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High p27^Kip1 Expression Predicts Superior Relapse-Free and Overall Survival for Premenopausal Women With Early-Stage Breast Cancer Receiving Adjuvant Treatment With Tamoxifen Plus Goserelin

Gudrun Pohl, Margaretha Rudas, Otto Dietze, Sigurd Lax, Eva Markis, Robert Pirker, Christoph C. Zielinski, Hubert Hausmaninger, Ernst Kubista, Hellmut Samonigg, Raimund Jakesz, Martin Filipits

From the Departments of Medicine I, Pathology, Surgery, and Gynecology, University of Vienna, Vienna; the Department of Pathology and the Third Medical Department, Salzburg Hospital, Salzburg; the Department of Pathology and the Medical Department, University of Graz, Graz; and the Department of Pathology, Wiener Neustadt General Hospital, Wiener Neustadt, Austria.

Address reprint requests to Martin Filipits, PhD, Associate Professor, Department of Medicine I, Clinical Division of Oncology, University of Vienna, Waehringer Guertel 18–20, A-1090 Vienna, Austria; e-mail: martin.filipits{at}akh-wien.ac.at.

	ABSTRACT

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Purpose: To determine the predictive value of p27^Kip1 in premenopausal women with early-stage hormone receptor–positive breast cancer.

Patients and Methods: We retrospectively examined tumor specimens from 512 patients with breast cancer who were enrolled onto Austrian Breast and Colorectal Cancer Study Group (ABCSG) Trial 5. In this trial, premenopausal, hormone receptor–positive breast cancer patients with stage I and II disease were randomly assigned to receive either 5 years of tamoxifen plus 3 years of goserelin or six cycles of cyclophosphamide, methotrexate, and fluorouracil. p27^Kip1 expression was assessed by immunohistochemistry, and its association with clinical outcome was determined. Statistical analyses were performed to test for interaction between p27^Kip1 status and treatment.

Results: High p27^Kip1 expression (nuclear p27^Kip1 staining in 50% of tumor cells) independently predicted superior relapse-free survival (RFS) and overall survival (OS) in both the total study population (RFS: relative risk [RR], 0.53; 95% CI, 0.34 to 0.82; P = .004; OS: RR, 0.29; 95% CI, 0.15 to 0.58; P < .001) and patients treated with combination endocrine therapy (RFS: RR, 0.32; 95% CI, 0.16 to 0.63; P = .001; OS: RR, 0.16; 95% CI, 0.05 to 0.53; P = .003). The interaction between p27^Kip1 expression and treatment was statistically significant for RFS (P = .04) but not for OS (P = .27).

Conclusion: High p27^Kip1 expression was an independent predictor of responsiveness to hormonal therapy and thus may be useful for the selection of premenopausal women with early-stage hormone receptor–positive breast cancer for adjuvant combination endocrine therapy.

	INTRODUCTION

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THE DEFINITION of accurate markers for the selection of the appropriate adjuvant therapy for patients with early-stage breast cancer would improve efficacy and avoid unnecessary toxicity and long-term side effects in patients not responsive to the selected adjuvant treatment. One of the most promising molecular markers currently being studied is the cell cycle regulator p27^Kip1.

Progression from G1 to the S phase of the cell cycle is regulated by the formation of cyclin/cyclin-dependent kinase (CDK) complexes.¹ CDK activity is inhibited by CDK inhibitory proteins, including the Cip/Kip family members p21^Waf1/Cip1, p27^Kip1, and p57^Kip2.^2,3 These proteins interact with complexes containing cyclin D, E, and A,^4–6 and recent data suggest they exert both positive and negative regulation of CDK activity at G1/S transition.^7–9 Various functions have been attributed to p27^Kip1, including promotion of apoptosis^10,11 and regulation of drug resistance.¹² In addition, decreased expression of p27^Kip1 is associated with poor clinical outcome in a variety of malignant diseases. Various groups have studied p27^Kip1 expression in primary breast cancer. Whereas p27^Kip1 protein reduction was a strong independent prognostic factor for disease-free survival (DFS) and overall survival (OS)^13–18 in most studies, others did not confirm these findings.^19,20

Preclinical data suggest that p27^Kip1 is an essential mediator of cell cycle arrest by tamoxifen and other antiestrogenic drugs. Results of a recent study suggest that, in addition to the estrogen receptor, a breast cancer cell must express functional p27^Kip1 for tamoxifen to mediate its cytostatic effects.²¹ This observation raises the hypothesis that deregulation and loss of p27^Kip1 may contribute to both hormone independence and tamoxifen resistance in breast cancer.

The present study was designed to determine whether p27^Kip1 could be used as a marker to identify a subgroup of patients more likely to benefit from adjuvant combination endocrine therapy likely than others. For this study, we have chosen patients enrolled onto the Austrian Breast and Colorectal Cancer Study Group (ABCSG) Trial 5, a prospective randomized trial comparing the efficacy of a combination endocrine treatment with cyclophosphamide, methotrexate, and fluorouracil (CMF) chemotherapy.

	PATIENTS AND METHODS

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ABCSG Trial 5
The objective of ABCSG Trial 5 was to compare the efficacy of a combination endocrine treatment with standard CMF chemotherapy.²² From December 1990 to October 1999, a total of 1,099 patients were entered, of whom 1,034 patients were assessable for the final analysis. Patients were stratified by tumor size, number of involved lymph nodes, type of curative surgery, tumor grade, and hormone receptor status. Patients with hormone receptor–positive breast cancer were randomly assigned to receive either five years of tamoxifen (Nolvadex; AstraZeneca Pharmaceuticals, Wilmington, DE) plus three years of goserelin (Zoladex; AstraZeneca Pharmaceuticals) or six cycles of CMF. None of the trial participants received tamoxifen after CMF treatment. The results of this study suggest that combination endocrine therapy is more effective than CMF in the adjuvant treatment of premenopausal patients with stage I or II breast cancer. The results of ABCSG Trial 5 are reported elsewhere.²²

All patients registered onto ABCSG Trial 5 were eligible for entry to the laboratory study, and the major participating centers were requested to provide tumor blocks of their patients.

Treatment Regimens
CMF was administered intravenously for six cycles, days 1 and 8, recycled on day 28, at the following doses: cyclophosphamide 600 mg/m², methotrexate 40 mg/m², and fluorouracil 600 mg/m². Goserelin was given subcutaneously at 3.6 mg per injection every 28 days for 3 years (39 injections). Tamoxifen was administered at 20 mg orally once a day for 5 years.

Immunohistochemistry
All tumor specimens were obtained at the time of surgery before the adjuvant therapy. Formalin-fixed, paraffin-embedded blocks from the primary breast lesions were used for p27^Kip1 immunostaining. A hematoxylin and eosin–stained slide was prepared from each block and used for pathologic confirmation of the presence of invasive breast cancer. All slides were reviewed by a pathologist (M.R.) who was blinded to clinical outcome. Immunohistochemical analysis reported in this study was carried out in a single laboratory (Clinical Division of Oncology, Department of Medicine I).

Tissue sections of 4 µm thickness were prepared, mounted on poly-l-lysine-coated slides, deparaffinized, and rehydrated with distilled water. Endogenous peroxidase activity was blocked by incubation in 0.06% hydrogen peroxide for 10 minutes at room temperature. After boiling for 10 minutes in 10 mmol/L citrate buffer (pH 6.0) for antigen retrieval, the tissues were preincubated for 20 minutes in normal serum (1:50; DakoCytomation, Glostrup, Denmark) before a 60-minute incubation with the anti p27^Kip1 monoclonal antibody (clone 57; antibody used at 1.25 µg/mL; Transduction Laboratories, Lexington, KY). Antibody binding was detected by the avidin-biotin-peroxidase method. Bound peroxidase was developed with 3,3'-diaminobenzidine (DakoCytomation). The slides were counterstained with Mayer’s hemalum and mounted with Aquatex (Merck, Darmstadt, Germany). All washes were performed in phosphate-buffered saline (pH 7.4).

Expression of p27^Kip1 in normal epithelial cells and small lymphocytes was used as internal positive control of immunostaining.¹⁴ In addition, negative controls without the primary antibody were performed as described above. Staining of tumor cells was examined independently by two observers (G.P., M.R.) without prior knowledge of the clinical outcome of the patients and the concordance of their evaluation was high. To explore the level of concordance between the two observers in greater detail, kappa statistics were used to assess interobserver reliability, and the observed kappa ratio was 0.91 (data not shown). The rare discrepant cases were reassessed together by both investigators using a double-headed microscope, and a consensus was reached. All invasive tumor cells on each slide were evaluated. Interpretation of the results was limited to the invasive portion of the tumor, and only nuclear staining was scored as positive. At least 200 tumor cells per case were evaluated and the result expressed as the percentage of p27^Kip1-labeled nuclei.

Comparisons of p27^Kip1 expression with clinical parameters and outcome were performed with p27^Kip1 expression as a dichotomized variable classified as low (nuclear p27^Kip1 staining in < 50% of tumor cells) or high (nuclear p27^Kip1 staining in 50% of tumor cells). This cutoff was based on previously published reports demonstrating the prognostic significance of p27^Kip1 expression in breast carcinomas.^14–19

Statistical Analysis
Associations of p27^Kip1 expression with age, tumor size, lymph node status, tumor grade, estrogen receptor (ER), and progesterone receptor (PgR) were assessed by the ² test. Survival probabilities were estimated with the Kaplan-Meier product limit method.²³ Survival time was defined as the period between the time of randomization and death (OS) or the period between the time of randomization and documented relapse (relapse-free survival [RFS]). Survival times of patients still alive were censored with the date of the last follow-up. Differences between survival curves were analyzed by the log-rank test. To describe the unadjusted effects of covariates on OS and RFS, univariate Cox proportional hazards regression models were used. Multiple Cox models were used to assess the independent effects of p27^Kip1 expression on OS and RFS.²⁴ All P values are results of two-sided tests. The SPSS 10.0 statistical software (SPSS Inc, Chicago, IL) was used for calculations.

	RESULTS

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Tumor blocks of 512 patients were available for p27^Kip1 immunohistochemical studies. The main clinical and laboratory parameters of these patients compared with all 1,034 patients enrolled in ABCSG Trial 5 are summarized in Table 1. Patients in each treatment group were balanced by variables listed in Table 1 and were also similar to those in the parent clinical trial. Therefore, the present study cohort was representative of the original study population enrolled in ABCSG Trial 5.

The median follow-up time of the total study population was 5.5 years, and the maximum follow-up time was 9.5 years. One hundred five patients (20.5%) relapsed (29 patients with low expression and 76 patients with high p27^Kip1 expression; P = .016), and 37 patients(7%) died as a result of cancer (14 patients with low and 23 patients with high p27^Kip1 expression; P = .003). The 5-year RFS and OS rates were 80% and 93%, respectively, for the studied population. Clinical parameters—age, tumor size, lymph node status, PgR, and p27^Kip1 expression—were significantly associated with relapse-free survival, as determined by univariate analysis (Table 3). Tumor size, lymph node status, tumor grade, and p27^Kip1 expression were significantly associated with OS as well (Table 3). Patients with high p27^Kip1 expression had significantly longer RFS and OS times than those with low p27^Kip1 expression (Fig 1A and B). The Kaplan-Meier estimate of the 5-year RFS rate was 83% in patients with high p27^Kip1 expression, compared with 65% in patients with low p27^Kip1 expression (P = .01). The 5-year OS rate was 95% for patients with high p27^Kip1 expression and 87% for those with low p27^Kip1 expression (P = .002). By multivariate Cox regression analyses, high p27^Kip1 expression was identified as an independent predictor for superior RFS (relative risk [RR], 0.53; 95% CI, 0.34 to 0.82; P = .004) and overall survival (RR, 0.29; 95% CI, 0.15 to 0.58; P < .001; Table 3).

View larger version (20K): [in this window] [in a new window]   Fig 1. Kaplan-Meier plots for (A) relapse-free survival and (B) overall survival. Survival data based on p27Kip1 expression are shown. Patients with low p27Kip1 expression had significantly shorter relapse-free survival and overall survival than patients with high p27Kip1 expression.

View larger version (23K): [in this window] [in a new window]   Fig 2. Relapse-free survival (A, B) and overall survival (C, D) for tamoxifen plus goserelin and CMF treatment arms in cohorts with low (A, C) and high (B, D) p27Kip1 expression. Relative risks of failure and P values shown on each plot are adjusted for age, tumor size, lymph node status, tumor grade, estrogen receptor, and progesterone receptor. CMF, cyclophosphamide, methotrexate, and fluorouracil chemotherapy; Tam, tamoxifen; Gos, goserelin; RR, relative risk.

	DISCUSSION

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The definition of accurate predictive factors to select the appropriate adjuvant therapy for patients with early-stage breast cancer is of immense importance. So far, the choice of adjuvant therapy is based on patients’ lymph node status and hormone receptor status. While there are many molecular markers with potential prognostic value in breast cancer, only few have been evaluated as predictors of response to specific treatments, and most of the currently available data are controversial and/or inconclusive.²⁵

In the present study, we examined p27^Kip1 expression in premenopausal, hormone receptor–positive breast cancer patients with stage I and II disease who were enrolled onto a prospective randomized trial. In this homogenous and well-defined patient population, we observed that low p27^Kip1 expression is an independent prognostic factor for poor RFS and OS, which is consistent with previous reports.^13–18 More importantly, however, we found an interaction between p27^Kip1 expression and a specific therapeutic regimen. Patients with high p27^Kip1 expression who were treated with combination endocrine therapy experienced a 48% relative reduction in relapse rate and a 49% relative decrease in mortality compared with those patients who received CMF. These differences translated to a 10% absolute improvement in RFS and a 4% absolute improvement in OS at 5 years. In contrast, patients with low p27^Kip1 expression experienced unfavorable outcome regardless of adjuvant combination endocrine therapy or CMF chemotherapy. These differences suggest that combination endocrine therapy may be more active in patients with high p27^Kip1 expression and that additional or other treatment strategies need to be developed for breast cancer patients with low p27^Kip1 expression.

Analyses of p27^Kip1 expression must be performed at the protein level because mutations in the human p27^Kip1 gene are rare,^26–28 in contrast to other cell cycle regulators (such as p16 or p53), and loss of p27^Kip1 expression is mainly due to increased proteolysis by the ubiquitin-proteasome pathway²⁹ and not to altered transcription or mRNA stability.³⁰ The immunohistochemical assay used in the present study can reliably be performed on formalin-fixed, paraffin-embedded tumor specimens and is a simple and appropriate detection method that has been widely used to assess p27^Kip1 expression in various malignant diseases, including breast cancer. Moreover, antibodies from different sources and comparison of immunohistochemistry results with Western blot gave similar results.¹⁴ In the majority of reports, p27^Kip1 levels have been classified as low (nuclear p27^Kip1 staining in <50% tumor cells) or high (nuclear p27^Kip1 staining in 50% tumor cells).^13–19 Therefore, we selected this cutoff in the present study. Nevertheless, we obtained comparable results when p27^Kip1 expression was analyzed as a continuous variable (data not shown). However, there is clearly a need to standardize the p27^Kip1 detection assays and scoring systems to ensure that determination of p27^Kip1 levels is comparable between laboratories before p27^Kip1 can become part of the routine processing of pathologic tumor specimens and used as a new predictive marker for specific treatments.

The treatment protocols of the present study represent reasonable treatment options with regard to the management of premenopausal women with early-stage hormone receptor–positive breast cancer. Various randomized trials have shown that ovarian ablation with or without tamoxifen and standard chemotherapy regimens like CMF have similar benefits for premenopausal women with early-stage receptor-positive breast cancer.³¹ Thus, both the panelists of the 2001 Consensus Meeting in St Gallen and the National Institutes of Health Consensus Development Panel have suggested that ovarian ablation is a reasonable adjuvant treatment option for those patients.^32,33 Moreover, the panelists at St. Gallen concluded that combined endocrine therapy may be regarded as a proper treatment option for premenopausal women with endocrine-responsive disease.³² Our present results may help to identify more precisely those patients who would benefit most from combined endocrine therapy.

In conclusion, our results suggest that p27^Kip1 may be a useful marker for the selection of patients for adjuvant combination endocrine therapy, but this requires further confirmation by prospective studies before clinical implementation.

	APPENDIX

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Members of the Austrian Breast & Colorectal Cancer Study Group who participated in Trial 5 were: M. Gnant, D. Kandioler, M. Schmidinger, G. Steger, S. Taucher (Departments of Surgery and Internal Medicine, Vienna University, Vienna); P. Mayer, C. Menzel, B. Mlineritsch, C. Rass, R. Reitsamer, G. Russ (Third Medical Department and Department of Special Gynecology, Salzburg Hospital, Salzburg); T. Bauernhofer, H.-J. Mischinger, M. Schmid, M. Smola, P. Steindorfer, H. Stöger (Departments of Internal Medicine and Surgery, Graz University, and Second Department of Surgery, Graz Hospital, Graz); E. Asseryanis, A. Galid, R. Möslinger-Gehmayr, M. Seifert (Division of Special Gynecology, Vienna University, Vienna); D. Depisch, K. Haider, W. Kwasny, A. Lenauer, T. Payrits (Department of Surgery, Wiener Neustadt Hospital, Wiener Neustadt); M. Fridrik, R. Greul, G. Hochreiner, G. Wahl (First Medical Department, Linz Hospital, Linz); G. Jatzko, V. Wette (Department of Surgery, Sankt Veit Hospital, Sankt Veit); H. Spoula, M. Stierer (Department of Surgery, Hanusch Hospital, Vienna); U. Schmidbauer, M. Wunderlich (Department of Surgery, BHS Hospital, Vienna); F. Hofbauer, M. Lang (Department of Surgery, Oberpullendorf Hospital, Oberpullendorf); P. Kier, K. Renner (Second Medical Department and Department of Surgery, SMZ Ost Hospital, Vienna); R. Kocher, F. Stangl (Department of Surgery, Leoben Hospital, Leoben); G. Luschin-Ebengreuth, R. Winter (Department of Gynecology, Graz University, Graz); W. Schennach, H. Zoller (Department of Surgery, Zams Hospital, Zams); F. Kugler, C. Tausch (Department of Surgery, BHS Hospital, Linz); E. Hanzal, C. Sam (Division of Gynecology and Obstetrics, Vienna University, Vienna); A. Haid, R. Köberle-Wührer (Department of Surgery, Feldkirch Hospital, Feldkirch); W. Döller, E. Melbinger (Department of Surgery, Wolfsberg Hospital, Wolfsberg); J. Berger, R. Lenzhofer (Medical Department, Schwarzach Hospital, Schwarzach); H. Ludwig, P. Sagaster (First Medical Department, Wilhelminenspital, Vienna); G. Reiner, D. Semmler (Department of Surgery, Mistelbach Hospital, Mistelbach); J. Omann (Department of Surgery, Klagenfurt Hospital, Klagenfurt); W. Neunteufel (Department of Gynecology, Dornbirn Hospital, Dornbirn).

	AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

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The authors indicated no potential conflicts of interest.

	NOTES

 
Supported by the Austrian Science Fund (Hertha-Firnberg Fellowship T71-MED and a Grant Project P15377).

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Submitted February 6, 2003; accepted July 9, 2003.

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