Cyclooxygenase-2 and HER2 expression in simultaneous breast ductal carcinoma in situ and invasive ductal carcinoma .

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Author: Adrienne Pratti Lucarelli

Cyclooxygenase-2 and HER2 expression in simultaneous breast ductal carcinoma in situ and invasive ductal carcinoma . Short Title: COX-2 and HER2 expression in simultaneous breast cancer Original Article Authors: Adrienne Pratti Lucarelli, MD. Instructor Professor, Maria Marta Martins, MD. Instructor Professor; Vilmar Marques de Oliveira,MD. Instructor Professor; Maria Antonieta L. Galvão Silva, MD. Assistant Professor ; Sebastião Piato, MD, Full Professor; José Francisco Rinaldi, Assistant Professor; Tsutomo Aoki, MD.Assistant Professor.

Instituition Mastology Unit of the Gynecology and Obstetrics Department of the Irmandade da Santa Casa de Misericórdia de São Paulo/ Santa Casa De São Paulo - Faculty of Medical Sciences, São Paulo, Brazil.

Corresponding Author: Adrienne Pratti Lucarelli Rua: Sóror Angélica 705, apto 11, CEP 02452060 Vila Ester, São Paulo, Brasil Phone: (55+11)62364827/ Fax: (55+11) 62585090 E-mail: adrilucarelli@terra.com.br

Total number of: Tables=1 / Abstract words= 162/ Text words=2712

ABSTRACT

The aim of this study was to verify the correlation between the cyclooxygenase-2 (COX-2) and HER2 expression in non-neoplastic ductos, ductal carcinoma in situ and invasive ductal carcinoma found in the same breast. Histologic slices of 47 surgicals fragments were evaluated. The technique used to detect the protein was immunohistochemistry, with anti-HER2 and anti-COX-2 antibodies. Tumors that were scored +2 for membranous staining of HER2 protein were subjected to FISH. COX-2 was expressed positively in IDC, DCIS, normal epithelium, stroma tumor, and normal stroma in 41(87%), 40 (85%) and 35 (74.5%). HER2 was positive in 31 (65.9%) and negative in 16 (34.1%) of 47 cases de IDC. Positivity in DCIS of HER2 was observed in 31 cases (65.9%) and negativity in others 16 (34.1%). Analysis of normal epithelium, stroma tumor and normal stroma did not show HER2 protein expression by immunohistochemical. We have not obtained statistical correlation between the COX-2 and HER2 in IDC, DCIS, normal epithelium, stroma tumor and normal stroma.

INTRODUCTION

Prostaglandins are know to participate in a multiple physiologic and pathologic processes, including wound healing, cardiovascular disease, inflammation, and in the development and growth of malignant tumors1. Cyclooxygenase (COX-2), the inducible isoform of prostaglandin H synthase, has been implicated in carcinogenesis of a variety of human cancers, including breast, colon, lung, gastric, skin, endometrial,ovarian and esophageal adenocarcinomas2,3. COX-2 seems to be involved in the processes of malignant transformation and tumor progression by affecting cell proliferation, mitosis, cell adhesion, apoptosis, immune surveillance, angiogenesis, and formation of carcinogenic metabolites such as malondialdehyde 4,5.

Breast cancer is the most common cancer in women and is second only to lung cancer as a cause of cancer-related mortality 6. Despite intensive efforts aimed primarily at early detection and therapy, the mortality rates of breast cancer have remained virtually constant for several decades. Innovative research efforts must therefore be redirect towards chemoprevention during the early stages of carcinogenesis. Epidemiological studies suggest that the regular use of non-steroidal anti-inflammatory drugs (NSAIDs) may protect against breast cancer 7.

NSAIDs and COX-2 inhibitors are found to be potent chemopreventive agents against some mammary carcinoma models 8-11. A significant reduction in the risk of human breast cancer was recently reported from the intake of selective COX-2 inhibitors12. Thus, COX-2 constitutes a rational target in chemoprevention. COX-2 expression is induced by proinflammatory cytokines, tumor promoters, growth factors, and viral transformation 13-14. How the over-expression of COX-2 results in tumorigenesis and how COX-2 selective agents mediate chemopreventive effects are issues that remain poorly understood. The HER2 protein is one of the four proteins of the HER family which has as its homologue the EGFR, also known as HER-1 15, 16. This protein presents a stimulating response upon the cell when it binds to specific activators that circulate in the extracellular space and are denominated as ligands 17. The overexpression of the HER2 protein usually results from the increase of its coding by the c-erb-b2 gene; therefore, it is commonly found to be associated to the amplification of this proto-oncogene 18-20. The increase of HER2 expression becomes even more important when this protein participates in the formation of heterodimers. When the latter is bound to the HER3 receptor, hyperactivity of cell signaling pathway occurs, leading to high levels of cell cycle regulation, with the emergence of a situation of uncontrolled growth and cell proliferation, with consequent tumorigenesis21.

The overexpression of this protein in breast cancer has been associated to disease aggressiveness, a more reserved prognosis, absence of estrogen and progesterone receptors, as well as resistance to hormonal and chemotherapy using cyclophosphamide, methotrexate and fluracil 22 Recent studies have reported that the protein HER2 regulates COX-2 expression23. Increased levels of cyclooxygenase-2 mRNA, protein,and prostaglandin E2 synthesis were detected in HER2/neu transformed human mammary ephithelial cells compared with its nontransformed partner cell line. HER-2/neu stimulated COX-2 transcription via the Ras→Raf→MAPK 23. In support of this hypothesis studies have demonstrated COX-2 is expressed in invasive breast cancer with HER2/neu overexpression 24,25. COX-2 and HER-2 gene expression in normal tissue is different from what is found in invasive. Many studies have already analyzed HER2/neu and COX-2 expression in ductal carcinoma in situ and invasive ductal carcinoma, but most of these studies were carried out in tissue from different women, thereby restricting the usefulness of these data for studying tumor progression. The purpose of this study was to evaluate COX-2 expression and HER2 expression in the successive steps of breast carcinogenesis within one same paraffin block.

MATERIALS AND METHODS

All of the 47 tissues used in this study were specimens obtained from patients who underwent breast cancer surgical treatment at Santa Casa Hospital of São Paulo,between June 2002 and July 2006. Clinical data were obtained by retrospective chart review. Only patients who presented invasive ductal carcinoma (IDC), ductal carcinoma in situ (DCIS) and normal epithelium in the same paraffin block were eligible for this study. The mean age of the patients was 52 years (range 31-85 years). None of the patients had received chemotherapy, hormonal treatment or radiation therapy before surgery or non-steroidal anti-inflammatory drugs 15 days preceding the procedure. The Hematoxylin and eosin slides for all case were reviewed and nuclear grade and presence or absence of comedonecrosis for DCIS as well as nuclear grade and histological type for IDC were determined 26,27.

Tissue preparation and Immunohistochemistry

Three-μm sections from specimens in formalin-fixed, paraffin-embedded blocks were prepared stepwise deparaffinized in xylene and rehydrated in descending alcohols. Antigen was retrieved using microwave oven (4 X 5 min in 700W in 00.1M sodium citrate buffer, pH 6.0). The slides were then immersed in 0.3% hydrogen peroxide in methanol for 30 min to block endogenous peroxidase activity and in blocking solution [1:5:100 normal horse serum in PBS (phosphate buffered saline)] for block nonspecific binding sites. Immunostaining was performed with COX-2 polyclonal antibody (3362- 100, Biovision Research Products Co., Mountais View, CA, USA) in dilution of 1:70 (15μg/ml). The HER2 immunostaining was performed with mouse monoclonal antihuman HER2 (clone CB11, DAKO A/S, Copenhagen, Denmark) in dilution of 1:150 for 18 hours. After slides were rinsed in PBS, the biotinylated secondary antibody was applied for 30min at room temperature (DAKO Corp., CA, USA). Incubated in streptovidin-biotin-peroxidase for 30 min at 37 C. Specimens were rinsed in 0.005% tween 20 in PBS then incubated with 3.3"- diaminobenzidine chromogenic substrate for five minutes. Sections were counterstained in Meyer`s hematoxylin and mounted.

HER2 FISH Analysis Tumors that were scored 2+ for membranous staining using the DAKO Herceptest kit were subjected to FISH analysis using the Vysis PathVysion kit, which incorporates a control probe for chromosome 17 as well as the test probe for the HER-2 gene, according to the manufacturer's instructions. In brief, 4-µm paraffin-embedded sections were dewaxed, taken to absolute ethanol, and air dried. They were then placed in 0.2 M HCl at room temperature for 20 min and in pretreatment solution at 80°C for 30 min and then underwent a proteolytic digestion at 37°C for 25 min. The sections were then denatured in formamide at 72°C for 5 min before incubation in the PathVysion HER-2/17 probe overnight in the dark at 37°C. The following day, the sections were washed in posthybridization buffer for 2 min at 72°C, air dried in the dark, and then mounted in 4',6-diamidino-2-phenylindole.

Evaluation of COX-2 and HER2 staining COX-2 and HER2 immunohistochemical staining were evaluated and scored independently and in a blind manner by two investigators. For COX-2, we used the same criteria adopted by Ristimaki et al 24: score 0, no staining; 1, weak diffuse cytoplasmatic staining (may contain stronger intensity in less than 10% of cells); 2, moderate to strong granular cytoplasmatic staining in 10-90% of cells; 3, over 90% of cells stained with strong intensity. COX-2 expression was considered positive with scores of 2 or 3 and negative with scores of 0 or 1. For HER2 overexpression was performed as recommended by the following Hercep Test scoring guidelines: 0, no staining or 10% of the tumor cells; +2, weak or moderately complete membrane staining in >10% of tumor cells; +3, strong complete membrane staining in >10% of tumor cells. For HER2, tumors that exhibited membranous staining of +3 intensity or were +2 but showed gene amplification by FISH analysis were considered positive. Sections were scored by one investigator and subjected to review by a second.

Statistical analysis

The correlation between COX-2 and HER2 was analysed using Spearman's Rank Coefficient. The Kruskal-Wallis test was used to analyse nuclear grade and histological grade, and the Mann-Whitney test to check for the presence or absence of comedonecrosis, with the aim of verifying possible differences between the positive percentages of the delineated categories. The chi-square test was used to analyze the age and tumor size groups, with the goal of verifying the possible differences between the two groupsA p value of References 1.

Dubois RN. Aspirin and breast cancer prevention: the estrogen connection. JAMA 2004;291:2488-2489. 2. Taketo MM. Cyclooxygenase-2 inhibitors in tumorigenesis (Part II). J Natl Cancer Inst 1998;90:1609-1620. 3. Soslow RA. Dannenberg AJ, Rush D. COX-2 is expressed in human pulmonary, colonic, and mammary tumors. Cancer 2000:89;2637-2645. 4. Singh B, Lucci A. Role of cyclooxygenase-2 in brest cancer. J Surg Res 2002;108:173-179. 5. Tsuji S, Tsujii M, Kawano S, Hori M. Cyclooxygenase-2 upregulation as a perigenetic change in carcinogenesis. J Exp Clin Cancer Res 2001;20: 117-129. 6. Greenlee RT, Hill-Harmon MB, Murray T, Thun M. Cancer statistics, 2001. CA Cancer J Clin 2001;51:15-36. 7.Sharpe CR, Collet JP, McNutt M, Belzile E, Bolvin JF, Hanley JA. Nested casecontrol study of the effects of non-steroidal anti-inflammatory drugs on breast cancer risk and stage. Br J Cancer 2000;83:112-120. 8.McCormick DL, Madigan MJ, Moon RC. Modulation of rat mammary carcinogenesis by indomethacin. Cancer Res 1985;45:1803-1808. 9.Noguchi M, Taniya T, Koyasaki N, Kumaki T, Miyazaki I, Mizukami Y. Effects of the prostaglandin synthetase inhibitor indomethacin on tumorigenesis, tumor proliferation, cell kinetics, and receptor contents of 7,12-dimethylbenz(a)anthraceneinduced mammary carcinoma in Sprague-Dawley rats fed a high- or low-fat diet. Cancer Res 1991;51:2683-2689. 10.Harris RE, Alshafie GA, Abou-Issa H, Seibert K. Chemoprevention of breast cancer in rats by celecoxib, a cyclooxygenase 2 inhibitor. Cancer Res 2000;60:2101-2103. 11.Harris RE, Beebe-Donk J, Doss H, Burr-Doss D. Aspirin, ibuprofen, and other nonsteroidal anti-inflammatory drugs in cancer prevention: a critical review of nonselective COX-2 blockade (review). Oncol Rep 2004,13:559-583. 12.Harris RE, Beebe-Donk J, Alshafie GA. Reduction in the risk of human breast cancer by selective cyclooxygenase-2 (COX-2) inhibitors. BMC Cancer 2006,6:27. 13. Smith WL, Langenbach R. Why there are two cyclooxygenase isozymes. J Clin Invest 2001;107:1491-5. 14.Thun MJ, Henley SJ, Patrono C. Nonsteroidal anti-inflamatory drugs as anticancer agents: mechanistic, pharmacologic, and clinical issues. J Natl Cancer Inst 2002;94:252-266. 15. Borg,A.; Baldetorp,B.; Ferno,M.; Killander,D.; Olsson,H.; Sigurdsson,H. ERBB2 amplification in breast cancer with a high rate of proliferation. Oncogene, 6(1):137-43, 1991. 16. Bertucci,F.; Borie,N.; Ginestier,C.; Groulet,A.; Charafe-Jauffret,E.; Adelaide,J.; Geneix,J.; Bachelart,L.; Finetti,P.; Koki,A.; Hermitte,F.; Hassoun,J.; Debono,S.; Viens,P.; Fert,V.; Jacquemier,J.; Birnbaum,D. Identification and validation of an ERBB2 gene expression signature in breast cancers. Oncogene 2004; 23(14): 2564-75. 17. Dickson, R.B., Lippman, M.E. Oncogenes and suppressor genes. In: Harris J., Lippman M.E , Morrow M.; Hellman S., editors. Diseases of the Breast, 2nd edition, Philadelphia PA, J.B. Lippincott, 2000. p.281-302. 18. Pinkas-Kramarski,R.; Shelly,M.; Guarino,B.C.; Wang,L.M.; Lyass,L.; Alroy,I.; Alimandi,M.; Kuo,A.; Moyer,J.D.; Lavi,S.; Eisenstein,M.; Ratzkin,B.J.; Seger,R.; Bacus,S.S.; Pierce,J.H.; Andrews,G.C.; Yarden,Y. ErbB tyrosine kinases and the two neuregulin families constitute a ligand-receptor network. Mol. Cel. Biol., 18(10):6090-101, 1998. 19. Van de Vijver,M.J.; Peterse,J.L.; Mooi,W.J.; Wisman,P.; Lomans,J.; Dalesio,O.; Nusse,R. R Neu-protein overexpression in breast cancer. Association with comedo-type ductal carcinoma in situ and limited prognostic value in stage II breast cancer. N. Engl. J. Med., 319(19):1239-45, 1988. 20. Borg,A.; Tandon,A.K.; Sigurdsson,H.; Clark,G.M.; Ferno,M.; Fuqua,S.A.; Killander,D.; McGuire,W.L. HER-2/neu amplification predicts poor survival in node-positive breast cancer. Cancer Res., 50(14):4332-7, 1990. 21. Tandon,A.K.; Clark,G.M.; Chamness,G.C.; Ullrich,A.; McGuire,W.L. HER-2/neu oncogene protein and prognosis in breast cancer. J. Clin. Oncol., 7(8):1120-8, 1989. 22.Adam,L.; Vadlamudi,R.; Kondapaka,S.B.; Chernoff,J.; Mendelsohn,J.; Kumar,R. Heregulin regulates cytoskeletal reorganization and cell migration through the p21-activated kinase-1 via phosphatidylinositol-3 kinase. J. Biol. Chem., 273(43):28238-46, 1998. 23. Subbaramaiah K, Norton L, Gerald W, Dannenberg AJ. Cyclooxygenase-2 is Overexpressed in HER-2/neu-positive Breast Cancer: Evidence for involvement of AP-1 and PEA3. J. Biol. Chem 2002; 277: 18649-18657. 24. Ristimäki A, Sivula A, Lundin J, Lundin M., Salminen T., Haglund C et al. Prognostic significance of elevated cyclooxygenase-2 expression in breast cancer. Cancer Res 2002; 62:632-35. 25. Half E, Tang XT, Gwyn K, Sahin A, Wathen K, Sinicrope FA. Cyclooxygenase-2 expression in human breast cancers and adjacent ductal carcinoma in situ. Cancer Res 2002; 62:1676-1681. 26. Dabbs DJ. Ductal carcinoma of breast: nuclear grade as a predictor of S-phase fraction. Human Pathol 1993;24:652-656. 27. Elston CW, Ellis IO. Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up. Histopathology 1991;19:403-410. 28. Boland GP, Butt IS, Prasad R, Knox WF, Bundred NJ. COX-2 expression is associated with an aggressive phenotype in ductal carcinoma in situ. Br J Cancer 2004;90:423-429. 29. Brower V. Feeding the flame: new research adds to role of inflammation in cancer development. News. J Natl Cancer Inst. 2005;97:251-253. 30.Davies G, Salter J, Hills M, Martin LA, Sacks N, Dowsett M Correlation between cyclooxygenase-2 expression and angiogenesis in human breast cancer. Clin Cancer Res 2003;9:2652-2656. 31.Shim V, Gauthier ML, Sudilovsky D, et al. Cycloosygenase-2 expression is related to nuclear grade in ductal carcinoma in situ and is increased in its normal adjacent epithelium. Cancer Res 2003;63:2347-2350. 32.Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL - Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogenese. Science 1987; 235: 177-82. 33.Yamada Y, Yoshimoto M, Murayama Y, Ebuchi M, Mori S, Yamamoto T et al. Association of elevated expression of the c-erbB-2 protein with spread of breast cancer. Jpn J Cancer Res 1989; 80: 1192-98. 34.Allred DC, Clark GM, Tandom AK, Molina R, Tormey DC, Osborne CK et al. HER-2/neu in node-negative breast cancer: prognostic significance of overexpression influenced by the presence of in situ carcinoma. J Clin Oncol 1992; 10:599-605. 35.Vadlamudi R., Mandal M., Adam L., Steinbach G., Mendelson J., Kumar R. Regulation of cyclooxygenase-2 pathway by HER2 receptor. Oncogene, 18: 305-314, 1999 36. Mann M., Sheng H., Shao J., Williams C. S., Pisacane P. I., Sliwkowski M. X., DuBois R. N. Targeting cyclooxygenase 2 and HER-2/neu pathways inhibits colorectal carcinoma growth. Gastroenterology, 120: 1713-1719, 2001. 37. Ranger GS, Thomas V ,Jewell A, Mokbel K. Elevated cyclooxygenase-2 expression correlates with distant metastases in breast cancer. Anticancer Research 2004: 24: 2349-52. 38. Schmitz KJ, Callies R, Wohlschlaeger J, Kimmig R, Otterbach F, Bohr J, et al. Overexpression of cyclo-oxygenase-2 is an independent predictor of unfavourable outcome in node-negative breast cancer, but is not associated with protein kinase B (Akt) and mitogen-activated protein kinase (ERK1/2, p38) activation or with Her-2/neu signalling pathways. J Clin Pathol. 2006; 59(7):685-91. 39. Liu CH, Chang SH, Narko K. Overexpression of cyclooxygenase COX)-2 is sufficient to induce tumorigenesis in transgenic mice. J Biol Chem 2001; 276: 18563-9. 40. Parrett ML, Harris RE, Joarder FS. Cyclooxygenase-2 gene expression in human breast cancer. Int J Oncol 1997; 10:503-07. 41. Perrone G, Santini D, Vincenci B, Zagami M, La Cesa A, Bianchi A, et al. COX-2 expession in DCIS: correlation with VEGF, HER-2/neu, prognostic molecular markers and clinicopathological features. Histopathology 2005; 46(5):561-8. 42. Cho MH, Yoon JH, Jaegal YJ, Choi YD, Lee JS, Lee JH, Nam JH, Choi C, Lee MC, Park CS, Woo JS, Min KW. Expression of cyclooxygenase-2 in breast carcinogenesis and its relation to HER-2 neu and p53 protein expression in invasive ductal carcinoma. Breast 2006: 390-8. 43.Tomozawa S, Nagawa H, Tsuno N, et al. Inhibition of haematogenous metastasis of colon cancer in mice by a selective COX-2 inhibitor, JTE-522. Br J Cancer 1999;81:1274-1279. 44. Xu XC. COX-2 inhibitors in cancer treatment and prevention, a recent development. Anticancer Drugs 2002;13:127-137. 45. Harris RE, Beebe-Donk J, Alshafie GA. Reduction in the risk of human breast cancer by selective cyclooxygenase-2 (COX-2) inhibitors. BMC Cancer 2006,6:27.

TABLE1

Association of expression HER2/neu and COX-2 in non-neoplastic ducts, ductal carcinoma in situ and invasive ductal carcinoma of the same breast Histologic tissuel Statistical analysis HER2 IDC HER2 DCIS HER2 normal epithelium COX-2 IDC Correlation coefficient 0,133 0,133 — Significant (p) 0,373 0,373 — n 47 47 47 COX-2 DCIS Correlation coefficient 0,242 0,242 — Significant (p) 0,102 0,102 — N 47 47 47 COX-2 normal epithelium Correlation coefficient 0,268 0,268 — Significant (p) 0,068 0,068 — N 47 47 47