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Altered Expression of FAS System Is Related to Adverse Clinical Outcome in Stage I-II Breast Cancer Patients Treated with Adjuvant Anthracycline-Based Chemotherapy

¹ Regina Elena Cancer Institute, Rome, Italy, and² University of Camerino, Camerino, Italy

	ABSTRACT

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Purpose: To determine the prognostic value of Fas receptor and Fas ligand (FasL) as apoptosis-related biomarkers in the context of chemoresponsiveness in breast cancer (BC) patients submitted to anthracycline-based adjuvant therapy.

Experimental Design: Fas and FasL were investigated by immunohistochemistry in surgical samples collected from 167 stage I-IIa-b BC patients enrolled in a prospective clinical trial using epirubicin plus cyclophosphamide in the adjuvant setting.

Results: Fas and FasL were significantly associated with tumor stage (P < 0.0001). Multivariate analysis indicated that stage, loss of Fas (relative risk, 8.5 and 9.12; P < 0.0001) and FasL up-regulation (relative risk, 2.38 and 2.88; P = 0.01) were independent prognostic variables influencing both disease-free survival (DFS) and overall survival (OS). A Cox analysis using a four-category Fas/FasL phenotype (+/-, +/+, -/+, -/-) as a stratification factor evidenced a highly positive association between Fas/FasL phenotype and the cumulative hazard of relapse and death in the entire series of patients. We also estimated the DFS and OS for different combinations of the pathological-tumor-node-metastasis (TNM) stage and Fas/FasL by using the K sample log-rank exact test demonstrating that significantly shorter DFS and OS were observed in Fas-negative and FasL-positive patients in both stage I-IIa and IIb.

Conclusions: Data presented herein demonstrated that, according to a number of in vitro studies, the prognosis for BC patients receiving adjuvant anthracycline-based chemotherapy strongly depends on the Fas/FasL status. Therefore, a concomitant altered pattern of Fas/FasL expression seems to configure an aggressive tumor phenotype linked to disease progression.

	INTRODUCTION

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Although the risk of mortality for breast carcinoma (BC) has been declining in the Western world over the last decade (1) , given the systemic nature of the disease at the time of diagnosis, an aggressive adjuvant chemotherapy (CT) is often required for stage I-II patients also (2) . Anthracyclines (doxorubicin or the 4-epimer epirubicin), considered the most effective drugs in advanced carcinomas, have been now introduced in the adjuvant setting and clinical data indicated that the inclusion of anthracyclines in adjuvant CT regimens produces a small but statistically significant improvement in survival over non-anthracycline-containing regimens (2) . Unfortunately most patients will develop an acquired resistance during treatment and die of progressive disease (3) . For this reason several studies have been devoted to the development of preclinical models aimed at understanding the molecular mechanisms producing treatment-resistant tumors. It is now well known that chemotherapeutic agents, irrespective of their intracellular target, act primarily through induction of apoptosis in susceptible cancer cells (4) . Thus, the efficacy of anticancer treatments does not depend on the DNA damage or other damage they cause, but is also related to the cellular capability to detect and respond to such damage (5) . However, the precise molecular requirements that characterize the drug-induced cell death in tumor cells are not completely understood (4 , 6) . Mitochondrial and cell-surface death receptor-mediated apoptosis are the two principal pathways leading to programmed cell death (7) . The mitochondrial pathway is thought to play a major role in the response to cancer treatments and is mediated by the bcl-2 protein family. In addition there is mounting evidence that cell surface receptor molecules of the tumor necrosis factor/nerve growth factor receptor/ligand family, such as Fas (CD95/APO-1) and Fas ligand (FasL; CD95L) molecules, studied mostly within the immune system (8) , play a pivotal role also in drug-induced apoptosis of both leukemia and solid tumor cells (9, 10, 11) . Recent in vitro studies demonstrated that apoptosis in response to different cytotoxic drugs is mostly triggered, through an autocrine/paracrine mechanism, by an intact Fas system activation pathway resulting in a correct cleavage and activation of effector caspases such as caspase-3 (12) . Therefore, interference with the FasL/receptor interaction by antagonist antibodies to the receptor or deficient activation of the Fas system may significantly reduce sensitivity to drug-mediated apoptosis (9) . These findings may suggest that, also in a clinical setting, an intact Fas-FasL apoptotic pathway system might play a pivotal role in determining sensitivity or resistance of tumor cells to anthracyclines. We have recently shown that BC is frequently associated with the down-regulation of Fas and the up-regulation of FasL (13) , demonstrating that a Fas-negative/FasL-positive phenotype, is significantly related to BC progression, strongly influencing patient outcome. Nevertheless in BC patients, no information is available on the relationship between the Fas/FasL system and response to anthracycline-based adjuvant therapy.

The aim of our study was to determine the clinical significance of Fas and FasL as apoptosis-related biomarkers in the context of chemoresponsiveness. To this end, we examined, by immunohistochemical (IHC) methods, the Fas and FasL expression in a series of 167 stage I-II BC patients enrolled in a prospective clinical trial using the epirubicin plus cyclophosphamide combination in the adjuvant setting.

	MATERIALS AND METHODS

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Patients.
The 167 stage I, IIa, and IIb BC patients included in this study were treated at the Regina Elena Cancer Institute between 1991 and 1993. They were drawn from a larger trial of adjuvant anthracycline-containing combination CT with high-dose epirubicin plus cyclophosphamide (14) . This series included 138 invasive ductal carcinomas and 29 invasive lobular carcinomas. Among these, 53 were stage I, 73 stage IIa, and 41 stage IIb. Tumors were staged according to the Unione Internationale Contre le Cancer tumor-node-metastasis (TNM) system criteria and histologically classified according to the WHO. According to the St. Gallen Consensus Conference, premenopausal women with invasive BC larger than 1 cm, grading 2–3, independent of nodal and hormonal receptors status, and postmenopausal patients with the same characteristics, but with negative estrogen and progesterone receptors were submitted every 3 weeks for four cycles to the following treatment: epirubicin (Farmorubicina; Pharmacia, Milan, Italy) 120 mg per m² of body surface area and cyclophosphamide (Endoxan; Asta Medica, Milan, Italy) 600 mg i.v. on day 1. Eligibility requirements for enrollment in the study included modified radical mastectomy or breast- conserving surgery (quadrantectomy) within 6 weeks before the start of CT; no prior CT or radiotherapy; age <65 years; adequate bone marrow, liver and renal functions; a resting left ventricular fraction (LVEF) >45%; no other malignancies; and informed consent. For patients treated with quadrantectomy, a standardized irradiation protocol was administered after the end of CT.

No patients received hormonal maintenance therapy (14) . Follow-up data were obtained from hospital charts and by corresponding with the referring physicians.

Antibodies and IHC.
Immunoreactivity for Fas protein was detected by using affinity-purified polyclonal antibody (Pab) C-20 (Santa Cruz Biotechnology, Santa Cruz, CA) raised against a peptide corresponding to amino acids 316–335, mapping at the COOH terminus of the Fas of human origin. FasL expression was evaluated by using FasL N-20 affinity-purified Pab (Santa Cruz Biotechnology), which recognizes epitopes corresponding to the amino acids 2–19 mapping at the amino terminus of the human FasL. Both Fas C-20 and FasL N-20 antibodies have been used for IHC detection of corresponding antigens in a variety of human tumors (15) . To confirm the results obtained with the Pabs, BC tissues were also stained with anti-Fas and anti-FasL monoclonal antibodies (Mabs clone GM30 and clone 5D1, respectively, Novocastra Laboratories, Newcastle, United Kingdom). A strong significant correlation between the two reagents has been found (anti-Fas Pab C-20 versus Mab GM30, P < 0.003; anti-FasL Pab N-20 versus Mab 5D1, P < 0.001); and the statistical analysis of clinical outcome, independently of the reagents used, displayed overlapping results.

As reported previously (13) , the antibody specificity was further confirmed by immunoprecipitation of whole-tissue extracts from breast tumor samples (six patients) performed either with C-20 or N-20 Pabs. Immunoprecipitated Fas and FasL proteins were identified in Western blots by using different specific anti-Fas (clone UB2; Immunotech, Hamburg, Germany) or anti-FasL (clone G247; PharMingen, San Diego, CA) Mabs. Western blot results demonstrated that Fas and FasL proteins, precipitated, respectively, by the Pabs C-20 and N-20, were specifically recognized by the anti-Fas Mab UB2 and the anti-FasL Mab G247.

IHC staining was carried out on 5-µm-thick sections on silane (APES; Sigma, St. Louis, MO)-treated slides from routinely fixed paraffin-embedded blocks. The deparafinized and rehydrated sections were pretreated twice in a microwave oven at 750 W for 5 min in citrate buffer and were incubated for 60 min at room temperature with primary Pabs (2 µg/ml) and Mabs (1:40 dilution). The reaction was visualized using a streptavidin-biotin immunoperoxidase system (LSAB 2 kit; Dakocytomation, Milan, Italy) and 3-amino-9-ethyl-carbazole solution (Dakocytomation) as chromogenic substrate. Sections were then slightly counterstained with Mayer hematoxylin and were mounted in aqueous mounting medium (Glycergel; Dakocytomation). Benign and malignant tumors displayed two kinds of immunoreactivity: (a) a well-detectable cell membrane staining prevalently accompanied by a granular cytoplasmic reactivity; (b) a diffuse cytoplasmic staining varying from moderate to strong. Cases with faint, uncertain cytoplasmic staining were regarded as negative. For the purpose of our study, Fas or FasL immunostaining was scored, independent of membrane or cytoplasmic staining localization, as follows: negative, no expression on tumor cells; heterogeneous expression, 10–50% positive tumor cells; homogeneous expression, >50% positive tumor cells. Normal breast tissues or benign lesions adjacent to invasive carcinomas were also carefully examined. Sections without primary antibodies or incubated with antibodies adsorbed with the immunizing peptides at a concentration of 20 µg/ml (FasL-amino-terminal, amino acids 2–19, and Fas-COOH-terminal, amino acids 316–335; Santa Cruz Biotechnology) served as negative controls. All of the immunostained slides were analyzed and scored independently by two investigators (M. M. and S. B.).

Estrogen and progesterone receptors were assayed immunohistochemically on formalin-fixed paraffin-embedded tumors with the use of commercially available antibodies (ER1D5 and 1A6; Immunotech, UCS, Rome, Italy). Tumors were defined positive for estrogen and progesterone receptors when 10% or more of the tumor cells showed unequivocal nuclear staining.

HER-2/neu protein expression was evaluated using both the mouse Mab 300G9 provided by the Immunology Laboratory of the Regina Elena Cancer Institute, Rome, Italy (16) and the Pab A0485 purchased from Dakocytomation. HER-2/neu was considered overexpressed only when at least 10% of the neoplastic cells displayed a distinct plasma membrane staining (++/+++ score).

Statistical Analysis.
Association between clinical and biopathological variables was evaluated using the ² test. All of these parameters were treated as dichotomous or categorical variables and were described using the Pearson statistic. The disease-free survival (DFS) and overall survival (OS) curves were estimated by the Kaplan-Meier product-limit method; the log-rank test was used to assess differences between subgroups. Significance was defined at the P < 0.05 level. The relative risk and the confidence limits were estimated for each variable by using the Cox univariate model and by adopting the most suitable prognostic category as referent group. A multivariate Cox proportional hazard model was also developed using stepwise regression (backward selection) with predictive variables that were significant in the univariate analyses. Enter limit and remove limit were P = 0.10 and P = 0.15, respectively. All of the analyses were conducted using the BMDP software package (Chicago, IL).

Coexpression of Fas and FasL was also analyzed by a log-rank test. Because of the relatively small number of patients, it was deemed convenient to perform the statistical analyses by exact methods using the StatXact4 for Windows software package (Cytel Corporation, Cambridge, MA).

	RESULTS

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Relationship among Biological and Clinicopathological Parameters.
To confirm that receptor and ligand Fas expression were inversely correlated in BC, as we have recently reported (13) , we analyzed the relationship between Fas and FasL in 167 stage-I/IIa-b patients included in this study. As summarized in Table 1 , the expression of receptor and ligand Fas antigens appeared to be significantly inversely related (P < 0.0001) with 69.0% of Fas- cases expressing a FasL+ phenotype and 70.8% of Fas+ tumors displaying a FasL- phenotype. Double-negative (Fas-/FasL-) and double-positive (Fas+/FasL+) phenotypes accounted for 31.0% and 29.2% of the samples, respectively.

The results of the univariate and multivariate analyses for DFS and OS in the 167 patients included in the study are summarized in Tables 3 and 4 .Univariate analyses (Cox model) identified nuclear grade, tumor stage, lack of Fas expression, and FasL positivity as significant predictors of DFS and OS (Table 3) . All of the variables that significantly affected survival in the univariate analysis were introduced into Cox proportional risk model. As summarized in Table 4 , a backward step procedure indicated that stage IIb, lack of Fas staining, and FasL up-regulation, also adjusted for stage, were independent prognostic variables influencing both DFS and OS. Of interest, the relative risk of relapse and death associated with loss of the Fas receptor was particularly high, namely 8.5 (95% confidence interval, 4.40–17.9; P < 0.0001) for DFS and 9.12 (95% confidence interval, 3.49–23.82; P < 0.0001) for OS.

View larger version (24K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Multivariate Cox analysis stratified according to Fas receptor and Fas ligand (FasL) status. A, cumulative hazard of relapse; B, cumulative hazard of death.

	DISCUSSION

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Experimental evidence suggests that the deregulation of the mechanisms involved in the programmed cell death plays a relevant role in the progression of disease and the resistance to anticancer drugs (18) .

The clinical impact of apoptosis-related molecules such as p53 and bcl-2 has been extensively evaluated in the adjuvant setting of BC. However, conflicting results have been published concerning their involvement in resistance to cytotoxic drugs (19 , 20) .

The Fas/FasL death pathway is another important mediator of apoptosis playing a pivotal role in the regulation of normal biological processes of breast glandular tissue modulating interaction between epithelial and nonepithelial cell populations (21) .

The new data presented herein demonstrate that the prognosis for patients with BC receiving adjuvant anthracycline-based CT strongly depends on the Fas/FasL status. In particular, concomitant down-regulation of Fas and up-regulation of FasL expression seems to configure an aggressive tumor phenotype the biological behavior of which can be poorly modified by cytotoxic drugs.

Our findings are strongly supported by a number of in vitro studies that demonstrated that Adriamycin resistance in BC cell lines may be associated with a lack of Fas antigen expression and subsequent resistance to Fas-mediated cytotoxicity (22) .

In particular, it has been reported that the chemosensitivity of epithelial tumor cell lines to doxorubicin is strictly related to the activation of the Fas system. In fact, on doxorubicin treatment, strong induction of FasL and caspase activity were found in responsive, but not in unresponsive, tumor cells. Of interest, a down-regulation of Fas expression, together with a blockade of caspases activation, was found in resistant tumor cell lines (9 , 12) . However, to date, there are only limited and conflicting clinical data to support any clear connection between the Fas system alteration and the response to a specific adjuvant cytotoxic therapy (i.e., anthracyclines).

In a recent study, Sjostrom et al. (23) analyzed, by means of an IHC method, the predictive value of Fas and FasL expression together with other biological factors (bcl-2, bax, bag-1) for CT response in 126 advanced BCs. The authors showed that neither Fas nor FasL predicted a response to CT in advanced BC, although FasL appeared the most significant predictor of survival. Similar results were observed by Pernick et al. (24) in 34 patients with locally advanced BC who received induction CT. Response to neo-adjuvant treatment was only marginally correlated with Fas and FasL status, but the recurrence rate was significantly correlated with Fas immunoreactivity. Reimer et al. (25) established that the FasL:Fas ratio in tumor tissue is a strong prognostic factor in BC, and, more recently, an extended follow-up of that cohort of patients allowed them to investigate whether the FasL:Fas ratio may predict response to cytotoxic or hormonal therapies (26) . The data reported indicated that the FasL:Fas ratio may be useful not only as a prognostic factor but also as a predictive factor for projecting response to the anti-estrogen tamoxifen. However, the FasL:Fas ratio had no significant predictive value in patients exclusively treated with CT. The lack of the predictive value of the FasL/Fas phenotype in these studies, apparently in contrast to our data, may be due to various reasons: different settings of patients; limited number of patients submitted to adjuvant CT (52 versus 167 in our study); different methods in evaluating Fas and FasL (IHC versus reverse transcription-PCR) with the possibility of analyzing residual normal glands and/or lymphocyte; and, last, the heterogeneity of the CT regimens.

Although functional data are lacking in this study, tumors displaying a Fas+/FasL- phenotype do seem to maintain an intact apoptotic pathway, thus becoming responsive to CT. On the contrary, BCs lacking Fas with FasL up-regulation experienced a particularly poor outcome. These findings can be related even to the role of FasL as a key effector molecule of T-cell-mediated cytotoxicity against neoplastic cells (27) . In this context, our group recently reported (28) that Fas+/FasL- BC with tumor-infiltrating lymphocytes lacking Fas expression, presented a higher apoptotic index and a more favorable outcome than patients bearing Fas-/FasL+ BC and Fas+ tumor-infiltrating lymphocytes.

These results, along with the recent observation that delivering a higher dosage of combination CT or adding newer compounds (e.g., taxanes) in the adjuvant setting failed to significantly improve BC outcome (29) , strongly support the belief that future therapeutic strategies should be directed at restoring the integrity of the apoptotic machinery.

In this context, our results are particularly relevant because the use of certain biological response modifiers, such as IFN-, may induce restoration of Fas-mediated apoptosis in cells lacking Fas expression (30 , 31) .

To the best of our knowledge, this is the first study that has investigated the prognostic value of Fas system in a cohort of BC patients homogeneously treated with anthracycline-based CT. Nevertheless, because of the relatively small number of patients, our results should be regarded as preliminary. However, the prospective nature of patient enrollment and the homogeneous treatment assignment make our study in line with Hayes’ recommendations (32) .

Hopefully the predictive value of Fas/FasL status will be confirmed in a large prospective study of adjuvant CT.

	ACKNOWLEDGMENTS

 
We thank Maria Assunta Fonsi for secretarial assistance and Paula Franke for the formal revision of the manuscript.

	FOOTNOTES

 
Grant support: Supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC), Ministero della Salute (to M. M. and to C. B.), Lega Italiana per la Lotta contro i Tumori.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Marcella Mottolese, Regina Elena Cancer Institute, Pathology Department, via Elio Chianesi 53, 00144, Rome, Italy. Phone: 39-6-52666139; Fax: 39-6-52666102; E-mail: mottolese{at}ifo.it

Received 7/15/03; revised 11/ 3/03; accepted 11/12/03.

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