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

The Veterinary Journal 193 (2012) 24–31

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Review

Prognostic studies of canine and feline mammary tumours: The need for 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, Portugal Multidisciplinary Unit for Biomedical Research (UMIB), University of Porto, Portugal c Institute for Biotechnology and Bioengineering, Centre of Genomics and Biotechnology, University of Trás-os-Montes and Alto Douro (IBB/CGB-UTAD), Vila Real, Portugal d Institute of Molecular Pathology and Immunology, University of Porto (IPATIMUP), Portugal e Utrecht University Clinic of Companion Animals, Utrecht, The Netherlands f Specialist Veterinary Centre De Wagenrenk, Utrecht, The Netherlands b

a r t i c l e

i n f o

Article history: Accepted 31 December 2011

Keywords: Canine Feline Mammary tumours Prognostic studies Standardization Comparative oncology

a b s t r a c t For several years, veterinary oncologists have been struggling with the prognosis of mammary tumours in dogs and cats. Translation of tumour characteristics into prognostic information is an invaluable tool for the 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 breast cancer. Collecting and interpreting information regarding the prognosis of canine and feline mammary tumours is difficult due to the fact that different methods have been applied to study various components and characteristics. This review identifies some of the challenges of prognostic studies of spontaneous canine and feline mammary tumours and suggests standardized procedures to overcome these challenges and facilitate reproducibility and assessment of results. Ó 2012 Elsevier Ltd. All rights reserved.

Introduction A recent publication (Webster et al., 2011) recommended guidelines for the conduct and evaluation of prognostic studies in veterinary oncology. The present article highlights challenges and possible solutions specifically related to the study of mammary tumours in companion animals, specifically dogs and cats. The most important information that is obtained from the examination of surgically excised canine and feline mammary tumours (cMGT and fMGT, respectively) is related to prognosis. The prognostic value of the clinico-pathological characteristics in cMGT and fMGT has led to a continued debate for over 30 years amongst veterinary oncologists. At the same time, companion animal longevity is increasing (Withrow, 2007), leading to a higher number of animals at risk for the development of cancer. Additionally, the awareness of the need for health care of those animals along with an improvement of the veterinary clinical services, increase the need for accepted high-value prognostic features that can be applied in routine veterinary practice. Recent publications point to the advantages of cMGT and fMGT as models of human breast cancer due to several similarities, such

⇑ Corresponding author at: Institute of Biomedical Sciences of Abel Salazar (ICBAS), University of Porto, Portugal. Tel.: +351 22 2062266. E-mail address: [email protected] (A.J.F. Matos). 1090-0233/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2011.12.019

as relative age of onset, incidence, risk factors, biological behaviour, 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 by Paoloni and Khanna (2008), cancer studies must be conducted under clear regulatory guidance, such that results can be correlated with studies in humans and benefit pets affected by these cancers. In studies of prognostic factors for a complex disease such as cMGT and fMGT, large size populations are required. As a general rule for multivariable models, the number of events should be at least 10 times the number of potential prognostic variables included in the model (Webster et al., 2011). Clinical trials with sufficient power regarding postoperative treatment of cMGT and fMGT are relatively scarce in the veterinary literature (Parodi et 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; Novosad et al., 2006; Simon et al., 2006) and, in contrast to human breast cancer trials, are conducted on populations selected on the basis of a suspected, but not proven similar prognosis or drug susceptibility. Indeed, these selections rely on clinical staging or histological grading systems designed decades ago. One initial step was taken when a group of veterinary and human medicine scientists met in 1966 to start a project under the auspices of the World Health Organization (WHO) that had as main purpose

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

‘to reveal similarities and differences between tumours in man and domestic animals and thus provide a sound basis for research in comparative oncology generally, and as secondary aim to help advance veterinary pathology’ (Beveridge and Sobin, 1974). Eight years later this group published the first International Histological Classification of Tumours of Domestic Animals (Hampe and Misdorp, 1974) and several 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 system (T, tumour diameter; N, regional lymph nodes involvement; M, distant metastasis) by a group of veterinary scientists with experience in oncology and/or pathology, once again under the auspices of the WHO (Owen, 1980). This proposal was a starting point, not an affirmed directive, since its validity and usefulness could only be tested with experience. In fact, several amendments were subsequently suggested in the light of information provided by clinical trials (Lana et al., 2007). When considered in retrospect, these trials were destined to suffer from the lack of clearly defined prognostic and predictive factors. One might even say that 40 years of research on cMGT and fMGT prognostic features have not been enough to identify widely accepted guidelines in their assessment. When trying to reach conclusions based upon therapeutic trials and prognostic studies, the reviewer is confronted with the multitude of methodologies in virtually all aspects of the work, hampering attempts to compare results and conclusions. In contrast, human breast cancer studies are currently focused on specific groups of patients with similar prognosis based on well-established prognostic and predictive factors (Bauer et al., 2007; Patani and Mokbel, 2009; Davoli et al., 2010). Lately, attention has been directed at the prognostic value of the activity of a broad category of genes by means of expression profiling which, in a recent metaanalysis of over 1000 breast cancer patients, has been shown to be a powerful tool (Györffy and Schäfer, 2009). Compared to experimental rodent cancer models, prognostic studies in spontaneous cMGT and fMGT are subject to more uncontrolled variables that can bias their results. However, spontaneous cancer cases are much more similar to real life and their study may have greater value for practical therapeutic guidelines or protocols. The reduction of methodological variables between studies would improve comparison of such studies and would facilitate the identification of prognostic factors for cMGT and fMGT. Here, our aim is to propose, based on the literature, adherence to methodological guidelines (Webster et al., 2011) and to perform a critical evaluation of essential methodologies that may help improve the design of prognostic studies of cMGT and fMGT. Such improvements are necessary to better understand the real prognostic value of host and tumour characteristics. Case selection It is common for dogs and cats with MGT to have a previous history of the same condition, leading to uncertainty when attributing the outcome to one specific malignancy. In addition, the length of time since first manifestation may vary widely which may influence post-treatment events. In many veterinary studies, it is not always clear whether such information was available or if the animals were excluded from the study. In our opinion, animals with a previous history of MGT may be included only if the previous tumours were classified as benign by expert histopathological analysis. Information sometimes relies upon pre-operative cytological diagnosis, but such cases should be excluded even if considered benign because of possible cytological under-estimation of the tumour’s malignant potential (Allen et al., 1986; Cassali et al., 2007). All animals with previous history of malignant MGT should be excluded from prospective studies regardless of the previous tumour characteristics or how much time elapsed between development of the

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two tumours, since postoperative events cannot be accurately attributed to the latest malignancy. Dogs also commonly present with more than one malignant MGT simultaneously and, when investigating tumour characteristics in relation to prognosis, published reports are not always clear on which tumour was considered. Often, it is stated that ‘the most malignant tumour’ was considered (Yamagami et al., 1996a; PérezAlenza 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), but few studies explain the basis for malignancy assessment and, in such cases, criteria are often unique to individual studies. To our knowledge, only one study reported that all animals with more than one malignant tumour type were excluded (Allen and Mahaffey, 1989). For those animals with more than one malignancy (and an uneventful follow-up) it may be acceptable to consider the characteristics of all malignant tumours, since none was able to recur or metastasize during the period. If one tumour is selected on the basis of its histological features of malignancy and considered responsible for later metastatic disease, there is clearly a strong potential bias since the inclusion criteria confuse the study objective. This malignancy selection assumes that (1) the microscopic features of malignancy have biological meaning and (2) all tumours developed at the same time. However, it is possible that an intrinsically less aggressive tumour that developed earlier compensated for its slower spread with increased time to metastasis and was responsible for metastatic disease in spite of the presence of a more recent and more aggressive primary tumour. Another reason for concern is the fact that histological assessment of malignancy is often partly based upon karyomegaly, meaning that the presence of large nuclei in tumours leads to a higher nuclear grade. Yet, especially in dogs, loss of DNA (DNAaneuploidy) that can lead to relatively small nuclei may be present in about 20% of mammary cancers (Rutteman et al., 1988; Hellmén et al., 1988), and such DNA-hypoploid cancers may have a high malignancy potential that is underestimated when applying histological nuclear grading systems. Thus, in animals with more than one histologically malignant tumour that developed metastatic disease during follow-up, it is not possible to determine with high confidence which primary tumour was the source of metastasis and such cases should be excluded from prospective studies. The prognostic value of ovariohysterectomy at the time of mastectomy 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 is omitted, the possible effect may interfere with the study of other prognostic factors and the efficacy of adjuvant post-operative therapeutic actions. Until this issue is clarified, bitches and queens should preferably be included in groups based on their ovariohysterectomy status. If numbers are not sufficient for this separation, then the number of spayed and non-spayed animals should be balanced in each group of animals. It may be argued that the administration of oestrous-preventive hormones should also be considered a factor when grouping cases, although previous studies 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 characteristics in prognostic studies of cMGT and fMGT. In essence, size correlates with number of tumour cell divisions and higher chance for the progression to a more malignant behaviour due to accumulation of mutations. This is a questionable assumption, as demonstrated by Andea et al. (2002) who concluded that the relationship between size and lymph node metastases in human breast cancer is not linear.

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

Table 1 Clinical malignant mammary tumour staging in dogs and cats. T: primary tumour maximum diameter

Dog

Cat

T1 T2 T3 T4

<3 cm 3–5 cm >5 cm Inflammatory carcinomaa

<1 cm 1–3 cm >3 cm

N: regional lymph node status

Dog and cat

N0 N1

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

M: distant metastasis

Dog and cat

M0 M1

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

Stage grouping I II III IV V

T1 N0 M0 T2 N0 M0 T3 N0 M0 Any T, N1 M0 Any T, any N, M1

a Excluding inflammatory carcinoma clinically recognized as ‘mastitis carcinomatosa’. The specific category of T4 for inflammatory carcinoma (recognized for its aggressiveness) 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 table is based (Lana et al., 2007).

One issue that should not be neglected is the time of tumour measurement. Currently, size is measured at pre-operative physical examination by palpation or with callipers, the largest diameter determining the T category. Such in vivo measurements may not reflect the real size of the tumour, since non-tumour tissues (e.g. skin, peri-tumour fibrosis, and inflammatory reaction) are included in these crude measurements. In human breast cancer, critical reviews have led to two staging systems. The clinical staging ‘is used to make local/regional treatment recommendations’ based on physical and imaging examinations complemented by histological biopsy of the primary cancer. In addition, there is the pathological staging system ‘to assess prognosis and to make recommendations for adjuvant treatment’. The pTNM system uses size and N-status as determined from pathological 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 regional node most often are obtained after surgical excision. This could be seen as an argument to rely more on pTNM staging in prognostic studies and therapeutic trials. Fixatives tend to reduce tumour volume, so measurements must be standardized in each study. This seems best accomplished by measurement of the primary tumour diameter after fixation at macroscopic pathological examination. A further problem may occur in animals with more than one histological tumour type diagnosed within one single mass, complicating the correct measurement of each tumour. For example, should the entire mass be measured for carcinomas in predominantly benign tumours or should the malignant fraction be estimated and only considered? Since carcinomas in benign tumours are rare entities, we suggest excluding them from prospective studies. The grouping of tumours according to size is variable among studies. If size is not statistically used as a continuous variable, tumours should be grouped according to the T status of the TNM classification of cMGT and fMGT (Table 1) (Lana et al., 2007). However, in our experience, MGT are currently being diagnosed and treated in less advanced stages, leading to an abundance of cases being classified as T1 (<3 cm) or T2 (3–5 cm) and fewer cases in the T3 (>5 cm) group. Experience in human breast cancer has clearly shown that in cancers <3 cm, further size subdivision has prognostic value (Narod, 2011). This should not come as a surprise

given the relationship between diameter and volume, which relates to tumour mass and risk of malignant progression (Table 2). The status of local and regional lymph nodes is among the most important prognostic factors in human breast cancer (Fitzgibbons et al., 2000). Although it has been demonstrated that intra-mammary lymph nodes exist in the dog (Matos et al., 2006a), only regional nodes (supra-mammary or superficial inguinal and accessory axillary nodes) are used in the clinical staging of cMGT and fMGT with the status being determined by pre-surgical cytological 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 most dogs and cats. If abnormalities of these nodes are palpable, it will be difficult but necessary to biopsy such nodes or to perform radical en bloc resection. For established metastases to the axillary node, it may be argued that they should be considered as distant nodal metastases instead of distant metastases. Clinical examination of companion animals presented with MGT should not overlook the status of other distant nodes, e.g. the prescapular and even popliteal nodes as well as the sternal and deep inguinal lymph nodes. Any change in size or consistency should be followed by FNABs for cytological examination. Whether distant lymph node involvement should be classified independently of other distant metastasis is still a matter of debate for human breast cancer (Singletary et al., 2002). This points to the need to thoroughly examine the literature on canine and feline mammary cancer and search for evidence for adjustments in the current 30 year-old TNM system to increase its prognostic value. In dogs (as in humans) occult nodal micrometastases (i.e., not detected by routine haematoxylin and eosin [HE] evaluation, but evidenced by techniques such as immunohistochemistry [IHC]) have been documented, although their significance is still unclear (Sobin and Wittekind, 2002; Matos et al., 2006a). However, while in human breast cancer nodal micrometastases (between 0.2 and 2 mm) or isolated tumour cells (<0.2 mm) are categorized for therapeutic purposes as non-metastatic tumours, clinical staging of these cases in cats and dogs classifies them as tumours with nodal metastases. Before this technique is considered in routine pathology laboratories, there is a need for studies to assess the prognostic and predictive value of micrometastases (Fig. 1). Optimal sampling strategies for lymph nodes are still being evaluated and must be standardized to minimize the chances of

A.J.F. Matos et al. / The Veterinary Journal 193 (2012) 24–31 Table 2 Relationship between tumour diameter (T) and tumour volume (V) assuming a spherical tumour. T (cm)

V (cm3)

1 2 3 4 5

0.52 4.19 14.14 33.51 65.45

undetected metastatic deposits. Currently, cases should preferably be grouped as presence of node metastases, presence of node micrometastases and lack of nodal metastases. Once the prognostic significance of node micrometastases is better understood, guidelines could be generated for classification of these cases with respect to TNM status. Such TNM staging could then be compared to the pTNM staging system of human breast cancer. The search for distant metastasis to internal organs is often restricted to standardized radiographic evaluation of the thorax. However, the presence of metastases in abdominal organs without thoracic involvement may occur (Misdorp and Hart, 1979). Therefore, prospective studies should also include ultrasonographic imaging of the abdomen in addition to three radiographic views of the thorax. The ultrasonographic suspicion of metastasis should be preferably confirmed by cytological analysis of FNABs. Computed tomography (CT) scans of the thorax and abdomen are considered to be the best and most reproducible current method to measure lesions selected for response assessment (Eisenhauer et al., 2009). However, CT scans cannot be expected to be performed at all study centres and CT can detect smaller (i.e. 64 mm) and likely earlier metastases than routine radiographic imaging. Since the efficacy of adjuvant therapy is at least in part related to tumour mass, patients diagnosed with CT scans may benefit more from treatment than those with larger metastases detected by radiography. Histological type cMGT and fMGT are classified by the WHO according to their phenotypic characteristics (Misdorp et al., 1999). According to this classification, ‘a prognostic element is added to the classification system’ despite the lack of specialized prognostic indicators. A new

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proposal with amendments to the 1999 classification has recently been published (Goldschmidt et al., 2011). It is our opinion that, until consensus is reached on the new classification, the 1999 WHO classification should be followed. For consistent and robust evaluations, histopathological examination and classification should be performed by certified veterinary pathologists and it is preferable to use a minimum of two pathologists for tumour classification with consensus being reached when disagreements occur. If necessary, immunohistochemical techniques should be used for classification when routine HE stain is insufficient to reach a diagnosis (Fig. 2). Infiltrative/invasive type of growth Four categories of tumour growth are recognized: (1) tumours with cellular evidence of malignancy but with an intact basement membrane in all sections examined (carcinoma in situ); (2) tumours that grow cohesively and push normal surrounding tissues, but without clear infiltration (expansive growth); (3) tumours with stromal infiltration (infiltrative growth); and (4) tumours with vascular invasion (vasoinvasive growth) (Matos et al., 2006b). Although invasive behaviour has been demonstrated to have prognostic significance (Misdorp and Hart, 1976; Gilbertson et 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; De las Mulas et al., 2005; Seixas et al., 2011), there are still discrepancies regarding the assessment of this feature. In particular, it is unclear whether vasoinvasive tumours should be combined with or separate from infiltrative tumours. We believe that there is the need for separation between tumours with expansive growth, tumours with infiltrative growth and tumours with demonstrated vascular invasion. As suggested for human breast cancer (Singletary and Conolly, 2006), in situ cancers may not be fully malignant and are better kept out of new therapeutic studies. Histological grade Several criteria are used to define the histological grade of cMGT and fMGT. Earlier studies (Misdorp and Hart, 1976; Shofer et al., 1989) used cellular and nuclear morphological characteristics to generate three histological grades based upon structural tissue differentiation, nuclear anaplasia and mitotic index. Gilbertson et al. (1983) added the type or presence of immune cells. Later, two studies (Pérez-Alenza et al., 1997; Peña et al., 1998) adopted the Scarff–Bloom–Richardson system also based on differentiation, nuclear pleomorphism and mitotic index, but with the addition of vascular invasion. Castagnaro et al. (1998), Karayannopoulou et al. (2005), and Seixas et al. (2011) utilized the Elston and Ellis grading system, while De las Mulas et al. (2005) used the Lagadic and Estrada system. Although all of these studies attributed a prognostic value to histological grade, it is hard to define methodological guidelines to be used for routine laboratory work. Furthermore, these are adaptations from human grading systems that use a different histological classification with a potential bias effect on tumour grading. We refrain from indicating a choice for a specific grading classification for cMGT and fMGT, but believe that this is an issue that deserves clarification in the standardization of procedures. Immunohistochemistry

Fig. 1. Lymph node with isolated tumour cells from a canine anaplastic mammary carcinoma identified by immunohistochemistry using an anticytokeratin AE1/AE3 antibody (Zymed Laboratories). Although identifiable, their prognostic significance remains unknown. Bar, 100 lm.

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

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

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.

validated antibodies for the detection of p53 and concluded that while the polyclonal antibody (CM1) appeared to be of value, several monoclonal antibodies did not although several reports using these antibodies can be found in the literature. To optimize and validate monoclonal antibodies for use in cat and dog tissues, the following steps can be used: (1) search for homology of the antibodies target molecules with selection of those with high cross-species homology; (2) use of positive controls, such as tissues from the species for which antibodies are produced and canine/feline tissues known to express the antigen; (3) use of an alternative detection method (e.g. Western blotting) to confirm antibody specificity. There should also be a clear definition of the method for slide assessment: quantitative vs. semi-quantitative; number of slides evaluated per tumour; number of microscopic fields assessed; criteria for selection of microscopic fields; cell types evaluated; cellular location of staining; labelling intensities used. If possible, figures should be included to illustrate the features used for assessment. All immunohistochemically stained slides should be independently classified by at least two observers. Genetic and related studies Prognostic studies in veterinary oncology increasingly use genomic, transcriptional and proteomic methods. This raises several considerations related to collection, preservation and processing of tissue samples for molecular studies. These studies are often limited by the availability of suitable materials. To acquire quality RNA is particularly challenging and requires standardized procedures to minimize degradation. Currently, there are RNA stabilization solutions designed to eliminate the need for immediate tissue processing or freezing (Mutter et al., 2004). Formalin fixed, paraffin embedded tissues (FPET) from archives provide an invaluable source for large-scale molecular genetic studies. However, their use is still challenged by poor DNA/RNA quality/quantity and amplification of small fragments (Imyanitov et al., 2002; Farrand et al., 2002; Lin et al., 2009). Studies using these samples are frequently hampered by partial nucleic acid degradation that occurs during formalin fixation and storage, especially due to increased formalin pH (Poljak et al., 2000). Such analytical procedures require tissue fixation in 10% buffered formalin. Therefore, it is important that practitioners involved in follow-up studies are aware of this requirement to properly collect tissue samples for processing. Additionally, formaldehyde is responsible for the cross-linkage between proteins and DNA or RNA, which in turn can limit further analysis of nucleic acids (Moller et al., 1977; Zsikla et al., 2004). More efficient and lower cost nucleic acid extraction kits are being developed to address these issues.

Placement of a carefully sectioned sample of the tumour (5 mm) in RNA-later solution enables future genetic studies of such samples. In our experience, samples stored in RNA-later overnight at 4 °C and immediately routinely processed yield good quality histological slides. It remains to be determined, however, whether IHC analyses can be performed on these tissues. Clinical follow-up Follow-up is one the most important variable in canine and feline MGT prognostic studies. Although some studies are based upon periodical clinical examinations, others rely on record reviews, questionnaires or phone calls to veterinarians or even owners. We believe that questionnaires or phone calls are unreliable follow-up methods. Record reviews are equally unreliable since it is unlikely that all animals were submitted to the same standardized procedures. Robust studies must be based on periodical extensive physical examinations and imaging procedures for a minimum of 24 months, equivalent in humans to a 8–10 year post-operative follow-up. If any abnormality is detected during these examinations, all available tests should be performed to clarify if it is related to the mammary tumour. For example, the mere detection of pulmonary masses should not be regarded as proof of metastasis unless cytologically or histopathologically confirmed. In our experience (unpublished data), about 5% of detectable lung masses are either non-neoplastic lesions or tumours of different origin (e.g. primary lung carcinoma). Therefore, when possible and ethically acceptable, biopsies should be used to confirm metastatic disease. Likewise, when possible, all animals that die or are euthanased during the follow-up period must be submitted to complete necropsies with careful search for metastases. Compliance of owners to post-mortem examinations can be assumed to be moderate at best, but the authors believe that prospective scientific studies must emphasize this need to owners when animals are enrolled. As outlined by the recently published guidelines for prognostic studies in veterinary oncology (Webster et al., 2011), specific endpoints should be defined including disease-free interval or time to tumour progression and type of relapse (local, regional, distant). Special attention should be given to discrimination between local recurrence and de novo primary tumour development. Like in human breast cancer (Yan et al., 2006), there is evidence that the development of mammary tumours in companion animals can represent an increased risk for new primary tumour development after excision of the first tumour (Stratman et al., 2008). Although both overall and disease-free survivals have been used in prognostic studies of cMGT and fMGT, pet owners have the option of electing euthanasia and there is inter-individual variability related to time of euthanasia and severity of metastatic disease, which is a potential bias in prognostic studies. Therefore,

A.J.F. Matos et al. / The Veterinary Journal 193 (2012) 24–31 Table 3 Summary of the proposed standardized procedures and suggestions for prognostic studies of feline and canine mammary gland tumours. Procedures Case selection 1. Based upon histological evaluation 2. No previous diagnosis of malignancy 3. 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 – exclude 4. Randomized in separate groups or balanced spayed and non-spayed at the time of mastectomy Tumour characteristics 1. Tumour size and lymph node status determined from pathological examination 2. Consider pathological staging system (pTNM) to assess prognosis and to make recommendations for adjuvant treatment 3. Exclude carcinomas in benign tumours 4. If size is not used as a continuous variable, group according to the T status of the TNM classification 5. Assess the status of distant lymph nodes (prescapular, popliteal, sternal and deep inguinal lymph nodes). Perform fine needle aspirate biopsies if size and consistency are altered 6. Search for nodal micrometastases using immunohistochemical techniques if negative by HE evaluation Histological type 1. Follow 1999 WHO classification 2. A minimum of two pathologists should be responsible for independent tumour classification Type of growth 1. Separate expansive, infiltrative and vasoinvasive growth 2. Exclude carcinomas in situ Histological grade 1. Need for specific canine and feline grading classifications Immunohistochemistry 1. Uniform, optimized and specific monoclonal antibodies 2. Clear definition and illustration of sample evaluation (quantitative or semi-quantitative; number of slides per tumour; number of microscopic fields per slide; criteria for slide and field selection) Genetic and related studies Standardized 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-up 1. Every 2–3 months for a minimum of 2 years after surgery 2. Complete physical examination; abdominal and thoracic imaging 3. Biopsies to confirm metastatic disease 4. Complete necropsies 5. Disease-free interval should be the preferred endpoint Proposal Creation of a task group of veterinary oncologists and pathologists to elaborate and periodically update standardized procedures for prognostic studies of canine and feline mammary tumours

disease-free interval, which exclusively depends on the stage of the disease and the aggressiveness of the primary tumour, is likely the best endpoint for cMGT and fMGT prognostic studies. Conclusions Comparing results and identifying solid prognostic factors for cMGT and fMGT is challenging because of inconsistent approaches in study designs and endpoints. Here, we suggest strategies to overcome these difficulties and to avoid potential biases in order to improve the strength and translational value of settings or therapeutic trials (Table 3). The creation of a task group of veterinary oncologists and pathologists would be useful to elaborate and periodically update standardized procedures related to clinical staging,

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