harijadi, Paramita DK, Fachiroh J et al.: Noninvasive diagnosis of nasopharyngeal carcinoma: nasopharyngeal brushings reveal high Epstein-Barr virus DNA load and carcinoma-specific viral BARF1 mRNA. Int J Cancer 2006, 119: 608-614.

39. Tune CE, Liavaag PG, Freeman JL, van den Brekel MW, Shpitzer T, Kerrebijn JD et al.: Nasopharyngeal brush biopsies and detection of nasopharyngeal cancer in a high-risk population. J Natl Cancer Inst 1999, 91: 796-800.

40. Tong JH, Tsang RK, Lo KW, Woo JK, Kwong J, Chan MW et al.: Quantitative Epstein-Barr virus DNA analysis and detection of gene promoter hypermethylation in nasopharyngeal (NP) brushing samples from patients with NP carcinoma. Clin Cancer Res 2002, 8: 2612-2619.

41. Ji MF, Yu YL, Cheng WM, Zong YS, Ng PS, Chua DT et al.: Detection of Stage I nasopharyngeal carcinoma by serologic screening and clinical examination. Chin J Cancer 2011, 30: 120-123.

42. Ali TS, Baig S: Evaluation of a cancer awareness campaign: experience with a selected population in Karachi. Asian Pac J Cancer Prev 2006, 7: 391-395.

43. Bhurgri H, Gowani SA, Itrat A, Samani S, Zuberi A, Siddique MS et al.: Awareness of cancer risk factors among patients and attendants presenting to a tertiary care hospital in Karachi, Pakistan. J Pak Med Assoc 2008, 58: 584-588.

Chapter 6

Photodynamic therapy in the therapy for recurrent/persistent nasopharyngeal cancer

Maarten AM Wildeman,

Heike J Nyst,

Baris Karakullukcu,

Bing I Tan

Head Neck Oncol. 2009 Dec 17;1:40.

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Abstract

To determine the efficacy of Photodynamic therapy of patients with recurrent Nasopharyngeal Carcinoma we reviewed all available literature. Since the treatment options for recurrent or persistent Nasopharyngeal Carcinoma are limited, the survival rates poor and the complications severe; there is definitely a place for alternative treatment modalities with more efficacy and less morbidity. Photodynamic therapy (PDT) has the potential to be a very effective local treatment modality for recurrent or persistent nasopharyngeal cancer, without the severe side effects seen with re-irradiation. This review shows all reported results of Photodynamic therapy in the treatment for Nasopharyngeal Carcinoma.


77

Introduction

Nasopharyngeal carcinoma (NPC) occurs sporadically in the Western world, but is endemic in certain parts of South-East Asia, such as southern China and the Indonesian archipelago [1]. The worldwide incidence of NPC exceeds 80.000 cases/year with 19,616 new cases each year in South-China [2], but for most developing countries data are lacking due to absent or inadequate registries. Treatment for primary NPC is radiotherapy, with chemotherapy for advanced stage disease. Despite the radiotherapy responsiveness of nasopharyngeal tumours, good long term survival is only achieved for patients who have early primary tumours with minimal neck disease with 67-71% 10 years disease free survival for T1, T2 and N0-1. Survival is poor for patients who have extended tumours and/or extended neck nodes 29-54% 10 years disease free survival for T3, T4 and N2, N3) [3-6]. Poor survival in the T4N0-1 category is chiefly the result of the high local recurrence rate (63.8%), where as for the T1-2N2-3 category, it is the result of the high distant metastases rate (approximately 50%). For the local recurrent or persistent tumours there are few treatment options left all with severe side effects.

Re-treatment of recurrent/persistent NPC

Usual treatment options for early stage recurrent or persistent NPC are surgery in combination with external radiotherapy, brachytherapy alone [7-9], or in combination with external re-irradiation [10-13] or stereotactic radiosurgery [14]. The standard treatment for advanced stages of recurrent or persistent disease is chemotherapy followed by re-irradiation, or concomitant chemo-re-irradiation [15]. Pryzant reported on 53 patients with locally persistent or recurrent NPC treated with re-irradiation. Forty-two patients were re-treated with external beam therapy alone and 11 with a component of brachytherapy. Local recurrence was confined to the nasopharynx in 27 patients, and persistent tumour in 26 patients. The five-year actuarial local tumour control rate was 35%, five-year disease free survival was 18%, and overall survival was 21%. Eight Patients developed severe complications from retreatment, two involving the brain, one the spinal cord, and two the cranial nerves, all of which were fatal. The five-year actuarial incidence of severe complications was 17%. The incidence of severe complications was related to the total cumulative dose of external irradiation [16]. Lee described the incidence of late complications after re-irradiation in 891 patients with local recurrence after definitive radiation therapy for nasopharyngeal carcinoma. After external re-irradiation, brachytherapy or a combination of both, a wide variety of serious complications, such as temporal lobe necrosis, cranial neuropathy, endocrine dysfunction, trismus and bone/soft tissue necrosis was reported, with an overall incidence of 23 to 29% and a treatment mortality from 1 to 3% [17]. Other authors also report these serious side effects [10-12]. A major determinant of post-treatment complication is the severity of damage sustained during the initial course. In case of re-irradiation after an interval of 2 years or more, the sum of total doses tolerated is higher than predicted based on results of single course irradiations [18]. Since the treatment options for recurrent or persistent NPC are limited, the survival rates poor and the complications severe; there is definitely a place for alternative treatment modalities with more efficacy and less morbidity.

Photodynamic Therapy

Photodynamic therapy (PDT) is a non-invasive treatment modality that might have a substantial role in treatment of recurrent nasopharyngeal carcinoma. PDT facilitates tumour destruction by the combination of a photosensitizer and laser light of a specific wavelength.

The therapeutic response of PDT depends on a complex combination of parameters that includes drug dose, drug light interval, tissue oxygenation, light dose and light intensity (the last two are more accurately referred

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to as fluence and fluence rate, respectively). PDT works through non thermal chemical pathways to damage cancer cells. The main agents of cell destruction are activated singlet oxygens. The cancer cells are destroyed by a process that might take up to several weeks and the treated area heals with normal mucosa advancing from adjacent tissue much like radiation treatment.

Photodynamic Therapy for Recurrent and Persistent NPC

Sun [19] has published the largest case series with 137 patients. Unfortunately this article is in Chinese. He has treated 137 NPC patients with hematoporphyrin derivative mediated PDT. Forty-eight and 72 hours after iv administration of 3-5 mg/kg HpD, laser treatment with either argon or dye laser was carried out. Dye laser (630 nm) with over 350 mw output transmitted through quartz fiber was given to 57 patients. Argon laser (488 nm and 514.5 nm) was delivered to 80 patients. The results were: complete response 76 cases (55.47%) and marked response 47 cases (34.31%), with an over-all marked response rate of 89.78% (123/137). These results are very successful. However, it is not clear if the patients had recurrent NPC or PDT is delivered as primary first choice treatment. In either case the results are very encouraging with such a large case series.

Kulapaditharom [20]et al reported in 1996 five patients treated with PDT for recurrent or residual NPC in Ramathidoni hospital in Bangkok. Three of these patients had a T1 tumour with no lymph node involvement and no distant metastasis. Two of these patients had a T3 tumour with no lymph node involvement and no distant metastases. 48 hours before illumination patients received Hematopirfyrin derivate with a dosage of 3 mg/kg and were illuminated (630 nm) by an optical fiber. The dose varied within 50-100 J/cm2. Power of the laser beam was adjusted to give a density within 100-150 mW/cm2 to avoid thermal effect. The T1 tumour patients had a complete response after one treatment. Complete response has been suggested by endoscopic examination and negative biopsy. The duration of response were: 24, 16 and 11 months. The T3 tumours responded partially. One patient received 4 treatments and the other one 2 treatments before they switched to chemotherapy. The partial response duration was respectively 19 and 12 months.

The side effects described for all treatment were oedema, pain and photosensitivity. All side effects would solve without treatment. There was one patient with severe pain after PDT. All other side effects were mild and moderate.

The same Kulapaditharom [21] et al reported in 2000 the treatment of 12 NPC patients. The patients received Hematopirfyrin derivate with a dosage of 3 mg/kg 48 hours before they were illuminated (630 nm) by an flat cut optical fiber (with a core diameter of 400 μm, Laserscope California) with a with a dose of 100 J/cm2 and a fluence rate of 100 mW/cm2. One patient was primary treated for an unknown primary. The primary tumour revealed in the nasopharynx (T1 NOMO) and because this patient already received radiotherapy and neck dissection he was treated with PDT. Complete response as observed for a follow up period of 31 months. Six patients were treated for recurrent or residual T1-T2 NPC without lymph nodes and distant metastasis. These patients had received chemo-radiation for initial treatment. All patients had a biopsy and endoscopic complete response after PDT treatment. Duration response was between 10 and 66 months, mean duration was 26,5 months. One patient died 12 months after PDT cause of an unrelated cause. One patient had a residual T3N0M0 adenoid cystic carcinoma instead of squamous cell carcinoma. Patient was treated with PDT and surgery. Patient had a partial response and has been symptom free for 22 months. Four patients received PDT as adjunct to conventional treatment; these patients were all T3-T4 patients. One received radiotherapy, the other three received 6 cycles of chemotherapy. Three patients had a partial response and one had a complete response.


79

Tong [22]et al reported 12 patients treated with PDT for recurrent NPC in Prince of Wales Hospital in Hong Kong. These patients were treated with hematoporphyrin derivate 5 mg/kg and exposed with 200 J/cm2 delivered from a gold laser vaper with a wave length of 630 nm. In eight patients cure of disease was deemed possible. The other four patients were treated for palliation of nasal obstruction. Four patients received surgical debulking before PDT. All patients showed radiology confirmed tumour regression six months after treatment. From the eight patients treated for cure five patients had a residual disease after 3-5 months, the other three remained disease free with a follow up between 9-12 months. Four patients had nasal regurgitation and two patients had their trismus aggravated, these side effects were temporarily. Two patients had mild skin hypersensitivity.

Lofgren [23]et al reported 5 patients treated with PDT for recurrent or persistent NPC in Orebro Medical Center Hospital, Sweden. Four patients were treated with hematoporphyrin derivative 2.5 mg/kg. One patient has been treated with potfimer sodium 2.0 mg/kg. The light dose varied from 50 to 100 J/cm2 and radiance varied from 100 to 150 mW/cm2. Four patients had SCC, two of them had a recurrence and two of them persistent disease. One patient had a persistent adenocarcinoma. All patients were initially treated with radiotherapy. Three patients remain tumour free with a follow up between 51 and 60 months. These patients had a tumour depth of 10 mm or less. One patient had persistent disease after PDT, a MRI-scan showed after treatment a tumour depth of 13 mm. One patient had a recurrence 6 months after treatment.All patients had significant headache (3 to 8 months) and middle ear effusion. Two patients experienced minor problems of photosensitivity after sun exposure five 40 days after treatment. Table 1 shows the overall treatment results.

Table 1 | Additional file 1: Overall Treatment Results

No of patients

TNM Residual/primary

Respons Duration Respons/FU In months

Remarks

Sun 137 unknown unknown 75 CR47 PR

unknown

Kulapaditharom 5 3x T1N0 residual 3 CR 24,16,11(DR)

2x T3N0 residual 2 PR 19,12(DR)

Kulapaditharom 12 1xT1N0 primary 1 CR 31(DR)

6xT1-2N0 residual 6 CR 10-66(DR)(mean26,5)

1xT3N0 residual PR 22(FU) Adeonocystic carcinoma, also received surgery

1xT3N1M1

residual CR 36(FU) Received 3 PDT treatments with 6 cycles of Chemotherapy

1xT3N0M0

residual PR 30 (FU) Received 2 PDT treatments with 6 cycles of chemotherapy

1xT4N0M0

residual PR 36 (FU) Received 2 PDT treatments with 6 cycles of chemotherapy

1xT4N0M0

residual PR 7 (FU) Received 3 PDT treatments with 12 Gy Radiotherapy

Tong 12 unknown residual 8 CR4PR

6-22 (FU)(mean 11)

Lofgren 5 unknown Residual & persistent

3 CR1 NR

51-60 (FU) Tumor depth <10mmTumor depth >13mm

1 PR 6 months another recurrence

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Discussion

PDT has the potential to be a very effective local treatment modality for NPC, without the severe side effects seen with radiotherapy. The articles above have shown that PDT is effective in destroying NPC, with good local control of tumor growth and complete responses in the majority of small recurrent or persistent disease (T1, T2) and long-term palliation in advanced stage (T3, T4) of the recurrence.

There are several advantages of PDT for management of recurrent NPC. Perhaps the most important advantage is its repeatability. PDT does not have cumulative effects. In case of a partial response the same area can be illuminated again. Secondly PDT is a local treatment rather than a systemic one. The photosensitizing drug accumulates in higher concentrations within cancer cells rendering them more susceptible to the toxic effects of light, compared to normal healthy cells and the light is delivered directly onto the tumour sparing the surrounding normal mucosa. Two other advantages of PDT are normal healing of the wound and illumination is only once.

In the papers reviewed above they all used the first generation photosensitizer hematoporphyrin. Yow [24] et al has shown in nasopharyngeal cancer cell lines that the uptake of second generation potosensitizer temaporfin is higher and efficiency is better compared with hematoporphyrin. Yow [25]et al also compared temoporfin and merocyanine 540, both second generation photosentisizers. They concluded that temoporfin-mediated PDT has a more potent effect in comparison with merocyanine- 540- mediated PDT. Based on these studies we hope for even better treatment results if temoporfin is used.

Illumination of nasopharyngeal cavity is a challenge, since access is difficult and the cavity has a complex and irregular geometry. Furthermore the cavity varies significantly in size and geometry between patients. The complex shape of the nasopharyngeal cavity makes it impossible to produce a homogenous field of illumination. In order to deliver a sufficient light distribution throughout the nasopharyngeal cavity over exposure cannot be avoided. Over exposure of tumour or surrounding normal mucosa is however not considered a problem. Some critical structures are well protected by bone others; however, like the soft palate and the dorsal oropharyngeal wall would suffer unacceptable damage by this approach. Hence, these areas must be shielded against the laser light. For this purpose we have developed a novel dedicated light delivery applicator that ensures proper light delivery to the target area and enables for proper shielding of the risk areas [26,27]. A new study has been commenced to use this applicator to deliver PDT to patients with recurrent NPC cancers by our group. How we illuminate the Nasopharynx is shown by figures 1, 2 and 3.

Figure 1 | A: Nasopharynx applicator, B: schematic view of positioning and illumination. 1. Cylindrical diffuser in shielding tube. 2. Target area. 3. Soft palate is shielded. Figure 1 have been published previously by Nyst et al, 2007 [26]


81

Figure 2 | Insertion of the Nasopharyngeal Applicator.

Figure 3 | Volunteer with Nasopharyngeal Applicator.

The side effects are much less frequent and severe compared to chemotherapy. Sunlight exposure have shown that photosensitivity is not a real burden for these patients and only limited adverse events were related to this problem [28]. In general, patients in this part of the world have a colored skin that makes them less prone to sunburn. The fact that people in the tropics generally avoid sun exposure also helps to minimize the problems around the photosensitivity.

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The immunologic reaction followed by PDT could be partly responsible for the good results. The theory is that an immunological response will be provoked by the use of PDT [29]. So in addition to these clinical trials several groups are working on the effectiveness of PDT in laboratory settings. PDT and the use of natural compounds become one of the new approaches in the investigation of NPC treatment. Such a group from Singapore has reported very encouraging results with hyperecin mediated PDT. This group has published extensively on biochemical pathways of cell damage caused by hypericin-PDT on NPC cell lines in vitro. Their excellent review titled “Hypericin lights up the way for the potential treatment of nasopharyngeal cancer by photodynamic therapy” by Olivo [30] et al summarizes this effort and proposes that hypericin-PDT is a potential treatment of NPC in humans. Koon [31] et al has chosen a different photosensitizing agent from the traditional Chinese medicine: curcumin. Their in vitro study with CNE2 NPC cells shows it to be a promising agent.

Xu et al[32] preferred to worked with photodynamic effects of pyropheophorbide-a methyl ester (MPPa) in CNE2 NPC cells, demonstrating the apoptotic effect and suggesting its clinical potential. Mak et al[33] worked with the same cell line inducing phototoxicity with sulphonamide derivatives of porphycene with successful results. Betz [34] et al obtained good results in their in vitro study with amore conventional photosensitizer: 5-aminolevulinic acid.

Bae [35] et al examined the immunotherapeutic significance of HPV-immortalitized tumour cell lysates induced by PDT and CpG-oligodeoxynucleotide(ODN). They found a significant induced IFN-β production and cytotoxic T- cell response in the PDT-cell lysate plus ODN immunized groups. Although PDT in combination with immunotherapy is still in experimental fase it offers a great promise as a new alternative treatment. On to this time no research has been done on PDT in combination with Immunotherapy in EBV related NPC. This combination could be very promising not only for local recurrence but also for advanced disease.

Acknowledgements

The authors greatly appreciate the cooperation of H.J. Sterenborg and R.L. van Veen from Erasmus Medical Centre with their help on the pictures and animations.

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Borneo Island. Cancer Epidemiol Biomarkers Prev. 2004;13:482-486. (2) Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin. 2005;55:74-108. (3) Lee AW, Poon YF, Foo W et al. Retrospective analysis of 5037 patients with nasopharyngeal carcinoma treated during

1976-1985: overall survival and patterns of failure. Int J Radiat Oncol Biol Phys. 1992;23:261-270. (4) Moench HC, Phillips TL. Carcinoma of the nasopharynx. Review of 146 patients with emphasis on radiation dose and

time factors. Am J Surg. 1972;124:515-518. (5) Chien CR, Chen SW, Hsieh CY et al. Retrospective comparison of the AJCC 5th edition classification for nasopharyngeal

carcinoma with the AJCC 4th edition: an experience in Taiwan. Jpn J Clin Oncol. 2001;31:363-369. (6) Kajanti MJ, Mantyla MJ. Carcinoma of the nasopharynx. A retrospective analysis of treatment results in 125 patients.

Acta Oncol. 1990;29:611-614. (7) Law SC, Lam WK, Ng MF, Au SK, Mak WT, Lau WH. Reirradiation of nasopharyngeal carcinoma with intracavitary mold

brachytherapy: an effective means of local salvage. Int J Radiat Oncol Biol Phys. 2002;54:1095-1113. (8) Kwong DL, Wei WI, Cheng AC et al. Long term results of radioactive gold grain implantation for the treatment of

persistent and recurrent nasopharyngeal carcinoma. Cancer. 2001;91:1105-1113. (9) Leung TW, Tung SY, Sze WK et al. Salvage radiation therapy for locally recurrent nasopharyngeal carcinoma. Int J

Radiat Oncol Biol Phys. 2000;48:1331-1338.


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(11) Teo PM, Kwan WH, Chan AT, Lee WY, King WW, Mok CO. How successful is high-dose (> or = 60 Gy) reirradiation using mainly external beams in salvaging local failures of nasopharyngeal carcinoma? Int J Radiat Oncol Biol Phys. 1998;40:897-913.

(12) Lee AW, Foo W, Law SC et al. Recurrent nasopharyngeal carcinoma: the puzzles of long latency. Int J Radiat Oncol Biol Phys. 1999;44:149-156.

(13) Hwang JM, Fu KK, Phillips TL. Results and prognostic factors in the retreatment of locally recurrent nasopharyngeal carcinoma. Int J Radiat Oncol Biol Phys. 1998;41:1099-1111.

(14) Chua DT, Sham JS, Kwong PW, Hung KN, Leung LH. Linear accelerator-based stereotactic radiosurgery for limited, locally persistent, and recurrent nasopharyngeal carcinoma: efficacy and complications. Int J Radiat Oncol Biol Phys. 2003;56:177-183.

(15) al Sarraf M, Reddy MS. Nasopharyngeal carcinoma. Curr Treat Options Oncol. 2002;3:21-32. (16) Pryzant RM, Wendt CD, Delclos L, Peters LJ. Re-treatment of nasopharyngeal carcinoma in 53 patients. Int J Radiat

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1985: survival after local recurrence. Int J Radiat Oncol Biol Phys. 1993;26:773-782. (18) Lee AW, Foo W, Law SC et al. Total biological effect on late reactive tissues following reirradiation for recurrent

nasopharyngeal carcinoma. Int J Radiat Oncol Biol Phys. 2000;46:865-872. (19) Sun ZQ. [Photodynamic therapy of nasopharyngeal carcinoma by argon or dye laser--an analysis of 137 cases].

Zhonghua Zhong Liu Za Zhi. 1992;14:290-292. (20) Kulapaditharom B, Boonkitticharoen V. Photodynamic therapy in the treatment of head and neck cancers: a two-year

experience. J Med Assoc Thai. 1996;79:229-235. (21) Kulapaditharom B, Boonkitticharoen V. Photodynamic therapy in management of head and neck cancers and

precancerous lesions. J Med Assoc Thai. 2000;83:249-258. (22) Tong MC, van Hasselt CA, Woo JK. Preliminary results of photodynamic therapy for recurrent nasopharyngeal

carcinoma. Eur Arch Otorhinolaryngol. 1996;253:189-192. (23) Lofgren LA, Hallgren S, Nilsson E, Westerborn A, Nilsson C, Reizenstein J. Photodynamic therapy for recurrent

nasopharyngeal cancer. Arch Otolaryngol Head Neck Surg. 1995;121:997-1002. (24) Yow CM, Chen JY, Mak NK, Cheung NH, Leung AW. Cellular uptake, subcellular localization and photodamaging effect

of temoporfin (mTHPC) in nasopharyngeal carcinoma cells: comparison with hematoporphyrin derivative. Cancer Lett. 2000;157:123-131.

(25) Yow CM, Mak NK, Szeto S et al. Photocytotoxic and DNA damaging effect of temoporfin (mTHPC) and merocyanine 540 (MC540) on nasopharyngeal carcinoma cell. Toxicol Lett. 2000;115:53-61.

(26) Nyst HJ, van Veen RL, Tan IB et al. Performance of a dedicated light delivery and dosimetry device for photodynamic therapy of nasopharyngeal carcinoma: phantom and volunteer experiments. Lasers Surg Med. 2007;39:647-653.

(27) van Veen RL, Nyst H, Rai Indrasari S et al. In vivo fluence rate measurements during Foscan-mediated photodynamic therapy of persistent and recurrent nasopharyngeal carcinomas using a dedicated light applicator. J Biomed Opt. 2006;11:041107.

(28) D’Cruz AK, Robinson MH, Biel MA. mTHPC-mediated photodynamic therapy in patients with advanced, incurable head and neck cancer: a multicenter study of 128 patients. Head Neck. 2004;26:232-240.

(29) Korbelik M, Sun J, Cecic I, Serrano K. Adjuvant treatment for complement activation increases the effectiveness of photodynamic therapy of solid tumors. Photochem Photobiol Sci. 2004;3:812-816.

(30) Olivo M, Du HY, Bay BH. Hypericin lights up the way for the potential treatment of nasopharyngeal cancer by photodynamic therapy. Curr Clin Pharmacol. 2006;1:217-222.

(31) Koon H, Leung AW, Yue KK, Mak NK. Photodynamic effect of curcumin on NPC/CNE2 cells. J Environ Pathol Toxicol Oncol. 2006;25:205-215.

(32) Xu CS, Leung AW. Photodynamic effects of pyropheophorbide-a methyl ester in nasopharyngeal carcinoma cells. Med Sci Monit. 2006;12:BR257-BR262.

(33) Mak NK, Kok TW, Wong RN et al. Photodynamic activities of sulfonamide derivatives of porphycene on nasopharyngeal carcinoma cells. J Biomed Sci. 2003;10:418-429.

(34) Betz CS, Lai JP, Xiang W et al. In vitro photodynamic therapy of nasopharyngeal carcinoma using 5-aminolevulinic acid. Photochem Photobiol Sci. 2002;1:315-319.

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(35) Bae SM, Kim YW, Kwak SY et al. Photodynamic therapy-generated tumor cell lysates with CpG-oligodeoxynucleotide enhance immunotherapy efficacy in human papillomavirus 16 (E6/E7) immortalized tumor cells. Cancer Sci. 2007;98:747-752.

Chapter 7

Temoporfi n mediated photodynamic therapy in patients with local persistent and recurrent

Nasopharyngeal Carcinoma after curative radiotherapy: A feasibility study

H.J. Nyst,

M.A. Wildeman,

S.R. Indrasari,

B. Karakullukcu,

R.L.P. van Veen,

M. Adham,

F.A. Stewart,

P.C. Levendag,

H.J.C.M. Sterenborg,

I.B. Tan

Photodiagnosis Photodyn Ther. 2012 Sep;9(3):274-81.

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88

Summary

Background

The treatment of persistent and recurrent nasopharyngeal carcinoma (NPC) remains a challenge, especially in Indonesia. We investigated the safety and efficacy of temoporfin mediated photodynamic therapy (PDT) for patients with local persistent and recurrent NPC.

Material and Methods

Twenty-two patients with persistent and recurrent NPC (maximum tumor depth <10 mm) underwent PDT under local anesthesia with use of a nasopharyngeal light applicator. Three different drug doses and light intervals have been administered: treatment arm A: 0.15 mg/kg Foscan®; 96 h drug-light interval; B: drug dose of 0.10 mg/, 48 h drug-light interval; C: drug dose of 0.075 mg/kg, 24 h drug-light interval. Toxicity was measured by using the CTCAE 3.1 scale.

Results

Arm A consisted of eight patients, arms B and C consisted of seven patients. The treatment procedure was well tolerable under local anesthesia. The most common grade III toxicities for all groups is headache (n = 7; 33%). No grade IV toxicity was seen. One patient died 2 days after treatment due to a misdiagnosed pneumonia. In 17 of the 22 patients a biopsy was performed after 40 weeks and showed no tumor in all biopsies. Arm A seems, in addition to comparable toxicity, clinically more effective than arms B and C.

Conclusion

The present study demonstrated that temoporfin mediated photodynamic therapy is a relatively simple technique that can be utilized to treat residual or recurrent nasopharyngeal cancer, restricted locally to the nasopharynx.

Temoporfin mediated photodynamic therapy in patients

89

Introduction

Nasopharyngeal cancer (NPC) is endemic in Southern China and most of South-East Asia with a yearly incidence reaching as high as 20—50 cases per 100,000 annually [1,2].

In Indonesia NPC is the most frequent cancer in the head and neck area and is the fourth most common tumor occurring in males. The incidence is estimated 6 per 100,000, leading to at least 14,000 new cases per year [3]. Unfortunately in Indonesia the majority of patients have advanced stage disease at initial diagnosis. In a recent study, 87% of Indonesian patients had stage III—IV disease at initial diagnosis and 18% presented with distant metastasis [4].

Radiotherapy alone or combined with chemotherapy is the treatment of choice for NPC and has a relatively high cure rate [5,6]. In 1999, Lin and Jan reported a failure rate for the primary tumor in patients with T3-4N0M0 NPC of 25% [5]. In 2000, Chang et al. reported a primary tumor control rate of 50—60% after radiotherapy in patients with advanced NPC [7]. During the last decade the treatment results have significantly improved; with disease-free and overall survival results of around 70% and 80%, respectively [8,9]. These improvements are due to the advances in diagnostic imaging, increased radiation dose [10], the different regimes of fractionation, the use of IMRT [11,12], and the use of concomitant chemotherapy [6]. These success percentages cannot be achieved in developing countries mainly due to a lack of radiotherapy facilities, imaging facilities, and poor patient compliance. In Indonesia these problems cause a high percentage of local persistent or recurrent tumor after radiotherapy [4].

Options for treatment of local recurrent NPC are brachytherapy, external re-irradiation, stereotactic radiosurgery and nasopharyngectomy. These treatment modalities can be used either alone or in combination [13—16]. Despite promising local response rates; re-irradiation causes a high incidence of major late complications, such as brain necrosis, cranial nerve palsies, and catastrophic hemorrhages [17—21]. In Indonesia re-irradiation and surgery for locally recurrent disease are not feasible because of insufficient techniques, personnel, equipment, and lack of allocated re-treatment time due to already long waiting times for treatment of primary tumors. In persistent disease, there is limited role for re-irradiation.

Photodynamic therapy (PDT) using a specially designed nasopharyngeal applicator may act as an alternative treatment option since it can be performed easily and does not require expensive equipment and extensive training of physicians and the medical staff.

PDT is a method that involves injection of a systemic photosensitizing drug that can be activated at the tumor site to produce reactive oxygen species, starting a cascade of oxidization of biomolecules and eventually causing tumor destruction. PDT has already been shown to be an effective modality for treatment of superficial lesions in previously irradiated sites [22—25].

PDT has been studied in the past to treat locally recurrent/ persistent NPC. The results from studies with first generation photosensitizers like hematoporphyrin and without a nasopharyngeal applicator for proper illumination, although quite promising, did not lead to established protocols for PDT for NPC [26]. Temoporfin mediated photo dynamic therapy (PDT) is a registered treatment in Europe for incurable head and neck cancer [22,24]. Temoporfin mediated PDT in combination with a specially designed nasopharyngeal applicator for proper illumination of the nasopharyngeal cavity might be a suitable therapy for local persistent or recurrent NPC, especially in developing countries where advanced radiotherapy options are limited [27].

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Yow et al. has shown in nasopharyngeal cancer cell lines that the uptake of second generation photosensitizer temoporfin is higher and the photodynamic reactions are more efficient compared with hematoporphyrin [28]. Since there are no data reported in the literature on temoporfin mediated PDT in NPC, we planned a study to investigate the feasibility of three different treatment regiments to be able to select one of these treatment regimes and conduct a phase II trial.

Methods

Patients

Eligible patients were aged 18 years or older with pathologically proven locally recurrent or residual nasopharyngeal carcinoma (type I, II or III), a discrete tumor ≤10 mm in depth measured by CT imaging (surface illumination with temoporfin has a effective penetration of 10 mm), which is endoscopically visible and accessible for unrestricted surface illumination using a nasopharyngeal applicator. Other eligibility criteria included a Karnofsky performance status of at least 70%. Local ethics committee approval was obtained for the study, and each patient gave written informed consent. All patients had previously been treated with in onset curative radiotherapy for their primary NPC.

Exclusion criteria were any disease which is caused or exacerbated by light and treatment within the prior 30 days with a light-activated therapy. Patients with distant metastasis and lymph-node involvement were also excluded from the study protocol.

Treatment regimes

Three treatment regimes were investigated to select the most safe and efficacious treatment scheme. Besides the standard dose for Head and neck tumors of 0.15 mg/kg, two lower drug doses were evaluated. The possible loss in effectiveness by reducing the drug dose was compensated by reducing the drug-light interval. The lower drug doses were evaluated to see if these would give less side effects and lower the costs with the same effectiveness. Any grade IV and V adverse event (according to CTC 3.1) was considered as SAE (serious adverse event). In case of two SAE from one dose group these parameters were considered too toxic.

Arm A: Eight patients received the dose level and the drug light interval that are recommended for the treatment of patients with squamous cell carcinoma of the head and neck. These parameters are drug dose: 0.15 mg/kg Foscan®; drug-light interval: 96 h; light dose: 20 J/cm2.

Arm B: 7 patients were treated at a drug dose of 0.10 mg/kg and a drug-light interval of 48 h.

Arm C: 7 patients were treated at a drug dose of 0.075 mg/kg and a drug-light interval of 24 h.

Drug administration

A dose of 0.15 mg, 0.10 mg or 0.075 mg of Foscan® per kilogram of body weight was administered by slow intravenous injection (into a proximal deep vein in at least 6 min, according to the producers instructions. Patients remained in a light-restricted room for 24 h after administration of temoporfin and then made a gradual return, with an increase of 100 lx per day, to unrestricted indoor light exposure over a period of 2 weeks. Guidelines for light exposure and a lux-meter to monitor light exposure were given to the patients, and treating physicians advised both patients and their families and friends about the importance of complying with a gradual return to normal light exposure. A booklet was also available for the patient to optimize adherence to these guidelines.


91

Laser Illumination

To ensure homogeneous light administration to the entire nasopharynx, a specially designed nasal light applicator (Rotterdam Nasopharyngeal Light Applicator; type 625, Wacker Chemie, Krommenie, The Netherlands) was used [27] (Fig. 1). The inner diameter of the silicon tubing can accommodate two linear light diffusers for light delivery and dosimetry.

After decongestion (R/xylometazoline hydrochloride 1%) and topical anesthesia, the applicator was introduced into the nasopharynx transorally over 2 guiding tubes (four French). By using a small silicone flange, the applicator remains fixed in a stable position during illumination.

Figure 1 | (A) Nasopharynx applicator, (B) schematic view of positioning and illumination. (1) Cylindrical diffuser in shielding tube. (2) Target area. (3) Soft palate is shielded. This figure has been used previously by Nyst et al. [27] and Wildeman [29].

The surface of the tumor was illuminated with 652 nm light, emitted from a 6-W Applied Optronics diode laser, inserted into one channel catheter [27]. The length of the diffuser is 5 cm, and the diameter of the catheter is 0.4 cm. The light dose administered was 20 J/cm at a fluence rate of 100 mW/cm. The total illumination time is 400 s for 2 diffusers.

Study Assessments

Following treatment with PDT, patients were evaluated on day 1 and 2, and week 1, 2, 4, 6, 8, 12 and 16 post PDT. Patients subsequently entered a followup phase, with visits every 4 weeks up to 40 weeks post-PDT. Thereafter, they were followed annually until death or lost to follow-up. The following assessments were performed to evaluate safety: physical examination including vital signs, Karnofsky performance status and weight, recording of adverse events according to CTCAE 3.1, concomitant medication (in particular use of analgesics).

The tumor response was monitored with endoscopic examination of the nasopharynx and biopsies of the nasopharynx. Overall survival was calculated from the time of illumination to death.

Table 1 | Patient characteristics.

Arm A (n = 8) mean (range) Arm B (n = 7) Arm C (n = 7)

Age 50 (19–68) 53 (45–61) 41 (43–57)

Male/female 6/2 5/2 2/5

Persistent/ recurrent 8/0 6/1 7/0

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In case of a partial response or recurrence, based on endoscopic examination and if pathologically proven, the patient was eligible for re-treatment with PDT.

Results

Patient characteristics

Most patients had pathologically proven residual NPC. Only one patient had recurrent disease. Tumor evaluation after the initial treatment, 12 weeks after finishing radiotherapy treatment, showed in the cases with persistent disease tumor in the nasopharynx and were included in this study. The patient with disease recurrence had, during regular follow up, a visible mass at endoscopy which after evaluation showed to be a local recurrence and was included. Tumor size was measured by CT-scan and all patients had a discrete tumor depth of 1 cm or less (Table 1).

Safety/adverse events/tolerability

All included patients were able to complete the treatment procedure. The treatment procedure was well tolerable under local anesthesia. There were no short term adverse events.

All grade I—IV adverse events scored, until 16 weeks after treatment, are presented in Table 2. All possible adverse events were scored but only the post treatment adverse events are presented in Table 2.

Three of eight patients in arm A had grade III toxicities for headache; no grade IV toxicity was seen in arm A. One patient in arm A died due to pneumonia 2 days after illumination of the tumor. This is considered a grade V adverse event. A chest X-ray prior to the treatment already showed pneumonia but this was misinterpreted by the radiologist and despite fever the patient was enrolled in the study.

In arm B two grade III toxicities were seen in three of seven patients; one patient had a grade III headache, one patient had a grade III tinnitus. There were no grade IV toxicities seen.

Arm C had a total of four grade III toxicities in three of the seven patients. Three patients had a grade III headache; one of these patients already suffered a grade II headache before treatment. One patient had a grade III musculoskeletal myositis of the neck muscles causing a grade III headache. There were no grade IV toxicities seen. One patient arm C started with a cranial neuropathy grade III that completely resolved one week after treatment.

The most common grade III toxicities for all groups is headache (n = 7; 33%). None of the patients had skin burns or other skin adverse reaction caused by the photosensitizer.


93

Tabl

e 2

| All

grad

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16

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all

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onia

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t.

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even

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rm A

(n =

8)*

Arm

B (n

= 7

)A

rm C

(n =

7)

Gra

de I

Gra

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Gra

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IG

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23

01

51

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61

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00

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00

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282

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224

0

Chapter 7

94

Clinical results

In 17 of the 22 patients a biopsy was performed after 40 weeks and showed no tumor in all 17 biopsies. The patients who did not receive a biopsy after 40 weeks already died (n = 3) or the biopsy could not be performed because the patient did not show up (n = 1) or was postponed (n = 1) because of the big earthquake in 2006 and the patient died before the biopsy could be performed [30].

In arm A one of eight patients died. One patient was lost to follow up but at his last visit, 32 months after treatment, he had no complaints and no signs of disease progression. In arm B six of seven patients died, three related to disease, one with unknown reason and two not related to the tumor. In arm C three out of seven patients died, all related to disease and four patients are still alive without any sign of disease.

Survival for all patients is shown in Figs. 2 and 3. With a mean follow up of 37.8 months (range: 2 days to 71 months), 10 patients are alive; two of them had a local recurrence and were successfully re-treated with PDT.

In total 10 patients died, two not related to disease or treatment, one of unknown cause, one treatment related and six related to disease. The 10 patients who are still alive have a mean follow up of 58 months (min 37 to max 71 months).

Figure 2 | Disease specific survival.


95

Figure 3 | Overall survival.

Discussion

The present study demonstrated that temoporfin mediated photodynamic therapy is a relatively simple technique that can be utilized to treat residual nasopharyngeal cancer, restricted locally to the nasopharynx. All patients tolerated PDT under local anesthesia in an outpatient clinic setting. Since this procedure takes less than half an hour, it has a significant advantage over other treatment options, which require extensive setups and experienced personnel. The treating physician can administer the PDT alone or with minimal nursing support. Furthermore PDT is a one-time treatment which makes patient compliance to treatment a non-issue, unlike re-radiation where the patient has to show up for multiple radiation sessions. In Indonesia it is frequently observed that patients do not comply with the radiation treatment schedules. Moreover, there is already a long waiting time for radiation treatment even for primary tumors. The same advantage applies over nasopharyngectomy approaches which require a time consuming specified surgical expertise, operating room facilities, time and a longstanding postoperative recovery.

Although this feasibility study is encouraging it is too early to talk about clinical efficacy, which needs to be demonstrated in a larger phase II trial. Considerable data is available over temoporfin mediated PDT of head and neck cancers [22—25]. However, this was not previously tested on NPC, which has a very different biological behavior than squamous cell cancers of the head and neck. The undifferentiated histological subtype, NPC WHO III, is the most prevalent NPC type in South-East Asia and Indonesia. This type of cancer is causally associated with the Epstein—Barr virus (EBV) [31]. Therefore we have compared the standard treatment regimen with two other regimens, which are in theory less toxic, to be able to select a regimen for a phase II trial. Regarding toxicity, there was no significant difference between the treatment groups. The adverse events are mostly local and could be disease related, as well as treatment-related. The most common grade III adverse events was headache. The headache seen in the patients can be caused by irritation of the deep neck muscles by PDT, since the prevertebral muscles are close to the nasopharynx and headache symptoms were

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accompanied by stiff neck feelings. The complaints of headache all disappeared eventually and irreversible damage of the prevertebral muscles is not likely since surface illumination has a penetration depth of 1 cm. The effusion of the middle ear, seen in 16 patients, was caused by massive edema of the nasopharynx after illumination, which caused occlusion of the Eustachian tube; around 6—8 weeks after illumination the edema disappeared and the effusion resolved. The death of one patient two days after treatment was probably caused by a missed pneumonia, which the patient already had before the injection of temoprofin.

Arm A seems, in addition to comparable toxicity, clinically more effective than arms B and C. Although clinical complete responses can also be observed in arms B and C, arm A seems to have a better overall survival and disease specific survival, but the numbers are too small to draw this conclusion. Except for the patient lost to pneumonia, all the patients in arm A are alive and disease free. Arms B and C both have three mortalities due to disease progression. At 40 weeks the biopsies taken from the nasopharynx did not show viable tumor cells. This suggests that all three treatment regimens are effective on the nasopharyngeal surface, but arms B and C might be less penetrating than arm A and missing deeper tumor tissues. The difference in effectiveness could be due to concentration of the photosensitizer. Lower doses of temoprofin might not provide sufficient concentration in the tumor tissues. Because the depth of the tumor receives a lower light fluence than the surface, the amount of activated photosensitizer might not be sufficient to cause cellular damage at certain depths. Whereas with higher concentrations, even though the activation percentage remains the same, the absolute amount of activated photosensitizer could be higher. Another consideration is the location of the photosensitizer in the tissues. Earlier research suggests that the maximal tumor intracellular concentration is reached 4 days after temoporfin injection and in shorter time points, temoporfin is located more in blood vessels [32]. Therefore in arms B and C the treatment effect could be necrosis due to vascular shutdown and in arm A more apoptosis due to intracellular damage. This could explain the better long-term disease control.

Whatever the reason might be, treatment arm A has a longer overall survival and disease-free survival with comparable toxicity/adverse events. Therefore this regimen is chosen to conduct the ongoing phase II study. Once this study is concluded we will be able to determine and compare the clinical efficacy of PDT in patients with small local residual/ recurrent NPC.

Based on our experiences so far it is not illogical to assume that temoporfin mediated PDT can also successfully be used in the future as primary treatment modality for superficially growing NPC, with still the availability of all other treatments and PDT for retreatment of small recurrences.

Conclusion

PDT could be a very suitable/attractive option for treatment of NPC, in terms of short waiting time, short procedure time which can be conducted under local anesthesia in the outpatient clinic; a simple procedure that can be learned and carried out easily by medical personnel.

Acknowledgements

The authors would like to thank Winrich Rauschning for helping with the study design. This project was supported by Biolitec Pharma Ltd., Dublin, Ireland.


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Borneo Island. Cancer Epidemiology, Biomarkers and Prevention 2004;13:482—6. [3] Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates of worldwide burden of cancer in 2008:

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July 23, 2011. [5] Lin JC, Jan JS. Locally advanced nasopharyngeal cancer: long-term outcomes of radiation therapy. Radiology

1999;211:513—8. [6] Zhang L, Zhao C, Ghimire B, et al. The role of concurrent chemoradiotherapy in the treatment of locoregionally

advanced nasopharyngeal carcinoma among endemic population: a meta-analysis of the phase III randomized trials. BMC Cancer 2010;10:558.

[7] Chang JTC, See LC, Liao CT, et al. Locally recurrent nasopharyngeal carcinoma. Radiotherapy and Oncology 2000;54:135—42.

[8] Al Sarraf M, LeBlanc M, Giri PG, et al. Chemoradiotherapy versus radiotherapy in patients with advanced nasopharyngeal cancer: phase III randomized Intergroup study 0099. Journal of Clinical Oncology 1998;16:1310—7.

[9] Wee J, Tan EH, Tai BC, et al. Randomized trial of radiotherapy versus concurrent chemoradiotherapy followed by adjuvant chemotherapy in patients with American Joint Committee on Cancer/International Union against cancer stage III and IV nasopharyngeal cancer of the endemic variety. Journal of Clinical Oncology 2005;23:6730—8.

[10] Leung TW, Wong VY, Sze WK, Lui CM, Tung SY. High-dose-rate intracavitary brachytherapy boost for early T stage nasopharyngeal carcinoma{private}. International Journal of Radiation Oncology, Biology, Physics 2008;70:361—7.

[11] Palazzi M, Orlandi E, Bossi P, et al. Further improvement in outcomes of nasopharyngeal carcinoma with optimized radiotherapy and induction plus concomitant chemotherapy: an update of the Milan experience. International Journal of Radiation Oncology, Biology, Physics 2009;74:774—80.

[12] Taheri-Kadkhoda Z, Pettersson N, Bjork-Eriksson T, Johansson KA. Superiority of intensity-modulated radiotherapy over three-dimensional conformal radiotherapy combined with brachytherapy in nasopharyngeal carcinoma: a planning study. British Journal of Radiology 2008;81:397—405.

[13] Wei WI, Sham JST. Nasopharyngeal carcinoma. Lancet 2005;365:2041—54. [14] Biel MA. Photodynamic therapy in head and neck cancer. Current Oncology Reports 2002;4:87—96. [15] Suarez C, Rodrigo JP, Rinaldo A, Langendijk JA, Shaha AR, Ferlito A. Current treatment options for recurrent

nasopharyngeal cancer. European Archives of Otorhinolaryngology 2010;267:1811—24. [16] Chen MK, Lai JC, Chang CC, Liu MT. Minimally invasive endoscopic nasopharyngectomy in the treatment of recurrent

T1-2a nasopharyngeal carcinoma. Laryngoscope 2007;117:894—6. [17] Lee AW, Law SC, Foo W, et al. Retrospective analysis of patients with nasopharyngeal carcinoma treated during 1976—

1985: survival after local recurrence. International Journal of Radiation Oncology, Biology, Physics 1993;26:773—82. [18] Wang CC. Re-irradiation of recurrent nasopharyngeal carcinoma—treatment techniques and results. International

Journal of Radiation Oncology, Biology, Physics 1987;13:953—6. [19] Yan JH, Hu YH, Gu XZ. Radiation therapy of recurrent nasopharyngeal carcinoma. Report on 219 patients. Acta

Radiologica Oncology 1983;22:23—8. [20] Teo PM, Kwan WH, Chan AT, Lee WY, King WW, Mok CO. How successful is high-dose (> or =60 Gy) reirradiation using

mainly external beams in salvaging local failures of nasopharyngeal carcinoma? International Journal of Radiation Oncology, Biology, Physics 1998;40:897—913.

[21] Chang JT, See LC, Liao CT, et al. Locally recurrent nasopharyngeal carcinoma. Radiotherapy and Oncology 2000;54:135—42.

[22] Tan IB, Dolivet G, Ceruse P, Vander Poorten V, Roest G, Rauschning W. Temoporfin-mediated photodynamic therapy in patients with advanced, incurable head and neck cancer: a multicenter study. Head & Neck 2010;32:1597—604.

[23] Copper MP, Triesscheijn M, Tan IB, Ruevekamp MC, Stewart FA. Photodynamic therapy in the treatment of multiple primary tumours in the head and neck, located to the oral cavity and oropharynx. Clinical Otolaryngology 2007;32:185—9.

[24] D’Cruz AK, Robinson MH, Biel MA. mTHPC-mediated photodynamic therapy in patients with advanced, incurable head and neck cancer: a multicenter study of 128 patients. Head & Neck 2004;26:232—40.

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[25] Karakullukcu B, van Oudenaarde K, Copper MP, et al. Photodynamic therapy of early stage oral cavity and oropharynx neoplasms: an outcome analysis of 170 patients. European Archives of Otorhinolaryngology 2011;268: 281—8.

[26] Wildeman MAM, Nyst HJ, Karakullukcu B, Tan BI. Photodynamic therapy in the therapy for recurrent/persistent nasopharyngeal cancer. Head & Neck Oncology 2009;1:40.

[27] Nyst HJ, van Veen RL, Tan IB, et al. Performance of a dedicated light delivery and dosimetry device for photodynamic therapy of nasopharyngeal carcinoma: phantom and volunteer experiments. Lasers in Surgery and Medicine 2007;39: 647—53.

[28] Yow CM, Chen JY, Mak NK, Cheung NH, Leung AW. Cellular uptake, subcellular localization and photodamaging effect of temoporfin (mTHPC) in nasopharyngeal carcinoma cells: comparison with hematoporphyrin derivative. Cancer Letters 2000;157:123—31.

[29] Wildeman MA, Nyst HJ, Karakullukcu B, Tan BI. Photodynamic therapy in the therapy for recurrent/persistent nasopharyngeal cancer. Head & Neck Oncology 2009;1:40.

[30] Gelling P. Indonesia hit by big quake near volcano. The New York Times; May 28, 2006. [31] Gulley ML. Molecular diagnosis of Epstein—Barr virus related diseases. The Journal of Molecular Diagnosis 2010; 3(1). [32] Lofgren LA, Ronn AM, Abramson AL, et al. Photodynamic therapy using m-tetra(hydroxyphenyl) chlorin. An animal

model. Archives of Otolaryngology—Head and Neck Surgery 1994;120:1355—62.

Chapter 8

Cytolytic virus activation therapy for Epstein-Barr virus driven tumors

Maarten A. Wildeman,

Zlata Novalic,

Sandra A.W.M. Verkuijlen,

Hedy Juwana,

Alwin D.R. Huitema,

I. Bing Tan,

Jaap M. Middeldorp,

Jan Paul de Boer,

Astrid E. Greijer

Clin Cancer Res. 2012 Sep 15;18(18):5061-70.

http://clincancerres.aacrjournals.org/content/18/18/5061

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Abstract

Purpose

Nasopharyngeal carcinoma (NPC) is causally linked to Epstein–Barr virus (EBV) infection. Because all tumor cells carry EBV, the virus itself is a potential target for therapy. In these tumor cells, EBV hides in a latent state and expresses only a few non-immunogenic proteins for EBV maintenance and contributes to tumor growth. We developed a cytolytic virus activation (CLVA) therapy for NPC treatment, reactivating latent EBV, triggering immune recognition, and inducing susceptibility to antiviral therapy.

Experimental Design

CLVA therapy combines gemcitabine (GCb) and valproic acid (VPA) for virus activation and tumor clearance with (val)ganciclovir (GCV) as the antiviral drug to block virus replication and kill proliferating virus-infected cells. CLVA treatment was optimized and validated in NPC cell lines and subsequently tested in 3 Dutch patients with NPC that was refractory to conventional treatment.

Results

In NPC cell lines, both GCb and VPA can induce the lytic cycle of EBV. Their combination resulted in a strong synergistic effect. The addition of GCV resulted in higher cytotoxicity compared with chemotherapy alone, which was not observed in EBV-negative cells. CLVA therapy was analyzed in 3 patients with end-stage NPC. Patients developed increased levels of viral DNA in the circulation originating from apoptotic tumor cells, had disease stabilization, and experienced improved quality of life.

Conclusions

Our results in the initial CLVA-treated patients indicate that the therapy had a biological effect and was well tolerated with only moderate transient toxicity. This new virus-specific therapy could open a generic approach for treatment of multiple EBV-associated malignancies. Clin Cancer Res; 18(18); 5061–70. ©2012 AACR.


103

Introduction

Epstein–Barr virus (EBV) is causally linked to multiple cancers, including several lymphomas, undifferentiated nasopharyngeal carcinoma (NPC), and approximately 10% of gastric cancers worldwide. Although NPC is an uncommon disease in most countries, NPC is the 4th most common tumor among males in Indonesia and has a high incidence in southern China, northern Africa, and Alaska (1). Early diagnosis is essential because disease-free survival declines for patients with late-stage NPC (2). Unfortunately, early symptoms for NPC, such as epistaxis and tinnitus, are nonspecific and the majority of patients come to the hospital with advanced stage disease. In Indonesia, 87% of patients have NPC stage III or higher and 17% have distant metastasis at first presentation (3).

Because all NPC tumor cells harbor EBV, this virus itself is a possible target for therapy. In these tumor cells, EBV “hides” in a latent state due to methylation of the viral promoters, expressing only a few non-immunogenic viral proteins that are essential for EBV maintenance and contribute to tumor growth (4). This state of latency enables NPC tumors cells to evade the immune system, even in the presence of an existing strong immune response to multiple viral antigens (5, 6). Recent studies have shown that the lytic cycle of EBV can be efficiently induced by using chemotherapeutic agents effecting DNA synthesis and drugs affecting host DNA methylation and histone deacetylation (HDAC; refs. 7–11). The reactivation of EBV leads to the expression of proteins involved in viral genome replication and formation of new virions. These newly expressed proteins are highly immunogenic and could induce a powerful immune response toward the tumor cells containing the reactivated virus (5). In addition, lytic induction enables expression of viral kinases sensitizing the cells to antiviral treatment (9).

Inducing the lytic phase with chemotherapy in combination with a HDAC inhibitor caused reduced tumor volume in EBV-positive tumor cells in a mouse lymphoma model (8). The addition of an antiviral component to this combination therapy did further decrease tumor development. This concept was first used in 1998 for the treatment of a patient with EBV-positive lymphoma in which a combination of an HDAC inhibitor with antiviral therapy was administered (12). This was followed by a study using valproic acid (VPA) instead of arginine butyrate (13). The combination of chemotherapy (5-FU) with an HDAC inhibitor (HDACi) for a stronger induction of the lytic cycle was evaluated in a Dutch patient with end-stage NPC. After 5 days, the patient received antiviral treatment with valganciclovir (GCV). This treatment resulted in an increase of viral DNA in the circulation, reflecting shedding of apoptotic fragments from the tumor, which was not observed before treatment (14).

In this study, a novel combination therapy was developed targeting EBV within the tumor cell by combining EBV reactivation with subsequent antiviral therapy. After validation in a single naturally EBV-infected NPC cell line and EBV-positive gastric cancer cell lines, this cytolytic virus activation (CLVA) therapy, comprising gemcitabine (GCb), VPA, and GCV, was applied as a new treatment modality in patients with EBV-positive NPC for which no curable treatment options were available. In addition to clinical parameters, biological response was analyzed by EBV viral load and serology.

Materials and Methods

Cell lines

The EBV-positive NPC cell line C666.1 was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; BioWitthaker) containing 10% fetal calf serum (FCS) and 1% penicillin/ streptomycin/glutamine (P/S/G). Cell culture plates were first coated with fibronectin (Calbiochem) in PBS (100µg/mL) for 1 hour at roomtemperature (rT).Gastric

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carcinoma cell lines of AGS and the EBV-positive cell line AGS-BX1were cultured in F12HAM(BioWitthaker) containing 10% FCS and 1% P/S/G. AGS-BX1 cells were cultured under G418 (geneticin; Life Science Technologies) selection. The AGS-BX1 harbors the EBV genome with an insertion of the neomycin-resistance gene and the GFP gene that disrupts the TK gene (kindly provided by L. Hutt-Fletcher).

Western blot analysis

Protein samples were obtained by lysing cells in a RIPA buffer in the presence of a protease inhibitor cocktail (Roche) for 30 minutes at 4°C. Total protein concentration was determined with the BCA protein assay kit (Thermo Scientific). Proteins were mixed with 4× sample buffer and boiled for 5 minutes at 95°C. Protein samples were run on SDS-PAGE and transferred to nitrocellulose sheets by blotting. Immunoblots were blocked with 5% dried milk powder in PBST (0.05% Tween20 in PBS) and incubated with the primary antibodies BZ-1 for detecting ZEBRA (a gift from P. Farrell) and actin-HRP (C4; Santa Cruz). After incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (DAKO) the proteins were visualized by the ECL detection kit (GE Healthcare) according to instructions provided in the manual.

Immunofluorescence

C666.1 cells were cultured on 10-mm coverslips in 24- well plates. Cells were induced with 3 µmol/L GCb and 0.3 mmol/L VPA for 2 days. Cells were fixed by incubation with methanol-acetone for 10 minutes. Subsequently, nonspecific binding was blocked by incubation in PBS containing 10% FCS. Glass slides were incubated with BZ1 antibody and, after washing with PBS containing 0.05% Tween-20, incubated with the secondary antibody conjugated with fluoroscein isothicyanate (FITC). Slides were mounted in Vectashield (Vector Laboratories Inc.) containing 0.3 µmol/ L 4‘,6-diamidino-2-phenylindole (DAPI). Immunofluorescence was analyzed by using a Leica microscope.

Cytotoxicity analysis

For CLVA treatment cytotoxicity screens, 25,000 C666-1 cells were seeded in a 96-well plate in 100 µL medium and were allowed to adhere for 24 hours at 37°C in5% CO2. The next day, serial dilutions of GCb in the presence of 0.3 mmol/L VPA were added to cells in triplicate for 6 days. After drug incubation, 5 µL of MTT (5 mg/mL; Roche) was added to each well and the mixture was incubated for 3 hours. Thereafter, 100 µL of isopropanol–HCl solubilization buffer was added and the plates were incubated at 37°C overnight. The OD595 was determined using a microplate reader (Molecular Devices).

Patients

Three patients with histologically confirmed residual, recurrent, and metastatic EBV-positive NPC were included in this study after they had failed conventional curative treatment options and were deemed incurable. All patients signed an informed consent for this experimental pilot study. Before treatment, patients had a strong and broadly reactive humoral immune response against EBV antigens, measured by ELISA and immunoblot as described below.

CLVA therapy

One treatment cycle extends over 42 days. Patients received GCb (1250 mg/m2; Fresenius Kabi Oncology Plc) on days 1 and 8 intravenously. Valproic acid (generic medicine) was administered orally (12.5 mg/kg per day) during the first 14 days of the treatment cycle. From days 9 to 22, patients were treated with GCV (Roche) daily (3 × 450 mg/day orally). This treatment cycle of 42 days was repeated 6 times.


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Clinical monitoring of patients

All patients had a baseline magnetic resonance imaging (MRI) scan of the head-and-neck region, an ultrasound of the neck region, and complete body PET-CT scan. After every 2 treatment cycles, the tumor response was measured by imaging. In case of response or stable disease, patients received a total of 6 cycles. For the nasopharyngeal and neck region, MRI was carried out. Distant metastases in the thoracic region were monitored with CT-imaging. After finishing the treatment, a PET-CT scan was conducted. Adverse events were graded on the basis of the National Cancer Institute Common Toxicity Criteria version 3.0. Blood samples for measuring EBV viral load and humoral antibody response were obtained every week. Brush sampling for measuring EBV DNA load was carried out every 3 weeks.

Nucleic acid isolation

DNA was isolated from nasopharyngeal brushing samples by silica-based nucleic acid extraction as described previously (15). One milliliter of lysate was used as input for the isolation procedure, and the nucleic acids were eluted in 100 µL water. Reagents for the isolation procedure were obtained from BioMerieux.

RNA was isolated from cell lines by addition of 250 µL TRIzol to 300,000 cells according to the manufacturer’s instructions (Invitrogen).

EBV viral load by real-time quantitative PCR

The EBV DNA load in whole blood was determined by a quantitative LightCycler480 amplifying a 99-bp part of EBNA1. The probe master mix used was supplemented with primer QP3 and 4, and hybridization probes were used for quantification as described previously (16). Real-time PCR (RT-PCR) reagents were obtained from Roche Diagnostics. Then, 10-fold serial dilutions of spectrophotometrically quantified plasmid DNA containing the EBNA1 target sequence were used to create a standard curve. To check for putative inhibition of PCR, EBV DNA-negative samples were spiked with 1,000 copies of EBV plasmid DNA. ß-Globin PCR was carried out with the primers PCO3 (5’-ACACAACTGTGTTCACTAGC-3’) and PCO5 (5’-GAAACCCAAGAGTCTTCTCT-3’), which generate a 209-bp PCR product. Reaction conditions were as described previously (17). Quantification of the amplification products was carried out with the second derivative software of the LC480 (Roche).

cDNA synthesis and quantitative RT-PCR

RNA was treated with 1 µL RQ RNase-free DNase (Qiagen), 1 µL RQ1 RNase-free DNase 10× Reaction Buffer, and precipitated with 1 µL 3 mol/L sodium acetate, 0.5 µL linear acrylamide (Ambion; 5 mg/mL), and 25 µL of 100% ethanol at –80°C. Precipitated RNA was reverse transcribed by gene-specific cDNA synthesis using a multiprimed approach for 10 minutes at 65°C. Subsequently, 2 µL RT buffer, 10 µL dNTPs (2 mMmol/L), 2 µL DTT(100 mmol/L), 0.5 µL H2O, 0.25 µL RNAsin, and 0.25 µL AMV reverse transcriptase was added to each sample and incubated for 1 hour at 42°C. EBV gene expression was quantified by quantitative LightCycler PCR using an LC480 system (Roche). Quantitative PCR was carried out using a LightCycler 480 SYBR Green I Master kit (Roche) in a total reaction mixture of 10 µL containing 2.5 µL of 10-times-diluted cDNA, 5 µL 2× LightCycler 480 SYBR Green Master Mix, 0.5 µL (10 pmol/µL) of the gene specific primers, and 1.5 µL H2O. Primers used in the quantitative LightCycler PCR are listed in supplementary Table S1. Absolute quantification was determined using a standard curve of the plasmid pool containing all targets for quantifying the exact amount of RNA molecules for each target.

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Serology

Humoral antibody responses were measured in the serum of the patients with NPC. Immunoglobulin A (IgA) reactivity was assessed by a synthetic peptide-based ELISA using immunodominant epitopes derived from EBNA1 and VCA-p18 as described previously (18). Immunoblot analysis was carried out on blot strips containing HH514.c16 nuclear antigen induced by TPA and sodium butyrate to produce the late lytic phase of EBV. Strips were prepared and analyzed as described previously (19, 20).

Results

Lytic induction by GCb in combination with VPA in vitro

To validate the concept of CLVA therapy, the combination of a chemotherapeutic agent and a histone deactylation inhibitor was tested on human EBV-positive NPC (C666.1) and gastric carcinoma (AGS-BX1) cell lines. C666.1 cells carry a tightly latent EBV, whereas AGS-BX1 cells are less latent, showing spontaneous lytic EBV reactivation in approximately 5% of cultured cells. For inducing the EBV, replicative phase cells were treated with 3 µmol/L GCb and 0.3 mmol/L VPA, either separately or simultaneously. Expression of lytic RNA and proteins was analyzed by quantitative RT-PCR and Western blot (Fig. 1). The ZEBRA protein, acting as the EBV lytic switch, is upregulated marginally by the HDAC inhibitor VPA alone in C666.1 cells. The addition of GCb to VPA resulted in a stronger lytic induction, as indicated by the increased level of ZEBRA protein (30-fold) in C666.1 cells and a 3-fold increase in the AGS-BX1 lines, which already showed detectable ZEBRA expression at baseline.

The combination GCb and VPA resulted in a steep increase in ZEBRA RNAlevels in both the C666.1 (150-fold) and AGS-BX1 cell line (10-fold; Fig. 1C) and was dosedependent for each drug (Fig. 2). Other EBV lytic cycle genes besides the ZEBRA switch protein, such as protein kinase (PK), thymidine kinase (TK), and the structurally small capsid protein VCA-p18, were also induced by GCb and VPA in a dose-dependent manner in C666.1 cells (Fig. 2). To analyze the percentage of cells entering the lytic phase, the ZEBRA protein was stained in C666.1 cells induced for 2 days by GCb and VPA. More than 80% of the cells showed ZEBRA expression in a typical staining of nuclear dots (Fig. 3). The intensity of the staining varied among the cells; however, in more than 10% of cells, a strong immunofluorescence was observed.

Cytotoxicity of lytic induction increased by antiviral treatment

Induction of the EBV lytic cycle can create susceptibility to antiviral treatment and consequently increases antiviral drug-induced cytotoxicity. In the early phase of EBV reactivation, the enzymes TK and PK are expressed, which convert the antiviral drug, ganciclovir, into its cytotoxic form (9, 21). The C666.1 cell line was used to induce the lytic phase of EBV by adding 0.3 mmol/L VPA and a serial dilution of GCb (Fig. 4A). After 3 days, the cells were reseeded and analyzed for cytotoxicity in the absence and presence of 20 µmol/L GCV. Cytotoxicity (MTT) assays were conducted 6 days after initial culture and the increase in cytotoxicity caused by the addition of GCV to GCb was determined. The IC50 values for GCb shifted under the addition of GCV from 6.1 to 3.1 mmol/L. In AGS-BX1, the addition of GCV lowered the IC50 values of GCb from 4.3 to 2.0 µmol/L (data not shown). The increased cytotoxicity provided byGCVwas not very high, butGCVshowed a clear effect on inhibition of EBV replication. We observed a total block in production of RNA encoding the viral capsid protein VCA-p18 above the 7.5 mmol/L GCV level (Fig. 4B). The absence of VCA-p18 reflects the inability of induced EBV to generate new virions, thus blocking virus spread.


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Figure 1 | Lytic induction in cells lines. EBV-carrying NPC (C666.1) and gastric carcinoma (AGS-BX1) cell lines were cultured in the presence of gemcitabine (GCb), valproic acid (VPA), and a combination of the 2 drugs. Lytic induction was indicated by: A, increase of ZEBRA protein on Western blot; B, quantification ZEBRA protein; and C, quantification of ZEBRA mRNA levels by RT-PCR.

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Figure 2 | Dose-dependent induction of EBV lytic cycle by GCb and VPA in C666.1 cells. Dose escalation of GCb was analyzed for lytic induction. A, RNA levels of lytic genes (ZEBRA, early genes TK, PK, and late gene VCA-p18 gene) were determined and normalized to a cellular housekeeping gene. B, Western blot analysis of the level of the proteins expressed from the immediate early gene ZEBRA was assessed with the loading control ß-actin. Dose escalation of GCb was analyzed for lytic induction after addition of 0.3 mmol/L VPA. C, RNA levels of lytic genes were determined and normalized to a cellular housekeeping gene. D, analysis and quantification of the ZEBRA protein levels was measured on Western blot assay. Dose escalation of VPA in presence of 3 µmol/L GCb was studied. E, RNA levels of lytic genes were determined and normalized to a cellular housekeeping gene. F, analysis and quantification of the ZEBRA protein levels was measured on Western blot assay.


109Figure 3 | Percentage of lytic-induced C666.1 cells by GCb VPA was visualized by ZEBRA protein staining. A, the percentage of ZEBRA-positive cells in untreated condition and after incubation for 48 hours with 3 µmol/L GCb and 0.3 mmol/L VPA. B, typical example of the punctuated ZEBRA staining in the lytically induced C666.1 cells (left), with the DAPI nuclear control (right). Although ZEBRA expression is (weakly) induced in most cells, brighter nuclear ZEBRA staining is observed in approximately 11% of the cells in a typical experiment.

CLVA therapy in patients with end-stage NPC

Patient 1

The first patient treated with CLVA therapy was a 48-year-old female (WHO performance status 1). She had a local recurrence with extension into the retropharyngeal recess surrounding the internal carotid artery. Twenty-two months before commencement of the CLVA treatment, she received chemoradiation for T4N1M0 NPC. Fifteen months after initial treatment, she received photodynamic therapy for local recurrence in the nasopharynx, but only had a partial response. This patient received a total of 6 cycles of CLVA therapy. During the first 2 cycles patient received 1.25 mg/kg VPA instead of 12.5 mg/kg due to logistic failure.

Tumor response

The patient showed a significant decrease of tumor in the nasopharynx after 2 courses (Fig. 5A and 5B). During the remaining treatment and for up to 11 months of the follow-up period, the patient had stable disease. An MRI scan after treatment showed disease progression of the tumor in the nasopharynx (Fig 5B).

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Figure 4 | Increased cytotoxicity and prevention of RNA expression of the structural gene VCA-p18 by ganciclovir in lytically induced C666.1 cell line. A, the additional toxic effect of GCV to GCb in the C666.1 cell line. IC50 for the C666.1 cells incubated with serial dilution of GCb in the presence of 0.3 mmol/L VPA was 6.1 µmol/L GCb and, after addition of 20 µmol/L GCV, the IC50 value decreased to 3.1 µmol/L GCb; the solid line shows group without GCV and the dotted line represents values in presence of GCV. B, the expression of the structural gene VCA-p18 is diminished by concentrations greater than 7.5 mmol/L GCV, indicating inhibition of viral replication. The lytic induction is not hampered as reflected by the stable ZEBRA gene expression.

Adverse events

This patient developed a grade 4 toxicity with platelet count decrease after course 3, but recovered completely before the start of course 4. After the first GCb course, the patient again developed a grade 3 toxicity platelet count; therefore, she received 50% of the GCb dose for the remaining courses. Due to grade 3 toxicity of hemoglobin levels, the patient received blood transfusions.

Patient 2

A 52-year-old male (WHO performance status 0) patient was referred to our hospital with disease progression after palliative radiotherapy for a T2aN3M1 NPC diagnosed 10 months previously. The disease progression consisted of cytologically proven positive lymph nodes high in the mediastinal region and a costal metastasis. In addition, 2 regional lymph nodes in the left neck were assessed and found to have persistent tumor after radiotherapy. This patient received a total of 6 cycles, with no dose reductions. Blood sampling for EBV viral load and antibody response were collected every 2 weeks.

Tumor response

After 2 courses of CLVA therapy, patient 2 was observed to have responded with a reduction of one of the tumor positive lymph nodes in the neck to normal size on CT-imaging. After 6 cycles, imaging showed the absence of PET activity. The remaining neck-node tumor remained stable after 6 courses of treatment. As there was an increase in quality of life and improvement of well-being, patient 2 received another course of


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adjuvant radiotherapy on the residual positive lymph node (36 Gray) in the left neck region and has been observed to have a persistent stable disease for 6 months in the follow-up period.

Adverse events

Patient 2 did not encounter any grade 3 or 4 adverse events.

Patient 3

The 3rd patient was a 48-year-old female (WHO performance status 1) with progressive disease in the nasopharynx, which was histologically proven as recurrent disease, and a cytologically proven lymph node in level V of the neck region. IN addition, PET-CT showed supraclavicular, intrathoracic, and left thyroid lobe metastases. Nine years previously, she underwent a modified radical neck dissection for a tumor in the neck with an unknown primary site. This patient had already received palliative treatment with capecitabine and radiotherapy before initiation of the present treatment with CLVA. Patient received a total of 5 treatment cycles, with 25% dose reductions during course 5. The patient refused to undergo cycle 6.

Tumor response

This patient had a significant decrease of tumor mass in the nasopharyngeal region (Fig. 5C and 5D). The lymph nodes and distant metastasis remained stable for 6 months during the foloow-up.

Adverse events

At the start of therapy, patient 3 had a grade 3 anorexia for which she received tube feeding during all courses of therapy and during follow-up. Patient 3 developed aspiration pneumonia grade 3 after cycle 4. During the 5th cycle, this patient was admitted to the hospital twice with two grade 3 infections. During the 5th course, she also suffered a grade 3 hepatotoxicity (increased SGPT).

EBV DNA load in the tumor cells and whole blood of CLVA-treated patients with NPC

The presence of high levels of EBV DNA in minimally invasive brushings, taken from the nasopharyngeal mucosa has been described to correlate with the presence of viable NPC tumor cells (22). In contrast, EBV viral DNA detected in the circulation of NPC patients may originate from apoptotic tumor cells rather than viable circulating tumor cells (29). The 3 patients treated with CLVA therapy were monitored for EBV viral load in nasopharyngeal brushing samples and in whole blood, drawn before, during, and after CLVA treatment (Fig. 6). Patients 1 and 2 did not have a visible tumor in the nasopharyngeal mucosa and, therefore, brushing samples were taken at longer intervals. The 3rd patient did have a locally visible tumor in the nasopharyngeal region and brushing samples were obtained every 2 weeks. The viral load measured in patient 3 before treatment was 4.3 × 106 EBV copies/ brushing sample. After an initial 200-fold decrease in viral load during the second cycle of treatment down to 2.4 × 104 EBV copies/brushing sample, the viral load showed a rise again mainly during a prolonged interval in the treatment schedule. Brushing samples of the other CLVA-treated patients showed an occasionally positive result up to 104 copies/brushing sample.

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Figure 5 | MRI scan of patients before and after CLVA treatment. A, MRI scan, axial slide of nasopharyngeal region of patient 1 before treatment and B, after treatment. C, MRI scan; axial slide of nasopharyngeal region of patient 3 before treatment and D, after 4 treatment cycles; imaging was not repeated after complete treatment because of claustrophobia noted in the patient. Arrows indicate the relevant area of response. E, humoral immune response against EBV antigens revealed by IgG immunoblot analysis [20] of sera from the CLVA patients before (1) and after (2) the completed therapy.

The viral DNA levels in blood were monitored to analyze the biological response to therapy. Before treatment, all patients had EBV copies below the clinical cutoff level, that is, 1.6 × 103 copies/mL of blood. At the start of therapy, patient 3 had slightly higher levels (1.1 × 104 copies/mL of blood). Viral load in blood during CLVA treatment showed a highly dynamic response, probably reflecting tumor apoptosis. The highest fluctuations were observed in patient 1, in whom it varied from below detection level up to 1.4 × 106 EBV copies/mL of blood. In patients 2 and 3, lower EBV DNA levels were measured in the blood. A common pattern in the blood that was observed for all 3 patients was the appearance of viral DNA in the periods following treatment (Fig. 6).

Humoral immune responses of CLVA-treated patients with NPC

The IgG response against immunodominant EBV epitopes was analyzed. The data confirmed that the patients had a molecularly diverse anti-EBV immune response before treatment, including reactivations against lytic viral antigens. A potent immune response is needed to allow identification of therapy-induced new immunogenic viral proteins. Two patients showed a strong immune response against a wide diversity of viral proteins (Fig. 5E). The first patient had a weaker response, mainly directed against ZEBRA and VCA-p18. The


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level of the immune response remained the same after treatment and the pattern of proteins recognized did not change.

Further, the level of the humoral immune response was determined by standardized peptide-based ELISA before and during therapy. High constant levels of VCA-p18 reactivity were observed, whereas the EBNA1 responses remained at a constant low level (data not shown).

Figure 6 | Effect of CLVA treatment on the viral load in whole blood (black line) and nasopharyngeal brushings (gray line) of the CLVA NPC-treated patients. In the boxes above the individual graphs, the therapy schedule is depicted. The arrows indicate the GCb infusions, 1250 mg/m2. However the last 3 GCb infusions in patient 1 comprised only half of the dose. The VPA was given at a concentration of 12.5 mg/kg per day, except for the first 4 cycles for patient 1, wherein 1.25 mg/kg per day was administered. The GCV concentration was 3 × 450 mg/day and administered at the indicated times. DNA load was determined in blood samples obtained weekly and in the tumor cells obtained by NP brushing at the indicated time points.

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Discussion

A new therapeutic approach was explored on the basis of reactivation of the latent viral genome in combination with antiviral therapy to target EBV within NPC tumor cells. This CLVA therapy proved to be effective in vitro and, subsequently, CLVA therapy was administered to 3 patients with end-stage NPC disease. CLVA treatment in patients with end-stage NPC was well tolerated and showed a clinical response and improvement in quality of life during and after therapy in all patients. Side effects of CLVA therapy were transient and only moderate. These first in vivo results indicate that the concept of epigenetic induction of viral antigens with subsequent administration of antiviral therapy may be a potent approach in patients with EBV-positive NPC, which can possibly be extended to other EBV-driven malignancies.

Gemcitabine is one of the most effective single-agent therapies in the treatment of NPC (23), and was recently studied in 31 patients where initial platinum-based chemotherapy failed and subsequent GCb therapy resulted in partial response in 43.8% of the patients and stable disease with minimal side effects in 28.1% of patients (24). Besides tumor cell apoptosis induced by GCb, it could reactivate EBV within the tumor cells. The potential for EBV lytic induction by GCbin EBV-positive NPC cell lines was shown to be higher than by 5-FU, which was used in a previous juvenile NPC case study (14, 25, 26). Activation of highly methylated DNA of latent EBV can be enhanced by epigenetic chromatin remodeling of the EBV genome induced by HDAC inhibitors and DNA-demethylating agents (7–11). In the naturally EBV-carrying infected NPC cell C666.1, the combination of histone deactylase inhibitor VPA with GCb showed a clear synergistic action on the expression of EBV lytic genes, ZEBRA, TK, PK, and VCA-p18, which was confirmed in the gastric carcinoma cell AGS-BX1 harboring a recombinant EBV (Figs. 1–3). Lytic induction sensitized the EBV-positive cell lines to the antiviral drug gancicolovir (GCV), which resulted in an increase of cytotoxicity compared to the toxicity induced by GCb and VPA alone (Fig 4A). Further, EBV lytic gene expression is known to result in an increase of apoptosis in combination with GCV (27). This additional toxic effect was observed in an EBV-positive lymphoma animal model (25). In addition, enhanced toxicity by GCV was observed in a recent study by Sides and colleagues in a study wherein they provoked lytic induction with low-dose arsenic (28). Importantly, GCV resulted in reduced production of RNA encoding the late viral capsid antigen p18 (VCA -p18) in lytically induced C666.1 cells (Fig. 2). This clearly demonstrates the effect of GCV treatment on inhibiting new viral particle formation after lytic induction, which is necessary for the safety of CLVA treatment in patients with NPC.

The choice of drugs already approved for human use resulted in the rapid translation of CLVA-based treatment into the clinic. Therapy was administered to 3 patients with end-stage NPC and was well tolerated. We observed some toxicity, mainly consisting of neutropenia, in only one case. However, previous chemotherapy administered to this patient may be more causal to the reduced bone marrow capability to recover from GCb. The other 2 patients did not encounter any detectable side effects of CLVA therapy.

All 3 patients showed a biological response to CLVA therapy indicated by an increase in viral DNA load in the blood, as observed in a previous case study (14). This increase reflects fragmented EBV DNA derived from apoptotic tumor cells (29). The dynamics of theDNAload in all patients showed a similar trend, that is, an immediate decline of EBV DNA loads during therapy cycles and an increase in EBV DNA load in the recovering periods. A decline of EBV load in blood has been described to predict a good clinical response in patients with NPC (30). The unexpected increase of viral load in the period without treatment could reflect the functional recovery of the immune system and its effect in eliminating lytic induced cells. We failed to detect an increase in anti-EBV immune reactivity as result of the CLVA therapy, although more detailed analysis may be needed for this, including analysis of T-cell responses.


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A residual (recurrent) tumor in the nasopharynx was only present in the 3rd patient. Viral load in noninvasive NP brushings taken regularly during and after therapy decreased, but did not disappear completely. Repeated biopsies in the nasopharynx during and after treatment were considered too invasive; therefore, it is unclear whether these viral loads represent shed tumor cells or viral reactivation in the nasopharynx.

Despite the small number of patients in this pilot study, which may prevent strong conclusions, we followed the clinical outcomes of these patients. All patients initially having progressive disease developed stable disease during and after treatment, and, in addition, a clear improvement of their clinical condition was observed.

In conclusion, virus-specific CLVA tumor therapy may provide a new and generic approach for treatment of multiple EBV-associated malignancies in both developed and developing countries worldwide. The 3 patients with endstage NPC all had a clinical response to CLVA therapy and an improved quality of life. A phase I/II trial was started recently (Eudract nr: 2010-022444-20). In this trial, additional PBMCs will be collected to obtain more insight in the immunological aspects of the CLVA therapy response of the patients.

The use of novel combination of existing drugs to activate EBV and eliminate virus-infected cells may open the way to more simplified, possibly oral therapies that would greatly benefit patients in developing countries where complex chemoradiation is not possible. Further studies on CLVA are needed for better insight in the molecular basis of tumor or EBV specific immune responses provoked by the lytic induction and the long-term effect of the treatment. The use of EBV as target in therapy could open up new approaches for other EBV-driven tumors.

Acknowledgements

The authors thank the patients who participated in this trial. The authors also thankMax Nobis and Chantal Kuijpers for excellent technical assistance and Renske Fles for organizing the logistics of patient care. The donation of the AGS-BX1 cell line by Dr. L. Hutt-Fletcher is greatly appreciated.

Translational relevance

Undifferentiated nasopharyngeal carcinoma (NPC) is causally associated with Epstein–Barr virus (EBV). As this virus is present in all tumor cells, EBV could serve as a target for therapy. In NPC cells, EBV hides in latency, expressing only a few essential proteins contributing to tumor formation although escaping immune elimination. Inducing the viral lytic phase makes tumor cells susceptible for immune recognition and antiviral therapy. The concept was confirmed in NPC cell lines using gemcitabine combined with a host DNA methylation and histone deacetylation (HDAC) inhibitor as lytic inducer. Subsequent addition of antiviral therapy (ganciclovir) increased specific cytolysis. The cytolytic virus activation (CLVA) therapy was evaluated in 3 patients with progressive end-stage NPC. All patients had stable disease during and for more than 6 months after therapy with improved quality of life. CLVA therapy resulted in increased shedding of viral DNA in the circulation. These data indicate that CLVA therapy may open up a new therapeutic approach for EBV-driven malignancies.

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13. Jones K, Nourse J, Corbett G, Gandhi MK. Sodium valproate in combination with ganciclovir induces lysis of EBV-infected lymphoma cells without impairing EBV-specific T-cell immunity. Int J Lab Hematol 2010;32:e169–74.

14. Stevens SJ, Zwaan CM, Verkuijlen SA, Middeldorp JM. Epstein–Barr virus (EBV) serology for predicting distant metastases in a white juvenile patient with nasopharyngeal carcinoma and no clinical response to EBV lytic induction therapy. Head Neck 2006;28:1040–5.

15. Boom R, Sol C, Beld M, Weel J, Goudsmit J, Wertheim-van DP. Improved silica-guanidiniumthiocyanate DNA isolation procedure based on selective binding of bovine alpha-casein to silica particles. J Clin Microbiol 1999;37:615–9.

16. Stevens SJ, Verkuijlen SA, Middeldorp JM. Quantitative detection of Epstein–Barr virus DNA in clinical specimens by rapid real-time PCR targeting a highly conserved region of EBNA-1. Methods Mol Biol 2005;292:15–26.

17. Hesselink AT, Van Den Brule AJ, Groothuismink ZM, Molano M, Berkhof J, Meijer CJ, et al. Comparison of three different PCR methods for quantifying human papillomavirus type 16 DNA in cervical scrape specimens. J Clin Microbiol 2005;43:4868–71.

18. Fachiroh J, Paramita DK, Hariwiyanto B, Harijadi A, Dahlia HL, Indrasari SR, et al. Single-assay combination of Epstein–Barr Virus (EBV) E. J Clin Microbiol 2006;44:1459–67.

19. Fachiroh J, Schouten T, Hariwiyanto B, Paramita DK, Harijadi A, Haryana SM, et al. Molecular diversity of Epstein–Barr virus IgG and IgA antibody responses in nasopharyngeal carcinoma: a comparison of Indonesian, Chinese, and European subjects. J Infect Dis 2004; 190:53–62.

20. Middeldorp JM, Herbrink P. Epstein–Barr virus specific marker molecules for early diagnosis of infectious mononucleosis. J Virol Methods 1988;21:133–46.

21. Meng Q, Hagemeier SR, Fingeroth JD, Gershburg E, Pagano JS, Kenney SC. The Epstein–Barr virus (EBV)-encoded protein kinase, EBV-PK, but not the thymidine kinase (EBV-TK), is required for ganciclovir and acyclovir inhibition of lytic viral production. J Virol 2010;84:4534–42.

22. Stevens SJ, Verkuijlen SA, Hariwiyanto B, Harijadi, Paramita DK, Fachiroh J, et al. Non-invasive diagnosis of nasopharyngeal carcinoma: nasopharyngeal brushings reveal high Epstein–Barr virus DNA load and carcinoma-specific viral BARF1 mRNA. Int J Cancer 2006;119:608–14.


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23. Bensouda Y, Kaikani W, Ahbeddou N, Rahhali R, Jabri M, Mrabti H, et al. Treatment for metastatic nasopharyngeal carcinoma. Eur Ann Otorhinolaryngol Head Neck Dis 2011;128:79–85.

24. Zhang L, Zhang Y, Huang PY, Xu F, Peng PJ, Guan ZZ. Phase II clinical study of gemcitabine in the treatment of patients with advanced nasopharyngeal carcinoma after the failure of platinum-based chemotherapy. Cancer Chemother Pharmacol 2008;61:33–8.

25. Feng WH, Israel B, Raab-Traub N, Busson P, Kenney SC. Chemotherapy induces lytic EBV replication and confers ganciclovir susceptibility to EBV-positive epithelial cell tumors. Cancer Res 2002;62: 1920–6.

26. Hsu CH, Hergenhahn M, Chuang SE, Yeh PY, Wu TC, Gao M, et al. Induction of Epstein–Barr virus (EBV) reactivation in Raji cells by doxorubicin and cisplatin. Anticancer Res 2002;22:4065–71.

27. Jung EJ, Lee YM, Lee BL, Chang MS, Kim WH. Lytic induction and apoptosis of Epstein–Barr virus-associated gastric cancer cell line with epigenetic modifiers and ganciclovir. Cancer Lett 2007;247: 77–83.

28. Sides MD, Block GJ, Shan B, Esteves KC, Lin Z, Flemington EK, et al. Arsenic mediated disruption of promyelocytic leukemia protein nuclear bodies induces ganciclovir susceptibility in Epstein–Barr positive epithelial cells. Virology 2011;416:86–97.

29. Stevens SJ, Verkuijlen SA, Hariwiyanto B, Harijadi, Fachiroh J, Paramita DK, et al. Diagnostic value of measuring Epstein–Barr virus (EBV) DNA load and carcinoma-specific viral mRNA in relation to anti-EBV immunoglobulin A (IgA) and IgG antibody levels in blood of nasopharyngeal carcinoma patients from Indonesia. J Clin Microbiol 2005;43: 3066–73.

30. Hassen E, Farhat K, Gabbouj S, Bouaouina N,Abdelaziz H, Chouchane L. Epstein–Barr virus DNA quantification and follow-up in Tunisian nasopharyngeal carcinoma patients. Biomarkers 2011;16:274–80.

Chapter 9

Summary and Discussion

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The management of patients with Nasopharyngeal Carcinoma (NPC) in Indonesia is currently not optimal. This is, to a great extent, due to the late presentation of patients in the health care system, the limited availability and quality of radiotherapy facilities, deficient treatment protocols, and poor doctor and patient compliance. In this thesis we have presented the challenges in NPC-care in Indonesia (chapter 3) and detailed possible solutions: (1) implementation of data management (chapter 2), (2) training of General Practitioners for better awareness and knowledge of NPC for earlier recognition of the disease (chapters 4 and 5), and (3) the introduction of new treatment modalities for NPC (chapters 6, 7, and 8).

Data management (Chapter 2)

In Indonesia, where current clinical record forms and data-registration are divided among different departments, it is difficult to obtain good insight into the treatment results and follow-up history of patients. In order to improve insight into treatment results, this project started with the introduction and implementation of an online data-management system to collect necessary data for treatment analysis. This new online data-management system has been tested for quality and accuracy of clinical data. In this chapter, we have illustrated how an online clinical data management service can improve data quality in a developing country. The digital nature of our system, as well as the online availability of the data, provides fast and easy insight with adherence to treatment protocols. Therefore, we expect less loss in follow-up and increased protocol compliance for future studies. To create more efficient medical care programs and studies in developing countries, we believe that an online data system is essential.

In addition to proper data quality and better protocol compliance, the same system can serve as a tool to train and educate medical physicians, resulting in an overall improvement of treatment protocols. With this in mind, we recommend further online data systems for international training collaborations between developing and western countries regarding cancer care. To further aid this endeavour, we suggest a training course prior to departure for a developing country, by a delegation of health care workers from overseas, to analyse the treatment online and develop a custom-made training program in advance, therefore insuring more efficient training visits.

This prescribed online data system resulted in the first prospective data collection of treatment results to-date for NPC in Indonesia.

Treatment results of Nasopharyngeal Carcinoma in Indonesia (Chapter 3)

Employing the online data-system, we analysed 78 patients treated with curative intent for primary NPC. The results revealed an unfavourable outcome regarding the current treatment of NPC within the Dr. Sardjito Hospital in Yogyakarta, Indonesia. It was revealed that the cure rates were not comparable to the treatment results currently found in literature with similar treatment protocols. Internationally, the treatment of primary NPC with radiotherapy and/or concurrent chemo-radiation is reported to yield good response rates with 3-year disease-free and overall survival results of 70% and 80%. In contrast, our study in Yogyakarta exposed an initial complete response rate of only 29% within a group of 78 NPC patients.

The advanced stage of disease within the patients at presentation is probably the leading reason for this poor treatment outcome. The second factor is the limited availability of radiotherapy facilities, resulting in a long treatment delay. To overcome this negative effect of an average waiting time of 120 days, patients are now


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treated with induction chemotherapy. Unfortunately, 23 (29%) of patients died before treatment response could be assessed, due to disease progression or the toxicity of the chemotherapy. The actual radiotherapy treatment delivery was another leading cause of poor treatment outcomes, with the overall treatment time appearing to be 62 days (instead of the prescribed 45) for delivering of the standard dose of 66Gy, therefore, rendering the treatment ineffective [1,2]. Reasons for this prolonged treatment time are presumably patient, doctor and hospital related. A current, on-going project focused on these factors will optimistically reveal the reasons for this prolonged treatment in more detail.

Typically, the simplest way to address the current concerns would be to establish a sufficient number of well-equipped radiotherapy facilities in Indonesia. However, given the current conditions, this would take decades. In the meantime, our goal is to assure that compliant patients with a high chance of cure are selected to receive an effective course of treatment. Early detection of NPC, new treatment regiment to overcome waiting time, a counselling system to support only those patients who have a realistic opportunity to complete the treatment, and improved maintenance for the radiotherapy facility are feasible short-term options for improving treatment outcomes in the near future.

Knowledge of general practitioners on Nasopharyngeal carcinoma (Chapter 4 and 5)

As previously described, earlier presentation of NPC-patients in the hospital will result in better treatment outcomes and overall survival. The knowledge and awareness of general practitioners (GPs) of NPC is crucial for patient referral to the hospital. Chapter 4 describes our methodology for testing the knowledge and awareness of GPs working within Yogyakarta. These tests revealed that GPs working in primary health care centers (PHCC) in Yogyakarta lacked sufficient knowledge on all aspects of NPC. GPs working in these PHHC are the most important providers of primary medical health care in Indonesia, therefore we enrolled these GPs in a training program to achieve earlier presentation of NPC in the treatment centers.

In chapter 5, we confirmed the results presented within Chapter 4, evaluated improvements post-training and compared the effectiveness of two different training formats: a centralized symposium versus local lectures in the PHCC. Both educational formats appeared equally effective in increased knowledge about key symptoms, in particular early-stage symptoms. In addition to raising the level of knowledge in primary health care workers, we will aim to raise public awareness about the importance of early-stage cancer recognition and how and when to consult a GP in all economic levels within Indonesian society. Similar campaigns to educate society on breast and cervical cancer have proven to be highly effective. Continued education of the local GPs and their surrounding society will ideally result in earlier detection of NPC, better treatment outcomes, and improved overall prognosis. Currently the NPC awareness program takes place in Yogyakarta, Jakarta, Surabaya and Makassar. In the future, we hope to expand this program to the rest of Indonesia.

Development of new treatment modalities for NPC

In Chapter 3 we demonstrated the poor prognosis rate for NPC patients in Indonesia. Most of these patients have persistent, progressive or metastatic disease after initial treatment, with limited realistic treatment options. This signifies an evident need for new treatment modalities, such as photodynamic therapy (PDT) and lytic induction chemotherapy.

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Photodynamic therapy Review: Photodynamic therapy in the therapy for recurrent/persistent nasopharyngeal cancer (Chapter 6)

Locally persistent or recurrent NPC is a recurring epidemic across Indonesia. As described in Chapter 3, 12 of the 48 NPC patients, who finished their treatment, continued to have persistent disease in the nasopharynx. In these circumstances, treatment options are limited. Re-irradiation of recurrent disease is not a realistic option to implement in a country with scarce radiotherapy facilities and the high risk of severe soft and osseous tissue damage. Surgical procedures are also complicated and require vast experience, which is not currently available.

Conversely, PDT has the potential to be a highly effective local treatment modality for recurrent or persistent nasopharyngeal cancer, without the severe side effects after re-irradiation. In this review, all reported results of PDT as an alternative treatment for NPC are summarized. Despite promising results with first generation photosensitizers, no new clinical studies have been enrolled in the past decade.

Temoporfin mediated photodynamic therapy in patients with local persistent and recurrent Nasopharyngeal Carcinoma: a feasibility study (Chapter 7)

In a feasibility study with 21 patients with locally-persistent NPC and one patient with locally-recurrent NPC, we investigated the safety and efficiency of temoporfin mediated PDT. PDT with the nasopharyngeal applicator is a relatively simple technique, which can be utilized to treat residual or recurrent NPC, restricted to the nasopharynx. The treatment is a suitable option for Indonesia because of the short waiting and treatment procedure times. Patients were treated under local anaesthesia with the use of a dedicated nasopharyngeal applicator. Three different drug doses and light intervals were administered. Overall the results are very promising; in 17 of the 22 patients a biopsy was performed 40 weeks after therapy and showed no tumor. One patient died two days after treatment due to a misdiagnosed pneumonia.

Based on our experiences and taking into account the poor treatment results currently for primary NPC in Indonesia, it is reasonable to assume that PDT can successfully be used in the future as part of the primary treatment. We hypothesize that PDT can be applied for NPC during the waiting time for radiotherapy, without compromising the options for all other possible treatment modalities. A current ongoing phase II study will hopefully confirm the effectiveness of PDT treatment for patients with locally persistent or recurrent NPC.

Cytolytic Virus Activaton Therapy (Chapter 8)

NPC is causally linked to Epstein-Barr virus (EBV). In EBV positive NPC all tumour cells carry EBV. This virus is a potential target for therapy. EBV hides in a latent state and expresses only a few non-immunogenic viral proteins essential for EBV maintenance, contributing to tumour growth. We developed a cytolytic virus activation (CLVA) therapy for NPC treatment, reactivating latent EBV by epigenetic modulation, triggering immune recognition and inducing susceptibility to antiviral therapy. CLVA therapy combines gemcitabine (GCb) and valproic acid (VPA) for virus activation and tumour clearance with (val)ganciclovir (GCV) as antiviral drug to block virus replication and kill proliferating virus infected cells. The CLVA treatment was optimized and validated in NPC cell lines and subsequently tested in three Dutch patients with recurrent NPC, refractory to conventional treatment. These patients developed increased levels of viral DNA in the circulation originating from apoptotic tumour cells, witnessed stabilization of the disease, and experienced improved quality of life.


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Our results in the first CLVA treated patients indicate that this therapy had a biological effect and was well tolerated.

This new virus specific therapy could open a generic approach for treatment of other EBV-associated malignancies. The effect of CLVA therapy on tumour behaviour remains to be proven in a phase I/II trial, which started in 2010 (Eudract nr: 2010-022444-20). In addition to a treatment option for other EBV-associated malignancies, it has the potential to be a valuable treatment modality during the waiting time for radiotherapy.

Future perspectives and projects

At present, the answer to locally advanced NPC, which is one of the most common cancers in Indonesia, is concurrent chemo-radiation. Chemotherapy can be administered in most well equipped hospitals within Indonesia, whereas radiotherapy for most patients is not a realistic option due to limited availability of radiotherapy facilities throughout the country. Although in several centres radiotherapy can be administered according to international standards, the scarcity makes this only available for a very limited number of patients. In 2008 there were 18 linear accelerators and 17 Cobalt-60 teletherapy available in Indonesia with a population of 229 million, whereas 6 machines were under commission, resulting in 0.13 accelerators per million inhabitants [3]. This is in stark contrast to Europe, where 5.5 accelerators are available per million inhabitants in high-resource countries, 3.5 in medium-resource countries, and 2 per million in low resource countries [4]. The recommended number of treatment units per population differs widely; European guidelines recommend on average 5.9 units per million inhabitants [5].

The vast lack of equipment in Indonesia results in excessive waiting lists, inadequate treatment procedures and preferential treatments for the elite. Many wealthy patients receive their treatment in hospitals abroad, resulting in an outflow of substantial health care dollars to adjacent countries such as Malaysia, Singapore, and Australia. The less fortunate individual therefore cannot receive this quality of treatment. Currently the Indonesian government offers a reimbursement for lower incomes, placing greater pressure on waiting lists. It is not unusual to wait 6 months or more before treatment. With radiotherapy remaining the cornerstone of cancer treatment today, this implies that many cancer patients, including NPC, cannot be treated effectively.

The best way to address these growing concerns would be to create sufficient radiotherapy facilities in Indonesia with well-trained staff, raise medical and public awareness, and provide proper health care insurance for all patients. The growing economy in Indonesia will expectantly, in time, enable these solutions, but in the meantime small-scale solutions can improve care for patients with NPC. For this reason, the main initiative in Yogyakarta for the coming years is to build an internationally supported cancer-training centre. By introducing health care at the international standard level, supported by committed overseas faculty, Indonesian doctors can be trained according to these international standards. This would reduce the need for higher income brackets to seek care abroad, therefore investing their money in the local and national medical systems and providing better overall heath care for the underprivileged (i.e. the ‘’Robin Hood principle’’). The minister of Health, the medical faculty, the University Hospital in Yogyakarta and foreign investors support this initiative, leading to the onset of international standards for cancer care in Indonesia.

Above and beyond the realization of an internationally supported cancer-training centre, clinical research projects must still be performed. The Dutch Cancer Society has granted two novel research projects. The first grant for a phase II study on cytolytic virus activation for NPC patients with distant metastasis or patients beyond cure. The effectiveness of lytic induction will hopefully be proven in this phase II trial. Future research will be needed in regard to other lytic inducing agents, which have even less side effects and can be taken

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orally. The role of lytic induction can be a very promising research line, since we expect less side effects compared with conventional chemotherapy regimes.

The second research grant will investigate how to decrease the number of patients presenting with late stage disease. One of the objectives of this study is to evaluate the effect of the training programmes for the GPs working in the PHCCs within Indonesia. Besides expected earlier presentation of NPC patients to the ENT-specialists, the incidence and the number of unnecessary referred patients will be evaluated. Study-specific questionnaires will be developed for the assessment of the reasons for patient’s delay in consulting the GP, and this information will be used to facilitating future customized public awareness campaigns.

The studies under this research grant will additionally focus on counselling and monitoring of NPC patients. This project will start with extensive monitoring of the patients during the waiting period and the radiotherapy treatment, providing further insight into the reason patients do not return to the hospital in time, or do not return at all. As the patients start the daily radiotherapy treatment, we will evaluate their return rate. In the case of a “no show”, the patient will be questioned to find out the reason for this non-compliance. Therefore, home visits will be part of this study. To prevent unnecessary treatments in the future, this project will analyse any necessary current diagnostic system adjustments. From this analysis it can be expected that patients will be screened (again) for distant metastasis after a long waiting time before radiotherapy. By only treating patients who still have a fair chance for cure, the overall waiting time will shorten and unnecessary use of the limited resources can be prevented. The final phase of this project will combine the above-mentioned projects and will optimistically lead to a new referral protocol, improved counselling structure, and an enhanced diagnostic system.

Furthermore, EBV markers can also be used for early-stage detection in defined populations, which, in combination with doctor’s awareness, may improve more effective treatment (better treatment response at early stage). The GP education program should include referencing the availability of improved diagnostic procedures for early detection. Improved education, combined with new diagnostic tests based on EBV, could be a cheap and sensitive method for detecting NPC in Indonesia and other high incidence countries. Importantly, the decision for serological diagnostic testing or EBV-DNA based analysis has to be based on complaints and duration of complaints registered and interpreted by the GP. Our on-going research is focussed on finding the best decision process to accomplish effective early stage testing in defined NPC risk groups, being either NPC family members or patients with characteristic head and neck complaints [6].

Another important issue to remember is the lack of proper data collection on a regular basis among local physicians, causing a lack of awareness regarding their treatment failures and complications. Because of this unawareness, there is no cause for self-evaluation or improvement of current treatments, creating a greater learning curve. This issue can hopefully be addressed with a newly funded program, making it possible to implement an online data-management system in all cancer centres across Indonesia, leading to better national insight and training programs.

Additional funding will be raised to investigate the role of PDT in primary treatment of NPC. In a phase I-II trial we hope to evaluate this treatment modality for NPC patients, taking the most advantage of the prolonged waiting time for standard (chemo)-radiation in developing countries like Indonesia. Patients who receive induction PDT during the waiting period for radiotherapy have the potential to increase the state of their health and limit further tumour progression.


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Reference List 1. Levendag PC, Lagerwaard FJ, Noever I, dePan C, vanNimwegen A, Wijers O, Schmitz PI, van Dieren E, Nowak PJ: Role

of endocavitary brachytherapy with or without chemotherapy in cancer of the nasopharynx. Int J Radiat Oncol Biol Phys 2002, 52:755-768.

2. Akimoto T, Mitsuhashi N, Hayakawa K, Sakurai H, Murata O, Ishizeki K, Ishikawa H, Nasu S, Yamakawa M, Niibe H: Split-course accelerated hyperfractionation radiotherapy for advanced head and neck cancer: influence of split time and overall treatment time on local control. Jpn J Clin Oncol 1997, 27:240-243.

3. Gondhowiardjo S, Prajogi G, Sekarutami S: History and growth of radiation oncology in Indonesia. Biomed Imaging Interv J 2008, 4:e42.

4. Slotman BJ, Cottier B, Bentzen SM, Heeren G, Lievens Y, van den Bogaert W: Overview of national guidelines for infrastructure and staffing of radiotherapy. ESTRO-QUARTS: work package 1. Radiother Oncol 2005, 75:349-354.

5. Bentzen SM, Heeren G, Cottier B, Slotman B, Glimelius B, Lievens Y, van den Bogaert W: Towards evidence-based guidelines for radiotherapy infrastructure and staffing needs in Europe: the ESTRO QUARTS project. Radiother Oncol 2005, 75:355-365.

6. Hutajulu SH: Clinical, Virological and Host Epigenetic Markers for Early Identification of Nasopharyngeal Carcinoma in High Risk Populations in Indonesia. PhD Thesis. 2012.

Chapter 10

Nederlandse Samenvatting

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In Indonesië zijn de behandelresultaten van patiënten met een nasofarynxcarcinoom (NPC) niet optimaal. Dit wordt voor een groot deel veroorzaakt door de late presentatie van patiënten bij de gezondheidsinstanties, het beperkte aantal radiotherapie voorzieningen, met tevens beperkte kwaliteit, en gebrekkige behandelprotocollen. Daarnaast zijn zowel arts als patiënt matig trouw aan gemaakte behandeling -en nazorgafspraken.

In het eerste deel van dit proefschrift worden de knelpunten in de NPC-zorg in Indonesië vastgesteld (hoofdstuk 3). Vervolgens worden mogelijke oplossingen beschreven, zoals de implementatie van een data management systeem (hoofdstuk 2), opleiding van huisartsen voor een betere bewustwording en kennis van NPC met het doel eerdere herkenning van de ziekte (hoofdstuk 4 en 5) en de introductie van nieuwe behandelingsmethoden voor NPC (hoofdstukken 6, 7 en 8).

Data management (hoofdstuk 2)

In Indonesië vormen de medische dossiers momenteel de enige vorm van data registratie. Deze zijn echter vaak onvolledig ingevuld en worden verspreid over verschillende afdelingen opgeslagen. Hierdoor is het moeilijk om een goed inzicht te krijgen in de gegeven behandeling, in de resultaten van de behandeling en in het recidief na de behandeling. Om hier verbetering in te brengen zijn we gestart met de invoering en implementatie van een online data management systeem. Dit systeem is essentieel voor het verzamelen van gegevens voor behandelanalyses. Dit nieuwe online data management systeem is getest op kwaliteit en geschiktheid voor opslag van data. In hoofdstuk 2 laten we zien dat dit online data management systeem voor klinische gegevens goed geïmplementeerd kan worden in een ontwikkelingsland en dat de kwaliteit van de gegevens voldoende is. Het digitale karakter van het systeem, evenals de online beschikbaarheid van de data, geven eenvoudig inzicht in de huidige protocollen en de therapietrouw aan deze protocollen. We verwachten hierdoor minder uitval tijdens de behandeling en een betere naleving van de protocollen in de toekomst. Voor betere en efficiëntere medische hulp aan ontwikkelingslanden zijn wij van mening dat een dergelijk online datasysteem van essentieel belang is.

Naast verbeterde kwaliteit van de klinische gegevens en een betere naleving van protocollen, kan hetzelfde systeem als instrument dienen om een op maat gemaakt trainingsprogramma aan te bieden wanneer westerse instanties hulp willen bieden aan ontwikkelingslanden. Op deze manier kan, voordat een delegatie van hulpverleners naar een ontwikkelingsland reist, een analyse gemaakt worden van de huidige behandelingen en behandeluitkomsten. Een bezoek van hulpverleners zal daardoor efficiënter verlopen. Het online data systeem uit deze studie resulteerde in de eerste prospectieve gegevensverzameling van de behandelingsresultaten voor NPC in Indonesië (hoofdstuk 3).

Behandelresultaten van het nasofarynxcarcinoom in Indonesië (hoofdstuk 3)

Met behulp van het online data systeem hebben we in het Dr. Sardjito ziekenhuis in Yogyakarta een prospectieve analyse kunnen opzetten van 78 patiënten die behandeld werden voor NPC met een curatieve intentie. Uit de resultaten bleek dat de huidige behandeling van NPC zeer matige uitkomsten opleverde in vergelijking tot de behandelresultaten in de literatuur met vergelijkbare behandelprotocollen. Het behandelprotocol dat internationaal wordt toegepast voor primair NPC bestaat uit radiotherapie. Voor NPC in een vergevorderd stadium is dat radiotherapie in combinatie met chemotherapie. Uit andere onderzoeken


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is gebleken dat deze behandeling een 3-jaars ziektevrije en een totale overleving geeft van respectievelijk 70 procent en 80 procent. Daarentegen vonden wij in onze studie in Yogyakarta maar initiële complete remissie in slechts 23 (29 procent) van de 78 patiënten.

Eén van de oorzaken voor deze slechte behandelresultaten is het late stadium waarin patiënten zich presenteren in het ziekenhuis. Zesenzeventig van de 78 patiënten hadden stadium IIB, III of IV bij presentatie. Daarnaast bleek er sprake van een gebrek aan voldoende radiotherapievoorzieningen, resulterend in een langdurige wachttijd. In ons onderzoek was de gemiddelde wachttijd voor radiotherapie 120 dagen. Om tumorprogressie gedurende deze wachttijd te voorkomen worden patiënten behandeld met chemotherapie. Helaas is deze behandeling niet zonder bijwerkingen. Voordat de respons op de behandeling überhaupt beoordeeld kon worden waren er in onze studie reeds 23 patiënten (29 procent) overleden door ziekteprogressie of door de bijwerkingen van chemotherapie.

Naast de lange wachttijd door het tekort aan radiotherapie faciliteiten is ook de behandelduur in Indonesië langer. Een verlengde behandelduur vermindert de effectiviteit van de bestraling en is daarmee een tweede oorzaak van de slechte behandelresultaten. De totale bestralingsduur voor de standaard dosis van 66Gy bleek in Yogyakarta gemiddeld 62 dagen te zijn (in plaats van de gebruikelijke 45 dagen). Deze verlengde bestralingsduur wordt meest waarschijnlijk veroorzaakt door een combinatie van patiënt, arts en ziekenhuis gerelateerde problemen (1, 2). In een lopend onderzoek worden de oorzaken van deze problemen onderzocht.

De eenvoudigste manier om de problemen in Indonesië aan te pakken zou het uitbreiden van de huidige radiotherapie voorzieningen zijn; echter, dit zal minstens tien jaar in beslag nemen. In de tussentijd moeten wij ervoor zorgen dat alleen geschikte kandidaten worden behandeld en dat de patiënten die worden behandeld een effectieve behandeling krijgen. Haalbare oplossingen op de korte termijn als vroege opsporing van NPC, ontwikkeling van nieuwe behandelingen om de wachttijd te overbruggen, goede begeleiding van patiënten tijdens en na de behandeling en beter onderhoud van de radiotherapie voorzieningen die nu in gebruik zijn, zullen leiden tot een evidente verbetering van de behandelresultaten.

Kennis van huisartsen over het nasofarynxcarcinoom (hoofdstuk 4 en 5)

Zoals hierboven beschreven zal eerdere presentatie in het ziekenhuis van NPC-patiënten leiden tot betere behandelingsresultaten en overleving. Kennis van huisartsen over NPC is van cruciaal belang voor het tijdig verwijzen van patiënten naar het ziekenhuis. In hoofdstuk 4 wordt beschreven hoe de kennis van huisartsen rond Yogyakarta is getest. Deze studie toonde aan dat huisartsen die werkzaam zijn in de eerstelijns gezondheidscentra in Yogyakarta een gebrek aan kennis hebben in alle aspecten van NPC. Deze artsen zijn de belangrijkste leveranciers van primaire medische zorg in Indonesië. Om ervoor te zorgen dat patiënten eerder worden gepresenteerd in het ziekenhuis hebben we een trainingsprogramma geschreven voor deze artsen. In hoofdstuk 5 bevestigen we de resultaten gepresenteerd in hoofdstuk 4; huisartsen werkend in de omgeving van Jakarta hadden, evenals in Yogyakarta, een gebrekkige kennis van de incidentie, de risicofactoren en de symptomen van het NPC. Tevens was nauwelijks bekend hoe patiënten doorverwezen worden bij verdenking op NPC. In dit hoofdstuk evalueren we het effect van training aan huisartsen en vergelijken we twee verschillende trainingsvormen: een gecentraliseerd symposium en een lezing in de lokale gezondheidscentra. Beide onderwijsmethodes bleken even effectief te zijn. Tevens bleek training de kennis te vergroten op het gebied van symptomen en in het bijzonder van symptomen die voorkomen bij een vroeg stadium van de ziekte.

Momenteel vindt het NPC onderwijsprogramma plaats in Jakarta, Yogyakarta, Surabaya en Makassar. Hopelijk kan dit programma in de toekomst worden uitgebreid naar de nog resterende gebiedsdelen van Indonesië.

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In een later stadium, wanneer huisartsen voldoende getraind zijn, willen we ook de bewustwording onder de bevolking vergroten door voorlichting te geven over klachten, waarvoor zij zich moeten melden bij de huisarts. Voordat we dit kunnen realiseren, is het essentieel dat eerst alle huisartsen voldoende getraind zijn in het tijdig verwijzen en in de verwijzing naar de juiste instantie. Voor het informeren van de bevolking, zijn vergelijkbare campagnes over borst- en baarmoederhalskanker, erg effectief gebleken. Onderwijs aan de huisartsen en de samenleving zal idealiter leiden tot vroegere detectie van NPC, betere behandelingsresultaten en verbeterde prognose.

Ontwikkeling van nieuwe behandelingsmethoden voor NPC

In hoofdstuk 3 hebben we aangetoond dat NPC een slechte prognose heeft in Indonesië. Het merendeel van de patiënten heeft na de behandeling voor primair NPC een tumorresidu, tumorprogressie, gemetastaseerde ziekte of krijgt later een recidief. Voor deze patiënten is geen genezende behandeling meer beschikbaar, zodat er met de huidige problematiek een duidelijke behoefte bestaat aan nieuwe behandelmethoden. Fotodynamische therapie (PDT) en lytische inductie chemotherapie zouden mogelijk beiden een effectief alternatief kunnen bieden.

Fotodynamische therapie Review: Fotodynamische therapie bij de behandeling van terugkerend / aanhoudend nasofarynxcarcinoom (Hoofdstuk 6)

Lokale residuen en recidieven na de primaire behandeling van NPC zijn een groot probleem in Indonesië. Zoals beschreven in hoofdstuk 3 hebben 12 van de 48 patiënten, die een behandeling hebben afgerond resterende tumor in de nasofarynx. De behandelingen hiervoor zijn beperkt. Her-bestraling is geen realistische optie, niet alleen omdat het moeilijk te implementeren is in een land met schaarste in radiotherapie faciliteiten, maar ook vanwege het hoge risico op ernstige bijwerkingen. In geval van persisterende ziekte is deze modaliteit gecontra-indiceerd, omdat er kort geleden nog in hetzelfde gebied bestraald is. Door de moeilijke anatomische verhoudingen in nasofarynx zijn ook chirurgische procedures voor het verwijderen van deze tumoren erg ingewikkeld en vereist deze veel expertise, welke niet aanwezig is in Indonesië.

PDT is een relatief eenvoudige behandelmodaliteit, zonder ernstige bijwerkingen zoals die worden waargenomen bij her-bestraling. Bovendien heeft het een zeer effectieve lokale werking bij recidiverend of persisterend NPC. In deze review zijn alle gepubliceerde resultaten van PDT in de behandeling van NPC samengevat. Ondanks de beschreven veelbelovende resultaten, zelfs met de eerste generatie fotosensitizers en zonder onze speciaal ontworpen nasofarynx applicator, zijn er tot op heden geen nieuwe klinische studies verschenen.

Temoporfin gemedieerde fotodynamische therapie bij patiënten met lokale persisterende en terugkerende nasofarynxcarcinoom: een feasability studie (hoofdstuk 7)

In een feasability studie met 21 patiënten met persisterend en 1 patiënt met recidiverend NPC, hebben we de veiligheid en werkzaamheid van Temoporfin gemedieerde PDT onderzocht. Patiënten werden onder plaatselijke verdoving behandeld met behulp van een speciaal ontworpen nasofarynx applicator. Er werden drie verschillende medicijn doseringen en licht intervallen toegediend. Eén patiënt overleed twee dagen na de behandeling als gevolg van een voor de behandeling gemiste longontsteking. De behandelresultaten met


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PDT zijn veelbelovend, in 17 van de 22 patiënten werd 40 weken na de behandeling in een biopsie uit het tumorgebied geen enkele patiënt werd vitaal tumor weefsel aangetroffen.

Momenteel loopt een fase II studie, waarin hopelijk de effectiviteit van de PDT-behandeling voor patiënten met lokale persisterende of terugkerende NPC wordt bevestigd.

Door gebruik te maken van de speciaal vormgegeven applicator is PDT een betrekkelijk eenvoudige techniek die kan worden toegepast om persisterende of terugkerende NPC, beperkt tot de nasofarynx, te behandelen. Daarnaast is het door de korte wachttijd en proceduretijd een uitermate geschikte behandeling voor de situatie in Indonesië.

In de toekomst kunnen we PDT mogelijk ook inzetten voor de behandeling van primair NPC in Indonesië, ervan uitgaande dat PDT effectief kan worden toegepast tijdens de wachttijd voor radiotherapie. Naast de reeds genoemde voordelen van PDT, zoals korte wachttijd en kortdurende eenvoudige procedure heeft PDT als voordeel dat andere behandelmogelijkheden niet verloren gaan. PDT tijdens de wachttijd is waarschijnlijk effectief in het voorkomen van tumorprogressie en daarnaast blijft de mogelijkheid aanwezig om tijdens de radiotherapie hoge dosis chemotherapie te verstrekken. Ons inziens zou dit tot een verbetering kunnen leiden in het voltooien van de behandeling binnen de beschikbare tijd en dus tot een effectievere behandeling kunnen leiden onder de huidige omstandigheden.

Cytolytische Virus Activaton Therapie (hoofdstuk 8)

Er bestaat een causaal verband tussen NPC en het Epstein-Barr virus (EBV). In EBV positieve NPC is in alle tumorcellen EBV aanwezig. Dit virus is een potentieel doelwit voor therapie. In NPC tumorcellen verbergt EBV zich in een latente toestand en komen er alleen enkele niet-immunogene virale eiwitten tot expressie die essentieel zijn voor EBV onderhoud en tumorgroei. Met de cytolytische virus activatie (CLVA) therapie hebben we een behandeling ontwikkeld waarbij het EBV gebruikt wordt als doelwit voor de behandeling van NPC. Door reactivering van het latente EBV door epigenetische modulatie, wordt het immuunsysteem geactiveerd en wordt NPC gevoelig gemaakt voor antivirale therapie. CLVA therapie combineert Gemcitabine (GCb) en Valproïnezuur (VPA) en (val) Ganciclovir (GCV). GCb en VPA voor virus activering en GCV) als antiviraal geneesmiddel om virusreplicatie te blokkeren en om de virus geïnfecteerde cellen te doden. De CLVA behandeling werd geoptimaliseerd en gevalideerd in NPC cellijnen en vervolgens getest in drie Nederlandse patiënten met NPC, die niet voldoende op conventionele behandeling hebben gereageerd. Deze patiënten lieten in de circulatie verhoogde concentraties zien van viraal DNA afkomstig van apoptotische cellen. Daarnaast hadden al deze patiënten gestabiliseerde ziekte en verbeterde de kwaliteit van leven. Uit de resultaten van de eerste patiënten behandeld met CLVA blijkt dat de therapie een biologisch effect had en goed werd verdragen.

Deze nieuwe virus specifieke therapie kan leiden tot een vernieuwde aanpak voor de behandeling van andere EBV-geassocieerde maligniteiten. Het effect van de CLVA therapie op het gedrag van de tumor wordt hopelijk verder bewezen in een fase I / II studie, die in 2010 is gestart (Eudract nummer: 2010-022444-20). Daarnaast kan deze behandeling mogelijk bij NPC ingezet worden in de wachttijd voor radiotherapie.

Toekomstperspectieven en nieuwe projecten

Radiotherapie in combinatie met chemotherapie is op dit moment de beste behandeling voor patiënten met NPC in een vergevorderd stadium van de ziekte. Chemotherapie kan worden toegediend in de meeste ziekenhuizen in Indonesië. Radiotherapie daarentegen is voor de meeste patiënten op korte termijn niet

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realistisch vanwege het ernstige tekort aan radiotherapeutische voorzieningen in het hele land. In 2008 waren er 18 lineaire versnellers en 17 Cobalt-60 teletherapie machines beschikbaar voor 229 miljoen inwoners in Indonesië. Zes machines daarvan werkten niet op dat moment. Dit resulteert in 0,13 versneller per miljoen inwoners(3). Vergeleken met Europa is dit erg weinig: in rijke Europese landen zijn er 5,5 versnellers per miljoen inwoners beschikbaar (4). Het aanbevolen aantal versnellers per miljoen inwoners verschilt sterk, in Europa is de aanbeveling 5,9 versnellers per miljoen inwoners (5).

Het enorme tekort aan faciliteiten in Indonesië resulteert in extreem lange wachtlijsten, inadequate behandelprocedures en preferentiële behandelingen. Als gevolg hiervan gaan veel rijke patiënten voor hun behandeling naar ziekenhuizen in het buitenland. Dit resulteert in een uitstroom van substantiële gezondheidszorg gelden naar aangrenzende landen als Maleisië, Singapore en Australië. De minder bedeelde patiënten kunnen geen geschikte behandeling krijgen. Nu de Indonesische regering een vergoeding voor de armen ter beschikking stelt, is de druk op de wachtlijsten nog meer toegenomen. De wachttijd van zes maanden voorafgaand aan de behandeling is in veel situaties standaard geworden. Aangezien radiotherapie nog steeds de belangrijkste behandeling is voor maligniteiten, betekent dit dat vele oncologische patiënten, waaronder NPC-patiënten, niet effectief behandeld kunnen worden.

De eenvoudigste manier om het huidige probleem op te lossen is voldoende radiotherapeutische capaciteit in Indonesië te creëren in combinatie met goed opgeleid personeel. De groeiende economie in Indonesië zal hopelijk uiteindelijk resulteren in voldoende faciliteiten, maar in de tussentijd zullen kleinschalige oplossingen de zorg voor patiënten met NPC moeten verbeteren. Eén initiatief is de bouw van een oncologisch-opleidingscentrum in Yogyakarta dat door een gespecialiseerd team van internationale artsen zal worden ondersteund. Met invoering van gezondheidszorg op een internationaal niveau, ondersteund door reeds toegezegde overzeese hulp, kunnen Indonesische artsen worden opgeleid volgens internationale normen. Rijke patiënten hoeven dan niet langer naar het buitenland te gaan voor een adequate behandeling. De opbrengst van dit initiatief kan worden geïnvesteerd in medische systemen en de behandeling voor armere patiënten (het zogenaamde Robin Hood-principe). De Minister van Volksgezondheid, de Medische Faculteit en het Universitair Ziekenhuis in Yogyakarta steunen dit initiatief. Ook hebben verschillende buitenlandse investeerders belangstelling getoond voor een dergelijk plan. Dit zou het begin kunnen zijn van een internationale standaard in de oncologische zorg in Indonesië.

Naast het realiseren van een internationaal gesteund oncologische-opleidingscentrum, zullen nieuwe klinische onderzoeksprojecten moeten worden opgestart. De Nederlandse Kankerbestrijding heeft subsidie verleend voor twee nieuwe onderzoeksprojecten. De eerste beurs is voor een fase II studie, de cytolytische virus activatie therapie voor NPC patiënten met metastasen op afstand. De effectiviteit van lytische inductie therapie zal hopelijk worden bewezen in deze fase II studie. Indien effectief, zal toekomstig onderzoek nodig zijn om andere lytische inducerende stoffen te onderzoeken, die mogelijk nog minder bijwerkingen hebben en mogelijk alleen oraal kunnen worden toegediend. De rol van de lytische inductie om de wachttijd te overbruggen kan een veelbelovende onderzoekslijn zijn, omdat we minder bijwerkingen in vergelijking met conventionele chemotherapie behandeling verwachten.

De tweede toegekende subsidie zal onderzoeken hoe het aantal patiënten met een vergevorderd stadium van de ziekte bij binnenkomst in het ziekenhuis is terug te dringen. Eén van de doelstellingen van deze studie is het evalueren van het effect van de opleidingsprogramma's voor de huisartsen die werkzaam zijn in de gezondheidscentra in Indonesië. Naast de verwachte eerdere presentatie van NPC patiënten bij de KNO-specialisten, zal de incidentie en het aantal onnodig verwezen patiënten worden geëvalueerd. Studiespecifieke vragenlijsten worden ontwikkeld voor de beoordeling van de oorzaak voor de vertraging


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van patiënten om de huisarts te consulteren. Dit zal de basis vormen voor toekomstige campagnes om de bevolking bewuster te maken van de incidentie en klachten van NPC.

De studie zal zich verder richten op de begeleiding en monitoring van NPC patiënten. Dit project zal starten met een uitgebreide monitoring van de patiënten tijdens de wachtperiode en de radiotherapie behandeling. Het doel is om meer inzicht te krijgen waarom patiënten niet op tijd terug komen naar het ziekenhuis of helemaal niet meer terugkomen. Na een uiteindelijke start van de radiotherapie, willen we bewerkstelligen dat patiënten elke dag terug komen om een effectieve behandeling te realiseren. In het geval een patiënt niet komt opdagen, wordt de patiënt opgespoord en wordt de reden van wegblijven uitgezocht.

Om onnodige behandelingen in de toekomst te voorkomen, zal dit project tevens evalueren of het huidige diagnostische systeem moet worden aangepast. Het meest waarschijnlijk zullen patiënten opnieuw moeten worden gescreend op het bestaan van metastasen op afstand na de lange wachttijd voor de radiotherapie. Patiënten, die metastasen op afstand hebben ontwikkeld tijdens de wachttijd, zullen niet behandeld worden met curatieve radiotherapie. Door alleen patiënten te behandelen die nog een kans hebben op genezing zal de totale wachttijd verkort kunnen worden en kan onnodig gebruik van de toch al beperkte middelen worden voorkomen. De laatste fase van dit project combineert alle resultaten van de bovengenoemde projecten en zal hopelijk leiden tot een nieuw verwijzingsprotocol, een verbeterd begeleidingssysteem en een verbeterd diagnostisch systeem.

Een ander belangrijk probleem dat aangepakt moet worden is de bewustwording van artsen van hun behandelingsresultaten en complicaties. De huidige onwetendheid leidt ertoe dat men zich niet realiseert welkt effect hun behandeling teweeg brengt. Het ontbreken van enige feed back staat verbetering van de behandeling in de weg. Hierdoor ontbreekt een leercurve. Dit probleem kan hopelijk worden aangepakt met nieuwe gelden die het mogelijk maken om een online datamanagement systeem te implementeren in alle oncologische centra in Indonesië.

Tot slot, zijn we van plan om extra fondsen te werven om de rol van PDT te onderzoeken in de primaire behandeling van NPC. In een fase I-II-studie willen we evalueren of deze behandeling modaliteit voor NPC patiënten de onvermijdelijke wachttijd voor standaard (chemo)-radiotherapie kan overbruggen in ontwikkelingslanden, zoals Indonesië.

Reference List 1. Levendag PC, Lagerwaard FJ, Noever I, dePan C, vanNimwegen A, Wijers O, Schmitz PI, van Dieren E, Nowak PJ: Role

of endocavitary brachytherapy with or without chemotherapy in cancer of the nasopharynx. Int J Radiat Oncol Biol Phys 2002, 52:755-768.

2. Akimoto T, Mitsuhashi N, Hayakawa K, Sakurai H, Murata O, Ishizeki K, Ishikawa H, Nasu S, Yamakawa M, Niibe H: Split-course accelerated hyperfractionation radiotherapy for advanced head and neck cancer: influence of split time and overall treatment time on local control. Jpn J Clin Oncol 1997, 27:240-243.

3. Gondhowiardjo S, Prajogi G, Sekarutami S: History and growth of radiation oncology in Indonesia. Biomed Imaging Interv J 2008, 4:e42.

4. Slotman BJ, Cottier B, Bentzen SM, Heeren G, Lievens Y, van den Bogaert W: Overview of national guidelines for infrastructure and staffing of radiotherapy. ESTRO-QUARTS: work package 1. Radiother Oncol 2005, 75:349-354.

5. Bentzen SM, Heeren G, Cottier B, Slotman B, Glimelius B, Lievens Y, van den Bogaert W: Towards evidence-based guidelines for radiotherapy infrastructure and staffing needs in Europe: the ESTRO QUARTS project. Radiother Oncol 2005, 75:355-365.

List of publications

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Radiotherapy in laryngeal carcinoma: can a panel of 13 markers predict response?Wildeman MA, Gibcus JH, Hauptmann M, Begg AC, van Velthuysen ML, Hoebers FJ, Mastik MF, Schuuring E, van der Wal JE, van den Brekel MW.Laryngoscope. 2009 Feb;119(2):316-22.

Molecular markers predict outcome in squamous cell carcinoma of the head and neck after concomitant cisplatin-based chemoradiation.van den Broek GB, Wildeman MA, Rasch CR, Armstrong N, Schuuring E, Begg AC, Looijenga LH, Scheper R, van der Wal JE, Menkema L, van Diest PJ, Balm AJ, van Velthuysen ML, van den Brekel MW.Int J Cancer. 2009 Jun 1;124(11):2643-50.

Photodynamic therapy in the therapy for recurrent/persistent nasopharyngeal cancer.Wildeman MA, Nyst HJ, Karakullukcu B, Tan BI.Head Neck Oncol. 2009 Dec 17;1:40. Review.

Knowledge of general practitioners about nasopharyngeal cancer at the Puskesmas in Yogyakarta, Indonesia.Fles R, Wildeman MA, Sulistiono B, Haryana SM, Tan IB.BMC Med Educ. 2010 Nov 18;10:81.

Photodynamic therapy of early stage oral cavity and oropharynx neoplasms: an outcome analysis of 170 patients.Karakullukcu B, van Oudenaarde K, Copper MP, Klop WM, van Veen R, Wildeman M, Bing Tan I.Eur Arch Otorhinolaryngol. 2011 Feb;268(2):281-8. Epub 2010 Aug 13.

Can an online clinical data management service help in improving data collection and data quality in a developing country setting?Wildeman MA, Zandbergen J, Vincent A, Herdini C, Middeldorp JM, Fles R, Dalesio O, van der Donk E, Tan IB.Trials. 2011 Aug 8;12:190.

Short-term effect of different teaching methods on nasopharyngeal carcinoma for general practitioners in Jakarta, Indonesia.Wildeman MA, Fles R, Adham M, Mayangsari ID, Luirink I, Sandberg M, Vincent AD, Fardizza F, Musa Z, Armiyanto, Middeldorp JM, Gerritsen G, Suwanto R, Tan IB.PLoS One. 2012;7(3):e32756. [Epub 2012 Mar 14]

Cytolytic virus activation therapy for epstein-barr virus-driven tumors.Wildeman MA, Novalic Z, Verkuijlen SA, Juwana H, Huitema AD, Tan IB, Middeldorp JM, de Boer JP, Greijer AE.Clin Cancer Res. 2012 Sep 15;18(18):5061-70. [Epub 2012 Jul 3]

A matched cohort comparison of mTHPC-mediated photodynamic therapy and trans-oral surgery of early stage oral cavity squamous cell cancer.Karakullukcu B, Stoker SD, Wildeman AP, Copper MP, Wildeman MA, Tan IB. Eur Arch Otorhinolaryngol. 2012 Jul 7. [Epub ahead of print]


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Temoporfin mediated photodynamic therapy in patients with local persistent and recurrent nasopharyngeal carcinoma after curative radiotherapy: A feasibility study.Nyst HJ, Wildeman MA, Indrasari SR, Karakullukcu B, van Veen RL, Adham M, Stewart FA, Levendag PC, Sterenborg HJ, Tan IB. Photodiagnosis Photodyn Ther. 2012 Sep;9(3):274-81. [Epub 2012 Aug 23]

Lower mortality from nasopharyngeal cancer in The Netherlands since 1970 with differential incidence trends in histopathology.Arnold M, Wildeman MA, Visser O, Karim-Kos HE, Middeldorp JM, Fles R, Bing Tan I, Coebergh JW. Oral Oncol. 2012 Oct 19. [Epub ahead of print]

Local control in advanced cancer of the nasopharynx: Is a boost dose by endocavitary brachytherapy of prognostic significance?Levendag PC, Keskin-Cambay F, de Pan C, Idzes M, Wildeman MA, Noever I, Kolkman-Deurloo IK, Al-Mamgani A, El-Gantiry M, Rosenblatt E, Teguh DN. Brachytherapy. 2012 Nov 3. pii: S1538-4721(12)00211-5.

Remarkable response after photodynamic therapy in residual T4N0M0 nasopharyngeal carcinoma: a case report.Indrasari SR, Timmermans AJ, Wildeman MA, Karakullukcu MB, Herdini C, Hariwiyanto B, I.Bing Tan.Photodiagnosis Photodyn Ther. 2012 accepted.

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Curriculum Vitae

Melchert Adrianus Martinus (Maarten) Wildeman werd geboren op 23 januari 1982 te Dordrecht. In juni 2000 behaalde hij zijn Gymnasium diploma aan het Johan de Witt Gymnasium te Dordrecht. Vanaf augustus 2000 studeerde hij geneeskunde aan de Universiteit van Amsterdam. Vanaf 2004 begon hij met onderzoek op de afdeling Hoofd- Hals Oncologie en Chirurgie in het Nederlands Kanker Instituut – Antoni van Leeuwenhoek Ziekenhuis onder leiding van prof. dr. M.W.M. van den Brekel. Zijn artsexamen behaalde hij in december 2007. Vanaf februari 2008 werkte hij gedurende een periode van 7 maanden als ANIOS op de afdeling Heelkundige Oncologische Disciplines in het Nederlands Kanker Instituut – Antoni van Leeuwenhoek Ziekenhuis te Amsterdam. Aansluitend begon hij in september 2009 als arts-onderzoeker aan zijn promotieonderzoek naar nasofarynxcarcinoom onder leiding van prof. dr. I. B. Tan en prof. dr. A.J.M. Balm wat heeft geleid tot deze promotie. Per 1 januari 2013 is hij in opleiding tot Keel Neus en Oorarts in het AMC (opleider prof. dr. S. van der Baan).

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Dankwoord

Het leukste om te schrijven en waarschijnlijk het best gelezen deel van dit proefschrift. Het is gelukt! Aan iedereen die me heeft geholpen en gesteund: veel dank!

Mijn promotor Professor I.B. Tan

Beste Bing, heel veel dank voor al je hulp, geduld en alle wijsheden in de afgelopen jaren. Ik heb heel veel bewondering voor hoe jij in het leven staat en hoop dat ik ooit zo’n geduldige, zorgzame en goede dokter zal worden. Ik heb veel van je geleerd, naast alle wetenschappelijke kennis mogelijk nog belangrijker veel levenswijsheden die mij goed van pas zullen komen. Ik ben heel blij en trots op ons resultaat.

Mijn promotor Professor A.J.M. Balm

Beste Fons, met veel bewondering kijk ik terug op uw betrokkenheid. Uw ideeën bij de voortgangsgesprekken op het moment dat het niet zo goed ging hebben mij fantastisch geholpen. Bovendien hebben uw adviezen en kritische blik aan het eind ervoor gezorgd dat mijn proefschrift nog aanzienlijk is verbeterd. Uw wijze woorden om geduld te hebben ervoor gezorgd dat ik nog trotser kan zijn op het eindresultaat.

Mijn copromotor dr. J.P. de Boer

Beste Jan Paul, zo blij dat jij mijn copromotor bent geworden na al je inspanning voor ons project. Je onbaatzuchtigheid, geduld en humor zal ik niet vergeten. Ik zal onze maandagochtend besprekingen missen.

Mijn copromotor Professor J.M. Middeldorp

Beste Jaap, heel veel dank voor al je fundamentele uitleg over het Epstein-Barr virus. Ik heb me heel erg welkom gevoeld in jouw lab waar ik veel heb geleerd. Ik heb enorme bewondering voor jouw gedrevenheid in onderzoek.

My copromotor Dr. Bambang Herwiyanto

Dear Tonki, thank you so much for all your help in Indonesia. Your team and department, which according to me is the best department of Sardjito, took care of everything during my absence. Also thank you for being here today and attending my defence.

De leden van mijn promotie commissie, Prof. Dr. S. Van der Baan, Prof dr. F.J.M. Hilgers, Prof. Dr. C. Rasch, Prof. Dr. D.L. Willems en Prof. Dr. B. De Gast wil ik graag bedanken voor de tijd die u allen heeft vrijgemaakt voor het beoordelen van mijn manuscript.

Geachte professor Van der Baan, ik kijk er heel erg naar uit om door u opgeleid te worden tot KNO-arts. Geachte professor Hilgers , ik heb enorme bewondering voor uw daadkracht en organisatorisch vermogen. Het was een grote eer om bij u op de afdeling te mogen werken.

Tevens wil ik het gehele team van de hoofd-hals chirurgie bedanken. Beste Michiel, bij jou is het allemaal begonnen: onderzoek doen. Bedankt voor al je inzichten. Beste Martin, aan het begin van mijn carrière gaf je mij advies over mijn houding. Deze adviezen zijn erg belangrijk geweest voor mij en ik denk dat ze ook van pas zullen komen in mijn verdere loopbaan. Beste Baris, mijn Turkse vriend, dank voor de gezellige en

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leerzame samenwerking. Zullen we dit jaar wel het rondje van Texel zeilen? Beste Lot, Ludi en Peter, bedankt voor de fijne samenwerking.

Lieve Renske, we kwamen wat laat op gang, maar wat heb ik veel leuke dingen met je meegemaakt. Heel veel dank voor de plezierige samenwerking. Dank voor het uitgeven van mijn boekje en dat je ook nog eens mijn paranimf geworden bent. Het brainstormen met jou en Dmitry aan de eettafel was vooral gezellig en ontspannen. Ik wens je veel succes met jouw onderzoek de komende vijf jaar. Ik stel graag mijn eettafel beschikbaar voor jullie om te brainstormen over jouw boek.

Lieve Sharon, kan jij je ons eerste bezoek aan Indonesië nog herinneren? Hoe we samen over de Marlioboro liepen? Zonder jou had ik hoofdstuk 3 mogelijk nooit afgeschreven. Ik ben blij dat jij het werk van mij gaat overnemen. Ik heb er veel vertrouwen in dat jij een goede draai gaat geven aan de lopende projecten.

Dear Yogya NPC-team, thank you so much for all your effort and keeping up with me. My typical Dutch blunt behaviour in the beginning didn’t make co-working any easier, but together we accomplished a lot and continued expanding with new projects. Dear Noni, thank you for your time and great discussions. Dear Yuni, keep up the good work! Without your data management skills this thesis would not have been accomplished. Dear Camelia and Sari, please continue treating patients with PDT with your enthusiasm. Dear Susan, Jajah and Dewi, I hope we will start with the lytic induction project soon. Dear professor Rika, thank you for your support. During times I thought we were really out of solutions you came to help and saved the day. Dear professor Maesadji, dr. Johan, dr. Kartika and dr. Retno, thank you so much for all your help. I hope you all will continue supporting our future projects.

Lieve Astrid, dankzij al jouw geduldige uitleg heb ik veel geleerd over NPC en de rol van het Epstein Barr Virus. Ik voelde me altijd erg welkom in jouw lab, mijn dank daarvoor. Goede herinneringen heb ik aan ons gezamenlijk bezoek aan Indonesië in juni. Wat fijn dat we daar samen hoofdstuk 8 voor de laatste keer hebben weggestuurd. Zlata, Octavia, Max, Sandra en Hedi, jullie hebben ervoor gezorgd dat de gekweekte kankercellen voor mij gingen leven, dank!

Beste Annemarie Vesseur, Marlies Maatje, Ilse Luirink, Mara Sandbergen, Jacqueline Timmermans, Charlotte Siegers en Fokko Smit, dank voor al jullie hulp in Indonesië. Ik hoop dat jullie reclame zullen maken voor onze projecten, zodat er meer coassistenten en jonge onderzoekers naar Indonesië kunnen afreizen.

Alle mede auteurs van al mijn artikelen wil ik graag bedanken voor hun bijdrage, betrokkenheid en hulp bij het schrijven van de artikelen. Dear Andrew, thank you so much for all your statistic analysis and improvement of my English writing.

Beste Olga, voor al mijn vragen over de radiotherapie kon ik bij jou terecht. Veel dank daarvoor. Ik vind het erg plezierig om met je samen te werken.

Lieve Marion, de bazen hebben maar geluk met zo’n goede secretaresse. Ben blij dat je mij ook zo goed hebt geholpen.

Het PDT-team, Hannah, Joan, Robbert, Jan, Ferrie, Josselyn en Ellen, wat waren het gezellige lunches elke woensdag. Misschien komt er na mijn vertrek wat meer structuur in.

Beste Robbert (“dr Bob”) en Heike, het was maar moeilijk om jullie rol over te nemen in Yogya. Heike, nog net voor jou de eindsprint gehaald. Hopelijk volg jij snel.

Dankwoord

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Geerten, de avondjes in de Desperado bar waren hilarisch! Dankzij jou heb ik twee, mij hele dierbare vrouwen leren kennen. Zullen we een keer Bali rond racefietsen?

Stichting Hoofdhals Kanker Indonesië, Peter Siegers, dank voor de steun. Ik hoop dat ik in de toekomst nog werk voor jullie mag doen.

Melani, your voluntarily editing helped improve my introduction and discussion significantly. I am so happy to have met people like you who invest so much time without expecting anything in return.

Dear Jakarta team, spending time in Jakarta was always great fun! Professor Bambang, Marlinda, Ibnu, Izzy and Mayang I really hope to see you soon! Marlinda, I know you will defend your PhD soon.

Dear Surabaya team, especially dr Romdhoni and professor Ario, I really hope our PDT project will soon start so I can come to see the progress and see you all again.

Dear Christoph thank you for all your support, I think that with your enthusiasm PDT will become a great success in Indonesia.

Beste collega-onderzoekers, de borrels en feestjes die ik heb meegepakt waren altijd leuk. Van O-gebouw naar hoofdgebouw en weer terug, het was altijd gezellig! Beste Jur en Eva, met jullie op de kamer was top: hard werken en gesprekken van hoge kwaliteit maar beperkte kwantiteit. Met jullie op de kamer zijn de meeste stukken tot stand gekomen! Lisette, dank voor al je steun tijdens mijn promotie en bij de laatste lootjes. Je hebt goed voor me gezorgd. Ik hoop dat we samen blijven rennen. Cindy, ik ben blij dat we weer collega’s worden in het AMC. Lieve Mila, ik zou bijna radiotherapeut worden om je collega te blijven.

Lieve vrienden en familie, door de 21 reizen naar Indonesië heb ik veel belangrijke dingen in jullie leven gemist. Dank voor jullie begrip de afgelopen vier jaar.

Tjeerd, paragraaf hierboven is zeker voor jou van toepassing. Zoals altijd zeg ik dan: “ik zal het goedmaken.”

De Meer 3, het mooiste team van allemaal! Dank voor al jullie begrip tijdens mijn spaarzame aanwezigheid! Willem, heerlijk om door jou over het veld geschreeuwd te worden. Philip, dank voor je steun. Jij bent met afstand mijn meest burgerlijke vriend. Thijs, als ik jou was zou ik stoppen met werpen en weer gaan ballen.

Beste Binken; Bart, Bram, Xander, Moos, Steven, Daan, Gerben en Dolf, ik heb eindelijk tijd om het weekendje te organiseren!

Beste Philip, altijd weer fijn om jou te zien na een reis. Bedankt voor al je adviezen en luisterend oor. Beste Joost, het heeft nu wel lang genoeg geduurd, wanneer kom jij terug? Zullen we dan met zijn drieën Dirk opzoeken?

Laurens, al 26 jaar vrienden (we worden oud). Sorry dat ik je de laatste vier jaar zo weinig heb gezien. Zullen we nog een keer met Pieter en Bart naar Winterberg gaan?

Lieve Thijs en Aukje, liever een goede buur als fijne vriend! Heerlijk dat ik altijd bij jullie kon aankloppen, ik zal nu weer zelf voor mijn plantjes zorgen.

Lieve Corrie, dank dat jij zo een goede vriend en buur bent voor mijn ouders. Lieve Eva, snel een spa rood met een jenevertje in de kroeg op de hoek?

Dolf wat jammer dat je niet naast me kan zitten vandaag, dan maar volgend jaar naar Lowlands.

Dankwoord

141

Berend, paranimf van het eerste uur, hoe hilarisch was het om met jou en Renske dit te organiseren. Ik ga jullie bij iedereen aanraden, in het bijzonder voor het uitzoeken van een feestlocatie!

Dear Lily, my Indonesian mother, thank you so much for all your love and hospitality in the last four years. Because of you I felt at home in Indonesia and I am truly sad I will see you less often in the coming years.

Lieve opa en oma, wat fijn dat jullie mailen! De fijne gesprekken bij jullie op de bank waren niet alleen gezellig maar ook leerzaam. Opa, ik hoop dat ik ooit op eenzelfde manier een toespraak kan geven als u.

Lieve Anne, misschien pest ik je soms een beetje, maar ik ben een hele trotse broer. Jij wordt een geweldige dokter. Lieve Jasper, ben blij dat je zo goed bent voor mijn zusje. Lieve Marieke, jouw zorgzaamheid en liefde is heel bijzonder. Jij hebt het hele gezin, inclusief mij, de afgelopen jaar enorm gesteund. Lieve Berend, maak eens wat tijd voor mijn zusje en houd eens op met feestjes te organiseren. Je bent een fijne vriend! Lieve Pieter, zo trots dat jij mijn broer bent. Jouw intelligentie en humor zijn van ongehoord niveau. Lieve Jocelyne, weet je nog met Wolf in je buik op beddenjacht? En nu dan eindelijk nog een doctor Wildeman. Lieve Wolf, met zulke ouders komt het wel goed met jou. Lieve papa, je bent er altijd voor mij. Heel fijn om zo’n slimme en goede arts als vader te hebben. Ik heb enorme bewondering voor hoe jij voor een ieder in ons gezin zorgt. Wel jammer dat je bij mijn 15de bezoek naar Indonesië, nadat je er zelf geweest was, nog steeds niet wist hoe het ook alweer met die tijdzones zat. Fijn dat je ongerust was over de Merapi uitbarsting, maar om me nou elke nacht wakker te bellen... Lieve mama, bedankt voor al je kleine knuffelbeertjes, briefjes en tekeningen die ik van je kreeg bij alle spannende dingen in mijn leven. Onthoud voor altijd dat ik van je hou.

Lieve Justine, ik ben zo blij dat ik je ontmoet heb en heb zo’n zin in onze toekomst. Ik kan niet wachten om jou Indonesië te laten zien.

Sponsor page

142

Sponsor page

Antoni van Leeuwenhoek Hospital

Biolitec Pharma

Dutch Cancer Society

Michel de Keijzer Foundation

Academic Medical Centre

Specsevers

Atos Medical

Roche

Daleco Pharma

Beter Horen

Entermed

Entercare

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