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Molecular-based tumour subtypes of canine mammary carcinomas assessed by immunohistochemistry

Molecular-based tumour subtypes of canine mammary carcinomas assessed by immunohistochemistry Background: Human breast cancer is classified by gene expression profile into subtypes consisting of two hormone (oestrogen and/or progesterone) receptor-positive types (luminal-like A and luminal-like B) and three hormone receptor-negative types [human epidermal growth factor receptor 2-expressing, basal-like, and unclassified ("normal-like”)]. Immunohistochemical surrogate panels are also proposed to potentially identify the molecular-based groups. The present study aimed to apply an immunohistochemical panel (anti-ER, -PR, -ERB-B2, -CK 5/6 and -CK14) in a series of canine malignant mammary tumours to verify the molecular-based classification, its correlation with invasion and grade, and its use as a prognostic aid in veterinary practice. Results: Thirty-five tumours with luminal pattern (ER+ and PR+) were subgrouped into 13 A type and 22 B type, if ERB-B2 positive or negative. Most luminal-like A and basal-like tumours were grade 1 carcinomas, while the percentage of luminal B tumours was higher in grades 2 and 3 (Pearson Chi-square P = 0.009). No difference in the percentage of molecular subtypes was found between simple and complex/mixed carcinomas (Pearson Chi- square P = 0.47). No significant results were obtained by survival analysis, even if basal-like tumours had a more favourable prognosis than luminal-like lesions. Conclusion: The panel of antibodies identified only three tumour groups (luminal-like A and B, and basal-like) in the dog. Even though canine mammary tumours may be a model of human breast cancer, the existence of the same carcinoma molecular subtypes in women awaits confirmation. Canine mammary carcinomas show high molecular heterogeneity, which would benefit from a classification based on molecular differences. Stage and grade showed independent associations with survival in the multivariate regression, while molecular subtype grouping and histological type did not show associations. This suggests that caution should be used when applying this classification to the dog, in which invasion and grade supply the most important prognostic information. Background different ways in the literature, namely HER2, c-ERB-2, Human breast cancer is considered a heterogeneous dis- neu, ERB-B2. This study uses the acronym ERB-B2. Fol- ease, and is classified by gene expression profile into low-up studies have shown these subtypes to be con- subtypes consisting of two hormone (oestrogen and/or served across diverse patient series and array platforms progesterone) receptor-positive types (luminal-like A [4,5], and that different gene expression-based predictors and luminal-like B) and three hormone receptor-nega- are likely tracking a similar common set of biological tive types [human epidermal growth factor receptor 2- subtypes, with significant agreement in predicting expressing, basal-like, and unclassified ("normal-like”)] patients’ clinical outcome [6]. [1-3]. Epidermal growth factor receptor 2 is indicated in Cost and complexity issues have to date rendered gene expression profiling impractical in laboratories equipped for routine diagnostic tests. However, some of the * Correspondence: giuseppe.sarli@unibo.it Department of Veterinary Public Health and Animal Pathology - Division of immunohistochemistry surrogate panels proposed can Veterinary Pathology, Faculty of Veterinary Medicine, University of Bologna, potentially identify the molecular-based groups Via Tolara di Sopra, 50 - 40064 - Ozzano dell’Emilia (Bologna), Italy © 2010 Sassi et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Sassi et al. BMC Veterinary Research 2010, 6:5 Page 2 of 9 http://www.biomedcentral.com/1746-6148/6/5 according to classification flowcharts [7] (figure 1). The the molecular classification to a series of canine mam- panels encompass at least anti-oestrogen receptor (ER), mary carcinomas, using a panel of antibodies apt to anti-progesterone receptor (PR), anti-ERB-B2 and anti- demonstrate a role in prognosis and identification of basal cytokeratin antibodies (CK 5/6 and 14) [8,9]. molecular groups similar to human breast cancer. Basal-like breast cancers are characterized by the lack of The present study aimed to apply an immunohisto- ER, PR and ERB-B2 expression and cytokeratin 5/6 and/ chemical panel (anti-ER, -PR, -ERB-B2, -CK 5/6 and or epidermal growth factor receptor expression [7], -CK14) in a series of canine malignant mammary whereas the luminal-like subtype is characterized by ER tumours to verify the molecular-based classification, its or PR expression [7] and further subgrouped into lumi- correlation with invasion and grade, and its use as a nal A or B depending on the absence or presence of prognostic aid in veterinary practice. ERB-B2 expression [10]. Negativity for luminal biomar- kers, positivity or negativity for basal markers and ERB- Methods B2 positivity characterizes the ERB-B2 overexpressing Cases and follow-up data group [10]. Forty-five tissue samples were obtained from female Canine mammary tumours are considered a sponta- dogs with malignant primary mammary tumours. All neous animal model of human breast cancer [11,12] the dogs underwent surgery at the University Clinic of prevalently on a histomorphological basis, but several Veterinary Surgery (Bologna, Italy) and in private clinics. differences between the tumours of the two species The tissue samples were immediately fixed in 10% buf- must be taken into account to make any comparisons fered formalin and routinely processed. Before surgery feasible. Myoepithelial cell proliferation is a frequent and every 2 mo over a 2-yr period, radiographs of the finding in the so-called complex and mixed patterns thorax and ultrasonograms of the spleen, liver, and kid- [13,14], but it is an uncommon feature of breast cancer neys were obtained. None of the dogs had distant in women [15]. Multiple tumours at the first clinical metastases (M0) at surgery. Follow-up data were col- presentation are very common in the female dog lected over this period and expressed as survival time [16,17], but not in women [15]. Do these differences (the time between surgery and death due to the tumour, indicate species-specific tumour genesis and develop- either spontaneous death or euthanasia). After 24 mo of ment? The molecular-based classification, recently follow-up, the animals still alive were considered adopted for breast cancer, seems to be a better tool assessed and those that had died non-assessed. than morphological features to establish objective simi- larities between the two models. In canine mammary Tumour grade and invasion tumours, the loss of hormone receptors in tumour pro- Histologic diagnoses were achieved on hematoxylin and gression is known [18,19], as is the overexpression of eosin stained slides according to Misdorp et al. [13], and ERB-B2 products in malignancies [20,21]. However, to tumour grade according to Lagadic and Estrada [23]. our knowledge, only one recent investigation merged Invasiveness (stage) was determined following a pre- these two features to devise a molecular-based sub- viously proposed system [24]: stage 0 = tumours without grouping of malignant tumours [22]. This study applied stromal invasion (in situ); stage I = tumours with stro- mal invasion; and stage II = tumours with neoplastic emboli in vessels. Immunohistochemistry For each tumour, five 4-micron-thick consecutive sections were used for anti-ER, -PR, -ERB-B2, -CK5/6 and -CK14 antibodies. Sections were dewaxed in toluene and rehy- drated. Endogenous peroxidase was blocked by immersion in 0.3% hydrogen peroxide for 20 min. Sections were then rinsed in Tris Buffer and antigen was retrieved with citrate buffer (2.1 g citric acid monohydrate/litre distilled water), pH 6.0, and heating for two 5 min periods in a microwave oven at 750 W, followed by cooling at room temperature for 20 min. The primary antibodies are summarized in Table 1. All antibodies were incubated with the tissue sec- tions overnight at 4°C, except for anti-PR incubated 1.5 Figure 1 Classification flowchart followed in the present study hours at 37°C, and were followed by a commercial strep- (from Conforti et al. [7] modified). toavidin-biotin-peroxidase technique (LSAB Kit, Dako, Sassi et al. BMC Veterinary Research 2010, 6:5 Page 3 of 9 http://www.biomedcentral.com/1746-6148/6/5 Amsterdam, The Netherlands). Diaminobenzidine (0.05% complex/mixed: comprising all the complex carcinomas for 10 min at room temperature) was used as chromogen. and carcinomas in mixed tumours; 3) others: including Slides were counterstained with Papanicolaou’shematoxy- the other histological types considered in the Misdorp lin. Negative controls were obtained substituting the pri- et al. [13] classification system. Kaplan-Meier survival mary antibody with an unrelated monoclonal antibody of curves were calculated. To test the influence of molecu- the same isotype. As positive controls to assess the cross- lar-based subgrouping (luminal-like A vs luminal-like B reactivity with canine tissues and the specificity of the vs basal-like), stage (0 vs I vs II) and grade (1 vs 2 vs 3 immunohistochemical stain, sections of canine normal separately for cases with and without vascular invasion) mammary gland (anti-ER, -PR and -CK14 antibodies), on survival, comparisons were verified by logrank test canine skin (anti-CK5/6) and bovine pancreas (anti-ERB- corrected for multiple comparisons. To investigate the B2 antibody) were used following the same protocols. simultaneous influence on survival, variables such as molecular-based subgrouping (luminal-like A vs lumi- Interpretation of immunohistochemical staining nal-like B vs basal-like), stage (0 vs I vs II), grade (1 vs 2 Staining data were classified semiquantitatively. All vs 3) and histotype (simple vs complex/mixed) were also immunohistochemical markers were accessed as nega- examined by multivariate regression with the propor- tive and positive groups. Specifically, negative cases were tional hazard Cox regression model for censored data. those that displayed no staining or staining in less than Analyses were performed by CSS software (Statsoft, a certain percentage of positive tumour cells, and posi- Tulsa, OK) statistics, and a conventional 5% level was tive cases were those with unequivocal staining in at used to define statistical significance. least a certain percentage of tumour cells. Positivity for CK5/6 and CK14, according to Kim et al. [9] was Results defined as the detection of at least 1% of invasive The 45 female dogs ranged from four to 15 years of age tumour cells showing strong cytoplasmic staining. (mean ± standard deviation = 9.66 ± 2.36; median = 10). Immunostaining for ERB-B2 was interpreted as positive Twenty-seven were crossbred and 17 purebred, namely when at least 10% tumour cells showed moderate to six German shepherds, five Yorkshire terriers, two poin- strong complete membranous staining [25]. Cases were ters, two dachshunds, two poodles. For one remaining considered positive for ER or PR when nuclear staining case breed and age were not known. was observed in at least 5% tumour cells [19]. Histologic diagnoses included seven in situ carcino- mas, 19 simple carcinomas (11 tubulo-papillary and six Grouping molecular subtypes solid types), 25 complex/mixed carcinomas (16 complex Based on a modified classification of Yang et al. [10], tubulo-papillary type and four carcinomas in mixed tumour subtypes were defined as luminal-like A (ER+ tumours) and one squamous carcinoma (included in the and/or PR+, ERB-B2 -, any CK5/6 or CK14), luminal- “other” group). Staging comprised seven non-infiltrating like B (ER+ and/or PR+, ERB-B2 +, any CK5/6 or tumours (in situ carcinomas, stage 0 i.e. the same identi- CK14); ERB-B2-expressing (ER-, PR-, ERB-B2 +, any fied by the morphological classification system), 24 car- CK5/6 or CK14); basal-like (ER- and PR-, ERB-B2 -, cinomas with stromal invasion (stage I), and 14 CK5/6 + and/or CK14+), and unclassified or normal-like carcinomas with vascular emboli and/or regional lymph (negative for all markers). node involvement (stage II). Grade assessment revealed 18 grade 1 (16 without and two with vascular invasion), Statistical analysis 14 grade 2 (11 without and three with vascular invasion) Pearson chi-squared statistic was used to test the asso- and13grade 3(four withoutand nine with vascular ciation of the molecular subtypes with numerosity of invasion) tumours. At 24 months after mastectomy, 33 cases homogeneous for invasion (0, I, II), grade (1, 2, 3) of 45 cases were still alive and 12 cases had died, either and histotype group. Histotype grouping was as follows: euthanized or spontaneously, in both cases due to 1) simple: including all the simple carcinomas; 2) tumour spread. Table 1 Primary antibodies used for immunohistochemistry in the current study. Antibody (anti-) Clone Manufacturer Working concentration Oestrogen Receptor polyclonal Zymed (South San Francisco, Ca) 1:50 Progesterone Receptor monoclonal PR 4-12 EMD Biosciences (San Diego, Ca) 1:10 ERB-B2 polyclonal Dako (Glostrup, Denmark) 1:50 Cytokeratin 14 monoclonal Ab-1 (LL002) NeoMarkers (Fremont, Ca) 1:300 Cytokeratins 5/6 monoclonal D5/16B4 Zymed (South San Francisco, Ca) 1:100 Sassi et al. BMC Veterinary Research 2010, 6:5 Page 4 of 9 http://www.biomedcentral.com/1746-6148/6/5 Following the score criteria, 35 cases were ER and/or superior to studies in a “diluted” population enrolling PR positive whereas ten were negative; 22 cases were breast cancer patients without distinction [27]. classified positive to ERB-B2 and ten positive for basal However, molecular-based sub-grouping according to cytokeratin expression. gene expression profile is impractical in laboratories According to the criteria adopted for molecular sub- equipped for routine diagnostic tests, and provides an type grouping, luminal B cases (n = 22 cases) accounted imprecise picture of the actual sub-classes [27] because for 49% of the tumours, followed by luminal A (n = 13, mRNA levels do not always correspond to changes in 29%), and basal-like (n = 10, 22%). No case was ERB-B2 protein expression [9]. The protein quality depends not expressing and no case was unassessed. Immunohisto- only on the amount and rate of transcription and trans- chemical expression of the panel of antibodies in the lation, but also on degradation and rate of transport different subtypes is reported in figure 2. from cells. In addition, tissues used for mRNA profiling No difference was present in the percentage of mole- include both tumour and stromal cells. Therefore, cular subtypes among the three invasion groups (Pear- immunohistochemistry may be beneficial in identifying son Chi-square P = 0.76). A higher percentage of the status of cellular expression and specific location of luminal A and basal cases was recorded among grade 1 the protein [9]. Immunohistochemical-based markers carcinomas (luminal A 50%, luminal B 12%, basal 38%). have been proposed to help identify the molecular cate- The percentage of luminal B cases was higher in grade 2 gories and define these sub-classes [9,10,7,8]. (luminal A 14%, luminal B 63%, basal 23%) and grade 3 In veterinary medicine, mammary gland tumours are (luminal A 15%, luminal B 77%, basal 8%) carcinomas classified morphologically providing good prognostic (Pearson Chi-square P = 0.009). No difference was indications [28,29] that may be enhanced by further found in the percentage of molecular subtypes between prognostic tools such as staging [24], histological grade simple and complex/mixed carcinomas (Pearson Chi- [23], proliferation indexes [30,31], hormone receptor sta- square P = 0.47). The group indicated as “others” was tus [19,32] and adhesion molecules expression [33,34]. not investigated because of the paucity of cases (one However, canine malignancies form a heterogeneous squamous carcinoma). These results are summarized in group of different molecular driven tumours which may table 2. benefit from a classification that takes such molecular Luminal-like A, luminal-like B and basal-like groups differences into account. revealed no difference by survival analysis (P = 0.85), The present study applied a panel of antibodies com- but basal-like tumours showed a better outcome than monly used to characterize the molecular groups in the luminal groups, and luminal B group had a slightly human pathology, and identified three tumour groups worse outcome than luminal A cases (figure 3A). Inva- (luminal-like A and B and basal-like) out of the five sion (P = 0.0025, figure 3B) and grade, separately for known groups (no ERB-B2 or normal-like cases were tumours with (P = 0.047, figure 3C) and without (P = present in our study, due to either the paucity of cases 0.03, figure 3D) vascular invasion, were significantly or to the epidemiological situation). Gama et al. [34], in associated with prognosis. a series of 102 canine mammary carcinomas, identified Stage and grade showed independent associations with four tumour groups (luminal-like A and B, basal-like, survival in the multivariate regression (table 3), while and HER2 overexpressing). Investigations in veterinary molecular subtype grouping and histological type did medicine seem to confirm the importance of a molecu- not show associations. lar characterization of canine mammary tumours, but further work is necessary to confirm the data from the Discussion present study and that of Gama et al. [34]. Human breast cancer is considered a heterogeneous dis- Our study showed that the molecular-based classifica- ease according to oestrogen receptor, tumour grade and tion of canine mammary cancer was related to grade, age [26]. Studies examining comprehensive gene-expres- but not to invasion and morphologic classification. sion patterns using hierarchical clustering disclosed four Luminal-like A group tumours included a significantly clusters and suggested a four-way classification of breast higher percentage of grade 1 tumours than luminal-like cancer: luminal-like (subsequently sub-classified as A B group in which grades 2 and 3 cancers prevailed. and B types), basal-like, HER-positive and normal-like Gama et al. [34] found that only the basal-like tumour [1,3]. The main goal of applying this innovative classifi- group was associated with grade and the presence of cation system is to demonstrate its role in prognosis. In vascular invasion. The molecular-based subgrouping of addition, this molecular-based classification of breast human breast cancer was associated with histological cancers provides an opportunity to investigate biological grade results in several investigations [10,9,7]. Simple or questions in homogeneous entities, and to enrich a spe- complex/mixed patterns do not seem to be correlated to cific subclass with a relevant signal. This might prove a specific molecular group in our study, whereas Gama Sassi et al. BMC Veterinary Research 2010, 6:5 Page 5 of 9 http://www.biomedcentral.com/1746-6148/6/5 Figure 2 Immunohistochemical expression of the panel of antibodies applied by IHC in canine mammary carcinoma. a-c PR staining; d- f ER staining; g-i ERB-B2 staining;j-l CK 14 staining; m-o CK 5/6 staining. Each column refers to a distinct molecular subtype. From left to right, each column represents luminal A, luminal B and basal subtypes. 400×. Sassi et al. BMC Veterinary Research 2010, 6:5 Page 6 of 9 http://www.biomedcentral.com/1746-6148/6/5 Table 2 Pearson Chi squared statistic. Luminal-like A Luminal-like B Basal-like P value (n. 13) (n. 22) (n.10) Invasion (stage) 0.76 Non-infiltrating (0) 1 4 2 Stromal invasion (I) 8 10 6 Vascular invasion (II) 4 8 2 Grade 0.009 Grade 1 9 3 6 Grade 2 2 9 3 Grade 3 2 10 1 Histotype 0.47 Simple 6 10 3 Complex/mixed 6 12 7 Other 1 0 0 Comparison between molecular phenotypes (luminal-like A, B, and basal-like) and invasion or grade or histotype groups. et al. [34] found the complex types associated with lumi- short survival. The difference between these studies can nal-like A tumours, and simple pattern and carcinosar- be explained by the different panels and criteria adopted comas to HER2 overexpressing and basal-like groups. to define the positivity to basal markers (cytokeratin 5/6 Application of this classification system in human and 14 used in our study, and cytokeratin 5, p63 and P- medicine has yielded conflicting results on the relation cadherin in Gama et al.’s [34] study). with different clinico-pathological variables and survival Stage and grade showed independent associations with where only the ERB-B2 overexpressing [9] or only the survival in the multivariate regression (table 3), while basal-like [2] tumours showed evidence of a significantly molecular subtype grouping and histological type did shorter survival. Our investigation failed to disclose any not show associations. These data suggest that, at the association between the molecular-based classification moment, caution should be used when applying this system and survival, whereas Gama et al. [34] found classification system to the dog, in which the most use- only the basal-like group significantly associated with a ful information for prognosis is obtained from invasion Figure 3 Kaplan-Meier overall survival curves of the different molecular (A), stage (B) and grade groups. The grade curves are presented separately for cases with (C) or without (D) vascular invasion. Sassi et al. BMC Veterinary Research 2010, 6:5 Page 7 of 9 http://www.biomedcentral.com/1746-6148/6/5 Table 3 Multivariate regression overexpressing and luminal-like tumours, and for this reason several different panels and score methods have Variable Beta* standard error hazard ratio* been proposed [36,37,9]. A second explanation is that Stage some of our basal-like cases could be luminal-like Stage 0 1.32 (0.05/2.59) 0.65 3.76 (1.05/13.38) tumours that were negative for ER and PR, but had Stage I positive myoepithelial cells that, in canine mammary Stage II tumours, are frequently expressed in the complex and Grade mixed patterns. The criterion we adopted was at least Grade 1 1.25 (0.29/2.21) 0.49 3.50 (1.34/9,12) 1% of cells positive to the basal markers in the invasive Grade 2 component, by virtue of the common myoepithelial pro- Grade 3 liferations that would induce a high number of false Histological type positive cases. Simple -0.72 (-1.97/0.53) 0.64 0.48 (0.14/1.71) It is assumed that cytokeratin 5 is more sensitive than Complex/mixed cytokeratin 14 as a basal marker because cytokeratin 5 Molecular group could be expressed by bipotential progenitor cells as Luminal-like A -0.31 (-1.43/0.81) 0.57 0.73 (0.24/2.24) well as basal-like cells, whereas expression of cytokeratin Luminal-like B 14 is limited to mature (basal) myoepithelial cells [9,36]. Basal-like To better characterize the basal-like tumours and to Proportional hazard Cox regression Chi = 20.20 - df = 4 - P = 0.00046. avoid confusion with cases belonging to other groups, a Groups are indicated for each variable. Only stage and grade show a higher coefficient of regression (beta) than their standard error, thereby attributing panel of antibodies raised to several basal markers them an independent prognostic significance.*(low/up) 95% confidence should be applied in canine mammary carcinomas. interval. and grade. However, when the dog is considered a spon- Conclusion taneous model of human breast cancer, similar tumour Application of a panel of antibodies, commonly used to types should be compared between the two species. characterize the molecular groups in human pathology Because the morphological basis does not guarantee the disclosed three tumour groups (luminal-like A and B and grouping of biologically homogeneous tumours, applica- basal-like) out of the five known (no ERB-B2 or normal- tion of a molecular-based system would improve the like cases were present in our samples). This finding comparison of homogeneous groups. This study and strengthens the assumption that canine mammary that of Gama et al. [34] have demonstrated the existence tumours are a model of human breast cancer, but to of molecular-categorized groups, but the similarities make this comparison more reliable, homogeneous groups should be identified and compared. Canine malig- between women and canine groups await confirmation nant tumours appear to be a heterogeneous group of dif- in future investigations. ferent molecular driven tumours which would benefit Application of the molecular-based classification sys- from a classification addressing molecular differences as tem provides additional information on the different cell in human medicine. However, the existence in the dog of origin of basal-like and luminal-like tumours. Perou et all the groups proposed for human breast cancer and al. [1] showed that basal-like tumours share some mole- their diversity in biological behaviour await confirmation. cular features with myoepithelial cells which constitute Stage and grade showed independent associations with the basal part of the normal epithelium, whereas lumi- survival in the multivariate regression, while molecular nal-like tumours have molecular features in common subtype grouping and histological type did not show with normal luminal cells. In veterinary medicine the associations. These data suggest that, at the moment, origin of the mesenchymal components in the so-called complex and mixed pattern of mammary tumours is caution should be used when applying this classification still a matter of debate, and the myoepithelial cell is the system to the dog, in which the most useful information most probable origin [35]. In our cases the basal-like for prognosis is obtained from invasion and grade. group shared some characteristics with luminal-like A tumours (high percentage of grade 1) that are not Acknowledgements known in human cancer and also conflict with the find- The authors are grateful to Anne Collins for English language editing. ings of Gama et al. [34]. This discrepancy may have two Author details explanations. Firstly, the panel of antibodies and the cri- Department of Veterinary Public Health and Animal Pathology - Division of teria adopted to identify the positivity to basal differed Veterinary Pathology, Faculty of Veterinary Medicine, University of Bologna, in the two studies. It is known that characterization of Via Tolara di Sopra, 50 - 40064 - Ozzano dell’Emilia (Bologna), Italy. Department of Veterinary Morphophysiology and Animal Production, the basal-like group is more difficult than that of HER2- Sassi et al. BMC Veterinary Research 2010, 6:5 Page 8 of 9 http://www.biomedcentral.com/1746-6148/6/5 Faculty of Veterinary Medicine, University of Bologna, via Tolara di Sopra 50- Institute of Pathology in cooperation with the American Registry of 40064 Ozzano Emilia - Bologna - Italy. Pathology and the World Health Organization Collaborating Centre for Worldwide Aderence on Comparative Oncology. Washington, DC 1999. Authors’ contributions 14. Misdorp W: Tumors of the mammary gland. Tumors in Domestic Animals CB and GS conceived the study, participated in its design and coordination Iowa State Press, Ames, IAMeuten DJ , 4 2002, 575-606. and drafted the manuscript. FS supervised the immunohistochemical stains 15. Rosen PP: Myoepithelial Neoplasms. Rosen’s breast pathology Lippincott and the scoring process. GS and GC performed the statistical analysis. All Williams & Wilkins, Philadelphia, PA, 2 2001, 425-453. authors read and approved the final manuscript. 16. 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Hum progesterone receptor expression and host and tumor factors as Pathol 2006, 37:1217-1226. predictors of disease-free period in mammary tumors of the dog. Vet 10. Yang XR, Sherman ME, Rimm DL, Lissowska J, Brinton LA, Peplonska B, Pathol 2005, 42:200-212. Hewitt SM, Anderson WF, Szeszenia-Dąbrowska N, Bardin-Mikolajczak A, 33. Brunetti B, Sarli G, Preziosi R, Monari I, Benazzi C: E-cadherin and b-catenin Zatonsky W, Cartun R, Mandich D, Rymkiewicz G, LIgaj M, Lukaszek S, reduction influence invasion but not proliferation and survival in canine Kordek R, García-Closas M: Differences in risk factors for breast cancer malignant mammary tumours. Vet Pathol 2005, 42:781-787. molecular subtypes in a population-based study. Cancer Epidemiol 34. Gama A, Paredes J, Gärtner F, Alves A, Schmitt F: Expression of E-cadherin, Biomarkers Prev 2007, 16(3):439-443. P-cadherin and b-catenin in canine malignant mammary tumours in 11. Misdorp W: Quelques aspects comparatifs des cancers de la mammelle relation to clinicopathological parameters, proliferation and survival. Vet chez la chienne, la chatte et la femme. Rec Med Vet Ec Alfort 1972, J 2008, 177:45-53. 148:583-590. 35. Hellmén E: Complex mammary tumours in the female dog: a review. J 12. Strandberg JD, Goodman DG: Animal model of human disease: Canine Dairy Res 2005, 72:90-97. mammary neoplasia. Am J Path 1974, 75(1):225-228. 36. Laasko M, Tanner M, Nilsson J, Wiklund T, Erikstein B, Kellokumpu- 13. Misdorp W, Esle RW, Hellmén E, Lipscomb TP: Histological Classification of Lehtinen P, Malmström P, Wilking N, Bergh J, Isola J: Basoluminal Mammary Tumors of the Dog and Cat. Published by the Armed Forces Sassi et al. BMC Veterinary Research 2010, 6:5 Page 9 of 9 http://www.biomedcentral.com/1746-6148/6/5 carcinoma: a new biologically and prognostically distinct entity between basal and luminal breast cancer. Clin Cancer Res 2006, 12(14):4185-4191. 37. Nielsen TO, Hsu FD, Jensen K, Cheang M, Karaca G, Hu Z, Hernandez- Boussard T, Livasy C, Cowan D, Dressler L, Akslen LA, Ragaz J, Gown AM, Gilks CB, Rijn van de M, Perou CM: Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma. Clin Cancer Res 2004, 10:5367-5374. doi:10.1186/1746-6148-6-5 Cite this article as: Sassi et al.: Molecular-based tumour subtypes of canine mammary carcinomas assessed by immunohistochemistry. BMC Veterinary Research 2010 6:5. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Veterinary Research Springer Journals

Molecular-based tumour subtypes of canine mammary carcinomas assessed by immunohistochemistry

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Springer Journals
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Copyright © 2010 by Sassi et al; licensee BioMed Central Ltd.
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Medicine & Public Health; Veterinary Medicine; Zoology; Transgenics
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1746-6148
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10.1186/1746-6148-6-5
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20109214
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Abstract

Background: Human breast cancer is classified by gene expression profile into subtypes consisting of two hormone (oestrogen and/or progesterone) receptor-positive types (luminal-like A and luminal-like B) and three hormone receptor-negative types [human epidermal growth factor receptor 2-expressing, basal-like, and unclassified ("normal-like”)]. Immunohistochemical surrogate panels are also proposed to potentially identify the molecular-based groups. The present study aimed to apply an immunohistochemical panel (anti-ER, -PR, -ERB-B2, -CK 5/6 and -CK14) in a series of canine malignant mammary tumours to verify the molecular-based classification, its correlation with invasion and grade, and its use as a prognostic aid in veterinary practice. Results: Thirty-five tumours with luminal pattern (ER+ and PR+) were subgrouped into 13 A type and 22 B type, if ERB-B2 positive or negative. Most luminal-like A and basal-like tumours were grade 1 carcinomas, while the percentage of luminal B tumours was higher in grades 2 and 3 (Pearson Chi-square P = 0.009). No difference in the percentage of molecular subtypes was found between simple and complex/mixed carcinomas (Pearson Chi- square P = 0.47). No significant results were obtained by survival analysis, even if basal-like tumours had a more favourable prognosis than luminal-like lesions. Conclusion: The panel of antibodies identified only three tumour groups (luminal-like A and B, and basal-like) in the dog. Even though canine mammary tumours may be a model of human breast cancer, the existence of the same carcinoma molecular subtypes in women awaits confirmation. Canine mammary carcinomas show high molecular heterogeneity, which would benefit from a classification based on molecular differences. Stage and grade showed independent associations with survival in the multivariate regression, while molecular subtype grouping and histological type did not show associations. This suggests that caution should be used when applying this classification to the dog, in which invasion and grade supply the most important prognostic information. Background different ways in the literature, namely HER2, c-ERB-2, Human breast cancer is considered a heterogeneous dis- neu, ERB-B2. This study uses the acronym ERB-B2. Fol- ease, and is classified by gene expression profile into low-up studies have shown these subtypes to be con- subtypes consisting of two hormone (oestrogen and/or served across diverse patient series and array platforms progesterone) receptor-positive types (luminal-like A [4,5], and that different gene expression-based predictors and luminal-like B) and three hormone receptor-nega- are likely tracking a similar common set of biological tive types [human epidermal growth factor receptor 2- subtypes, with significant agreement in predicting expressing, basal-like, and unclassified ("normal-like”)] patients’ clinical outcome [6]. [1-3]. Epidermal growth factor receptor 2 is indicated in Cost and complexity issues have to date rendered gene expression profiling impractical in laboratories equipped for routine diagnostic tests. However, some of the * Correspondence: giuseppe.sarli@unibo.it Department of Veterinary Public Health and Animal Pathology - Division of immunohistochemistry surrogate panels proposed can Veterinary Pathology, Faculty of Veterinary Medicine, University of Bologna, potentially identify the molecular-based groups Via Tolara di Sopra, 50 - 40064 - Ozzano dell’Emilia (Bologna), Italy © 2010 Sassi et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Sassi et al. BMC Veterinary Research 2010, 6:5 Page 2 of 9 http://www.biomedcentral.com/1746-6148/6/5 according to classification flowcharts [7] (figure 1). The the molecular classification to a series of canine mam- panels encompass at least anti-oestrogen receptor (ER), mary carcinomas, using a panel of antibodies apt to anti-progesterone receptor (PR), anti-ERB-B2 and anti- demonstrate a role in prognosis and identification of basal cytokeratin antibodies (CK 5/6 and 14) [8,9]. molecular groups similar to human breast cancer. Basal-like breast cancers are characterized by the lack of The present study aimed to apply an immunohisto- ER, PR and ERB-B2 expression and cytokeratin 5/6 and/ chemical panel (anti-ER, -PR, -ERB-B2, -CK 5/6 and or epidermal growth factor receptor expression [7], -CK14) in a series of canine malignant mammary whereas the luminal-like subtype is characterized by ER tumours to verify the molecular-based classification, its or PR expression [7] and further subgrouped into lumi- correlation with invasion and grade, and its use as a nal A or B depending on the absence or presence of prognostic aid in veterinary practice. ERB-B2 expression [10]. Negativity for luminal biomar- kers, positivity or negativity for basal markers and ERB- Methods B2 positivity characterizes the ERB-B2 overexpressing Cases and follow-up data group [10]. Forty-five tissue samples were obtained from female Canine mammary tumours are considered a sponta- dogs with malignant primary mammary tumours. All neous animal model of human breast cancer [11,12] the dogs underwent surgery at the University Clinic of prevalently on a histomorphological basis, but several Veterinary Surgery (Bologna, Italy) and in private clinics. differences between the tumours of the two species The tissue samples were immediately fixed in 10% buf- must be taken into account to make any comparisons fered formalin and routinely processed. Before surgery feasible. Myoepithelial cell proliferation is a frequent and every 2 mo over a 2-yr period, radiographs of the finding in the so-called complex and mixed patterns thorax and ultrasonograms of the spleen, liver, and kid- [13,14], but it is an uncommon feature of breast cancer neys were obtained. None of the dogs had distant in women [15]. Multiple tumours at the first clinical metastases (M0) at surgery. Follow-up data were col- presentation are very common in the female dog lected over this period and expressed as survival time [16,17], but not in women [15]. Do these differences (the time between surgery and death due to the tumour, indicate species-specific tumour genesis and develop- either spontaneous death or euthanasia). After 24 mo of ment? The molecular-based classification, recently follow-up, the animals still alive were considered adopted for breast cancer, seems to be a better tool assessed and those that had died non-assessed. than morphological features to establish objective simi- larities between the two models. In canine mammary Tumour grade and invasion tumours, the loss of hormone receptors in tumour pro- Histologic diagnoses were achieved on hematoxylin and gression is known [18,19], as is the overexpression of eosin stained slides according to Misdorp et al. [13], and ERB-B2 products in malignancies [20,21]. However, to tumour grade according to Lagadic and Estrada [23]. our knowledge, only one recent investigation merged Invasiveness (stage) was determined following a pre- these two features to devise a molecular-based sub- viously proposed system [24]: stage 0 = tumours without grouping of malignant tumours [22]. This study applied stromal invasion (in situ); stage I = tumours with stro- mal invasion; and stage II = tumours with neoplastic emboli in vessels. Immunohistochemistry For each tumour, five 4-micron-thick consecutive sections were used for anti-ER, -PR, -ERB-B2, -CK5/6 and -CK14 antibodies. Sections were dewaxed in toluene and rehy- drated. Endogenous peroxidase was blocked by immersion in 0.3% hydrogen peroxide for 20 min. Sections were then rinsed in Tris Buffer and antigen was retrieved with citrate buffer (2.1 g citric acid monohydrate/litre distilled water), pH 6.0, and heating for two 5 min periods in a microwave oven at 750 W, followed by cooling at room temperature for 20 min. The primary antibodies are summarized in Table 1. All antibodies were incubated with the tissue sec- tions overnight at 4°C, except for anti-PR incubated 1.5 Figure 1 Classification flowchart followed in the present study hours at 37°C, and were followed by a commercial strep- (from Conforti et al. [7] modified). toavidin-biotin-peroxidase technique (LSAB Kit, Dako, Sassi et al. BMC Veterinary Research 2010, 6:5 Page 3 of 9 http://www.biomedcentral.com/1746-6148/6/5 Amsterdam, The Netherlands). Diaminobenzidine (0.05% complex/mixed: comprising all the complex carcinomas for 10 min at room temperature) was used as chromogen. and carcinomas in mixed tumours; 3) others: including Slides were counterstained with Papanicolaou’shematoxy- the other histological types considered in the Misdorp lin. Negative controls were obtained substituting the pri- et al. [13] classification system. Kaplan-Meier survival mary antibody with an unrelated monoclonal antibody of curves were calculated. To test the influence of molecu- the same isotype. As positive controls to assess the cross- lar-based subgrouping (luminal-like A vs luminal-like B reactivity with canine tissues and the specificity of the vs basal-like), stage (0 vs I vs II) and grade (1 vs 2 vs 3 immunohistochemical stain, sections of canine normal separately for cases with and without vascular invasion) mammary gland (anti-ER, -PR and -CK14 antibodies), on survival, comparisons were verified by logrank test canine skin (anti-CK5/6) and bovine pancreas (anti-ERB- corrected for multiple comparisons. To investigate the B2 antibody) were used following the same protocols. simultaneous influence on survival, variables such as molecular-based subgrouping (luminal-like A vs lumi- Interpretation of immunohistochemical staining nal-like B vs basal-like), stage (0 vs I vs II), grade (1 vs 2 Staining data were classified semiquantitatively. All vs 3) and histotype (simple vs complex/mixed) were also immunohistochemical markers were accessed as nega- examined by multivariate regression with the propor- tive and positive groups. Specifically, negative cases were tional hazard Cox regression model for censored data. those that displayed no staining or staining in less than Analyses were performed by CSS software (Statsoft, a certain percentage of positive tumour cells, and posi- Tulsa, OK) statistics, and a conventional 5% level was tive cases were those with unequivocal staining in at used to define statistical significance. least a certain percentage of tumour cells. Positivity for CK5/6 and CK14, according to Kim et al. [9] was Results defined as the detection of at least 1% of invasive The 45 female dogs ranged from four to 15 years of age tumour cells showing strong cytoplasmic staining. (mean ± standard deviation = 9.66 ± 2.36; median = 10). Immunostaining for ERB-B2 was interpreted as positive Twenty-seven were crossbred and 17 purebred, namely when at least 10% tumour cells showed moderate to six German shepherds, five Yorkshire terriers, two poin- strong complete membranous staining [25]. Cases were ters, two dachshunds, two poodles. For one remaining considered positive for ER or PR when nuclear staining case breed and age were not known. was observed in at least 5% tumour cells [19]. Histologic diagnoses included seven in situ carcino- mas, 19 simple carcinomas (11 tubulo-papillary and six Grouping molecular subtypes solid types), 25 complex/mixed carcinomas (16 complex Based on a modified classification of Yang et al. [10], tubulo-papillary type and four carcinomas in mixed tumour subtypes were defined as luminal-like A (ER+ tumours) and one squamous carcinoma (included in the and/or PR+, ERB-B2 -, any CK5/6 or CK14), luminal- “other” group). Staging comprised seven non-infiltrating like B (ER+ and/or PR+, ERB-B2 +, any CK5/6 or tumours (in situ carcinomas, stage 0 i.e. the same identi- CK14); ERB-B2-expressing (ER-, PR-, ERB-B2 +, any fied by the morphological classification system), 24 car- CK5/6 or CK14); basal-like (ER- and PR-, ERB-B2 -, cinomas with stromal invasion (stage I), and 14 CK5/6 + and/or CK14+), and unclassified or normal-like carcinomas with vascular emboli and/or regional lymph (negative for all markers). node involvement (stage II). Grade assessment revealed 18 grade 1 (16 without and two with vascular invasion), Statistical analysis 14 grade 2 (11 without and three with vascular invasion) Pearson chi-squared statistic was used to test the asso- and13grade 3(four withoutand nine with vascular ciation of the molecular subtypes with numerosity of invasion) tumours. At 24 months after mastectomy, 33 cases homogeneous for invasion (0, I, II), grade (1, 2, 3) of 45 cases were still alive and 12 cases had died, either and histotype group. Histotype grouping was as follows: euthanized or spontaneously, in both cases due to 1) simple: including all the simple carcinomas; 2) tumour spread. Table 1 Primary antibodies used for immunohistochemistry in the current study. Antibody (anti-) Clone Manufacturer Working concentration Oestrogen Receptor polyclonal Zymed (South San Francisco, Ca) 1:50 Progesterone Receptor monoclonal PR 4-12 EMD Biosciences (San Diego, Ca) 1:10 ERB-B2 polyclonal Dako (Glostrup, Denmark) 1:50 Cytokeratin 14 monoclonal Ab-1 (LL002) NeoMarkers (Fremont, Ca) 1:300 Cytokeratins 5/6 monoclonal D5/16B4 Zymed (South San Francisco, Ca) 1:100 Sassi et al. BMC Veterinary Research 2010, 6:5 Page 4 of 9 http://www.biomedcentral.com/1746-6148/6/5 Following the score criteria, 35 cases were ER and/or superior to studies in a “diluted” population enrolling PR positive whereas ten were negative; 22 cases were breast cancer patients without distinction [27]. classified positive to ERB-B2 and ten positive for basal However, molecular-based sub-grouping according to cytokeratin expression. gene expression profile is impractical in laboratories According to the criteria adopted for molecular sub- equipped for routine diagnostic tests, and provides an type grouping, luminal B cases (n = 22 cases) accounted imprecise picture of the actual sub-classes [27] because for 49% of the tumours, followed by luminal A (n = 13, mRNA levels do not always correspond to changes in 29%), and basal-like (n = 10, 22%). No case was ERB-B2 protein expression [9]. The protein quality depends not expressing and no case was unassessed. Immunohisto- only on the amount and rate of transcription and trans- chemical expression of the panel of antibodies in the lation, but also on degradation and rate of transport different subtypes is reported in figure 2. from cells. In addition, tissues used for mRNA profiling No difference was present in the percentage of mole- include both tumour and stromal cells. Therefore, cular subtypes among the three invasion groups (Pear- immunohistochemistry may be beneficial in identifying son Chi-square P = 0.76). A higher percentage of the status of cellular expression and specific location of luminal A and basal cases was recorded among grade 1 the protein [9]. Immunohistochemical-based markers carcinomas (luminal A 50%, luminal B 12%, basal 38%). have been proposed to help identify the molecular cate- The percentage of luminal B cases was higher in grade 2 gories and define these sub-classes [9,10,7,8]. (luminal A 14%, luminal B 63%, basal 23%) and grade 3 In veterinary medicine, mammary gland tumours are (luminal A 15%, luminal B 77%, basal 8%) carcinomas classified morphologically providing good prognostic (Pearson Chi-square P = 0.009). No difference was indications [28,29] that may be enhanced by further found in the percentage of molecular subtypes between prognostic tools such as staging [24], histological grade simple and complex/mixed carcinomas (Pearson Chi- [23], proliferation indexes [30,31], hormone receptor sta- square P = 0.47). The group indicated as “others” was tus [19,32] and adhesion molecules expression [33,34]. not investigated because of the paucity of cases (one However, canine malignancies form a heterogeneous squamous carcinoma). These results are summarized in group of different molecular driven tumours which may table 2. benefit from a classification that takes such molecular Luminal-like A, luminal-like B and basal-like groups differences into account. revealed no difference by survival analysis (P = 0.85), The present study applied a panel of antibodies com- but basal-like tumours showed a better outcome than monly used to characterize the molecular groups in the luminal groups, and luminal B group had a slightly human pathology, and identified three tumour groups worse outcome than luminal A cases (figure 3A). Inva- (luminal-like A and B and basal-like) out of the five sion (P = 0.0025, figure 3B) and grade, separately for known groups (no ERB-B2 or normal-like cases were tumours with (P = 0.047, figure 3C) and without (P = present in our study, due to either the paucity of cases 0.03, figure 3D) vascular invasion, were significantly or to the epidemiological situation). Gama et al. [34], in associated with prognosis. a series of 102 canine mammary carcinomas, identified Stage and grade showed independent associations with four tumour groups (luminal-like A and B, basal-like, survival in the multivariate regression (table 3), while and HER2 overexpressing). Investigations in veterinary molecular subtype grouping and histological type did medicine seem to confirm the importance of a molecu- not show associations. lar characterization of canine mammary tumours, but further work is necessary to confirm the data from the Discussion present study and that of Gama et al. [34]. Human breast cancer is considered a heterogeneous dis- Our study showed that the molecular-based classifica- ease according to oestrogen receptor, tumour grade and tion of canine mammary cancer was related to grade, age [26]. Studies examining comprehensive gene-expres- but not to invasion and morphologic classification. sion patterns using hierarchical clustering disclosed four Luminal-like A group tumours included a significantly clusters and suggested a four-way classification of breast higher percentage of grade 1 tumours than luminal-like cancer: luminal-like (subsequently sub-classified as A B group in which grades 2 and 3 cancers prevailed. and B types), basal-like, HER-positive and normal-like Gama et al. [34] found that only the basal-like tumour [1,3]. The main goal of applying this innovative classifi- group was associated with grade and the presence of cation system is to demonstrate its role in prognosis. In vascular invasion. The molecular-based subgrouping of addition, this molecular-based classification of breast human breast cancer was associated with histological cancers provides an opportunity to investigate biological grade results in several investigations [10,9,7]. Simple or questions in homogeneous entities, and to enrich a spe- complex/mixed patterns do not seem to be correlated to cific subclass with a relevant signal. This might prove a specific molecular group in our study, whereas Gama Sassi et al. BMC Veterinary Research 2010, 6:5 Page 5 of 9 http://www.biomedcentral.com/1746-6148/6/5 Figure 2 Immunohistochemical expression of the panel of antibodies applied by IHC in canine mammary carcinoma. a-c PR staining; d- f ER staining; g-i ERB-B2 staining;j-l CK 14 staining; m-o CK 5/6 staining. Each column refers to a distinct molecular subtype. From left to right, each column represents luminal A, luminal B and basal subtypes. 400×. Sassi et al. BMC Veterinary Research 2010, 6:5 Page 6 of 9 http://www.biomedcentral.com/1746-6148/6/5 Table 2 Pearson Chi squared statistic. Luminal-like A Luminal-like B Basal-like P value (n. 13) (n. 22) (n.10) Invasion (stage) 0.76 Non-infiltrating (0) 1 4 2 Stromal invasion (I) 8 10 6 Vascular invasion (II) 4 8 2 Grade 0.009 Grade 1 9 3 6 Grade 2 2 9 3 Grade 3 2 10 1 Histotype 0.47 Simple 6 10 3 Complex/mixed 6 12 7 Other 1 0 0 Comparison between molecular phenotypes (luminal-like A, B, and basal-like) and invasion or grade or histotype groups. et al. [34] found the complex types associated with lumi- short survival. The difference between these studies can nal-like A tumours, and simple pattern and carcinosar- be explained by the different panels and criteria adopted comas to HER2 overexpressing and basal-like groups. to define the positivity to basal markers (cytokeratin 5/6 Application of this classification system in human and 14 used in our study, and cytokeratin 5, p63 and P- medicine has yielded conflicting results on the relation cadherin in Gama et al.’s [34] study). with different clinico-pathological variables and survival Stage and grade showed independent associations with where only the ERB-B2 overexpressing [9] or only the survival in the multivariate regression (table 3), while basal-like [2] tumours showed evidence of a significantly molecular subtype grouping and histological type did shorter survival. Our investigation failed to disclose any not show associations. These data suggest that, at the association between the molecular-based classification moment, caution should be used when applying this system and survival, whereas Gama et al. [34] found classification system to the dog, in which the most use- only the basal-like group significantly associated with a ful information for prognosis is obtained from invasion Figure 3 Kaplan-Meier overall survival curves of the different molecular (A), stage (B) and grade groups. The grade curves are presented separately for cases with (C) or without (D) vascular invasion. Sassi et al. BMC Veterinary Research 2010, 6:5 Page 7 of 9 http://www.biomedcentral.com/1746-6148/6/5 Table 3 Multivariate regression overexpressing and luminal-like tumours, and for this reason several different panels and score methods have Variable Beta* standard error hazard ratio* been proposed [36,37,9]. A second explanation is that Stage some of our basal-like cases could be luminal-like Stage 0 1.32 (0.05/2.59) 0.65 3.76 (1.05/13.38) tumours that were negative for ER and PR, but had Stage I positive myoepithelial cells that, in canine mammary Stage II tumours, are frequently expressed in the complex and Grade mixed patterns. The criterion we adopted was at least Grade 1 1.25 (0.29/2.21) 0.49 3.50 (1.34/9,12) 1% of cells positive to the basal markers in the invasive Grade 2 component, by virtue of the common myoepithelial pro- Grade 3 liferations that would induce a high number of false Histological type positive cases. Simple -0.72 (-1.97/0.53) 0.64 0.48 (0.14/1.71) It is assumed that cytokeratin 5 is more sensitive than Complex/mixed cytokeratin 14 as a basal marker because cytokeratin 5 Molecular group could be expressed by bipotential progenitor cells as Luminal-like A -0.31 (-1.43/0.81) 0.57 0.73 (0.24/2.24) well as basal-like cells, whereas expression of cytokeratin Luminal-like B 14 is limited to mature (basal) myoepithelial cells [9,36]. Basal-like To better characterize the basal-like tumours and to Proportional hazard Cox regression Chi = 20.20 - df = 4 - P = 0.00046. avoid confusion with cases belonging to other groups, a Groups are indicated for each variable. Only stage and grade show a higher coefficient of regression (beta) than their standard error, thereby attributing panel of antibodies raised to several basal markers them an independent prognostic significance.*(low/up) 95% confidence should be applied in canine mammary carcinomas. interval. and grade. However, when the dog is considered a spon- Conclusion taneous model of human breast cancer, similar tumour Application of a panel of antibodies, commonly used to types should be compared between the two species. characterize the molecular groups in human pathology Because the morphological basis does not guarantee the disclosed three tumour groups (luminal-like A and B and grouping of biologically homogeneous tumours, applica- basal-like) out of the five known (no ERB-B2 or normal- tion of a molecular-based system would improve the like cases were present in our samples). This finding comparison of homogeneous groups. This study and strengthens the assumption that canine mammary that of Gama et al. [34] have demonstrated the existence tumours are a model of human breast cancer, but to of molecular-categorized groups, but the similarities make this comparison more reliable, homogeneous groups should be identified and compared. Canine malig- between women and canine groups await confirmation nant tumours appear to be a heterogeneous group of dif- in future investigations. ferent molecular driven tumours which would benefit Application of the molecular-based classification sys- from a classification addressing molecular differences as tem provides additional information on the different cell in human medicine. However, the existence in the dog of origin of basal-like and luminal-like tumours. Perou et all the groups proposed for human breast cancer and al. [1] showed that basal-like tumours share some mole- their diversity in biological behaviour await confirmation. cular features with myoepithelial cells which constitute Stage and grade showed independent associations with the basal part of the normal epithelium, whereas lumi- survival in the multivariate regression, while molecular nal-like tumours have molecular features in common subtype grouping and histological type did not show with normal luminal cells. In veterinary medicine the associations. These data suggest that, at the moment, origin of the mesenchymal components in the so-called complex and mixed pattern of mammary tumours is caution should be used when applying this classification still a matter of debate, and the myoepithelial cell is the system to the dog, in which the most useful information most probable origin [35]. In our cases the basal-like for prognosis is obtained from invasion and grade. group shared some characteristics with luminal-like A tumours (high percentage of grade 1) that are not Acknowledgements known in human cancer and also conflict with the find- The authors are grateful to Anne Collins for English language editing. ings of Gama et al. [34]. This discrepancy may have two Author details explanations. Firstly, the panel of antibodies and the cri- Department of Veterinary Public Health and Animal Pathology - Division of teria adopted to identify the positivity to basal differed Veterinary Pathology, Faculty of Veterinary Medicine, University of Bologna, in the two studies. It is known that characterization of Via Tolara di Sopra, 50 - 40064 - Ozzano dell’Emilia (Bologna), Italy. Department of Veterinary Morphophysiology and Animal Production, the basal-like group is more difficult than that of HER2- Sassi et al. BMC Veterinary Research 2010, 6:5 Page 8 of 9 http://www.biomedcentral.com/1746-6148/6/5 Faculty of Veterinary Medicine, University of Bologna, via Tolara di Sopra 50- Institute of Pathology in cooperation with the American Registry of 40064 Ozzano Emilia - Bologna - Italy. Pathology and the World Health Organization Collaborating Centre for Worldwide Aderence on Comparative Oncology. Washington, DC 1999. Authors’ contributions 14. Misdorp W: Tumors of the mammary gland. Tumors in Domestic Animals CB and GS conceived the study, participated in its design and coordination Iowa State Press, Ames, IAMeuten DJ , 4 2002, 575-606. and drafted the manuscript. FS supervised the immunohistochemical stains 15. Rosen PP: Myoepithelial Neoplasms. Rosen’s breast pathology Lippincott and the scoring process. GS and GC performed the statistical analysis. All Williams & Wilkins, Philadelphia, PA, 2 2001, 425-453. authors read and approved the final manuscript. 16. 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Nielsen TO, Hsu FD, Jensen K, Cheang M, Karaca G, Hu Z, Hernandez- Boussard T, Livasy C, Cowan D, Dressler L, Akslen LA, Ragaz J, Gown AM, Gilks CB, Rijn van de M, Perou CM: Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma. Clin Cancer Res 2004, 10:5367-5374. doi:10.1186/1746-6148-6-5 Cite this article as: Sassi et al.: Molecular-based tumour subtypes of canine mammary carcinomas assessed by immunohistochemistry. BMC Veterinary Research 2010 6:5. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit

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