Altered Expression of FAS System Is Related to Adverse Clinical
Outcome in Stage I-II Breast Cancer Patients Treated with
Adjuvant Anthracycline-Based Chemotherapy
Claudio Botti,1Simonetta Buglioni,1
Maria Benevolo,1Diana Giannarelli,1
Paola Papaldo,1Francesco Cognetti,1
Patrizia Vici,1Franco Di Filippo,1
Franca Del Nonno,1Franco Maria Venanzi,2
Pier Giorgio Natali,1and Marcella Mottolese1
1Regina Elena Cancer Institute, Rome, Italy, and2University of
Camerino, Camerino, Italy
Purpose: To determine the prognostic value of Fas re-
ceptor and Fas ligand (FasL) as apoptosis-related biomark-
ers in the context of chemoresponsiveness in breast cancer
(BC) patients submitted to anthracycline-based adjuvant
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 clin-
ical trial using epirubicin plus cyclophosphamide in the
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 chemo-
therapy 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 dis-
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 anthra-
cyclines in adjuvant CT regimens produces a small but statisti-
cally significant improvement in survival over non-anthracy-
cline-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 charac-
terize the drug-induced cell death in tumor cells are not com-
pletely understood (4, 6). Mitochondrial and cell-surface death
receptor-mediated apoptosis are the two principal pathways
leading to programmed cell death (7). The mitochondrial path-
way 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 (9–11). Recent
in vitro studies demonstrated that apoptosis in response to
different cytotoxic drugs is mostly triggered, through an auto-
crine/paracrine mechanism, by an intact Fas system activation
pathway resulting in a correct cleavage and activation of effec-
tor 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 signifi-
cantly reduce sensitivity to drug-mediated apoptosis (9). These
Received 7/15/03; revised 11/3/03; accepted 11/12/03.
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 In-
stitute, Pathology Department, via Elio Chianesi 53, 00144, Rome, Italy.
Phone: 39-6-52666139; Fax: 39-6-52666102; E-mail: email@example.com
1360 Vol. 10, 1360–1365, February 15, 2004
Clinical Cancer Research
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 anthracy-
clines. 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 pheno-
type, is significantly related to BC progression, strongly influ-
encing patient outcome. Nevertheless in BC patients, no infor-
mation 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 signif-
icance 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 expres-
sion in a series of 167 stage I-II BC patients enrolled in a
prospective clinical trial using the epirubicin plus cyclophos-
phamide combination in the adjuvant setting.
MATERIALS AND METHODS
The 167 stage I, IIa, and IIb BC patients in-
cluded 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 sta-
tus, 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 treat-
ment: epirubicin (Farmorubicina; Pharmacia, Milan, Italy) 120
mg per m2of body surface area and cyclophosphamide (En-
doxan; Asta Medica, Milan, Italy) 600 mg i.v. on day 1. Eligi-
bility requirements for enrollment in the study included modi-
fied radicalmastectomy or
(quadrantectomy) within 6 weeks before the start of CT; no
prior CT or radiotherapy; age ?65 years; adequate bone mar-
row, liver and renal functions; a resting left ventricular fraction
(LVEF) ?45%; no other malignancies; and informed consent.
For patients treated with quadrantectomy, a standardized irradi-
ation 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
breast- conserving surgery
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, Novo-
castra Laboratories, Newcastle, United Kingdom). A strong signif-
icant 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 overlap-
As reported previously (13), the antibody specificity was
further confirmed by immunoprecipitation of whole-tissue ex-
tracts 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, Ger-
many) 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-?m-thick sections on
silane (APES; Sigma, St. Louis, MO)-treated slides from rou-
tinely 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 ?g/ml) and Mabs
(1:40 dilution). The reaction was visualized using a streptavidin-
biotin immunoperoxidase system (LSAB 2 kit; Dakocytoma-
tion, Milan, Italy) and 3-amino-9-ethyl-carbazole solution (Da-
kocytomation) as chromogenic substrate. Sections were then
slightly counterstained with Mayer hematoxylin and were
mounted in aqueous mounting medium (Glycergel; Dakocyto-
mation). 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 cy-
toplasmic staining localization, as follows: negative, no expres-
sion on tumor cells; heterogeneous expression, 10–50% positive
tumor cells; homogeneous expression, ?50% positive tumor
cells. Normal breast tissues or benign lesions adjacent to inva-
sive carcinomas were also carefully examined. Sections without
primary antibodies or incubated with antibodies adsorbed with
the immunizing peptides at a concentration of 20 ?g/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 ana-
lyzed and scored independently by two investigators (M. M.
and S. B.).
Estrogen and progesterone receptors were assayed immu-
nohistochemically 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
Clinical Cancer Research
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 neo-
plastic cells displayed a distinct plasma membrane staining
Association between clinical and
biopathological variables was evaluated using the ?2test. 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 step-
wise 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
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 pack-
age (Cytel Corporation, Cambridge, MA).
Relationship among Biological and Clinicopathological
To confirm that receptor and ligand Fas expres-
sion 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? pheno-
type 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
Table 2 summarizes the correlation between the Fas/FasL
immunoreactivity and the bio-pathological parameters. Both
receptor and ligand were significantly related to tumor stage, the
majority (73.5%) of stage I patients being Fas positive (P ?
0.01) and the majority (61%) of stage IIb patients FasL positive
(P ? 0.04). Furthermore, FasL was associated to HER-2/neu
overexpression (P ? 0.03). In contrast no correlation was found
between Fas/FasL expression and menopausal status, nuclear
grade or hormonal receptor status.
Impact of Biological Parameters on DFS and OS.
median length of follow-up of surviving patients was 69 months
(range, 16–97 months). The DFS and OS percentages were 72%
(SE, ?0.060) and 78% (SE, ?0.036), respectively. Thirty-six
deaths, due to cancer-related causes, and 46 recurrences, 3 of
which were locoregional recurrences, 40 distant metastases, and
3 contralateral BC had occurred.
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 proce-
dure indicated that stage IIb, lack of Fas staining, and FasL
up-regulation, also adjusted for stage, were independent prog-
nostic 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.
To obtain a better clinical understanding of the predictive
Relationship between Fas and Fas ligand (FasL) expression
in 167 breast cancer patients treated with anthracyclinea
Total (%) Negative (%) Positive (%)
Total90 (53.9) 77 (46.1)167 (100)
aCorrelation between Fas and FasL tumor expression, P ? 0.0001.
Relationship between Fas and Fas ligand (FasL) expression
and biopathological variables
Frequency of immunoreactive cases
bER, estrogen receptor; PgR, progesterone receptor.
1362 Fas/FasL in Anthracycline-Treated BC
value of response to adjuvant epirubicin-plus-cyclophospha-
mide treatment of these apoptosis-related molecules, we per-
formed a Cox analysis using a four-category Fas/FasL pheno-
type (?/?,?/?, ?/?, ?/?) as a stratification factor both in the
stage I-IIa and in the stage IIb groups of patients. As evidenced
in Fig. 1, a highly positive association between Fas/FasL phe-
notype and the cumulative hazard of relapse (Fig. 1A) as well as
death (Fig. 1B), was observed.
On the basis of these results, we also estimated the DFS
and the 5-year survival rate for different combinations of the
p-TNM stage and Fas/FasL covariates. Because of the relatively
small number of events recorded in some of these subgroups, we
estimated the statistical significance of our data by using the K
sample log-rank exact test. As summarized in Table 5, 95.6% of
stage I-IIa patients bearing Fas?/FasL? tumors were disease
free within 5 years from the end of CT against 53.7% of patients
with Fas?/FasL? tumors (P ? 0.0001). In addition, 5 years OS
evidenced that 98.3% of Fas?/FasL? patients were alive in
comparison with 73.1% of Fas?/FasL? patients (P ? 0.0013).
In stage IIb, 77.8% of patients with Fas?/FasL? tumors were
disease free, whereas all of the Fas?/FasL? patients relapsed
(P ? 0.0005). With regard to OS, none of the patients died when
BC displayed a Fas?/FasL? phenotype; in contrast, only
14.9% of women with a Fas?/FasL? tumor phenotype were
alive (P ? 0.0001) within 5 years.
Despite aggressive adjuvant treatment, the appearance of
metastatic spread is frequently seen during the clinical course of
patients with BC. For this reason, knowledge of the processes
underlying the inherent or acquired drug resistance of the tumor
is a priority for modern biomedical research (17).
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
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 inter-
action between epithelial and nonepithelial cell populations
The new data presented herein demonstrate that the prog-
nosis 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
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 treat-
ment, 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,
Univariate analysis of negative prognostic factors for survival
RR (95% CI)
RR (95% CI)
Menopausal status (post vs. pre)
Histotype (ductal vs. lobular)
Nuclear grade (3 vs. 2)
Stage (IIb vs. I–IIa)
ER status (positive vs. negative)
PgR status (positive vs. negative)
HER-2/neu (positive vs. negative)
FAS (negative vs. positive)
FAS ligand (positive vs. negative)
aDFS, disease-free survival; OS, overall survival; RR, relative risk; 95% CI, 95% confidence interval; ER, estrogen receptor; PgR, progesterone
Multivariate analysis of negative prognostic factors for survival
RR (95% CI)
RR (95% CI)
Stage (IIb vs. I–IIa)
FAS (negative vs. positive)
FASL (positive vs. negative)
aDFS, disease-free survival; OS, overall survival; RR, relative risk; 95% CI, 95% confidence interval; FasL, Fas ligand.
Clinical Cancer Research
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 cyto-
toxic 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 expres-
sion 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 signif-
icant 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 heter-
ogeneity 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 experi-
enced 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 con-
text, our group recently reported (28) that Fas?/FasL? BC with
tumor-infiltrating lymphocytes lacking Fas expression, pre-
sented a higher apoptotic index and a more favorable outcome
than patients bearing Fas?/FasL? BC and Fas? tumor-infil-
These results, along with the recent observation that deliv-
ering a higher dosage of combination CT or adding newer
compounds (e.g., taxanes) in the adjuvant setting failed to sig-
nificantly improve BC outcome (29), strongly support the belief
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.
Five years disease-free and overall survival according to Fas/Fas ligand status and p-TNM stage
Fas system status
No. of cases
% (95% CI)
% (95% CI)
9 77.8 (36.4–93.9)
11 68.1 (28.6–88.9)
aDFS, disease-free survival; OS, overall survival; FasL, Fas ligand; 95% CI, 95% confidence interval.
bStage I–IIa were grouped based on the results of univariate analysis.
1364 Fas/FasL in Anthracycline-Treated BC
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 lack-
ing 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. How-
ever, the prospective nature of patient enrollment and the ho-
mogeneous 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.
We thank Maria Assunta Fonsi for secretarial assistance and Paula
Franke for the formal revision of the manuscript.
1. Peto, R., Boreham, J., Clarke, M., Davies, C., and Beral, V. UK and
USA breast cancer deaths down 25% in year 2000 at ages 20–69 years.
Lancet, 355: 1822, 2000.
2. National Institutes of Health Consensus Development Panel. Na-
tional Institutes of Health Consensus Development Conference state-
ment: adjuvant therapy for breast cancer, November 1–3, 2000. J. Natl.
Cancer Inst. (Bethesda), 93: 979–989, 2001.
3. Hortobagyi, G. N., and Budzar, A. U. Current status of adjuvant
systemic therapy for primary breast cancer progress and controversy.
CA Cancer J. Clin., 45: 199–226, 1995.
4. Rowan, S., and Fisher, D. E. Mechanisms of apoptotic cell death.
Leukemia (Baltimore), 11: 457–466, 1997.
5. Reed, J. C. Dysregulation of apoptosis in cancer. Cancer J. Sci. Am.,
4 (Suppl. 11): S8–S14, 1998.
6. Debatin, K. M. Cytotoxic drugs, programmed cell death and the
immune system: defining new roles in an old play. J. Natl. Cancer Inst.
(Bethesda), 89: 750–751, 1997.
7. Kaufmann, S. H., and Earnshaw, W. C. Induction of apoptosis by
cancer chemotherapy. Exp. Cell Res., 256: 42–49, 2000.
8. Griffith, T. S., and Ferguson, T. A. The role of FasL induced
apoptosis in immune privilege. Immunology Today, 18: 2240–2244,
9. Fulda, S., Los, M., Friesen, C., and Debatin, K. M. Chemosensitivity
of solid tumor cells in vitro is related to activation of the CD95 system.
Int. J. Cancer, 76: 105–114, 1998.
10. Fulda, S., Meyer, E., Friesen, C., Susin, S. A., Kroemer, G., and
Debatin, K. M. Cell type specific involvement of death receptor and
mitochondrial pathways in drug induced apoptosis. Oncogene, 20:
11. Friesen, C., Herr, I., Krammer, P. H., and Debatin, K. M. Involve-
ment of the CD95 (APO-1/Fas) receptor/ligand system in drug induced
apoptosis in leukemia cells. Nat. Med., 2: 574–577, 1996.
12. Micheau, O., Solary, E., Hamman, A., Martin, F., and Dimanche-
Boitrel, M. T. Sensitization of cancer cells treated with cytotoxic drugs
to fas-mediated cytotoxicity. J. Natl. Cancer Inst. (Bethesda), 89: 783–
13. Mottolese, M., Buglioni, S., Bracalenti, C., Cardarelli, M. A., Cia-
bocco, L., Giannarelli, D., Botti, C., Natali, P. G., Concetti, A., and
Venanzi, F. M. Prognostic relevance of altered Fas (CD95)-system in
human breast cancer. Int. J. Cancer, 89: 127–132, 2000.
14. Papaldo, P., Lopez, M., Cortesi, E., Camilluzzi, E., Antimi, M.,
Terzoli, E., Lepidini, G., Vici, P., Barone, C., Ferretti, G., et al. Addition
of either lonidamine or granulocyte colony-stimulating factor does not
improve survival in early breast cancer patients treated with high-dose
epirubicin and cyclophosphamide. J. Clin. Oncol., 21: 3462–3468,
15. Walker, P. R., Saas, P., and Dietrich, P. Y. Tumor expression of Fas
ligand (CD95L) and the consequences. Curr. Opin. Immunol., 10:
16. Digiesi, G., Giacomini, P., Fraioli, R., Mariani, M., Nicotra, M. R.,
Segatto, O. ,and Natali, P. G. Production and characterization of murine
Mabs to the extracellular domain of human neu oncogene product
gp185HER2. Hybridoma, 11: 519–527, 1992.
17. Baum, M. The changing face of breast cancer-past, present and
future perspectives. Breast Cancer Res. Treat., 75S: S33–S35, 2002.
18. Robert, J. New concepts for the study of anticancer drug resistance
(French). Bull. Cancer, 89: 17–22, 2002.
19. Hamilton, A., and Piccart, M. The contribution of molecular mark-
ers to the prediction of response in the treatment of breast cancer: a
review of the literature on HER-2, p53 and BCL.-2. Ann. Oncol., 11:
20. Munster, P. N., and Norton, L. Predictive factor for the response to
adjuvant therapy with emphasis in breast cancer. Breast Cancer Res., 3:
21. Keane, M. M., Ettenberg, S. A., Lowrey, G. A., Russell, E. K., and
Lipkowitz, S. Fas expression and function in normal and malignant
breast cell. lines Cancer Res., 56: 4791–4798, 1996.
22. Cai, Z., Stancou, R., Korner, M., and Chouaib, S. Impairment of
Fas-antigen expression in Adriamycin-resistant but not TNF-resistant
MCF7 tumor cells Int. J. Cancer, 68: 535–546, 1996.
23. Sjostrom, J., Blomqvist, C., Von Bogulawski, K., Bengtsson, N. O.,
Mjaaland, I., Malmstrom, P., Ostenstadt, B., Wist ,E., Valvere, V.,
Takayama, S, et al. The predictive value of bcl-2, bax, bcl-xL, bag-1,
Fas and FasL for chemotherapy response in advanced breast cancer.
Clin. Cancer Res., 8: 811–816, 2002.
24. Pernick, N. L., Biernat, L., Du, W., and Visscher, D. W. Clinico-
pathologic analysis of Fas, Fas ligand, and other biomarkers in locally
advanced breast carcinoma. Breast J., 6: 233–241, 2000.
25. Reimer, T., Herrnring, C., Koczan, D., Ricgter, D., Gerber, B.,
Kabelitz, D., Friese, K., and Thiesen, H. J. FasL:Fas ratio-a prognostic
factor in breast carcinomas. Cancer Res., 60: 822–828, 2000.
26. Reimer, T., Koczan, D., Muller, H., Friese, K., Thiesen, H. J., and
Gerber, B. Tumor FasL:Fas ratio greater than 1 is an independent
marker of relative resistance to tamoxifen in hormone receptor positive
breast cancer. Breast Cancer Res., 4: R9, 2002.
27. O’Connel, J., Houston, A., Bennett, M. W., O’Sullivan, G. C., and
Shanahan, F. Immune privilege or inflammation? Insights into the Fas
ligand enigma. Nat. Med., 7: 271–274, 2001.
28. Buglioni, S., Benevolo, M., Giannarelli, D., Di Modugno, F., Cione,
A., Botti, C., Nistico `, P., Natali, P. G., and Mottolese, M. The prognostic
value of Fas/FasL system in breast cancer is related to tumor infiltrating
lymphocytes Fas/FasL phenotype. Proc. Am. Ass. Cancer Res., 42: 577,
29. Hortobagyi, G. N. Adjuvant systemic therapy for early breast
cancer: progress and controversies. Clin. Cancer Res., 7: 1839–1842,
30. Ahn, E. Y., Pam, G., Vickers, S. M., and McDonald, J. M. IFN-?
upregulates apoptosis-related molecules and enhances Fas-mediated apo-
ptosis in human cholangiocarcinoma. Int. J. Cancer, 100: 445–451, 2002.
31. Li, J. H., Kluger, M. S., Madge, L. A., Zheng, L., Bothwell,
A. L., and Pober, J. S. Interferon-? augments CD95(APO-1/Fas) and
pro-Caspase-8 expression and sensitizes human vascular endothelial
cells to CD95-mediated apoptosis. Am. J. Pathol., 161: 1485–1495,
32. Hayes, D. F., Isaacs, C., and Stearns, V. Prognostic factors in breast
cancer: current and new predictors of metastasis. J. Mammary Gland
Biol. Neoplasia, 6: 375–292, 2001.
Clinical Cancer Research