Cross-talk between clinico-histopathological traits of malignant canine mammary tumors and their prognostic relevance Gautam Siddharth1,*, Chauhan Indrasen1, Joshi Chitra2, Gupta Kuldip3, Sood Naresh Kumar3 1Division of Temperate Animal Husbandry, ICAR-IVRI, Mukteshwar-263138, Nainital, Uttarakhand 2Department of Animal Husbandry, Almora-263601, Uttarakhand 3Department of Veterinary Pathology, GADVASU-141004, Ludhiana, Punjab, India. *Address for Correspondence, Dr Siddharth Gautam, Division of Temperate Animal Husbandry, ICAR-Indian Veterinary Research Institute, Mukteshwar Campus-263138, Nainital, Uttarakhand, India, E-mail: gautam.ivri.m@gmail.com
Online Published on 28 July, 2022. Abstract Malignant canine mammary tumors (mCMT) are one of the leading non-infectious causes of death in canines. The present study aimed primarily at delineating the association between various clinico-histopathological attributes of 30 spontaneous malignant canine mammary tumors (mCMT) along with their prognostic relevance. Thorough clinical and histopathological assessment of mCMT was followed by correlation, univariable (cox regression), multivariable (elastic net), and survival analysis. A great many mCMT showed hard consistency (63%), > 5 cm phenotype (70%), pulmonary metastases (70%), and involvement of multiple glands (40%). Carcinosarcoma was the most common histopathological subtype (40%), followed by complex carcinoma (33%). Tumour-infiltrating lymphocytes (TIL; 19/30), neovascularization (29/30), lymphangiogenesis (29/30), haemorrhages (19/30), intravascular embolism (5/30), lymphatic embolism (6/30), necrosis (22/30), inflammation (21/30), and microscopic cysts (7/30) were vital intratumoral lesions. Larger tumors were prone for pulmonary metastasis (p = 0.05) and showed hard consistency (p = 0.03). Cases with multiple glands showed a higher rate of intratumoral angiogenesis (p = 0.01), lymphatic invasion (p = 0.03) and pulmonary metastasis (p = 0.02). Also, TIL was significantly associated with lymphatic embolism (p = 0.02), and inflammation with vascular (p = 0.01) and lymphatic embolism (p = 0.04). A significant association between intratumoral vascular changes, viz:-angiogenesis, lymphangiogenesis, haemorrhages, and lymphovascular invasion, was noticed. A follow-up of 510 days revealed the median overall survival of 144 ± 75 days, which differed significantly by angiogenesis, TIL, vascular invasion, histopathological type, grade and cutaneous ulcers, suggesting these factors as independent prognosticators of mCMT. Top Keywords Canine mammary tumor, Lymphangiogenesis, Lymphovascular invasion, Metastases, Survival analysis, Tumor-infiltrating lymphocytes. Top |
Introduction Cancer is one of the leading non-infectious causes of death among humans and companion animals across the globe1,2. Mammary gland tumors are the most frequently encountered neoplastic condition in both species2,3, with nearly 50% malignancy rate in canines4. Surgical resection of the affected mammary gland and regional lymph nodes is the first choice of treatment in malignant CMT (mCMT)3, and adjuvant chemotherapy is recommended only in cases with metastasis and poor prognosis5. Despite this, 41-47% of mCMT affiliated population generally met a fatal outcome within one year post-surgery6. Therefore, discerning the early prognostic markers of mCMT and understanding the correlation between them becomes imperative for the timely therapeutic management of mCMT affiliated dogs. |
Different variables viz: clinical manifestations, intra-and peritumoral histopathological features, and expression of tumor-specific and non-specific proteins, have been evaluated for their prognostic relevance in mCMT. Despite the prognostic significance of several molecular biomarkers in mCMT patients, none has been adopted in routine veterinary practices due to the lack of universal optimization, high cost, a requirement of sophisticated techniques and laboratory set-up, and the inability to quantify results accurately. |
In order to identify more thriving and practical prognosticators of mCMT, researchers in recent years have focussed on the clinico-histopathological variables7-9. Moreover, most of these studies have used a bivariable selection model to select variables in multivariable analysis. This approach can introduce an uncontrolled bias by inappropriately rejecting potentially critical confounding variables10. Only a limited number of studies have analyzed the survival factors of mCMT without univariable prefiltering7,9,11. |
Another less explored facet of CMT biology is the association between clinical features and the mCMT microenvironment characters. The relationships between different molecular markers and clinico-histopathological parameters have been studied comprehensively in mCMT7,9,12. However, despite being the crux for morphological diagnosis, little attention has been paid towards delineating the cross-talk between different clinical and intratumoral histopathological traits of mCMT, and the baseline information on the clinical-histopathological correlation of mCMT is primarily lacking. |
Clinical manifestations and histopathological features of mCMT are easy to record and are routinely used in Veterinary Medicine. Studying the correlation between all the plausible important clinico-histopathological features of mCMT would enhance our understanding of mCMT biology. In this context, 30 mCMT cases were clinically and histopathologically characterized. Also, the possible association between different clinical and intratumoral histopathological attributes and their prognostic relevance were evaluated. |
Top Materials And Methods Animal subjects The present study was conducted on the spontaneous mCMT cases presented between February 2012 and January 2013 to the Small Animal Clinics, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India. All the cases presented with suspected tumor were examined clinically, and the tentative diagnosis was made based on clinical presentation and cytological evaluation of fine-needle aspirates. Thirty cases without any prior history of tumor (benign or malignant) with clinical and cytological characters suggesting mCMT were included in the study. The confirmatory diagnosis of mCMT was done by the histopathological examination of the surgically excised tumor mass. All the activities were performed as per the Institute Ethics Committee guidelines for Animal Welfare, GADVASU, Ludhiana, India. Furthermore, the study did not involve animal experimentation and included only clinically excised tumor samples. Clinical examination On clinical presentation, the bitches were carefully examined for the presence of gross pathologies, including haemorrhages, ulceration, signs of inflammation and the consistency (hard or soft) of the tumor mass. The size of the tumor mass was noted at the largest diameter [T1 (< 3 cm), T2 (3–5 cm) and T3 (> 5 cm)] after removal of peritumoral fat and skin. The involvement of single or multiple pair of glands along with tumor location was recorded. The local metastasis [N1 (present) and N0 (absent)] was assessed by the clinical palpation of the draining lymph nodes and, whenever possible, histopathological evaluation. Thoracic radiographic examination (two projections; lateral and ventrodorsal), abdominal ultrasonography, and respiratory signs were used to evaluate pulmonary (distant) metastasis [M1 (present) and M0 (absent)]. Only radiographic patterns characteristic to pulmonary metastasis as described previously were taken into account13. The mammary tumors were staged using the TNM system as per the modified WHO staging system for CMT14. The physiological attributes of canine patients, including age, body weight, breed, feed, pregnancy status, parturition, and lactation, were collected through a questionnaire and reported elsewhere15. None of the bitches received any anticancer drug pre-or postmastectomy, and the bitches underwent tumor resection as the only therapeutic intervention following written consent from the animal owners. Sample collection and processing The mammary tumors were excised by regional or radical mastectomy with or without the draining lymph nodes. In cases presented with multiple tumor masses, the entire tumor chain was resected, and representative tissue samples were collected from multiple (at least 3) sites from each tumor mass. The samples were fixed immediately in 10% neutral buffered formalin, processed for histopathology and stained with routine H&E16. In cases with multiple tumor masses, the tumor with the highest histopathological grade (primary criterion) or largest size (secondary criterion) was selected for evaluation and analysis. Histopathological evaluation The tumors were classified as per the WHO classification of dogs’ mammary tumors and WHO-AIFP classification of canine mammary tumors17. Three criteria were used for histopathological grading of canine mammary carcinoma: 1) tubule formation; 2) hyperchromasia and mitoses; and 3) nuclear polymorphism17. Histopathological classification, grading and histopathological characterization of mCMT were done consensually by a panel of two certified veterinary pathologists. Increased blood and lymphatic vessel density in the neoplastic areas compared to adjacent normal tissue was assigned as angiogenesis and lymphangiogenesis, respectively. The presence of atypical hyperchromatic cells in the vascular and lymphatic vessels was considered as metastatic tumor emboli. Inflammatory cells, particularly tumor-infiltrating lymphocytes (TIL), were characterized in the peri-and intra-tumoral regions based on the histo-morphological features. The presence of intra-tumoral necrosis, haemorrhages and cysts were recorded, and surgical margins were examined. All the histopathological observations within intra-tumoral areas were scored semi-quantitatively on a 4-point scale (0 to 3+) based on the distribution as follows: 0 = absent; 1+ = focal; 2+ = multifocal; and 3+ = diffuse. Images were captured using an Olympus BX 53F upright light microscope. Follow-up and survival data The study started in February 2012, and the data was collected in a 73 weeks (510 days) follow-up period lasting June 2013. The survival data were collected by post-operative follow-up examination (physical examination and thoracic radiography) and by contacting the animal owners every two months after the initial surgery. The overall survival (OS) was calculated from the day of tumor resection to the day of death or the last follow-up. Unfortunately, the animal owners did not consent to conduct a post-mortem examination, and tumour-related death was diagnosed based on the clinical signs and progressive deterioration in systemic condition. Cases with mortality not related to mCMT or where the actual cause of death was unknown were right-censored at the time of death. Also, cases that were lost to follow-up or were alive at the last follow-up/clinical examination were right-censored. Only two cases experienced tumor reoccurrence, and therefore, the disease-free survival was not estimated. Statistical analysis Exploratory data analysis was done to see whether a correlation existed among the study variables. Correlation among the variables was explored using R package ‘corrplot’, version 0.84. Univariable Cox regression was performed to estimate the hazard of each variable on the survival of canine patients with R package ‘survival’, version 3.1-12. The multivariable selection was performed with elastic net penalized Cox regression method. Coxnet function from R package ‘glmnet’, version 4.0-2 was used to fit Cox model regularized by an elastic net penalty. The optimum hyper-parameter alpha of elastic net was decided by 5-fold cross-validation. A sequence of (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9) of alpha was used and separate calls to cross-validation were made with different values of alpha. The value of another hyper-parameter lambda giving minimum mean cross-validated error was taken as optimum, and the value of alpha corresponding to this lambda was chosen as the optimum alpha. Kaplan– Meier curves were generated using R package prodlim, version 0.4.8. C-index, calibration plot, and integrated Brier score were employed for model evaluation. C-index was derived from R package ‘glmnet’, while calibration plot and integrated Brier score were derived from R package ‘pec’, version 2020.11.17. Model evaluation was performed on the whole dataset with variables selected by the multivariable elastic net penalized Cox regression method. All the analyses were performed in R programming environment, version 4.0.2. For analysis, following clinical and histopathological variables were considered: tumor grade (grade 1, 2 versus grade 3); involvement of glands (single pair versus multiple pair); tumor consistency (soft versus hard); ulceration (present versus absent); tumor size (≤ 5 cm versus > 5 cm); tumor stage (stage 1, 2 and 3 versus stage 4 and 5); pulmonary metastasis (present versus absent); histopathological tumor classification; angiogenesis and lymphangiogenesis (absent/focal versus multifocal/diffuse); vascular and lymphatic emboli (present versus absent); cystic spaces (present versus absent); TIL (absent versus focal versus multifocal/diffuse); necrosis, inflammation and haemorrhages (absent/focal versus multifocal/diffuse). Top Results Clinical characteristics of the study population Marked variation in the size of the mammary gland tumor was observed, which varied from 1 to 30 centimetres (cm) with 21 (70%) cases displaying T3 (> 5 cm) phenotype (Fig. 1a). Multifocal/multicentric involvements of mammary glands ranging from single nipples to multiple glands or even the entire chain of mammary glands were observed (Fig. 1b). The inguinal glands, either unilateral or bilateral, were the most frequently affected (24/30), followed by abdominal (14/30) and thoracic glands (5/30). Multiple pairs of glands were involved in 12 (40%) cases. Nineteen tumor (63%) masses were hard, and 11 (37%) showed soft consistency on palpation and the surface varied from nodular (Fig. 1c) to smooth. Besides, four tumor masses were cystic with varying fluid levels, and the same number of cases demonstrated superficial to deep cutaneous ulcerations (Fig. 1d). Radiographic examination of the thoracic cavity to unravel pulmonary metastasis revealed varying magnitude of miliary to nodular to focal or patchy radioopacities, indicating secondary lesions in the lungs in 21 (70%) cases. Concurrently, 21 cases (70%) showed malignancy of stage 5, followed by stage 4, stage 3, stage 2 and stage 1 malignancies in 0, 4 (13%), 3 (10%), and 2 (7%) cases, respectively. Histopathological classification of mCMT Carcinosarcoma was the most frequently encountered mCMT (n = 12) in the present study. This was followed by complex carcinoma (n = 10) and simple carcinoma (n = 5). Two cases were identified as extra-genital transmissible venereal tumors (TVT) of the mammary gland, and one case was classified as sarcoma. Simple carcinomas (n = 5) were characterized by the proliferation of either glandular epithelial cells or surrounding basal cells (Fig. 2a). Complex carcinomas (n = 10) exhibited involvement of both glandular and basal/myoepithelial cells. The epithelial components were arranged in various patterns, including ductular, tubulo-acinar, cystic papillary, solid and adenosquamous (Fig. 2b). The myoepithelial component was organized in a more or less stellate and reticular pattern. The sarcomatous pattern within the carcinosarcomas comprises varying degrees of malignant chondroid and osteoid components (Fig. 2c). In one case, the presence of multinucleated giant cells was also observed (Fig. 2d), particularly at the osseous and chondroid matrix interface, indicating an active or dynamic transformation. The sarcoma’s sole case was classified as osteochondrosarcoma and was characterized by malignant osseous and cartilaginous cells with basophilic chondroid and osseous matrix (Fig. 2e). Neoplastic cells were arranged in a haphazard pattern with a low mitotic index. Focal calcification areas were also observed in the mesenchymal components of the carcinosarcomas and sarcoma cases. TVT cases revealed round cells with centrally located, large, oval or round nuclei and a single prominent nucleolus with coarse chromatin (Fig. 2f). Histopathological characterization of mCMT Five cases each of grades 1, 2, and 3 were recorded among carcinoma cases. Neoangiogenesis (n = 29/30), lymphangiogenesis (n = 29/30), haemorrhages (n = 19/30), necrosis (n = 22/30), inflammation (n = 21/30) and cysts (n = 7/30) ranging from absent to focal to diffuse were significant intratumoral histopathological lesions. Nineteen cases also depicted varying degrees of TIL. Histopathological features of vascular and lymphatic tumor embolism were observed in five and six cases, respectively. Except for one case where surgical margins demonstrated ductal hyperplasia, the rest of the cases revealed clear resection margins. Infection and hemosiderin-laden macrophages in the intra-tumoral areas were observed in one and two cases, respectively. Two cases also depicted mild fibrosis and encapsulation. The histopathological attributes of mCMT are depicted in Fig. 3(1-d) and detailed in Table 1. Correlation between clinical and histopathological variables A significant correlation between several clinical and histopathological variables was determined. A strong positive association of tumor stage with pulmonary metastasis and tumor size is self-explanatory. A high proportion of mCMT patients with tumor size > 5 cm exhibited pulmonary metastasis (p = 0.05). A relationship between tumor size and tumor texture was noted where larger tumor masses demonstrated hard consistency (p = 0.03). Also, tumors with hard consistency were in advanced clinical stages (p = 0.03); however, showed significantly lesser vascular embolism (p = 0.03). The involvement of multiple mammary glands emerged as an important clinical parameter, and a high proportion of cases with multifocality/multicentricity showed advanced tumor stages (p = 0.02). The incidence of cases with multiple glands involvement showed higher intratumoral angiogenesis (p = 0.01) and lymphatic invasion (p = 0.03). Intratumoral angiogenesis was significantly correlated with histopathological evidence of vascular and lymphatic embolism (p< 0.001), lymphangiogenesis (p = 0.001) and haemorrhages (p = 0.01). Moreover, lymphatic embolism was also influenced by lymphangiogenesis ((p = 0.03), vascular embolism (p = 0.01) and intratumoral TIL (p = 0.02). Additionally, intratumoral inflammation was also associated with vascular invasion (p = 0.01) and lymphatic invasion (p = 0.04). Prognostic relevance of clinical parameters in mCMT The median OS time of 30 canine patients suffering from mCMT after a follow-up of 510 days (median followup = 157 ± 60 days) was 144 ± 75 days (Fig. 4a). None of the clinical parameters could be associated with the OS of the mCMT patients in the univariable analysis. However, the presence of superficial to deep cutaneous ulcers over tumor masses was significantly associated with survival time in multivariable analysis and was recognized as a poor prognostic marker for mCMT (Table 2). Prognostic relevance of intratumoral histopathological parameters in mCMT The univariable analysis demonstrated neoangiogenesis within intratumoral lesions as a negative prognostic indicator of OS in mCMT affiliated dogs. Furthermore, neoangiogenesis was also associated with significant differences in survival time in multivariable analysis, suggesting its independent prognostic potential. Likewise, cases with minimal TIL and no vascular invasion had a higher probability of survival, as indicated by both univariable and multivariable data. We also found low histopathological grade as a favourable prognosticator of mCMT, showing a higher survival rate than highgrade mammary tumors. Multivariable analysis revealed a significant difference in the survival time between complex carcinoma and carcinosarcoma cases. Complex carcinoma showed a higher survival probability than other tumor types, whereas carcinosarcoma showed a grave prognosis. Briefly, angiogenesis, TIL, vascular invasion, histologic tumor type and histologic tumor grade turned up as independent prognostic factors for survival in mCMT cases (Table 3). The Kaplan-Meier survival curve for the independent prognostic variables is depicted in Figs. 4b-4d. Top Discussion The present study deals with delineating the clinically relevant prognostic factors of mCMT using a multivariable approach. A significant association between several clinical and histopathological characters of mCMT was determined. Also, the independent prognostic significance of routinely used clinico-histopathological parameters of mCMT was discerned. |
Univariable prefiltering before multivariable analysis to eliminate the non-significant confounding factors can draw biasness in interpretation by omitting significant prognostic factors with low weightage10. To overcome this limitation, both univariable and multivariable approaches were without any bivariable selection. Among histopathologic attributes, angiogenesis, vascular emboli and TIL were identified as prognostic factors in both univariable and multivariable analysis, whereas histopathologic tumor grade and tumor type were exclusive to multivariable analysis. It was further confirmed that superficial to deep skin ulcerations on the tumor masses has substantial poor prognostic value, as suggested in previous studies7,11. Several studies highlighted that histopathological grade advancement is associated with poor survival outcomes8,11. However, others revealed that this association is significant only in univariable analysis and not multivariable analysis18,19. The results of the present study demonstrated that histopathological tumor grades 1 and 2 had a lower death risk than grade 3. It was further noted that canine mammary complex carcinomas have a favourable prognosis and higher survival probability, whereas carcinosarcomas showed the highest risk of CMT related deaths. The present study results are in line with previous studies depicting similar outcomes in canine mammary gland tumors9,18-20. |
Tumor size is one of the well-acknowledged prognostic factors for CMT4,8,21 and CMT greater than 5.0 cm are generally malignant, show a higher proliferation index, and have a poor prognosis22. Present study results also suggested that tumor size and involvement of multiple mammary glands contributes critically towards the biological outcome of mCMT and should always be considered during the prognostic assessment of mCMT. |
Intratumoral vascular aberrations are a prerequisite for the progression of mCMT and are traits of aggressive tumors23. The malignant cells may induce neovascularisation during cancer progression with24 or without25 inducing endothelial cell proliferation. These newly formed vessels have leaky and defective endothelium and facilitate vascular invasion26. Yet, little is known about the affiliations between different vascular and lymphatic changes in the mCMT microenvironment. To this end, the study demonstrated a significant association between intratumoral angiogenesis, lymphangiogenesis, lymphovascular invasion and haemorrhages. Evaluation of neovascularization as a potential prognostic factor further revealed a positive association between intratumoral angiogenesis and a higher risk of tumor-specific mortality. |
Additionally, vascular tumor invasion, but not lymphatic invasion, was found as a strong unfavourable prognosticator of mCMT, as suggested earlier9,27. Intra-and peri-tumoral blood vessel density and endothelial cell proliferation have been revealed as crucial survival predictors of CMT28. Collectively, the present study findings suggested that all the cancer-induced vascular aberrations are linked and interdependent within the tumor microenvironment and should be looked at attentively while evaluating the probable outcome of mCMT. |
Intratumoral necrosis and inflammation are indicators of aggressive carcinomas with poor prognosis29. Necrosis and inflammation were not associated with the CMT specific OS in the present study; however, considering that inflammation was positively linked with vascular and lymphatic invasion, utmost care should be taken while interpreting the prognostic implications of intratumoral inflammation. Previous studies also suggested no influence of intratumoral necrosis on the survival of mCMT patients but indicated inflammatory signs as a poor prognosticator of mCMT11,30. |
Meagre information is available on the status of lymphangiogenesis in CMT cases31, and less so, its prognostic relevance32. Varying levels of intratumoral lymphangiogenesis in 97% cases (29/30) was detected in the present study; however, no difference in the OS of CMT patients with no/focal versus multifocal/diffuse lymphangiogenesis could be determined. Similar findings with no prognostic value of lymphatic vessel density, mean lymphatic vessel perimeter and the relative area occupied by lymphatic vessels have been reported in CMT cases32. |
In the recent past, attention has been paid towards understanding the tumor microenvironment as the immune response within the tumoral lesions plays a vital role in tumor progression or regression33. The role of TIL in modulating tumor progression has been explored in CMT patients34,35. In the present study, a positive association between the TIL and the intratumoral lymphatic invasion was noted. Also, TIL was determined as a strong unfavourable prognostic indicator of mCMT with poor OS. The present study findings align with investigations suggesting an association between TIL and vascular invasion, higher lymph node metastasis, low OS and poor prognosis34-36. |
Top Conclusion The present study explored and revealed a significant association between the different clinical and histopathological parameters of spontaneous mCMT. Additionally, the study disclosed ulceration of tumor mass, intratumoral angiogenesis and vascular invasion, histopathological grade and tumor type, and TIL as decisive, independent prognostic factors of spontaneous mCMT. |
Top Acknowledgements The authors are grateful to the Vice-chancellor and Director of Research, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, for providing the necessary facilities for the research. |
Top Figures | Fig. 1.: Clinical characteristics of dogs with spontaneous malignant canine mammary gland tumors. a. Surgically excised tumor mass depicting T phenotype (> 5 cm) and nodular appearance. b. Canine mammary tumor involving the entire unilateral chain of mammary glands. c. Tumor mass showing nodular appearance. d. Deep ulcer in the inguinal mammary gland exposing the underlying tumor tissue.
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| | Fig. 2.: Histopathological classification of malignant canine mammary gland tumors. a. Simple carcinoma with epithelial cell proliferation revealing tubulo-acinar pattern, H&E ×200. b. Complex carcinoma showing multifocal areas of lymphangiogenesis (arrow), H&E ×200. c. Carcinosarcoma case depicting the epithelial cell proliferation (solid pattern) along with malignant osteoid component (star), H&E ×200. d. Giant cell tumor (carcinosarcoma) showing numerous multinucleated giant cells (arrow), H&E ×400. e. Osteochondrosarcoma with malignant osseous and chondroid matrix, H&E ×100. f. primary transmissible venereal tumor of mammary gland showing round cells with abundant cytoplasm and centrally placed nuclei with single prominent nucleolus, H&E ×400.
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| | Fig. 3.: Histopathological attributes of spontaneous malignant canine mammary tumors. a. Canine mammary tumor tissue revealing lymphatic invasion and embolism (arrowhead) along with lymphangiogenesis. Also, a focal aggregation of tumor infiltrating lymphocytes is present, H&E ×200. b. Severe metastases in inguinal lymph node, characterized by replacement of lymphocytes by malignant cancer cell (arrow) and hemosiderin laden macrophages (arrowhead), H&E ×200. c. A case of carcinosarcoma showing neoangiogenesis, particularly within the malignant osteoid matrix, H&E ×100. d. Early carcinoma lesions showing multifocal areas of haemorrhages, H&E ×100.
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| | Fig. 4.: The Kaplan-Meier curve showing the overall survival of canine patients suffering from spontaneous malignant canine mammary gland tumors. a. Kaplan-Meier graph depicting the overall survival of thirty dogs against time (days post-surgery). b. Cases with no/focal intratumoral angiogenesis had higher overall survival than those with multifocal/diffuse angiogenesis, Log-rank p = 0.006. c. Overall survival of canine patients with vascular invasion and embolism is significantly lower than cases with no vascular embolism, Log-rank p = 5e-04. d. Dogs with no tumor infiltrating lymphocytes survived longer than cases showing multifocal/diffuse intratumoral TIL.
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Tables | Table 1.: Histopathological characteristics of the neoplastic canine mammary tumors.
| | Characteristics | No. of cases | Characteristics | No. of cases | | Histopathological classification (n = 30) | | Vascular invasion (n = 30) | | | Carcinoma | | Absent | 25 | | Simple | 5 | Focal | 2 | | Complex | 10 | Multifocal | 2 | | Carcinosarcoma | 12 | Diffuse | 1 | | Osteochondrosarcoma | 1 | Lymphatic invasion (n = 30) | | | TVT | 2 | Absent | 24 | | Tumor resection margins (n = 30) | | Focal | 2 | | Clean/clear | 29 | Multifocal | 4 | | Ductal hyperplasia | 1 | Tumor infiltrating lymphocytes (n = 30) | | | Tumor grade (n = 15) | | Absent | 11 | | Simple carcinoma | | Focal | 15 | | Grade I | 3 | Multifocal | 1 | | Grade II | 0 | Diffuse | 3 | | Grade III | 2 | Intra-tumoral hemorrhages (n = 30) | | | Complex carcinoma | | Absent | 11 | | Grade I | 2 | Focal | 13 | | Grade II | 5 | Multifocal | 4 | | Grade III | 3 | Diffuse | 2 | | Angiogenesis (n = 30) | | Intra-tumoral necrosis (n = 30) | | | Absent | 1 | Absent | 8 | | Focal | 25 | Focal | 12 | | Multifocal | 3 | Multifocal | 6 | | Diffuse | 1 | Diffuse | 4 | | Lymphangiogenesis (n = 30) | | Cystic spaces (n = 30) | | | Absent | 1 | Absent | 23 | | Focal | 20 | Focal | 5 | | Multifocal | 5 | Multifocal | 2 | | Diffuse | 4 | | |
| | | Table 2.: Prognostic significance of the clinical parameters for overall survival in spontaneous malignant canine mammary tumors by univariable and multivariable analysis (n = 30).
| | Variables | | Univariable analysis (cox regression) | | Multivariable analysis (elastic net) | | | Hazard ratio | 95% CI | p | Coefficient | Inference | | Histological grade | Grade 1 & 2 | 0.2129 | 0.0255-1.774 | NS | -0.347 | Lower risk of death | | Grade 3 | 0.9498 | 0.1908-4.728 | NS | | | | Glands involved | Single paira | | | | | | | Multiple pair | 1.904 | 0.5102-7.106 | NS | | | | Consistency | Harda | | | | | | | Soft | 1.484 | 0.3925-5.61 | NS | | | | Cutaneous ulcers | Absenta | | | | -0.005 | Lower risk of death | | Present | 2.0003 | 0.4141-9.662 | NS | 0.005 | Higher risk of death | | Tumor size | ≤ 5 cma | | | | | | | > 5 cm | 1.812 | 0.3745-8.77 | NS | | | | Tumor stage | Low stages (1 to 3)a | | | | | | | High stages (4 & 5) | 1.119 | 0.2791-4.49 | NS | | | | Pulmonary metastasis | Absenta | | | | | | | Present | 1.119 | 0.2791-4.49 | NS | | |
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| reference variable in cox regression analysis ; NS – non-significant | |
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