**4. Acknowledgments**

224 A Bird's-Eye View of Veterinary Medicine

al, 2006). He suggested that heat shock proteins are important prognostic factors. Presence of Hsp70 was also reported in normal gastrointestinal tissues, but an increased expression of this protein was observed in gastrointestinal tumors (Isomoto et al, 2003). Isomoto (2003) believed that Hsp70 plays an important role in the degree of cellular differentiation in tumors, which would indicate that Hsp70 is an important prognostic factor. Similar studies are conducted in veterinary medicine, but to a lesser extent (Isomoto et al, 2003). A similar study was conducted by Rommanucci (2005) in mammary gland tumors in female dogs (Romanucci et al, 2006). She found Hsp70 and Hsp90 expression in simple and complex, as well as solid-type adenocarcinomas. Expression of these proteins was observed in the cytoplasm, as well as in the nuclei of tumor cells. Rommanucci (2006), in her study of breast cancers in female dogs, has not attempted to verify the possible correlations between heat shock proteins and other tumor markers (Rommanucci et al, 2006). She focused on establishing the degree of protein expression and their localization in tumor cells. Basing on the results of her study, the author suggested that these proteins may play an important role in the process of carcinogenesis. In our study, Hsp70 protein expression was found in 86.4% of tumors, while the expression of Hsp90 was observed in 66.2% of tumors. The aim of our study was to demonstrate the relationship between the expression of heat shock proteins and other prognostic factors. When comparing the expression of Hsp70 to the expression of Hsp90 in different types of tumors, we found a high statistical significance between the expression of both proteins. Analysis of the relationship between the expression of nuclear antigen Ki-67 and the expression of heat shock proteins showed that the highest expression of Ki-67 as well as Hsp90 was present in solid tumors. The highest expression of Hsp90 protein and Ki-67 was also found in cancers of the highest histological grades. Based on these data it can be concluded that Hsp90 is an important factor, which could be considered a marker of malignancy and which may be useful in the diagnostics of cancers. Analysis of the results showed high expression of Hsp70 in cancers with grade 1 and 3 of COX-2 expression; the lowest expression of Hsp70 was found in cancers with grade 2 of COX-2 expression. Statistical significance of P = 0.009 was found between the expression of Hsp70 and expression of COX-2. An increased expression of COX-2 was observed in tumors with a low mean number of cells showing positive reaction to Hsp90 protein. Correlation between the expression of Hsp70 protein and p53 protein in tumors of epithelial origin was also confirmed. Based on the analysis of expression of both proteins and taking into account the mean apoptotic index, it was found that in cancers with the third (the highest) histological grade, expression of p53 protein is significantly lower than the expression of Hsp70, and the value of apoptotic index in these cancers was

Occurrence of mammary tumors in female dogs and their potential use as a model in comparative pathology makes it an interesting research material. It is known that the degree of malignancy is positively correlated with proliferative activity. The relationship between expression of estrogen receptors and the degree of malignancy is not entirely clear, although most authors believe that the expression of these receptors is higher in benign tumors.

the lowest.

**3.2 Conclusions** 

Study was carried out at the Institute of Pathology, Department of Clinical Sciences, Faculty of Veterinary Medicine, Warsaw School of Life Sciences, 159C Nowoursynowska Street, 02-766 Warsaw. Research was conducted as a part of a doctoral dissertation, partially funded by a grant from the Ministry of Science and Information Technology, No N30800632/0667

### **5. References**


Prospective Study of Tumor Markers as Prognostic Factors

pp. 377-383.

168-176.

5127.

81-84.

pp. 225-232.

Vol. 12, pp. 6161-6169.

77, pp. 1892-1898.

in the Histopathological Differential Diagnosis of Mammary Gland Neoplasms in Female Canines 227

Koda, M.; Reszec, J.; Sulkowska, M.; Kanczuga – Koda, L.; Sulkowska, S. (2004). Expression

Kumaraguruparan, R.; Kurunagaran, D.; Balachandran, C.; Manohar, B.M.; Nagini, S. (2006).

Lanza-Jacoby, S.; Burd, R.; Rosato, F.E.J.; McGuire, K.; Little, J.; Nougbilly, N.; Miller, S.

Lee, J.; Hughes, C.S.; Fine, R.L.; Page, L.R. (1996). P-glycoprotein Expression in Canine

Liu, X.H.; Rose, D.P. (1996). Differential expression and regulation of cyclooxygenase-1

Lőhr C.V., Teifke, J.P.; Failing, K.; Weiss, E. (1997). Characterization the standarized AgNOR

Martin, P.M.; Cotard, M.; Mialot, J.P.; Andre, F.; Rayanaud J.P. (1984) Animal models for

Mayr, B.; Schellander, K.; Schlleger, W.; Reifinger, M. (1994). Sequence of exon of the

McEwen, E.G.; Patnaik, A.K.; Harvey, H.J.; Panko, W.B. (1092). Estrogen receptor in canine

Millanta, F.; Calandrella, M.; Bari, G.; Niccolini, M.; Vannozzi, I.; Poli, A. (2005).

Mirecka, J.; Korabiowska, M.; Schauer, A. (1993). Correlation between the occurrence of Ki-

Misdorp, W.; Else, R.W.; Hellmen, E.; Lipscomb, T.P. (1999). Histological classification of mammary tumors of the dog and cat, *World Health Organization*, Geneva Misdorp, W.W.; Meuten, D. (edit.). (2002). Tumors in Domestic Animals. Iowa State Press,

Mohammed, S.I.; Khan, K.N.; Sellers, R.S.; Hayek, MG.; DeNicola, D.B.; Wu, L.; Bonney,

*Veterinary Pathology*, Vol. 3, No. 4, pp. 212-221.

*Chemotherapy and Pharmacology,* Vol. 12, pp. 13-70.

*Cytobiologica*; Vol. 31, No.2, pp. 83-86.

Black Publishing Company. 4th ed., 575-606.

mammary tumors. *Cancer Research*, Vol. 42, pp. 2255-2259.

Kubiak, J.Z. (2001). Cancer and cell cycle. *Advances in Cell Biology*, Vol. 28, pp. 97-307.

of the insulin-like growth factor-I receptor and proapoptotic Bax and Bak proteins in human colorectal cancer. *Annals of the New York Academy of Sciences* , Vol.1030,

Of humans and canines: a comparative evaluation of heat shock and apoptosisassociated proteins in mammary tumors. *Clinica Chimica Acta*, Vol. 365, No.1-2, pp.

(2006). Effect of simultaneous inhibition of epidermal growth factor receptor and cyclooxygenase-2 in HER-2/neu-positive breast cancer. *Clinical Cancer Research*,

Lymphoma a Relevant, Intermediate Model of Multidrug Resistance. *Cancer*, Vol.

and –2 in two human breast cancer cell lines. *Cancer Research*, Vol. 56, pp. 5125-

method with postfixation and immunohistologic detection of Ki-67 and PCNA.

hormone-dependent human breast cancer. Relatioship between steroid receptor profiles in canine and feline mammary tumors and survival rate. *Cancer* 

canine p53 gene-mutation in a papilloma. *British Veterinary Journal*, Vol. 150, pp.

Comparison of steroid receptor expression in normal, dysplastica and neoplastic canine and feline mammary tissues. *Research in Veterinary Science*, Vol. 70, No.3,

67 antigen and clinical parameters in human breast carcinoma. *Folia Histochemica et* 

P.L.; Knapp, D.W. (2004). Expression of cyclooxygenase-1 and 2 in naturally-


Ciocca, D.R.; Oesterreich, S.; Chamness, C.; Mcguire, W.L.; Fuqua, S.A. (1993). Biological and

Dempke, W.; Rie, C.; Grothey, A.; Schmoll, H.J. (2001). Cyclooxygenase-2: a novel target for

Doré, M.; Lanthier, I.; Sirois, J. (2003). Cyclooxygenase-2 expression in canine mammary

Gamblin, R.M.; de Maria, R.; Piccoli, M. (1994). Cd44 triggering enhanced human NK cell cytotoxic function. The *Journal of Immunology,* Vol. 153, pp. 4399-4407. Geraldes, M.; Gärtner, F.; Schmitt, F. (2000). Immunohistochemical study of hormonal

spontaneous mammary tumors. *Veterinary Record*, Vol. 146, pp. 403-406 Gilbertson, S.R.; Kurzaman, I.D.; Zachran, R.E.; Hurvitz, H.J.; Black, M.M. (1983). Canine

Ginn, PE. (1996). Immunohistochemical detection of P-glycoprotein in formalin - fixed and

Giziński, S.; Boryczko, Z.; Katkiewicz, M.; Bostedt, H. (2003). Ki-67 protein as a prognostic

Hampe, J.F.; Misdorp, W. (1973) Tumours and dysplasias of the mammary gland. *Bull World* 

Heller, D.A,; Clifford, C.A; Goldschmidt, M.H.; Holt, D.E.; Shofer, F.S.; Smith, A.;

Hellmen, E.; Bergstrom, R.; Holmberg, L.; Spanberg, I.B.; Hansson, K.; Lindgren, A. (1993).

Host, H.; Lund, E. (2006). Age as prognostic factor in breast cancer. *Cancer*, Vol. 57, pp. 2217-

Howlin, J.; McBryan, J; Martin, F. (2006) Pubertal mammary gland development: insights

Isomoto, H.; Oka, M.; Yano, Y.; Kanazawa, Y.; Soda, H.; Terada, R.; Yasutake, T.; Nakayama,

Johnson, A.S.; Couto, C.G.; Weghorst, C.M. (1998). Mutation of the p53 tumor suppressor

consecutive cases. *Veterinary Pathology*, Vol. 30, pp. 20-27.

assessed in 232 dogs. *Veterinary Pathology,* Vol. 20, pp. 127-142

*National Cancer Institute*, Vol. 85, pp. 1558-1570

tumors. *Veterinary Pathology*, Vol.40, pp. 207-212

411-417

33, pp. 533-54

*Health Organ,* Vol. 50, pp. 111-133.

891.

780.

2221.

283-297.

pp. 213- 217.

Vol. 198, No.2, pp. 219-228.

clinical implications of heat shock protein 27000 (Hsp27): a Review. *Journal of the* 

cancer chemotherapy? *Journal of Cancer Research & Clinical Oncology*, Vol. 127, p.

receptors and cell proliferation in normal canine mammary glands and

mammary epithelial neoplasms: biologic implications of morfrologic characteristics

paraffin-embedded normal and neoplastic canine tissues. *Veterinary Pathology*, Vol.

indicator in breast cancer in females. *Veterinary Medicine*, Vol. 59, No. 10, pp. 888-

Sorenmo, K.U. (2005). Cyclooxygenase-2 expression is associated with histologic tumor type in canine mammary carcinoma. *Veterinary Pathology,* Vol. 42, pp. 776-

Prognostic factors in canine mammary tumors: a multivariate study of 202

from mouse models. *Journal of Mammary Gland Biology and Neoplasia*, Vol. 11, pp.

T.; Shikuwa, S.; Takeshima, F.; Udano, H.; Murata, I.; Ohtsuka, K.; Kohno, S. (2003). Expression of heat shock protein Hsp70 and Hsp40 in gastric cancer. *Cancer Letters*,

gene in spontaneously occuring osteosarcomas of the dog. *Carcinogenesis*, Vol. 19,


Prospective Study of Tumor Markers as Prognostic Factors

*International Journal of Cancer*, Vol. 49, pp. 44-49.

breast cancer. *Cancer Research* , Vol. 62, pp. 632-635.

*Veterinary Research*, Vol. 78, pp. 817-820.

Agricultural University, Warsaw.

Rotkiewicz, (Ed.), 100-107, Olsztyn

*Veterinary Medicine*, Vol. 58, No. 40, pp. 261-264.

*Cancer*, Vol. 89, pp. 2637-2645.

570-573

ed., Philadelphia

424-429.

in the Histopathological Differential Diagnosis of Mammary Gland Neoplasms in Female Canines 229

Ramalho, L.N.Z.; Ribeiro-Silva, A.; Cassali, G.D. (2006). Zucoloto S. The Expression of p63

Rilke, F.; Colnaghi, M.I.; Cascinelli, N.; Andreola, S.; Baldini, M.T.; Bufalino, R.; Della Porta,

Ristimäki, A.; Sivula, A.; Lundin, J.; Lundin, M.; Salminen, T.; Haglund, C.; Joensuu, H.;

Rodo, A. (2007). Expression of HER-2 receptors. E cadherin, and p53 protein in mammary gland tumors in bitches. Doctoral thesis, Agricultural University, Warsaw. Romanucci, M.; Marinelli, A.; Giuseppe, S.; Dell Salda, L. (2006). Heat shock protein expression in canine malignant mammary tumours. *BMC Cancer*, Vol. ,6, pp. 171. Roses, D.F.; Bell, D.A.; Flotte, T.J.; Taylor, R.; Ratech, H.; Dubin, N. (1982). Pathologic

Rungsipipata, A. (1999) Immunohistochemical analysis of c-yes and c-erbB-2 oncogene

Rutteman, G.R.; Withrow, S.J.; MacEwen, E.G. (2001). Tumors of the mammary gland. W:

Sartin, E.A.; Barnes, S.; Kwapien, R.P.; Wolfe, L.G. (1992). Estrogen and progesterone

Saymour, L.; Bezwoda, W.R.; Meyer, K. (1990) Tumor factors predicting for prognosis in

Skrzypczak, M. (2004). Angiogenesis in mammary gland tumors in bitches. Doctoral thesis,

Sobczak-Filipiak, M.; Malicka, E.(1997). Diagnosis of mammary gland tumors including

Soslow, R.A.; Dannenberg, A.J.; Rush, D.; Werner, B.M.; Khan, K.N.; Masferrer, J.; Koki, A.T.

Storm, F.K.; Mahvi, D.M.; Gilchrist, K.W. (1993). Heat shock protein 27 overexpression in

Szczubiał, M.; Łopuszański, W. (2002). Prognosis in mammary gland tumors in bitches.

dogs. *American Journal of Veterinary Research,* Vol. 53, pp. 2196-2200.

*Journal of Veterinary Medical Science*; Vol.61, No.1, pp. 27-32.

duration of survival. *Cancer*; Vol.66, No.11, pp. 2390-2394.

and cytokeratin 5 in mixed tumors of the canine mammary gland provides new insights into the histogenesis of these neoplasms. *Veterinary. Pathology*, Vol. 43, pp.

G.; Menard, S.; Pierotti, M.A.; Testori, A. (1991). Prognostic significance of HER-2/neu expression in breast cancer and its relationship to other prognostic factors.

Isola, J. (2002) Prognostic significance of elevated cyclooxygenase-2 expression in

predictors of recurrence in stage 1 (T1NOMO) breast cancer. *American Journal of* 

products and p53 tumor suppressor protein in canine mammary tumors. The

*Small Animal Clinical Oncology.* S. J. Withrow; E. G. MacEwen, (Ed.), 455-477, 3rd

receptor status of mammary carcinomas and correlation with clinical outcome in

metastatic breast cancer. The presence of P24 predicts for response to treatment and

immumocytochemistry methods*. Materials Conference. Veterinary Oncology*. T.

(2000). COX-2 is expressed in human pulmonary, colonic and mammary tumours.

breast cancer lymph node metastasis. *Annals of Surgical Oncology*; Vol. 3, No.6, pp.

occurring canine cancer. *Prostaglandins Leukotrienes and Essential Fatty Acids*, Vol. 70, pp. 479-483.


Moulton, J.E. (1990). Tumors of mammary gland, W: *Tumors in Domestic Animals. 3rd ed.,*

Mulas, J.M.; Millán, Y.; Dios, R. (2005). A prospective analysis of immunohistochemically

Muto, T.; Wakui, S.; Takahashi, H.; Maekawa, S.; Masaoka, T.; Ushigome, S.; Furusato, M.

Nerurkar, V.R.; Naik, S.N.; Lalitha, V.S.; Chitale, A.R.; Ishwad, C.S;, Jalnapurkar, B.V. (1990).

Nieto, A.; Péna, L.; Pérez-Alenza, M.D.; Sànchez, M.A.; Flores, J.M.; Castańo, M. (2000).

Niwińska, A (1995). New prognostic factors in patients with breast cancer. *Journal of* 

Nowak, M.; Madej, J.A.; Dzięgiel, P. (2005). Immunohistochemical localization of cox-2 in

grade. The *Bulletin of the Veterinary Institute in Pulawy*, Vol. 49, pp. 433-437. Nowak, M.; Madej, J.A.; Dzięgiel, P. (2007). Comparison of expressions of estrogen and

Olszewski, W.(1994). Selected aspects of the pathology of breast cancer. *Journal of Oncology*,

Parl, F.F.; Dupont, W.D. (1982). Retrospective cohort study of histologic risk factor in breast

Peńa, L.; Nieto, A.; Perez-Alenza, D. (1998). Immunohistochemical detection of Ki-67 and

variables. *Journal of Veterinary Diagnostic Investigation*, Vol.10, pp. 237-246. Petterino, C.; Rossetti, E.; Bertoncello, D.; Martini, M.; Zappulli, V.; Bargelloni, L.;

Philibert, J.; Snyder, P.W.; Glickman, N.; Glickman, L.T.; Knapp, D.W.; Waters D.J. (2003).

Queiroga, FL.; Alves, A; Pires, I; Lopes, C. (2007). Expression of Cox-1 an Cox-2 in canine mammary tumours. *Journal of Comparative Pathology*, Vol. 136, pp 177-185.

tumors. *Journal of Veterinary Internal Medicine*, Vol. 17, pp. 102-106.

mammary tumors of the dog. *Vet Pathol*, Vol. 37, pp. 248-253.

*University of California Press*, pp. 518-549, Berkeley.

dog. *Veterinary Pathology*, Vol. 42, pp. 200-212.

pp. 479-483.

35-40, NIN, ICMR, India

*Pathology*, Vol. 37, pp. 239-247.

*Oncology,* Vol. 45, pp. 459-469.

Vol. 44, No.2, pp. 10-16.

cancer patient. *Cancer*, Vol. 50, pp. 2410-2416.

*Physiology Pathology Clinical Medic*, Vol. 53, pp. 174-178.

1215.

occurring canine cancer. *Prostaglandins Leukotrienes and Essential Fatty Acids*, Vol. 70,

determined estrogen receptor α and progesterone receptor expression and host and tumor factors as predictors of disease-free period in mammary tumors of the

(2000). p53 Gene Mutations occurring in spontaneous benign and malignant

Mammary tumours in dogs and their similarities with human breast cancer. W: Bamji M.S. (edit.).: *Proceedings of Symposium on Animal Information Service Center*, pp.

Immunohistologic detection of estrogen receptor alpha in canine mammary tumors: clinical and pathologic associations and prognostic significance. *Veterinary* 

cells of mammary adenocarcinomas in bitches as related to tumour malignancy

progesterone receptors in adenocarcinomas of the mammary gland in bitches with mitotic activity of neoplastic cells. *Veterinary Medicine*, Vol. 63, No.10, pp. 1211-

PCNA in canine mammary tumors: Relationship to clinical and pathologic

Castagnaro, M. (2006). Immunohistochemical detection of P-glycoprotein (clone C494) in canine mammary gland tumours. *Journal of Veterinary Medicine Series A -* 

Influence of host factors in survival in dogs with malignant mammary gland


**Part 3** 

**Veterinary Care in Livestock** 

