[Cancer Research 60, 3132-3136, June 15, 2000]
© 2000 American Association for Cancer Research
Vaccine-induced Apoptosis: A Novel Clinical Trial End Point?1
Schwann Amin,
R. Adrian Robins,
Charles A. Maxwell-Armstrong,
John H. Scholefield and
Lindy G. Durrant2
Departments of Surgery [S. A., C. A. M-A., J. H. S.], Immunology [R. A. R.], and Clinical Oncology [L. G. D.], University of Nottingham, Nottingham NG5 1PB, United Kingdom
 |
ABSTRACT
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The functional end point of immunotherapy is to induce tumor regression.
Because immune effector mechanisms usually result in apoptosis, the aim
of this study was to determine whether measurement of tumor apoptosis
ex vivo is a good end point to evaluate the efficacy of
cancer vaccines. A prototype vaccine, 105AD7, was administered to
colorectal cancer patients before resection of their primary tumors.
There was a significant increase in apoptosis of tumor cells within
immunized patients compared with control patients as assessed by
immunohistochemistry (P = 0.005;
n = 16) or by flow cytometry
(P = 0.003; n
= 34). Preoperative immunization and measurement of tumor cell
apoptosis may be a valuable clinical end point for evaluation of new
vaccine and other biological approaches.
|
Introduction
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A better understanding of the molecular basis for immune
recognition has led to the development of many new and exciting
approaches to cancer immunotherapy (1)
. There is an
increasing need to adopt a rational trial design to assess the
antitumor effects of these approaches. Phase I toxicity trials are
necessary to verify the safety of any new agent. However, trials
designed to assess maximum tolerated doses are not applicable for
immunotherapeutics because increasing the dose does not necessarily
increase the effectiveness of the agent (2)
. It may be
possible to optimize immunotherapy by monitoring immune responses
induced in patients. However, in several recent trials in melanoma,
tumor regression was observed in the absence of peripheral blood immune
responses (3
, 4)
. Because immune cells only transit in the
blood en route to target tissues, the most effective
antitumor immunization would result in immune responses within the
tumor; however, this may be difficult to assess in advanced solid
tumors. A further complication is that very few immunotherapeutics
induce objective regression of advanced cancer. This may be because the
immune response stimulated is insufficient to have a significant impact
on bulky disease, although other factors associated with advanced
disease such as immunosuppressive effects and evolution of escape
mechanisms by the tumor may also be involved. Immunotherapy may be more
successful when administered to patients with minimal residual disease
(5)
. For example, the
mAb3
17-1A was able to reduce recurrence and enhance survival when
administered to Dukes’ C cancer patients (6)
.
Unfortunately, this trial required 189 patients and took 5 years to
evaluate. Thus, large numbers of patients would be involved in studies
lasting several years, making it unfeasible to assess numerous new
approaches simultaneously. There is therefore a need to design rapid
trials in patients with early-stage tumors to allow selection of the
most promising agents. New therapies that have been shown to be
nontoxic in Phase I clinical trials could be used to immunize patients
at diagnosis before surgical resection. This would allow direct
monitoring of immune responses on the resected tumor.
105AD7 is a human anti-idiotypic antibody that mimics the CD55 antigen
(7)
. In a Phase I clinical trial in advanced colorectal
cancer patients, both helper and cytotoxic immune responses were
induced in the peripheral blood (8)
. T cells that
proliferated to either 105AD7 or to tumor cells expressing CD55 antigen
were measured (9)
. Enhanced plasma interleukin 2 was
detected, and an accumulation of CD4/CD45RO and CD8/CD45RO cells was
seen with successive immunizations. However, although three patients
showed prolonged periods of stable disease, no regression of any liver
metastases was observed. We therefore assessed immune infiltration in
metastatic colon cancer by evaluating hepatic metastases; however,
insufficient tissue was obtained from most patients to give a
representative view of the lesion. A new trial was therefore designed
whereby patients were immunized at diagnosis and before resection of
their primary tumor. The resection specimen could then be analyzed for
immune effector cells. Enhanced infiltration of CD4 and NK cells was
observed in 105AD7-immunized tumors as compared with grade- and
stage-matched tumors from control patients (10)
.
Furthermore, the infiltrating cells within the immunized tumors
expressed significantly more CD25 than control tumors
(11)
. This suggested that the immune cells did reach the
tumor and remain active. However, the real goal was to show that these
activated cells could induce tumor cell death. Immune effector cells
cause apoptosis of their target cells either directly or by cytokine
release. We therefore investigated whether measuring tumor cell
apoptosis could be a sensitive method to assess the antitumor
effectiveness of the 105AD7 vaccine and whether this may represent a
rational approach for evaluation of other immunotherapies.
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Materials and Methods
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Patients.
This trial was run under the auspices of the CRC, United Kingdom, Phase
I Targeting Trial Committee. Local ethical approval was obtained from
the recruiting hospital. Patients with histopathologically proven
colorectal adenocarcinoma who were scheduled for elective surgery were
recruited. Patients had to have a WHO performance status of 0–2, a
hemoglobin of >10 g/dl, a WBC count of >2 x
109/liter, and platelets >
50 x 109/liter. All patients had
normal renal and liver function (no more than 25% deviation from
normal values). All patients gave written informed consent and were
registered with the CRC data center. Patients with any acute
intercurrent illness, with autoimmune or chronic hematological
disorders, or receiving other concomitant anticancer therapy were
excluded. No women of child-bearing age or receiving planned
preoperative radiotherapy to primary rectal tumors were included.
Patients could receive postoperative chemotherapy if indicated.
However, 3 months after the completion of chemotherapy, they
completed their postoperative 105AD7 course.
Human mAb.
Clinical grade human mAb was produced as described previously
(9)
using the guidelines of the CRC (12)
.
Samples of the seed lots passed testing for sterility and viral
contamination. Antibody for clinical use was prepared as either 10 µg
of antibody in sterile saline for skin test doses or as an aluminum
hydroxide gel (alHydrogel 85; Superphos Biosector, Vebaek, Denmark)
precipitated i.m. doses of 100 µg antibody/ml. The antibody can be
stored at 4°C. Stability studies have shown that the antibody can be
stored at 4°C for a minimum of 5 years with no loss in binding
activity.
Clinical Protocol.
Twenty-one of the total 50 patients recruited were immunized with the
human mAb 105AD7. The clinical protocol initially stated that patients
should receive an intradermal skin test of 105AD7 (10 µg), and if
there was no adverse reaction after 24 h, they could then receive
the i.m. dose of 105AD7 precipitated on alum (50 or 100 µg). Patients
received an initial dose of 105AD7 at diagnosis of their colorectal
cancer and then were boosted at 6-weeks, 12-weeks, and then at
3-month intervals after their surgical resection. The remaining
patients were not immunized and were treated as a control group.
Immunohistochemistry.
A pathologist took samples from two edges of the tumor where possible.
Tumor tissue from both immunized and nonimmunized (age- and
gender-matched) patients was stored in liquid nitrogen. Tumor blocks
from both the edges of an immunized tumor and a stage-, grade-, and
site-matched control tumor selected from the tumor bank were selected.
Sections (5 µm) were fixed in acetone for 10 min and labeled with mAb
APO 2.7 (Ref. 13
; Beckman Coulter, Luton, United Kingdom)
or normal mouse IgG1 (Sigma, Dorset, United Kingdom), using an indirect
avidin-biotin complex technique (Dako, Ely, United Kingdom).
Image Analysis.
Sections were viewed under x125 magnification, and the image was
digitized and transferred by camera to an Apple Macintosh Quadra 660AV
computer. Using the NIH image program, it was possible to quantify the
degree of staining and express it as a pixel count. Sections were
analyzed from two edges of the tumor. Infiltration was quantified on
five randomly selected areas for each section. Immunized and
nonimmunized tumors were coded before staining and analysis to ensure
that there was no observer bias. Pixel counts on immunized and matched
nonimmunized tumors were decoded and analyzed for significance by a
two-tailed paired Wilcoxon signed rank test.
Flow Cytometry.
Fresh samples were obtained from four growing edges of the tumor and
finely minced. They were then dissociated in 0.05% collagenase
(Boehringer Mannheim, Lewes, United Kingdom) and 0.1% DNase
(Boehringer Mannheim). Cells for Apo2.7 staining were fixed immediately
in 0.5% formaldehyde, and an aliquot of unfixed cells was stained with
FITC-labeled annexin V (Biowhittaker, Workingham, Berks, United
Kingdom) and propidium iodide according to the manufacturer’s
protocol. Saponin (0.1%; Sigma Chemicals, Poole, United
Kingdom)-permeabilized cells were stained with
Apo2.7-PE-conjugated mAb (Beckman Coulter, Luton, United
Kingdom) or PE-conjugated mouse IgG in control tubes, together
with the epithelial marker BerEp4 FITC-conjugated mAb (Dako) or the
leukocyte marker CD45 FITC-conjugated mAb (Dako). BerEp4 binds to both
normal and malignant epithelial cells. Analysis was performed on a
Becton Dickinson FACScan, and 5,000–10,000 events were collected;
Apo2.7 staining was measured in epithelial cells and leukocytes gating
on the BerEp4-positive cells and CD45-positive cells, respectively.
The percentage of apoptotic cells within immunized tumors was compared
with stage-, grade-, and site-matched tumors from nonimmunized patients
and analyzed for significance by a two-tailed paired Wilcoxon signed
rank test. Nonpaired data were analyzed for significance using a
nonparametric Wilcoxon signed rank test.
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Results
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The cancer vaccine 105AD7 had been shown to induce enhanced
infiltration of T cells and NK cells within the tumors of immunized
patients as compared with control patients matched for age, gender, and
tumor location, grade, and stage. These tumors were therefore analyzed
for apoptosis. Tumor sections were cut and stained by indirect
immunoperoxidase staining with mAb Apo2.7. Three sections were analyzed
from each block, and five fields were quantified by image analysis on
each section (Table 1)
. There was a significant increase in apoptosis in the 105AD7-immunized
tumors compared with control tumors (median, 3.12 pixel count
versus 1.78 pixel count; P
< 0.005) when analyzed by a Wilcoxon two-tailed test paired
comparison for immunized versus control tumors for each
region. Tumors from immunized and control patients were also stained
with an IgG1 control antibody. Staining was low, and there was no
significant difference between immunized and control tumors. The pixel
counts for Apo2.7 for each tumor were then summated, and Fig. 1
shows the cumulative pixel count for each immunized tumor as compared
with its matched control. Six of eight immunized patients had higher
levels of apoptosis than matched controls. To attempt to exclude
sampling error as a possible explanation for this, a new prospective
study was designed whereby all tumor tissue not necessary for
pathological evaluation was disaggregated, stained, and analyzed by
flow cytometry.
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Table 1 Apo2.7 immunohistochemical staining and image analysis of tumor from
patients immunized with 105AD7 or matched control tumors
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Cell death within resected tumors was initially measured by Apo2.7,
which measures the mitochondrial antigen 7A6 that is exposed as an
early event of apoptosis. Tumor cells were disaggregated by a 1-h
incubation with collagenase and then fixed before Apo2.7 staining, thus
minimizing the time and processing steps to which the unfixed cells
were subjected. Apo2.7 staining also had the advantage that dual
staining with cell type-specific antibodies could be used to identify
the cells undergoing apoptosis. Thirteen patients were immunized with
105AD7 preoperatively (median, 14 days between immunization and
surgery). There were eight males and five females; six patients had
rectal tumors, and seven patients had colon cancers. All tumors were of
moderate grade. One patient had Dukes’ A cancer, five patients had
Dukes’ B cancer, and seven patients had Dukes’ C cancer. Twenty-one
tumors from nonimmunized patients were also processed. There were 12
males and 9 females; 8 patients had rectal tumors, and 13 patients had
colon tumors. All tumors were of moderate grade. One patient had
Dukes’ A cancer, 7 patients had Dukes’ B cancer, 12 patients had
Dukes’ C cancer, and 1 patient had Dukes’ D cancer. Similar
proportions of epithelial cells (53% versus 56%) and
leukocytes (12% versus 10%) were released by collagenase
from the immunized and control tumors. Fig. 2
shows a scatter plot of the proportion of apoptotic cells from
immunized and control tumors. There is an increase in apoptosis in the
tumor cells of the immunized patients as compared with nonimmunized
tumor cells (22% versus 7.8%; P
= 0.0023). However, there was no difference in apoptosis of
leukocytes from either immunized or control tumors (7.2%
versus 5.5%; P = 0.55).
Interestingly, the level of apoptosis in the tumors of nonimmunized
patients was similar to the level of apoptosis of the intratumor
leukocytes (7.8% versus 5.5%;
P = 0.50) from these tumors, suggesting that
both cell types had a similar turnover.
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Fig. 2. Tumors from 105AD7-immunized (I) or
nonimmunized control patients (C) were disaggregated
with collagenase/DNase. Tumor cells stained with the antiepithelial
antibody BerEp4 (T) or leukocytes stained with the
antileukocyte antibody CD45 (L) were gated and analyzed
for costaining with the antiapoptotic mAb Apo2.7.
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Thirteen nonimmunized patients were selected as age-, gender-, site-,
stage-, and grade-matched controls for the vaccine-immunized patients
(Fig. 3
). Apoptosis was significantly elevated in the tumors of immunized
patients compared with control tumors (22% versus 7.0%;
P = 0.003). However, there was no difference
in the proportion of apoptotic leukocytes from immunized tumors as
compared with control tumors.
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Discussion
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Although early trials with nonspecific immunotherapy including
interleukin 2 therapy, adoptive transfer of tumor-infiltrating
lymphocytes, and whole cell vaccines have shown some encouraging
results (14)
, these results were frequently seen on only a
small subset of cancers and usually in melanoma patients. In the
last decade, a much better understanding of the molecular basis for
immune recognition and activated has been elucidated. This has led to
the identification of new antigens recognized by T cells and to many
new approaches to specifically activate antitumor immunity (1
, 14)
. However, it is still unclear how these new agents should be
evaluated in the clinic because few Phase I trials have resulted in
regression of advanced disease. This may be a reflection of a numerical
balance between tumor cells and immune effector cells that may be
irrevocably tipped in favor of tumor in patients with bulky disease
and/or the tumors acquiring resistance to immune attack.
An effective cancer vaccine must stimulate all arms of the immune
response including helper T cells that aid both antibody and cytotoxic
T-cell production but also release cytotoxic cytokines (IFN-
and
tumor necrosis factor ß) and recruit nonspecific effector cells such
as NK cells and tumoricidal macrophages. All of these immune mediators
may induce apoptosis in their target tumor cells. Measurement of tumor
cell apoptosis may therefore prove to be a good method of assessing the
overall efficacy of a vaccine. In colorectal cancer patients, there is
a window of opportunity of 2–3 weeks between diagnosis and resection
of the primary tumor to allow for such an evaluation to be made. This
study shows that if patients are immunized with the prototype vaccine
105AD7 immediately after diagnosis of their tumor, it is possible to
measure antitumor effects of the vaccine by measuring immune cell
infiltration and by enumerating the degree of apoptosis of tumor cells
in the resected cancer.
The 105AD7-immunized tumors exhibited enhanced apoptosis compared with
matched control tumors as assessed by immunohistochemistry with mAb
Apo2.7 (13)
. This suggested that 105AD7 stimulated immune
cells that infiltrated the primary tumor and caused extensive cell
death. However, quantitative image analysis is difficult because
infiltration can vary greatly in different regions of the tumors, and
slight variations in tissue thickness can also have dramatic effects on
results. To avoid these problems, and also because it was unclear
whether the enhanced apoptosis was a result of the immune effector cell
or tumor cell death, a prospective study of 13 patients immunized with
105AD7 and 21 nonimmunized control patients was therefore performed to
address these issues. Immunized patients were injected with
105AD7 at diagnosis of their colorectal tumor, and control patients
were not immunized. Apoptosis in this study was measured on
disaggregated tumor samples collected at resection.
Apoptosis was initially measured by annexin V staining, but the
processing time and the necessity of staining, washing, and analyzing
unfixed cells resulted in increased tumor cell death. Apo2.7 staining
was possible on cells processed and fixed much more quickly, and the
percentage of apoptotic tumor cells in control tumors using this
protocol more closely correlated with levels reported by terminal
deoxynucleotidyl transferase-mediated nick end labeling staining of
colorectal tumor tissue sections (3.6–11%; Ref. 15
). The
105AD7 cancer vaccine induced a 3-fold increase in the proportion of
apoptotic tumor cells in 13 immunized patients compared with stage-,
grade-, and site-matched nonimmunized controls, and the degree of
apoptosis in control tumors was similar in all Dukes’ stages. Earlier
studies have shown that apoptosis decreases in the transition from
normal mucosa to polyp to carcinoma, but that subsequent tumor
progression has little effect (16)
. It was of interest
that the proportion of apoptotic tumor cells was similar to the
proportion of apoptotic leukocytes in control tumors, suggesting that
both populations have a similar turnover. Also there was no difference
in the number of apoptotic leukocytes between immunized and control
tumors, suggesting that there was no evidence for dramatic killing of
infiltrating leukocytes in the tumor environment. In contrast,
immunization with 105AD7 selectively results in tumor cell apoptosis.
There has been much debate as to whether tumor vaccines targeting
overexpressed self-antigens can stimulate antitumor immune responses.
However, there is now a plethora of data to support this. There has
also been a lack of direct evidence that the immune response observed
in the blood of immunized patients would be mirrored in the tumor
environment. In fact, there is evidence that the tumor environment is
particularly hostile to cell-mediated immunity, with reports of tumors
expressing transforming growth factor ß and Fas ligand, although
recent reports have suggested that Fas ligand is more likely to
stimulate rather than suppress an inflammatory response
(17, 18, 19)
. In this study, it has been possible to
show, at least in primary colorectal cancer, that a vaccine mimicking
CD55 antigen can induce immune responses that result in a significant
increase in tumor cell apoptosis. We would suggest that this trial
design (using an apoptosis index) could represent a valid end point for
Phase II trials assessing immunotherapy. It would also be possible to
assess tumor infiltration by immune effector cells. 105AD7 induced
infiltration of NK cells and T cells that expressed the activation
marker CD25 as assessed by quantitative immunohistochemistry
(11)
. However, recent advances in immunology may allow a
more accurate quantification of vaccine-specific T cells within both
the blood and the tumor of immunized patients. MHC tetramers
(20)
or MHC Fc chimeric molecules (21)
can be
folded around specific T-cell epitopes and then used to stained for
epitope-specific T cells. Similarly, short-term in vitro
culture with antigen and measurement of intracellular cytokines can
give accurate frequencies of antigen specific T cells
(22)
.
The neoadjuvant approach can measure immune infiltration of tumors and
can be used to assess the ability of the induced immune response to
kill tumor cells within primary lesions. This in itself will justify
for each individual patient a rationale for continuing immunization.
However, it may not predict survival. We do not know the effects of
this therapy on micrometastases that may be left after surgery. The
effect on overall survival will need to be assessed in classical Phase
III trials with tumor recurrence and patient survival as the primary
end points. Likewise, to achieve long-term survival, it may be
necessary to induce a sustained immune response over several years to
induce a continuous response to what is a self-antigen. Therefore,
although measuring tumor cell apoptosis in response to therapy may not
necessarily predict outcome, it may be sufficient to justify large
Phase III randomized trials to evaluate new immune therapies that
result in increased apoptosis at the primary tumor site. These trials
can be performed rapidly because response is evaluable within 3–4
weeks at tumor resection. Measuring tumor apoptosis with Apo2.7 is
sensitive, accurate, and simple, and in this trial, using colorectal
cancer vaccine 105AD7, Apo2.7 shows significant differences between
treated and control patients. Additional studies are needed to
test this approach in the evaluation of other immunotherapies.
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FOOTNOTES
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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.
1 Supported by CRC Program Grant SP2220/0501 and a
grant from the Lewis Charitable Trust. 
2 To whom requests for reprints should be
addressed, at Academic Department of Clinical Oncology, City Hospital,
Hucknall Road, Nottingham NG5 1PB, United Kingdom. Phone:
0044-115-9628033; Fax: 0044-115-9627923;
3 The abbreviations used are: mAb, monoclonal
antibody; NK, natural killer; CRC, Cancer Research Campaign.
Received 2/ 2/00.
Accepted 5/ 1/00.
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ABSTRACT
Introduction
)
or nonimmunized controls (
) were stained by indirect
immunoperoxidase staining with the antiapoptotic mAb Apo2.7. Cumulative
pixel counts for each tumor are presented.