immunohistochemical detection of cd 95 (fas) & fas ligand (fas-l) in plasma cells of multiple...
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ORIGINAL ARTICLE: CLINICAL
Immunohistochemical detection of CD 95 (Fas) & Fas ligand (Fas-L) inplasma cells of multiple myeloma and its correlation with survival
MINE HEKIMGIL1, SECKIN CAGIRGAN2, MUSTAFA PEHLIVAN2,
BASAK DOGANAVSARGIL1, MURAT TOMBULOGLU2, & SALIHA SOYDAN1
1Department of Pathology, 2Department of Haematology, Ege University Faculty of Medicine, Izmir, Turkey
AbstractMultiple myeloma (MM) is a malignant disease resulting from an uncontrolled proliferation of a neoplastic plasma cell clonein the bone marrow, which might also be induced by the loss of control on apoptosis. Fas ligand (Fas-L), a member of thetumor necrosis factor family, induces apoptosis mediated via its transmembrane death receptor Fas (Apo-1/CD95) antigen.In the present study, immunostaining was performed on the initial diagnostic bone marrow biopsies of 36 MM patients(1 stage I, 5 stage II, 30 stage III), to evaluate the distribution of Fas receptor and Fas-L on malignant plasma cells. Both Fasand Fas-L were positive in 13 cases and negative in 3, whereas 10 cases were Fas-negative, Fas-L-positive and 10 wereFas-positive, Fas-L-negative. Although no association was found between the expression of Fas receptor or Fas-L and overallsurvival, Fas-L positivity was significantly associated with a shorter event-free survival (p¼ 0.0335). In this study, it has beenshown that the expression of Fas-L, in malignant plasma cells of myeloma patients significantly shortens the event-freesurvival, indicating that the defect in apoptosis might be associated with disease progression in MM.
Keywords: Fas, Fas ligand, multiple myeloma, plasma cell
Introduction
Apoptosis, programmed cell death, is an exquisitely
controlled cell selection mechanism playing a major
role in the maintenance of homeostasis and in vivo
growth control of lymphoid cells. Signaling cascades
involved in apoptosis are now being delineated.
Members of tumor necrosis factor (TNF) family
are type II transmembrane proteins, involved in the
development of some hematological malignancies,
predominantly lymphoproliferative disorders [1]. Fas
(Apo-1/CD95) antigen, a member of the tumor
necrosis factor/nerve growth factor (TNF/NGF)
receptor supergene family, plays an important
role in the apoptotic cell death [2,3]. Fas-ligand
(CD95L/Fas-L) is a 40 kD type 2 glycoprotein that is
excepted as a member of TNF, because of its
significant homology in the organization of extra-
cellular domains with other members of the TNF-
family like TNF-a, CD30 ligand, CD40 ligand and
lymphotoxin [4,5]. The functional Fas-L recognizes
and cross-links its natural membrane receptor Fas,
resulting in the formation of Fas/Fas-L complex,
triggering the cascade of signals for the induction of
apoptosis in the Fas-positive, Fas-L sensitive target
cells [2,4,5]. Although Fas-L is usually active in its
membrane-bound form [5], the molecule may also
be functional in its soluble form, after being released
by the cell membrane [6].
Fas has been an almost ubiquitously found
transmembrane death receptor that is expressed in
a variety of normal and neoplastic tissues including
hematopoetic cell lines, activated normal T- and
B-cells and lymphoma cells [7], whereas activated
T-cells and cytotoxic T-cells were thought to be
the major source of Fas-L molecules [2,6,8,9]. Fas/
Fas-L system is actively involved in the negative
selection of autoreactive T- and B-cells and acquisi-
tion of self-tolerance by clonal deletion of thymocytes
[10 – 12]. Furthermore, it has been shown that Fas-L
plays the major role in the development of auto-
immunity [13,14] and homeostasis of immune
responses [15]. Previous studies have also proved
that Fas-L also participate in the cytotoxic T-cell-
mediated target cell killing as part of the host-defense
against virally infected or transformed cells [16 – 18].
Correspondence: Mine Hekimgil, Department of Pathology, Ege University Faculty of Medicine, Bornova, Izmir 35100 Turkey. Tel: þ90 (232) 388 10 25.
Fax: þ90 (232) 3736143. E-mail: mine.hekimgil@ege.edu.tr
Received for publication 28 June 2005.
Leukemia & Lymphoma, February 2006; 47(2): 271 – 280
ISSN 1042-8194 print/ISSN 1029-2403 online � 2006 Taylor & Francis
DOI: 10.1080/10428190500286218
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Until recently, T-cells were thought to be the
only source of active Fas-L molecule, mediating the
process of Fas-induced target cell killing. However,
recent identification of Fas-L in Sertoli cells of testes
[19,20] and cells of retina [21] and uterus [22]
revealed that the induction of apoptosis of tissue
infiltrating lymphocytes via Fas/Fas-L might suppress
cellular immune responses and inflammation
in some non-lymphoid tissues expressing Fas-L
[21,23,24]. The Fas-L-mediated killing of tissue
infiltrating Fas-positive T-cells protects these
immune-privileged tissues with Fas-L-positive cells
from immune attacks. The recognition of Fas-L
molecule also in macrophages [25] and neutrophils
[26] has led to the hypothesis that Fas-L is not
restricted to T-cells, questioning a more ubiquitous
distribution within the hematopoetic system. The Fas
gene has been investigated in various tissues, but the
expression and localization of Fas-L gene product in
the normal lymphoid tissue has not been defined
until recently [27,28]. The distribution of Fas-L in
normal human lymphoid tissue such as thymus,
spleen, lymph nodes, tonsils and mucosa-associated
lymphoid tissue (MALT) containing organs such
as stomach and appendix was studied by immuno-
histochemistry, immunofluorescence, in situ hybri-
dization and flow cytometry and it has been
surprisingly demonstrated that plasma cells are the
most prominent producers of Fas-L [28]. It has been
proposed that Fas-L expression of plasma cells, in
addition to a probable pivotal role in the down-sizing of
immune responses at sites of immunoglobulin secre-
tion, may also be directed against locally activated,
apoptosis sensitive T-cells. Besides, in immunohisto-
chemical examination, a paranuclear cap-like staining
pattern, consistent with Golgi compartment, sug-
gested that plasma cells might shed a soluble form of
Fas-L to the micro-environment. The autocrine or
paracrine Fas/Fas-L-mediated apoptosis mechanism
of T-cells is unlikely for mature plasma cells, since
untransformed plasma cells do not express Fas [29].
The Fas gene has been defined as a tumor-
suppressor gene [30] and Fas expression of trans-
formed cells varies according to tissues, silenced in
many tumor entities [7]. Various lymphoid malig-
nancies demonstrate an oncofetal transition, fetal
cells are Fas-positive, adult normal cells lose their
Fas and tumor cells regain their Fas expression, are
thus Fas-positive. With tumor progression, malig-
nant cells frequently lose their susceptibility to Fas-
mediated apoptosis. As a result, Fas-deficient tumor
cells escape immunosurveillance [31] and absence of
functional Fas, frequently leads to aberrant expres-
sion of Fas-L in neoplastic cells [32]. Accumulating
evidence in current reports reveal Fas-L expression
in a growing number of malignancies and neoplastic
cell lines and it has been proposed that these
malignancies use the Fas/Fas-L interaction to escape
host defense mechanisms by attacking tumor infiltrat-
ing lymphocytes. The malignancies described to
have an impairment in Fas and/or Fas-L expression
are melanoma [33], colon carcinoma [34], renal
cell carcinoma [35], some T-cell tumors [36], astro-
cytoma [37], esophagus carcinoma [38], breast
carcinoma [39] and prostate adenocarcinoma [40].
The over-expression of Fas-L in these tumors have led
to the hypothesis that the induction of apoptosis of
activated T lymphocytes and NK cells results in
immune escape of malignant cells [41]. Therefore,
Fas-L not only causes transformed target cell death by
effector T lymphocytes as previously well-defined, but
also has a ubiquitous role in the suppression of cellular
immune responses against malignant cells by trigger-
ing a counterattack on Fas-positive effector cells. The
elimination of T-cells by transformed cells is an active
immunosuppression strategy, divergent from the
other proposed mechanisms of immune escape.
In this study, we presented for the first time the
immunohistochemical distribution of Fas receptor
and Fas-L proteins in malignant plasma cells of
multiple myeloma (MM) patients and correlated the
results with prognostic factors, response to standard
therapeutic protocols and survival. Over one-third of
cases showed loss of Fas protein and one-third
presented Fas-L in malignant plasma cells of initial
diagnostic tissue samples. The event-free survival
was statistically significantly shorter in Fas-L-positive
cases, providing evidence of disease progression in
MM when there is a deregulation in the apoptotic
cascade of Fas/Fas-L.
Materials and methods
Patients and tissue samples
The initial bone marrow biopsies of 36 patients
diagnosed as MM (1 stage I, 5 stage II, 30 stage III)
between 1995 – 2002 were studied retrospectively by
immunohistochemistry to identify the extent of Fas
and Fas-L distribution in malignant plasma cells.
Five patients received VAD (vincristine, Adriamy-
cin, dexamethasone) regimen, meanwhile 31 were
treated with autologous peripheral blood stem cell
transplantation. The relationship of the distribution
Fas receptor and Fas-L were correlated with each
other and overall survival, event-free survival,
response to therapy and other prognostic factors
such as age, sex, stage, performance status, serum
creatinine, CRP, albumin, b2-microglobulin and
LDH levels at diagnosis and pre-transplant renal
involvement.
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Immunohistochemical staining
Serial 5-mm thick paraffin sections of B5-formalin
fixed tissues were deparaffinized, rehydrated and the
endogenous peroxidase activity was blocked by
incubation in 3% hydrogen peroxide/methanol for
5 min. For antigen retrieval, the sections were
incubated in a microwave oven in 0.1 mM EDTA
(pH 8.0) solution, for 5 min at high temperature
(800 W) and 20 min at low temperature (650 W).
The sections were then treated with blocking solu-
tion, followed by overnight incubation at 48C with
monoclonal primary mouse antibodies against Fas
(1:15 dilution, Neomarkers, CA, USA) and Fas-L
(1:10 dilution, Neomarkers, CA, USA). Immuno-
histochemical staining for Fas and Fas-L was perfor-
med by Dako EnVisionTM kit (Dako, Denmark).
Diaminobenzidine (DAB, Dako, Denmark) in the
presence of hydrogen peroxide was used as the
chromogen and Gill’s hematoxylin for counterstain-
ing. Small intestine was studied as a positive control
for anti-Fas antibody and prostate tissue for anti-
Fas-L antibody. Negative controls were run in
parallel with each batch by omitting the primary
antibodies.
Evaluation of immunohistochemical staining
First, the extent and intensity of plasma cell
infiltration were evaluated in serial sections of bone
marrow biopsy samples at diagnosis. Then, the
percentage of cells that exhibited staining in each
specimen was recorded as negative if 45% and
positive when 45%. The evaluation was performed
by 2 independent blinded observers (MH and BD)
and the average score of their data was calculated as
the final score.
Statistical analysis
Anti-Fas and anti-Fas-L staining were correlated
with factors related to survival by Pearson correlation
test and survival analysis was performed by Kaplan
Meier (log rank) analysis.
Results
The study group included 36 patients, 18 men and
18 women, with a median age of 52 years (range
30 – 75 years). The clinical characteristics are sum-
marized in Tables I – III.
Stem cell autografting was performed on 31 cases
and 2 patients were lost on short-term after trans-
plantation. Results of the clinical evaluation after
therapy is summarized in Table IV and results of
survival analysis are given in Figure 1.
A cytoplasmic staining was identified with anti-Fas
(Figure 2), meanwhile both cytoplasmic and cytoplas-
mic membrane staining were noted with anti-Fas-L
(Figure 3). The mean scores of Fas (median 96%,
SD 20.82%, range 14 – 100%) and Fas-L (median
89%, SD 18.80%, range 21 – 100%) positivity were
Table I. The distribution of immunoglobulins.
Ig sub-types n k l %
IgG 18 10 8 50
IgA 7 5 1 19
IgD 1 – 1 3
Light chain 10 6 4 28
Total 36 21 14 100
Table II. Stages of patients at diagnosis.
Stage n A B %
Stage I 1 1 0 3
Stage II 5 4 1 14
Stage III 30 17 13 83
Total 36 22 14 100
Table III. The distribution of prognostic factors.
Prognostic factor n Total %
ECOG 4 1 6 28 21
LDH 4460 U L71 8 32 25
CRP 4 0.3 mg dL71 15 18 83
b2M 4 4 mg L71 17 22 77
ECOG, performance evaluation at diagnosis; LDH, lactic
dehydrogenase; CRP, C-reactive protein; b2M, b2-microglobulin.
Table IV. Follow-up results of MM patients.
n %
Total number of patients 36
Transplant-related mortality
(5100 days)
2/31 6.5
Evaluation after therapy
Complete response 14 39
Partial response 18 50
Refractory 3 8
Relapse/progression 14 39
Death due to progressive disease 10 28
Still in complete remission 9 25
Still alive 24 67
Median follow-up (months) 36.7 (2.9–91.9)
Total survival expectancy
(7 years)
43%
Overall survival (median) 70.1 months
Event-free survival expectancy
(5 years)
34%
Event-free survival (median) 13.3 months
Fas & Fas-L in multiple myeloma 273
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88.72% and 76.50%, respectively. Cases were
divided into 4 groups according to the distribution
of Fas and Fas-L (Table V).
No significant correlation was found between Fas
receptor or Fas-L and the other prognostic factors,
an inverse correlation, was noted between Fas and
Fas-L, though statistically not significant (p¼ 0.066,
r¼70.319). Although no association was found
between the expression of Fas receptor or Fas-L
and other prognostic factors and overall survival,
Fas-L positivity was significantly associated with a
shorter event-free survival (p¼ 0.0335) (Figure 4 and
Tables VI and VII).
Discussion
Plasma cell disorders, characterized by the abnormal
proliferation and expansion of a single clone of
Figure 1. The results of survival analysis performed by Kaplan Meier (log rank) analysis.
Figure 2. Cytoplasmic Fas positivity in myeloma cells infiltrating the bone marrow; (a) hematoxylin-eosine, (b) Fas, immunoperoxidase 61000.
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plasma cells, most commonly present as MM.
Although the survival factors (IL-6, bcl-2, NF-KB,
etc) have been fully characterized, the mechanisms
inducing programmed cell death have not been
thoroughly examined in MM. Suppression of natural
programmed cell death might be one of the major
mechanisms of myeloma pathogenesis, resulting in
longer survival of neoplastic plasma cells. Elementary
studies using flow cytometry on purified cells of
plasma cell dyscrasias as well as MM derived cell
lines have shown that Fas antigen is variably expressed,
but only some of these Fas-positive cell clones were
sensitive to Fas-mediated apoptosis upon cross-
linking by anti-Fas monoclonal antibody [42 – 44].
Cell clones with high levels of Fas expression had
significantly higher cell death in response to antibody
[45]. Thus, resistance to Fas-induced apoptosis in
some purified MM cells were explained by either
impairment or lack of surface membrane expression
of Fas antigen or high bcl-2 content in these cells [46].
In addition to these 2 mechanisms, the resistance of
Fas-positive myeloma cells in patient bone marrow
mononuclear cell cultures to anti-Fas-mediated
apoptosis have suggested the presence of a soluble
protective factor(s) in human serum, inhibiting Fas
pathway of plasma cells [42]. IL-6 has been shown to
be one soluble inhibitory factor of Fas-induced
apoptosis of MM cells [47] and the amplitude of
Fas-induced cell death was found to be parallel to the
sensitivity of the cells to the cytokine [48]. The latter
in vitro study on myeloma cell clones has concluded
that highly malignant cells are insensitive to IL-6,
lack CD38 expression, show up-modulated expres-
sion of Fas-L and are resistant to Fas-induced
apoptosis [48].
Taken together, recent studies have already
answered the concerns on the concept by which
Fas-sensitive tumor cells protect themselves from
Fas-L-mediated attack of tumor infiltrating T-cells.
As with myeloma progression, loss of Fas antigen
acts as a mechanism of immune escape, causing
insensitivity of malignant cells to Fas-L present in
tumor infiltrating T-cells [48]. In addition to the
absence of a functional Fas antigen on myeloma
cells, Fas up-regulation has been described in
activated T-cells of MM patients, raising their
susceptibility to apoptosis [49]. Furthermore, recent
in vitro studies have demonstrated Fas-L expression
on myeloma cells and tumor cell-induced suppres-
sion of host cellular immune response via function-
ally active Fas-L on malignant plasma cells [50,51].
Villunger et al. [50] have confirmed the ability of
neoplastic plasma cells to induce apoptosis in Fas sen-
sitive activated T-cells, regardless of the myeloma
Figure 3. Cytoplasmic and cytoplasmic membrane localization of Fas-L positivity in bone marrow infiltration of myeloma; (a) hematoxylin-
eosine, (b) Fas-L, immunoperoxidase 61000.
Table V. Grouping of patients according to the distribution of Fas
and Fas-L.
Group Fas Fas-L n %
I þ þ 13 36
II 7 þ 10 28
III þ 7 10 28
IV 7 7 3 8
Total 23 23 36 100
Fas & Fas-L in multiple myeloma 275
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cells expression level of the Fas receptor and their
own sensitivity to Fas-mediated signaling. The
authors have also concluded that the Fas-L-positive
myeloma cells aid in suppression of immunosurveil-
lance by triggering secretion of granzyme and
perforin, resulting in cytotoxic T-cell death [50].
These mechanisms of immune escape via Fas-L
might be used by highly malignant myeloma cells
[48]. The shorter event-free survival in our Fas-L-
positive cases could be explained by the attack of
malignant Fas-L-positive cell clones to kill the
cytotoxic Fas-positive T cells, leading to suppression
of host defense mechanisms. Thirteen of our 36
cases were Fas-negative and of these 10 (Group II)
expressed Fas-L. The findings in these cases support
the hypothesis of disappearance of Fas and acquisi-
tion of Fas-L expression in MM.
Signs of clinical progression in MM include the
widespread proliferation of malignant plasma cells in
skeletal system and normochromic/normocytic ane-
mia. Studies have shown that Fas-L plays an active
role in the regulation of erythropoiesis [52,53] and
over-expression of Fas-L in highly malignant myelo-
ma cells is involved in the pathogenesis of severe
anemia associated with progression of disease
[54,55]. The proerythroblasts at prebasophilic/baso-
philic stage are Fas-L-negative and very sensitive to
Fas stimulation. Thus, Fas-L-positive differentiated
mature erythroblasts induce apoptosis in these
Fas-L-negative immature erythroblasts, acting as
negative regulators of erythroid maturation through
Fas/Fas-L interaction [53]. It has been strongly
supported that as Fas declines, myeloma cells acquire
Fas-L and, in patients with aggressive disease, the
malignant plasma cells with up-regulated expression
of Fas-L use the same cytotoxic mechanism to
induce apoptosis in Fas-positive erythroblasts, result-
ing in progressive destruction of erythroid matrix
and severe anemia [54 – 57]. It has recently been
reported that withdrawal of erythropoietin or stimu-
lation of death receptors such as Fas or TRAIL-Rs
leads to apoptosis of erythroid cells, under physiolo-
gic and several pathologic conditions including MM,
thalassemia, myelodysplasia and aplastic anemia
Figure 4. The difference of event-free survival in Fas-L-positive cases vs Fas-L-negative ones.
Table VI. The correlation of prognostic factors and Fas and
Fas-L positivity in MM patients.
Fas Fas-L
Pearson correlation
test (p)
Pearson correlation
test (p)
Fas 0.066 R¼7319
Fas-L 0.066 R¼7319
Age 0.553 1.00
Stage 0.778 0.815
ECOG 0.173 0.323
LDH 0.210 0.998
CRP 0.282 0.629
b2M 0.960 0.538
ECOG, performance evaluation at diagnosis; LDH, lactic dehy-
drogenase; CRP, C-reactive protein; b2M, b2-microglobulin.
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[58]. Because osteoblasts are sensitive to Fas
stimulation, induction of apoptosis of osteoblasts by
highly malignant Fas-L-positive myeloma cells could
also account for progression of bone lesions [59,60].
We believe that all these mechanisms, which could be
summarized as destruction of host defense, suppres-
sion of erythropoiesis and appearance or extension of
osteolytic lesions, are the consequences of Fas-L up-
regulation in highly malignant myeloma cell clones.
Thus, the shortened event-free survival of these cases
could be attributed to expansion of an immune-
privileged clone triggering apoptosis in tumor infil-
trating cytotoxic T cells, early erythroblasts and
osteoblasts.
In our immunohistochemical study, both Fas recep-
tor and Fas-L were positive in 13 cases (Group I) and
10 cases were Fas-negative, Fas-L-positive (Group II).
Whereas, 10 cases were Fas-positive, Fas-L-negative
(Group III), both antibodies were negative in 3 cases
(Group IV). These Fas and Fas-L expression
patterns might provide a strategic approach to the
therapy of multiple myeloma, since Fas/Fas-L inter-
action might play a major role in the autologous
control of neoplastic cell proliferation. There is no
previously done immunohistochemical study in
literature describing Fas and Fas-L distribution in
multiple myeloma patient samples as demonstrated
in this study. In previous studies, the extent of Fas
and Fas-L proteins in neoplastic plasma cells have
been investigated on myeloma culture cells and
patient blood and bone marrow aspiration samples
by immunoblot and flow cytometric single cell
analysis, proving that, although some cases express
Fas antigen, the Fas-L molecule is functionally active
in some, capable of killing Fas-positive target T cells
[42 – 51,54]. The only immunohistochemical study
investigating Fas-L expression is performed on
normal lymphoid tissues, to describe the distribution
of Fas-L in normal lymphoid cells [28]. In this
report, the authors have stated that a sub-set of
plasma cells turned out to be the most prominent
producers of Fas-L and that the sensitivity of in situ
hybridization to detect Fas-L expression is superior
to immunostaining. Since the authors have concluded
that negative results in immunohistochemistry
should be interpreted with caution, studies using
in situ hybridization might reveal a higher percentage
of cases with Fas-L positivity.
The only study in the literature correlating serum
soluble Fas-L levels with the clinical prognostic
factors did not reveal any correlation of Fas-L
expression with poor clinical course of disease in
myeloma cases [61]. This observation is not con-
sistent with findings reported by this present study,
mainly because of the technical differences. Deter-
mining serum-soluble Fas-L levels is probably not as
reliable as immunostaining of Fas-L in tissue
sections. The Fas and Fas-L phenotyping of malig-
nant plasma cells in tissue sections of newly
diagnosed cases may be a step to determine the
apoptotic pathway of tumor cells and even outline
prognostically and therapeutically high risk patient
group.
Data from the present study revealed that over one
third of patients present with malignant cells expres-
sing both Fas and Fas-L (Group I, n¼ 13). As
postulated by Villunger et al. [50], highly active
intrinsic control mechanisms may prevent suicide of
neoplastic cells via autologous cell/cell contact or via
soluble Fas-L shed by the neoplastic cells. First, as
also demonstrated in more than one third of the cases
(Groups II and IV, n¼ 13) in the present study, loss
of Fas antigen could account for their resistance to
Fas-induced apoptosis. In addition, although Fas
antigen could be demonstrated immunohistochemi-
cally, intrinsic deficiencies in the Fas signaling
pathway could explain the insensitivity of tumor cells
to Fas-L [34]. Secondly, additional signals delivered
by cell/cell or myeloma/T-cell contact might also
contribute to the suppression of Fas-sensitive plasma
cell death [62]. Thirdly, some cytokines released by
myeloma cells or the micro-environment might
protect myeloma cells from suicide or the cytotoxic
T-cell attack. The defective or deregulated expres-
sion of Fas, signals delivered during cell contact or
immunomodulatory cytokines could explain the
resistance of Fas-positive myeloma cells to death
induced by both Fas-L-positive myeloma or T-cells
Table VII. The correlation of Fas and Fas-L positivity with survival in MM patients.
n
OS
(months)
TSE 7 years
(%) p n
EFS
(months)
EFSE 5
years (%) p
Fas 7 13 88.1þ 60 0.5819 12 9.6 49 0.7279
þ 23 70.1 40 23 13.3 33
Fas-L 7 11 70.1 0 0.1654 11 61.3þ 62 0.0335
þ 23 67.2 42 22 11.2 0
OS, overall survival (median); TSE, total survival expectancy; EFS, event-free survival (median); EFSE, event-free survival expectancy.
Fas & Fas-L in multiple myeloma 277
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[50] and this interesting aspect raises the question of
whether resistance to Fas-mediated apoptosis of
myeloma cells has any contribution to resistance to
therapy. Two recent studies by Landowski et al.
[63,64] have supported the hypothesis that apoptotic
pathways mediated by both chemotherapeutic agents
and physiologic stimuli share a common downstream
effector initiated by Fas/Fas-L interaction, but
resistance to Fas-mediated apoptosis does not
indicate cross-resistance to cytotoxic drugs. It has
been recently shown that IFN-induced up-regulation
of Fas sensitizes MM cells to Fas-mediated apoptosis
[65]. However, although Fas expression cannot be
assessed as a sign of resistance to chemotherapy, it
should be kept in mind that, similar to the develop-
ment of mechanisms of drug resistance, myeloma
cells might develop mechanisms of resistance to
Fas-mediated apoptosis.
With the aid of improvements in therapy of MM,
such as the use of high dose chemotherapy and
autologous peripheral blood stem cell transplanta-
tion, the complete remissions have raised from 5% to
30% and 50% and median event-free survival from
1 – 2 to 3 – 4 years, median overall survival from 3 – 4
to 5 – 7 years [66]. However, the cases with aggres-
sive disease enter an accelerated refractory period
within early stages of disease and the highly
malignant clones manifest uncontrolled proliferation
and expansion, which is unresponsive to therapy. On
the contrary to the research in this field, the
molecular phenotypes of these aggressive cases have
not been defined yet. As pointed out, the inhibition
or suppression of apoptosis in neoplastic cells plays a
pivotal role both in myeloma pathogenesis and drug
resistance mechanisms of MM. A better under-
standing of apoptotic mechanisms will provide a
more reliable approach to the therapy of MM. Since
myeloma cells may exert an autocrine and paracrine
control on their proliferation via Fas/Fas-L and the
highly malignant Fas-negative myeloma cells may
escape cytotoxic T-cell attack by up-regulation of
Fas-L [50,51], a therapeutic strategy aimed at down-
regulation of Fas-L in neoplastic cells, might, by
lowering their resistance to host immune system,
prolong the event-free survival of these aggressive
cases. The goal of immunotherapy approaches might
also be directed towards induction of Fas-mediated
apoptosis of tumor cells, as well as increasing
the activity of cytotoxic T lymphocytes against
tumor cells.
Acknowledgement
Supported by the Ege University Scientific Research
Projects (2001/TIP/005).
References
1. Fiumara P, Younes A. CD40 ligand (CD154) and tumour
necrosis factor-related apoptosis inducing ligand (Apo-2L) in
haematological malignancies. British Journal of Haematology
2001;113:265 – 274.
2. Itoh N, Yonehara S, Ishii A, Yonehara M, Mizushima S,
Sameshima M, et al. The polypeptide encoded by the cDNA
for human cell surface antigen Fas can mediate apoptosis. Cell
1991;66:233 – 239.
3. Oehm A, Behrmann I, Falk W, Pawlita M, Maier G, Klas C,
et al. Purification and molecular cloning of the Apo-1 cell
surface antigen, a member of the tumor necrosis factor/nerve
growth factor receptor superfamily: sequence identity with the
Fas antigen. The Journal of Biological Chemistry 1992;267:
10709 – 10715.
4. Nagata S, Goldstein P. The Fas death factor. Science
1995;267:1449 – 1456.
5. Suda T, Takahashi T, Golstein P, Nagata S. Molecular
cloning and expression of the Fas ligand, a novel member
of the tumor necrosis factor family. Cell 1993;75:1169 –
1178.
6. Tanaka M, Suda T, Takahashi T, Nagata S. Expression of the
functional soluble form of human Fas ligand in activated
lymphocytes. The EMBO Journal 1995;14:1129 – 1135.
7. Leithauser F, Dhein J, Mechtersheimer G, Koretz K,
Bruderlein S, Henne C, et al. Constitutive and induced
expression of APO-1, a new member of the nerve growth
factor/tumor necrosis factor receptor superfamily, in normal
end neoplastic cells. Laboratory Investigation 1993;69:415 –
429.
8. Suda T, Okazaki T, Naito Y, Yokota T, Arai N, Ozaki S, et al.
Expression of the Fas ligand in cells of T cell lineage. Journal
of Immunology 1995;154:3806 – 3813.
9. Glass A, Walsh CM, Lynch DH, Clark WR. Regulation of the
Fas lytic pathway in cloned CTL. Journal of Immunology
1996;156:3638 – 3644.
10. Watanabe-Fukunaga R, Brannan CI, Copeland NG, Jenkins
NA, Nagata S. Lymphoproliferation disorder in mice ex-
plained by defects in Fas antigen that mediates apoptosis.
Nature 1992;356:314 – 317.
11. Takahashi T, Tanaka M, Brannan CI, Jenkins NA, Copeland
NG, Suda T, Nagata S. Generalized lymphoproliferative
disease in mice, caused by a point mutation in the Fas ligand.
Cell 1994;76:969 – 976.
12. Muschen M, Rajewsky K, Kronkr M, Kuppers R. The origin
of CD95-gene mutations in B-cell lymphoma. Modern
Trends in Immunology 2002;23:75 – 80.
13. Vignaux F, Golstein P. Fas-based lymphocyte-mediated
cytotoxicity against syngeneic activated lymphocytes: a reg-
ulatory pathway. European Journal of Immunology 1994;24:
923 – 927.
14. Alderson MR, Tough TW, Davis-Smith T, Braddy S, Falk B,
Schooley KA, et al. Fas ligand mediates activation-induced
cell death in human T lymphocytes. The Journal of Experi-
mental Medicine 1995;181:71 – 77.
15. Lynch DH, Ransdell F, Alderson MR. Fas and Fas-L in the
homeostatic regulation of immune responses. Immunology
Today 1995;16:569 – 574.
16. Kagi D, Vignaux F, Ledermann B, Burki K, Depraetere V,
Nagata S, et al. Fas and perforin pathways as major
mechanisms of T cell-mediated cytotoxicity. Science 1994;
265:528 – 530.
17. Lowin B, Hahne M, Mattmann C, Tschopp J. Cytolytic T-cell
cytotoxicity is mediated through perforin and Fas lytic
pathways. Nature 1994;370:650 – 652.
278 M. Hekimgil et al.
Leu
k L
ymph
oma
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Uni
vers
ity o
f B
ritis
h C
olum
bia
on 1
0/29
/14
For
pers
onal
use
onl
y.
18. Rensing-Ehl A, Frei K, Flury R, Matiba B, Mariani SM,
Weller M, et al. Local Fas/Apo-1 (CD95) ligand-mediated
tumor cell killing in vivo. European Journal of Immunology
1995;25:2253 – 2258.
19. Bellgrau D, Gold D, Selawry H, Moore J, Franzusoff A, Duke
RC. A role for CD95 ligand in preventing graft rejection.
Nature 1995;377:630 – 632.
20. Sugihara A, Saiki S, Tsuji M, Tsujimura T, Nakata Y, Kubota
A, et al. Expression of Fas and Fas ligand in testis and
testicular germ cell tumors: an immunohistochemical study.
Anticancer Research 1997;17:3861 – 3865.
21. Griffith TS, Brunner T, Fletcher SM, Gren DR, Ferguson
TA. Fas ligand-induced apoptosis as a mechanism of immune
privilege. Science 1995;270:1189 – 1192.
22. Hunt JS, Vassmer D, Ferguson TA, Miller L. Fas ligand is
positioned in mouse uterus and placenta to prevent trafficking
of activated leucocytes between the mother and the conceptus.
Journal of Immunology 1997;158:4122 – 4128.
23. Griffith TS, Ferguson TA. The role of Fas-L-induced apoptosis
in immune privilege. Immunology Today 1997;18:240 – 244.
24. Green DR, Ware CF. Fas-ligand: privilege and peril.
Proceedings of the National Academy of Sciences (USA)
1997;94:5986 – 5990.
25. Badley AD, McElhinny JA, Leibson PJ, Lynch DH, Alderson
MR, Paya CV. Upregulation of Fas ligand expression by
human immunodeficiency virus in human macrophages
mediates apoptosis of uninfected T lymphocytes. Journal of
Virology 1996;70:199 – 206.
26. Liles WC, Kiener PA, Ledbetter JA, Aruffo A, Klebanoff SJ.
Differential expression of Fas (CD95) and Fas ligand on
normal human phagocytes: implications for the regulation of
apoptosis in neutrophils. The Journal of Experimental
Medicine 1996;184:429 – 440.
27. Kondo E, Yoshino T, Nishiuchi R, Sakuma I, Nishizaki K,
Kayagaki N, et al. Expression of Fas ligand mRNA in germinal
centers of the human tonsil. Journal of Pathology 1997;183:
75 – 79.
28. Strater J, Mariani SM, Walczak H, Rucker FG, Leithauser F,
Krammer PH, Moller P. CD95 Ligand (CD95L) in normal
lymphoid tissues: a subset of plasma cells are prominent
producers of CD95L. American Journal of Pathology 1999;
154:193 – 201.
29. Leithauser F, Dhein J, Merchtersheimer G, Koretz K,
Bruderlein S, Henne C, et al. Constitutive and induced
expression of APO-1, a new member of the nerve growth
factor/tumor necrosis factor receptor superfamily, in normal and
neoplastic cells. Laboratory Investigation 1993;69:415 – 429.
30. Muschen M, Warskulat U, Beckmann MW. Defining CD95
as a tumor suppressor gene. Journal of Molecular Medicine
2000;78:312 – 325.
31. Muschen M, Moers C, Warskulat U, Even J, Niederacher D,
Beckmann MW. CD95-ligand expression as a mechanism
of immune escape in breast cancer. Acta Microbiologica
et Immunologica Hungarica 2000;99:69 – 77.
32. Muschen M, Warskulat U, Schmidt B, Schulz WA,
Haussinger D. Regulation of CD95 (Apo-1/Fas) ligand and
receptor expression in human embryonal carcinoma cells by
interferon g and all-trans retinoic acid. Biological Chemistry
1998;379:1083 – 1091.
33. Hahne M, Rimoldi D, Schroter M, Romero P, Schreier M,
French LE, et al. Melanoma cell expression of Fas (Apo-1/
CD95) ligand: implications for tumor immune escape.
Science 1996;274:1363 – 1366.
34. O’Connell J, O’Sullivan GC, Collins JK, Shanahan F. The Fas
counterattack: Fas-mediated T cell killing by colon cancer
cells expressing Fas ligand. The Journal of Experimental
Medicine 1996;184:1075 – 1082.
35. Nonomura N, Mili T, Yokoyama M. Fas/APO-1 mediated
apoptosis of human renal cell carcinoma. Biochemical and
Biophysical Research Communications 1996;225:945 – 951.
36. Tanaka M, Suda T, Haze K, Nakamura N, Sato K, Kimura F,
et al. Fas ligand in human serum. Nature Medicine 1996;
2:317 – 322.
37. Saas P, Walker PR, Hahne M, Quiquerez AL, Schnuriger V,
Perin G, et al. Fas ligand expression by astrocytoma in vivo:
maintaining immune privilege in the brain? The Journal of
Clinical Investigation 1997;99:1173 – 1178.
38. Gratas C, Tohma Y, Barnas C, Taniere P, Hainaut P, Ohgaki
H. Up-regulation of Fas (APO-1/CD95) ligand and down-
regulation of Fas expression in human esophageal cancer.
Cancer Research 1998;58:2057 – 2062.
39. Mullauer L, Mosberger I, Grusch M, Rudas M, Chott A. Fas
ligand is expressed in normal breast epithelial cells and is
frequently up-regulated in breast cancer. The Journal of
Pathology 2000;190:20 – 30.
40. Jiang J, Ulbright TM, Zhang S, Eckert GJ, Kao C, Gardner
TA, et al. Fas and Fas ligand expression is elevated in prostatic
intraepithelial neoplasia and prostatic adenocarcinoma. Can-
cer 2002;95:296 – 300.
41. Walker PR, Saas P, Dietrich PY. Role of Fas ligand (CD95L)
in immune escape: the tumor cell strikes back. Journal of
Immunology 1997;158:4521 – 4524.
42. Westendorf JJ, Lammert JM, Jelinek DF. Expression and
function of Fas (APO-1/CD95) in patient myeloma cells and
myeloma cell lines. Blood 1995;85:3566 – 3576.
43. Hata H, Matsuzaki H, Takeya M, Yoshida M, Sonoki T,
Nagasaki A, et al. Expression of Fas/Apo-1 (CD95) and
apoptosis in tumor cells from patients with plasma cell
disorders. Blood 1995;86:1939 – 1945.
44. Shima Y, Nishimoto N, Ogata A, Fujii Y, Yoshizaki K,
Kishimoto T. Myeloma cells express Fas antigen/APO-1
(CD95) but only some are sensitive to anti-Fas antibody
resulting in apoptosis. Blood 1995;85:757 – 764.
45. Shain KH, Landowski TH, Buyuksal I, Cantor AB, Dalton
WS. Clonal variability in CD95 expression is the major
determinant in Fas-medicated, but not chemotherapy-
medicated apoptosis in the RPMI 8226 multiple myeloma
cell line. Leukemia 2000;14:830 – 840.
46. Tu Y, Xu FH, Liu J, Vescio R, Berenson J, Fady C,
Lichtenstein A. Upregulated expression of Bcl-2 in multiple
myeloma cells induced by exposure to doxorubicin, etoposide,
and hydrogen peroxide. Blood 1996;88:1805 – 1812.
47. Chauhan D, Kharbanda S, Ogata A, Urashima M, Teoh G,
Robertson M, et al. Interleukin-6 inhibits Fas-induced
apoptosis and stress-activated protein kinase activation in
multiple myeloma cells. Blood 1997;89:227 – 234.
48. Frassanito MA, Silvestris F, Silvestris N, Cafforio P, Camarda
G, Iodice G, Dammacco F. Fas/Fas ligand (Fas-L)-deregu-
lated apoptosis and IL-6 insensitivity in highly malignant
myeloma cells. Clinical and Experimental Immunology 1998;
114:179 – 188.
49. Massaia M, Borrione P, Attisano C, Barral P, Beggiato E,
Montacchini L, et al. Dysregulated Fas and Bcl-2 expression
leading to enhanced apoptosis in T cells of multiple myeloma
patients. Blood 1995;85:3679 – 3687.
50. Villunger A, Egle A, Marschitz I, Kos M, Bock G, Ludwig H,
et al. Constitutive expression of Fas (Apo-1/CD95) ligand on
multiple myeloma cells: a potential mechanism of tumor-
induced suppression of immune surveillance. Blood 1997;
90:12 – 20.
51. Greil R, Egle A, Villunger A. On the role and significance of
Fas (Apo-1/CD95) ligand (Fas-L) expression in immune
privileged tissues and cancer cells using multiple myeloma as a
model. Leukemia and Lymphoma 1998;31:477 – 490.
Fas & Fas-L in multiple myeloma 279
Leu
k L
ymph
oma
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Uni
vers
ity o
f B
ritis
h C
olum
bia
on 1
0/29
/14
For
pers
onal
use
onl
y.
52. Dai CH, Price JO, Brunner T, Krantz SB. Fas ligand is
present in human erythroid colony-forming cells and interacts
with Fas induced by interferon-g to produce erythroid cell
apoptosis. Blood 1998;91:1235 – 1241.
53. De Maria R, Testa U, Luchetti L, Zeuner A, Stassi G, Pelosi
E, et al. Apoptotic role of Fas/Fas ligand system in the
regulation of erythropoiesis. Blood 1999;93:796 – 803.
54. Silvestris F, Tucci M, Cafforio P, Dammacco F. Fas-L up-
regulation by highly malignant myeloma plasma cells: role in
the pathogenesis of anemia and disease progression. Blood
2001;97:1155 – 1164.
55. Silvestris F, Cafforio P, Tucci M, Dammacco F. Negative
regulation of erythroblast maturation by Fas-L-positive/
TRAILþ highly malignant plasma cells: a major pathogenetic
mechanism of anemia in multiple myeloma. Blood 2002;
99:1305 – 1313.
56. Silvestris F, Cafforio P, Grinello D, Dammacco F. Upregula-
tion of erythroblast apoptosis by malignant plasma cells: a new
pathogenetic mechanism of anemia in multiple myeloma.
Reviews in Clinical and Experimental Hematology 2002;
6(Suppl 1):39 – 46.
57. Tucci M, Grinello D, Cafforio P, Silvestris F, Dammacco F.
Anemia in multiple myeloma: role of deregulated plasma cell
apoptosis. Leukemia and Lymphoma 2002;43:1527 – 1533.
58. Testa U. Apoptotic mechanisms in the control of erythropoi-
esis. Leukemia 2004;18:1176 – 1199.
59. Nakashima T, Sasaki H, Tsuboi M, Kawakami A, Fujiyama
K, Kiriyama T, et al. Inhibitory effect of glucocorticoid for
osteoblast apoptosis induced by activated peripheral blood
mononuclear cells. Endocrinology 1998;139:2032 – 2040.
60. Silvestris F, Cafforio P, Tucci M, Grinello D, Dammacco F.
Upregulation of osteoblast apoptosis by malignant plasma
cells: a role in myeloma bone disease. British Journal of
Haematology 2003;122:39 – 52.
61. Kanda Y, Ara C, Chizuka A, Yamamoto R, Hamaki T,
Suguro M, et al. Lack of correlation between clinical
characteristics and serum soluble Fas ligand levels in patients
with multiple myeloma. Leukemia and Lymphoma
2001;40:351 – 356.
62. Hahne M, Renno T, Schroeter M, Irmler M, French L,
Bornard T, et al. Activated B cells express functional Fas
ligand. European Journal of Immunology 1996;26:721 – 724.
63. Landowski TH, Gleason-Guzman MC, Dalton WS. Selection
for drug resistance results in resistance to Fas-mediated
apoptosis. Blood 1997;89:1854 – 1861.
64. Landowski TH, Shain KH, Oshiro MM, Buyuksal I, Painter
JS, Dalton WS. Myeloma cells selected for resistance to
CD95-mediated apoptosis are not cross-resistant to cytotoxic
drugs: evidence for independent mechanisms of caspase
activation. Blood 1999;94:265 – 274.
65. Dimberg LY, Dimberg AI, Ivarsson K, Stromberg T,
Osterborg A, Nilsson K, et al. Ectopic and IFN-induced
expression of Fas overcomes resistance to Fas-mediated
apoptosis in multiple myeloma cells. Blood 2005;106:1346 –
1354.
66. Attal M, Harousseau J, Stoppa AM, Sotto JJ, Fuzibet JG,
Rossi JF, et al. A prospective, randomized trial of autologous
bone marrow transplantation and chemotherapy in multiple
myeloma. The New England Journal of Medicine 1996;
335:91 – 97.
280 M. Hekimgil et al.
Leu
k L
ymph
oma
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Uni
vers
ity o
f B
ritis
h C
olum
bia
on 1
0/29
/14
For
pers
onal
use
onl
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