2006;12:3355-3360.

Clin Cancer Res

Gerhard G. Grabenbauer, Godehard Lahmer, Luitpold Distel, et al.

Cells Predict Outcome in Anal Squamous Cell Carcinoma
Tumor-Infiltrating Cytotoxic T Cells but not Regulatory T

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Tumor-Infiltrating CytotoxicT Cells but not RegulatoryTCells
Predict Outcome in Anal Squamous Cell Carcinoma
Gerhard G. Grabenbauer,
1
Godehard Lahmer,
1
Luitpold Distel,
1
and Gerald Niedobitek
2
Abstract Purpose: Tumor-infiltrating lymphocytes (TIL) are a possible prognostic factor in solid tumors.
CytotoxicTILs are generally considered as prognostically favorable, whereas regulatoryTcells
(Treg) may have adverse effects by virtue of their ability toinhibit effector cells.We have evaluated
the effect of T-cell subsets on survival in patients with anal squamous cell carcinoma following
radiochemotherapy.
Methods: Biopsy specimens from38 patients with anal carcinomas were evaluatedusing tissue
microarrays and immunohistochemistry for the presence of tumor-infiltrating immune cells using
CD3, CD4, CD8, and CD68 antibodies. Treg were identified using an antibody directed against
the transcriptionfactor FoxP3, andgranzyme Bservedas a marker for cytotoxic cells. Intratumoral
immune cells were enumerated using a semiautomatic image analysis program. Prognostic effect
of TIL subsets was evaluated by the log-rank test comparing no evidence of disease survival for
groups with high and lownumbers using median values as cutoff.
Results: CD3
+
and CD4
+
TILs influenced no evidence of disease survival: 3-year rates for
patients with low numbers were 89% and 95%, respectively, and 54% (P = 0.02) and 48%,
(P = 0.01), respectively, in cases with high numbers. Large numbers of tumor-infiltrating gran-
zyme B
+
cytotoxic cells had a significant negative prognostic effect (P = 0.008), whereas no
effect was observed forTreg.
Conclusions: TILs were identified as negative prognostic indicators in anal squamous cell carci-
nomas with granzyme B
+
cytotoxic cells showing highest effect on outcome. This is possibly
explained by the selection of therapy-resistant tumor cell clones. No prognostic influence of Treg
was found. Knowledge of local immune responses is important for the development of immuno-
therapeutic strategies.
Anal squamous cell carcinoma is a comparatively rare
neoplasm with an incidence for men and women of f2.1
per 100,000 in the United States (1). Anal squamous cell
carcinoma is highly radiosensitive and chemosensitive. In
patients with early-stage disease, local control rates and 5-year
survival rates with no evidence of disease (NED) in the range of
70% to 80% are achieved. In advanced-stage disease, cure rates
of around 40% to 50% may be obtained following radical
radiochemotherapy (2, 3). Nevertheless, a proportion of
patients fails to achieve long-term remission.
Human papillomaviruses (HPV), most commonly the high-
risk HPV types, have been detected in >80% of cases (4, 5). This
suggests that HPV may be involved in the pathogenesis of anal
squamous cell carcinoma. In addition, the virus may provide a
target for immunotherapeutic approaches. Indeed, vaccination
using a vaccinia virus construct expressing the transformation-
associated HPV-encoded proteins E6 and E7 has been shown to
be immunogenic in patients with advanced cervical carcinoma
and in patients with vulvar intraepithelial neoplastic (6, 7). In
the latter study, 8 of 18 patients showed a reduction in lesional
size following vaccination. This was associated with higher
numbers of intralesional immune cells (Langerhans cells and
CD4
+
and CD8
+
T cells) before vaccination, suggesting that
boosting of a preexistent immune response may have been
instrumental (7).
Tumor-infiltrating lymphocytes (TIL) are found in a variety of
solid cancers and have been considered to be a manifestation of
a host immune response directed against cancer cells. In support
of this notion, several studies have shown that the presence of
large numbers of tumor-infiltrating CD8
+
T cells or natural killer
T cells are associated with a favorable prognosis in colorectal
(810), ovarian (11), pancreatic (12), and esophageal carcino-
ma (13, 14). In colorectal carcinoma, TILs are particularly
numerous in cases associated with microsatellite instability
(9, 15). In these cases, microsatellite instabilityassociated
mutations may lead to the expression of altered proteins and
may render the tumor cells more immunogenic, thus offering
an explanation for the favorable clinical outcome (15). This
Imaging, Diagnosis, Prognosis
Authors Affiliations:
1
Department of Radiation Oncology and
2
Institute for
Pathology, Friedrich Alexander University, Erlangen, Germany
Received11/9/05; revised 3/4/06; accepted 3/24/06.
Grant support: Interdisciplinary Centre for Clinical Research of the Friedrich
Alexander University of Erlangen Nuremberg (G.G. Grabenbauer and G. Niedobitek).
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
with18 U.S.C. Section1734 solely to indicate this fact.
Requests for reprints: Gerhard G. Grabenbauer, Department of Radiation
Oncology, University Hospital, Universita tsstr. 27, 91054 Erlangen, Germany.
Phone: 49-9131-8534086; Fax: 49-9131-8534185; E-mail: gg@strahlen.
imed.uni-erlangen.de.
F2006 American Association for Cancer Research.
doi:10.1158/1078-0432.CCR-05-2434
www.aacrjournals.org Clin Cancer Res 2006;12(11) June1, 2006 3355
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scenario should also apply to virus-associated cancers because
virus-encoded antigens expressed in the neoplastic cells may
represent neoantigens targeted by the immune system. Indeed,
immunotherapeutic strategies targeting HPV- or EBV-encoded
proteins are currently being developed for virus-associated
tumors (16, 17). However, Oudejans et al. have shown a rapid
fatal outcome among patients with EBV-associated nasopha-
ryngeal carcinoma displaying high levels of granzyme B/CD8
+
TILs (18). This observation might be explained by a disruption
of proapoptotic pathways (19). Similar results have also been
reported for renal carcinoma and nonsmall cell lung cancers
(20, 21). The recent description of CD4
+
/CD25
+
regulatory T
cells (Treg) has added another level of complexity to this issue
(22). Treg have the ability to inhibit effector T cells by cell-
cell contact or through the secretion of cytokines, such as
interleukin-10 or transforming growth factor-h (22). In animal
experiments, depletion of Treg from a murine fibrosarcoma
rendered the tumors immunogenic and led to tumor rejection
(23). Curiel et al. have shown that intratumoral Treg are
predictive of poor prognosis in patients with ovarian cancer
(24). Thus, the development of immunotherapeutic
approaches to the treatment of solid tumors targeting tumor
antigens (viral or cellular) requires a thorough knowledge
of local immune responses. We have, therefore, studied the
prognostic significance of TILs in a series of anal carcinomas
focusing on T-cell subsets, including Treg.
Materials and Methods
Patient selection. Between 1985 and 2001, a total of 112 patients
with anal carcinoma were admitted for treatment by combined
radiochemotherapy to this University Hospital. For this study, a
subgroup of 38 patients was selected according to the criteria given in
Fig. 1. This project received approval from the Interdisciplinary Clinical
Research Center of the University Hospital of the Friedrich Alexander
University.
All patients included in the study were HIV negative. Following
clinical and rectal examination, a biopsy was taken under general
anesthesia. All patients had rectoscopy, including endorectal ultra-
sound, whenever feasible. Staging was completed by computed
tomography scans of the abdomen and pelvis, chest X-rays, and liver
Fig. 1. Patient selection criteria.
Fig. 2. Biopsy specimens were processed
into tissue microarrays using a 2-mmneedle
core (A). Immunohistochemistry was
used to identify CD3
+
Tcells (B), FoxP3
+
regulatoryTcells (C, red nuclear labeling),
and granzyme B
+
cytotoxic cells
(D, red granular cytoplasmic labeling).
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ultrasound. Histopathologic diagnosis was established according to
WHO criteria (25). T categories were T1 (6), T2 (17), T3 (11), and
T4 (4). No patients with small perianal lesions (<2 cm) treated by
complete excision were included in this protocol. Twenty-four patients
had no evidence of nodal disease, whereas 14 initially presented
clinically with involved regional nodes. Nodes of z1.5 cm in largest
diameter were judged as positive.
Treatment protocol. Radiation treatment was directed to the primary
tumor region, inguinal, perirectal, and internal iliac/presacral nodes
using a four-field box technique with 10 to 15 MV photons. Dose
prescription followed the International Commission on Radiation
Units report no. 50 guidelines with single fractions of 180 cGy and a
total dose of 5,040 cGy. An additional local boost was applied to larger
(>4 cm) primaries either by external multiple-field techniques (540-900
cGy) or interstitial radiation using Iridium-192 low-dose-rate or pulsed
dose rate (1,200-1,600 cGy) depending on circumferential extension of
the disease. All patients were scheduled for simultaneous chemotherapy
with two cycles of 5-fluorouracil at a dose of 1,000 mg/m
2
/24 hours as
a 120-hour continuous i.v. infusion on days 1 to 5 and 29 to 33 and
mitomycin C at 10 mg/m
2
/d on days 1 and 29 as a single i.v. bolus
Table1. Relation of clinical and immunohistochemical
variables to overall survival and NED survival
Factors* Overall
survival (3 y) %
P NED
survival (3 y) %
P
Tcategory
T1/T2 (n = 24) 89 0.2 79 0.25
T3/T4 (n = 14) 75 63
Ncategory
N0, X (n = 24) 94 0.07 83 0.13
N+(n = 14) 64 51
Age
>62.4 (n = 12) 93 0.77 80 0.57
V62.4 (n = 26) 72 65
Gender
Male (n = 10) 60 0.08 80 0.61
Female (n = 28) 91 69
CD3
V3.8 (n = 19) 84 0.06 89 0.02
>3.8 (n = 19) 81 54
CD4
V1.5 (n = 19) 90 0.06 95 0.01
>1.5 (n = 19) 76 48
CD8
V2.1 (n = 19) 85 0.4 89 0.09
>2.1 (n = 19) 81 53
Granzyme B
V0.6 (n = 19) 92 0.08 89 0.008
>0.6 (n = 19) 74 47
FoxP3
V2.0 (n = 19) 88 0.22 67 0.97
>2.0 (n = 19) 78 76
CD68
V4.4 (n = 19) 95 0.2 70 0.75
>4.4 (n = 19) 72 74
Activated caspase-3
V1.9 (n = 19) 89 0.7 70 0.7
>1.9 (n = 19) 77 74
*
Figures givenfor CD3, CD4, CD8, granzyme B, FoxP3, and CD68 refer to the
number of TIL per 100 tumor cells and for activated caspase-3 to the percent-
age of labeled tumor cells. Median values were used as cutoff (see text for
details).
Fig. 3. NEDsurvival rates in relation to the intratumoral accumulation of Tcells for
CD3
+
Tcells (top), CD4
+
Tcells (middle), and CD8
+
Tcells (bottom).
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injection. Six weeks after radiochemotherapy, tumor response was
evaluated clinically and by means of proctoscopy, endorectal ultra-
sound, and computed tomography scans of the pelvis. All patients had
regular follow-up in 3-month intervals during the first 2 years and in
6-month intervals thereafter. The median follow-up was 25.5 months
with a range between 2 and 147 months.
Tissue microarray and immunohistochemistry. Paraffin-embedded
biopsy samples obtained from 38 patients with anal squamous cell
carcinoma before therapy were processed into a tissue microarray
(BioCat, Heidelberg, Germany) using a core diameter of 2 mm
(Fig. 2A). Between two and seven cores were taken per patient (mean
= 2.8), resulting in a total of 120 cores. All materials had been
submitted for diagnostic purposes and were used in accordance with
national ethical principles. Immunohistochemistry of paraffin sections
was carried out using a standard streptavidin biotinylated alkaline
phosphatase (ABC-AP, DakoCytomation, Hamburg, Germany) method
or tyramide signal amplification followed by ABC-AP (only for FoxP3).
The following antibodies were used: CD3 (DakoCytomation; Fig. 2B),
CD4 (Novocastra, Newcastle upon Tyne, United Kingdom), CD8,
CD68 (DakoCytomation), FoxP3 (Abcam, Cambridge, United King-
dom; Fig. 2C), granzyme B (DakoCytomation; Fig. 2D), total caspase-3
(DakoCytomation), and activated caspase-3 (Cell Signaling Technolo-
gy, Danvers, MA). Using a standard light microscope, images were
acquired by a CCD camera, transferred to a Personal computer, and
counted using the image analysis software COUNT (Biomas, Erlangen,
Germany). Numbers of labeled tumor-infiltrating cells were deter-
mined per 100 tumor cells (labeling index). The average number tumor
cells counted per patient for each immunohistologic marker was 762
(range, 116-2,862). Between 0 and 17.3 CD3
+
T cells were detected
per 100 tumor cells (median = 3.8). For CD4
+
T cells, the median was
1.5 per 100 tumor cells (range, 0-11.4); for CD8
+
T cells, the median
was 2.1 per 100 tumor cells (range, 0-9.7); for FoxP3
+
Treg, the median
was 2 per 100 tumor cells (range, 0.3-7.3); for granzyme B
+
cytotoxic
cells, the median was 0.6 per 100 tumor cells (range, 0-9.1); and for
CD68
+
macrophages, the median was 4.4 per 100 tumor cells (range,
0.3-29.2). Average variation of labeling index within the two to seven
samples for all patients was quantitatively assessed. Mean labeling
index with SD was 0.044 F 0.018 for CD3, 0.02 F 0.008 for CD4,
0.025 F 0.011 for CD8, 0.009 F 0.003 for granzyme B, 0.064 F 0.025
for CD68, 0.022 F 0.007 for FoxP3, and 0.021 F 0.008 activated
caspase-3.
Statistical methods. Overall survival and NED survival rates were
calculated according to Kaplan-Meier (26). The log-rank test was used to
compare survival rates (27) between subgroups of patients. For all
immunohistochemical markers, cutoff for definition of subgroups was
the median value. Follow-up time was calculated as the interval between
end of chemoradiation and last follow-up or death. The following events
were defined as end points: death (overall survival) and the appearance
of local, nodal, or distant recurrence (NED survival).
Results
Overall survival and NED survival rates for the study
population were 83% and 72% at 3 years and 70% and 65%
at 5 years, respectively. Influence of clinical as well as
immunohistochemical variables on both survival end points
is shown in Table 1.
NED survival was strongly influenced by the intratumoral
accumulation of T cells (Fig. 3; Table 1). Intratumoral CD3
+
and
CD4
+
TIL had a significant effect on NED survival: 3-year rates
for patients with lower numbers of TIL (V3.8 CD3
+
per 100
tumor cells and V1.5%CD4
+
per 100 tumor cells) were 89%and
95%, respectively. Patients with higher numbers of TIL (>3.8%
CD3
+
per 100 tumor cells and >1.5% CD4
+
per 100 tumor
cells), by comparison, had survival rates of 54% (P = 0.02) and
Fig. 4. NEDsurvival rates in relation to the intratumoral accumulation of CD68
+
macrophages (top), FoxP3
+
regulatoryTcells (middle), and granzyme B
+
cytotoxic
cells (bottom).
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48%(P = 0.01), respectively. Similarly, higher numbers of CD8
+
TIL (>2.1 per 100 tumor cells) were associated with lower NED
survival of 53% versus 89% for patients with lower numbers
(V2.1 per 100 tumor cells), although this did not reach statistical
significance (Table 1; Fig. 3).
No prognostic influence was observed for the numbers of
tumor-infiltrating CD68
+
macrophages and of FoxP3
+
regula-
tory T cells (Table 1; Fig. 4). The TIL subpopulation showing
highest effect on outcome were granzyme B
+
cytotoxic cells.
Patients with comparatively large numbers (>0.6 cells per 100
tumor cells) showed a 3-year NED survival rate of 47%,
whereas patients with lower numbers fared significantly better
(3-year NED survival rate of 89%, P = 0.008; Table 1; Fig. 4).
Expression of total caspase-3 was detected in the neoplastic cells
of all cases. The number of tumor cells expressing the activated
caspase-3 had no influence on prognosis (Table 1). None of the
variables investigated showed a significant effect on overall
survival, most likely due to the relatively small number of cases.
Discussion
Here, we show that large numbers of tumor-infiltrating CD3
+
T cells are a significant negative prognostic marker in patients
with anal carcinoma. This is unlikely to be an epiphenomenon
(e.g., reflecting an inflammatory response to a more aggressive
invasive tumor growth) because numbers of CD68
+
macro-
phages showed no correlation with disease-free survival. Thus,
the observed effect seems to be T cell specific. Further subtyping
of T cells revealed that both CD4
+
and CD8
+
T cells were
associated with an unfavorable prognosis, although this reached
statistical significance only for CD4
+
cells. We, therefore,
investigated the possibility that increased numbers of Treg may
account for this observation. Treg are CD4
+
/CD25
+
T cells with
the ability to inhibit effector T cells (22, 28). In ovarian cancer,
the presence of Treg has been shown to be associated with poor
prognosis (24). Treg express various surface antigens, including
CD25, CD103, OX-40, CTLA-4, and the glucocorticoid-induced
tumor necrosis factor receptor, none of which seems to be
entirely specific for Treg (22). The transcription factor FoxP3 is
crucial for the development of Treg and is currently considered to
be the best Treg marker (29). To identify Treg in tissue sections,
we have, therefore, employed immunohistochemistry for the
detection of FoxP3. Using this approach, no relationship
between the number of tumor-infiltrating Treg and prognosis
was observed, suggesting that the effects of Treg observed in
ovarian cancer may not be relevant to other tumor models.
Previous studies have identified high numbers of granzyme
B
+
/CD8
+
TIL as a significant predictor of fatal outcome in EBV-
associated nasopharyngeal carcinoma as well as in Hodgkins
lymphoma (18, 30). This observation is confirmed here for
anal carcinoma. That large numbers of granzyme B
+
TIL were a
statistically significant adverse prognostic factor, whereas this
was not the case for CD8
+
TIL, probably indicates that the state
of activation rather than the mere presence of CTL is important
(30). The mechanism underlying this observation remains
uncertain. It has been speculated that T cell induced selection
of tumor cell clones resistant to apoptosis may explain this
phenomenon (19). However, in our study, tumor cells in all
cases expressed caspase-3, and the proportion of tumor cells
positive for activated caspase-3 showed no relation to
prognosis. Thus, in contrast to a previous study of nasopha-
ryngeal carcinoma (19), the absence of activated caspase-3 is no
indicator of poor clinical outcome in anal carcinoma. It
remains to be determined if other components of apoptotic
pathways are disrupted in anal cancer.
Previous studies addressing the possible prognostic signifi-
cance of TIL have determined T-cell subsets as a proportion of
the total TIL. Because our original hypothesis was that TILs
represent an immune response directed against the neoplastic
cells, we have chosen to determine the numbers of TIL subsets
in relation to the number of tumor cells, thus establishing an
effector/target ratio. Using this approach, we found that the
number of tumor-infiltrating CD3
+
, CD4
+
, CD8
+
, or granzyme
B
+
T cells compared with the number of neoplastic cells was
extremely small (medians = 3.8, 1.5, 2.1, and 0.6 cells per 100
neoplastic cells, respectively). It remains unclear if such a small
number of T cells would be able to mount a sufficient
antitumor immune response, assuming that they were directed
against the tumor cells. It is conceivable, however, that an
immune response insufficient to mediate effective tumor cell
killing may still be able to drive the selection of more aggressive
tumor cell subclones (e.g., resistant to apoptosis; ref. 18).
Negative prognostic effects of large numbers of activated
cytotoxic TIL have been reported for nasopharyngeal carcino-
mas and Hodgkins lymphomas, both representing EBV-
associated tumors (18, 30). By contrast, a positive prognostic
effect has been described for other malignancies [e.g., colorectal
(810), ovarian (11), pancreatic (12), and esophageal carci-
noma (13, 14)], which are not known to be associated with a
viral infection. It is interesting to note in this context that anal
carcinoma is commonly associated with HPV (4). It is possible,
therefore, that viral proteins expressed in the tumor cells may
render the neoplastic cells more resistant to apoptosis, or that
the expression of viral neoantigens in the tumor cells may alter
the nature of the local immune responses.
In summary, we show that tumor-infiltrating T cells are an
adverse prognostic marker in anal carcinoma. Contrary to
expectations, this is not attributable to the presence of Treg but
rather to granzyme B
+
cytotoxic cells. The detailed study of local
immune responses in carcinomas is important for the
development of immunotherapeutic strategies. Furthermore,
the composition of local T-cell infiltrates may be open to
therapeutic modulation (e.g., by eliminating Treg; ref. 28), thus
providing an additional tool in the treatment of carcinomas.
Acknowledgments
We thank ChristaWinkelmann for excellent technical assistance.
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