b^mr h A^fZmheh`r - Saudi Scientific Society of Hematology · agulant drugs include oral direct...

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Editorial Board:Editor-in- Chief

Abdul Kareem Al-Momen

Associate EditorsAbdel Galil M. Abdel Gader

Mohamed QariTarek Owaidah

Editors

Fahad Al-MoharebAbdullah Al-JefriAbdul Rahman Al-MusaFaten Sayes

Zakaria HawsawiAhmed TarawaMohamed AbdulaalHind Al-Humaidan

Anisa Abboud (Yemen)Anwaar Al-Kordofani (Sudan)Abdulla Owaidi (Jordon)Ali Taher (Lebanon)Ahmed Ibrahim (Lebanon)Salem Al- Shemmari (Kuwait)Ahmed Rustomani (UAE)Salam Al-Kindi (Oman)Naima Al-Mulla (Qatar)Abdullah Al-Ajmi ( Bahrain)Ashraf Abu El-Naga (Egypt)Shaker Musa (USA)Ghulam Mufti (UK)

Michele Samama (France)Giuseppe Saglio (Italy)George Rivard (Canada)Michael Copeman (Australia)Orhan Ulutin (Turkey),Aris Hashim (Malaysia)Abdullah Kutlar (USA)Armand Keating (Canada)Thomas Ortel (USA)Assad Haffar (Canada)Alison Street ( Australia)Ayalew Tefferi (USA)

Advisory Board:Local Members:

International Members:

Mirghani Ali M. AhmedGhazi DamanhoriSalem KhalilHazzaa Al-ZahraniSoad Jaoni

Ahmed Al-AskarAbdul Aziz Al-AbdulaalySaud Abu-HarbeshBassem Al-BeirutiBaker Al-Awamy

Ahmed Al-SulaimanMohamad Al-ShahraniFatimah Al-Batniji

Scientist Publishing Group P.O. Box 91409 Riyadh 11633Saudi Arabia . Tel . : 4780312a a l e m f o r p r e s s @ y a h o o . c o m

Scientist Publishing Group

Aims and Scope:

The Journal of Applied Hematology is an international journal that publishes articles covering both clinical and experimental research in hematology. The journal welcomes original as well as review articles, manuscripts, case reports, short communications, images and clinical pictures, letters and correspondence to the Editor; on non-malignant and malignant hematological diseases, hemostasis and thrombosis, immunology, stem cells biology, and transfusion medicine. Clinical studies describing novel therapeutic approaches to the diagnosis and treatment of hematological diseases are welcomed and encouraged as well.

Author Guidelines:

Manuscripts are considered for publication with the understanding that they have not been published or considered for publication elsewhere. The submission letter must be signed by all co-authors. The official language of the journal is English (with Arabic and French summaries).Articles submitted for publication should be typewritten, using only one side of an A4-size paper, in double spacing throughout, with a margin of 3.5 cm all round. Until the electronic submission of articles is established, the submitted manuscript should be accompanied by a copy on a CD or computer disk in Microsoft format.

Manuscript Preparation:

The format of The Journal of Applied Hematology complies with the Uniform Requirement for Manuscripts Submitted to Biomedical Journals (The Vancouver Style) (see www.icmje.org/).

The contents of all submitted articles should be arranged in the following order: title page, abstract and key words, introduction, materials and methods, results, discussion, acknowledgement, references, tables, glossary photos and/or figures, legends to figures and tables. The number of words in the abstract should not exceed 250 words and the key words should not be more than six)

References:

References should be listed, double-spaced, consecutively in the order of their citation in the text. Journal names should be abbreviated according to the Index Medicus. Standard international unit (SI units) abbreviations are to be used instead of full words (for example:cm, mm, m, km, ml, g, kg, %).The author(s) are fully responsible for the accuracy of all statements and data in the submitted manuscript.

Send all correspondence to:

Editor-in-Chief: Journal of Applied [email protected]

The journal is also included in the Directory ofOpen Access Journals(http://www.doaj.org)ISSN: 1658-5127

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Dear Colleagues,

It is our pleasure to introduce to you a new refereed international journal, the Journal of Applied Hematology, which is the official scientific journal of the Saudi Society of Hematology.The Journal of Applied Hematology publishes high-quality scientific papers, in-cluding review articles, editorials, case reports, and letters to the editor, that meet the high standards of the journal and the journal’s peer-review system. Clinical research papers may cover various aspects of the wide spectrum of hematology, including but not limited to, hemoglobinopathies, anemias, bleeding disorders, thrombosis, hematological malignancies, stem cell transplantation, transfusion medicine and hematopathology.Manuscripts submitted to the journal must follow the Uniform Requirements for Manuscripts Submitted to Biomedical Journals (http://www.icmje.org/). Manuscripts can be submitted online or submitted as an email. Members and non-members of the Saudi Society of Hematology, including clinical hematologists, hematopathologists, oncologists, internists, pediatricians, surgeons, pharmacists, nurses and technologists, are encouraged to submit their papers to the Journal.The Journal will be published four times a year (January, April, July and October), but twice a year temporarily. The Journal will be available in print, as well as on-line, to allow easy access for physicians and students to benefit from the scien-tific contents of the journal.

Editorial Board

LETTER FROM THE EDITORIAL BOARD

4

Table of Contents

Review Articles

5 Novel Anticoagulant Therapy from Oral Warfarin to Oral Anti-Xa/Oral Anti-IIa: Principles and PracticeShaker Mousa

19 Chronic Myeloid LeukemiaCarmen Fava, Giovanna Rege Cambrin, Stefano Ulisciani, Daniela Cilloni, Giuseppe Saglio

Original Reports

28 Significance of sCD117 as a Tumor Marker in Acute Myeloid LeukemiaHuda AlSayed, Faisal Rawas, Amal AlSuraihi, Fahad AlShareef, Lila Bashawri, Tarek Owaidah

36 Serum CA-125 correlates with staging, prognosis and survival of Non-Hodgkin Lymphoma (NHL) Tarek Ashour, Mohamad Qari

43 Prevalence of thalassemia disorders and hemoglobinopathies -in Jeddah, western Saudi ArabiaSoad Khalil Al Jaouni

47 Transfusion related non-infectious complication: implication for an appropriate reporting (hemovigilance) system Leila Kasraian, Alireza Tavassoli

52 Human leukocyte antigen (HLA) allele associations in idiopathic recurrent spontaneous abortion patients from Saudi ArabiaMohammed Gad El Rab, Abdulwahab Al-Jabbari, Abdulaziz Al-Mishari, Abdel Galil Gader, Abdulkareem Al-Momen, Zainab Habib

Case Reports

59 Acquired Prothrombin Inhibitor Saud Abu-Harbesh, Hisham Yousef

63 Validation of Automated and Rapid Method for Measurement of ADAMTS13 Activity and AntibodiesMansoor Ahmed, Rasheed Nasr, Randa Alnounou, Tarek Owaidah

66 Pioneers in HematologyAbul Qasim Khalaf ibn al-Abbas al-Zahrawi (Abulcasis)Mohamad Qari

Proven efficacy for the management of bleeding in previously treated and • previously untreated pediatric and adult patients with hemophilia A24.A safety profile with no reported incidence of pathogen transmission • 2,3,7.Convenient and easy to store-up to 6 months room temperature storage • 4.Consistent and uninterrupted worldwide supply • 4.

1. Data on file as of December 2009. Westlake Village, CA: Baxter Healthcare Corporation. 2. White GC 2nd, Courter S, Bray GL, Lee M, Gomperts ED for The Recombinate Previously Treated Patient Study Group. A multicenter study of recombinant factor VIII (Recombinate) in previously treated patients with hemophilia A. Thromb Haemost. 1997;77(4):6603 .667-. Bray GL, Gomperts ED, Courter S, et al. The Recombinate Study Group. A multicenter study of recombinant factor VIII (Recombinate): safety, efficacy, and inhibitor risk in previously untreated patients with hemophilia A. Blood. 1994;83(9):24284 .2435-. RECOMBINATE SPC. 5. Rocino A, Santagostino E, Mancuso ME, Mannuci PM. Immune tolerance induction with recombinant factor VIII in hemophilia A patients with high responding inhibitors. Haematologica. 2006;91(4):5586 .561-. Borel-Derlon A, Lambert T, Rocino A, et al. Retrospective assessment of immune tolerance induction with RECOMBINATE rAHF, a highly purified, full-length recombinant FVIII (rFVIII), in hemophilia A patients with inhibitors. Poster presented at: XXVI International Congress of the World Federation of Hemophilia; October 172004 ,21-; Bangkok, Thailand. 7. Data on file as of January 14, 2010. Westlake Village, CA: Baxter Healthcare Corporation.

Baxter A.G CompanyAhsaa st, Rabwa dist, Dareen BuildingP.O Box 246968 Riyadh 11312Saudi Arabia

RECOMBINATErecombinant coagulation factor VIII

Step into the future of hemophilia therapy

review

Journal of Applied Hematology 2010 5

Novel Anticoagulant Therapy from Oral Warfarin to Oral Anti-Xa/Oral Anti-IIa: Principles and Practice

Shaker Mousa, PhD, MBA, FACC, FACB

The Pharmaceutical Research Institute (PRI), Albany College of Pharmacy and Health Sciences,Rensselaer, NY and Center of Excellence for Thrombosis and Hemostasis, King Saud University, Riyadh, Saudi Arabia

Key Words: Anticoagulants; direct thrombin in-hibitors; factor Xa inhibitors; factor IXa inhibitors; pentasaccharide; tissue factor/factor VIIa complex inhibitors; venous thromboembolism

Corresponding Author:

Dr. Shaker MousaPharmaceutical Research Institute Albany College of Pharmacy and Health Sciences1 Discovery Drive, Rensselaer, NY [email protected] T: 518-694-7397 F: 518-694-7567

Currently, there are several lines of evidence of the interplay between coagula-

tion and inflammation in the propagation of various disease processes, including

venous thromboembolism (VTE) and inflammatory diseases. The vitamin K an-

tagonists (VKAs) such as warfarin have been the standard treatment of throm-

boembolic diseases for over 60 years. However, the level of anticoagulation with

VKAs is frequently outside the therapeutic range, potentially compromising

safety and efficacy because of the food/drug interactions, and other variables.

These limitations have prompted development of new oral anticoagulants that

target Factor IIa or Factor Xa and that are given in fixed doses without routine

anticoagulation monitoring. Major advances in the development of oral antico-

agulants have resulted in considerable progress towards the goal of safe and

effective oral anticoagulation that does not require frequent monitoring or dose

adjustment, and has minimal food/drug interactions. Indirect inhibitors of factor

Xa and factor IIa such as low-molecular-weight heparin (LMWH) and the penta-

saccharide fondaparinux represent improvements to unfractionated heparin/for

acute treatment of VTE, constituting a more targeted anticoagulant approach,

with predictable pharmacokinetic profiles and no requirement for monitoring.

The VKAs, with their inherent limitations in terms of multiple food and drug in-

teractions and frequent need for monitoring, remain the only oral anticoagulant

approved for long-term secondary thromboprophylaxis in VTE. Newer antico-

agulant drugs include oral direct thrombin inhibitors (dabigatran), oral direct fac-

tor Xa inhibitors (rivaroxaban, apixaban, and others), and tissue factor/factor VIIa

complex inhibitors that have been “tailor-made” to target specific pro-coagulant

complexes, and have the potential to greatly expand oral antithrombotic targets

for both acute and long-term treatment of VTE, and for the prevention of stroke

in atrial fibrillation patients.

Inflammation plays a key role in triggering a pro-thrombotic state through the activation of plate-lets, induction of coagulation, and vascular insult.

venous thromboembolism (VTE) continues to be a major cause of morbidity and mortality in the western world.1 VTE represents 1 in 10 hospital deaths, and post-thrombotic syndrome and pulmonary hyperten-sion occur in 10% of deep-vein thrombosis (DVT) and 5% of pulmonary embolism (PE) patients, re-spectively.2 For more than 50 years, traditional drugs such as unfractionated heparin (UFH) were used par-enterally for acute treatment, followed by oral vitamin

K antagonists (VKAs) such as warfarin for long-term treatment (Figure 1). These drugs exert their anti-thrombotic effects by inhibiting multiple steps of the coagulation cascade, but there are inherent limitations for each drug.

For acute VTE treatment, the limitations of UFH include a less than predictable anticoagulant response with the need for frequent monitoring, and the po-tential for severe toxicity, especially heparin-induced thrombocytopenia (HIT) in up to 3% of patients.3 Over the past fifteen years, the use of LMWH, and more recently, the synthetically-derived pentasaccha-

review oral anticoagulants

Journal of Applied Hematology 20106

Figure 1. Advances in anticoagulants in the United States. There have been important advances in injectable anticoagulants in the last decade, but no new class of oral anticoagulant has been introduced since 1954 in the United States. Vitamin K antagonists such as warfarin are the only currently available oral anticoagulants. As outlined in the review, a number of very promising oral anti-Xa and oral anti-IIa (direct thrombin inhibitors or DTIs) are approved in the European and other worldwide markets and awaiting FDA approval in the United States.

ride fondaparinux has improved the acute management of VTE. A more targeted approach to pro-coagulant complex inhibition, predictable pharmacodynamic characteristics, and improved safety profiles have en-abled complete treatment of VTE on an outpatient basis for selected patients without the need for anti-coagulant monitoring. Other parenteral drugs, such as the direct thrombin inhibitors (DTIs) lepirudin and ar-gatroban, have achieved only limited use in acute VTE treatment, namely in thrombosis associated with HIT.

Optimal long-term treatment of VTE is defined by the limitations of VKAs, the only oral anticoagulants currently approved for use. These limitations include a slow onset of action and the need for bridging an-ticoagulation with a parenteral drug in the acute set-ting, multiple food and drug interactions, and a narrow therapeutic window, necessitating frequent coagulation monitoring and dose adjustment.4 Additionally, some patient subgroups cannot tolerate VKA, such as preg-nant patients requiring anticoagulation, in whom VKA is associated with a risk of teratogenicity,5 or patients in whom VKA is associated with higher risks of re-current thromboembolism and major bleeding, such as those with active cancer.6,7 In both of these patient groups, emerging data support the use of long-term lowmolecular weight heparin (LMWH),8-10 with limited parenteral use.

An improved understanding of the molecular

mechanisms of coagulation and thrombosis, and the potential to apply this knowledge at the clinical level to different patient subgroups has led to the develop-ment of newer antithrombotic drugs for use in VTE treatment. Many of these drugs are orally active, syn-thetically derived, and target specific pro-coagulant complexes within the coagulation cascade.11 These drugs can broadly be categorized as interfering with the initiation of coagulation [tissue factor/factor VIIa (TF/FVIIa) complex], propagation of coagulation [indirect and direct inhibitors of activated factor X (FXa) or FIXa], and thrombin activity (DTIs). This review will focus on these investigational drugs for VTE treatment, with an emphasis on those undergo-ing or that have recently completed phase II or III clinical studies (Table 1). The role of anticoagulants on stroke in atrial fibrillation patients will also be dis-cussed. Atrial fibrillation (AF) is an epidemic, affect-ing 11.5% of the population in the developed world. Its significance lies predominantly in that AF patients have a five-fold increased risk of stroke. Stroke as-sociated with AF is usually more severe and confers increased risk of morbidity and mortality, and poor functional outcome. Despite advances in the devel-opment of promising experimental approaches for selected patients with acute stroke, primary preven-tion with pharmacological agents remains the best ap-proach to reducing the burden of stroke.

revieworal anticoagulants


2. Anticoagulant Targets That Inhibit the Initiation Phase

2.1 TF/FVIIa Complex Inhibitors The TF/FVIIa complex, as part of the extrinsic sys-tem of the coagulation cascade, is considered to be the key system for the initiation of coagulation. In the venous vascular system, exposure of TF in orthopedic surgery and in subsets of cancer patients is believed to be responsible for the high risk of VTE in these patient groups,12,13 making pharmacological inhibition of the TF/FVIIa complex important.14 The function of TF can be blocked using several approaches, such as antibodies that prevent the binding of FVIIa to TF, active-site-inhibition of FVIIa, small molecules or an-tibodies that block TF/FVIIa complex function, and molecules that inhibit the active site of FVIIa in the TF/FVIIa complex after binding to FXa.15,16 Moreover, TF pathway inhibitor (TFPI), a naturally occurring in-hibitor, forms a neutralizing complex with TF/FVIIa and FXa.14 Thus TFPI, either by up-regulation of en-dogenous TFPI pools or exogenous administration of recombinant TFPI (rTFPI), represents an attractive anticoagulant that acts by blocking the pathological im-pact of TF/VIIa and Xa in coagulation and beyond.17

Nematode Anticoagulant Proteins:The hematophagous hookworm Ancylostoma caninum produces a family of small, disulfide-linked protein an-ticoagulants (75-84 amino acid residues) referred to as nematode anticoagulant proteins (NAPs), which have been the focus of antithrombotic drug development efforts due to their ability to inhibit the TF/FVIIa com-plex. One of these nematode anticoagulant proteins, NAP5, inhibits the amidolytic activity of FXa with a Ki=43 pM, and is the most potent natural FXa inhibi-tor identified to date. NAP5 does not inhibit FVIIa in the TF/FVIIa complex. Rather, it either binds to FXa alone, or, as is the case for its family member NAPc2, in combination with a protein exosite, resulting in potent inhibition of the TF/FVIIa complex. NAPc2 has been tested subcutaneously for VTE prophylaxis in a phase I/II clinical trial using mandatory unilateral venography in 293 patients undergoing total knee re-placement surgery. At a dose of 3 µg/kg administered within one hour after surgery, NAPc2 was associ-ated with an overall DVT rate of 12.2%, a proximal DVT rate of 1.3%, and a major bleed rate of 2.3%.18 Further clinical trials with NAPc2 are in progress.19,20 Recombinant NAPc2, like other inhibitors of TF/FVIIa, including TFPI and active site-blocked FVIIa (ASIS, FFR-rFVIIa or FVIIai), have a promising role

in the prevention and treatment of venous and arte-rial thrombosis, and could potentially be efficacious in the management of disseminated intravascular co-agulopathies, due to their ability to selectively inhibit TF/FVIIa.19,20

Anti-TF/VIIa:FVIIa is a key serine protease involved in the initiation of the coagulation cascade. It is a glycosylated disul-fide-linked heterodimer comprised of a heavy chain and a light chain. The light chain contains an amino-terminal gamma-carboxyglutamic acid-rich (Gla) do-main and two epidermal growth factor (EGF)-like do-mains, and there is a chymotrypsin-like serine protease domain in the heavy chain.21 TF, a membrane bound protein, is an essential cofactor of FVIIa that is re-quired for maximal activity towards its biological sub-strates (FX, FIX and FFVII). As such, the TF/FVIIa complex plays an important role in normal physiol-ogy as well as in thrombotic diseases such as unstable angina (UA), disseminated intravascular coagulation (DIC), and DVT. In addition to its function as an initia-tor of coagulation, TF/FVIIa plays an important role in inflammation and angiogenesis.22,23 A wide array of strategic approaches to inhibiting the biochemical and biological functions of the TF/FVIIa complex has been pursued. These have been greatly enhanced by elucidation of the structures of TF, FVII, FVIIa, and the TF/FVIIa complex, resulting in inhibitors that are directed specifically towards either FVIIa or TF. Antagonists of the TF/FVIIa complex include active site inhibited FVIIa, TF mutants, anti-TF antibodies, anti-FVII/FVIIa antibodies, naturally occurring pro-tein inhibitors, peptide exosite inhibitors, and protein and small molecule active site inhibitors. These an-tagonists can inhibit catalysis directly at the active site as well as impair function by binding to exosites that interfere with substrate, membrane, or cofactor bind-ing. Several different small molecule potent inhibitors of TF/FVIIa have been shown to reduce thrombus weight in animal models and decrease the level of in-terleukin-6 (IL-6) in a LPS-stimulated mouse model of endotoxemia.22-25 A study designed to evaluate the antithrombotic efficacy and bleeding propensity of a selective, small-molecule inhibitor of TF/VIIa in comparison to small-molecule, selective inhibitors of factor Xa and thrombin in a nonhuman primate model of thrombosis was reported by Suleymanov et al (2003).25 The data indicated that TF/VIIa inhibi-tion effectively prevents arterial thrombosis, with less impact on bleeding parameters than equivalent doses of factor Xa and thrombin inhibitors.25



Figure 1: Advances in Anticoagulants in the USA. There have been important advances in injectable anticoagulants in the last decade, but no new class of oral anticoagulant has been introduced since 1954 in the USA. Vitamin K antagonists, such as warfarin, are the only currently available oral anticoagulants. As outlined in the review a number of very promising oral anti-Xa and oral anti-IIa (Direct thrombin inhibitors, DTI) are approved in the European and other worldwide markets and awaiting FDA approval.

Tissue Factor Pathway Inhibitor (TFPI):The anticoagulant effects of TFPI in animals and the role of TFPI release in secondary anticoagulant mechanisms of LMWH action in humans have been demonstrated.17,26,27 To date, rTFPI has been tested only in experimental models. TFPI is a natural (that is, endogenous) inhibitor of TF coagulant and signaling activities. TFPI exerts anti-angiogenic and anti-meta-static effects in vitro and in vivo.28,29 In animal mod-els of experimental metastasis, circulating and tumor cell-associated TFPI significantly reduce tumor cell-induced coagulation activation and lung metastasis.28 Heparins and heparin derivatives, which induce the release of TFPI from the vascular endothelium, also exhibit antitumor effects, and TFPI likely contributes significantly to these effects.29 Recently, a non-antico-agulant LMWH with intact TFPI-releasing capacity was shown to have significant anti-metastatic effects in an experimental mouse model.30 Evidence of dual in-hibitory functions of TFPI on TF-driven coagulation and signaling strengthen the rationale for considering TFPI a potential anticancer agent.26

TFPI-2, a member of the Kunitz-type serine pro-teinase inhibitor family, is a structural homologue of TFPI. The expression of TFPI-2 in tumors is inversely related to the degree of malignancy, suggesting a role for TFPI-2 in the maintenance of tumor stability and inhibition of neoplastic growth.31 TFPI-2 inhibits the TF/VIIa complex and a wide variety of serine pro-teinases, including plasmin, plasma kallikrein, FXIa, trypsin, and chymotrypsin. Aberrant methylation of TFPI-2 promoter cytosine-phosphorothioate-guanine (CpG) islands in human cancers and cancer cell lines results in decreased expression of TFPI-2 mRNA and decreased synthesis of TFPI-2 protein during cancer progression.31 TFPI-2 has been shown to induce apop-tosis and inhibit angiogenesis, thereby potentially con-tributing significantly to inhibition of tumor growth. Restoration of TFPI-2 expression in tumor tissue in-hibits invasion, tumor growth, and metastasis, lending support to the use of TFPI-2-targeted therapeutics as novel treatments for cancer.31

3. Inhibitors of Coagulation Propagation It is widely accepted that FXa, as part of a prothrom-binase complex with FVa, has a central role in clot formation, given that it is generated by the extrinsic and intrinsic pathways of coagulation as they converge into the final common pathway. In addition, within the prothrombinase complex, one molecule of FXa can exponentially generate 138 molecules of thrombin per minute. Theoretically, FXa inhibitors might have an

advantage over thrombin inhibitors by preventing the activation of coagulation amplification mechanisms as well as thrombin generation in both platelet-rich arterial thrombosis and fibrin-rich venous thrombo-sis, making FXa a prime target for anticoagulant drug design. However, in animal models of thrombosis, selective FXa inhibition was less potent than direct thrombin inhibition in arterial and venous models of thrombosis, and associated with increased global clot-ting times, indicating that inhibition of FXa is less ef-fective than direct thrombin inhibition in controlling thrombin formation.32

The possibility that selective inhibition of FXa upstream could result in a safer bleeding profile can-not be entirely ruled out; in the absence of thrombin inhibition, small amounts of thrombin would escape neutralization and facilitate hemostasis.

Inhibitors of FXa include both indirect (anti-thrombin-mediated) and direct antithrombin (AT)-in-dependent selective inhibitors. Other possible targets of coagulation propagation, through the prothrom-binase complex or other routes, include FIXa inhibi-tors, FVIIIa and FVa inhibitors, activated protein C, or soluble thrombomodulin.

3.1. Indirect FXa Inhibitors The synthetically derived pentasaccharide fondaparinux and idraparinux represent the most advanced selective indirect FXa inhibitors. These agents exert their action through high-affinity binding and activation of AT, which then inhibits free FXa. Fondaparinux contains the pentasaccharide sequence of heparin, and selec-tively binds to and induces a conformational change in AT, increasing the anti-Xa activity of AT nearly 300-fold in a catalytic fashion. Fondaparinux has a linear pharmacokinetic profile and predictable anticoagulant response, with a plasma half-life of approximately 18 hours and >95% bioavailability after intravenous or subcutaneous injection, allowing non-monitored once daily subcutaneous dosing. In addition, it does not bind to platelet factor 4, and has not been associated with drug-induced thrombocytopenia. Fondaparinux is currently approved for acute treatment of DVT and PE based on the recently completed MATISSE studies in VTE.33,34 As such, fondaparinux represents the first of a class of selective indirect FXa inhibitors to pro-vide proof of concept that FXa inhibitors can be used to treat thrombosis in the acute stage as efficaciously as drugs with established AT activity. Fondaparinux is also the first of a new class of antithrombotic drugs designed specifically to inhibit a single target or pro-coagulant complex in the coagulation cascade.



3.1.1. Idraparinux (Sanofi-Aventis)Idraparinux is a hypermethylated, long-acting penta-saccharide that can be administered with once-weekly dosing. Idraparinux sodium is a second generation pentasaccharide with sulfated side chains, which re-sults in a 30-fold higher binding affinity to AT as compared to fondaparinux, and a 120 hour elimina-tion half-life, allowing once-weekly administration.35 It has similar clinical properties as fondaparinux, with 100% bioavailability after parenteral administration, linear pharmacokinetics, a predictable anticoagulant response with no need for monitoring, does not in-duce platelet aggregation, has no effects on platelet factor 4, and no evidence of induction of thrombo-cytopenia. The major drawback, as for fondaparinux, has been the lack of an antidote, although the im-portance of an antidote in clinical practice is contro-versial. However, it should be noted that biotinylated idraparinux, discussed in detail later, does have an antidote.

The PERSIST study, a randomized phase II, dose-ranging study, compared idraparinux with warfarin over a 12-week course of treatment for DVT (after initial studies of enoxaparin with idraparinux demon-strated efficacy at all doses that was similar to warfa-rin). No clear dose-response relationship for efficacy was shown with idraparinux, but a significant dose-re-sponse relationship for major bleeding was shown.36 A large phase III trial comparing the efficacy and safety of idraparinux with heparin or fondaparinux and dose-adjusted warfarin in both acute and long-term treatment of DVT and PE has recently been completed.36,37

3.1.2. SSR126517E (biotinylated idraparinux) This synthetic pentasaccharide being developed by Sanofi-Aventis exerts antithrombotic properties through AT-mediated inhibition of FXa activity. It is identical to idraparinux with the exception of a biotin moiety covalently affixed through a linker to the pen-tasaccharide structure so that anti-FXa activity can be neutralized in vivo by avidin. In vitro studies revealed that SSR126517E binds with high-affinity (Kd=10-9

mol/l) to human AT and inhibits FXa in a concen-tration-dependent manner. It does not inhibit platelet aggregation or cross-react with antibodies from sera of patients with HIT. In phase I studies, the medi-an time to reach maximum concentration was four hours, with an absolute bioavailability of 100% and a half-life of approximately 200 hours. Exposure of SSR126517E to avidin resulted in a rapid decrease of anti-Xa activity and no serious adverse events.37

3.2. Selective Direct Factor Xa Inhibitors (Table 1)Advantages of direct FXa inhibitors include the ab-sence of intermediary molecules such as AT that may potentially contribute to inconsistent anticoagulation, particularly in acute or inflammatory states. DX-9065a was the first of a class of small, synthesized, selective direct FXa inhibitors to undergo phase II clinical trials in arterial thrombosis.38 Efforts to develop orally avail-able selective FXa inhibitors for VTE and prevention of stroke in AF patients are underway (Table 1).

3.2.1. Razaxaban Razaxaban (BMS-561389), developed by Bristol-Myers Squibb (formerly DuPont) is a small molecule oral direct FXa inhibitor that does not require antico-agulant monitoring. In phase I trials involving young and elderly volunteers, it was well tolerated and well absorbed, with only nuisance bleeding reported; FXa inhibition and dose-dependent anticoagulation were noted. Razaxaban was investigated for proof of prin-ciple DVT prevention in patients undergoing total knee replacement. In phase IIb trials, Razaxaban at doses of 25, 50, 75, or 100 mg twice daily adminis-tered starting 8 hours after surgery was compared to enoxaparin at a dose of 30 mg twice daily initiated 12-24 hours after surgery. The study revealed efficacy but an unacceptable risk/benefit profile at higher doses.39 Razaxaban was discontinued for further development in March 2005 in lieu of apixaban, an oral FXa inhibi-tor under development by the same company with a more favorable safety profile.

3.2.2. Apixaban Apixaban (formerly BMS-562247 or DPC-AG0023) is an orally active, small-molecule direct FXa inhibitor being developed by Bristol-Myers Squibb with a more favorable safety profile than Razaxaban. It is a highly potent inhibitor of human FXa, with a Ki of 0.08 ± 0.01 nM, and binds to serum proteins at a rate of 87%. It has a consistent oral absorption profile and linear pharmacokinetics, with a maximal plasma concentra-tion achieved within 3 hours and an effective half-life of 9 hours for twice daily administration, and 14 hours for once daily administration. It is eliminated via both hepatic and renal routes. Apixaban had only modest effects on two traditional markers of anticoagulation, International Normalized Ratio (INR) and activated partial thromboplastin time (aPTT). Phase I clinical studies revealed mild bleeding and prolonged bleed-ing time, with no evidence of elevated transaminases (alanine transaminase or aspartate transaminase ≥ 5 times the upper limit of the normal range). The safety



profile of apixaban remains to be determined in phase II/III trials, and it is presently undergoing phase III clinical studies in elective total knee replacement sur-gery patients and patients with acute DVT. The effi-cacy and safety of apixaban as a thromboprophylaxis in patients following total knee replacement was de-termined.40 Apixaban in doses of 2.5 mg b.i.d. or 5 mg q.d. exhibited a promising benefit-risk profile as compared to current standards of care following total knee replacement.40

Apixaban is also in Phase III trials studying the pre-vention of stroke and other thromboembolic events in patients with AF. The AF program consists of two trials. The ARISTOTLE trial is investigating apixaban compared to warfarin in approximately 15,000 pa-tients with AF. The AVERROES trial is investigating apixaban compared to aspirin in approximately 5,600 patients with atrial fibrillation who are ineligible for VKA treatment or haven’t tolerated previous VKA treatment. The VTE treatment program consists of two trials. The AMPLIFY trial is a 6-month trial in-vestigating apixaban compared to enoxaparin plus warfarin in approximately 4,800 patients with acute DVT or PE. The AMPLIFY-EXT trial is a 12-month trial investigating apixaban compared to placebo for extended treatment to prevent recurrent VTE in ap-proximately 2,400 patients who have completed 6 to 12 months of treatment VTE.

3.2.3. Rivaroxaban (Bayer HealthCare AG and J&J/Scios, Inc.)Rivaroxaban (BAY 59-7939) is a small molecule, se-lective oral direct FXa inhibitor developed by Bayer for the prevention and treatment of thrombosis. Rivaroxaban is an oxazolidinone derivative opti-mized for inhibiting both free Factor Xa and Factor Xa bound in the prothrombinase complex. It is al-ready marketed in a number of countries by Bayer as Xarelto and if approved by the United States FDA, it will be marketed by Ortho-McNeil Pharmaceutical. It is the first available orally active direct inhibitor of coagulation Factor Xa. Oral rivaroxaban may be given in fixed once-daily doses, with potentially no coagula-tion monitoring. These properties, along with results from preclinical and clinical studies, suggest that the use of rivaroxaban might be more advantageous than current treatments. Studies in arterial and venous ani-mal models have demonstrated that rivaroxaban has potent antithrombotic properties without prolonging bleeding times. In healthy subjects, rivaroxaban was well tolerated with a predictable pharmacological pro-file and a low propensity for clinically relevant drug-

drug interactions. In preclinical studies, endogenously generated FXa was inhibited with an IC50 of 21±1 nM.41 This antithrombotic effect was demonstrated in different thrombosis models at doses of 0.6-10 mg/kg, depending on the model and species, administered orally. In dogs, bioavailability was determined to range from 60-86%.

In phase I studies, rivaroxaban was rapidly ab-sorbed (maximum concentration [Cmax] reached af-ter 30 minutes) and well tolerated (up to 80 mg single dose in healthy people).42 Elimination occurred with a terminal half-life of 4.86-9.15 hours (steady state). Prothrombin time (PT), aPTT, and HepTest were pro-longed in a dose-dependent manner, and there was no effect on bleeding time. In elderly men and women (>60 years), mean area under the concentration-time curve and Cmax tended to be about 20% higher. There were no drug-drug interactions or induction of major cytochrome P450 (CYP) isoforms, with the exception of strong CYP3A4 inhibitors, and no prolongation of QTc.

Four large dose-ranging studies of rivaroxaban (ODIXa-HIP, ODIXa-HIP2, ODIXa-KNEE, and ODIXa-OD-HIP) have been completed, covering a 12-fold increase in dose, from 2.5 to 30 mg b.i.d. and 5 to 40 mg once daily (q.d.) for VTE prevention in major orthopedic surgery.43,44 The open-label phase IIa ODIXa-HIP trial using mandatory venography confirmed proof of principle for rivaroxaban use for VTE prophylaxis. Other studies have confirmed the efficacy and safety of rivaroxaban as compared to enoxaparin under double-blind, double-dummy con-ditions for VTE prevention in patients undergoing orthopedic surgery.43 Phase II studies of rivaroxaban for the prevention of VTE after major orthopedic surgery support these findings, and suggest that a to-tal daily dose range of 5 to 20 mg rivaroxaban has similar efficacy and safety as enoxaparin, with 10 mg rivaroxaban once daily the optimal dose.45

The drug development plan for rivaroxaban is ag-gressive, with a program of simultaneous investiga-tion of multiple indications rather than a sequential approach. At present, over 24,000 patients (12,000 are predominantly post-operative orthopedic pa-tients) have been evaluated in completed phase II and III trials of rivaroxaban for thromboprophylaxis and treatment of DVT. By the time all the currently enrolling trials have concluded, more than 50,000 pa-tients will have been evaluated in randomized con-trolled trials of rivaroxaban. The advantages of riva-roxaban include the potential for once daily dosing for all indications, no required dose-adjustment for



body-weight, no known interactions with common cardiovascular medications, a relatively safe pharma-codynamic profile with respect to bleeding risk and hepatotoxicity, no clinically significant interaction with aspirin, and the ability to bridge with LMWH when necessary. On the other hand, rivaroxaban is partially renally cleared and will require dose-adjust-ment in those with grade III chronic kidney disease, and is not being studied in patients with a creatinine clearance less than 30 mL per minute. In addition, since rivaroxaban metabolism is affected by potent cyp P450 3A4 inhibitors such as ketoconazole and clarithromycin and protease inhibitors, use will be re-stricted in certain special populations. Nonetheless, after extensive phase II, and now emerging phase III trial data, it appears that rivaroxaban is effective in preventing and treating VTE with a bleeding risk comparable to other anticoagulants. The results of randomized trials evaluating rivaroxaban for the pre-vention of stroke and non-central nervous system (CNS) embolism in AF and secondary prevention of acute coronary syndromes are currently ongoing.

While there are several other oral anticoagulants in development, none have been evaluated as extensive-ly or in as many patients as rivaroxaban. Rivaroxaban has the advantage that it can be dosed once a day, which has been shown to improve patient compli-ance and outcomes.46,47 Despite once daily dosing, ri-varoxaban has a half-life that is considerably shorter than other oral FXa inhibitors, which is advantageous in the event of bleeding or an urgent need to discon-tinue anticoagulation. Unlike the direct thrombin in-hibitor dabigatran, the bioavailability of rivaroxaban is very good, and there is low risk of drug-drug inter-actions, including with medications that alter gastric pH, which are taken chronically by 3% of the US population.48 Perhaps more importantly, rivaroxaban has been shown to have no effect on platelet aggre-gation, and its pharmacokinetic profile is unaffected by aspirin and other notable cardiovascular medica-tions such as digoxin. Finally, after clinical investiga-tion in thousands of patients, rivaroxaban appears to have no significant hepatotoxicity and a bleeding risk comparable to other conventional anticoagulants.

Rivaroxaban could potentially be used in several clinical indications and disease states, including VTE prophylaxis, long term treatment of DVT, PE, and the prevention of stroke and non-CNS embolism in patients with AF and possibly acute coronary syn-dromes. In order to understand the efficacy and safety of such a wide range of patient populations, several large simultaneous studies evaluating rivar-

oxaban in both venous and arterial thromboembo-lism are under way. The greatest therapeutic impact of rivaroxaban might be in providing a much-needed and attractive alternative to warfarin. While formal cost-effective analyses are not yet available, avoid-ing the intense, costly, and frequent monitoring re-quired with VKAs, as well as the potential to reduced adverse vascular events precipitated by the narrow therapeutic window of VKAs, will most likely trans-late into a significant improvement in quality-of-life and cost-savings. While further data (especially large phase III trials) and caution is required, there is rea-son for optimism. Rivaroxaban may very well be the long-awaited alternative to warfarin.

The RECORD4 study concluded that rivaroxaban was significantly more effective in reducing the oc-currence of venous blood clots following knee re-placement surgery than enoxaparin. In addition, ri-varoxaban is being studied in phase III clinical trials for stroke prevention in non-valvular AF (ROCKET-AF), prevention of VTE in hospitalized medically ill patients (MAGELLAN), treatment and secondary prevention of VTE (EINSTEIN), and secondary prevention of major cardiovascular events in patients with acute coronary syndrome (ACS) (ATLAS ACS TIMI 51).

3.2.4. YM-150 Astellas (formerly Yamanouchi) has developed YM-150, an oral selective FXa inhibitor for DVT preven-tion. The compound has an immediate antithrom-botic effect after oral administration, with a dose-de-pendent response and prolongation of PT; no signifi-cant food interactions have been noted. In a phase II dose-escalation study in patients undergoing elective primary hip replacement surgery, YM-150 (3, 10, 30, or 60 mg PO q.d.) given 6-10 hours after surgery for 7-10 days was compared to enoxaparin administered at a dose of 40 mg SQ q.d. 12 hours before surgery.49 There was no major bleeding, and the median inci-dence of VTE ranged from 52% in the 3-mg group to 19% in the 60-mg group. Overall, the drug ap-pears to be safe and well tolerated. A dose escalation study of YM150 in the prevention of VTE in elec-tive primary hip replacement surgery was also carried out.50 YM150, administered at doses of 10-60 mg daily starting 6-10 hours after primary hip replacement was shown to be safe, well tolerated and effective.

3.2.5. DU-176b Daiichi Sankyo is developing DU-176b, an oral FXa inhibitor for the treatment of thrombotic disorders.



Table 1. Oral anticoagulants in phase II/III trials.

Drugs* Target Dosing Adjustment and Interactions Indications

Rivaroxaban (Bayer Schering) Factor Xa Once or

Twice daily

· Avoid or monitor with CrCl <30 ml/min · Dose reduction for CrCl 30-49 ml/min · Avoid or monitor with strong CYP450 3A4 inhibitors

· Phase III for VTE prophylaxis after joint replacement completed (n=2531). · Phase II for secondary prevention after ACS enrolling (n=1350) · Phase II for treatment of DVT completed, phase III enrolling (n=2900) · Phase III for prevention of stroke in non valvular AF enrolling (n=14,000) · Phase III for treatment of PE enrolling (n=3300)

Apixaban (Bristol-Myers Squibb and Pfizer)

Factor Xa Twice daily · Avoid or monitor with strong CYP450 3A4 inhibitors

· Phase II trial for treatment of acute DVT completed. · Phase III trials for VTE prevention after joint replacement enrolling (n=7258). · Phase III trial for prevention of VTE in medical patients (n=6524) · Phase III trial for prevention of stroke in non valvular AF enrolling (n=15,000).

PRT054021 (Portola Pharmaceuticals Factor Xa Twice daily · Undefined yet · Phase II trial for VTE prophylaxis after joint

replacement completed.

LY517717 (Eli Lilly), Factor Xa Once daily · Undefined yet · Phase II trial for VTE prophylaxis after joint

replacement completed.

Dabigatran (Boehringer Ingelheim)

Factor IIa (thrombin)

Once or Twice daily

· Avoid or monitor with proton pump inhibitors

· Phase III trial for VTE prevention after joint replacement completed. · Phase III trial for treatment of VTE enrolling (n=2554). · Phase III trial for prevention of stroke in non valvular AF enrolling (n=15,000).

*Additional oral anticoagulants in preclinical or early clinical developments are not listed.

Preclinical data in mouse models revealed potent an-tithrombotic effects in AT-positive and AT-deficient mice.51 In rat models, DU-176b at doses of 0.05-1.25 mg/kg/hour prevented arterial and venous throm-bosis.

In terms of clinical studies, DU-1766 in a single 60 mg dose was given to healthy males. The drug inhib-ited FXa activity, reduced thrombin generation, pro-longed PT, aPTT and INR, and reduced venous and arterial thrombosis by 28% and 26%, respectively, in a Badimon chamber. Further studies are planned.

3.2.6. LY-517717 LY-517717, an indol-6-yl-carbonyl derivative, is the lead in a series of oral selective FXa inhibitors being developed by Eli Lilly as part of a research collabora-tion with other partners. It is 1000-fold more selective as an FXa inhibitor than other serine proteases, with a Ki of 5 nM. The oral bioavailability of LY-517717 is approximately 25-82%, with a plasma half-life of 7-10 hours. In a rat atrioventricular shunt model, the me-dian effective dose was 5-10 mg/kg PO, and absorp-

tion in dogs indicated no bleeding issues. In a phase I study, LY-517717 was found to be well tolerated and suitable for once daily administration. In a dose-esca-lating study, 511 patients undergoing hip or knee re-placement surgery were randomized to receive one of six oral doses of LY-517717 (25, 50, 75, 100, 125, or 150 mg), or enoxaparin (40 mg SQ daily), started pre-operatively, for 6-10 doses. The 100, 125, and 150 mg dose groups were non-inferior to enoxaparin in the incidence of symptomatic or venographically proven DVT or PE.52 The compound produced a dose-de-pendent prolongation of PT and was well tolerated, and there were no differences in bleeding risk as com-pared to enoxaparin.52

3.3. Selective, Direct Factor IXa Inhibitors Although the development of direct FIXa inhibitors is at an earlier phase than direct FXa inhibitors, the theo-retical advantages are similar. TTP 889, manufactured by Trans Tech Pharma, is an oral, direct Factor IXa inhibitor with a half-life of 20 hours, enabling once daily dosing. A phase II proof of principle study for



VTE prevention in hip fracture surgery, the FIXIT tri-al, recently completed enrollment of 206 patients who received standard in-hospital thromboprophylaxis. Efficacy and safety are being compared between pa-tients randomized to receive TTP 889 versus placebo for up to 3 weeks post-discharge.53

3.4. Factor XIa inhibitorsDevelopment of FXIa inhibitors is currently at the preclinical level. BMS-262084 is an irreversible and selective small molecule inhibitor of FXIa with an IC50 of 2.8 nM against human factor Xia. The effect of inhibiting activated blood coagulation FXIa with BMS-262084 has been determined in rat models of thrombosis and hemostasis.54 BMS-262084 doubled aPTT in human and rat plasma at concentrations of 0.14 and 2.2 µM, respectively. Consistent with FXIa inhibition, the PT was unaffected at concentrations up to 100 µM. BMS-262084 administered by intravenous loading with sustained infusion was effective against FeCl (2)-induced thrombosis in both the vena cava and carotid artery. In contrast, doses of up to 24 mg/kg+24 mg/kg/h had no effect on TF-induced venous thrombosis or ex vivo PT. Doses of up to 24 mg/kg+24 mg/kg/h did not significantly prolong bleeding time provoked by puncture of small mesenteric blood vessels, template incision of the renal cortex, or cuticle incision. These results demonstrate that pharmacolog-ic inhibition of FXIa achieves antithrombotic efficacy with minimal effects on provoked bleeding.54

4. Inhibitors of Thrombin Activity Thrombin is the central serine protease in hemosta-sis. The mechanisms of action of thrombin involve coagulation, platelet activation, fibrinolysis, and vascu-lar cell biology. In addition to its major role in fibrin formation and activation of FXIII, which cross-links fibrin, thrombin is essential for feedback activation of other coagulation factors such as FV, FVIII, and FIX.55 Thrombin is involved in platelet activation and subse-quent aggregation,52 and can act as an anticoagulant by binding to thrombomodulin, which converts pro-tein C to its active form, inactivating FVa and FVIIIa. Thrombomodulin-bound thrombin regulates coagula-tion through activation of thrombin-activatable fibri-nolysis inhibitor (TAFI) and subsequent down-regula-tion of fibrinolysis. Predictions that thrombin inhibi-tors would be more effective than FXa inhibitors in arterial thromboembolic disease (where thrombin has a key role in platelet activation) and less effective in VTE have not been borne out by clinical data. Given its central role in the coagulation cascade, inhibitors of

thrombin activity--whether mediated by AT or acting directly on the active site--represent an important class of anticoagulant drugs in our armamentarium.

4.1. Indirect Thrombin Inhibitors

SNAC/Heparin SNAC (sodium N-[8(2-hydroxybenzoyl)amino] capry-late), developed by Emisphere Technologies, enables macromolecule delivery of the large negatively charged and poorly absorbed heparin molecule through a non-covalent complex with heparin, allowing passive, tran-scellular absorption. SNAC itself has no pharmaco-logical activity. Phase I studies revealed that in doses of up to 10.5 g SNAC/150,000 U heparin, the compound is well tolerated, with nausea being the only significant adverse event observed. Dose-dependent increases in aPTT and anti-FXa levels were also observed, suggest-ing that both AT-mediated thrombin and FXa inhi-bition play a role in the anticoagulant effects of the drug.56 In addition, there was an apparent food and diurnal effect, but no age effect.

The PROTECT study was a large phase III study in 2,264 hip-replacement patients with two SNAC treatment arms (low-dose SNAC and high-dose SNAC) for 30 days versus 10 days of enoxaparin at a dose of 30 mg SC every 12 hours.57 Mandatory ve-nography on days 27-30 revealed an overall VTE rate of 31.8% in the low-dose SNAC group, 29.7% in the high-dose group, and 26.1% in the enoxaparin group. The rates of proximal DVT/PE were 18.6% in the low-dose group and 13.8% in the high-dose group, both of which were significantly higher than in the enoxaparin group. There was overall poor compli-ance in 22.1% of the patients on the low-dose regi-men and in 31.4% of the patients on the high-dose regimen, suggesting that substance compliance was a key factor in failure to achieve proof of principle for the use of SNAC/heparin in VTE prevention as an indirect FIIa/Xa inhibitor. Improved formulations of heparin in solid dosage form are currently under clini-cal investigation.58,59 An orally administrable chemical conjugate of heparin and hydrophobic deoxycholic acid (DOCA), referred to as LHD, has also been de-veloped. LHD was pre-formulated with dimethyl sulf-oxide (DMSO) as solubilizer to further improve oral bioavailability (9.1% in monkeys). LHD was absorbed mainly in the jejunum and ileum of the small intestine, although it is in the ileum that absorption was most notable. Mechanistic studies of LHD absorption us-ing Caco-2 cell monolayers, which mimic the intestine, demonstrated that the high permeability of LDH is



mediated by passive diffusion through the transcellular route, and permeation is affected in part by bile acid transporters. These results demonstrated the feasibil-ity of using chemically modified heparin for long-term oral administration as an effective therapy for VTE.58 A more recent clinical study determined the true PK for injectable versus oral heparin.59

4.2. Direct Thrombin Inhibitors (DTIs) (Table 1) The development of DTIs was driven by three ma-jor factors: increasing recognition of immune throm-bocytopenia as a potentially severe complication of heparin use,60 the notion that heparin-AT inhibition of thrombin produces only weak inhibition of cell-sur-face- or clot-bound thrombin, which is active and can be released during fibrinolysis,61 and the non-specific binding properties of heparin, necessitating frequent monitoring. Hence, non-AT-based thrombin inhibi-tors with improved safety profiles over heparin, the ability to inhibit surface- or clot-bound thrombin, and predictable dose-responses would be advantageous in the clinical setting. Furthermore, oral formulations of these drugs would be a major advantage. DTIs could be ideal drugs for the treatment of HIT, as this condition is characterized by the generation of large amounts of thrombin. A theoretical concern about DTIs is that they could inhibit the anticoagulant properties of thrombin, namely inhibition of the thrombin-throm-bomodulin-mediated negative feedback mechanism of the protein C system, with the possibility of rebound hyper-coagulability.62

Four parenteral DTIs have emerged: lepirudin, bi-valirudin, argatroban, and melagatran, the first three of which have been approved for clinical use. Lepirudin is a naturally occurring bivalent DTI indicated for thromboprophylaxis of HIT. Argatroban is a proto-type non-covalent, reversible, small molecule DTI in-dicated for thromboprophylaxis or treatment of HIT. Melagatran is the active form of the oral, prodrug, small molecule DTI ximelagatran, discussed below. All of these drugs have limitations in terms of parenteral use, need for frequent monitoring, and high cost.

4.3. Selective Oral DTIs

4.3.1. Ximelagatran Ximelagatran, developed by AstraZeneca, represents the first of a new class of orally active, small-molecule DTIs to reach late-stage development with limited clinical indications for VTE prevention. Ximelagatran is a hydrophilic prodrug that is converted by a cyp-P450-independent liver enzyme system to its active

form melagatran. Bioavailability of ximelagatran is ap-proximately 20%, and the half-life is 4-5 hours in pa-tients. It can be administered twice daily and does not require anticoagulant monitoring or dose adjustment. Ximelagatran was studied extensively in a large phase III trial for VTE prevention and treatment and was found to be either superior or equivalent to warfarin in terms of efficacy.63-68 However, initial long-term data with ximelagatran revealed elevated liver enzymes (ap-proximately 6%). Based upon this and other consid-erations, it was not approved by the US FDA. It was, however, approved in other countries for short-term, post-orthopedic thromboprophylaxis. In February 2006, AstraZeneca withdrew ximelagatran from the world market due to continuing concerns about severe liver toxicity with long-term use.

4.3.2. Dabigatran Dabigatran etexilate is another small molecule, oral-ly active, prodrug DTI developed by Boehringer Ingelheim that has reached late-stage clinical devel-opment. It is being studied for various clinical indica-tions and may replace warfarin as the preferred anti-coagulant in many cases. It is orally administered and is currently marketed as Pradaxa since April 2008 in European countries and Pradax in Canada. It is rapidly absorbed and converted to the active form, dabiga-tran. It has linear characteristics in terms of concen-tration and global coagulation parameters, including thrombin clotting time, INR, and ecarin clotting time. Dabigatran has a Ki of 4.5±0.2 nmol/l, peak plasma concentration of two hours post-dose, and a half-life of approximately 14-17 hours after multiple dose ad-ministration.69 It is metabolized mainly (80-85%) by renal excretion.

The BISTRO II study was a multicenter, paral-lel group, double-blind, dose-finding study for VTE prevention in 1,949 patients undergoing total hip or knee replacement.70 Patients were randomized to re-ceive dabigatran (50 mg, 150 mg, or 225 mg b.i.d., or 300 mg q.d.) starting 14 hours after surgery. The com-parator was enoxaparin (40 mg q.d.) initiated 12 hours prior to surgery. A significant dose-dependent de-crease in DVT was observed with increasing doses of dabigatran (P>.001). Compared to enoxaparin, DVT was significantly lower in patients receiving dabigatran 150 mg b.i.d. (odds ratio [OR] 0.47, P=.0007), 300 mg q.d. (OR 0.61, P=.02), and 225 mg b.i.d. (OR 0.47, P=.0007). Major bleeding was lower with the low dose of 50 mg b.i.d. (0.3% vs. 2.0%, P=.047), but elevated at higher doses, with trends almost reaching statistical significance in those receiving the 300 mg dabigatran



dose (4.7%, P=.051). In terms of adverse events, the incidence of elevated alanine aminotransferase (> 3 times ULN) was lower in the dabigatran groups (1.5-3.1%) than in the enoxaparin group (7.4%). There were no cases of clinically relevant thrombocytopenia. The authors concluded that dabigatran started early in the postoperative period was effective and safe across a wide range of doses. In addition, the frequency and extent of severe hepatic abnormalities were lower than those observed with ximelagatran. Dabigatran is currently undergoing extensive phase III evaluation in VTE prevention, treatment, and secondary thrombo-prophylaxis through the RE-VOLUTION program.

A randomized, double-blind, non-inferiority trial was conducted comparing dabigatran etexilate to enoxaparin for prevention of VTE after total hip re-placement.71 Patients (3494 total) undergoing total hip replacement were randomized into treatment for 28-35 days with dabigatran etexilate 220 mg (n=1157) or 150 mg (n=1174) once daily, starting with a half-dose 1-4 hours after surgery, or subcutaneous enoxaparin (40 mg) once daily (n=1162), starting the evening be-fore surgery. The primary efficacy outcome was the composite of total VTE (venographic or symptom-atic) and death from all causes during treatment. On the basis of the absolute difference in rates of VTE with enoxaparin versus placebo, the non-inferiority margin for the difference in rates of thromboembo-lism was defined as 7.7%. Both doses of dabigatran were non-inferior as compared to enoxaparin. There was no significant difference in major bleeding rates with either dose of dabigatran etexilate as compared with enoxaparin.71 The frequency of increased liver enzyme concentrations and acute coronary events during the study did not differ significantly between the groups. The study concluded that oral dabigatran etexilate was as effective as enoxaparin in reducing the risk of VTE after total hip replacement surgery, with a similar safety profile.71

A phase III study, RE-LY, evaluated the efficacy and safety of two different doses of dabigatran rela-tive to warfarin in over 18,000 patients with AF. A total of 18,113 patients with AF were randomized to one of three arms: (1) adjusted dose warfarin, (2) dabigatran 110 mg twice daily, or (3) dabigatran 150 mg twice daily. The warfarin arm was open label, but adverse events were adjudicated by reviewers blinded to treatment. Dabigatran 110 mg was non-inferior to warfarin for the primary efficacy endpoint of stroke or systemic embolization, while dabigatran 150 mg was significantly more effective than warfarin or dabi-gatran 110 mg. Major bleeding occurred significantly

less often with dabigatran 110 mg than warfarin; dabi-gatran 150 mg had similar bleeding to warfarin.72 A large (2539 patients), randomized, double-blind trial by the RE-COVER study group demonstrated non-inferiority of dabigatran when compared to warfarin in the treatment of acute VTE, with a similar rate of major bleeding and a lower rate of combined major plus non-major bleeding. Patients randomized to dabi-gatran had fewer minor bleeds but more dyspepsia and more drug discontinuation. Dabigatran-treated patients did not require coagulation testing.73

4.3.3. TGN-167 TGN-167 (TRI-50c-04) is an oral thrombin inhibi-tor being developed by Trigen Holdings for the po-tential treatment of thrombosis. A controlled-release formulation of the drug is also being developed with Eurand for long-term treatment of thrombosis. The compound produces a marked increase in thrombin clotting time, with minimal effects on aPTT. A dou-ble-blind, phase I, dose-escalation study with 20 vol-unteers showed the drug to be well tolerated, with no significant adverse events reported.74 At 600 mg, all dosed subjects achieved effective anticoagulant activ-ity in vitro and phase II studies are being planned.

5. Expert Opinion and Conclusions The antithrombotic management of VTE will undergo significant changes in the next 5-10 years. Limitations of existing parenteral and oral anticoagulants have led to the development of new agents designed to target specific pro-coagulant complexes in the coagulation pathway, inhibiting coagulation initiation, coagulation propagation, or thrombin activity. With respect to ef-ficacy, during acute treatment of VTE, newer anti-thrombotic agents must exhibit at least non-inferiority in a methodologically sound study as compared to the existing parenteral agent of choice, LMWH, and the emerging agent fondaparinux. This is true particularly in high-risk venous thromboses, such as ileo-femoral VTE, PE, or VTE associated with cancer. For long-term VTE treatment, there is a need to improve upon existing oral anticoagulants, namely, VKAs. Target-selective oral agents must exhibit an improved safety profile (especially as it pertains to major or clinically significant bleeding), ease-of-use, and tolerability as compared to VKAs. If successful, emerging oral an-ticoagulants could negate the traditional distinction of acute versus long-term treatment of VTE, as they could potentially be used throughout the spectrum of disease, without the need for overlap with parenteral therapies.51 Lastly, any new long-term anticoagulant



must be safely tolerated in combination with anti-platelet agents, as an increasingly aging population will be prone to arterial as well as venous thrombo-embolic disease. Cost considerations are also impor-tant, especially from a population perspective.

Newer agents should, in theory, fulfill the following requirements of an ideal anticoagulant: a rapid onset with predictable response characteristics, predictable pharmacokinetics, pharmacodynamics with low plas-ma protein binding, no required monitoring, a half- life that provides both safety and ease of use (partic-ularly during temporary withdrawal), lack of food or drug interactions, an excellent safety profile (particu-larly with respect to immune-mediated thrombocy-topenia, hepatotoxicity, and potential for thrombotic rebound phenomenon), and reversibility or availabil-ity of an antidote. In addition, oral agents with pre-dictable intestinal absorption/bioavailability used in a simple, fixed-dose once or twice daily regimen and for which compliance can be monitored would be even more advantageous. At this time, drugs at the most advanced stage of development with respect to VTE management include the parenteral indirect FXa in-hibitor idraparinux and biotinylated idraparinux, the oral DTI dabigatran, and the oral selective direct FXa inhibitors rivaroxaban and apixaban. Whether there

are inherent advantages in blocking initial thrombin formation via the prothrombinase complex early in the coagulation system or blocking thrombin directly and preventing feedback amplification is still a matter of debate, as is the notion of whether there is any clinically meaningful effect of small molecule DTIs that target both clot-bound and free thrombin. Long-term clinical data with respect to efficacy of anti-Xa inhibitors will be available shortly, while long-term data is currently available on the efficacy of direct thrombin inhibition. The lessons from ximelagatran reveal the importance of long-term safety data in dif-ferent patient populations. Ximelagatran had shown significant potential as a possible replacement to war-farin therapy, but was withdrawn because of poten-tial liver toxicity. Its contrast, dabigatran appears to have a better safety profile and has recently entered a phase III randomized clinical trial in AF. Oral di-rect FXa inhibitors (rivaroxaban, apixaban, and oth-ers) may prove to be more potent and safe. Selective inhibitors of specific coagulation factors involved in the initiation and propagation of the coagulation cascade (FIXa, FVIIa, circulating TF) are at an early stage of development. Additional new agents in clini-cal development include nNAPc2, protein C deriva-tives, and soluble thrombomodulin.



1. Anderson FA, Jr., Spencer FA. Risk factors for venous thromboembolism. Circulation 2003;107:I9-16.2. Kearon C. Natural history of venous thromboembolism. Circulation 2003;107:I22-30.3. Warkentin TE, Levine MN, Hirsh J, et al. Heparin-induced thrombocytopenia in patients treated with low-molecular-weight heparin or unfractionated hepa-rin. N Engl J Med 1995;332:1330-1335.4. Spyropoulos AC. Managing oral anticoagulation requires expert experience and clinical evidence. J Thromb Thrombolysis 2006;21:91-94.5. Bates SM, Ginsberg JS. Anticoagulants in pregnancy: fetal effects. Baillieres Clin Obstet Gynaecol 1997;11:479-488.6. Hutten BA, Prins MH, Gent M, Ginsberg J, Tijssen JG, Buller HR. Incidence of recurrent thromboembolic and bleeding complications among patients with venous thromboembolism in relation to both malignancy and achieved inter-national normalized ratio: a retrospective analysis. J Clin Oncol 2000;18:3078-3083.7. Palareti G, Legnani C, Lee A, et al. A comparison of the safety and efficacy of oral anticoagulation for the treatment of venous thromboembolic disease in patients with or without malignancy. Thromb Haemost 2000;84:805-810.8. Bates S, Greer I, Hirsch J, Ginsberg J. Use of antithrombotic agents during pregnancy. The Seventh ACCP Conference on Antithrombotic and Thrombo-lytic Therapy. Chest 2004;126:627S-644S.9. Greer IA, Nelson-Piercy C. Low-molecular-weight heparins for thrombo-prophylaxis and treatment of venous thromboembolism in pregnancy: a system-atic review of safety and efficacy. Blood 2005;106:401-407.10. Lee AY, Levine MN, Baker RI, et al. Low-molecular-weight heparin versus a coumarin for the prevention of recurrent venous thromboembolism in patients with cancer. N Engl J Med 2003;349:146-153.11. Weitz JI, Hirsh J, Samama M. New anticoagulant drugs. The Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy. Chest 2004;126:265S-286S.12. Dahl OE. Mechanisms of hypercoagulability. Thromb Haemost 1999;82:902-906.13. Fernandez PM, Patierno SR, Rickles FR. Tissue factor and fibrin in tumor an-giogenesis. Semin Thromb Hemost 2004;30:31-44.14. Girard TJ, Nicholson NS. The role of tissue factor/factor VIIa in the patho-physiology of acute thrombotic formation. Curr Opin Pharmacol 2001;1:159-163.15. Hirsh J, O>Donnell M, Weitz JI. New anticoagulants. Blood 2005;105:453-463.16. Weitz JI, Bates SM. New anticoagulants. J Thromb Haemost 2005;3:1843-1853.17. Mousa SA, Fareed J, Iqbal O, Kaiser B. Tissue factor pathway inhibitor in thrombosis and beyond. Methods Mol Med 2004;93:133-155.18. Lee A, Agnelli G, Buller H, et al. Dose-response study of recombinant factor VIIa/tissue factor inhibitor recombinant nematode anticoagulant protein c2 in prevention of postoperative venous thromboembolism in patients undergoing total knee replacement. Circulation 2001;104:74-78.19. Giugliano RP, Wiviott SD, Stone PH, et al. Recombinant nematode antico-agulant protein c2 in patients with non-ST-segment elevation acute coronary syndrome: the ANTHEM-TIMI-32 trial. J Am Coll Cardiol 2007;49:2398-2407.20. Moons AH, Peters RJ, Bijsterveld NR, et al. Recombinant nematode anticoag-ulant protein c2, an inhibitor of the tissue factor/factor VIIa complex, in patients undergoing elective coronary angioplasty. J Am Coll Cardiol 2003;41:2147-2153.21. Edgington TS, Mackman N, Brand K, Ruf W. The structural biology of expres-sion and function of tissue factor. Thromb Haemost 1991;66:67-79.22. Arnold CS, Parker C, Upshaw R, et al. The antithrombotic and anti-inflam-matory effects of BCX-3607, a small molecule tissue factor/factor VIIa inhibitor. Thromb Res 2006;117:343-349.23. Lazarus RA, Olivero AG, Eigenbrot C, Kirchhofer D. Inhibitors of Tissue Fac-tor. Factor VIIa for anticoagulant therapy. Curr Med Chem 2004;11:2275-2290.24. Buckman BO, Chou YL, McCarrick M, et al. Solid-phase synthesis of naph-thylamidines as factor VIIa/tissue factor inhibitors. Bioorg Med Chem Lett 2005;15:2249-2252.25. Suleymanov OD, Szalony JA, Salyers AK, et al. Pharmacological interruption of acute thrombus formation with minimal hemorrhagic complications by a small molecule tissue factor/factor VIIa inhibitor: comparison to factor Xa and thrombin inhibition in a nonhuman primate thrombosis model. J Pharmacol Exp Ther 2003;306:1115-1121.26. Bajaj MS, Bajaj SP. Tissue factor pathway inhibitor: potential therapeutic ap-plications. Thromb Haemost 1997;78:471-477.27. Sandset PM, Abildgaard U, Larsen ML. Heparin induces release of extrinsic coagulation pathway inhibitor (EPI). Thromb Res 1988;50:803-813.28. Amirkhosravi A, Meyer T, Amaya M, et al. The role of tissue factor pathway

inhibitor in tumor growth and metastasis. Semin Thromb Hemost 2007;33:643-652.29. Mousa SA, Mohamed S. Inhibition of endothelial cell tube formation by the low molecular weight heparin, tinzaparin, is mediated by tissue factor pathway inhibitor. Thromb Haemost 2004;92:627-633.30. Mousa SA, Linhardt R, Francis JL, Amirkhosravi A. Anti-metastatic effect of a non-anticoagulant low-molecular-weight heparin versus the standard low-mo-lecular-weight heparin, enoxaparin. Thromb Haemost 2006;96:816-821.31. Sierko E, Wojtukiewicz MZ, Kisiel W. The role of tissue factor pathway inhibi-tor-2 in cancer biology. Semin Thromb Hemost 2007;33:653-659.32. Biemond BJ, Friederich PW, Levi M, Vlasuk GP, Buller HR, ten Cate JW. Com-parison of sustained antithrombotic effects of inhibitors of thrombin and factor Xa in experimental thrombosis. Circulation 1996;93:153-160.33. Buller HR, Davidson BL, Decousus H, et al. Fondaparinux or enoxaparin for the initial treatment of symptomatic deep venous thrombosis: a randomized trial. Ann Intern Med 2004;140:867-873.34. Buller HR, Davidson BL, Decousus H, et al. Subcutaneous fondaparinux ver-sus intravenous unfractionated heparin in the initial treatment of pulmonary embolism. N Engl J Med 2003;349:1695-1702.35. Herbert JM, Herault JP, Bernat A, et al. Biochemical and pharmacological properties of SANORG 34006, a potent and long-acting synthetic pentasac-charide. Blood 1998;91:4197-4205.36. Reiter M, Bucek RA, Koca N, Heger J, Minar E. Idraparinux and liver enzymes: observations from the PERSIST trial. Blood Coagul Fibrinolysis 2003;14:61-65.37. Buller HR, Cohen AT, Davidson B, et al. Idraparinux versus standard therapy for venous thromboembolic disease. N Engl J Med 2007;357:1094-1104.38. Alexander JH, Dyke CK, Yang H, et al. Initial experience with factor-Xa inhibi-tion in percutaneous coronary intervention: the XaNADU-PCI Pilot. J Thromb Haemost 2004;2:234-241.39. Lassen M, Davidson B, Gallus A, Pineo G, Ansell J, Deitchman DBaA. A phase II randomized, double-blind, five-arm, parallel group, dose-response study of a new oral directly-acting factor Xa inhibitor, Razaxaban, foe the prevention of deep vein thrombosis in knee replacement surgery. Blood 2003;102:Abstract 41.40. Lassen MR, Davidson BL, Gallus A, Pineo G, Ansell J, Deitchman D. The ef-ficacy and safety of apixaban, an oral, direct factor Xa inhibitor, as thrombo-prophylaxis in patients following total knee replacement. J Thromb Haemost 2007;5:2368-2375.41. Perzborn E, Strassburger J, Wilmen A, et al. In vitro and in vivo studies of the novel antithrombotic agent BAY 59-7939--an oral, direct Factor Xa inhibitor. J Thromb Haemost 2005;3:514-521.42. Kubitza D, Becka M, Voith B, Zuehlsdorf M, Wensing G. Safety, pharmacody-namics, and pharmacokinetics of single doses of BAY 59-7939, an oral, direct factor Xa inhibitor. Clin Pharmacol Ther 2005;78:412-421.43. Eriksson BI, Borris L, Dahl OE, et al. Oral, direct Factor Xa inhibition with BAY 59-7939 for the prevention of venous thromboembolism after total hip replacement. J Thromb Haemost 2006;4:121-128.44. Turpie AG, Fisher WD, Bauer KA, et al. BAY 59-7939: an oral, direct fac-tor Xa inhibitor for the prevention of venous thromboembolism in patients after total knee replacement. A phase II dose-ranging study. J Thromb Haemost 2005;3:2479-2486.45. Laux V, Perzborn E, Kubitza D, Misselwitz F. Preclinical and clinical charac-teristics of rivaroxaban: a novel, oral, direct factor Xa inhibitor. Semin Thromb Hemost 2007;33:515-523.46. Claxton AJ, Cramer J, Pierce C. A systematic review of the associations between dose regimens and medication compliance. Clin Ther 2001;23:1296-1310.47. Richter A, Anton SE, Koch P, Dennett SL. The impact of reducing dose fre-quency on health outcomes. Clin Ther 2003;25:2307-2335; discussion 2306.48. Jacobson BC, Ferris TG, Shea TL, Mahlis EM, Lee TH, Wang TC. Who is using chronic acid suppression therapy and why? Am J Gastroenterol 2003;98:51-58.49. Eriksson BI, Turpie AG, Lassen MR, et al. A dose escalation study of YM150, an oral direct factor Xa inhibitor, in the prevention of venous thromboembolism in elective primary hip replacement surgery. J Thromb Haemost 2007;5:1660-1665.50. Eriksson BI, Turpie A, Lassen M, et al. YM150, an oral direct factor Xa inhibitor, as prophylaxis for venous thromboembolism in patients with elective primary hip replacement surgery. A dose escalation study. Bood 2005;106:1865.51. Fukuda F, Honda Y, Matsumoto C, et al. Impact of antithrombin deficien-cy on efficiencies of DU-176b, a novel orally active direct factor Xa inhibitor, and antithrombin dependent anticoagulants, fondaparinux, and heparin. Blood 2005;106:1874.52. Agnelli G, Haas S, Krueger K, Bedding A, Brandt J. A phase II study of the safety and efficacy of a novel oral fXa inhibitor (LY517717) for the prevention of venous thromboembolism following TKR or THR. Blood 2005;106:85a.

References



53. Rothlein R, Shen J, Naser N, et al. TTP889, a novel orally active partial inhibi-tor of FIXa inhibits clotting in two A/V shunt models without prolonged bleed-ing. Blood 2005;106:1886.54. Schumacher WA, Seiler SE, Steinbacher TE, et al. Antithrombotic and he-mostatic effects of a small molecule factor XIa inhibitor in rats. Eur J Pharmacol 2007;570:167-174.55. Lundblad RL, Bradshaw RA, Gabriel D, Ortel TL, Lawson J, Mann KG. A re-view of the therapeutic uses of thrombin. Thromb Haemost 2004;91:851-860.56. Baughman RA, Kapoor SC, Agarwal RK, Kisicki J, Catella-Lawson F, FitzGerald GA. Oral delivery of anticoagulant doses of heparin. A randomized, double-blind, controlled study in humans. Circulation 1998;98:1610-1615.57. Hull D, Kakkar A, Marder V, Pineo GF, Goldberg M, Raskob G. PROTECT trial: Oral SNAC-heparin vs enoxaparin for preventing venous thromboembo-lism following total hip replacement. Blood 2001;100:148a-149a.58. Kim SK, Lee DY, Lee E, et al. Absorption study of deoxycholic acid-heparin conjugate as a new form of oral anti-coagulant. J Control Release 2007;120:4-10.59. Mousa SA, Zhang F, Aljada A, et al. Pharmacokinetics and pharmacodynam-ics of oral heparin solid dosage form in healthy human subjects. J Clin Pharmacol 2007;47:1508-1520.60. Kelton JG. The pathophysiology of heparin-induced thrombocytopenia: bio-logical basis for treatment. Chest 2005;127:9S-20S.61. Weitz JI, Hudoba M, Massel D, Maraganore J, Hirsh J. Clot-bound thrombin is protected from inhibition by heparin-antithrombin III but is susceptible to in-activation by antithrombin III-independent inhibitors. J Clin Invest 1990;86:385-391.62. Mattsson C, Menschik-Lundin A, Nylander S, Gyzander E, Deinum J. Effect of different types of thrombin inhibitors on thrombin/thrombomodulin modu-lated activation of protein C in vitro. Thromb Res 2001;104:475-486.63. Eriksson BI, Agnelli G, Cohen AT, et al. The direct thrombin inhibitor mela-gatran followed by oral ximelagatran compared with enoxaparin for the pre-vention of venous thromboembolism after total hip or knee replacement: the EXPRESS study. J Thromb Haemost 2003;1:2490-2496.

64. Eriksson BI, Agnelli G, Cohen AT, et al. Direct thrombin inhibitor melagatran followed by oral ximelagatran in comparison with enoxaparin for prevention of venous thromboembolism after total hip or knee replacement. Thromb Hae-most 2003;89:288-296.65. Fiessinger JN, Huisman MV, Davidson BL, et al. Ximelagatran vs low-molecu-lar-weight heparin and warfarin for the treatment of deep vein thrombosis: a randomized trial. JAMA 2005;293:681-689.66. Francis CW, Berkowitz SD, Comp PC, et al. Comparison of ximelagatran with warfarin for the prevention of venous thromboembolism after total knee replacement. N Engl J Med 2003;349:1703-1712.67. Francis CW, Davidson BL, Berkowitz SD, et al. Ximelagatran versus warfarin for the prevention of venous thromboembolism after total knee arthroplasty. A randomized, double-blind trial. Ann Intern Med 2002;137:648-655.68. Schulman S, Wahlander K, Lundstrom T, Clason SB, Eriksson H. Secondary prevention of venous thromboembolism with the oral direct thrombin inhibitor ximelagatran. N Engl J Med 2003;349:1713-1721.69. Stangier J, Rathgen K, Gansser D, Kohlbrenner V, Stassen J. Pharmacokinetics of BIBR 953 ZW, a novel low molecular weight direct thrombin inhibitor in healthy volunteers. Abstract. Thromb Haemost 2001;86:OC2347.70. Eriksson BI, Dahl OE, Buller HR, et al. A new oral direct thrombin inhibitor, dabigatran etexilate, compared with enoxaparin for prevention of thromboem-bolic events following total hip or knee replacement: the BISTRO II randomized trial. J Thromb Haemost 2005;3:103-111.71. Eriksson BI, Dahl OE, Rosencher N, et al. Dabigatran etexilate versus enoxa-parin for prevention of venous thromboembolism after total hip replacement: a randomised, double-blind, non-inferiority trial. Lancet 2007;370:949-956.72. Connolly SJ, Ezekowitz MD, Yusuf S, et al. Dabigatran versus warfarin in pa-tients with atrial fibrillation. N Engl J Med 2009;361:1139-1151.73. Schulman S, Kearon C, Kakkar AK, et al. Dabigatran versus warfarin in the treatment of acute venous thromboembolism. N Engl J Med 2009;361:2342-2352.74. Coombe S, Allen G, Kennedy A. A phase I double-blind, ascending dose study of an oral synthetic direct thrombin inhibitor, TGN167. Blood 2005;106:530a.

review


Chronic myeloid leukemia (CML) is a rare dis-ease. Although its incidence is low, its preva-lence is increasing. CML is the first malignant

disease for which a direct genetic link has been found and, as a result, there has been much recent progress in understanding the biology of the disease.1-4 The cy-togenetic hallmark of CML is the Philadelphia chro-mosome (Ph), created by a reciprocal translocation be-tween chromosomes 9 and 22 [t(9;22)(q34;q11)].5 The conjugation of the breakpoint cluster region gene on chromosome 22 and the Abelson kinase gene on chro-mosome 9 creates the chimeric BCR-ABL oncogene, which codes for the deregulated tyrosine kinase, bcr-abl.6 Bcr-abl displays transforming activity owing to its constitutive kinase activity, which results in multiple signal transduction pathways, including the Ras/Raf/mitogen-activated protein kinase (MAPK), phospha-tidylinositol 3-kinase, STAT5/Janus kinase, and Myc, leading to uncontrolled cell proliferation and reduced apoptosis, and resulting in the malignant expansion of pluripotent stem cells in bone marrow.7,8 The bcr-abl

Chronic Myeloid Leukemia

Carmen Fava, Giovanna Rege Cambrin, Stefano Ulisciani, Daniela Cilloni, Giuseppe Saglio

From the Division of Hematology and Internal Medicine, Department of Clinical and Biological Sciences, University of Turin, Turin, Italy

Corresponding author:

Giuseppe Saglio, MDDepartment of Clinical and Biological Sciences of the University of Turin, San Luigi Hospital, 10043 Orbassano, Torino, Italy;T: +39 011 670 5473/76; +39 011 902 6610F: +39 011 903 8636; [email protected]

The successful introduction of the tyrosine kinase inhibitors has revolution-

ized the treatment of patients with chronic myeloid leukemia (CML). Imatinib

mesylate drastically improves survival in CML. Patients achieving a major mo-

lecular response within 12 months of treatment experience longer cytogenetic

remission. Resistance to imatinib represents a clinical challenge. Changes in the

levels of BCR-ABL transcripts are predictive of response or relapse. Vigilant

monitoring of a patient’s response to current treatment is imperative to the

management of CML. Early identification of treatment failure may increase the

probability that therapy will be effective. For patients with primary or second-

ary imatinib resistance or intolerance, dasatinib or nilotinib may be prescribed.

Novel targeted agents have been designed to overcome imatinib resistance,

including bosutinib and INNO406. These agents have demonstrated activity in

patients harboring imatinib-resistant mutants, except for the T315I substitu-

tion. Other approaches are exploring new drugs with different mechanisms

of action. Several trials are ongoing, with the aim of improving on standard

imatinib 400 mg/day therapy, including imatinib combination therapy, and results

are controversial. In multicenter studies, investigators are trying to assess the

superiority of dasatinib and nilotinib as front-line therapy, in terms of higher

12-month complete cytogenetic response, major molecular response rates and

fewer progressions.

kinase itself enhances genomic instability leading to accumulation of secondary genetic errors, which may be responsible for resistance to drugs, and transfor-mation of the typically indolent chronic phase (CP), to advanced phases (accelerated phase [AP] and blast phase [BP]).6 One of the major differences between the CP and BC phases of CML is their differential responsiveness to anti-leukemia treatment. Whereas most CP patients respond well to the current front-line treatment and responses are usually durable, the majority of BC patients respond, but then relapse fairly rapidly. Historically, CML was treated with bu-sulfan or hydroxyurea, and was associated with a poor prognosis.9 These agents controlled the hematologic manifestations of the disease, but did not delay disease progression. Treatment with interferon- alfa (IFN-alfa) produced complete cytogenetic responses (CCyR) in 5% to 25% of patients with CML in CP, and improved survival compared with previous treatments.10 With IFN-alfa, a survival benefit was seen compared to what was achieved with chemotherapy. This survival

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benefit was dependent on the achievement of cyto-genetic responses: patients achieving a CCyR have a 10-year survival rate of 78%, falling to 39% in patients with a partial cytogenetic response (PCyR) and 25% in those without any cytogenetic response.8,9 Combining IFN-alfa? with cytarabine produced additional ben-efits.11 Allogeneic stem cell transplantation (SCT) may be curative in CML. However, compatible donors are not available for the majority of patients, and even when a fully matched donor is available, transplant-re-lated morbidity and mortality can limit this approach.

The tyrosine kinase inhibitor imatinib mesylate (Gleevec or Glivec, Novartis Pharmaceuticals, East Hanover, NJ) was developed to specifically inhibit the bcr-abl fusion protein12 and it has become the golden standard frontline therapy for all CML patients in early CP, on the basis of the impressive response rates and good tolerability shown in numerous clinical trials. This conclusion is mainly based on the results of the International Randomized Study of Interferon and STI571 (IRIS) trial. In fact, in the IRIS trial arm with patients receiving imatinib 400 daily, the best observed rates for major CyR and CCyR at 7 years were 89% and 82%, respectively.13 The estimated event-free Survival (EFS) at 8 years was 81% and freedom from progres-sion to AP/BC was 92%; estimated overall survival (OS) was 85%. It was 93% when only CML-related deaths and those prior to SCT were considered. The annual rates of progression to AP/BC in years 4 to 8 after initiation of therapy were 0.9%, 0.5%, 0%, 0%, and 0.4%, respectively.14 Only 15 (3%) patients who achieved CCyR progressed to AP/BC, all but one within 2 years of achieving CCyR.

Definition of optimal and suboptimal responseThe European Leukemia Net (ELN) has published in-ternationally accepted clinical practice recommenda-tions, including monitoring practice, and formal defi-nitions of optimal responses, warning signs, subopti-mal responses, and treatment failure, using hemato-logic, cytogenetic, and molecular criteria.15 Commonly used time-based landmarks of optimal and suboptimal responses, and treatment failure, are shown in Table 1. The evaluation of the response is based on blood counts and differential (hematologic response, HR), on the examination of marrow cell metaphases (cy-togenetic response) and on a quantitative assessment of BCR-ABL transcripts level (molecular response). An optimal response means that there is no indication that a change of therapy may improve a survival that is currently projected as close to 100% after 6 to 7 years.16 An optimal response to imatinib is defined by com-

plete HR and at least minimal CyR (Ph+< 95%) at 3 months, at least partial PCyR (Ph+<35%) at 6 months, CCyR at 12 months and major molecular response (MMR=BCR-ABL/ABL<0.1%) at 18 months.16 The value of the ELN recommendations in predicting the prognosis of patients in early CP CML has been dem-onstrated. In fact, after 8 years, achieving landmark cytogenetic responses (minor CyR at 3 months, partial CyR (PCyR) at 6 and 12 months, CCyR at 18 months) were prognostic indicators of stable CCyR and lack of events, whereas suboptimal responders had a poorer prognosis.14 Patients with minor to PCyR at 3 months and those with PCyR at 6 and 12 months were more likely to achieve a stable CCyR than have an event. Among patients with less than CCyR at 18 months, the probability of an event was comparable to the probability of achieving stable CCyR. The IRIS study also confirmed that the achievement of CCyR and MMR at 12 months predicted for a low risk of events or progression in the long term and no patients with MMR at 12 months progressed to AP/BP, emphasiz-ing the value of achieving a molecular response early in the treatment course. Thus failure is defined by in-complete HR at 3 months, no CyR (Ph+> 95%) at 6 months, less than PCyR (Ph+> 35%) at 12 months, less than CCyR at 18 months and loss of a complete HR or a complete CyR; treatment failure indicates that treatment at the current schedule is no longer appro-priate for a patient and a change in therapy is indi-cated.15 In any other situation, the response is defined as suboptimal:16 suboptimal responses indicate that although patients may continue to receive a benefit from imatinib treatment with the present schedule, the chances of an optimal outcome are reduced, so that the patient may be eligible for alternative approaches.16 Suboptimal responders defined at 6 and 12 months had a significantly poorer PFS and lower probability of CCyR. Suboptimal responders by the 12-month criteria also had significantly worse survival. The 18-month criteria failed to identify patients with worse OS or PFS.17 Following initial therapy with imatinib, patients should be routinely assessed for response, and therapy should be adjusted to maximize the chance of response for patients with lack of response or sub-optimal response. The US National Comprehensive Cancer Network (NCCN) offers another widely rec-ognized set of recommendations, but does not include time-based molecular response landmark.

Molecular Response MonitoringVigilant monitoring aids prompt detection of imatinib failure, and ensures that patients receive the most suit-

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Table 1. Time-based landmarks of optimal and suboptimal responses, and treatment failure.16

Optimal Response Suboptimal Response Failure

3 months CHR, and at least minor CyR No CyR Less than CHR

6 months At least PCyR Less than PCyR No CyR

12 months CCyR PCyR Less than PCyR

18 months MMR Less than MMR Less than CCyR

Any Time Stable or improving MMR

Loss of MMR Loss of CHR

Mutations still sensitive to imatinib

Mutations poorly sensitive to imatinib

Additional chromosomal abnormalities on the Ph+clone

CHR: complete hematological response, CyR: cytogenetic response, CCyR: complete cytogenetic response, PCyR: partial cytological response, MMR: molecular response

able therapy as early in the disease course as possible. ELN guidelines recommend measuring BCR-ABL transcript levels using RT-PCR every three months following treatment start.16 There is clinical evidence to support the use of the MMR as a treatment goal. In particular, BCR-ABL transcript levels at the time of CCyR have been shown to predict the duration of cy-togenetic remission. In one study, patients with stable CCyR had significantly lower BCR-ABL transcript lev-els at the time of achieving CCyR compared with those suffering cytogenetic relapse.18 Furthermore, achiev-ing a MMR at 12 or 18 months has also been shown to be associated with longer cytogenetic remission than in patients who do not achieve this goal.17 Achieving a MMR may also affect survival outcomes. In the IRIS trial, no patient who had both a CCyR and MMR at 12 months was shown to progress to advanced dis-ease over five years.19 The MMR rates increased over time, and in patients who achieved an MMR at any time point, progression was rare. Achievement of a CCyR correlated well with a molecular 1% from 6 months onwards. At 12 months, patients with a mo-lecular >1% or without CCyR, fared more poorly than those with a molecular of 1% or those in CCyR. At 18 months patients with a molecular 1% had excellent long term outcomes, with the best outcomes seen in those with a molecular 0.1%. The comparison at 18 months was the only one that was statistically signifi-cant.20 However, in another study, the achievement of MMR at 12 or 18 months had no significant effect on 5-year PFS or OS in patients with early CP CML.17 Failure to achieve a MMR within 18 months from start of treatment, or loss of MMR at any time, is regarded as a suboptimal response (Table 1).16 There is cur-rent controversy regarding the significance of a com-plete molecular response (CMR). Firstly, the rate of

such responses, defined by the absence of detectable BCR-ABL transcripts, is inevitably dependent on the sensitivity of the molecular assay used. Furthermore quiescent stem cells may lodge in marrow niches and be relatively occult, and in contrast to the bulk of the CD34+ cells, leukemia stem cells may be transcrip-tionally silent and require a genomic PCR for detec-tion. On the other hand, some transplanted patients never relapsed as well as about 50% of patients who discontinued TKIs.21,22 Studies are ongoing to assess the depth of molecular response for patients treated with more potent inhibitors. Continued molecular monitoring throughout treatment is recommended, even if a sustained CCyR has been achieved. It is now becoming apparent that the dynamics of BCR-ABL transcript levels vary among patients with CCyR, and that this may affect response. In fact, transcript levels in patients who have achieved CCyR may progressively decline, plateau, or rise.23 A significant increase in tran-script levels warrants closer monitoring. The prognos-tic value of loss of MMR is not clear, but is considered a suboptimal response and more frequent monitoring is needed, including cytogenetic analysis and mutational analysis.16,17 Rising levels of BCR-ABL transcripts (not outright loss of MMR) have been described as indicat-ing a loss of response, with more rapid doubling times associated with relapse into accelerated or blast crisis.24 A later study also demonstrated that a half-log increase in BCR-ABL transcripts is significantly predictive of shortened cytogenetic remission.25 Monitoring BCR-ABL transcripts may therefore aid early identification of loss of CCyR in such patients. However, such rises are not always associated with treatment failure. In an-other study, increasing levels of subscripts predicted a loss of cytogenetic response only in patients who did not achieve a MMR.26 In the case of suboptimal

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response, there are no published data to suggest that switching treatment in response to rising BCR-ABL levels in CCyR patients improves long-term outcome. Enrollment in a clinical trial examining early detec-tion and treatment is recommended. Few studies have examined the effect of dose escalation in subopti-mal molecular responders, showing the achievement of MMR by 5% to 38% of patients with high-dose imatinib, but the small size of the samples precludes firm conclusions being drawn.27-29 Furthermore, dose escalation to ≥600 mg/day is associated with lower patient compliance, most likely due to associated tox-icity. Second-generation TKIs may provide a better benefit-risk assessment than imatinib in cases of sub-optimal response but data are still not available. The association between rising BCR-ABL transcript levels and the emergence of imatinib-resistant mutations un-derlines the requirement for molecular monitoring.24 Mutation analyses are recommended by the ELN fol-lowing treatment failure, a suboptimal response, or if BCR-ABL transcript levels are observed to rise.16 An increasing BCR-ABL value should also prompt an in-quiry into compliance.

Resistance and progressionIt is well recognized that with continued imatinib treatment a significant number of patients show re-sistance to imatinib.3,30 The incidence of primary (i.e., patients who never respond to imatinib) and second-ary (i.e., patients who become resistant after an initial response) resistance increases with more advanced phases of CML. In a 4.5-year follow up of patients with CP, AP or myeloid BP-CML (total n=300), primary resistance occurred in 3%, 9% and 51% of patients, respectively. Secondary resistance (i.e., a he-matological recurrence) was noted in 22, 32 and 41% of patients, respectively.31 Patients classified as failure according to the ELN recommendations had a signif-icantly lower OS, PFS, and probability of achieving CCyR than the patients classified as responders. For example, based on the assessment at 12 months, the 5-year survival was 87.1% versus 95.1% (P=.02), PFS 76.% versus 90% (P=.002), and CCyR rate 26.7% ver-sus 94.1% (P<.001).17 Patients classified as failure at any time point who did eventually achieve CCyR (after 18 months) had a significantly higher probability of losing their CCyR. For example, at 12 months, the cu-mulative incidence of loss of CCyR for these patients was significantly higher than that of responders (51% vs 10.3%, P=.001).17

Various mechanisms, bcr-abl dependent or inde-pendent, may contribute to imatinib resistance, includ-

ing increased expression of bcr-abl kinase through gene amplification, decreased intracellular imatinib concentrations caused by drug efflux proteins, ima-tinib binding by plasma proteins, and clonal evolu-tion.3 Activation of members of the Src-kinase fam-ily or of other pathways downstream of bcr-abl (like the PI3K-AKT pathway) has been reported and it is increasingly becoming evident that multiple mecha-nisms and events can be involved in the development of imatinib resistant subclones.32 All types of resis-tance can be ascribed to the high degree of genomic instability characterizing the Ph-positive clone.33 So far the molecular mechanisms leading to this instability are only in part understood and, although it is proved that bcr-abl activation is able to induce some degree of genomic instability, at least in some cases, a stem cell disease causing genomic instability and pre-exist-ing to the acquisition of the Ph-chromosome cannot be excluded. Most of the mechanisms of resistance, however, involve bcr-abl kinase domain mutations, with resulting impairment of the ability of imatinib to bind the ATP binding pocket of the bcr-abl tyrosine kinase domain. Up to now, more than 100 different mutants of bcr-abl have been described, but some of them show a higher frequency than others.34 Most of the clinically relevant mutations develop at just a few residues in the in the P-loop (G250E, Y253F/H, and E255K/V), contact site (T315I), and catalytic domain (M351T and F359V).35

Another example of imatinib failure is intolerance, which is the discontinuation of treatment necessitated by drug toxicity. Intolerance to imatinib is a significant clinical issue, with 29% of patients requiring dose in-terruption, and 26% of these patients discontinuing therapy.36 Intolerance is more frequently observed in patients in advanced stages of CML who have experi-enced a long duration of therapy.

Imatinib dose escalation is typically the first option for CML-CP patients with cytogenetic resistance or relapse to 400 mg/day imatinib. Support for imatinib dose escalation is provided in a retrospective analysis of imatinib patients dose escalated in the IRIS trial.37 A dose increase to 600-800 mg/day was reported for 106 of 551 (19%) of patients enrolled in the study. Clinical responses were achieved within 12 months for 21 of 48 (44%) patients dose escalated per ELN rec-ommendations15 with 6 of 11 (55%) achieving a ma-jor cytogenetic response (MCyR) within 12 months of dose escalation and with 2 of 10 (20%) patients with failure at 18 months attaining CCyR 12 months after dose increase. For the entire cohort of 106 patients who had imatinib dose escalation, the estimated PFS

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was 89% and the OS was 84% at 36 months after es-calation of imatinib dose. In another study, 84 patients with CML-CP were dose escalated to imatinib 600-800 mg/day following hematologic (n=21), or cytogenetic failure (resistance: n=30; relapse: n=33) to imatinib standard dose.38 Among patients that met the criteria for cytogenetic failure, 75% responded to imatinib dose escalation. For patients dose escalated due to loss of hematologic response, 48% achieved a CHR and 14% attained a CyR after imatinib dose increase.

Strategies to cope with resistance have led to the development of second-generation bcr-abl inhibitors. The largest experience with these second-generation TKIs has been gained with dasatinib and nilotinib.39-41 Nilotinib and dasatinib have a higher affinity for bcr-abl, as compared with imatinib, and inhibit the major-ity of clinically relevant imatinib-resistant mutant sub-clones; they have similar potencies in inhibiting bcr-abl, but significantly differ in their target specificity. Dasatinib inhibits a large number of tyrosine kinases, including the Src kinases and several receptor tyrosine kinases, whereas nilotinib, like imatinib, inhibits mainly Abl, c-KIT and PDGFR. Both these two agents have been used mostly in the context of patients who are either resistant to or intolerant of imatinib, and have included patients in all stages of the disease, as well as patients with Ph-positive acute lymphoblastic leuke-mia. Both agents have resulted in hematologic and cy-togenetic responses in a large proportion of patients, particularly those treated still in the CP.42 Dasatinib was approved by EMEA and FDA for the second-line treatment of all phases of disease on the basis of its ef-ficacy and safety profiles shown in a series of phase II trials in patients who failed or were intolerant to first-line imatinib therapy (Table 2).43-45 Recent 24-month follow-up data demonstrated a 2-year MCyR rate of 62% and a CCyR rate of 53%.46 The PFS at 2 years was 80% and the OS rate was 94%. Responses to dasatinib in patients with imatinib-resistant CP CML enrolled

in phase II studies of dasatinib have been assessed by baseline mutational status.28,47 CCyR rates were simi-lar among patients with unmutated bcr-abl and those harboring mutations, with the exception of the T315I and the F317L mutations. Importantly, responses to dasatinib did not appear to be diminished among pa-tients harboring the P-loop mutations. Nilotinib was approved following an open-label phase II study in patients with CML who failed or who were intolerant to imatinib therapy.48,49 Follow-up data confirm the ef-fectiveness of this compound particularly in CP and AP CML (Table 2).50-52 No CCyR were observed in pa-tients harboring T315I, L248V, Y253H, or E255K/V and F359C/V mutations most, frequently associated with progression.53,54 Unfortunately, neither nilotinib or dasatinib are affective against the T315I mutation.55 A number of new TKIs are in development, includ-ing bosutinib (SKI-606 , Wyeth Pharmaceuticals, NJ, USA), a dual BCR-ABL and SFK inhibitor currently in phase II and III trials. Bosutinib is 30-fold more potent than imatinib against BCR-ABL and inhibits Src ki-nases, but not c-KIT and PDGFR.56 In a phase II trial, CCyR rates after a median 8 months of treatment in patients who had received imatinib (but not dasatinib or nilotinib) were 29% in imatinib-resistant patients and 50% in imatinib-intolerant patients.57 INNO-406 is also a dual BCR-ABL/SFK inhibitor, with activity against PDGFR and c-Kit.58 Moreover AP24534 is an orally available multiple TKI that potently inhibits the enzymatic activity of BCR-ABL-T315I, the native enzyme and all other tested variants. Not all effective therapies for CML are kinase inhibitors. Omacetaxine is a first-in-class cetaxine with clinical activity against Ph+ CML and a mechanism of action independent of tyrosine kinase inhibition.59

Bone marrow transplantAccording to the ELN recommendations, an alloge-neic stem cell transplant (allo-SCT) is suggested for

Table 2. Phase II trials in patients who failed or were intolerant to first-line imatinib therapy.43-45,50-52

Cytogenetic Response (%) Overall survival

Major CompleteN D

N D N D

CML Chronic 58 59 42 49 91% (18 months) 96% at 15 months

CML Accelerated 32 64 19 39 82% (12 months) 76% at 10 months

CML Blastic 40Ly:33

29Ly:26

42% (12 months)Ly: 11.8 months

My:52 My:46 My: 5.3 months

D=dasatinib; N=nilotinib; Ly=Lymphoblastic; My=Myeloblastic

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patients in AP or BP or with the T315I mutation and for patients who experience failure on second-line TKIs.16 Allo-SCT is also a significant option in the patients who have a suboptimal response to da-satinib or nilotinib second line (Table 3). Treatment recommendations are different for patients who are referred in AP or BP. These patients should preferen-tially receive an allo-SCT, if eligible, after a pretreat-ment with imatinib 600 or 800 mg if TKI naive, or with second-generation TKIs if imatinib resistant. In fact, prior imatinib therapy is not exclusive to the ef-ficacy of ASCT, and indeed the two approaches may be favorably combined.60-62 The German CML-Study IV considered allo-SCT in three instances: early allo-SCT (in case of low EBMT score, high risk patients, or patient request); allo-SCT after imatinib failure or intolerance in first CP, and allo-SCT in second CP or accelerated phase or blast crisis. Overall survival rate at two years for the first group was 87.8%, for the second group was 93.8%, and for the last group 49.5%. By EBMT score, survival rates were 100% for risk score 0-2, 82.2% for risk score 3-4, and 43.5% for risk score 5.62

First-line therapy optimizationAccording to the IRIS and other studies,14,19,63 a no-table proportion of patients either do not respond ad-equately or do not achieve a durable response to ima-tinib. At 8 years, 45% of imatinib-treated patients had discontinued the IRIS study, including 14% for resis-tance and 5% for intolerance.14 To improve responses to imatinib, imatinib 400 mg was compared with ima-tinib 800 mg in the TOPS study, but the higher 12-month MMR and faster response with imatinib 800 mg did not confer a long-term survival.64 Randomized phase III trials are ongoing to compare imatinib monotherapy with combination treatment.65,66 In the French SPIRIT trial, patients were randomized 1:1:1:1 to receive imatinib 400 mg/day, imatinib 600 mg/day, imatinib 400 mg/day plus cytarabine, or imatinib 400 mg/day plus pegylated IFN. Twelve-month CCyR rates were 55%, 62%, 63%, 65%, respectively (dif-ferences not statistically significant);67,68 were ob-

served, whereas at 18 months MMR was 41%, 52%, 53%, and 62% (P=.0001), and CMR was 4%, 7%, 5% and 15%, respectively (P=.0013), with a potential advantage for the imatinib plus IFN arm. However, further follow-up is needed to establish whether these early differences will confer a long-term survival ad-vantage. Overall, 45% of the patients discontinued IFN during the first 12 months, with higher rates of MMR, optimal molecular response and undetect-able minimal residual disease in patients who were treated with IFN for more than 12 months. A similar study in more than 1300 patients is being performed by the German CML Study Group (Study IV), with patients randomized to imatinib 400 mg/day or 800 mg/day alone, or imatinib 400 mg/d in combina-tion with IFN.69 In the imatinib 400 mg, imatinib 800 mg, and imatinib 400 mg plus IFN arms, 12-month CCyR rates were 52%, 65%, and 51%, respectively, and 12-month MMR rates were 30%, 54% and 35%, respectively. After 5 years of follow-up, no difference was reported between arms in PFS or OS, as seen in the TOPS study.69 Following the success of nilotinib and dasatinib in the second-line setting, both drugs have been assessed in phase 2 studies with newly diag-nosed CML patients. Sixty-two newly-diagnosed CP CML patients were treated with dasatinib 50 mg twice daily (BID) or 100 mg once daily (QD) at the MD Anderson Cancer Center, with a median follow-up of 24 months. Most patients achieved a rapid CCyR (94% at 6 months), with a cumulative CCyR ratio of 98%. After 12 and 18 months, a MMR was achieved by 71% and 79% of patients.70 Two phase II studies demonstrated substantial activity of nilotinib 400 mg BID in newly diagnosed CP CML patients. Among 61 patients treated at the MD Anderson Cancer Center with nilotinib with a median follow-up 17 months (range 1-43) MCyR was achieved by 96% of patients at 6 months, with a cumulative CCyR of 98%, and after 12 and 18 months, 81% and 79% had achieved a MMR.71 Seventy-three newly-diagnosed CP-CML pa-tients were treated with nilotinib in Italy, with a CCyR of 78% at 3 months, 96% at 6 and 12 months and MMR rates were 52%, 66%, and 85%, respectively.72,73 The toxicity profile with dasatinib and nilotinib front line was favourable. In order to perform a full compar-ison of imatinib and next-generation TKIs, random-ized phase III trials have been initiated. In CA180-056 (DASISION), an international trial, patients are randomized to receive either dasatinib 100 mg QD or imatinib 400 mg QD. CAMN107A2303 (ENESTnd) is an international phase III trial with 3 arms: 846 CP CML patients were randomized to receive nilotinib

Table 3. Suboptimal response to dasatinib or nilotinib second line.16

Suboptimal response

3 months minor CyR

6 months PCyR

12 months less than MMR

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300 mg BID vs nilotinib 400 mg BID vs imatinib 400 mg/day.74 Preliminary data from ENESTnd have shown that nilotinib (300 mg or 400 mg BID) was superior to imatinib, with a significantly higher 12-month CCyR (80% [P<.0001] vs 78% [P=.0005] vs 65%), MMR (44% [P<.0001] vs 43% [P<.0001] vs 22%) and, most importantly, a significantly lower rate of progressions (0.7% vs 0.4% vs 3.9%). Bosutinib has also been proposed as first line in a phase III trial of bosutinib vs imatinib.

ConclusionsCML is an important model for understanding on-cologic diseases. It is the first malignant disease for which a direct genetic link has been found and there has been much recent progress in understanding the pathophysiology and finding a cure. Targeted agents

have significantly improved the prognosis in CML. Imatinib represented a significant turning-point in the natural history of the disease and in patient sur-vival. However, a considerable proportion of patients (up to 45%) discontinue imatinib due to resistance or intolerance. The achievement of CCyR at 12 months predicted for a low risk of events or progression in the long term and the achievement a MMR represents a ‘safe haven’ for CML patients. In fact, no patients with MMR at 12 months progressed to AP/BC. Targeted therapies with TKIs in CML need molecular evalua-tions throughout the whole time of therapy to maxi-mize the chance of response for patients with lack of response or suboptimal response. The recent avail-ability of highly potent TKIs has further improved the outcome of many patients. The advent of novel agents, with the possibility of combining drugs with

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Efficacy and Safety of Bosutinib (SKI-606) in Patients with Chronic Phase (CP) Ph+ Chronic Myelogenous Leu-kemia (CML) with Resistance or Intolerance to Imatinib. ASH Annual Meeting Abstracts. 2008;112:1098-.58. Craig AR, Kantarjian HM, Cortes JE, et al. A phase I study of INNO-406, a dual inhibitor of Abl and Lyn kinases, in adult patients with Philadelphia chromo-some positive (Ph+) chronic myelogenous leukemia (CML) or acute lympho-cytic leukemia (ALL) relapsed, refractory, or intolerant of imatinib. J Clin Oncol (Meeting Abstracts). 2007;25:7046-.59. Cortes J, Talpaz M, Deininger M, et al. A Phase 1 Trial of Oral AP24534 in Patients with Refractory Chronic Myeloid Leukemia and Other Hematologic Malignancies: First Results of Safety and Clinical Activity against T315I and Resis-tant Mutations. ASH Annual Meeting Abstracts. 2009;114:643-.60. Lee SJ, Kukreja M, Wang T, et al. Impact of prior imatinib mesylate on the outcome of hematopoietic cell transplantation for chronic myeloid leukemia. Blood. 2008;112:3500-3507.61. Oehler VG, Gooley T, Snyder DS, et al. The effects of imatinib mesylate treat-ment before allogeneic transplantation for chronic myeloid leukemia. Blood. 2007;109:1782-1789.62. Saussele S, Lauseker M, Gratwohl A, et al. Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) in the Imatinib-Era: High Survival Rate Following Allogeneic HSCT after Imatinib Failure: Results of the German CML Study IV. ASH Annual Meeting Abstracts. 2008;112:448-.63. Lucas CM, Wang L, Austin GM, et al. A population study of imatinib in chron-ic myeloid leukaemia demonstrates lower efficacy than in clinical trials. Leukemia. 2008;22:1963-1966.64. Pletsch N, Saussele S, Lauseker M, et al. Optimaization of Imatinib Therapy by Combination. 5 Year Survival and Response Results of the Pilot Phase of the Randomized German CML STUDY IV. ASH Annual Meeting Abstracts. 2009;114:862-.65. Gardembas M, Rousselot P, Tulliez M, et al. Results of a prospective phase 2 study combining imatinib mesylate and cytarabine for the treatment of Phila-delphia-positive patients with chronic myelogenous leukemia in chronic phase. Blood. 2003;102:4298-4305.66. Baccarani M, Martinelli G, Rosti G, et al. Imatinib and pegylated human re-combinant interferon-alpha2b in early chronic-phase chronic myeloid leukemia. Blood. 2004;104:4245-4251.67. Guilhot F, Preudhomme C, Guilhot J, et al. Significant Higher Rates of Unde-tectable Molecular Residual Disease and Molecular Responses with Pegylated Form of Interferon a2a in Combination with Imatinib (IM) for the Treatment of Newly Diagnosed Chronic Phase (CP) Chronic Myeloid Leukaemia (CML) Patients (pts): Confirmatory Results at 18 Months of Part 1 of the Spirit Phase III Randomized Trial of the French CML Group (FI LMC). ASH Annual Meeting Abstracts. 2009;114:340-.68. P. Rousselot CP, J. Guilhot, V. Dubruille, T. Lamy, M. Delain, H. Tilly, P. Cony-Mak-houl, C. Allard, H. Jardel, S. Cheze, F.X. Mahon, F. Guilhot. Molecular Responses Of The Spirit Phase Iii Trial Of The French Cml Group Comparing Imatinib 400 Mg To Higher Dose Imatinib Or Combination With Interferon Or Cytarabine For The Treatment Of Newly Diagnosed Chronic Phase (Cp) Chronic Myeloid Leukaemia (Cml) Patients. Haematologica. 2009;94[suppl.2]:abs. 1093.69. Hehlmann R, Jung-Munkwitz S, Lauseker M, et al. Randomized Comparison of Imatinib 800 Mg Vs. Imatinib 400 Mg +/- IFN in Newly Diagnosed BCR/ABL Positive Chronic Phase CML: Analysis of Molecular Remission at 12 Months; The German CML-Study IV. ASH Annual Meeting Abstracts. 2009;114:339-.70. Cortes JE, Jones D, O’Brien S, et al. Results of Dasatinib Therapy in Patients With Early Chronic-Phase Chronic Myeloid Leukemia. J Clin Oncol;28:398-404.71. Cortes JE, Jones D, O’Brien S, et al. Nilotinib As Front-Line Treatment for Patients With Chronic Myeloid Leukemia in Early Chronic Phase. J Clin On-col;28:392-397.72. Rosti G, Palandri F, Castagnetti F, et al. Nilotinib for the frontline treatment of Ph+ chronic myeloid leukemia. Blood. 2009;114:4933-4938.73. Rosti G, Castagnetti F, Palandri F, et al. Nilotinib 800 Mg Daily as Front-line Therapy of Ph + Chronic Myeloid Leukemia: Dose Delivered and Safety

reviewcml


Profile for the GIMEMA CML Working Party. ASH Annual Meeting Abstracts. 2009;114:2205-.74. Saglio G, Kim D-W, Issaragrisil S, et al. Nilotinib Demonstrates Superior Ef-

ficacy Compared with Imatinib in Patients with Newly Diagnosed Chronic My-eloid Leukemia in Chronic Phase: Results From the International Randomized Phase III ENESTnd Trial. ASH Annual Meeting Abstracts. 2009;114:LBA-1.

original report

Journal of Appplied Hematology 201028

Acute leukemias account for approximately 10% of all human cancers.1 Acute leukemia is classi-fied into two categories based on the lineage of

the blast cells: acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML).

The diagnosis of AML is enhanced by identification of characteristic cluster of designation (CD) antigens expressed on the surface and/or in the cytoplasm of the myeloblasts that can be identified by flowcytometry.

CD117 is coded by the c-kit proto-oncogene on chromosome 4(q11) which is a tyrosine kinase trans-membrane receptor (TKR). CD117 is present on ap-proximately 4% of normal bone marrow mononuclear cells and in AML, but rarely in ALL.2 During malignant proliferation, poteolytic cleavage of cell surface recep-tor results in soluble CD117 (sCD117).

Elevated levels of sCD117 have been reported in nonmalignant and malignant conditions.3-6 Kamel, et.al had shown elevated sCD117 in AML patients and sug-gested its possible use as a tumor marker.6

The aim of this study was to investigate the diagnos-tic and prognostic role of cell surface CD117 antigen

Significance of sCD117 as a Tumor Marker in Acute Myeloid Leukemia

Huda AlSayed,* Faisal Rawas,** Amal AlSuraihi,*** Fahad AlShareef,**** Lila Bashawri,* Tarek Owaidah**

From the *King Faisal University, Dammam, **Department of Pathology & Laboratory Medicine, ***Department of Pediatric Hematology Oncology & ****Department of Oncology, King Faisal Specialist Hospital & Research Center, Riyadh, Saudi Arabia

Correspondening author:

Tarek Owaidah, MDDepartment of Pathology and Laboratory Medicine (MBC 10)King Faisal Specialist Hospital and Research Center P.O. Box 3354, Riyadh 11211, Saudi ArabiaT: +966-1-442-4328; F: [email protected]

Background: The diagnosis of acute myeloid leukemia (AML) is en-

hanced by identification of antigens that are expressed in the cytoplasm and/or

on the surface of myeloblasts. Anti-CD117 monoclonal antibody is useful in the

immunophenotyping of acute leukemia cells that are committed to the myeloid

lineage regardless of the subtype. The proteolytic cleavage of this cell surface

receptor results in soluble CD117.

Materials and Methods: We have investigated the diagnostic and

prognostic role of sCD117 in Saudi patients with AML. Detailed immunopheno-

typing of the bone marrow blast cells of 20 patients was performed by flowcy-

tometry, and the serum c-kit level was measured using the ELISA assay.

results: CD117 surface expression showed no significant correlation with

sCD117, but there was a significant elevation of sCD117 pre-chemotherapy and

a significant decrease post-chemotherapy.

conclusion: sCD117 was had good diagnostic value for early AML cas-

es and a prognostic value post-chemotherapy. This suggests the usefulness of

sCD117 as a tumor marker in AML patients.

keywords: Immunophenotyping, sCD117, AML, tumor marker.

and the sCD117 form in sera of AML patients based on a local patient population (Saudi Arabia), and to eval-uate its role in monitoring post-chemotherapy patients.

Materials and Methods

Study GroupA cohort of 20 cases out of 75 acute leukemias that presented to King Faisal Specialist Hospital and Research Center, Riyadh (KFSHRC) from January 2007 to July 2007 was studied for CD117. The diagnosis of acute leukemia was based on clinical history, clinical ex-amination, complete blood picture and bone marrow smear examination and evaluation. Prior to undertak-ing this study, ethical approval was obtained from the Local Ethical Committee and Research Advisory of KFSHRC. Patients or guardians in the case of children consented to be in the study.

Samples Collection and ProcessingBlood and Blood Marrow samples were collected us-ing a convenient nonrandom sampling technique from

original report


AML patients who were positive for CD117 on immu-nophenotyping. Peripheral blood (PB) or bone mar-row (BM) samples from each patient were collected in sodium heparin or EDTA tubes. CBC was performed on Cell Dyne 4000 (Abbott). A sample of whole blood was collected from each patient in a serum separator tube (SST), which was allowed to clot for 30 minutes before centrifugation for 15 minutes at 1000g. Serum was then separated and kept in a deep freezer (-80ºC) for subsequently measuring the sCD117 by ELISA. A second sample was collected from all patents at day 28 post-induction chemotherapy and at 3 months or more during follow up.

A total of 50 normal Saudi donors of different age groups and sex were used as controls for the ELISA method.

ImmunophenotypingThe diagnosis of acute leukemia was confirmed us-ing detailed immunophenotyping including 32 mono-clonal antibodies (obtained from Becton Dickinson, San Jose, CA, USA) performed on each bone mar-row sample at KFSHRC, Riyadh using FACSCalibur instrument (Becton Dickinson, San Jose, CA, USA). The monoclonal antibodies included: CD2, CD3, CD4, CD7, CD8, CD9, CD10, CD19, CD20, CD22, CD25, CD38, CD79a, kappa and lambda light chains, terminal deoxynucleotide transferase enzyme (TdT); (T cell and B cell lymphoid markers), CD13, CD33, CD117 and myeloperoxidase enzyme (MPO); (my-eloid cell markers), CD11b, CD11c, CD14, CD15, CD64, CD65 (monocytic markers), others include CD34, HLA-DR, CD58, CD66 and NG2, CD45 been used for gating. Three- and four-color staining meth-ods were used through the following combinations of fluorochrome-conjugated monoclonal antibodies: flu-orescein isothiocyanate (FITC), phycoerythrin (PE), peridin clorophyll protein (PerCP) and allophycocya-nin (APC) directed against surface antigens.

An aliquot from the BM aspirate was washed three times using phosphate buffered saline (PBS) with 1% bovine serum albumin (BSA) and 0.1% sodium azide (NaN3). After the last wash the aspirate was filtered and the count adjusted to 0.5-1×109/L of which 50 uL was placed in each tube and incubated with the appropriate combination of monoclonal antibodies for 15-20 minutes in the dark at room temperature. After the incubation period, the red cells were lysed using lysing kit (Immunolyse from Beckman Coulter Company) followed by a single washing step. The cells were re-suspended in 0.5 mL of 1% paraformal-dehyde (PFD) and analyzed by a flowcytometer in-

strument. In addition, the expression of MPO, TdT, CD3, IgG, IgM and CD79a was also performed at the cytoplasmic level. For staining of the cytoplasmic antigens, cells were permeabilized using Intra-Prep Permeabilization Reagent Kit (from Beckman Coulter Company) according to the recommendations of the manufacturer.

Data Acquisition and Analysis of FlowcytometryFlowcytometry was performed on a FACS Calibur (Becton Dickinson) equipped with a 15.10±5% mW Argon laser. Non-specific antibody binding was monitored using an FITC and/or PE conjugated mouse IgG1 isotype control (obtained from Becton Dickinson). The forward scatter (FSC) and the side scatter (SSC) parameters were adjusted so that cells with high FSC and high SSC could be observed. The cell acquisition rate was adjusted to approximately 500-1000 events/second. Small particles up to 7 µm were excluded by FSC gate. A fluorescence threshold was set at 100 so that the background fluorescence was not greater than 0.5%. Data acquisition and analy-sis were performed using Cell Quest Pro software (Becton Dickinson) following daily calibration of the instrument using Calibrite Beads (Becton Dickinson) and a two-level control for CD3/CD8/CD45/CD4 and CD3/CD16+56/CD45/CD19 to ensure the con-sistency in the instrument function. Data analysis was performed by Cell Quest Pro software using CD45 versus side scatter as a gating strategy to gate on the blast cells. Gating is critical to isolate the abnormal cells because the leukemic phenotype should be de-termined on as pure a population as possible. The fre-quency of positive cells was determined after channel-by-channel subtraction of the background (isotype control). The criteria for scoring the positivity of blast cells for a given marker were defined as follows: posi-tive if more than 20% of the cells had expression of the marker, and negative if less than 20%.

Measurement of sCD117 by ELISAThe frozen serum of the 20 patients as well as healthy control samples were assayed for sCD117 by quantita-tive ELISA kit (Solid Phase Sandwich Enzyme Linked Immunosorbent Assay) using Quantikine Human SCF sR/c-kit Immunoassay (obtained from R&D Systems, Inc. Company, Minneapolis, USA). In this assay a monoclonal antibody specific for CD117 has been pre-coated onto a microplate. Standards, controls, and serum samples, 100 µL each, were pipetted into the wells and coated by the immobilized antibody during the 2-hour incubation period at room temperature for

sCD117 IN AML

original report


binding sCD117, if any. After washing off any un-bound substances, an enzyme-linked monoclonal an-tibody specific for sCD117 was added and incubated for another 2 hours at room temperature. Following a wash to remove any unbound antibody-enzyme re-agent, a substrate solution was added and color de-veloped within 30 minutes incubation in proportion to the amount of sCD117 bound. The color develop-ment was stopped by stopping solution and the in-tensity of the color was measured; the optical density of each well was determined within 30 minutes us-ing a microplate reader set to 450 nm using ETI Max 3000 ELISA reader (from DiaSorin company, Italy). According to the manufacturer, the sensitivity of this method was evaluated and the minimum detectable level of sCD117 ranged from 0.057 ng/mL. As to the specificity of this assay method, it showed no signifi-cant cross-reactivity or interference. The mean was used as a cut-off value as there was no other meth-od to compare the sensitivity and accuracy with the ELISA method used in the study.

Statistical AnalysisCollected data was revised and entered into the statis-tical software (SPSS version 10). Data are described by percentage, mean, range and by graphs. Data was analyzed using student t-test, paired t-test, one-way ANOVA and simple correlation.

ResultsTable 1, depicts the characteristics of the 20 patients studied: 16 AML cases and 4 biphenotypic acute leu-kemia (BAL) cases. There were 13 adults (>14 years) with ages ranging from 18 to 53 years and 7 children with ages ranging from 2 to 10 years with an overall median age of 21 years; 9 of the patients were fe-males and 11 were males. The most frequent clinical presentation was lymphadenopathy in 9 (45%) pa-tients followed by hepatomegaly in 5 (25%) patients, while 6 (30%) of the patients presented with bleeding symptoms. More than 50% of the cases had fever at presentation. The WBC count at presentation ranged from 0.84 to 96.8×109/L with a mean of 23×109/L. The blast count at presentation ranged from 20-95% with a mean of 69%. The most common FAB classification of AML was M2 in 5 (25%), while the other cases were as follow: M3 (n=3); M1 (n=3); M4 (n=2), M5 (n=1), M0 (n=1). The BAL cases were B/myeloid in 2 patients and T/myeloid in the other 2 patients. Four cases (20%) had a normal karyotype, 4 (20%) cases had various chromosomal deletions and 4 (20%) had a complex karyotype. All patients, ex-

cept 2 (10%), had achieved complete remission by day 28 post-chemotherapy.

Surface markers expressionAs shown in Table 2, all cases were positive for CD117 surface antigen. The myeloid antigens CD33, CD13, CD15 and MPO were expressed in 19 (95%), 17 (85 %), 9 (45%) and 11 (55%) respectively. CD34 was expressed in 14/20 (70 %) of cases. B-cell mark-ers, CD19 was positive in 8/20 cases (40%), CD22 and CD79a were positive in 2 cases (10 %). T-cell markers, CD2 and CD3 were positive in 2/20 cases (10%).

The expression of surface CD117 and soluble CD117The mean fluorescence intensity (MFI) of surface CD117 at diagnosis was 434.5 MFI ranging from 51-1193 MFI; 15 cases (75 %) had MFI of <500 (dim expression), while 5 cases (25 %) had MFI of >500 (moderate to bright expression). (Table 3).

The serum level of sCD117 in 50 normal sub-jects was of 7.2 to 17.5 ng/mL with a mean value of 12 ng/mL in the patient group. The concentra-tion of sCD117 at diagnosis ranged from 3.3 to 50 ng/mL with a mean of 20.6 ng/mL. Five cases (25 %) showed a concentration of sCD117 <12 ng/mL, while 15 cases (75 %) showed concentration of sCD117 more than 12 ng/mL at time of diagnosis (Table 4). No significant correlation was detected between MFI of surface CD117 and sCD117 be-fore chemotherapy (P= .054). All the 15 cases with sCD117 level in their serum at presentation more than the cut off (>12) were studied for sCD117 on day 28 post-chemotherapy. All these cases showed decreased sCD117 concentration in their serum, with sCD117 concentration was ranging from 1.8 to 18.9 ng/mL (P=.0001). (Table 3).

Eighteen patients (90 %) showed complete remis-sion on day 28 defined by BM blasts <5%. All these patients showed a decrease in the level of sCD117 concentration compared to the level at diagnosis. The MFI of CD117 for the two patients with no remission and another two with suspected residual leukemia was performed at day 28. In comparison with the changes that had been seen with sCD117, there was no clear relationship between the level at presentation and at end of induction.

DiscussionManagement of acute leukemia requires risk strati-fication to tailor the chemotherapy according the disease severity. Most of the risks stratifying factors

sCD117 IN AML

original report


tab

le 1

. Sum

mar

y of

clin

ical

and

labo

rato

ry c

hara

cter

istic

s.

Se

rial

#A

cute

Leu

kem

ia s

ubty

pes

Sex

Age

Sign

s/Sy

mpt

oms

WBC

(x

109 /L

)BL

AST

%K

aryo

typi

ng

1

AM

L M

1F

50H

epat

omeg

aly,

Lym

phad

enop

athy

, fev

er

61.7

93

Nor

mal

2

AM

L M

2M

41Bl

eedi

ng/B

ruisi

ng L

ymph

aden

opat

hy, f

ever

18.9

85

Mon

osom

y 8

3

BAL

(B-M

yelo

id)

F10

Lym

phad

enop

athy

0.84

90

Com

plex

Kar

yoty

pe

4

AM

L M

0M

25 B

leed

ing/

Brui

sing

Lym

phad

enop

athy

, fev

er25

.170

de

l(5)(

q31)

,del

(16)

(q22

)[16

]

5

A

ML

M4

M6

Hep

atom

egal

y, Ly

mph

aden

opat

hy, f

ever

4.88

30

Mon

osom

y 7

6

AM

L M

4F

29Ly

mph

aden

opat

hy, f

ever

55.3

95

Nor

mal

7

BAL

( B-

Mye

loid

)M

2Ly

mph

aden

opat

hy, f

ever

1.91

90

inv(

19)(

p13.

1q13

.1)[

14]

8

AM

L M

5F

53Fl

u-lik

e sy

mpt

oms

79.3

90

del(5

)(q3

1),d

el(7

)(q3

1)

9

AM

L M

3 (A

PL)

F47

Gen

eral

wea

knes

s1.

6650

t(

15;1

7)

10

AM

L M

3 M

32Bl

eedi

ng/B

ruisi

ng L

ymph

aden

opat

hy11

.390

t(

15;1

7)

11

AM

L M

1M

4H

epat

o-sp

leno

meg

aly,

feve

r7.

4320

C

ompl

ex K

aryo

type

12

BAL

( T-M

yelo

id)

M20

feve

r1.

4290

C

ompl

ex K

aryo

type

13

AM

L-M

5M

52Bl

eedi

ng/B

ruisi

ng26

.680

t(

3;21

)

14

AM

L M

2F

18Bl

eedi

ng/B

ruisi

ng,fe

ver

31.1

30

t(8;

21;9

)(p2

3;q2

2;q2

2)[1

4]/

46xx

[1]

15

BAL

( T-

Mye

loid

)M

9H

epat

o-sp

leno

meg

aly

Lym

phad

enop

athy

5.85

90

Nor

mal

16

AM

L M

1M

21fe

ver

9.64

90

del(7

)(q2

2)[6

],del

(3)(

q22q

25)

[5]

17

AM

L M

3 F

25Bl

eedi

ng/B

ruisi

ng fe

ver

96.8

95

Nor

mal

18

AM

L M

2F

26fe

ver

7.52

30

del(9

q)(q

12q2

2)[1

5]

19

AM

L M

2F

2R

epea

ted

vom

iting

16

.150

t(

8;21

)

20

AM

L M

2M

7he

pato

meg

aly

2.6

40

Com

plex

Kar

yoty

pe

sCD117 IN AML

original report


tab

le 2

. Flo

wcy

tom

etry

imm

unop

heno

typi

ng r

esul

ts S

how

ing

Ant

igen

exp

ress

ion

of d

iffer

ent A

ML

subt

ypes

:

Se

rial

#D

iagn

osis

CD

2C

D3

CD

4C

D7

CD

10C

D11

cC

D11

bC

D13

CD

14C

D15

CD

19C

D22

CD

33C

D34

CD

79a

CD

117

TdT

MPO

1

AM

L M

1-

--

--

++

+-

--

-+

--

+-

+

2

AM

L M

2-

-+

--

++

+-

+-

-+

--

+-

+

3

BAL

(( B

-Mye

loid

)-

--

+-

--

--

+-

++

++

--

4

AM

L M

0-

--

+-

++

+-

++

++

+-

+-

-

5

AM

L M

4-

--

--

--

+-

--

-+

+-

+-

-

6

AM

L M

4-

--

--

++

++

+-

-+

--

+-

+

7

BAL

( B

-Mye

loid

)-

--

-+

--

+-

-+

++

++

+-

-

8

AM

L M

5-

--

--

-+

+-

+-

-+

-+

-+

9

AM

L M

3 -

-+

+-

--

+-

-+

-+

+-

+-

+

10

A

ML

M3

--

--

--

-+

--

--

+-

-+

-+

11

AM

L M

1-

--

--

-+

+-

--

-+

+-

+-

-

12

BAL

( T-

Mye

loid

)-

+-

+-

++

+-

+-

-+

+-

++

+

13

AM

L-M

5+

-+

--

++

++

--

-+

+-

+-

-

14

AM

L M

2-

--

--

+-

+-

++

-+

+-

+-

+

15

BAL

( T-

Mye

loid

)+

++

+-

-+

+-

+-

--

+-

++

-

16

AM

L M

1-

--

+-

--

+-

--

-+

+-

+-

-

17

AM

L M

3 -

--

--

+-

--

--

-+

-+

-+

18

AM

L M

2-

--

--

--

+-

++

-+

+-

+-

-

19

AM

L M

2-

--

--

+-

--

-+

-+

+-

+-

+

20

A

ML

M2

--

--

-+

-+

-+

+-

++

-+

-+

sCD117 IN AML

original report


are based on molecular and genetic changes. The diagnosis of acute leukemia is enhanced by “immu-nophenotyping” that is performed by flowcytometry which can be used as a tool to monitor the response to treatment, however, this technique is demanding. It requires a skillful technologist to detect the minimal amount of residual disease and has to be done on a fresh sample.7

CD117 is considered to play a crucial role in he-matopoiesis.8,9 It was first identified in a subgroup of AML using monoclonal antibody against AML blast cells. CD117 monoclonal antibody is claimed to be especially useful in the immunophenotyping of acute

myeloid leukemia with specificity and sensitivity of CD117 for AML as 100% and 69%, respectively.10 Although CD117 has a high specificity, it is not as sen-sitive as CD13 or CD33 for detecting myeloid leuke-mic processes. The prognostic value of CD117 has been suggested in adult AML, but was not of value in pediatric AML.11,12

CD117 can be proteolytically shed from the cell surface to produce soluble c-kit. Soluble CD117 levels have been determined in a number of clinical condi-tions. Among the hematopoietic neoplasms examined, acute myeloid leukemia subtypes M1 and M2 were associated with a modest increase in sCD117 levels.13

table 3. The mean fluorescence intensity (MFI) of surface CD117 and concentration of sCD117 and during follow-up.

Serial # MFI of CD117At diagnosis

ConcentrationOf CD117In Serum.

At diagnosis

Remission Status

MFI of CD117At 28 days post-chemotherapy

Concentrationof CD117In Serum

At 28 days post-chemotherapy

Concentrationof CD117In Serum

At 3 months or more post- chemotherapy

1 86 10.4 263 2.9 NA**

2 818 16.4 Remission NA* 6.8 10

3 51 9.3 Remission NA* 2 16

4 316 42.6 Remission NA* 18.9 11

5 162 10.2 Remission NA* 1.7 NA**

6 289 3.3 Remission NA* 2.8 NA**

7 57 15 Remission 55 4.2 12.8

8 266 17.5 Remission 385 6.6 NA**

9 974 13.6 Remission NA* 7.4 10.8

10 496 18.3 No Remission 405 7.8 8.7

11 122 9.8 Remission NA* 5.7 11.3

12 413 18.9 Remission NA* 12.5 NA**

13 419 14.7 Remission NA* 8.5 4

14 416 29.3 Remission NA* 11.5 16.5

15 276 29.5 No Remission NA* 6.4 11.6

16 1193 >50 Remission NA* 1.8 7.2

17 436 37.4 Remission NA* 7.8 6.5

18 455 22.3 Remission 138 12.8 13.6

19 595 27.3 Remission NA* 14.7 16.7

20 850 16.7 Remission NA* 7.4 8.5

MEAN 434.5 20.6 263 7.5 10.9

RANGE 51 to1193 3.3 to >50 55 to 405 1.8 to 18.9 4 to 16.7

NA*: not done because it is not indicated (no residual blasts). NA**: not done (no sample collected).

sCD117 IN AML

original report


Figure 1. Mean concentration of the sCD117 before and after chemotherapy in different FAB subtypes compared to control.

CD117 expression does not seem to be a reliable pre-dictor of FAB AML subtype. In the present study, the highest frequency of CD117 expression was found in the AML M2 category, which is similar to previously reported12,13 studies and may indicate over expression in more immature myeloid leukemia.

The level of sCD117 in normal controls varies, al-though in a previous study it was as high as 719 ng/mL.14 In our study the normal range was 7.2 to 17.5 ng/mL which was in agreement with another study reported from Egypt with a closer range 4.05-5.45 ng/mL, which suggests some correlation with ethnic-ity.6 In this study sCD117 was detected in all 20 cases, but using a cut off value of 12 mg/mL only 15 sub-jects showed an elevated level at diagnosis, which had dropped in all patients at day 28 of chemotherapy.

Despite the high specificity of CD117 in acute myeloid leukemia, the intensity of surface CD117 ex-pression was dim in 75% of the cases in our study. (The correlation between cellular and soluble forms of CD117 was not significant (P=.054). The mean con-centration of sCD117 before chemotherapy was 20.6 ng/mL while its mean concentration after chemother-apy was 7.5 ng/mL, and the difference was statistically

significant (P<.0001). No significant correlation was detected between

the mean fluorescence intensity of surface CD117 and sCD117 before chemotherapy. This finding is the same as that reported by Kamel et al.6

There was no significant difference in the level of sCD117 found among the different FAB subtypes of AML whether before or after chemotherapy. CD117 expression does not seem to be a reliable predictor of FAB AML subtype. This finding is compatible with the study of Kamel et al.6 In contrast, Rajima, et.al. reported an increased level of sCD117 in M1 and M2 cases.15

In conclusion, although the study group is small, we could show that surface expression of CD117 in comparison to the soluble CD117 has no significant correlation. Yet sCD117 could be considered a good indicator for response to therapy as its level in the se-rum dropped significantly post-chemotherapy in most cases, but it could not detect the presence of residual leukemia nor can it be used as a predictor of relapse. (Further studies have to be done to evaluate the signifi-cance of sCD117 as in monitoring AML patients on chemotherapy marker for remission).

sCD117 IN AML

original report


1. Hoffbrand AV, Pettit JE, Moss PAH. Essential Haematology.5th edition. Oxford. London Edinburgh Boston; 2006: Chapter 12:157-173.2. Wells SJ, Bray RA, Stempora LL, Farhi DC. CD117/CD34 expression in leuke-mic blasts. J Clin Pathol .1996; 106(2): 192-195.3. Akin C, Schwartz LB, Kitoh T, Obayoshi H, Worbec AS. Soluble stem cell factor receptor (CD117) and IL-2 receptor alpha chain (CD25) in plasma of patients with mastocytosis, relationship to disease severity and bone marrow pathology. Blood. 2000; 96(4): 1267-1273.4. Makowska JS, Cieslak M, Kowalski ML Stem cell factor and its soluble receptor (c-kit) in serum of asthmatic patients- correlation with disease severity. BMC Pulm Med. 2009 Jun 1;9:27. 5. Takeshima H, Kuratsu J. A review of soluble c-kit (s-kit) as a novel tumor marker and possible molecular target for the treatment of CNS germinoma. Surg Neurol. 2003 Oct;60(4):321-4; discussion 324-5.6. Kamel A, Sharkawy N, Khalaf M, Galal S, Ghaleb F, Ghazaly T.CD117 Expres-sion and Serum CD117 in Acute Non Lymphoid Leukemia. Journal of the Egyp-tian Nat. Cancer Inst.2002; 14(3): 243-249.7. Langerbrake C, Creutzig U, Dworzak M, Hrusak O, Mejstrikova E, Griesinger F, Zimmermann M, Reinhardt D. Residual Disease Monitoring in Childhood Acute Myeloid Leukemia by Multiparameter Flow Cytometry: The MRD- AML-BFM Study Group. Journal of Clinical Oncology. 2006; 24(22):3686-3692.8. Ashman LK. The biology of stem cell factor and its receptor c-kit. Int J biol.1999; 31(10): 1037-1051.9. Di Noto R, Lo Pardo C, Schiavone EM, Manzo C, Vacca C, Ferrara F, Del

vcchio L. Stem cell factor receptor (c-kit, CD117) is expressed on blast cells from most immature types of acute myeloid malignancies but is also a char-acteristic of a subset of acute promyelocytic leukemia. British J.Haematol.1996; 92(3): 562-564.10. Nomdedeu JF, Matue R, Altes A, L Iorente A, Rio C, Estivill C, Lopez BJ, Rubiol E. Enhanced myeloid specificity of CD117 compared with CD13 and CD33.Leuk Res .1999; 23(4): 341-347. 11. Ashman LK, Roberts MM, Gadd SI, Cooper SJ, Juttner CA: Expression of a 150-kD cell surface antigen identified by monoclonal antibody YB5.B8 is as-sociated with poor prognosis in acute nonlymphoblastic leukaemia. Leuk Res 12:923, 1988.12. Smith FO, Broudy VC, Zsebo KM, Lampkin BC, Buckley CV, Buckley JD, Opie T, Woods WG, Hammond GD, Bernstein ID. Cell surface expression of c-kit receptors by childhood acute myeloid leukemia blasts is not of prognostic value: a report from the Childrens Cancer Group. Blood. 1994 Aug 1;84(3):847-52.13. Christine P. Hans, William G. Finn, Timothy P. Singleton, Bertram Schnitzer and Charles W. Ross. Usefulness of Anti-CD117 in the Flow Cytometric Analysis of Acute Leukemia. Am J Clin Pathol 2002;117:301-305.14. Makowska JS, Cieslak M, Kowalski ML. Stem cell factor and its soluble recep-tor (c-kit) in serum of asthmatic patients- correlation with disease severity. BMC Pulm Med. 2009 Jun 1;9:27.15. Tajima F, Kawatani T, Ishiga K, Nanba E, Kawasaki H. Serum soluble c-kit recep-tor and expression of c-kit protein and mRNA in acu te myeloid leukemia. Eur J Haematol. 1998 May;60(5):289-96.

References

sCD117 IN AML

original report


Non-Hodgkin lymphomas (NHLs) are het-erogeneous tumors with different patterns of clinical behavior and response to che-

motherapy.1 The treatment strategy is based on the independent prognostic factors of the International Prognostic Index (IPI) namely B symptoms (fever, night sweats, weight loss), performance status, age, serum lactate dehydrogenase (LDH) level, serum ß2 microglobulin, tumor bulk, and number of nodal and extranodal sites of disease.2,3. The IPI is a widely ac-cepted prognostic tool.3 However, some patients do not respond well to the standard therapy and thus more potent treatments are used. The information

Serum CA-125 correlates with staging, prognosis and survival of Non-Hodgkin Lymphoma (NHL)

Tarek Ashour,* Mohamad Qari **

From the *Department of Hematology, College of Medicine, Umm Al Qura University and the **Dept of Hematology, College of Medicine, King Abdulaziz University


Dr. Mohamad QariHematology Dept.College of MedicineKing Abdulaziz UniversityPO Box 80215Jeddah 21589Saudi [email protected]

Background: The clinical importance of the serological markers in NHL

is based on their role in staging, prognostic assessment and in monitoring NHL

patients pre- or post-treatment.

oBjective: To assess the correlation of CA-125 with the clinical and patho-

logic features of NHL. To evaluate its role in the staging, follow up of patients,

monitoring of treatment and whether it has independent predictive value on

survival of patients.

Patients and methods: Serum levels of CA-125 were determined in

50 patients with NHL. Serum samples were obtained from all patients at diagno-

sis, during chemotherapy and at remission. Thirty age- and sex-matched healthy

individuals were included as a control.

results: The serum levels of CA-125 were significantly increased when

compared to the control group (P<.05) with 48% patients having levels above the

median. The high values were significantly associated with stage III-VI disease. The

event-free survival (EFS) was significantly highest in the group with low sCA-125

(76.9%) versus 52.5% in the group with the high levels. The EFS was even better

when both sCA-125 and LDH were low.

conclusion: sCA-125 might be employed to aid in diagnosis, staging and

prognostic assessment of NHL. It can be used not only in monitoring the re-

sponse to primary therapy, but also in planning treatment duration. It has inde-

pendent influence on the survival of patients as proved by the Cox proportional

hazard test. Combination of both sCA-125 and sLDH could improve the prog-

nostication of patients.

keywords: Non Hodgkin’s lymphoma, Prognosis, serum CA-125, lactate

dehydrogenase, survival, tumor marker, staging,

derived from this novel prognostic factors combined with the previously known ones, should be added to the existing basic system, to establish treatment strate-gies.

The CA125 antigen is a glycoprotein expressed by epithelial ovarian cancer.4 CA125 serum levels are used to monitor response to therapy and for follow-up of patients with ovarian cancer. Serum levels may be elevated in many other malignant and nonmalignant diseases, including lymphoma.5,6

The aim of this work was to assess the serum levels of CA125 in correlation with the clinical and patho-logic features of NHL. Also, we evaluated its role

original reportCA-125 AND NHL


in the staging, follow up of patients , monitoring of treatment response, and whether it has independent predictive value on survival of patients.

Subjects and MethodsThis prospective controlled study was carried out at the Hematology/Oncology Unit, Ain Shams University Hospital in Cairo, Egypt. All of the patients who were newly diagnosed were informed on the nature of the study and consented to participation. These patients were enrolled from 2006-2008. Patients with any coex-isting disease like tuberculosis or HIV infection were excluded from the study

Reclassification of the patients according to the levels of sCA125 using the median as a cut off value was done. Also, the patients were divided into 4 groups by combination of sCA125 with sLDH (very low, low, intermediate and high risk group). Very low risk=Low sCA125+low sLDH, low risk=low sCA125+high sLDH, intermediate risk=high sCA125+low sLDH and high risk group=high sCA125 and high sLDH.

Group II: Control group: Thirty age- and sex-matched apparently healthy individuals with a mean age of 30±10.4 years and a median age of 25 years were chosen as a control from individuals free from known diseases.

At diagnosis all the patients were subjected to the following:

1. Full clinical history and physical examination.2. Plain chest x-ray, chest and abdominal computed

tomography and abdominal ultrasonography.3. Bone marrow aspiration and examination.4. Lymph node and trephine biopsy and histological

classification. All these investigations (1-4) were performed as a staging examination according to the Ann Arbor staging classification (8).

5. Routine investigations included a complete blood count and differential blood count on Coulter S plus Cell Counter (Coultronics, FI, USA). Total protein, albumin, uric acid and LDH on a Synchron CX7 (Beckman-Coulter, Nyon, Switzerland).

6. Immunophenotyping: Cell surface markers were evaluated by flow cytometry (FCM) on a Coulter EPICS XL (Coulter electronics, Healeh Fl, USA). The immunophenotyping was performed on whole blood, lysis of red blood cells was done by using IQ lyse (IQ products, Zernikepark, Groningen, The Netherland). The panels of FITC/PE-labeled monoclonal antibodies used are: CD5/CD19,CD10/CD20,CD23/CD19,CD103/CD22,CD25/CD19,Kappa/

CD19,CD19/Lambda, CD4/CD8,CD3/CD56 A sample was considered positive when >20% of cells showed the marker. Monoclonal anti-bodies were purchased from Coulter.

7. Cytogenetic analysis: bone marrow heparinized samples were taken aseptically and processed im-mediately following the direct preparation tech-nique (9).

8. Serum CA125 assay: it was determined by en-zyme immunoassay (EIA) supplied by Roche Diagnostics Products Ltd (Welwyn Garden City, Hertfordshire, USA) (10). The values were ex-pressed in µ/mL.

N.B.: The routine laboratory investigations and assay of CA125 were done for control subjects too.

Statistical analysisStatistical analysis was performed using an IBM per-sonal computer with statistical program SPSS version 8 for Windows. Differences between groups were eval-uated by the Mann-Whitney U test and the Wilcoxon ranked-sign test. Qualitative data are presented and the comparison between the two groups was done us-ing the chi- square test. Also, the multiple regression analysis and Spearman correlation were done. Survival analysis was done using the Kaplan-Meir method. A P value less than .05 was considered statistically sig-nificant. Multivariate analysis of the prognosis was performed using Cox proportional hazards regression model.

ResultsA total of 80 subjects were included and divided into 2 groups. Group I included 50 patients (29 males and 21 females) with a male-to-female ratio of 1.3:1. Their ages ranged from 23 to 70 years with a mean (SD) age of 46.7±10.6 years. These 50 patients included 26 with indolent type and 24 with aggressive lymphoma according to the revised European American classi-fication of lymphoid neoplasm (REAL) scheme (7). The indolent lymphoma included 14 with chronic lymphocytic leukemia (CLL), 2 with hairy cell leuke-mia (HCL), 2 with splenic lymphoma, 4 with mucosa-associated lymphoid tissue (MALT) and 4 with fol-licular lymphoma grade 1. The aggressive lymphoma group included 9 with diffuse large cell lymphoma (DLCL), 4 with follicular large cell lymphoma grade III (FLCL), 4 with mantle cell lymphoma (MCL) and 7 with prolymphocytic leukemia (PLL). Reclassification of the patients according to the levels of sCA125 us-ing the median as a cut off value was done. Also, the

original report CA-125 AND NHL


patients were divided into 4 groups by combination of sCA125 with sLDH (very low, low, intermediate and high risk group). Very low risk=Low sCA125+ low sLDH, low risk=low sCA125+high sLDH, inter-mediate risk=high sCA125+low sLDH and high risk group=high sCA125 and high sLDH.

Group II: Control group: Thirty age- and sex-matched apparently healthy individuals with a mean age of 30 ±10.4 years and a median age of 25 years were chosen as a control from individuals free from known diseases. laboratory findings of all patients versus control group are summarized in Table (1). Immunophenotyping was carried out in 37 cases only, the remaining of the 50 patients were diagnosed by histopathology only. Cytogenetic analysis was done in 10 cases only. The analyzed cases revealed normal karyotype in 5 cases (50%), numerical aberration in 3 cases (30%) and structural aberration in 2 cases (20%). When serum CA125 was compared to the con-trol group in general, it showed a significant increase (P<.05) (Table 1).

The frequency of cases with elevated sCA125in NHL histological subtypes are illustrated in Table 2. In the indolent group the lymphoma of the MALT type showed the highest frequency of elevation (100%), followed by the chronic lymphocytic leukemia group (57%), and follicular lymphoma grade I (25%). While patients with splenic lymphoma and HCL showed no elevation of sCA125 levels. In the aggressive group the follicular large cell lymphoma had the highest el-evation (100%), followed by diffuse large cell lympho-ma (44%) and by prolymphocytic leukemia (29%) and lastly by MCL (25%).

The elevated sCA125 was found to be associated with certain clinical features at presentation. Twenty-four (48%) patients had serum CA125 above the

median. These high values were significantly associ-ated with stage III-IV (P<.05), aggressive histology (P<.05), abdominal involvement (P<.05), high LDH (P<.01), high tumor burden (P<.05), presence of effu-sion (P<.01) and high IPI score (P<.01) (Table 3).

Correlations between sCA125 and other param-eters using multiple regression analysis are demon-strated in Table 4. sCA125 showed a highly significant positive correlation with CD19, uric acid, IPI and ef-fusion (P<.01) and a significant positive correlation with GIT infiltration (P<.05). It also showed a highly significant negative correlation with CD5 (P<.01).

In the group of patients found to have a high level of sCA125, 13 patients (54.2%) showed complete re-

table 1. Laboratory investigations in patients and control group at presentation.

Parameters studiedControl group Patients Groups

P and sig.Mean±SD Median Mean±SD Median

T. protein (g/dL) 6.8± 0.6 6.5 7.2±1.6 7.3 >.05 NS

Albumin (g/dL) 4.1±0.6 4 2.9±0.5 2.9 <.01 HS

Uric acid (mg/dL) 3.6±1.0 4 5.8±3.0 4.8 <.01 HS

LDH (IU/L) 346±62 390 827±86 558 <.01 HS

Hb (g/dL) 13.1±1.4 13 7.7±2.8 6.5 <.01 HS

TLCx109/L 6.2±1.3 6 44.3±26.9 30 <.01 HS

Plateletsx109/L 216.6±54 200 83.9±79.1 80 <.01 HS

sCA125 (U/mL) 13±8 12 110.3±153.3 20 <.05 S

table 2. The frequency of cases with high sCA125 (u/ml) in various subtypes of NHL.

sCA125%

No. (24/50)

Indolent

MALT 4/4 100

CLL 8/14 57

FL ¼ 25

HCL 0/2 0

SLVL 0/2 0

Agressive

FLCL 4/4 100

DLCL 4/9 44

PLL 2/7 28.6

MCL 1/4 25



mission, 2 patients (8.3%) were resistant to therapy and 9 patients (37.5%) died. While in the group with a low level, 17 patients (65.4%) showed complete remis-sion, 3 patients (11.5%) were resistant to therapy and 6 patients (23%) died. Using the Cox F test to compare the survival time in patients with higher and lower val-ues than the median, the event-free survival were con-secutively 52.5% versus 76.9% (P<.05) (Table 5).

In the group of patients with values higher than the median of sCA125, all those who achieved com-plete remission (13/24) had normalization of sCA125 by the end of the treatment, while none of the pa-tients failing first line of therapy (2/5) had a return of sCA125 to normal levels. On the other hand, in the group of patients with values lower than the median levels of sCA125 (17/26) at presentation, all respond-ers maintained normal values at final restaging (Table 6). Of the nonresponder patients (3/5) showed elevat-ed values at final restaging.

To detect which of the variables studied is asso-ciated with an influence on the survival of the pa-tients (Table 7), the Cox proportional hazard test was performed. It showed that sCA125, serum uric acid (P<.01), bone marrow infiltration, IPI score, lympho-cyte count, sLDH and age of the patient (P<.05) had an independent influence on survival.

The role of combined sCA125 and sLDH as an accurate prognostic factor was investigated; thus, pa-tients were divided into 4 risk groups based on this combination (very low, low, intermediate and high-risk group). The very low risk group=low sCA125+low sLDH; (EFS 100%). The low risk group=low sCA125+high sLDH; (EFS 85%), intermediate group=high sCA125+low sLDH; (EFS 66.7%). High risk group=high sCA125+high sLDH; (EFS 50%) (Figure 1).

Discussion The clinical importance of the serological markers in NHL is based on their role in staging, prognostic as-sessment and in monitoring malignancy progression following therapy. In this study, sCA125 was studied beside sLDH which is an old serological marker.

In the present study, patients with NHL had signifi-cantly elevated levels of sCA125 when compared to the control group. This is in agreement with many pre-vious reports.11,12 The frequency of elevated sCA125 in this study revealed increased levels of sCA125 in both the indolent and aggressive form of lymphoma. Patients were classified into two groups above and be-low median by this parameter. It was found that 48% of patients had elevated levels of sCA125 above the

table 3. Serum CA125 levels (u/ml) in relation to the presenting features in NHL..

sCA125

>20 (Median) No. 24

<20 (Median)No. 26

B symptom Yes 12 6 6

No 38 18 20

P value >.05 NS

Stage I-II 5 0 5

III=IV 45 24 21

P value <.05 S

Histology Indolent 26 8 18

Agressive 24 18 6

P value <.05 S

Abdominal LN Yes 4 4 0

No 46 20 26

P value <.05 S

Peripheral LN Yes 40 19 21

No 10 5 5

P value <.05 NS

BM infiltration Yes 39 20 19

No 11 4 7

P value >.05 NS

LDH >500 29 18 11

<500 21 6 15

P value <.01 HS

Tumor burden Yes 45 24 21

No 5 0 5

P value <.05 S

Effusion Yes 17 14 3

No 33 10 23

P value <.01 HS

IPI score <2 10 0 10

≥2 40 24 16

P value <.01 HS



table 4. Correlation between serum CA125 levels (µ/mL) and all the parameters studied using multiple regression analysis in NHL.

Dependent variables Beta F P value

sCA125

CD19 0/91

6.1

<.01 HS

CD5 -0.9 <.01 HS

Uric acid 0.46 <.01 HS

IPI score 0.4 <.01 HS

Effusion 0.32 <.01 HS

GIT infiltration 0.3 <.01 S

table 5. Response to treatment and comparison of survival between patients with higher and lower than the median of sCA1.

Remission (30) Resist to treatment (5) Died (15) Event-free survival (EFS)

P valueNo. % No. % No. %

Total no. (50) 30 60 5 10 15 30

sCA125

>Median (24) 13 54.2 2 8.3 9 37 52.5%

<Median (26) 17 65.4 3 11.5 6 23 76.9% <.05 S

EFS: event free survival

table 6. Comparison of sCA125 levels at diagnosis and at remission with the control group.

At diagnosis At remission Control group P value

>Median 13/24 230.6±138.4 14.8±5.6 13±8.8

P1<.01 HSP2>.05 NS

<Median 17/26 11.6±5.0 11.3±5.1 P1>.05NS P2>.05 NS

P1: between at diagnosis and at remission. P2: between remission and control group.

median value. This is in agreement with previous stud-ies.13-15

An analysis of the relationship between pretreat-ment levels of sCA125 and the clinical and pathologi-cal features of NHL showed that a statistically signifi-cant association between high sCA125 levels and dis-ease stage III-IV, with aggressive clinical presentation such as effusion, abdominal involvement, aggressive histology, high IPI score, high LDH and high tumor burden. The significant association with tumor bur-den is confirmed in our work by the significant posi-tive correlation between sCA125 and each of CD19, serum uric acid and high IPI score and also by the significant negative correlation with CD5, which are all a close reflection of tumor load and tumor growth. No patients with disease stage I-II or with low tumor burden had an abnormal level of sCA125. This is in agreement with many authors.14-18 sCA125 is a glyco-

protein produced by mesothelial cells, not released by lymphoma cells.14,19 For that reason, the increased sCA125 may represent the response of pleuroperi-cardial or peritoneal mesothelial cells to the direct in-volvement by the tumor.13,20 On the other hand, Pui and Ludwig21 proposed that the lymphokines released by the tumor might represent the stimulus for sCA125 production. Thus, sCA125 could reflect the patient’s response to the invasiveness potential of the tumor acting as a measure of its infiltrative activity. It may also be an index of the clinically undetected extra-nodal extension of the tumor; therefore, it is a useful marker that may improve staging and be a promising complementary parameter to other tumor markers in the initial staging of NHL.13

In our study, the EFS of patients with lower than the median sCA125 was significantly better than EFS in those with higher levels (76.9% versus 52.5%). Also,



table 7. Proportional hazard (Cox) regression analysis in NHL.

Parameters Beta t value P value

Ca-125 -0.02 -3.4 <.01 HS

Uric acid 0.5 2.4 <.01 HS

BM infiltration -4.4 -2.3 <.05 S

IPI score 1.8 2.4 <.05 S

Lymphocyte count 0.05 2.1 <.05 S

LDH -0.002 -2.0 <.05 S

Age -0.08 -1.9 <.05 S

this applies to the complete remission, which was sig-nificantly better when the sCA125 was lower (65.4%) versus when the sCA125 is higher (54.2%).

It is clear thus that the elevated levels of sCA125 influence complete remission duration and survival negatively. Patients with normal levels exhibited lon-ger remission and survival than those with elevated or borderline levels. Several investigators have ad-dressed the prognostic significance of sCA125, and all reported that serum CA125 levels at diagnosis had a strong correlation with event-free and overall survival where patients with increased levels had worsened sur-vival.14,15,22

Among the patients presenting with abnormal sCA125, all complete responders had normalization of the values by the end of the treatment. In contrast, the nonresponding patients maintained sCA125 above the normal limit throughout therapy. On the other hand, for those with values less than the median at diagnosis, all responders switched back to normal val-ues, while the nonresponders showed elevated values at final restaging. This indicates that there is a close relationship between this marker and disease activity and points to its usefulness, not only in predicting the response to primary therapy, but also in determining the outcome of treatment. It also can help to adjust the optimal treatment duration following the disap-pearance of sCA125 from the patient’s serum. These results are confirmatory to those reported by previous studies.13,14,23

To detect if sCA125 has an independent influence on the survival of patients, the Cox proportional haz-ards test was performed. It revealed that sCA125, IPI score, uric acid, TLC and sLDH had an independent predictive value on survival. In contrast to our results, Bonnet et al15 reported that sCA125 does not enhance the performance of standard prognostic markers in the management of patients with NHL or HD.

In our work, in order to attempt to improve the prognostication we have examined the value of com-bining the sCA125 and sLDH. It was found that in the group of patients who had the best EFS, (100%, very low risk group) both parameters were low, fol-lowed by a low risk group with an EFS of 85%; the sCA125 was low while the sLDH was high. This was followed by the intermediate risk group with an EFS of 66% where the sCA125 was high and the sLDH was low. The worst risk group was associated with a rise in both parameters and EFS of 50%. It was ob-vious that when the two markers were low the EFS was good. When one marker was elevated the EFS was variable, while the EFS was poorest when both mark-

Figure 1. The event free survival in the 4 groups of sCA125 with sLDH.

ers were elevated. In only one study was the combination of sCA125

with sLDH used and it was reported that the high risk group had a shorter survival time and the combina-tion of both markers were found to be an important prognostic factor in low grade lymphoma.11

In conclusion, sCA125 might be employed for diag-nosis, staging and prognostic assessment of different grades of NHL. It can be used not only in monitoring the response to primary therapy , but also in determin-ing the number of chemotherapy cycles that should be given to patients to achieve cure and also the optimal treatment duration. The combination of both sCA125 and S LDH could improve the prognosis and assess-ment of the outcome of treatment.



1. Armitage JO. Treatment of non-Hodgkin’s lymphoma. N Engl J Med 1993; 328: 1023-1030.2. Shipp MA. Prognostic factors in aggressive non-Hodgkin’s lymphoma: who has “high-risk” disease? Blood 1994; 83: 1165-1173.3. A predictive model for aggressive non-Hodgkin’s lymphoma. The Inter-national Non-Hodgkin’s Lymphoma Prognostic Factors Project. N Engl J Med 1993; 329: 987-994. 4. Canney PA, Moore M, Wilkinson PM et al. Ovarian cancer antigen CA125: a prospective clinical assessment of its role as tumor marker. Br J Cancer 1984; 50: 765-769.5. Imai A, Itoh T, Niwa K et al. Elevated CA125 serum levels in a patient with tuberculosis peritonitis. Arch Gynecol Obstet 1991; 248: 157-159.6. Halila H, Stenman UH, Seppala M. Ovarian cancer antigen CA 125 levels in pelvic inflammatory disease and pregnancy. Cancer 1986;57:1327-1329.7. Harris NL, Jaffe ES and Stein H(1994): A revised European - American classification of lymphoid neoplasms: a proposal from the international lymphoma study group. Blood 84: 1361-1392.8. Carbone PP, Kaplan HS, Musshof K (1971): Report of the committee on HD staging classification. Cancer res 31: 1860.9. Rooney DE and Czepulk-owski PH (1992): Human cytogentic. A practi-cal approach vol.2. Malignancy and acquired abnormality 2nd ed. Oxford university press 1-25.10. Kubonishi I, Bandobashi K,Miyoshi I (1997): High serum levels of CA125 and IL-6 in patient with Ki-1 lymphoma. Br J Hematol 98: 452.11. Benboubker L, Valat C, Linassier C, Cartron G, Delain M, Bout M, Fetis-sof F, Lefranq T, Lamagnere JP, Colombat P(2000): A new serologic index for low-grade non-Hodgkin’s lymphoma based on initial CA125 and LDH serum levels. Ann Oncol 11(11): 1485-91.12. Zacharos ID, Efstathiou SP, Petreli E et al. The prognostic significance of CA 125 in patients with non-Hodgkin’s lymphoma. Eur J Haematol 2002;

69: 221-226.13. Lazzarino M, Orlandi E, Klersy C et al. (1998): Serum CA 125 is of clinical value in the staging and follow-up of patients with non-Hodgkin’s lymphoma: correlation with tumor parameters and disease activity. Cancer 1998; 82: 576-582.14. Bairey O, Blickstein D, Stark P, Prokocimer M, Nativ HM, Kirgner I, Shaklai M.(2003): Serum CA 125 as a prognostic factor in NHL. Leukemia lymphoma Oct; 44(10): 1733-8.15. Bonnet C, Beguin Y, Fassotte MF, Seidel L, Luyckx F, Fillet G(2007): Lim-ited usefulness of CA125 measurement in the management of Hodgkin’s and NHL. Eur J Hematol , May 78(5): 399-404.16. Dilek I, Ayakta H, Demir C, Meral C, Ozturk M.(2005): CA 125 levels in patients with non-Hodgkin lymphoma and other hematologic malignan-cies. Clin Lab Hematol, Feb; 27(1): 51-5.17. Fehm T, Beck E, Valerius T, Gramatzki M, Jäger W(1998):CA 125 eleva-tions in patients with malignant lymphomas. Tumor boil 19(4): 283-9.18. Kubonishi I, Bandobashi K, Murata N, Daibata M, Ido E, Sonobe H, Oht-suki Y, Miyoshi I.(1997): High serum levels of CA125 and interleukin-6 in a patient with Ki-1 lymphoma. Br J Hematol, Aug; 98(2): 450-2.19. Apel RL and Fernandes BJ (1995): Malignant lymphoma presenting with an elevated serum CA125 level. Arch Pathol Lab Med; 13: 530.20. Burney IA,Siddiqui T and Siddiqui I(1999):Serum CA125 is of clinical value in staging and follow up of patients with NHL: Correlation with tumor parameters and disease activity. Cancer 1; 85(3): 755.21. Pui T and Ludwig C(1995): CA125 a tumor marker in NHL. J of clinical oncology; 13: 1827. 22. Kutluk T, Varan A and Erlas B (1999): Serum CA125 levels in children with NHL. Pediatr Hematol Oncol; 16(4): 311.23. Vlasveld LT, Ermes AA, Sonnenberg AA and Pauwels P (2000): Elevated sCA125 concentration due to expression by a diffuse large cell lymphoma . Ann Clin Biochem Jul; 37(pt4): 545-8.

References

brief report


The hemoglobin disorders are one of the most common single gene disorders encountered in the region and in the Kingdom of Saudi

Arabia. They are major health problems, associated with chronic illness and complications which place a considerable burden on health services. In the Kingdom of Saudi Arabia, the incidence of thalas-semias and hemoglobinopathies has shown wide geo-graphical variation.1-5 Interest in the study of the ge-netic epidemiology of the hemoglobin disorders goes back almost four decades to 1963 when Lehmann and his co workers reported on high prevalence of sickle cell trait in the Eastern Province.6

Abnormalities in the biosynthesis of globin chains have traditionally been divided into those with defi-cient quantity [thalassemia (thal) syndromes] or struc-tural variation [abnormal hemoglobin (Hbs), each as-sociated with a distinct phenotype expression.7,8

Prevalence of thalassemia disorders and hemoglobinopathies in Jeddah, western Saudi Arabia

Soad Khalil Al Jaouni

From the Hematology Department, King Abdulaziz University Hospital, Faculty of Medicine King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia


Soad Khalil Al Jaouni, MD, FRCPC Associate Professor of Hematology Consultant Hematologist Hematology Department King Abdulaziz University HospitalFaculty of MedicineKing Abdulaziz University PO Box 80215 Jeddah 21589 Kingdom of Saudi [email protected]: +966 (2) 640-2000 ext. 17047F: +966 (2) 6060200

The inherited hemoglobinopathies are a large group of disorders that include

the thalassemias and sickle cell diseases. The hemoglobin disorders are the most

common single gene disorders encountered in the region and in the Kingdom

of Saudi Arabia. The hemoglobinopathy genes occur with a variable frequency

in different regions of the Kingdom. Genetic screening is an important tool to

control and prevent genetic disorders. The aim of this study was to estimate

the frequency of hemoglobin disorders among the Saudi population at Jeddah,

in western Saudi Arabia. A total of 7,584 candidates were screened, divided

into two groups. Group I: The general population, 6,750 unrelated Saudi adult

volunteers of both sexes (5,050 males, 1,700 females). Group II: Cord blood

samples of 834 Saudi neonate (422 males, 412 females). Our results showed the

prevalence of sickle cell trait was 5.4%, (-thalassemia trait 4.69%, Hb E trait 0.85%

and 1 (0.12%) of 834 neonates screened had sickle cell anemia. The prevalence

of alpha-thalassemia in this study was 40.0%. The outcome of this study indi-

cated that the Saudi population in this area is at risk for hemoglobin disorders.

Screening programs are essential and should be implemented in the prevention

program as a routine practice.

Keywords: Prevalence of Sickle Cell Disease, Thalassemia Disorders,

Hemoglobinopathies, Diagnosis Hb variants, Western Saudi Arabia.

In the thalassemias, excess of the normally syn-thesized globin chain results in ineffective erythro-poiesis, phenotypically most apparent in homozy-gotes or compound heterozygotes for thalassemia mutations.

In this paper our aim was to estimate the preva-lence of hemoglobin disorders and compare two methods of screening for hemoglobin variants by using standard hemoglobin electrophoresis and high performance liquid chromatography among the Saudi population from the Jeddah area, western Saudi Arabia.

MethodsA total of 7,584 healthy candidates were screened, to estimate the frequency of abnormal hemoglobin in this area, divided into two groups. Group 1: repre-senting the general population, consisted of a total of

brief report THALASSEMIA IN JEDDAH


6,750 unrelated Saudi adult volunteers of both sexes (5,050 males, 1,700 females) There ages ranged from 20-45 years old, with a mean age of 28 years. Blood samples were collected from blood donors, students, and health care workers. Group II were 834 con-secutive cord blood samples prospectively analyzed from Saudi neonates of both sexes (422 male, 412 fe-males). The study was conducted at the Hematology Laboratory and Hematology Research Lab at King Abdulaziz University (KAU) in Jeddah. Five mL of venous blood samples were collected in ethylene di amine tetra-acetic acid (EDTA) and processed within 2 hour after the collection to obtain hematological analysis and blood films. Routine hematology was carried out using a Coulter LH 750 analyzer (Coulter Electronics, USA) with blood smears by Wright stain, H inclusions bodies using brilliant cresyl blue stain (BCB) to detect alpha thalassemia disorders. Data interpretation including family history were examined by an expert hematologist. Hemoglobin studies were analyzed by both twin-tier hemoglobin electrophoresis (alkaline/acid) Hydrasys Sebia and by cation exchange high performance liquid chro-matographies (HPLC) using a variant TM. Using Hemoglobin Testing system with the b-Thalassemia Short Program, we followed the analytical protocol developed by the manufacturer’s instruction and the Genetic Disease Laboratory.9-15 Hemoglobin A2 (HbA2) was estimated by microcolumn chromatog-raphy. All specimens from cord blood were screened by the b-thalassemia variant program (Bio-Rad Laboratories, USA). The b-thalassemia program was therefore able to provide a reliable quantitative Hb

Bart’s and to detect alpha thalassemia genotype in cord blood. To detect Hb Bart’s, required a slightly longer run time. Cord blood of normal infants has <1% Hb Bart’s, but that of infants with b-thalasse-mia II has levels of 1-3% Hb Bart’s and b-thalasse-mia I had levels of >3-10% Hb Bart’s. Hemoglobin H disease has 15-30% of Hb Bart’s. After one year of age Hb Bart’s cannot be detected except for Hb H disease with 1-5%. We followed the analytical proto-col developed by the manufacturer’s instruction and the Genetic Disease Laboratory.9,16-18

Results of hemoglobin studies were used to clas-sify the individuals as normal (HbAA), sickle cell trait/carrier (HbAS), and sickle cell anemia (HbSS). A sickle cell trait was diagnosed if the proportion of the sickle cell hemoglobin (HbS) was 35% to 45% and the sickling test was positive. If the HbS was less than 35% of the individual with HbAS was supposed to be investigated for co-existing b-thalassemia.20-22 Subjects were considered to have thalassemia trait if a blood indices compatible with the thalassemia pic-ture (high RBC count, low MCV <79fl, low MCH <27 pg with mild alteration of the erythrocyte mor-phology), and the individual was declared to have b-thalassemia trait if he had a thalassemic blood pic-ture and hemoglobin A2 (HbA2) level >3.5%. An in-dividual was diagnosed to have b-thalassemia trait if he had a blood indices compatible with thalassemia trait and the presence and detection with H inclusion bodies. The diagnosis of hemoglobin H (Hb H dis-ease) was made on the basis of the presence of Hb H, Hb Bart’s on the electrophoresis/HPLC, supple-mented by the demonstration of Hb H inclusion in the red blood cell.

ResultsThe initial screen of 6,750 healthy candidates showed that about 11.2% of the general populations in this area were heterozygous for one of the abnormal Hb genes: 365 (5.4%) had sickle cell trait with Hb S quan-tity (range 23.4-45%) and 109 (30%) of this popu-lation also had (-thalassemia trait. 316 (4.69%) had b-thalassemia trait, with HbA2 (range 3.6 - 7.6%). 57 (0.85%) had Hb E trait, with (range of HbA2/E 19.2 -31%). The HbA2 quantity in b-thalassemia trait did not exceed 8.0% in our population while the HbA2 quantity in HbE traits ( 19%. (0.19%) diagnosed as Hb H disease, and 2 (0.03%) had Hb C trait. One (0.015%) was a symptomatic student was diagnosed as heterozygous for hereditary persistence of fetal hemoglobin (HPFH) and confirmed by Kleihauer-Betke Acid elution test. The screening result of he-

Table 1. Hemoglobin studies of 6750 Saudi Adult in Jeddah, Western of Saudi Arabia.

Screening Result

Total number of adult screened and percentage 6750 100.00%

Normal Screen (Hb AA) 5996 88.8%

Abnormal Result 754 11.2%

Sickle Cell Trait (Hb AS) 365 5.4%

b-Thalassemia Trait (Hb AA2) 316 4.69%

Hb E Trait (Hb A E) 57 0.85%

Hemoglobin H disease 13 0.19%

Hb C Trait (Hb A C) 2 0.03%

Heterozygous HPFH 1 0.015%

HPFH=Hereditary persistence of fetal hemoglobin

brief reportTHALASSEMIA IN JEDDAH


moglobin studies of 6750 candidates is shown in Table 1.

Neonatal screening showed an additional finding of the high prevalence of b-thalassemia gene 39.57% with a predominance of b-thalassemia II 35.86%. One neonate was diagnosed as having sickle cell ane-mia out of 834 screened (0.12%) (Table 2).

DiscussionScreening programs for congenital diseases espe-cially for detection of hemoglobin disorders have been available in the Kingdom since early 1980.23 This present study tested the possibility of deploying modern high performance liquid chromatography (HPLC) as a successful approach from the efficiency and cost effectiveness point of view in the detection and screening of the general population—including neonates and adults who are carriers or patients for genetic hemoglobin disorders. The results show that standard electrophoresis and HPLC are comparable. HPLC is efficient and cost effective in detecting he-moglobin disorders. HPLC, coupled with the devel-opment of an automated and rapid method. achieves excellent sensitivity and specificity, while adding the very important quantitative element to the analysis. It also has proven to be clinically accurate, and in line with other studies it confirms the occurrence of vari-ous genetic hemoglobin disorders at varying rates.9-15

One of the limitations of most screening meth-ods including HPLC in the diagnosis of thalassemia major and persistent fetal hemoglobin, is that there is

often confusion between sickle cell anemia and sickle cell b-thalassemia. A similar screening limitation is true of other compound disorders such as E/b-thal-assemia. HPLC is good for neonatal screening, which requires consistent demonstration of the presence or absence of small quantities of HbA, which can be detected at 0.5%. The common variants. HbS, HbC, HbE and HbD, were also detected consistently at the level of 1%.14-16

In our communities, with the cultural preference for consanguinity, gene variants are spread among families and tribes. Hence, an affected child is a mark-er for a group at a high genetic risk. In the Kingdom the percentage of consanguinity varies from 37 per-cent to 50 percent, which increases the incidence of these disorders.5,24

The results of the present study indicated that the use of HPLC is efficient and cost-effective in detect-ing subjects who are carriers or patients of genetic hemoglobin disorders at various rates. Our finding confirms the previous studies in the area with the additional characterization of the various Hb disor-ders. 1,5,22

Summary of the finding The prevalence of b-thalassemia among the popula-tion in the Jeddah area was high (40%), with alpha thalassemia II the highest at 35.86% (Table 2). Sickle cell trait was 5.4% with 30% co-existence with (-thal-assemia trait, followed by b thalassemia trait (4.69%), Hb E trait (0.85%), and others non common variants

Table 2. Distribution of hemoglobinopathies in neonates in Jeddah, Saudi Arabia by high performance liquid chromatography.

Newborn Pattern Screening Result Male Female

Total number of neonates screened and percentage 834 (100.00%) 422 (50.6%) 412 (49.4%)

Normal Screen (Hb FA) 441 ( 52.88%) 196 (23.5% ) 245 (29.38)

Abnormal Result 393 (47.12%) 193 (23.14%) 200 (23.98%)

Total a-thalassemia (Hb AF Bart’s) 330 (39.57%) 143 (17.15%) 187 (22.42%)

a-thalassemia II (a a/ a-) 299 ( 35.86%) 133 (15.95%) 166 (28.73%)

a-thalassemia I (a-/-a) 30 (3.60%) 13 (1.56%) 17 (2.04%)

Hb H Disease ( - -/ -a) 1 (0.12%) 0 (0.00%) 1 (0.12%)

Sickle Cell Trait (Hb FAS) 37 (4.44%) 18 (2.16%) 19 (2.28%)

Sickle Cell Anemia (Hb FS) 1 (0.12%) 1 (0.12%) 0 (0.00%)

Hb E Trait (Hb F A E) 16 (1.92%) 7 (0.84%) 9 (1.08%)

Hb C Trait (Hb F A C) 1 (0.12%) 1 (0.12%) 0 (0.00%)

b-Thalassemia Trait (Hb F A A2) 8 ( 0.96%) 5 (0.6%) 3 (0.36%)

brief report THALASSEMIA IN JEDDAH


1. Acquaye J, Ganeshaguru K, Sejeny SA, Selchouk S, Hassounah, F, Samuel, APW, Omer, A. A study of Thalassaemia Families in Western Saudi Arabia. J. Trop. Geog. Med., 1985; 37:319.2. El-Hazmi M. Haemoglobinopathies, thalassaemias and enzymophathies in Saudi Arabia. Saudi Med J., 1992; 13(6): 488-499.3. Niazi, G. Rowland, H. Haemoglobinopathies- A review. Saudi: Med J., 1989; 10 (5): 340-352.4. Al-Awamy, BH. Thalassemia syndromes in Saudi Arabia: Meta-analysis of local Studies- Review Articles. Saudi Medical Journal, 2000; 21 (1):8-17.5. Al-Jaouni, SK. Qari, MH., Damanhouri, GA., Fadlalh, MF. and Jarullah, JM. Chromatography Phenotypes Distribution of Neonatal Thalassemia and Sickle Cell Anemia in Hospitals Births in Jeddah, Saudi Arabia: Egyptian Journal of Paediatrics, 2003; 20, No 324, 595-605.6. El-Hazmi MA. F. Warsy AS. Genetic epidemiology of sickle cell and thal-assemia genes in Saudi Arabia 9Abstract). 8th symposium on Genetics in health and Disease, Riyadh, Saudi Arabia, 2002.7. Poyart C. and Wajcman, H. Hemolytic anemias due to hemoglobinopa-thies. In: Erythrocyte Pathophysiology, edited by D. Bossi. Molec. Aspects Med., 1996; 17; 129- 142.8. Adams, JG. and Coleman, MB. Structural hemoglobin variants that pro-duce the phenotype of thalassemia. Seminal. Hematol, 1990; 27:229-238.9. Eastman, JW., Wong, R., Liao, CI., Morales, DR. Automated HPLC screen-ing of newborns for sickle cell anemia and other hemoglobinopathies. Proc 8th Nat Neonatal Screening symp. 1991; Jan. Feb. 10. Bassett, P., Braconnier, F., Rosa, J. An update on electrophoretic and chromatographic methods in the diagnosis of hemoglobinopathies J. Chro-matogr., 1982; 227 (2): 267-304. 11. Wilson, J., Bm Headlee ME, Huisman THJ. A new high performance Liquid chromatography procedure for the separation and quantitation of various hemoglobin variants in adults and newborn babies. J Lab Clin Med., 1983; 102(2): 174-186.12 . Huisman THJ. Usefulness of cation exchange high performance liquid chromatograpgy as a testing procedure. Pediatrics, 1989; 83 (5): 849-851. 13. Johnson, JP., Vichinsky, E., Hurst, D., Camber, A., Lubin, B., Louie, E. Dif-ferentiation of homozygous hemoglobin E from compound heterozygous

hemoglobin E-beta O-thalassemia by hemoglobin E mutation analysis. J. Pediatr., 1992; 120(5):775-779.14. Kutlar, A., Kutlar, F., Wilson, JB., Headlee, MG., Huisman, THJ. Quantitation of hemoglobin components by high performance cation-exchange liquid chromatography: its uses in diagnosis and in the assessment of cellular dis-tribution of hemoglobin variants. Am J Hematol 1894; 17(1): 39-53.15. Lorey, FW., Cunningham, GC., Vichinsky, E., Lubin, B., Shafer, F., Eastman, J. Detection of Hb E/beta-thalassemia versus homozygous EE using high per-formance liquid chromatography results from newborn. Biochem Metab Biol., 1992; 49(1): 67-73.16. Lorey, F., Cunningham, G., Shafer, F., Lubin, B., Vichinsky, E. Universal screening for hemoglobinopathies using high performance liquid: clinical results of 2.2 million screens. EU J Hum Genet, 1994; 2(4): 262-271.17. Chapman, CS., Neonatal screening for hemoglobinopathies Review, Clin Lab haematol, 1999; 21(4): 229-234. 18. Bunn, HF., Forgel, BG. The inheritance of (-thalassemia hemoglobin mo-lecular genetic & clinical aspects. Philadelphia, W. B. Saunders Co., 1986; p. 329. 19. Henry, JB. Clinical diagnosis and management by laboratory methods. HB. Jovanorich (ed.) Philadelphia, WIB. Saunders Company, 1991, 646-666.20. Wintrobe, MM. Clinical Hematology 11th ed. Philadelphia, Lippincott W & Wilkins, 2004.21. Sergeant, GR. Sergeant BE: Sickle cell disease in Saudi Arabia: The Asian Haplotype. Ann Saudi Medi, 2004, 24, 166-168.22. Sejeny, S., Ganeshaguru, K., Samuel, A., Acquaye, J., Agyei-Obese, S., Has-souneh, F. The diagnostic value of globin biosynthesis in thalassemia. Saudi Med J., 1985; 6:546-53. 23 . Abu-Osba, YK., Mallouh, A., Salamah, M., Hann, R. Comprehensive new-born screening program, National need and recommendations, Annals of Saudi medicine, Volume 12, No. 3, 1992:235-240. 24. El-Hazmi, MFA., Al-Sawailem, AR., Warsy, AS., Al-Swailem, AM., Sulaiman, R., Al-Meshari, AA. Consanguinity among the Saudi Arabian population. J Med Genet., 1995, 32, 623-626. 25. Al Shahrani, M. Steps toward the prevention of hemoglobinopathies in the Kingdom of Saudi Arabia, Hemoglobin, 2009;33 Suppl 1:S21-4.

e.g. heterozygous Hb C and heterozygous HPFH. An accurate prevalence of sickle cell anemia in this area re-quires a larger and multi-center study. Compared with the results of previous studies in this area, the preva-lence of sickle cell carrier seems to the same but the fre-quency of b-thalassemia carrier is substantially higher with the additional presence of hemoglobin E carrier. Genetic screening is an important tools to control, min-imize, and prevent genetic disorders.25

AcknowledgmentsThe author would like to thanks King Abdulaziz University for partial grant support for neonatal Screening in this study, and would like to acknowledge Dr. Majdi Anwar, for review-ing all the hematological studies (blood films, hemoglobin elec-trophoresis and BCB methods) and thanks for Ms. Mofida Tahlawi and Mrs. Olfat Sheite Omer for their technical effort in hemoglobin studies and Mrs. Jummanah Jarullah in data entry.

References

original report


Safe transfusion therapy is a basic requirement in advanced medical care. Although blood transfu-sion saves lives and reduces morbidity in many

clinical situations, it is associated with certain risks. An adverse transfusion reaction is any unfavorable event occurring in a patient during or after receiving a blood transfusion.1,2 In spite of enormous progress in blood safety over time, leading to a decline in the risk of the infectious hazard of blood, non-infectious adverse events still remain the most common compli-cation associated with transfusion. About 0.5% to 3% of all transfusion results in some adverse events, but most are minor without any significant consequence.3,4

Transfusion related non-infectious complications: implications for an appropriate reporting (hemovigilance) system

Leila Kasraian,* Alireza Tavassoli**

From the Shiraz Blood Transfusion Organization, Shiraz, Iran


Dr. Leila KasraianIran Blood Transfusion Research CenterShiraz Blood Transfusion OrganizationAsiab Ghavami, Ghasrodasht Ave.Shiraz, Iran, [email protected]

Background: Blood transfusion may be associated with adverse events

in recipients. The current incidence of non-infectious complication of blood is

unknown.This study aimed to investigate the reporting system for recognized

transfusion reactions, and the type, cause and clinical outcome of these re-

ported errors.

patients and Methods: This cross-sectional study performed in all

hospitals in Shiraz from 23 September 2008 to 23 September 2009. The nurses

filled out a questionnaire that consisted of demographic characteristics, the

type of blood product, the time of occurrence of transfusion reaction, patient

symptoms and signs, the type of reaction, and the modalities performed for

managing the patient and the severity of any reactions. The number of units of

blood transfused was recorded for estimating the risk of adverse events when

the transfusion reaction occurred.

results: Of 86 849 blood units transfused, 100 adverse reactions were

reported (0.01%). Most (90.9%) patients had a history of previous transfusion.

The most frequent symptoms observed were urticaria, dyspnea and fever. The

most frequent reaction was major allergic reaction and next was febrile non-

hemolytic transfusion reaction. The largest proportion of adverse reaction’s

(43%) occurred in patients aged less than 19 years of age.

conclusion: The rate of transfusion reaction was lower than that of

other studies, showing the deficiency in distinguishing and reporting of these

events. It seems that implantation of a hemovigilance system to improve re-

porting of transfusion reaction mortality and morbidity and the effect of new

procedures on it, is essential

key words: Transfusion reaction, blood transfusion, prevalence rate

Most transfusions are safe and without complications. However, mild reactions occur frequently; severe and even fatal reactions occur rarely. The most common reactions are allergic reactions (hypersensivity) which occur in about 1 to 2 percent of transfusions.5 The acute reaction of blood transfusion consist of acute hemolytic reaction (AHTR), transfusion-related acute lung injury (TRALI), febrile non-hemolytic transfu-sion reaction (FNHTR), urticaria reaction and ana-phylaxis.3,6 FNHTR and urticaria reaction are the most frequent non-life-threatining acute transfusion reactions. AHTR is the most frequent severe reaction and the leading cause of death associated with transfu-

original report


sion.5 TRALI and transfusion associated graft-versus-host disease (TA-GVHD) are the most fatal transfu-sion reactions.7,8

A significant proportion of adverse events may oc-cur as a result of errors in the preparation, ordering or administration of blood products,2 causing many complications in blood recipients. Despite careful typ-ing and cross-matching of blood, mismatches can still cause the transfused red blood cell to be destroyed shortly after the transfusion (hemolytic reaction). These reactions can have multiple causes; some are re-lated to transfusion practice at the hospital, others to the clinical condition of the recipient or they may be due to interaction among the biological products being transfused and the recipient characteristics.2

In one study, it was shown that the number of errors rose from 1238 to 2052 from 1993 to 1999.9 It is par-ticularly alarming that errors involving patient samples had a 4-fold increase from 1993 to 1999.9 These errors caused a lot of morbidity and mortality associated with hospital-based errors. The research has focused on in-fectious complications of blood so far, but there are very real non-infectious hazards of transfusion.9

A previous study has shown that in reports of ad-verse occurrences in the United Kingdom mistransfu-sion accounted for over 50% of hazards of transfusion and non-infectious hazards accounted for over 95% of adverse events.10 France and Canada suggested that in-novation to address non-infectious hazards should be given high priority.11

Any adverse reaction to the transfusion of blood or blood products should be reported immediately. Rapid distinction of non-infection hazard is essential because of the possible life threatening effect of these events. Hospitals should report these adverse events to blood banks. Blood banks must evaluate the rate, type and the cause of non-infection hazards. A blood bank physi-cian should be consulted for evaluation of the appro-priate blood component for future transfusions.11

Improvement in transfusion reaction reporting

can make us aware of the frequency of transfusion reactions and assist in planning for the reduction of transfusion risks. This research aimed to collect data on adverse reactions of transfusion to estimate the magnitude of the risks and measure them in relation to epidemiological characteristics.

Patients and MethodsThis was a cross-sectional study conducted in Shiraz from 23 September 2008 to 23 September 2009. This surveillance targeted 33 hospitals (all hospitals) in Shiraz. Collaboration between hospitals and blood transfusion organizations was necesssary. The man-ager of blood banks and the head nurses of all Shiraz hospitals were invited and educated about the defini-tion of transfusion reactions and how to distinguish, manage and report them. Then, a questionnaire was distributed among them. It consisted of demographic characteristics, the type of blood product, the time of occurrence of transfusion reaction, the patient symp-toms and signs, the type of reaction, the modalities performed for managing the patient, and the severity of these reactions. These hospitals transfused 100% of blood products in Shiraz. Then, the numbers of units of blood transfused was recorded for estimating the risk of adverse events when transfusion reaction oc-curred. The hospitals filled out the form and initiated the investigation of the reactions. The forms were sub-mitted to the blood bank. We entered and analyzed the data by SPSS software. Descriptive analysis of the data included the number and proportion of each category of adverse events, their distribution by age, sex, sever-ity, and their relationship to transfusion.

ResultsOf 86 849 blood units were transfused, 100 adverse reactions were reported (0.01%). Sixty-four percent of the reactions occurred in women. The average age of all patients was 22±12.05 years. A history of previous transfusion was reported for 90.9%.The re-

table 1. Adverse transfusion reaction by probable relationship to transfusion.

Adverse reactionsRelationship to Transfusion

Probable Definite

Major allergic 5 (10.9%) 41 (89.1%)

ABO incompatibility 0 1 (100%)

Febrile non hemolytic transfusion reaction 0 7 (100%)

Bacterial contamination 0 1 (100%)

Anaphylaxis 1 (25%) 3 (75%)

NON-INFECTIOUS HEMOVIGILANCE

original report


lationship of adverse reaction to blood transfusion is shown in Table 1. The most frequent symptoms of patients were urticaria (59.6%), dyspnea (23.1%), chills (32.3%), fever (21.2%), nausea (11.1%), tachycardia (11.1%) and hypotension (5.1%). The most frequent reaction was major allergic reaction and the next, fe-brile non-hemolytic transfusion reaction (Table 2). The mean time of occurrence of transfusion reaction was 93±64 minutes. The largest proportion of adverse reaction (43%) occurred in patients aged less than 19 years. The diagnosis of the patients who developed reactions were 90.6% thalassemia, 2.1% leukemia, and 7.3% were in patients that received blood during oper-ations. The severity of the outcome is shown in Table 3. The type of blood product that caused a transfusion reaction was 93.2% packed RBC, 5.7% platelets and 1.1% fresh frozen plasma.

DiscussionThis study was conducted to evaluate the report-ing system of transfusion reactions, and evaluate the efficacy of this system. Any adverse reaction to the transfusion of blood or blood products should be reported immediately. Rapid distinction of non-infec-tion hazards of blood is essential because of the pos-

sible life-threatening effects of these events. Hospitals should report these adverse events to blood banks. Blood banks must evaluate the rate, type and cause of non-infection hazards. A blood bank physician should be consulted regarding the evaluation of the appropri-ate blood component for future transfusions.11,12 The current incidence of non -infectious complications of blood is unknown. Improvement in the transfusion re-action reporting can make us aware of the frequency of transfusion reactions and assist us in planning for the reduction of transfusion risks.

The risks of blood transfusion should always com-pare to the benefits of blood transfusion to treat criti-cal clinical diseases.Nevertheless, much effort has been made to provide a zero-risk blood supply to eliminate both infectious and noninfectious complications of transfusion. With the improvements in reducing the transfusion of infectious diseases, the non-infectious complications still cause serious morbidities or mor-talities following transfusion. Patients should be ac-curately informed about the risk of blood transfusion and the possible consequences of refusing transfusion and how those risks compared with those for other common medical therapies.

The rate of transfusion reactions in this study was

table 2. Diagnosis of adverse transfusion reactions reported to Shiraz blood bank.

Adverse reactions Valid percent Proportion Number

Major allergic 77% 47% 47

ABO incompatibility 1.6% 1% 1

Viral infection 0% 0% 0

Bacterial contamination 1.6% 1% 1

Febrile hemolytic transfusion reaction 13.1% 8% 8

Anaphylaxis 6.6% 9% 4

Not mention the type 0% 39% 39

table 3. Adverse transfusion reaction by severity of outcome.

Adverse reactionsSeverity of Outcome

Total Not Determined Minor/No Sequel Life-threatening

Febrile hemolytic transfusion 7 (100%) 0 5 (71.4%) 2 (28.6%)

ABO incompatibility 1 (100%) 0 0 1 (100%)

Major allergic 42 (100%) 1 (2.4%) 39 (92.9%) 2 (4.8%)

Bacterial contamination 1 (100%) 0 1 (100%) 0

Anaphylaxis 4 (100%) 0 3 (75%) 1 (25%)


original report


0.001% that was lower than the prevalence rate of this reaction in other settings. Other studies showed that about 0.5% to 3% of all transfusion results in some adverse events.3,4 In another study in France the rate of transfusion reaction was 4.02 per 1000 for platelet transfusion, 1.71 per 1000 for RBC transfusion and 0.34 per 1000 for FFP transfusion.13 The lower inci-dence of reported transfusion reaction in compare to other studies showed the deficiency in reporting sys-tem. Probably because most of the transfusion reac-tions were not distinguished or reported well and mild reactions are possibly not reported. Perhaps, the only reactions being reported to Shiraz blood transfusion organization (BTO) were the severe ones.

Most of the reactions reported were in thalasse-mic patients, probably due to greater prevalence of adverse reactions among these patients as a result of repeated blood transfusions and better supervision given to them. In this study, the most frequent reac-tion was allergic reaction that was similar to a previous study.5 Because there is no specific sign or symptom for distinguishing the type of reactions; therefore, the type of reaction may not distinguished well. In this study, we had only one report of a bacterial complica-tion of blood due to the prevalence of this reaction in other settings. It seems that in our setting, no attention was paid to its signs and symptoms and they are not regarded as a cause of fever and sepsis in patients.

We had only one death report in the transfusion reactions, which may be due to lack of consideration of this problem as the possible cause of death and It might be due to the fact that delayed transfusion reaction did not report because of transfusion his-tory in patients and not assume it as a leading cause of death.

These data should not be considered as national figures and must be interpreted cautiously. These fig-ures only showed the defect in reporting system but only represented a substantial underestimate of in-cidence of important failures in transfusion process. This data are inadequate to determine the true inci-dence of transfusion reactions. Due to lower reported reaction, we could not do any statistical analysis on data. This is a new surveillance system and the coop-eration of Shiraz hospitals may have been low. There was too little information transferred to Shiraz BTO to interpret the diagnoses and to be assumed as valid

It is not possible to evaluate the extent of under-reporting since a clinical condition must be recognized first at the primary level as a transfusion reaction with-in a hospital and it needs education and some of the

reactions were possibly missed.However, these data probably do not reflect real

transfusion reaction rates, but it shows the importance of implementation of a surveillance system and should certainly be included for future endeavors in order to enable the estimation of risks of adverse reactions.

This study has limitations in the capacity to capture transmission of viral infections because of the cur-rent inability to link with public health data. Such a link is necessary, as these infections are often not rec-ognized until weeks or months later when the patient is no longer in hospital. Estimating this risk needs a longitudinal study.

There is, currently, an initiative to develop and pilot a system for error surveillance that would eventually be incorporated into blood bank so that the national surveillance system would be more comprehensive observes the patient during transfusion. It seems that implantation of hemovigilance network to improve knowledge of blood transfusion-related mortality and morbidity is essential.That needs education regarding the observation, diagnosing, distinguishing, managing and reporting these events. Finally, the system for re-porting serious hazard of transfusion should expand in the future to include surveillance of major errors in transfusion medicine

Understanding the real transfusion reaction rate can help blood banks to plan preventive measures and consider appropriate blood products according to pa-tient clinical status. It is important to share informa-tion and develop standards in transfusion medicine in Iran.

ConclusionThe rate of transfusion reaction was lower than that of other studies, showing the deficiency in distin-guishing and reporting of these events. It seems that implantation of hemovigilance system to improve knowledge of transfusion-related mortality and mor-bidity and measure the effect of new procedures on it, is essential.

AcknowledgmentWe would like to appreciate from Asad Asadi and Mehri Khalife for nice help in data collection and the head nurses and managers of Shiraz hospital blood banks for reporting the reactions. In addition, we would like to thank Maryam Shirmohammadi for entering data, Fariba Farhadi for typing the manuscript. I re-ally appreciate Dr. Mehrdad Vosugh for data analysis and Dr. Nasrin Shokrpoor for editing the manuscript.


original report


1. Dzik Sunny.Blood developments. Non-infectious serious hazards of trans-fusion. www.bloodcenters.org June 2002 ; No 17.2. Standards for Blood Banks and transfusion Services. Ed 18. American As-sociation of Blood Banks, MD, 1997; 41, K2.000.3. Kleinman S, Chiavetta J, Hindieh F, Pi D, Ricketts M, Robillard P, Sher G, Chan P. The Surveillance and Epidemiology of Transfusions Working Group Final Report. Ottawa , 1999 .4. Kicklighter EJ , Klein HG. Hemolytic transfusion reactions. In: Linden JV and Bianco C, editors. Blood Safety and Surveillance. New York: Marcel Dekker, Inc. 2001;47-705. Kopko PM and Holland PV. Mechanisms of severe transfusion reactions. Transfus Clin Biol 2001;8:278-281.6. Gresens CJ , Holland PV. Other reactions and alloimmunization. In: Linden JV and Bianco C, editors. Blood Safety and Surveillance. New York: Marcel Dekker, Inc. 2001;71-867. Popovsky MA. Transfusion and lung injury . Transfus Clin Biol 2001;8:272-

277.8. Webb IJ , Anderson KC . Graft - versus - host disease . In : Linden JV and Bianco C , editors . Blood Safety and Surveillance . New York : Marcel Dekker, Inc . 2001; 109-124.9. Butch SH. Comparison of seven years of occurrence reports (abstract). Transfusion 2000;40(supp 1):159S10. Williamson L, et al. the serious hazards of transfusion (SHOT) initiative: the UK approach to haemovigilance. Vox Sang 2000; 78 (supp 2): 291-5.11. 7 Adverse reactions to transfusion . www.pathology.med.umich.edu/bloodbank/manual/bbch-7/ Version July 2004, Revised 5/26/05.12. Hiroko Otsubo , Kazunari Yamaguchi. Current Risks in Blood Transfusion in Japan. Jpn . J . Infect . Dis 2008; . 61, 427-43313. Andreu G,Morel P,Forestier F ,Debeir J,Rebibo D,Janvier G,Herve P.Hemovigilance network in France;organization and analysis of immedi-ate transfusion incidence report from 1994-1998.transfusion volume 2002(42)10;1356-1364

References


original report


In his classic 1953 paper, Medawar pointed out that survival of the genetically disparate (allogeneic) mammalian conceptus contradicts the laws of tis-

sue transplantation. Mammalian reproduction poses an immunological paradox because fetal alloantigens en-coded by genes inherited from the father should pro-voke responses by maternal T cells leading to fetal loss. Medawar proposed the concept of the “fetal allograft” to explain the immune relationship between mother and fetus.1 In this model, immunological interaction between mother and fetus is suppressed, either through lack of fetal-antigen presentation to maternal lympho-cyte or maternal lymphocyte functional suppression. In a further advance, Wegmann et al(2) suggested that even if fetal antigen were presented, maternal immune

Human leukocyte antigen (HLA) allele associations in idiopathic recurrent spontaneous abortion patients from Saudi ArabiaMohammed Gad El Rab,* Abdulwahab Al-Jabbari,** Abdulaziz Al-Mishari,** Abdel Galil Gader,** Abdulkareem Al-Momen,*** Zainab Habib**

From the Departments of *Pathology, **Obstetrics and Gynaecology College of Medicine and King Khalid University Hospital, King Saud University, ***Centre of Excellence for Thrombosis and Hemostasis, King Saud University, Riyadh, Saudi arabia.


Gad El Rab M.O.Immunology Unit, Department of Pathology,College of MedicineKing Khalid University Hospital,King Saud University PO Box 2925 (32) Riyadh [email protected]

The prevalence of recurrent fetal loss and its relationship to sharing of hu-

man leukocyte antigens within couples was examined in a large, ethnically

homogeneous Saudi population in Riyadh. Out of 300 couples, HLA-antigen

sharing, at one or more loci, was detected in 225 of the cases (75%). A com-

parison of HLA antigen sharing in patients with history of primary abortion

versus patients with secondary abortion, revealed that HLA-sharing at the

A-locus was higher among the primary abortus patients (65.2%) as compared

to secondary aborters (57.9%). The same trend was observed at the DR-locus

(76.1%) for primary aborters versus (68.4%) for secondary aborters, but the

difference between these prevalence rates was not statistically significant (P

. 0.05). HLA-sharing at the B-locus was greater in the secondary aborters

(52.6) as compared to primary aborters (43.5%). Analysis of HLA-CW6 and

CW7, as forming a protective layer on the extra-villous cytotrophoblast, in

the control and patient group showed interesting findings. More patients with

unexplained fetal loss lack both antigens (41.5%) as compared to 33.3% in the

control group. On the other hand, a very small number of the patients (5.7%)

have both antigens present as compared to 33.3% in the normal-child bear-

ing group. These studies provide substantial support for the role of recessive

genes linked to the major histocompatability complex in the pathogenesis of

recurrent spontaneous abortion.

responses would be biased to the less damaging, anti-body-mediated T-helper 2 (Th2) - type(2)pathway.

Recurrent spontaneous abortion (RSA) is defined as a sequence of three or more consecutive sponta-neous abortions. RSA is a heterogeneous condition, which may have many possible causes; more than one contributory factor is suggested to underlie the recur-rent pregnancy losses. The major causes are attributed to genetic, endocrinological, or immunological basis. In the immunological causes, it may be autoimmunity or alloimmunity. In autoimmune disease, a woman may develop antiphospholipid antibodies, antithyroid anti-bodies, antinuclear antibodies, and antineutrophilic cy-toplasmic antibodies. Alloimmune cases of RSA show involvement of human leukocyte antigen (HLA) and

original reporthla in recurrent abortion


blood group antigens. The literature review reveals as-sociation of HLA alleles with RSA in various popula-tions. Strong association of HLA DR alleles have been shown with RSA.3-8

The Hypothesis of HLA sharing and Recurrent AbortionThe MHC is a region of highly polymorphic genes whose products are expressed on the surfaces of a va-riety cells. Benacraf and McDevitt and their colleagues9 showed that immune responsiveness is an autosomal dominant mapped on the MHC region. The genes that control such immune responses are called immune re-sponse (Ir) genes.

There are two different types of MHC products, called class I and class II MHC molecules. Because they are expressed on human leukocytes, the alloan-tigens recognized became known as human leukocyte antigens (HLA). Family studies were used to map these genes. The first 3 genes defined by serologic ap-proaches were called HLA-A, HLA-B, and HLA-C. The second three to be mapped on an adjacent region were called HLA-D, which was originally detected by mixed lymphocyte reactions. The first gene product detected by alloantibodies that mapped to the HLA-D region was called “HLA-D related” or HLA-DR. The final two genes were subsequently called HLA-DQ and HLA-DP.

HLA sharing and recurrent abortionEarly investigators reported compatibility or sharing of histocompatibility antigens (HLA) in some couples with unexplained recurrent abortion at the HLA-A and HLA-B loci. The early observations of an increased sharing of HLA antigens between husband and wife were both confirmed and extended by studies conduct-ed by McIntyre and Fauk,10,11 and by Beer et al.12 They included in their studies the HLA-A, -B, -C and DR loci. The postulated mechanism is that histoincompatibility between husband and wife permits sufficient maternal-fetal disparity in inherited HLA loci to induce maternal blocking antibodies, which prevent the rejection of the foreign trophoblastic material. The concept behind the immunologic mechanism as explained by Beer and co-workers postulates that the sharing of HLA antigens is simply the detectable marker for the segment of chro-mosome 6 which carries recessive MHC-linked genes responsible for chronic spontaneous abortions.

The protective function of HLA-GAt least three additional class I genes, HLA-E, HLA-F and HLA-G (known as non-classical Class I genes) have been identified which are highly homologous

to classical HLA genes. HLA-E and HLA-F are ex-pressed in many fetal and adult tissues. By contrast, HLA-G is only expressed on fetal placental tissues at the materno-fetal interface where classical HLA-class I and II antigens are absent. This restricted expression suggests that HLA-G products may play an important role in the immune tolerance of the semi-allogenic fe-tus by the mother.13 HLA-G is strongly expressed on trophoblasts from the first trimester and this suggests that HLA-G is strongly involved in materno-fetal im-mune tolerance.14

Interaction between NK cells and HLA-GMany investigators have demonstrated that NK activ-ity can be regulated by HLA class I antigens.15 Some HLA-C and HLA-B alleles present on target cells act as protective molecules against NK lysis and consti-tute the specific ligands for P58 and bp 43, respective-ly and HLA-B51, B58 and B27 are specific ligands for NKB1. With regard to the materno-fetal barrier, it was demonstrated that HLA-G is capable of inhibiting NK activity of decidual large granular leukocytes against the trophoblast from the first trimester of gestation. Human trophoblast tissue lacks classical HLA-A, -B, -C molecules, except for limited HLA-C expression during the first trimester. These class I molecules are ligands for killer-cell inhibitory receptors (KIRs) and their absence would normally triggers cytolytic NK-cell response. Since this does not happen it indicates the presence of a separated trophoblast KIR ligand. It has been shown that HLA-CW6 and HLA-CW7 are protective against lysis by NK cells only with the rel-evant KIR specificity, while HLA-G confer protection against all NK-cell lines. Therefore HLA-G inactivates NK-cell mediated lysis and its protective function for the trophoblast may be paramount in maternal toler-ance of the fetus.

Many studies indicate that there is much sharing of HLA antigens in couples with RSA. In 1977, Komlos and his associates demonstrated the HLA allele-shar-ing hypothesis concerning RSA.16 Several studies have confirmed HLA antigen-sharing between spouses in RSA.17-19 Further meta-analysis of selected case-con-trol studies suggested a slightly increased and signifi-cant risk of RSA among couples who shared at least one allele in the HLA DR locus.12 Other studies have also found non-sharing of HLA antigens.20,21 The fe-tus, in part, evades maternal immune rejection because the trophoblast does not express class I (HLA-A and HLA-B) or class II molecules. The expression of non-classical HLA-G on extravillous cytotrophoblast does not appear to stimulate cytotoxic T-lymphocyte (CTL)

original report hla in recurrent abortion


activity22 and actively inhibits natural killer (NK) cells.23 Furthermore, Th1 responses are suppressed by placental products such as progesterone, prostaglan-din E2 and cytokines such as interleukin 4 (IL-4) and interleukin 10 (IL-10).24,25 Such modulation is apparent in maternal peripheral blood lymphocyte.26

Unexplained RSAs have many etiologies. Many of them involve immune dysfunctions, which include the presence of cytotoxic antibodies, an absence of ma-ternal blocking antibodies, sharing of HLA antigens, and disturbances of killer cell immunoglobulin-like re-ceptor dysfunction. The roles of immunological and genetic characters have been implicated in RSA. Very little information has been obtained in Saudi Arabia. Therefore, the present study was conducted to evalu-ate the role of HLA in RSA.

Materials and Methods

Two hundred and twenty-five, consecutive couples, attending the Obstetric & Gynecology clinic at King Khalid University Hospital, with a history of unex-plained fetal loss were selected for this case-control study. Couples with diagnosed anatomic, hormonal, chromosomal or infectious cause were excluded. Fifteen normal child-bearing couples were recruited as controls.

Twenty-five mL blood samples were obtained by venepuncture from each husband and wife. 20 mL blood was collected in ACD tubes for immunologi-cal studies and 5 mL in red top tubes for serological studies.

HLA typing, for both husband and wife, was per-formed using Lambda Monoclonal HLA class I and Class II tissue typing trays (one Lambda, Inc. 21001 Kittredge Street, Canoga Park, CA 91303-2801, USA). The trays are kept at –70°C until use.

1. T-lymphocytes and B-lymphocytes. Lymphocytes were isolated by magnetic heads.

2. Trays were thawed at room temperature (20-25°C ) for 15 minutes.

3. To each well, 1 µL of a 2×106/mL suspension of either T or B lymphoscytes to the class I tray or B-lymphocytes to the class II tray were added.

4. The micro droplets microplates were mixed us-ing an electrostatic mixer.

5. The trays were incubated at room temperature (20-25°C) for 1 hour.

6. For fluorescence testing, 5 µL of fluoro quench Acridine Orange/Ethiduim Bromide (FQAE-500) were added.

7. The trays were covered with Terasaki Insta Seal

(T1s250) and left to stand at room temperature for 15 minutes to allow lymphocytes to settle.

8. The trays were then examined by a Lambda Scan Plus II Fluorescent Microscope.

Since the lymphocytes were incubated with a mix-ture of complement-binding monoclonal antibodies and complement. In a negative reaction the lympho-cytes remain viable. If the lymphocytes have an an-tigen recognized by a monoclonal antibody, comple-ment is bound and this leads to cell lysis. Under the microscope, lymphocytes will be dead in a positive reaction. The results are then analyzed by a computer program and results obtained in a print out for further analysis.

ResultsHLA-antigen sharing, at one or more loci, was detect-ed in 225 couples (75%) (Table 1). The distribution of sharing at one or more locus are presented in Table 2. A comparison of HLA antigen sharing in patients with a history of primary abortion versus patients with secondary abortion, is presented in Table 3. HLA-sharing at the A-locus was higher among the primary aborters patients (65.2%) as compared to secondary aborters (57.9%). The same trend was observed at the DR-locus (76.1%) for primary aborters versus (68.4%) for secondary aborters, but the difference between these prevalence rates was not statistically significant. HLA-sharing at the B-locus was greater in the second-ary aborters (52.6) as compared to primary aborters (43.5%).

HLA-sharing in relation to intra-uterine fetal death (IUFD) appear in Table 4. The result show that the prevalence of HLA-sharing at the three loci was high-er in patients with a history of IUFD’s than in patients without such history, but the differences between the prevalence rates were not statically significant (P>.05) due to the small number of patients with history of IUFD Furthermore, the highest (81.8%) prevalence rate of a history of I.U.F.Ds occurred in couples hav-ing HLA-sharing at the DR locus.

Results comparing normal child-bearing women and couples with unexplained fetal loss are presented in Table 5. Although the degree of sharing was almost identical in the 2 groups (73.3%), the pattern of shar-ing was different. The following observations were noted:

1. None of the normal child-bearing couples shared all the four antigens. This was shown by two pa-tients in the group with recurrent abortion.

2. HLA-DR3 and HLA-DR7 occurred with more



Table 1. The frequency of sharing HLA antigens in patients with unexplained recurrent abortion.

Number of patients Patients with sharing Patients with no sharing

(Couples) Number % Number %

300 225 75% 75 25%

Table 2. Distribution of HLA sharing in couples with unexplained recurrent abortion.

Degree of HLA-sharing Number Percentage

Sharing at one locus 86 38.2%

Sharing at two loci 67 29.8%

Sharing at three loci 42 18.7%

Sharing at four loci 30 13.3%

Total 225 100%

Table 3a. Sharing of HLA antigens in Patients with unexplained recurrent fetal loss.

Shared HLA Antigens

Couple No. HLA-A HLA-B HLA-C HLA-DR

1 19 (29) - - 4

2 2 7.21 (50) W7 3.7

3 - 35 W4 -

4 19 (30) - - 3.7

5 - - - 7

6 2 - - -

7 68 8 W6 3

8 - - - 4

9 - 21 (49) - 8

10 - - - 13

11 2 - W7 3

Number of Antigens Number of patients

One antigen 4

Two antigens 4

Three antigens 1

Four antigens 2

frequency in the group with unexplained fetal loss. HLA-DR3 did not appear in the group with normal child-bearing.

3. In the control group there was more sharing at the HLA-B locus as compared to the patient group.

4. These findings are in agreement with the report by Kishore et al, 19967 where they documented significant HLA-sharing in couples with recur-rent spontaneous abortion at the HLA-A and HLA-DR loci compared to normal controls. Other workers could not detect significant dif-ferences between the 2 groups of patients.8 In our study group, although the prevalence rates were not statistically significant, however, we ob-served greater sharing at the HLA-A and HLA-DR locus for patients with primary abortion.

Analysis of HLA-CW6 and CW7, as forming a protective layer on the extra-villous cytotrophoblast, in the control and patient group showed interesting findings. The comparison appears in Table 6. More patients with unexplained fetal loss lack both antigens (41.5%) as compared to 33.3% in the control group. On the other hand, a very small number of the pa-tients (5.7%) have both antigens present as compared to 33.3% in the normal-child bearing group.

In the light of the importance of certain HLA an-tigens acting as protective molecules against NK cell activity, we analyzed our HLA data for these antigens. The result is shown in Table 7. All protective antigens, HLA- B51,-B58, -B27 and -B7 appeared in very low frequencies in abortion cases.

DiscussionOur analysis revealed that 75% of couples with re-current fetal loss shared HLA antigens. The most frequent HLA alleles shared were HLA- A2 (22.9%) followed by HLA-DR7 (16.9%). Many studies have associated recurrent fetal loss with the Dr. Locus. A study by Takamizawa et al 1987 associated recurrent abortion with locus HLA-DRW8.27 Another study associated recurrent abortion with HLA-DR5.28 An investigation by Christiansen, 1999 concluded that HLA-DR1, - DR5 and DR4 show association to re-current miscarriage in Caesarian women.29 However, an important point to be noted is that the discrepan-cies in antigen and haplotype frequencies among the different reports could be explained by ethnic differ-ences in HLA frequencies.30

In this study, we also attempted to analyze further the pattern of HLA-antigen sharing in the different



Table 3b. HLA-sharing in normal child-bearing couples.

Shared HLA Antigens

Couple No. HLA-A HLA-B HLA-C HLA-DR

1 2 - - -

2 - 35 W6 5

3 2.11 W52 (5) - 2

4 - 7 - -

5 1 - - 7

6 - W63 (15) - -

7 - - - 7

8 2 7 - W11 (5)

9 2 - W7 -

10 - W57 (17), 7 - W15 (2)

11 31 (W19) W51 (5) - W13 (W6)

Table 4. Comparison of HLA-sharing between primary and secondary aborters.

Parity

Locus of HLA-Sharing 0+Fetal Losses (n=46) >1+Fetal Losses (n=57) P Value

HLA-A 30 (65.2%) 33 (57.9%) .5791

HLA-B 20 (43.5%) 30 (52.6%) .4680

HLA-DR 35 (76.1%) 39 (68.4%) .5224

Table 5. Loci of HLA-sharing in relation to history of IUFDs.

I U F D s

Locus of HLA-Sharing Yes (n=11) No (n = 88) P Value

HLA-A 8 (72.7%) 55 (62.5%) .3791

HLA-B 7 (63.6%) 41(46.6%) .4553

HLA-DR 9 (81.8%) 61 (69.3%) .3179

Table 6. Comparison of HLA-CW6 and HLA-CW7 in patients with recurrent fetal loss and normal child-bearing women.

Patients with recurrent fetal loss (n=176) Normal child-bearing women (n=15)

(Couples) Number % Number %

Both antigens present 10 5.7% 5 33.3%

Both antigens absent 73 41.5% 5 33.3%

HLA-CW6 only 25 14.2% 3 20%

HLA-CW7 only 68 38.6% 2 13.4%



Table 7. Frequency of protective HLA antigens in patients with RFL.

Frequency of protective HLA-antigens (abortion cases) HLA antigens (Frequency in population)

Number Percentage Percentage

HLA-B51 20 8.9 36.1

HLA-B58 2 0.8 11.2

HLA-B27 1 0.4 2.6

HLA-B7 6 2.7 11.4

groups within the patients with recurrent fetal loss. These include patients with primary abortion, patients with secondary abortion and patient with history of intra-uterine fetal death (IUFD).

Extensive studies have been performed in an at-tempt to associate HLA-antigen sharing with recurrent abortion. An interesting Danish study1 relates several specific haplotypes of the HLA loci with a susceptibil-ity to recurrent pregnancy loss. It was unclear which exact loci were important, but they concluded that these alleles were less efficient at initiating a Th2-type response (beneficial) while still generating a Th1-type response (embryotoxic). This link remains intriguing but unproven.31

Analysis of HLA-CW6 and CW7, as forming a protective layer on the extra-villous cytotrophoblast, in the control and patient group showed interesting findings. More patients with unexplained fetal loss lack both antigens (41.5%) as compared to 33.3% in the control group. On the other hand, a very small num-ber of the patients (5.7%) have both antigens pres-ent as compared to 33.3% in the normal-child bearing group.

In the light of the importance of certain HLA an-tigens acting as protective molecules against NK cell activity, we analyzed our HLA data for these antigens. All protective antigens, HLA- B51,-B58, -B27 and -B7 appeared in very low frequencies in abortion cases. Varla-Leftherioti et al,32 have demonstrated that NK activity can be regulated by HLA class I antigens: some HLA-C and HLA-B alleles present on target cells act as protective molecules against NK lysis, and consti-tute the specific ligand for NK-cell receptors. HLA-CW4 and HLA-B7 are the specific ligand for p58 and kp43 respectively. HLA-B51, HLA-B58 and HLA-B27 are specific legands for NKB1.The hypothesis is that the numerous uterine NK-like cells may act to control placentation. This provide a mechanism whereby ma-ternal leukocytic recognition of fetal trophoblast may result in maternal cytokine production which would act locally in a paracrine manner important for tro-

phoblast growth and differentiation. On the other hand, any basic MHC-non restricted cytolytic activity of NK-cells would be suppressed by their expression of the killer-cell inhibitory receptor (KIR) molecules. HLA antigens such as HLA-C and HLA-G would act as KIR ligands. Therefore, trophoblast would be susceptible to cytolysis by cells lacking both NK1 and NK2 KIRs. It has also been demonstrated that pre-conception natural-killer (NK) cell activity could be a predictor of miscarriage and that women with high preconception NK activity, had a significantly higher abortion rate in the next pregnancy.

A comparative analysis of HLA in RSA patients reported from the world revealed a differential associ-ation among our patients. HLA B17 has been reported to be associated in Italian RSA patients.33

Various studies on HLA DR and DP loci have shown associations with RSA in Italian, Danish,34 and Japanese35 populations. Recently, HLA DRB1*1502 has been reported to be significantly associated in Japanese RSA patients. The reports on antigen shar-ing in couples with recurrent miscarriage led to sev-eral studies assessing the role of HLA antigens and their influence on the outcome of pregnancy. In fact, couples sharing at HLA A, HLA B, HLA C, HLA DR, and HLA DQ loci has been reported to be positively associated with the risk of RSA. Since 1977, increased HLA sharing among spouses has been associated with RSAs; later more specific HLA DR and/or DQ anti-gens were suggested. HLA sharing has also been re-ported in couples that fail to achieve pregnancy with multiple cycles of assisted reproductive techniques. Studies examining the association of HLA sharing with a risk of RSA have yielded inconsistent results, in terms of whether or not couple sharing is signifi-cantly related to the outcome and which particular HLA gene may be responsible. Meta-analysis of se-lected case-control studies suggested a significant risk of RSA among couples who shared at least one allele at the HLA-DR locus, but not at the other HLA loci. The discrepant results in the HLA and RSA studies



1. Medawar, P. B. Some immunological and endocrinological problems raised by the evolution of viviparity in vertebrates. Symp. Soc. Exp. Biol. 1953. 7:320-338.2. Wegmann TG. Fetal protection against abortion: is it immuno suppression or immuno stimulation? Ann Immunol Inst Pasteur 1984; 135D: 309-11.3. Takakuwa K, Adachi H, Hataya, Ishik, Tamura M, Tanaka K. Molecular genetic studies of HLA -DRB1 allele in patents with unexplained recurrent abortion in the Japanese population. Hum Reprod 2003;18:728-33. 4. Takakuwa K, Adachi H, Hataya, Ishik, Tamura M, Tanaka K. Molecular genetic studies of HLA -DRB1 allele in patents with unexplained recurrent abortion in the Japanese population. Hum Reprod 2003;18:728-33. 5. Takakuwa K, Adachi H, Hataya, Ishik, Tamura M, Tanaka K. Molecular genetic studies of HLA -DRB1 allele in patents with unexplained recurrent abortion in the Japanese population. Hum Reprod 2003;18:728-33. 6. Takakuwa K, Adachi H, Hataya, Ishik, Tamura M, Tanaka K. Molecular genetic studies of HLA -DRB1 allele in patents with unexplained recurrent abortion in the Japanese population. Hum Reprod 2003;18:728-33. 7. Takakuwa K, Hataya M, Arakawa M, Kikuchi A, Higashino M, Yasuda M, et al . Possible susceptibility of the HLA-DPB1*0402 and HLA-DPB1*04 alleles to unexplained recurrent abortion: analysis by means of polymerase chain reac-tion-restricted fragment length polymorphism method. Am J Reprod Immunol 1999;42:2338. Christiansen OB, Vlcova-Gallova Z, Mohapeloa H, Krauz V. Studies on associa-tions between Human Leukocyte antigen (HLA) class II alleles and antiphospho-lipid antibodies in Daniish and Czech women with recurrent miscarriages. Hum Reprod 1998;13:3326-31. 9. Hataya I, Takakuwa K, Tanaka K. Human leukocyte antigen class II genotype in patients with recurrent foetal miscarriage who are positive for anticardiolipin antibody. Ferti Steril 1998;70:919-23. 10. Saski T, Yamada H, Kato EH, Sudo S, Kishida T, Nishigaki F, et al . Increased frequency of HLA-DR4 allele in women with unexplained recurrent spon-taneous abortions, detected by the method of PCR-SSP. J Reprod Immunol 1997;32:273-9. 11. Christiansen OB, Pedersen B, Mathiesen O, Husth M, Grunnet N. Maternal HLA class II alleles predispose to pregnancy losses in Danish women with re-current spontaneous abortion and their female relatives. Am J Reprod Immunol 1996;35:239-44.12. Baruj Benacerraf and Hugh O. McDevitt Histocompatibility-Linked Immune Response Genes ,Science 1972:Vol. 175. no. 4019, pp. 273 - 279.13. McIntyre JA, Faulk WP. Recurrent spontaneous abortion in human preg-nancy: results of immunogenetical, cellular, and humoral studies. Am J Reprod Immunol. 1983 Dec;4(4):165-170. 14. McIntyre JA, Faulk WP. Histocompatibility and recurrent abortion. Fertil Steril. 1984 Apr;41(4):653-654. 15. Beer AE, Quebbman JF, Ayers JW, Haines RF. Major histocompatibility com-plex antigens, maternal and paternal immune responses and chronic habitual miscarriage. Am J Obstet Gynecol. 1981;141:987-99. 16. Rouas Freiss, N., Goncalves, R.M., Menier, C., Dausset, J. and Carosella, E.D. Direct evidence to support the role of HLA-G in protecting the fetus from maternal uterine natural killer cytolysis. Proc. Natl Acad. Sci. USA, 1997, 94, 11520-11525.17. Van Wijk, I.J., Griffioen, S., Tjoa, M.L., Mulders, M.A., Van Vugt, J.M., Loke, Y.W. and Oudejans, C.B. HLA-G expression in trophoblast cells circulating in ma-ternal peripheral blood during early pregnancy. Am. J. Obstet. Gynecol 2001., 184, 991-997.18. Emmer, P.M., Nelen, W.L., Steegers, E.A., Hendriks, J.C., Veerhoek, M. and Joosten, I. Peripheral natural killer cytotoxicity and CD56+CD16+ cells increase during early pregnancy in women with a history of recurrent spontaneous abortion. Hum. Reprod.,2000, 15, 1163-1169.

19. KomLos L, Zamir R,Joshuah H, Halbrecht I. HLA antigens in couples withre-peated miscarriages. Clin Immunol Immunopathol 1977:7:330-5. 20. Takakuwa K, Ishil K, Adachi H, Higashino M, Kurata H, et al . HLA-class I antigens in patients with unexplained recurrent abortion. J Perinat Med 2001;29:427-32.21. Vojvodic S, Belic B. The HLA antigen system in married couples with recur-rent spontaneous abortion. Med Pregl 2001;54:75-9. 22. Beydoun H, Saftlas AF. Association of human leukocyte antigen sharing with recurrent spontaneous abortions. Tissue Antigens 2005;65:123-35. 23. Caudle MR, Rote NS, Scott JR, De Witte C, Barney MF. Histocompatibility complex antigens, maternal and paternal immune responses and chronic ha-bitual miscarriage. Am J Obstet Gynecol 1985;141:987-99. 24. Jaitinen T, Koskimies S, Westman P. Foeto-maternal compatibility in HLA-DR, DQ and DP loci in Finnish couples suffering from recurrent spontaneous abor-tions. Eur J Immunogenet 1993;20:249-58. 25. Kishore R, Agarwal S, Halder A, Das V, Shukla BR, Agarwal SS. HLA sharing, anti-Paternal cytotoxic antibodies and MLR blocking factors in women with re-current spontraneous abortion. J Obstet Gynecol Res 1996;22:177-83. 26. Varla-Leftherioti M, Spyropoulou-Vlachou M, Niokou D, Keramitsoglou T, Darlamitsou A, Tsekoura C, et al . Natural Killer (NK) cell receptors reper-toire in couples with recurrent spontaneous abortions. Am J Reprod Immunol 2003:49:183-91. 27. Kelso A.Th1 and Th2 subsets: paradigms lost. Immunol Today 1995;16:374-9.28. Vince GS, Johnson PM. Is there a Th2 bias in human pregnancy? J Reprod Immunol 1996;32:101-4.29. Sacks GP, Redman CWG, Sargent IL. Monocytes are primed to produce the Th1 type cytokine IL-12 in normal human pregnancy: an intracellular flow cytometric analysis of peripheral blood mononuclear cells. Clin Exp Immunol 2003;131:490-7.30. Takamizawa M., Juji T., Tsunegoshi H., Neida M. et al. Recurrent spontaneous abortion and human leukocyte antigen DRw8. Amm J obstet gynaecol. 1987; 157: 514 - 515.31. Smith JB., Cowchock FS., Hauskinson B., Iftekar A. Association of HLA-DR5 with recurrent spontaneous abortion in women treated with paternal leuko-cytes. Arthritis Rheum. 1989; 32: 1572 - 1576.32. Christiansen OB. The possible role of classical human leukocyte antigens in recurrent miscarriage. AmJReprod Immunol 1999; 42(2):110-5.33. Baur MP., Danilovs, JA. Population analysis of HLA-A, B, C, DR and other genetic markers. Histocompatibility testing 1980, ed. Terasaki, P.I., pp. 955 - 1210, ULCA. Tissue Typing Laboratory, Los Angeles, California34. Christiansen, OB., Rasmussen KL., Jersild C., Crunnet N: HLA class II alleles confer susceptibility to recurrent fetal losses in Danish women. Tissue Antigns 1994; 44: 225 - 233.35. Varla-Leftherioti M, Spyropoulou-Vlachou M, Niokou D, Keramitsoglou T, Darlamitsou A, Tsekoura C, et al. Natural Killer (NK) cell receptors reper-toire in couples with recurrent spontaneous abortions. Am J Reprod Immunol. 2003;49:183-91.36. Vanoli M, Fabio G, Bonara P, Eisera N, Pardi G, Acaia B, et al . Histocompat-ibility in Italian couples with recurrent spontaneous abortions of unknown origin and with normal fertility. Tissue Antigens 1985;26:227-33. 37. Christiansen OB, Pedersen B, Mathiesen O, Husth M, Grunnet N. Maternal HLA class II alleles predispose to pregnancy losses in Danish women with re-current spontaneous abortion and their female relatives. Am J Reprod Immunol 1996;35:239-44. 38. Takakuwa K, Adachi H, Hataya, Ishik, Tamura M, Tanaka K. Molecular genetic studies of HLA -DRB1 allele in patents with unexplained recurrent abortion in the Japanese population. Hum Reprod 2003;18:728-33.

were attributed to inconsistent definitions of recur-rent miscarriage and of the control groups, the small

number of samples, and the different tissue typing methods used to investigate the HLA loci.

References

case report


Autoantibodies can be directed against a vari-ety of clotting factors, with variable clinical manifestations. Prothrombin antibodies are

most often detected in patients with antiphospho-lipid antibodies.1-3 The prothrombin antibodies, which can cause significant clinical bleeding, usually bind a non active portion of the molecule, resulting in ac-celerated clearance of prothrombin.2 Laboratory test-ing is most consistent with a factor deficiency rather than an inhibitor, since the functional activity of pro-thrombin is not impaired in a clotting assay when ad-ditional prothrombin is added. The possible presence of prothrombin antibodies should be suspected if a patient with antiphospholipid antibodies develops bleeding rather than the expected thrombotic event.3,4 An autoantibody directed against prothrombin has been recognized in one patient who did not have a lupus anticoagulant.5 The autoantibody bound to a different epitope on the prothrombin molecule,but still led to depletion of prothrombin without affect-ing prothrombin function. Specific immunochemical measurement of the prothrombin concentration is re-quired to establish the diagnosis.3-5

Case The patient was a 19 years old female, from a small town about 200 km from the capital Riyadh. She pre-sented to our hospital on 31/01/2005- with left lower

Acquired prothrombin inhibitor

Saud Abu-Harbesh, Hisham Yousef

From the Hematology and Oncology DepartmentSecurity Forces Hospital


Saud Abu-HarbeshHematology & OncologySecurity Forces HospitalPO Box 3643Riyadh 11481Saudi [email protected]

Acquired inhibitors of coagulation are antibodies that either inhibit the activity

or increase the clearance of a clotting factor. A common clinical manifestation

in an affected patient is a hemorrhagic diathesis. Antiphospholipid antibodies

represent a different problem as these antibodies prolong certain clotting as-

says but result in thrombosis rather than bleeding. We describe a patient with

an initial presentation of deep venous thrombosis, found to be secondary to

antiphospholipid syndrome (APS), which is well known to be a hypercoagu-

lable state. However, her clinical presentation changed to a bleeding disorder,

as she developed hematuria; that was only manageable with immunosuppres-

sive medications. Patients with APS should be warned about probable bleeding.

Antiprothrombin antibodies should be looked for if the patient continues to

bleed in spite of stopping anticoagulant therapy. Immunosuppressive medications

are successful in the correction of prolonged coagulation assays and in treating

the bleeding clinically.

limb pain and swelling. She was known to be com-pletely healthy before, with no significant past medical or surgical problems and was receiving no medications. She denied any cardiac , respiratory , gastrointestinal , urinary or neurological symptoms. She had normal and regular menstrual cycles. She was single with no histroy of use of oral contraceptive pills.

On examination she was hemodynamically stable,and chest and cardiac examinations were normal, with no signs of pulmonary hypertension. Abdominal and neurological examinations were unremarkable. She was afebrile and oxygen saturation was 97% on room air. There was no malar rash, discoid skin rash, photosensitivity, mouth ulcers, serositis or arthritis. The left lower limb was swollen on examination up to midthigh, with a 5 cm difference below the knee and a 4 cm difference above the knee when compared to the other limb. The swollen limb was red, hot and tender up to the midthigh. Doppler ultrasound study of left lower limb confirmed the presence of thrombosis of the deep venous system involving the popliteal vein and reaching up to the femoral vein (deep venous sys-tem).

Laboratory Studies showed a CBC-WCC=6.5, hemoglobin=12.6, platelets=320,000, INR=1.2, APTT=59.6. A mixing study for APTT showed NO correction. The patient was treated with heparin fol-lowed by warfarin aiming for an INR 2-3. She was

case report acquired prothrombin inhibitor


positive anticardilipin antibodies IgG level=76 GPL/ml and high APTT=59.6 which didn’t correct in mix-ing study suggesting lupus anticoagulant. She was di-agnosed antiphospholipid syndrome (APS) by 2 lab. and 1 clinical criteria. A hypercoagulable state which was complicated by deep venous thrombosis of the left lower limb. She also had low protein C level. The ANA was positive (titer 1/360); but anti DNA was negative.Urine analysis was free of any abnormality.She was discharged 08/02/2005, on warfarin 1 mg PO OD, to be followed up in the anticoagulation clinic, aiming for an INR 2-3 Elastic stockings were pre-scribed.

Last clinic before the next admission was on 08/03/2005. INR was 2.3 , and APTT was 64.8. The patient was clinically better with less swelling of the left lower limb with no more pain and less tenderness.The impression was therapeutic range INR, and the decision was to continue anticoagulation for total of 6-12 months, and to continue on elastic stocking with regular follow up.

However she presented to our hospital ER on 26/06/2005, with a 20-day history of hematuria. She sought medical advice in her local town hospital 20 days before when she developed the hematuria, and physicians there stopped warfarin , gave her medica-tion and she was transfused, but she continued to have hematuria.

In our hospital her hemoglobin found to be 76 g/l (HB drop), platelet count 165,000. Inspite of no war-farin for 20 days the INR was 3.8 and APTT was 148.

Hematuria did not show any response to vitamin K, and FFP transfusion showed only a slight and tran-sient clinical effect then she continued to bleed again.

A mixing study for PT and APTT showed correc-tion of the PT but no correction for APTT,which sug-gested either a high titre antiphospholipid antibodies or antiprothrombin antibodies. In this clinical setting of bleeding antiprothrombin antibodies was the most likely diagnosis. On 03/07/2005 factor II level was <3%. On 04/07/2005 INR was 3.6 and APTT was 139.1 Inferior vena cava filter was inserted.

On 04/07/2005, methylprednisolone 500 mg IV OD for 5 days, followed by PO prednisolone 60 mg PO OD, aiming to suppress the inhibitor.

On 10/07/2005, INR was 1.2 and APTT was 36.6, there was no more bleeding and she was discharged asymptomatic on a tapering dose of steroids.

ANA was positive since her first presentation on January 2005, but anti- DNA was negative at that time. On June 2005 both ANA and anti- DNA were posi-tive. In June 2005 C3 and C4 were low. DAT was posi-

tive for both IgG and C3d. SLE was suspected and valuated by the rheumatology team as well.

Two years later, on October 2007 she developed proteinuria during clinic follow up, 24- hour urine pro-teins=3.7 g/l. There was enough criteria to diagnose systemic lupus erythematosus and lupus nephritis. She was given high dose pulse steroid therapy and monthly cyclophosphamide 500 mg IV-for 6 cycles-last cycle was on 08/03/2008.

Azathioprine (Imuran) was added as a steroid sparing agent.She was then followed in the clinic on 12/08/2009, Hb was 115 g/l, INR=1.1, APTT=33.9; she was on prednisolone 7.5 mg PO OD and azathio-prine 100 mg PO OD. She was asymptomatic.

DiscussionThis young patient was diagnosed with antiphospho-lipid syndrome based on the presence of one clinical criterion (vascular thrombosis), and two lab. criteria (moderately elevated anticardiolipin antibodies of the IgG type and the presence of lupus anticoagulant). Lupus anticoagulant was evidenced by the prolonged APTT that didn’t correct upon adding an equal amount of normal plasma in the mixing study.

Antiphospholipid syndrome is considered to be a hypercoagulable state inspite of the prolonged APTT by the effect of the inhibitor lupus anticoagulant. There are several explanations for the hypercoagulable state of the antiphospholipid syndrome ; these include platelet activation by the antibodies, endothelial dam-age by the antibodies, inhibition of the protein C path-way in endogenous anticoagulation, and decreased production of prostacyclin. These factors may work alone or together to produce the hypercoagulable state commonly found in the antiphospholipid syndrome.

As may be expected this patient developed deep ve-nous thrombosis of the left lower limb, which reached proximally up to the femoral vein.

This patient had a prolonged APTT by the effect of the lupus anticoagulant, but the INR was normal.

Treatment of the DVT started with LMWH and warfarin aiming for INR 2-3, when INR was above 2 for two days, LMWH was discontinued and the pa-tient was continued on warfarin. LMWH was a good choice in this setting when compared to unfractionat-ed heparin, because following of the anticoagulant ef-fect of the later by APTT was expected to be difficult as the APTT was already prolonged by the effect of the lupus anticoagulant. Warfarin anticoagulant effect monitoring using the INR was easy as the patient had normal INR before anticoagulation.

When the patient was followed up in the clinic, she

case reportacquired prothrombin inhibitor


was found to have a therapeutic INR and the patient was improving clinically with decrease in the pain and swelling in the left lower limb.

Then she developed hematuria which led her to seek medical advice in her local town hospital 20 days before coming to our hospital ER. Warfarin was stopped , but the patient still continued to bleed. Twenty days later she presented to our hospital with hematuria, INR = 3.8 and APTT =148.

With the mixing study the INR showed correction but APTT showed no correction. This pattern in mix-ing studies can be seen in two clinical situations:

(1) High titre of antiphospholipid antibodies. Because phospholipids are essential for both the intrinsic and extrinsic pathways of the co-agulation cascade; it theoretically expected to have prolongation of both PT and APTT when phospholipids are attacked by the antiphospho-lipid antibodies. But because the reagent used to measure the PT contains higher amount of phospholipids when compared to the reagent use to measure the APTT , this renders the PT more liable to be corrected when even more amount of phospholipids in the normal plasma of the mixing study is added during the test. However the APTT will fail to be corrected as the reagent used for its measurement contains less phospholipids.

(2) In case of antiprothrombin antibodies in the setting of antiphospholipid syndrome. Prothrombin being in the final common path-way of the coagulation cascade, a deficiency will lead to prolongation of both PT and APTT. APTT will not be corrected by a mix-ing study due to the presence of lupus antico-agulant. However, PT will be corrected as the antiprothrombin antibodies behave like factor deficiency rather than an inhibitor.

In this clinical setting of bleeding, the presence of antiprothrombin antibodies was the most likely di-agnosis. The patient had a normal platelet count all the time. Even when patients with antiphospholipid syndrome have thrombocytopenia they rarely bleed as the younger newly released platelets are thought to be more hemostatically effective. The diagnosis was con-firmed by finding a low factor II level.

Our aim was then to eradicate the inhibitor, but

we considered that if this happened successfully, the DVT of the left lower limb may worsen and be complicated by pulmonary embolism, so an inferior vena cava filter was inserted. The patient responded perfectly to the treatment with high dose methylpred-nisolone followed by oral prednisolone 1 mg/kg/day. INR returned back to normal with no more bleeding.

More criteria developed with time from January to June/2005 that added weight to the diagnosis of SLE; anti DNA became positive, C3 and C4 were found to be low, also direct antiglobulin test turned to be positive but without evidence of hemolysis. However, the patient continued to be asymptomatic untill October/2007 when she developed significant proteinuria and lupus nephritis that was managed with high dose steroids and cyclophosphamide with good response.

One report monitored 70 patients with apparent primary antiphospholipid syndrome over a 5-year pe-riod, and none developed SLE.7 In another observa-tional study, 3 of 80 patients with primary antiphos-pholipid syndrome developedovert SLE during follow up (mean of 6.5 years ).8

Patients with SLE and APS have an increased inci-dence of thrombosis; this association was illustrated in a review of 21 studies of 1400 patients with SLE ( 39% of whom had antiphospholipid antibodies ).Twenty-four percent of all patients with SLE had thromboses. The thromboses occurred in 42% of those antiphospholipid antibodies, 51% with lupus anticoagulant and 31% with anticardiolipin antibod-ies.6

ConclusionPatients with APS should be warned about prob-able bleeding. Antiprothrombin antibodies should be looked for if a patient with APS continues to bleed in spite of stopping the anticoagulant therapy. Immunosuppressive medications aiming to eradicate the antibodies are successful in correction of pro-longed coagulation assays and in treating the bleeding clinically.

Patients with primary APS may develop SLE with time and regular follow up with a high level of suspi-cion is critical in diagnosing the natural progression of the disease process. Patients with SLE and APS have an increased incidence of thrombosis.

case report acquired prothrombin inhibitor


1. Laurence, J ,Wong, JE , Nachman, R. The cellular hematology of systemic lupus erythematosus. In:Systemic Lupus Erythematosus, 2nd, Lahita, RG(ED), Churchill Livingstone, New Yoork, 1992.2. Bajaj , SP, Rapaport , SI,Fierer, DS ,et al. A mechanismfor the hypoprothrom-binemia of the acquired hypoprothrombinemia-lupus anticoagulant syndrome.Blood 1983;61:684.3. Vivaldi, P ,Rossetti, G , Galli ,M , Finazzi, G. Severe bleeding due to acquired hypoprothrombinemia-lupus anticoagulant syndrome. Case report and review of literature.Haematologica 1997; 82:345.4. Erkan, D , Bateman, H,Lockshin, MD. lupus anticoagulant- hypoprothrombin-emia syndrome associated with systemic lupus erythematosus: report of 2 cases and review of literature. Lupus 1999;8:560.5. Bajaj , SP, Rapaport , SI , Barclay, S , et al. Acquired hypoprothrombinemia due

to nonneutralizing antibodies to prothrombin: mechanism of management. Blood 1985; 65:1538.6. McNeil, HP, Chesterman, CN, Krilis, SA. Immunology and clinical impor-tance of antiphospholipid antibodies. Adv Immunol 1991;49:193.7. Rouby, RA. Autoantibodies to phospholipid-binding plasma proteins: A new view of lupus anticoagulant and other antiphospholipid autoantibodies. Blood 1994;84:2854.8. Mujic, F, Cuadrado, MJ, Lloyd, M, et al. Primary antiphospholipid anti-body syndrome evolving into systemic lupus erythematosus.J Reumatol 1995;22:1589.9. Wilson, WA, Gharavi, AE, Koike, T, et al. Iternational consensus statement on the preliminary classification criteria for definite antiphospholipid syndrome: report of an international workshop. Arthritis Rheum 1999;42:1309.

References

brief report


Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disorder that is characterized by microangiopathy and hemolytic anemia. It

has been linked to a severe deficiency in ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 motif, member 13) activity that targets a cleav-age sequence in von Willebrand factor (VWF). VWF normally circulates as multimers of 500-20 000 kDa. Ultra-large VWF (ULVWF) multimers (>20 000 kDa) have been detected in endothelial cells, but are not usually present in plasma. However, they are found in plasma from some patients with TTP.1 Congenital or acquired deficiency of ADAMTS13 is associated with intravascular platelet aggregation manifested clinically as TTP.

Since the identification of ADAMTS13 and its role in TTP, several novel ADAMTS13 activity, antigen and autoantibody assays have been developed.2-5 These assays make indirect measurements and involve the incubation of patient plasma samples with a source of VWF and then measuring the amount of residual.

Validation of Automated and Rapid Method for Measurement of ADAMTS13 Activity and Antibodies

Mansoor Ahmed, Rasheed Nasr, Randa Alnounou, Tarek Owaidah

Most of these assays generally take 2-3 days and are technically difficult to perform. We are reporting the validation of a new automated rapid test for measuring of ADAMTS13 activity and Inhibitor in the plasma.

Materials and MethodsSamples from 14 patients who presented with classical clinical picture of TTP were collected prior to initia-tion of plasma exchange during a period of 5 years from 2003-2008. An aliquot of each sample was fro-zen while one aliquot was sent to a reference labo-ratory for measurement of ADAMTS13 activity and inhibitors based on a previously reported methodol-ogy.2,6 All samples were tested after removal of patient identification.

All the materials were bought locally except the VWF concentrate Wilfactin® from Laboratoire Fraincas du Fractionnement et des Biotechnologies LFB) with a final concentration of 100 u/ml , Pefabloc from (Merck®, Germany) and BC von Willebrand re-agent from ( Siemens).

From the Department of Pathology & Laboratory Medicine, King Faisal Specialist Hospital & Research Center, Riyadh, Saudi Arabia


Tarek Owaidah, MDDepartment of Pathology and Laboratory Medicine (MBC 10)King Faisal Specialist Hospital and Research Center and Center of Excellence for Thrombosis and Homeostasis, King Saud of University PO Box 3354, Riyadh 11211, Saudi ArabiaT: +966-1-442-4328; F: [email protected]

Background: ADAM-TS 13 activity targets a cleaving sequence in von

Willebrand factor (vWF). Deficiency of ADAM TS 13 leaves the vWF intact

causing intravascular platelet aggregation which manifested clinically as TTP.

Many tests have been developed to measure ADAM TS 13 activities and auto-

antibodies. A new rapid assay had been developed. It involves the incubation of

patient plasma samples with a source of vWF for indirect measurement of the

residual factor.

Materials and Methods: We tested 14 patient samples who pre-

sented with classical clinical picture of TTP by two methods, the new rapid

method with a standard reference method based on SDS agarose.

results and conclusion: There was good concordance between

the new method and the referenced method. The obtained r2 value was 0.98.

The new method is valid and useful in clinical management of TTP and can be

used as a follow up marker for detecting relapse.

keywords: ADAMTS13, TTP, vWD, Inhibitors

brief report automated method for adamts13


The assay protocol was set up in the instrument BCS (Siemens). For each batch of samples a calibra-tion curve is plotted using the Normal Pooled Plasma (NPP) with concentrations of 6.25%, 12.5%, 25%, 50% and 100%. Heat inactivated NPP (ADAM-TS13 depleted) was used for dilution. Both controls (Normal and Pathological) from Siemens were run in each batch. ADAMTS13 activity is determined by measuring the decrease in VW Ristocetin co-factor activity (VWF:RCo) of VWF substrate. After dilution of plasma with low ionic tris buffer and activation of the protease with barium chloride, vWF concentrate is denatured in the presence of urea prior to digestion. Digestion occurs during overnight incubation of the denatured substrate with diluted test plasma at 37°C. Subsequently, the residual VWF:RCo of the samples is assessed using the BC von Willebrand Reagent

table 1. Results of ADAMTS13 by both methods.

Reference Lab Activity KFSH&RC Activity Agreement

Specimen - 1 Normal Normal (87%) Yes

Specimen - 2 Normal Normal (>100%) Yes



Specimen - 5 Severely decreased Decreased (39.6%) No



Specimen - 8 Moderately decreased Decreased (33%) Yes


Specimen - 10 Moderately decreased Decreased (32%) Yes

Specimen - 11 Metalloprotease absent Severely decreased (<6.2%) Yes




table 2. Comparison between the reference method and new method for ADAM TS 13 Antibodies.

Specimen Reference Lab KFSH&RC

Specimen-1 Inhibitor present

Inhibitor present

Specimen-2 Inhibitor present

Inhibitor present

Specimen-3 Inhibitor absent

Inhibitor absent

(Siemens) and the ADAM-TS13 activity in the test sample is calculated and the results were expressed in % activity.

For detecting ADAMTS13 inhibitors, the sample was depleted from endogenous ADAMTS13 activ-ity by incubating test sample for 30 min at 56º and then centrifuge for 15 min at 15000g to separate the supernatant. A test sample is considered positive for ADAMTS13 inhibitors if it reduces the ADAMTS13 activity of NPP to less than 75% of the activity in the reference mixture.

We tested 24 normal subjects (13 male and 11 Females) for ADAMTS13 activity and identified a ref-erence range (51%-149%).

ResultsThe test results from both methods were entered into an Excel spread sheet and analyzed. The result from both the reference lab and the new validated method was reported as normal or reduced. In addition we are reporting percentage of activity with the new method. For comparison reason, the sample was considered normal if there were more than 51% ac-tivity. Out of the 14 cases studied by both methods, 10(71%) were reported as normal and 4 (29%) were reported as reduced with various degrees of activ-ity, (Table 1). Good agreement (100%) was observed in samples with decreased and normal activity of ADAMTS13 levels. All samples with reduced activ-

brief reportautomated method for adamts13


1. Richard Starke, Samuel Machin, Marie Scully, Gordon Purdy and Ian Mackie. The clinical utility of ADAMTS13 activity, antigen and autoantibody assays in thrombotic thrombocytopenic purpura. British Journal of Haematology 2006 ,136, 649-6552. Furlan M, Robles R, Solenthaler M, Lammle B. Acquired deficiency of von Wil-lebrand factor-cleaving protease in a patient with thrombotic thrombocytopenic purpura. Blood 1998;91: 2839-46.3. Bohm M, Vigh T, Scharrer I. Evaluation and clinical application of a new method for measuring activity of von Willebrand factor-cleaving metalloprotease (AD-AMTS13). Ann Hematol 2002;81: 430-5.4. Obert B, Tout H, Veyradier A, Fressinaud E, Meyer D, Girma JP. Estimation of the von Willebrand factor-cleaving protease in plasma using monoclonal anti-bodies to vWF. Thromb Haemost 1999; 82:1382-5.

5. Whitelock JL,NolascoL,Bernardo A, Moake J,Dong JF,CruzMA. ADAMTS-13activityinplasmaisrapidlymeasuredbyanewELISA method that uses recombi-nant VWF-A2 domain as substrate. JThrombHaemost2004;2: 485-91.6. Sahud,M. et al. VWF cleaving protease inhibitor in patients with immunodefi-ciency syndrome associated with TTP. British journal of hematology 20027. Sadler JE, Moake JL, Miyata T, George JN. Recent advances in thrombotic thrombocytopenic purpura. Hematology (Am Soc Hematol Educ Program) 2004;1: 407-23.8. Tripodi A, Chantarangkul V, Böhm M, Budde U, Dong JF, Friedman KD, Galbus-era M, Girma JP, Moake J, Rick ME, Studt JD, Turecek PL, Mannucci PM. Measure-ment of von Willebrand factor cleaving protease (ADAMTS-13): results of an international collaborative study involving 11 methods testing the same set of coded plasmas. J Thromb Haemost. 2004 Sep;2(9):1601-9.

References

ity for ADAMTS13 were tested for inhibitors except one due to insufficient quantity. We had detected in-hibitors in all the tested 3 samples by both methods (Table 2)

DiscussionRapid recognition of the disease along with timely treatment has greatly improved the clinical outcome by inducing remission in more than 80% of patients.7 The specificity of low ADAMTS13 levels for the di-agnosis of TTP, HUS, or other thrombotic micro-angiopathies is still debated.8 Although the current recommendation does not mandate having the result of ADAMTS13 to initiate management, yet the value of this test is in addition to establishment of the dif-ferential diagnostic criteria for the microangiopathic haemolytic anaemias. It is useful in the daily clinical management of TTP and as a follow up marker for

detecting relapse. There are many methods that have been developed for testing ADAMTS13 levels and inhibitor. Although most of the methods displayed a linear relationship when studied in comparison to a reference method, yet the degree of agreement be-tween them was variable. Tripodi A al showed in a large international collaborative study involving 11 different methods that the best concordance between observed and expected levels was achieved by two methods.8 One is our newly validated method using VWF ristocetin cofactor with r2 value of 0.98.

In our study we confirmed the good concordance between this method and another reference method based on SDS-agarose in measuring the cleavage of ultralarge VWF by the protease.

In conclusion, we are reporting validity and sensi-tivity of an automated and rapid method for testing ADAMTS13 activity with ability to detect inhibitors.

Trusted to sustain activity

Baxter and Feiba are trademarks of Baxter International Inc. its subsidiaries and affiliates. Copyright (June 2009). GBL768

Up to 12 hours between doses to enjoy his day1

• Offers patients a long dosing interval, which can provide a greater sense of freedom1

Proven in on-demand therapy• 93% (153/165 episodes) efficacy in stopping bleeds in a prospective study2

• Results from the FENOC study showed comparable (but not equivalent) bleed resolution to rFVIIa at all time points3

Demonstrated efficacy in prophylaxis• Up to 83% reduction in bleeding episodes (per 6 months)4

• Improved or stabilized joint status in 85% to 100% of patients4,5

Integral to therapy• Patient responses may vary, so both agents play an important role in inhibitor management3,6,7

A heritage you can trust• Physicians in more than 50 countries rely on FEIBA therapy• FEIBA therapy has been used successfully for more than 30 years8

Help your patients by making FEIBA therapy an integral part of your inhibitor management strategy.

References: 1. FEIBA VH [package insert]. Westlake Village, Calif: Baxter Healthcare Corp; 2006. 2. Hilgartner MW, Knatterud GL, and the FEIBA Study Group. The use of factor eight inhibitor by-passing activity (FEIBA Immuno) product for treatment of bleeding episodes in hemophiliacs with inhibitors. Blood. 1983; 61:36-40. 3. Astermark J, Donfield SM, DiMichele DM, et al; for the FENOC Study Group. A randomized comparison of bypassing agents in hemophilia complicated by an inhibitor: the FEIBA NovoSeven Comparative (FENOC) Study. Blood. 2007;109:546-551. 4. Leissinger CA, Becton DL, Ewing NP, Valentino LA. Prophylactic treatment with activated prothrombin complex concentrate (FEIBA) reduces the frequency of bleeding episodes in paediatric patients with haemophilia A and inhibitors. Haemophilia. 2007;13:249-255. 5. DiMichele D, Négrier C. A retrospective postlicensure survey of FEIBA efficacy and safety. Haemophilia. 2006;12:352-362. 6. National Hemophilia Foundation. MASAC Recommendation 167 Regarding the Use of Bypassing Agents in Patients with Hemophilia A or B and Inhibitors. Available at: http://www.hemophilia.org/NHFWeb/MainPgs/MainNHF.aspx?menuid=57&contentid=579. Accessed April 2009. 7. Berntorp E. Differential response to bypassing agents complicates treatment in patients with haemophilia and inhibitors. Haemophilia. 2009;15:3-10. 8. Turecek PL, Váradi K, Gritsch H, Schwarz HP. FEIBA: mode of action. Haemophilia. 2004;10(suppl 2):3-9.

FEIBA therapy gives him extra hours to enjoy his dayUp to 12 hours between doses

Baxter A.G CompanyAhsaa st, Rabwa dist, Dareen BuildingP.O Box 246968 Riyadh 11312Saudi Arabia

IXth congress of the Saudi Societyof Hematology

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ΩódG ¢VGôeC’

22-23 December 201016-17 Moharram 1432

Gulf Meridien HotelAl Khobar

Topics Include:

Hematological malignancies• Hemoglobinopathies• Transfusion Medicine• Thrombosis & Hemostasis•

Registration:Symposium Secretary: Miss Randa Ramadan

hematology [email protected]

+966553622677

pioneers in hematology


Abul Qasim Khalaf ibn al-Abbas al-Zahrawi (known in the west as Abulcasis) was born in 936 C.E. in Zahra in the neighbourhood of

Cordova. He became one of the most renowned sur-geons of the Muslim era and was physician to King Al-Hakam-II of Spain. After a long medical career, rich with significant original contribution, he died in 1013 C.E.

Abulcasis wrote the first description on hemo-philia, an inherited genetic disease, in his Al-Tasrif, in which he wrote of an Andalusian family whose males died of bleeding after minor injuries.

He is best known for his early and original break-throughs in surgery as well as for his famous Medical Ecyclopaedia called Al-Tasrif, which is composed of thirty volumes covering different aspects of medi-cal science. The more important part of this series comprises three books on surgery, which describe in detail various aspects of surgical treatment as based on the operations performed by him, including cau-terization, removal of stone from the bladder, dis-section of animals, midwifery, stypics, and surgery of eye, ear and throat. He perfected several delicate operations, including removal of the dead foetus and amputation.

Al-Tasrif was first translated by Gherard of Cremona into Latin in the Middle Ages. It was fol-lowed by several other editors in Europe. The book contains numerous diagrams and illustrations of surgical instruments, in use or developed by him, and comprised a part of the medical curriculum in European countries for many centuries. Contrary to the view that the Muslims fought shy of surgery, Al-Zahrawi’s Al-Tasrif provided a monumental collec-tion for this branch of applied science.

Al-Zahrawi was the inventor of several surgical instruments. A special medical instrument called a cauter, used for the cauterization of arteries, was first described by Abu al-Qasim in his Kitab al-Tasrif. Abu al-Qasim also introduced the use of ligature for the blood control of arteries in lieu of cauteriza-tion. He was the first surgeon to make use of cotton (which itself is derived from the Arabic word qutn) as a medical dressing for controlling hemorrhage.

Al-Zahrawi became an eminent surgeon. He was appointed as the Court-Physician of King Abdel-

Mohamad Qari

Abul Qasim Khalaf ibn al-Abbas al-Zahrawi (Abulcasis)

From the Department of Hematology, College of Medicine, King Abdulaziz University,

President of Saudi Society of Hematology

Key Words: Pioneers, Al-Zahrawi, Abulcasis


Dr. Mohamad QariHematology Dept.College of MedicineKing Abdulaziz UniversityPO Box 80215Jeddah 21589Saudi [email protected]

pioneers in hematology


Rahman III. He spent a productive life in practic-ing medicine, especialy in surgery and medical writ-ings. He died at the age of 83. He wrote mainly four books. One of them is “Al-Tastif Liman Ajiz’an Al-Ta’lif ’ which is the best medieval surgical encyclope-dia. It was used in Europe until the 17th century. He stressed the importance of basic sciences: “... Before practicing, one should be familiar with the science of anatomy and the functions of organs so that he will understand them, recognize their shape, understand their connections, and know their borders. Also he should know the bones, nerves, and muscles, their numbers, their origin and insertions, the arteries and the veins, their start and end. These anatomical and physiological bases are important, and as said by Hippocrates: ‘These are many physicians by title and a few by practice.’ ... If one does not comprehend the anatomy and physiology, he may commit a mistake that can kill the patient. I have seen someone, who pretended to be a surgeon, incised an aneurysm in the neck of a woman, mistaking it for an abscess.The woman bled to death.”

Heller stated that Al-Zahrawi described the liga-ture of arteries long before Ambrose Pare (Khairallah 1942). Al-Zahrawi also used cautery to control bleed-ing. He used wax and alcohol to stop bleeding from the skull during cranial surgery. Sprengel said that Al-Zahrawi was the first to teach the lithotomy position for vaginal operations (Khairallah 1942). Al-Zahrawi also described the tracheotomy operation and per-formed it as an emergency on one of his servants. He was the first to write on orthodontia. He showed evidence of great experience from details of clinical picture and surgical procedures e.g. his description of varicose veins stripping, even after ten centuries, is almost like modern surgery (Al-Okbi 1971): “... Have the leg shaved if it is much hairy. The patient gets a bath and his leg is kept in hot water until it becomes red and the veins dilate; or he exercises vigorously. Incise the skin opposite the varicose vein longitu-dinally either at the ankle or at the knee. Keep the skin opened by hooks. Expose, dissect, and separate the vein. Introducc a spatula underneath it. When the vein is elevated above the skin level, hang it with a blunt rounded hook. Repeat the procedure about three fingers from the previous site and hang the vein with another hook as previously done. Repeat the procedure at as many sites along the varicose vein as necessary. At the ankle, ligate and strip it by pulling it from the incision just above. When it reaches there,

repeat at the higher incision until all of it is stripped. Ligate the vein and then excise it. If difficulty is en-countered in pulling it, ligate its terminal part with a string and pass it under the spatula and dissect it further. Pull gently and avoid its tearing because if it does, it becomes difficult to strip all of it and can cause harm to the patient. When you have stripped it all, put alcohol spanges at the sites of the skin inci-sions and take care of the incisions until they heal. If the varicose vein is tortuous, you have to incise the skin more frequently, at each change of direction. Dissect it and hang it with the hooks and strip it as previously described. Do not tear the vein or injure it. If this happens, it becomes difficult to strip it. The hooks used should be blunt, eyeless, and rounded, otherwise it can injure the vein”.

During the time of Al-Zahrawi, surgery in the Islamic world became a respected specialty practiced by reputable physicians. On the contrary in Europe, surgery was belittled and practiced by barbers and butchers. In 1163 A.D., the Council of Tours declared the following resolution “Surgery is to be abandoned by the schools of medicine and by all decent physi-cians.”

al-zahrawi (abulcassis)

Saudi Society of Hematology is based in the College of Medicine, King Abdul Aziz University, Jeddah, Saudi Arabia as an official league for Clinical Hematologists (Adults and Pediatrics), Hematopathologists, Immunohematologists, Internists, Scientists, Technologists, Nurses, and all of those who have interest in any aspect of blood disorders.

The society encourages all members and non-members to support the journal by submitting their research papers and clinical experiences for publication in the journal.

Mohammed H. Qari, MD, FRCPA,President,

[email protected],www.hematology-sa.org

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