Fas Ligand Expression in Metastatic Renal Cell Carcinoma During
Interleukin-2 Based Immunotherapy: No In vivo Effect of Fas
Ligand Tumor Counterattack
Frede Donskov,1,4Hans von der Maase,1
Niels Marcussen,2Stephen Hamilton-Dutoit,2
Hans Henrik Torp Madsen,3Jens Jorgen Jensen,3
and Marianne Hokland4
Departments of1Oncology,2Pathology, and3Radiology, Aarhus
University Hospital, Aarhus, Denmark; and4Department of Medical
Microbiology and Immunology, University of Aarhus, Aarhus,
Purpose: It has been hypothesized that tumor cells ex-
pressing Fas ligand (FasL) might be able to counterattack
and neutralize tumor-infiltrating lymphocytes. We assessed
the effect of FasL tumor counterattack on the clinical out-
come of interleukin-2 (IL-2)–based immunotherapy in met-
astatic renal cell carcinoma.
Experimental Design: Tumor core needle biopsies were
obtained before IL-2–based immunotherapy in 86 patients
and repeated within the first cycle in 57 patients. Tumor
cells expressing FasL and intratumoral lymphocyte subsets
expressing CD4, CD8, CD56, and CD57 were analyzed by
Results: At baseline, negative FasL staining in tumor
cells was seen in 10 of 86 (12%) biopsies, whereas intense
FasL staining was seen (a) in fewer than 10% of tumor cells
in 26 (30%) biopsies; (b) in 11 to 50% of tumor cells in 25
(29%) biopsies; (c) in 51 to 90% of tumor cells in 18 (21%)
biopsies; and (d) in >90% of tumor cells in 7 (8%) biopsies.
On treatment, tumor FasL expression did not change from
baseline levels. Moreover, tumor FasL expression was not
correlated with objective response or survival whereas the
absolute number of CD4?, CD8?, CD56?, and CD57?cells
per mm2tumor tissue at baseline was significantly higher in
responding patients compared with nonresponding patients
(P ? 0.01, P ? 0.008, P ? 0.015, and P < 0.001, respec-
tively). During the first course of immunotherapy, the abso-
lute number of CD4?, CD8?, and CD57?cells per mm2
tumor tissue was significantly higher in responding patients
compared with nonresponding patients (P ? 0.034, P <
0.001, and P < 0.001, respectively). However, no correlation
was observed between the number of intratumoral lympho-
cytes and tumor FasL expression level.
Conclusion: These observations do not support the hy-
pothesis that FasL tumor “counterattack” has an effect on
the clinical outcome in metastatic renal cell carcinoma dur-
ing IL-2–based immunotherapy.
The explanation for the low levels of responses seen after
interleukin-2 (IL-2)– and interferon-? (IFN-?)–based immuno-
therapy is probably extremely complex. However, the hypoth-
esis that tumors might create a zone of immune “privilege” by
expressing Fas ligand (FasL, CD95L) and counterattack tumor-
infiltrating lymphocytes by delivering apoptotic death signals
may represent one of several factors of importance for treatment
FasL, cloned in 1993, was initially thought to be expressed
in immune cells only, including activated T-lymphocytes and
natural killer cells, and to play a key role in cytotoxicity and
immune homeostasis. However, FasL expression in the eye and
placenta was noted, and thus the concept of immune privilege
was established (1).
Furthermore, studies subsequently reported the expression
of FasL by human malignancies, including melanoma (2, 3),
astrocytomas (4) and colorectal (5), esophageal (6), lung (7),
ovarian (8), head and neck (9), and pancreas carcinoma (10).
Although controversial (11–15), these studies led to the hypoth-
esis that tumor tissues could represent sites of immune privilege
that enable cancers to “counterattack” the immune system (16,
In 1999, Uzzo et al. (18) provided the first evidence that
FasL was expressed in renal cell carcinoma cell lines and renal
tumor tissue, and they also suggested that FasL expression was
functional. The observation that FasL is expressed in renal cell
carcinoma has subsequently been confirmed by others (19–25).
Metastatic renal cell carcinoma (mRCC) is a treatment-
resistant disease, that, however is responsive to IL-2– and IFN-
?–based immunotherapy. The antineoplastic actions of IL-2 and
IFN-? are associated with infiltration of immune effector cells
within the tumor tissue (26, 27). However, functional interaction
occurring in vivo between tumor cells expressing FasL and
tumor-infiltrating lymphocytes during IL-2–based immunother-
apy has not been investigated previously. Thus, in the present
Received 6/7/04; revised 8/16/04; accepted 9/1/04.
Grant support: from the Danish Research Council, Max and Inger
Woerzner Foundation, Radiumstationens Forskningsfond, Gerda and
Aage Haench?s Foundation, The Beckett Foundation, Preben and Anna
Simonsens Foundation, Agnes Niebuhr Anderssons Foundation, Jo-
hannes Fogh-Nielsen Foundation, Agnes and Poul Friis Foundation,
Erland Richard Frederiksen Foundation, Jens C. Christoffersen Foun-
dation, Kristian Kjaer Foundation, Hans and Nora Burchard?s Founda-
tion, the Danish Medical Association Research Fund, and The Danish
Cancer Society (M. Hokland).
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: Frede Donskov, Department of Oncology, Aarhus
University Hospital, Norrebrograde, Aarhus, 8000, Denmark. Phone: 8949-
3333; Fax: 011-45-8949-2530; E-mail: email@example.com.
©2004 American Association for Cancer Research.
Vol. 10, 7911–7916, December 1, 2004
Clinical Cancer Research
study, we have assessed tumor cells expressing FasL and tumor-
infiltrating lymphocyte subsets in situ in repeated tumor tissue
core needle biopsies obtained at baseline and within the first
cycle of IL-2–based immunotherapy in patients with mRCC,
and correlated the findings with the clinical outcome.
MATERIALS AND METHODS
Patients and Response Evaluation.
secutive single-institution patients with inoperable histologi-
cally confirmed mRCC were treated on an outpatient basis with
IL-2–based immunotherapy from February 1999 to August
2002. Twenty-six of these patients were enrolled in a multi-
center prospective phase II trial of IL-2, IFN-?, and histamine
dihydrochloride (28). A further 23 patients received standard
treatment with the same schedule but without histamine (26).
Sixty-three patients were enrolled in a randomized phase II trial
of IL-2 ? histamine dihydrochloride, and the final eight patients
were treated with IL-2 alone. The local ethical committee and
the Danish Medical Agency approved the studies.
For the interferon-containing regimens, the treatment plan
consisted of 1 priming-week of daily IFN-? and up to 9 treat-
ment cycles of 4 weeks with IFN-? (human leukocyte IFN-?,
Interferon Alfanative, BioNative, Umea, Sweden, for the phase
II trial, otherwise Introna, Schering-Plough, Farum, Denmark)
3.0 million IU as a fixed dose subcutaneously once daily, 7 days
per week; IL-2 (Aldesleukin, rIL-2, Proleukin, Chiron, Amster-
dam-Zuidoost, the Netherlands) 2.4 million IU/m2subcutane-
ously twice daily, 5 days per week, weeks 1 and 2 every cycle;
and histamine dihydrochloride (Ceplene, Maxim Pharmaceuti-
cals Inc, San Diego, CA) 1.0 mg in 1.0 mL by a 20-minute slow
subcutaneous injection, twice daily, 5 days per week throughout
the study. Patients were evaluated for objective response every
For the noninterferon-containing regimens, the treatment
plan consisted of up to 4 treatment cycles of 5 weeks with IL-2,
18 million IU as a fixed dose subcutaneous injection once daily,
5 days per week for 3 weeks followed by 2 weeks rest. In case
of randomization, 1.0 mg of histamine dihydrochloride was
added twice daily, concomitantly with IL-2. Patients were eval-
uated for objective response every 2 cycles.
Objective response was defined according to standard
World Health Organization criteria: (a) complete response (CR),
defined as total disappearance of all clinical disease; (b) partial
response (PR), defined as a reduction of ?50% in the bidimen-
sional product diameter; (c) stable disease (SD), defined as a
reduction of ?50% or an increase in size of ?25%; and (d)
progressive disease (PD), defined as an increase in size of
?25% in the bidimensional product diameter.
Collection of Samples.
Core needle biopsies (18G cut-
ting needle) were collected by standard ultrasound-guided pro-
cedures at baseline and before the start of week 3 (non-IFN-
containing schedules) or week 5 (IFN-containing schedules).
These time points for repeated biopsies were selected to coin-
cide with routine outpatient clinical visits, according to the
immunotherapy schedule. For the present study analysis, week 3
and week 5 biopsies were classified as on-treatment biopsies.
The local ethical committee had approved the biopsy study. Of
the 120 consecutive patients, written informed consent and
A total of 120 con-
baseline biopsies were obtained from 101 patients. Four patients
did not complete one course of therapy because of toxicity and
were not evaluable for objective response. Two patients had
only fine needle biopsies done. These six patients were excluded
from all further analyses. Thus, 95 patients were included in the
study (Table 1). Three patients had biopsies containing necrotic
tissue only and six patients had biopsies with insufficient tumor
tissue. Thus at baseline, 86 patients had evaluable biopsies.
On-treatment biopsies were only evaluable in 57 of the 95
patients for the following reasons: (a) 21 patients refused re-
peated biopsies; (b) 8 had biopsies with insufficient tumor
tissue; (c) 5 had biopsies showing necrosis only and (d) 4 had no
biopsies done for safety reasons. On the basis of well-known
prognostic factors of Memorial Sloan Kettering Cancer Center
(29), there were no significant differences between the 95-
patient group and the 86-patient group or the 57-patient group,
P ? 0.24 and P ? 0.27, respectively, Fisher‘s exact test.
Biopsies were done from accessible tumor locations: kid-
ney, n ? 68; abdominal soft tissue, n ? 16; liver, n ? 17;
pleura/chest wall, n ? 10; lymph node, n ? 9; muscle, n ? 5;
kidney bed, n ? 4; subcutis, n ? 5; and lung, n ? 3 (some
patients had biopsies from more than one location). No complete
responding patients had accessible tumors for core needle biop-
sies. On-treatment biopsy was obtained from the same tumor as
the baseline biopsy.
fixed, paraffin-embedded biopsies were mounted on ChemMate
slides (S2024, DakoCytomation, Glostrup, Denmark), dried for
1 hour at 60°C, deparaffinized, and rehydrated. After endoge-
nous peroxidase blocking (0.5% hydrogen peroxidase in water
for 30 minutes), antigens were retrieved by microwave oven
Sections (2 ?m) of formalin-
Patient characteristics (N ? 95)
Median age, years (range)
Karnofsky performance status
Excision of metastatic lesions
Number of disease sites
Most common sites of disease
Primary kidney tumor
Local recurrence kidney bed
Lung metastasis alone
MSKCC prognostic criteria *
* Memorial Sloan-Kettering Cancer Center (29)
7912 FasL in mRCC During IL-2–Based Immunotherapy
heating [3 ? 5 minutes at 850 W in Tris/EGTA retrieval buffer
(pH 9.0)]. The tissue sections were incubated for 1 hour with the
following antibodies: anti-FasL [F 37720, clone 33 (Transduc-
tion Laboratories, Newington, NH), diluted 1:40; and G247-4
(PharMingen San Diego, CA), diluted 1:10, 1:25, and 1:50 with
and without the copper signal enhancement; anti-CD4 (NCL-
CD4–1F6, 1:50, NovoCastra); anti-CD8 (M7103, 1:100, Dako-
Cytomation); anti-CD56 (NCL-CD56–1B6, 1:40, NovoCastra)
and anti-CD57 (33251A, 1:500, PharMingen). Sections were
then incubated for 30 minutes with peroxidase-conjugated En-
Vision (K4000, DakoCytomation). Staining was visualized with
3,3?-diaminobenzidine solution. Sections were counterstained in
Mayer‘s hematoxylin and mounted with Aqutex (64912–50,
Struers KEBO Lab, Albertslund, Denmark). All staining was
done in a TechMate automatic immunohistochemistry staining
machine (DakoCytomation). Sections from normal tonsil, nor-
mal kidney, and from various kidney tumors were used as
positive controls. Negative controls consisted of either relevant
isotype controls from PharMingen and DakoCytomation or
stains in which the primary antibody was omitted from the
staining was assessed in a semiquantitative fashion, incorporat-
ing both the intensity and distribution of specific staining: (0) no
intensely stained cells; (1?) 1 to 10% intensely stained cells,
almost all tumor cells positive; (2?) 11 to 50% intensely stained
cells, almost all tumor cells positive; (3?) 51 to 90% intensely
stained cells, almost all tumor cells positive; (4?) ? 90%
intensely stained cells, almost all tumor cells positive. Tumor
cells were identified based on morphology. Semiquantitative
scoring was assessed blinded by two observers (F. D. and
N. M.). Intratumoral immune cells were enumerated by a ste-
reological method (30). In short, this was done with a morpho-
metric system consisting of an Olympus AH-3 microscope with
a motorized stage that was controlled by a computer for manual
interactive counting on the computer screen. The software used
was CAST-grid version 2.0, developed by Olympus (Al-
bertslund, Denmark). Each microscopic field of vision was
projected onto the computer screen with a video camera, and the
computer generated an unbiased counting frame in which the
measurements were done. On the projected image of the section,
the tumor area was encircled. Necrosis, artifacts and fibrous
areas were omitted. The first field of vision was chosen at
random, after which the computer sampled systematically the
following fields of vision within the entire encircled area. Using
a ?40 objective, we counted a total number of 40 fields (4,951
?m2each), if the size of the tumor allowed for it. The entire core
needle biopsy was assessed. Only a cell with staining restricted
to the plasma membrane, a visible nucleus, and located within
the counting frame was counted as positive. The mean number
of cells per mm2tumor tissue was assessed for each patient.
Staining was analyzed blinded by one observer. Selected sec-
tions were counted blinded by a senior histopathologist (N. M.)
and a high level of reproducibility was found, as reported
Overall survival time was measured from first
day of treatment until death or last follow-up evaluation. We
evaluated the relationship between assessed variables and ob-
jective response using the nonparametric Mann-Whitney U test.
Tumor cell FasL
The Spearman correlation test was used to evaluate the relation-
ship between tumor FasL expression and intratumoral lympho-
cyte subsets. The Wilcoxon signed rank test for paired samples
was used to assess the significance of changes from baseline to
week 5. We evaluated the relationship between assessed vari-
ables and survival using the Kaplan-Meier method and the
log-rank test. Dichotomy of the patients was done at the median
value for each evaluated variable. The median follow-up period
was 30.2 months (range 18–61 months). No patients were lost
to follow-up. Data were updated March 11, 2004. SPSS v11.0
(SPSS, Chicago, IL) was used to do statistical analyses.
Clinical Treatment Results.
with IL-2–based immunotherapy were evaluable for consecutive
tumor biopsies. Table 1 lists patient characteristics. Of these, seven
patients achieved PR, thirty-five patients achieved SD and 53
patients had PD. Median survival was 13.3 months (range 0.5–
60.9? months). Four patients (3 with PR and 1 with SD) had no
evidence of disease (NED) and were alive at 44?, 53?, 59?, and
61? months, respectively, after immunotherapy followed by sub-
sequent resection of residual tumor.
Correlation between Tumor FasL Staining and Objec-
Baseline and on-treatment tumor FasL expres-
sion was evaluated and correlated with objective response. We
could not find detectable levels of FasL using the monoclonal
G247-4 antibody. However, with the clone 33 antibody, tumor
cells stained positive for FasL with a cytoplasmatic, often gran-
ular, perinuclear reaction. At baseline, FasL tumor staining was
negative (0?) in 10 of 86 (12%) biopsies; with ?10% intensely
stained tumor cells (1?) in 26 of 86 (30%) biopsies; with 11 to
50% intensely stained cells (2?) in 25 of 86 (29%) biopsies;
with 51 to 90% intensely stained cells (3?) in 18 of 86 (21%)
biopsies; and with ?90% intensely stained cells (4?) in 7 of 86
(8%) biopsies (Fig. 1). On-treatment, tumor FasL expression did
not change from baseline (P ? 0.31). When FasL staining of
responding (PR) and nonresponding (SD?PD) patients at base-
line or on-treatment were compared, no significant differences
were noted (P ? 0.46 and P ? 0.16, respectively). Patients with
NED did not show any significant difference in FasL staining
compared with non-NED patients at baseline or on-treatment
(P ? 0.9 and P ? 0.4, respectively). In this group of four
patients, we also analyzed FasL expression in the resected
“residual” tumors. However, there were no significant differ-
ences in FasL staining in the residual tumors compared with
Correlation between Tumor FasL Staining and Sur-
Baseline and on-treatment tumor FasL expression were
examined and compared with survival. However, no statistically
significant correlations with survival were shown (log-rank P ?
0.89 and 0.64, respectively).
Correlation between Tumor-Infiltrating Lymphocytes
and Objective Response.
Baseline and on-treatment intratu-
moral lymphocyte subsets defined by immunohistochemistry
were evaluated and correlated with objective response. At base-
line, the absolute number of CD4, CD8, CD56, and CD57 cells/
mm2tumor tissue was significantly higher in responding pa-
tients (PR) compared with nonresponding patients (SD?PD);
A total of 95 patients treated
Clinical Cancer Research
P ? 0.01, P ? 0.008, P ? 0.015, and P ? 0.001, respectively;
Fig. 1). During the first course of immunotherapy, the absolute
number of CD4, CD8, and CD57 cells per mm2tumor tissue
was significantly higher in responding patients compared with
nonresponding patients (P ? 0.034, P ? 0.001, and P ? 0.001,
respectively), whereas no significant difference was observed
for intratumoral CD56 cells (P ? 0.53; Fig. 1).
Correlation between Tumor-Infiltrating Lymphocytes
and Tumor Cells Expressing FasL.
baseline or on-treatment tumor-infiltrating CD4, CD8, CD56, or
CD57 lymphocyte subsets and FasL expressing tumor cells were
evaluated by use of the Spearman’s correlation test. However,
no statistically significant correlations were found (data not
Fig. 1 A–D, baseline and on-treatment intratumoral immune cells as predictors of response in 57 patients with metastatic renal cell carcinoma
undergoing IL-2–based immunotherapy for patients obtaining PR, SD, and PD. The data points represent the absolute number of lymphocyte subsets
per mm2tumor tissue in individual patients for A, CD4?; B, CD8?; C, CD56?; and D, CD57?. E, tumor cell FasL staining with no correlation to
response, assessed in a semiquantitative fashion: 0, no intensely stained cells; 1?, 1 to 10% intensely stained cells; 2?, 11 to 50% intensely stained
cells; 3?, 51 to 90% intensely stained cells; 4?, ? 90% intensely stained cells. Only patients with a complete set of two data points are displayed
in Fig. 1.
7914 FasL in mRCC During IL-2–Based Immunotherapy
This is to our knowledge the first in vivo serial assessment
of tumor tissue FasL expression during IL-2–based immuno-
therapy. Although the explanation for the low level of responses
seen in IL-2– and IFN-?–based immunotherapy is probably
extremely complex, the hypothesis that tumor cells expressing
FasL may escape the immune response and counterattack
tumor-infiltrating lymphocytes by delivering apoptotic death
signals has intuitive appeal. However, this hypothesis is not
supported by our data. We showed that tumor FasL expression
was not correlated with objective response or survival, whereas
high numbers of intratumoral lymphocyte subsets at both base-
line and during treatment were significantly positively corre-
lated with objective response. Moreover, no correlation, neither
negative nor positive, was observed between the number of
intratumoral lymphocytes and tumor FasL expression. Thus, in
the tumor milieu during IL-2–based immunotherapy, FasL-
mediated counterattack seems to be less important than would
be predicted from in vitro assays, suggesting that the FasL
counterattack hypothesis is of no significance in vivo.
After the initial publications in 1996 (5), many types of
tumor have been reported to express FasL (2, 4–10). Moreover,
FasL expression has been reported to be negatively correlated
with prognosis in melanoma (31) and in breast (32), ovarian
(33), liver (34), and renal cell carcinoma (RCC; ref. 25). How-
ever, the counterattack hypothesis is controversial and has been
questioned recently, primarily based on major concerns about
laboratory methods and reagents (13, 35) including the use of
possible nonspecific antibodies in the analyses (36, 37). A
revised hypothesis has been proposed, suggesting that FasL is
expressed by T lymphocytes upon activation after tumor cell
recognition, causing them to kill themselves (“suicide”) and also
each other (“fracticide”; ref. 13). It has also been suggested
that melanoma cells express FasL intracellularly confined to
lysosomal-like microvesicles and that these FasL bearing mi-
crovesicles can be released extracellularly mediating FasL cell
death that does not necessarily imply cell-to-cell contact (3).
Nevertheless, in all these models, it is the presence of the tumor
that initiates the events culminating in immune cell death (17).
In 1999, Uzzo et al. (18) provided the first evidence that
FasL is expressed in RCC cell lines and renal tumor tissues, and
they also suggested that FasL was functional. Because of the
controversy over the specificity of certain commercially avail-
able anti-FasL antibodies (36), Uzzo et al. (18) documented
FasL in RCC by multiple techniques. For the immunohisto-
chemical analyses, they used the clone 33 antibody. Therefore,
we also used this antibody in the present study. For comparison,
we also used the G247-4 antibody (37). However, we were not
able to find detectable levels of FasL using G247-4. Gerharz et
al. (21) were also unable to detect FasL using the G247-4
antibody in RCC cell lines that had been shown to be positive
for FasL expression by RT-PCR. Only after immunoprecipita-
tion before Western blotting, G247-4 antibodies revealed ex-
pression of FasL in RCC cells (21). Therefore, they concluded
that tumor FasL was present, but only weakly in RCC, and that
its detection depended on the sensitivity of the analytical meth-
ods used (21).
Although the ultimate test of a molecules role in disease
pathology is whether its expression correlates with the patients
outcome, we assessed the role of tumor FasL expression in vivo
during “extreme conditions,” (i.e., during manipulation of the
immune system by IL-2 and IFN-? to induce tumor regression).
Thus, objective response was selected as end point whereas
tumor shrinkage seems a more valid end point than numbers of
apoptotic tumor-infiltrating lymphocytes. Moreover, this end
point better reflects the “attack-counterattack” model used.
However, independently of tumor FasL expression levels, we
observed in situ both at baseline and during the first course of
IL-2–based immunotherapy, a significantly higher number of
tumor-infiltrating lymphocytes in patients who subsequently
achieved response compared with nonresponding patients. This
observation challenges the FasL tumor “counterattack” hypoth-
esis during IL-2–based immunotherapy.
Indeed, immune privilege cannot be associated with a
single protective FasL-dependent mechanism but rather in-
volves an intricate orchestration of different processes (38).
Moreover, it has been shown that tumor specific CD4?and
CD8?T cells that are isolated from melanoma lesions seem to
be resistant to tumor FasL expression, and thus they have
developed strategies for overcoming FasL escape mechanisms
(39). This indicates that the role of FasL in the homeostatic
regulation of immune responses seems to be much more com-
plex than initially thought. A direct impact on tumor FasL
expression for IL-2, IFN-?, or histamine has, to the best of our
knowledge, not been reported.
Repeated attempts have been made to identify variables
important for objective response and survival in patients with
mRCC undergoing IL-2–based immunotherapy to select those
patients most likely to benefit from treatment. However, our
data suggests that the fate of a patient with mRCC before IL-2–
and IFN-?–based immunotherapy cannot be determined by
measuring tumor FasL expression. Either mRCC tumor FasL
expression in vivo is not functional or tumor-infiltrating lym-
phocytes have developed strategies for overcoming this escape
mechanism, thus supporting the development of immunothera-
peutic strategies for the treatment of mRCC.
In summary, we provide the first quantitative analysis of
the clinical relevance of tumor FasL expression in mRCC at
baseline and during IL-2–based immunotherapy. Our observa-
tions do not support the hypothesis that FasL tumor “counter-
attack” has an effect on the clinical outcome in mRCC during
Thanks go to Karin Vestergaard for cutting sections and to Tom
Nordfeld for help with immunohistochemistry. Staff members at the
Department of Oncology are acknowledged for careful management of
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7916 FasL in mRCC During IL-2–Based Immunotherapy