ANTICANCER RESEARCH 25: 1649-1654 (2005)

Heat Shock Protein 27 is Associated with Decreased Survival in Node-negative Breast Cancer Patients

F. THANNER1 M.W. SÜTTERLIN1, M. KAPP1, L. RIEGER1, A.K. MORR1, P. KRISTEN1, J. DIETL1, A.M. GASSEL2 and Th. MÜLLER1

1Department of Obstetrics and Gynecology and 2Institute of Pathology, University of Würzburg, Germany

Abstract. Background: Heat shock protein 27 (hsp27) is a pathways (1, 2). By interference with the Ras-1 pathway, it molecular chaperone which supports cells to keep their has been shown to block apoptosis (3, 4). homeostasis under stressful conditions. It is associated with Because hsp27 has cytoprotective and anti-apoptotic resistance to chemotherapeutics, radiation and hyperthermia. capacities, it has been investigated in many tumors and The aim of this retrospective study was to investigate the tumor models: hsp27 is expressed at higher levels in prognostic value of hsp27 for patients with node-negative malignancies compared to normal tissues (5, 6). Storm et al. breast cancer. Materials and Methods: Paraffin sections of 191 found an increased expression in lymph node metastases patients were stained immunohistochemically with a than in the corresponding primary tumors (7). The protein monoclonal antibody against hsp27. Median follow-up was is further overexpressed in different well established tumor 177 months. The results were correlated with clinical and cell lines (8). histopathological parameters using the Chi-square test. Results: Transfection of the hsp27 gene into tumor cells is There was no significant correlation between hsp27 expression associated with an alteration of cell growth and and standard histopathological features or the proliferation differentiation (9-11). Additionally, tumorigenicity is marker ki-67. Disease-free survival (DFS) was not altered for elevated and the transfected tumor cells exhibit a resistance patients expressing hsp27-positive tumors, whereas overall to different chemotherapeutics and radiation (12-16). In survival (OS) [p=0.02] and survival after first recurrence (SR) gynecologic oncology, hsp27 has been shown to be [p=0.01] were significantly decreased. Conclusion: The correlated with a decreased survival after chemotherapy in expression of hsp27 in primary breast cancers is associated with ovarian cancer, but with parameters of good prognosis in a short survival for node-negative patients. endometrial cancer (17, 18). The prognostic significance of hsp27 has also been Heat shock protein 27 (hsp27) is a member of a studied for lymph node-negative (N0) breast cancer patients. heterogeneous protein family whose expression is induced At least four clinical studies with a follow-up from 53 to 91 as a cellular response to various kinds of harmful and months showed contradicting data for survival analysis. Two physiological conditions. It works as a molecular chaperone studies found a significant correlation of hsp27 on disease- which supports cells to keep their homeostasis under free survival (DFS) or survival after first recurrence (SR), extrinsic or intrinsic stress conditions. Moreover, hsp27 has while the others were not able to confirm this intriguing been found to be a regulative protein in growth and correlation (19-22). differentiation, interacting with different signal transduction The aim of this study was to verify the exsisting data about the prognostic value of hsp27 for a well-defined group of N0 breast cancer patients with a follow-up-period of almost fifteen years. Correspondence to: Dr. med. F. Thanner, University of Würzburg, Department of Obstetrics and Gynecology, Josef-Schneider-Str.4, 97080 Würzburg, Germany. Tel: 0049/931/20125621, Fax: Materials and Methods 0049/931/20125406, e-mail: [email protected] Breast cancer tissues of 191 unselected N0 patients were obtained Key Words: Heat shock protein, hsp27, prognosis, immuno- by surgery, between January 1980 and December 1986, at the histochemistry, breast cancer. Department of Gynaecology, University of Wuerzburg, Germany.

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Figure 1. Cytoplasmic expression in cells of a primary node-negative breast cancer after immunohistochemical staining with antibody against hsp27. Magnification: x 100.

The median clinical follow-up time was 177 months. The standard For statistical analysis, the Chi-square-Test was used to compare histopathological examination was performed in the Institute of hsp27 expression with dichotomized prognostic variables. Analyses Pathology, University of Wuerzburg. for overall survival (OS), DFS and SR were performed using the For immunhistochemical staining, paraffin sections were cut, Kaplan-Meier method. All computations were implemented with applied to slides and deparaffinized overnight. The slides were the use of SPSS 11.0 software. subsequently immersed in phosphate-buffered saline (PBS) and in methanol with 3% hydrogen peroxide in order to block endogenous Results peroxidase activity. The slides were preincubated with 1% Periglobinì solution in PBS to inactivate unspecific antibody Clinical follow-up. The median age of the 191 patients was 58 binding in the tissue. As a next step we applied the primary years (range: 24-86). 96.9% were treated with mastectomy monoclonal antibody against hsp27 (StressGen Biotechnologies Corp.: SPA-800, Victoria, BC, Canada) in a dilution of 1:100 and and all patients received axillary lymphadenectomy. A incubated for one hour at room temperature in a humid chamber. median 7 (range: 1-20) lymphatic nodes were obtained. From After rinsing with PBS, the LSAB Kit HRP (DAKO: Ko 675, an initial 191 patients, 83 (43.5%) had died by the end of the Hamburg, Germany) for detection of the primary antibody was used. follow-up period, 41 (21.5%) of these because of recurrent Diaminobenzidine (SIGMA: D-4293, Taufkirchen , Germany) was breast cancer. For all women in our study, we calculated a used as chromogen, whereupon a counterstaining with haemalaun median OS of 177 months with a range of 2 to 254 months. was performed. During the observed period, 56 (29.3%) patients suffered We analyzed the immunostained slides by examination of at least five high-power fields (x 100 magnification) by light microscopy from local or systemic metastatic reactivation of the disease. using the scoring system suggested by Cano et al. (5). For each slide, This resulted in a median DFS of 171 months. The median we obtained a value for cytoplasmic staining (Figure 1). SR was 35 months (range: 2 - 178 months).

1650 Thanner et al: hsp27 in Patients with Node-negative Breast Cancer

Table I. Correlation of hsp27 expression in No breast cancer patients and histopathologically evaluated prognostic parameters.

hsp27-positive hsp27-negative P-value

Tumor size <2 cm 119 27 0.652 >2 cm 38 7

Grading G1/G2 104 24 0.077 G3 46 4 Typing invasive- ductal 117 25 0.190 others 32 3

Estrogen receptor Figure 2. Overall survival for all patients with primary node-negative positive 149 32 0.852 breast cancer after 120 months (53 events) as a function of hsp27 negative 8 2 positivity in cytoplasm. The survival curve was constructed by the method of Kaplan and Meier. Progesterone receptor positive 141 28 0.217 negative 16 6

Ki-67 content <20% 74 19 0.440 >20% 59 11

Table II. Correlation of hsp27 expression in No breast cancer patients and clinically evaluated survival data.

hsp27-positive hsp27-negative P-value

Disease-free survival (DFS) <90 months 48 7 0.244 >90 months 109 27

Overall survival (OS) <120 months 49 4 0.022 Figure 3. Survival after recurrence for 56 patients with relapsed primary node negative breast cancer as a function of hsp27 positivity in cytoplasm. >120 months 108 30 The survival curve was constructed by the method of Kaplan and Meier.

Survival after first recurrence (SR) <30 months 29 2 0.013 >30 months 17 8 Correlation of hsp27 with survival data. In our samples there was no significant correlation (p=0.24) between expression of hsp27 in primary cancers and DFS. Hsp27 expression was associated with a shorter SR (p=0.01) and a shorter OS (p=0.02). The Correlation of hsp27 with other prognostic factors. None results are shown in Table II, Figure 2 and Figure 3. of the investigated standard histopathological parameters (tumor size, grading, typing, hormone receptor status), Discussion nor the additionally stained proliferation marker ki-67, showed a statistically significant correlation to the Heat shock protein 27 is a molecular chaperone with expression of hsp27 in breast cancer tissues, as is shown cytoprotective and anti-apoptotic capacities (1-4). Its in Table I. overexpression in malignant tissues and various tumor cell

1651 ANTICANCER RESEARCH 25: 1649-1654 (2005) lines is associated with altered cell growth and elevated advanced breast cancer (30). Moreover, we observed a tumorigenicity (6, 8, 10, 12). For that reason, the prognostic shorter SR in hsp27-positive tumors, confirming the results and predictive properties of hsp27 have been investigated in of Love et al. (19). As the patients in our study were various malignancies. It is correlated with decreased survival operated in the early eighties, 97% of them were treated in ovarian cancer, but also with favorable outcome in with primary mastectomy and received chemotherapy or patients with malignant fibrous histiocytoma or endometrial radiation first in case of relapse. The worse outcome for cancer (17, 18, 23). patients with recurrent disease which express hsp27 might Hsp27 has been studied extensively in patients with breast be explained by its known cytoprotective effect on cancer cancer. Some authors found a significant correlation to cells, resulting in resistance to cytotoxic drugs like estrogen receptor (ER) status, which may be explained by the doxorubicin (1, 31). fact that hsp27 expression is regulated by estrogen (2, 5, 24). This study confirms former observations that the In comparison to other prognostic variables, the expression expression of heat shock protein 27 in node-negative breast of ER was not significantly associated with the appearance of cancer is associated with a worse outcome for the patients hsp27 in our group of patients. We attribute this result to the (19, 20, 22). Nevertheless, further studies are warranted to small number of ER-negative women in this study. Ciocca et evaluate the definite prognostic value of this protein and al. described "a lack of correlation with respose to tamoxifen" could also concentrate on a possible predictive value of for the expression of hsp27 in breast cancer tissues (25). In hsp27 with regards to cancer chemotherapy or radiation. contrast to this finding, the same investigators showed that hsp27 was associated with resistance to chemotherapeutics, References both in vitro and in vivo (16, 26). The prognostic significance of hsp27 for patients with 1 Ciocca DR, Oesterreich S, Chamness GC, McGuire WL and early or advanced breast cancer has been the subject of Fuqua SA: Biological and clinical implications of heat shock various clinical studies. For both groups, contradictory protein 27000 (Hsp27): a review. J Natl Cancer Inst 85: 1558- results exist about the influence of hsp27 on survival. 1570, 1993. 2 Benndorf R and Bielka H: Cellular stress response: stress Darmstrup et al. and Tetu et al. found no effect on DFS or proteins – physiology and implications for cancer. Rec Results OS in nodal-positive patients, while other authors detected Cancer Res 143: 129-144, 1997. a worse outcome for the subgroup of patients with one to 3 Garrido C, Gurbuxani S, Ravangnan L and Kroemer G: Heat three affected lymph nodes (19, 22, 27, 28). In comparison, shock proteins: endogenous modulators of apoptotic cell death. two other studies described a favorable outcome for patients Biochem Biophys Res Com 286: 433-442, 2001. with hsp27-positive breast cancers (29, 30). 4 Jolly C and Morimoto RI: Role of heat shock response and At least four studies investigated the prognostic value of molecular chaperones in oncogenesis and cell death. J Natl hsp27 expression, as measured by immunohistochemistry or Cancer Inst 92(19): 1564-1572, 2000. 5 Cano A, Coffer AI, Adatia R, Millis RR, Rubens RD and King Western blotting, for patients with node-negative breast RJB: Histochemical studies with an estrogen receptor-related cancer. The clinical follow-up of the women included in protein in human breast tumors. Cancer Res 46: 6475-6480, 1986. these studies ranged from 53 to 92 months. As for patients 6 Ciocca DR, Adams DJ, Edwards DP, Bjercke RJ and McGuire with advanced breast cancer, the results were contradictory. WL: Distribution of an estrogen-induced protein with a While two studies did not observe an effect of hsp27 on molecular weight of 24,000 in normal and malignant human DFS or OS, Chamness et al. and Love et al. found a tissues and cells. Cancer Res 43: 1204-1210, 1983. significantly shortened disease-free period after operation. 7 Storm FK, Mahvi DM and Gilchrist KW: Heat shock protein The latter authors also showed a lower survival after first 27 overexpression in breast cancer lymph node metastasis. Ann Surg Oncol 3: 570-573, 1996. recurrence (19-22). 8 Morino M, Tsuzuki T, Ishikawa Y, Shirakami T, Yoshimura M, In this study, a group of 191 node-negative breast cancer Kiyosuke Y, Matsunaga K, Yoshikumi C and Saijo N: Specific patients with a long median follow-up period of 177 months expression of hsp27 in human tumor cell lines in vitro. In Vivo were presented. As Oesterreich et al. and Thor et al., we did 11: 179-184, 1997. not detect a significant influence on DFS (21, 22). On the 9 Walsh D, Grantham J, Zhu X, Wei Lin J, Ososterum M, Taylor other hand, we found a statistically significant and R and Edwards M: The role of heat shock proteins in disadvantageous effect on OS for those women expressing mammalian differentiation and development. Environmental hsp27 in their primary breast cancer. As we first Med 43: 79-87, 1999. 10 Kindas-Mugge I, Micksche M and Trautinger F: Modification dichotomized our patients for OS after ten years, it is of growth in small heat shock (hsp27) gene transfected breast conceivable that this effect was not uncovered in the studies carcinoma. Anticancer Res 18(1A): 413-417, 1998. mentioned above because of a shorter period of follow-up. 11 Calabrese V, Scapagnini G, Ravagna A, Giuffrida Stella AM We regard this result as interesting, since Seymour et al. also and Butterfield DA: Molecular chaperones and their roles in reported a shortened OS for hsp27-positive patients with neural cell differentiation. Dev Neurosci 24: 1-13, 2001.

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12 Garrido C, Fromentin A, Bonnotte B, Favre N, Moutet M, 24 Adams DJ and McGuire WL: Quantitative enzyme-linked Arrigo A, Mahelen P and Solary E: Heat shock protein 27 immunosorbent assay for the estrogen-regulated Mr24,000 enhances the tumorgenicity of immunogenic rat colon protein in human breast tumors: correlation with estrogen and carcinoma cell clones. Cancer Res 58: 351-366, 1998. progesterone receptors. Cancer Res 45: 2445-2449, 1985. 13 Lemieux P, Oesterreich S, Lawrence JA, Steeg PS, Hilsenbeck 25 Ciocca DR, Green S, Elledge RM, Clark GM, Pugh R, Ravdin SG, Harvey JM and Fuqua SA: The small heat shock protein P, Lew D, Martino and Osborne CK: Heat shock proteins hsp27 hsp27 increases invasiveness but decreases motility of breast and hsp70: lack of correlation with response to tamoxifen and cancer cells. Invasion Metastasis 17(3): 113-123, 1997. clinical course of disease in estrogen receptor-positive 14 Oesterreich S, Weng CN, Qiu M, Hilsenbeck SG, Osborne CK metastatic breast cancer (A Southwest Oncology Group Study). and Fuqua SA: The small heat shock protein hsp27 is correlated Clin Cancer Res 5: 1263-1266, 1998. with growth and drug resistance in human breast cancer cell 26 Ciocca DR, Fuqua SA, Lock-Lim S, Toft DO, Welch WJ and lines. Cancer Res 53: 4443-4448, 1993. McGuire WJ: Response of human breast cancer cells to heat 15 Hansen RK, Parra I, Lemieux P, Oesterreich S Hilsenbeck SG shock and chemotherapeutic drugs. Cancer Res 52: 2648- and Fuqua SA: Hsp27 overexpression inhibits doxorubicin- 3654, 1992. induced apoptosis in human breast cancer cells. Breast Cancer 27 Darmstrup L, Andersen J, Kufe DW, Hayes DF and Skovgaard Res Treat 56: 187-196, 1999. Poulsen H: Immunocytochemical determination of the 16 Vargas-Roig LM, Gago FE, Tello O, Aznar JC and Ciocca R: estrogen-regulated proteins Mr 24000, Mr 52000 and DF3 Heat shock protein expression and drug resistance in breast breast cancer associated antigen: clinical value in advanced cancer patients treated with induction chemotherapy. Int J breast cancer and correlation with estrogen receptor. Ann Cancer 79: 468-465, 1995. Oncol 3: 93-97, 1992. 17 Langdon SP, Rabiasz GJ, Hirst GL, King JB, Hawkins RA, 28 Tetu B, Brisson J, Landry J and Huot J: Prognostic significance Smyth JF and Miller WR: Expression of the heat shock protein of heat shock protein 27 in node-positive breast carcinoma: an hsp27 in human ovarian cancer. Clin Cancer Res 1: 1603-1609, immunohistochemical study. Breast Cancer Res Treat 36: 93- 1995. 97, 1995. 18 Geisler JP, Geisler HE, Tammela J, Miller GA, Wiemann MC 29 Hurliman J, Gebhard S and Gomez F: Estrogen receptor, and Zhou Z: A study of heat shock protein 27 in endometrial progesterone receptor, pS2, ERD5, Hsp27 and cathepsin D in carcinoma. Gynecol Oncol 72: 347-350, 1999. invasive ductal breast cancer. Histopathology 23: 239-248, 1993. 19 Love S and King R: A 27kDa heat shock protein that has 30 Seymour L, Bezwoda WR and Meyer K: Tumor factors anomalous prognostic powers in early and advanced breast predicting for prognosis in metastatic breast cancer: the cancer. Br J Cancer 69: 743-748, 1994. presence of p24 predicts for response to treatment and duration 20 Chamness GC, Ruiz A, Fulcher L, Clark GM, Fuqua SA and of survival. Cancer 66: 2390-2394, 1990. McGuire WL: Estrogen-inducible heat shock protein Hsp27 31 Hansen RK, Parra I, Lemieux P, Oesterreich S, Hilsenbeck SG predicts recurrence in node-negative breast cancer. Proc Am and Fuqua SA: Hsp27 overexpression inhibits doxorubicin- Ass Cancer Res 30: 252, 1989. induced apoptosis in human breast cancer cells. Breast Cancer 21 Oesterreich S, Hilsenbeck SG, Ciocca DR, Allred DC, Clark Res Treat 56: 187-196, 1999. GM, Chamness GC, Osborne CK and Fuqua SA: The small heat shock protein Hsp27 is not a independent prognostic marker in axillary lymph node-negative breast cancer patients. Clin Cancer Res 2: 1199-1206, 1996. 22 Thor A, Benz C, Moore D, Goldman E, Edgerton S, Landry J, Schwartz L, Mayall B, Hickey E and Weber L: Sress response protein (srp-27) determination in primary human breast carcinomas: clinical, histologic, and prognostic implications. J Natl Cancer Ins 83: 170-178, 1991. 23 Tetu B, Lacasse B, Bouchard HL, Lagace R, Huot J and Landry J: Prognostic influence of hsp27 expression in malignant fibrous histiocytoma: a clinicopathological and immunohistochemical Received August 2, 2004 study. Cancer Res 52: 2325-2328, 1992. Accepted February 8, 2005

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