The Veterinary Journal 193 (2012) 24–31

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The Veterinary Journal

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Review

Prognostic studies of canine and feline mammary tumours: The needfor standardized procedures

A.J.F. Matos a,b,⇑, C.S. Baptista a,c, M.F. Gärtner a,d, G.R. Rutteman e,f

a Institute of Biomedical Sciences of Abel Salazar (ICBAS), University of Porto, Portugalb Multidisciplinary Unit for Biomedical Research (UMIB), University of Porto, Portugalc Institute for Biotechnology and Bioengineering, Centre of Genomics and Biotechnology, University of Trás-os-Montes and Alto Douro (IBB/CGB-UTAD), Vila Real, Portugald Institute of Molecular Pathology and Immunology, University of Porto (IPATIMUP), Portugale Utrecht University Clinic of Companion Animals, Utrecht, The Netherlandsf Specialist Veterinary Centre De Wagenrenk, Utrecht, The Netherlands

a r t i c l e i n f o a b s t r a c t

Article history:Accepted 31 December 2011

Keywords:CanineFelineMammary tumoursPrognostic studiesStandardizationComparative oncology

1090-0233/$ - see front matter � 2012 Elsevier Ltd. Adoi:10.1016/j.tvjl.2011.12.019

⇑ Corresponding author at: Institute of Biomedic(ICBAS), University of Porto, Portugal. Tel.: +351 22 2

E-mail address: ajmatos@icbas.up.pt (A.J.F. Matos)

For several years, veterinary oncologists have been struggling with the prognosis of mammary tumours indogs and cats. Translation of tumour characteristics into prognostic information is an invaluable tool forthe use of the most appropriate therapies, as well as for planning innovative therapeutic trials. Moreover,canine and feline spontaneous mammary gland tumours are good models for the study of human breastcancer. Collecting and interpreting information regarding the prognosis of canine and feline mammarytumours is difficult due to the fact that different methods have been applied to study various componentsand characteristics. This review identifies some of the challenges of prognostic studies of spontaneouscanine and feline mammary tumours and suggests standardized procedures to overcome these chal-lenges and facilitate reproducibility and assessment of results.

� 2012 Elsevier Ltd. All rights reserved.

Introduction

A recent publication (Webster et al., 2011) recommendedguidelines for the conduct and evaluation of prognostic studies inveterinary oncology. The present article highlights challenges andpossible solutions specifically related to the study of mammarytumours in companion animals, specifically dogs and cats.

The most important information that is obtained from theexamination of surgically excised canine and feline mammarytumours (cMGT and fMGT, respectively) is related to prognosis.The prognostic value of the clinico-pathological characteristics incMGT and fMGT has led to a continued debate for over 30 yearsamongst veterinary oncologists. At the same time, companion ani-mal longevity is increasing (Withrow, 2007), leading to a highernumber of animals at risk for the development of cancer. Addition-ally, the awareness of the need for health care of those animalsalong with an improvement of the veterinary clinical services,increase the need for accepted high-value prognostic features thatcan be applied in routine veterinary practice.

Recent publications point to the advantages of cMGT and fMGTas models of human breast cancer due to several similarities, such

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al Sciences of Abel Salazar062266..

as relative age of onset, incidence, risk factors, biological behav-iour, metastatic pattern, histopathological and molecular features,and responses to therapy (Pang and Argyle, 2009; Uva et al., 2009;Baptista et al., 2010; Rivera and von Euler, 2011). As pointed out byPaoloni and Khanna (2008), cancer studies must be conducted un-der clear regulatory guidance, such that results can be correlatedwith studies in humans and benefit pets affected by these cancers.

In studies of prognostic factors for a complex disease such ascMGT and fMGT, large size populations are required. As a generalrule for multivariable models, the number of events should be atleast 10 times the number of potential prognostic variablesincluded in the model (Webster et al., 2011). Clinical trials withsufficient power regarding postoperative treatment of cMGT andfMGT are relatively scarce in the veterinary literature (Parodiet al., 1983; MacEwen et al., 1984a,b, 1985; Rutten et al., 1990;Morris et al., 1993, 1998; Fox et al., 1995; Yamagami et al.,1996a; Teske et al., 1998; Karayannopoulou et al., 2001; Novosadet al., 2006; Simon et al., 2006) and, in contrast to human breastcancer trials, are conducted on populations selected on the basisof a suspected, but not proven similar prognosis or drug suscepti-bility. Indeed, these selections rely on clinical staging or histologi-cal grading systems designed decades ago.

One initial step was taken when a group of veterinary and humanmedicine scientists met in 1966 to start a project under the auspicesof the World Health Organization (WHO) that had as main purpose

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‘to reveal similarities and differences between tumours in man anddomestic animals and thus provide a sound basis for research in com-parative oncology generally, and as secondary aim to help advance vet-erinary pathology’ (Beveridge and Sobin, 1974). Eight years later thisgroup published the first International Histological Classification ofTumours of Domestic Animals (Hampe and Misdorp, 1974) and sev-eral variant classification systems have been proposed since then(Gama et al., 2008; Sassi et al., 2010; Goldschmidt et al., 2011).

A second step was the proposal of the clinical TNM-staging sys-tem (T, tumour diameter; N, regional lymph nodes involvement;M, distant metastasis) by a group of veterinary scientists withexperience in oncology and/or pathology, once again under theauspices of the WHO (Owen, 1980). This proposal was a startingpoint, not an affirmed directive, since its validity and usefulnesscould only be tested with experience. In fact, several amendmentswere subsequently suggested in the light of information providedby clinical trials (Lana et al., 2007). When considered in retrospect,these trials were destined to suffer from the lack of clearly definedprognostic and predictive factors. One might even say that 40 yearsof research on cMGT and fMGT prognostic features have not beenenough to identify widely accepted guidelines in their assessment.

When trying to reach conclusions based upon therapeutic trialsand prognostic studies, the reviewer is confronted with the multi-tude of methodologies in virtually all aspects of the work, hamper-ing attempts to compare results and conclusions. In contrast,human breast cancer studies are currently focused on specificgroups of patients with similar prognosis based on well-estab-lished prognostic and predictive factors (Bauer et al., 2007; Pataniand Mokbel, 2009; Davoli et al., 2010). Lately, attention has beendirected at the prognostic value of the activity of a broad categoryof genes by means of expression profiling which, in a recent meta-analysis of over 1000 breast cancer patients, has been shown to bea powerful tool (Györffy and Schäfer, 2009).

Compared to experimental rodent cancer models, prognosticstudies in spontaneous cMGT and fMGT are subject to more uncon-trolled variables that can bias their results. However, spontaneouscancer cases are much more similar to real life and their study mayhave greater value for practical therapeutic guidelines or protocols.The reduction of methodological variables between studies wouldimprove comparison of such studies and would facilitate the iden-tification of prognostic factors for cMGT and fMGT.

Here, our aim is to propose, based on the literature, adherenceto methodological guidelines (Webster et al., 2011) and to performa critical evaluation of essential methodologies that may help im-prove the design of prognostic studies of cMGT and fMGT. Suchimprovements are necessary to better understand the real prog-nostic value of host and tumour characteristics.

Case selection

It is common for dogs and cats with MGT to have a previous his-tory of the same condition, leading to uncertainty when attributingthe outcome to one specific malignancy. In addition, the length oftime since first manifestation may vary widely which may influencepost-treatment events. In many veterinary studies, it is not alwaysclear whether such information was available or if the animals wereexcluded from the study. In our opinion, animals with a previoushistory of MGT may be included only if the previous tumours wereclassified as benign by expert histopathological analysis. Informa-tion sometimes relies upon pre-operative cytological diagnosis,but such cases should be excluded even if considered benignbecause of possible cytological under-estimation of the tumour’smalignant potential (Allen et al., 1986; Cassali et al., 2007). All ani-mals with previous history of malignant MGT should be excludedfrom prospective studies regardless of the previous tumour charac-teristics or how much time elapsed between development of the

two tumours, since postoperative events cannot be accuratelyattributed to the latest malignancy.

Dogs also commonly present with more than one malignantMGT simultaneously and, when investigating tumour characteris-tics in relation to prognosis, published reports are not always clearon which tumour was considered. Often, it is stated that ‘the mostmalignant tumour’ was considered (Yamagami et al., 1996a; Pérez-Alenza et al., 1997; Peña et al., 1998; Nieto et al., 2000; Itoh et al.,2005; Karayannopoulou et al., 2005; De las Mulas et al., 2005), butfew studies explain the basis for malignancy assessment and, insuch cases, criteria are often unique to individual studies.

To our knowledge, only one study reported that all animals withmore than one malignant tumour type were excluded (Allen andMahaffey, 1989). For those animals with more than one malig-nancy (and an uneventful follow-up) it may be acceptable to con-sider the characteristics of all malignant tumours, since none wasable to recur or metastasize during the period. If one tumour is se-lected on the basis of its histological features of malignancy andconsidered responsible for later metastatic disease, there is clearlya strong potential bias since the inclusion criteria confuse thestudy objective. This malignancy selection assumes that (1) themicroscopic features of malignancy have biological meaning and(2) all tumours developed at the same time. However, it is possiblethat an intrinsically less aggressive tumour that developed earliercompensated for its slower spread with increased time to metasta-sis and was responsible for metastatic disease in spite of the pres-ence of a more recent and more aggressive primary tumour.

Another reason for concern is the fact that histological assess-ment of malignancy is often partly based upon karyomegaly,meaning that the presence of large nuclei in tumours leads to ahigher nuclear grade. Yet, especially in dogs, loss of DNA (DNA-aneuploidy) that can lead to relatively small nuclei may be presentin about 20% of mammary cancers (Rutteman et al., 1988; Hellménet al., 1988), and such DNA-hypoploid cancers may have a highmalignancy potential that is underestimated when applying histo-logical nuclear grading systems. Thus, in animals with more thanone histologically malignant tumour that developed metastaticdisease during follow-up, it is not possible to determine with highconfidence which primary tumour was the source of metastasisand such cases should be excluded from prospective studies.

The prognostic value of ovariohysterectomy at the time of mas-tectomy in animals with malignant MGT is still under debate(Yamagami et al., 1996b; Morris et al., 1998; Sorenmo et al.,2000; Philibert et al., 2003; Overley et al., 2005). If this factor isomitted, the possible effect may interfere with the study of otherprognostic factors and the efficacy of adjuvant post-operative ther-apeutic actions. Until this issue is clarified, bitches and queensshould preferably be included in groups based on their ovariohys-terectomy status. If numbers are not sufficient for this separation,then the number of spayed and non-spayed animals should be bal-anced in each group of animals. It may be argued that the admin-istration of oestrous-preventive hormones should also beconsidered a factor when grouping cases, although previous stud-ies suggested that it is not a prognostic factor (Hellmén et al.,1993; Peña et al., 1998; Nieto et al., 2000).

Tumour characteristics

Tumour size or volume is one of the most studied characteristicsin prognostic studies of cMGT and fMGT. In essence, size correlateswith number of tumour cell divisions and higher chance for the pro-gression to a more malignant behaviour due to accumulation ofmutations. This is a questionable assumption, as demonstrated byAndea et al. (2002) who concluded that the relationship betweensize and lymph node metastases in human breast cancer is notlinear.

Table 1Clinical malignant mammary tumour staging in dogs and cats.

T: primary tumour maximum diameter Dog Cat

T1 <3 cm <1 cmT2 3–5 cm 1–3 cmT3 >5 cm >3 cmT4 Inflammatory carcinomaa

N: regional lymph node status Dog and cat

N0 Histological or cytological – no signs of metastasisN1 Histological or cytological – metastasis present

M: distant metastasis Dog and cat

M0 Histological or cytological – no signs of metastasisM1 Histological or cytological – metastasis present

Stage groupingI T1 N0 M0II T2 N0 M0III T3 N0 M0IV Any T, N1 M0V Any T, any N, M1

a Excluding inflammatory carcinoma clinically recognized as ‘mastitis carcinomatosa’. The specific category of T4 for inflammatory carcinoma (recognized for itsaggressiveness) was included in the WHO staging system (Owen, 1980), but was erroneously excluded in the 4th edition of Small Animal Clinical Oncology on which this tableis based (Lana et al., 2007).

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One issue that should not be neglected is the time of tumourmeasurement. Currently, size is measured at pre-operative physi-cal examination by palpation or with callipers, the largest diameterdetermining the T category. Such in vivo measurements may notreflect the real size of the tumour, since non-tumour tissues (e.g.skin, peri-tumour fibrosis, and inflammatory reaction) are includedin these crude measurements.

In human breast cancer, critical reviews have led to two stagingsystems. The clinical staging ‘is used to make local/regional treat-ment recommendations’ based on physical and imaging examina-tions complemented by histological biopsy of the primary cancer.In addition, there is the pathological staging system ‘to assess prog-nosis and to make recommendations for adjuvant treatment’. ThepTNM system uses size and N-status as determined from patholog-ical examination (Singletary et al., 2002; Singletary and Conolly,2006).

For mammary tumour management in companion animals,data on the pathological nature of the primary tumour and the re-gional node most often are obtained after surgical excision. Thiscould be seen as an argument to rely more on pTNM staging inprognostic studies and therapeutic trials.

Fixatives tend to reduce tumour volume, so measurementsmust be standardized in each study. This seems best accomplishedby measurement of the primary tumour diameter after fixation atmacroscopic pathological examination. A further problem may oc-cur in animals with more than one histological tumour type diag-nosed within one single mass, complicating the correctmeasurement of each tumour. For example, should the entire massbe measured for carcinomas in predominantly benign tumours orshould the malignant fraction be estimated and only considered?Since carcinomas in benign tumours are rare entities, we suggestexcluding them from prospective studies.

The grouping of tumours according to size is variable amongstudies. If size is not statistically used as a continuous variable, tu-mours should be grouped according to the T status of the TNMclassification of cMGT and fMGT (Table 1) (Lana et al., 2007). How-ever, in our experience, MGT are currently being diagnosed andtreated in less advanced stages, leading to an abundance of casesbeing classified as T1 (<3 cm) or T2 (3–5 cm) and fewer cases inthe T3 (>5 cm) group. Experience in human breast cancer hasclearly shown that in cancers <3 cm, further size subdivision hasprognostic value (Narod, 2011). This should not come as a surprise

given the relationship between diameter and volume, which re-lates to tumour mass and risk of malignant progression (Table 2).

The status of local and regional lymph nodes is among the mostimportant prognostic factors in human breast cancer (Fitzgibbonset al., 2000). Although it has been demonstrated that intra-mam-mary lymph nodes exist in the dog (Matos et al., 2006a), only re-gional nodes (supra-mammary or superficial inguinal andaccessory axillary nodes) are used in the clinical staging of cMGTand fMGT with the status being determined by pre-surgical cyto-logical examination of fine-needle aspiration biopsies (FNABs)and by pathological examination after surgery (Lana et al., 2007).

The status of the axillary node is difficult to ascertain in mostdogs and cats. If abnormalities of these nodes are palpable, it willbe difficult but necessary to biopsy such nodes or to perform rad-ical en bloc resection. For established metastases to the axillarynode, it may be argued that they should be considered as distantnodal metastases instead of distant metastases. Clinical examina-tion of companion animals presented with MGT should not over-look the status of other distant nodes, e.g. the prescapular andeven popliteal nodes as well as the sternal and deep inguinallymph nodes. Any change in size or consistency should be followedby FNABs for cytological examination.

Whether distant lymph node involvement should be classifiedindependently of other distant metastasis is still a matter of debatefor human breast cancer (Singletary et al., 2002). This points to theneed to thoroughly examine the literature on canine and felinemammary cancer and search for evidence for adjustments in thecurrent 30 year-old TNM system to increase its prognostic value.

In dogs (as in humans) occult nodal micrometastases (i.e., notdetected by routine haematoxylin and eosin [HE] evaluation, butevidenced by techniques such as immunohistochemistry [IHC])have been documented, although their significance is still unclear(Sobin and Wittekind, 2002; Matos et al., 2006a). However, whilein human breast cancer nodal micrometastases (between 0.2 and2 mm) or isolated tumour cells (<0.2 mm) are categorized for ther-apeutic purposes as non-metastatic tumours, clinical staging ofthese cases in cats and dogs classifies them as tumours with nodalmetastases. Before this technique is considered in routine pathol-ogy laboratories, there is a need for studies to assess the prognosticand predictive value of micrometastases (Fig. 1).

Optimal sampling strategies for lymph nodes are still beingevaluated and must be standardized to minimize the chances of

Table 2Relationship between tumour diameter (T) andtumour volume (V) assuming a sphericaltumour.

T (cm) V (cm3)

1 0.522 4.193 14.144 33.515 65.45

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undetected metastatic deposits. Currently, cases should preferablybe grouped as presence of node metastases, presence of nodemicrometastases and lack of nodal metastases. Once the prognosticsignificance of node micrometastases is better understood, guide-lines could be generated for classification of these cases with re-spect to TNM status. Such TNM staging could then be comparedto the pTNM staging system of human breast cancer.

The search for distant metastasis to internal organs is often re-stricted to standardized radiographic evaluation of the thorax.However, the presence of metastases in abdominal organs withoutthoracic involvement may occur (Misdorp and Hart, 1979). There-fore, prospective studies should also include ultrasonographicimaging of the abdomen in addition to three radiographic viewsof the thorax. The ultrasonographic suspicion of metastasis shouldbe preferably confirmed by cytological analysis of FNABs.

Computed tomography (CT) scans of the thorax and abdomenare considered to be the best and most reproducible current meth-od to measure lesions selected for response assessment (Eisenhau-er et al., 2009). However, CT scans cannot be expected to beperformed at all study centres and CT can detect smaller (i.e.64 mm) and likely earlier metastases than routine radiographicimaging. Since the efficacy of adjuvant therapy is at least in part re-lated to tumour mass, patients diagnosed with CT scans may ben-efit more from treatment than those with larger metastasesdetected by radiography.

Histological type

cMGT and fMGT are classified by the WHO according to theirphenotypic characteristics (Misdorp et al., 1999). According to thisclassification, ‘a prognostic element is added to the classification sys-tem’ despite the lack of specialized prognostic indicators. A new

Fig. 1. Lymph node with isolated tumour cells from a canine anaplastic mammarycarcinoma identified by immunohistochemistry using an anticytokeratin AE1/AE3antibody (Zymed Laboratories). Although identifiable, their prognostic significanceremains unknown. Bar, 100 lm.

proposal with amendments to the 1999 classification has recentlybeen published (Goldschmidt et al., 2011). It is our opinion that,until consensus is reached on the new classification, the 1999WHO classification should be followed. For consistent and robustevaluations, histopathological examination and classificationshould be performed by certified veterinary pathologists and it ispreferable to use a minimum of two pathologists for tumour clas-sification with consensus being reached when disagreements oc-cur. If necessary, immunohistochemical techniques should beused for classification when routine HE stain is insufficient to reacha diagnosis (Fig. 2).

Infiltrative/invasive type of growth

Four categories of tumour growth are recognized: (1) tumourswith cellular evidence of malignancy but with an intact basementmembrane in all sections examined (carcinoma in situ); (2) tu-mours that grow cohesively and push normal surrounding tissues,but without clear infiltration (expansive growth); (3) tumours withstromal infiltration (infiltrative growth); and (4) tumours with vas-cular invasion (vasoinvasive growth) (Matos et al., 2006b).

Although invasive behaviour has been demonstrated to haveprognostic significance (Misdorp and Hart, 1976; Gilbertsonet al., 1983; MacEwen et al., 1984a,b; Shofer et al., 1989; Bostock,1975; Ito et al., 1996; Castagnaro et al., 1998; Itoh et al., 2005; Delas Mulas et al., 2005; Seixas et al., 2011), there are still discrepan-cies regarding the assessment of this feature. In particular, it is un-clear whether vasoinvasive tumours should be combined with orseparate from infiltrative tumours. We believe that there is theneed for separation between tumours with expansive growth, tu-mours with infiltrative growth and tumours with demonstratedvascular invasion. As suggested for human breast cancer (Singl-etary and Conolly, 2006), in situ cancers may not be fully malig-nant and are better kept out of new therapeutic studies.

Histological grade

Several criteria are used to define the histological grade of cMGTand fMGT. Earlier studies (Misdorp and Hart, 1976; Shofer et al.,1989) used cellular and nuclear morphological characteristics togenerate three histological grades based upon structural tissue dif-ferentiation, nuclear anaplasia and mitotic index. Gilbertson et al.(1983) added the type or presence of immune cells. Later, twostudies (Pérez-Alenza et al., 1997; Peña et al., 1998) adopted theScarff–Bloom–Richardson system also based on differentiation, nu-clear pleomorphism and mitotic index, but with the addition ofvascular invasion. Castagnaro et al. (1998), Karayannopoulouet al. (2005), and Seixas et al. (2011) utilized the Elston and Ellisgrading system, while De las Mulas et al. (2005) used the Lagadicand Estrada system.

Although all of these studies attributed a prognostic value tohistological grade, it is hard to define methodological guidelinesto be used for routine laboratory work. Furthermore, these areadaptations from human grading systems that use a different his-tological classification with a potential bias effect on tumour grad-ing. We refrain from indicating a choice for a specific gradingclassification for cMGT and fMGT, but believe that this is an issuethat deserves clarification in the standardization of procedures.

Immunohistochemistry

Comparing immunohistochemical studies in cMGT and fMGTcan be challenging. The rarity of anti-dog or cat antibodies leadsto the use of anti-human or anti-mouse antibodies with question-able specificity. For example, a recent study (Zacchetti et al., 2007)

Fig. 2. Lymph node metastases from a canine mammary complex carcinoma identified by immunohistochemistry. (A) AE1/AE3 (Zymed Laboratories). (B) CK14 (clone LL002;Serotec Laboratories). The use of different immunohistochemical markers allows for the correct identification of the cell types (A, epithelial cells; B, myoepithelial cells). Bar,100 lm.

28 A.J.F. Matos et al. / The Veterinary Journal 193 (2012) 24–31

validated antibodies for the detection of p53 and concluded thatwhile the polyclonal antibody (CM1) appeared to be of value, sev-eral monoclonal antibodies did not although several reports usingthese antibodies can be found in the literature.

To optimize and validate monoclonal antibodies for use in catand dog tissues, the following steps can be used: (1) search forhomology of the antibodies target molecules with selection ofthose with high cross-species homology; (2) use of positive con-trols, such as tissues from the species for which antibodies are pro-duced and canine/feline tissues known to express the antigen; (3)use of an alternative detection method (e.g. Western blotting) toconfirm antibody specificity.

There should also be a clear definition of the method for slideassessment: quantitative vs. semi-quantitative; number of slidesevaluated per tumour; number of microscopic fields assessed; cri-teria for selection of microscopic fields; cell types evaluated; cellu-lar location of staining; labelling intensities used. If possible,figures should be included to illustrate the features used for assess-ment. All immunohistochemically stained slides should be inde-pendently classified by at least two observers.

Genetic and related studies

Prognostic studies in veterinary oncology increasingly usegenomic, transcriptional and proteomic methods. This raises sev-eral considerations related to collection, preservation and process-ing of tissue samples for molecular studies. These studies are oftenlimited by the availability of suitable materials. To acquire qualityRNA is particularly challenging and requires standardized proce-dures to minimize degradation. Currently, there are RNA stabiliza-tion solutions designed to eliminate the need for immediate tissueprocessing or freezing (Mutter et al., 2004).

Formalin fixed, paraffin embedded tissues (FPET) from archivesprovide an invaluable source for large-scale molecular geneticstudies. However, their use is still challenged by poor DNA/RNAquality/quantity and amplification of small fragments (Imyanitovet al., 2002; Farrand et al., 2002; Lin et al., 2009). Studies usingthese samples are frequently hampered by partial nucleic acid deg-radation that occurs during formalin fixation and storage, espe-cially due to increased formalin pH (Poljak et al., 2000). Suchanalytical procedures require tissue fixation in 10% buffered for-malin. Therefore, it is important that practitioners involved in fol-low-up studies are aware of this requirement to properly collecttissue samples for processing. Additionally, formaldehyde isresponsible for the cross-linkage between proteins and DNA orRNA, which in turn can limit further analysis of nucleic acids (Mol-ler et al., 1977; Zsikla et al., 2004). More efficient and lower costnucleic acid extraction kits are being developed to address theseissues.

Placement of a carefully sectioned sample of the tumour (5 mm)in RNA-later solution enables future genetic studies of such sam-ples. In our experience, samples stored in RNA-later overnight at4 �C and immediately routinely processed yield good quality histo-logical slides. It remains to be determined, however, whether IHCanalyses can be performed on these tissues.

Clinical follow-up

Follow-up is one the most important variable in canine and fe-line MGT prognostic studies. Although some studies are basedupon periodical clinical examinations, others rely on record re-views, questionnaires or phone calls to veterinarians or even own-ers. We believe that questionnaires or phone calls are unreliablefollow-up methods. Record reviews are equally unreliable since itis unlikely that all animals were submitted to the same standard-ized procedures. Robust studies must be based on periodical exten-sive physical examinations and imaging procedures for a minimumof 24 months, equivalent in humans to a 8–10 year post-operativefollow-up. If any abnormality is detected during these examina-tions, all available tests should be performed to clarify if it is re-lated to the mammary tumour. For example, the mere detectionof pulmonary masses should not be regarded as proof of metastasisunless cytologically or histopathologically confirmed. In our expe-rience (unpublished data), about 5% of detectable lung masses areeither non-neoplastic lesions or tumours of different origin (e.g.primary lung carcinoma). Therefore, when possible and ethicallyacceptable, biopsies should be used to confirm metastatic disease.

Likewise, when possible, all animals that die or are euthanasedduring the follow-up period must be submitted to complete necr-opsies with careful search for metastases. Compliance of owners topost-mortem examinations can be assumed to be moderate at best,but the authors believe that prospective scientific studies mustemphasize this need to owners when animals are enrolled.

As outlined by the recently published guidelines for prognosticstudies in veterinary oncology (Webster et al., 2011), specific end-points should be defined including disease-free interval or time totumour progression and type of relapse (local, regional, distant).Special attention should be given to discrimination between localrecurrence and de novo primary tumour development. Like in hu-man breast cancer (Yan et al., 2006), there is evidence that thedevelopment of mammary tumours in companion animals can rep-resent an increased risk for new primary tumour developmentafter excision of the first tumour (Stratman et al., 2008).

Although both overall and disease-free survivals have been usedin prognostic studies of cMGT and fMGT, pet owners have the op-tion of electing euthanasia and there is inter-individual variabilityrelated to time of euthanasia and severity of metastatic disease,which is a potential bias in prognostic studies. Therefore,

Table 3Summary of the proposed standardized procedures and suggestions for prognosticstudies of feline and canine mammary gland tumours.

Procedures

Case selection1. Based upon histological evaluation2. No previous diagnosis of malignancy3. Animals with more than one surgically treated

mammary malignancy:(a) No local recurrence or metastasis during follow-up –consider the characteristics of all tumours

(b) Recurrence or metastasis during follow-up – exclude4. Randomized in separate groups or balanced spayed

and non-spayed at the time of mastectomy

Tumour characteristics1. Tumour size and lymph node status determined

from pathological examination2. Consider pathological staging system (pTNM) to assess

prognosis and to make recommendations for adjuvant treatment3. Exclude carcinomas in benign tumours4. If size is not used as a continuous variable, group according

to the T status of the TNM classification5. Assess the status of distant lymph nodes (prescapular, popliteal,

sternal and deep inguinal lymph nodes). Perform fine needle aspiratebiopsies if size and consistency are altered

6. Search for nodal micrometastases using immunohistochemicaltechniques if negative by HE evaluation

Histological type1. Follow 1999 WHO classification2. A minimum of two pathologists should be responsible

for independent tumour classification

Type of growth1. Separate expansive, infiltrative and vasoinvasive growth2. Exclude carcinomas in situ

Histological grade1. Need for specific canine and feline grading classifications

Immunohistochemistry1. Uniform, optimized and specific monoclonal antibodies2. Clear definition and illustration of sample evaluation (quantitative

or semi-quantitative; number of slides per tumour; number ofmicroscopic fields per slide; criteria for slide and field selection)

Genetic and related studiesStandardized collection of samples in order to avoid nucleic

acid degradation:1. RNA stabilization solutions (5 mm sample)2. Tissue fixation in 10% buffered formalin (remaining tissues)

Clinical follow-up1. Every 2–3 months for a minimum of 2 years after surgery2. Complete physical examination; abdominal and thoracic imaging3. Biopsies to confirm metastatic disease4. Complete necropsies5. Disease-free interval should be the preferred endpoint

ProposalCreation of a task group of veterinary oncologists and pathologists to

elaborate and periodically update standardized procedures for prognosticstudies of canine and feline mammary tumours

A.J.F. Matos et al. / The Veterinary Journal 193 (2012) 24–31 29

disease-free interval, which exclusively depends on the stage of thedisease and the aggressiveness of the primary tumour, is likely thebest endpoint for cMGT and fMGT prognostic studies.

Conclusions

Comparing results and identifying solid prognostic factors forcMGT and fMGT is challenging because of inconsistent approachesin study designs and endpoints. Here, we suggest strategies toovercome these difficulties and to avoid potential biases in orderto improve the strength and translational value of settings or ther-apeutic trials (Table 3). The creation of a task group of veterinaryoncologists and pathologists would be useful to elaborate and peri-odically update standardized procedures related to clinical staging,

pathological classification and staging, as well as histological grad-ing of malignancy.

Conflict of interest statement

None of the authors of this paper has a financial or personalrelationship with other people or organisations that could inappro-priately influence or bias the content of the paper.

Acknowledgements

The authors express their sincere gratitude to Professor StephenWithrow (Animal Cancer Centre, Department of Clinical Sciences,Colorado State University, USA) for his constructive review of themanuscript.

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