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

y.