Clinical Cancer Research Vol. 6, 4663-4673, December 2000
© 2000 American Association for Cancer Research
Locoregional Cellular Immunotherapy for Patients with Advanced Esophageal Cancer1
Uhi Toh,
Hideaki Yamana2,
Susumu Sueyoshi,
Toshiaki Tanaka,
Fumihiko Niiya,
Katsuko Katagiri,
Hiromasa Fujita,
Kazuo Shirozou and
Kyogo Itoh
Departments of Surgery [U. T., H. Y., S. S., T. T., F. N., H. F., K. S.] and Immunology [U. T., K. K., K. I.], Kurume University School of Medicine, Kurume 830-0011, Fukuoka, Japan
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ABSTRACT
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The objectives of the present study were to determine the safety of
locoregional administration of autologous lymphocytes stimulated with
autologous tumor cells and interleukin (IL) 2 in vitro
and to find laboratory markers to predict either clinical toxicity or
clinical response. Eleven patients with advanced (n = 4) or recurrent (n = 7) esophageal cancers
received the locoregional administration of these activated lymphocytes
every 2 weeks for two to nine times (mean, 5.6 times), and mean numbers
of the administered cells were 0.8 x 109 cells
per treatment. The activated lymphocytes that were pretested for their
surface markers and CTL activity were endoscopically injected into
primary tumor sites (n = 4) or directly injected
into metastatic lymph nodes (n = 2), pleural
(n = 4) or ascitic (n = 1)
regions. Grade 3 hypotension, grade 2 diarrhea, and grade 1
fever were observed in 1, 1, and 6 patients, respectively, and there
was no adverse effect in the remaining three patients. The clinical
outcome was as follows: one, complete response (CR); three, partial
response (PR); two, stable response (SR); and five, progressive
disease (PD). CTL activity in the administered cells was observed in 5
of the 11 patients (1 CR, 3 PR, and 1 PD) and was not observed in the
remaining 6 patients (2 SR and 4 PD). Percentages of CD16+
cells in the peripheral blood of the responder group (CR+PR)
significantly increased when compared with those before treatment or
with those of the nonresponder group before as well as after treatment.
Because the clinical toxicity was moderate and tolerable, this new
method of locoregional immunotherapy will be applicable for use in
treatment of patients with advanced and recurrent esophageal cancers.
Both CTL activity in the administered cells and the percentages of
CD16+ cells in the peripheral blood may be useful
laboratory markers for predicting of clinical response.
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INTRODUCTION
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Cancer in the esophagus is one of most common malignant neoplasms
in the world, particularly in the Pacific countries. Surgery remains
the standard approach for patients with locoregional advanced disease
that is resectable. Curative resection is feasible in only 50% of
cases, and local or distant failure is common after resection
(1, 2, 3)
. The 5-year survival is only 
30% for stage-III
and -IV patients undergoing surgery. Some adjuvant multimodality
therapies have been attempted to control both local and systemic
disease (4, 5, 6)
. However, unresectable and relapsed
esophageal cancers are still resistant to the presently available
chemotherapy or radiation therapy regimens, and there is almost no
clear advantage from these regimens for overall survival. Consequently,
the development of a new effective therapeutic approach such as
immunotherapy could be valuable to expand treatment modalities
(7, 8, 9)
. Recently several reports presented the clinical
efficacy of immunotherapy for advanced cancer in the digestive tract,
but little clinical experience has been reported for advanced
esophageal cancer (10, 11, 12)
. We have reported the presence
of precursors of HLA class I-restricted and
SCC3
-specific CTLs in both PBMCs and TILs of patients with esophageal cancer
(13, 14, 15)
. In the present study, we investigated the
clinical toxicity and clinical response of locoregional administration
of PBMCs stimulated with autologous tumor cells in advanced and
recurrent esophageal cancer patients. Laboratory markers for the
prediction of toxicity or response were also determined.
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MATERIALS AND METHODS
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Patients.
Patients with unresectable primary or recurrent metastatic esophageal
cancer were eligible to this pilot study. Patients were required to
have disease assessable by physical or radiographic examination and
life expectancies of at least 2 months. Patients’ characteristics are
shown in a Table 1. No patients had been
receiving corticosteroids or any prior immunotherapy; however, six
patients had received prior chemotherapy and radiotherapy. The
intervals between these prior treatments and immunotherapy were at
least 1 month (range, 1–6 months). The protocol was approved by the
Institutional Review Committee of the Kurume University. The protocol
was explained to each patient, and written informed consent to
participate in the study was obtained from all of the patients who
entered this study. Thirteen patients fulfilled the eligibility
criteria, but two patients were eliminated before treatment because of
inadequate growth of cultured PBMCs. Eleven patients with unresectable
esophageal cancer (n = 4) or recurrent metastatic
cancer (n = 7) received this treatment (Table 2)
. These tumors were histologically confirmed as SCCs by pathological
examination. The mean age of the patients was 67.7 years. Six of the 11
patients (cases 4–8 and 10) received prior radiotherapy (48, 50, 52,
50, 50, and 50 Gy, respectively; mean, 50 Gy) combined with two cycles
of the following chemotherapy: 110 mg/m2/day of
cisplatin and 700 mg/m2/5 days of 5-fluorouracil.
The remaining five patients (cases 1–3, 9, and 11) did not receive any
chemotherapy or radiotherapy before the immunotherapy.
Cells.
Tumor samples used for the stimulation of PBMCs were obtained as
follows: original esophageal tumors that were biopsied through an
endoscope (n = 4), resected metastatic left
supraclavicular LNs (n = 2), carcinomatous
pleural effusion (n = 4), and carcinomatous ascitic
fluid (n = 1; Table 2
). The mean weight of the biopsied
specimens was 0.29 ± 0.43 g, and each contained the mean of
0.5 to 2.0 x 107 viable tumor cells. The
tumor cells were irradiated with a dosage of 50 Gy over 10 min.
Autologous tumor cells were prepared from biopsied samples by mincing
and by enzymatic digestion with stirring in 50 ml of PBS containing 10
mg of type IV collagenase and 5 mg of DNase type I (Sigma) for 3–4 h
at room temperature, followed by filtration through a layer of 100
nylon mesh and then by washing twice with PBS. The samples and effusion
were applied on Ficoll-Hypaque solution and were centrifuged to isolate
live cells from dead cells and aggregates. Tumor cells were
discriminated from inflammatory mononuclear cells on the basis of the
size and the other morphological features under the microscope, as
reported previously (15)
. These cells were also
cryopreserved in 90% human AB serum (Blood Center of
Japanese Red Cross) plus 10% DMSO (Sigma) at -178°C in liquid
nitrogen for restimulation and subsequent immunological assay.
Heparinized peripheral blood samples (50–100 ml; mean, 78 ml) were
collected from patients for 2 weeks to prepare cells for the treatment.
The yield of PBMCs that were obtained from the blood samples with the
Ficoll-Hypaque gradient method (14)
was 0.38–1.3 x
106 cells/ml (mean, 0.54) or 0.5–1.5 x
106 cells (mean, 1.0) in patients or healthy
volunteers, respectively. The tumor cell lines and normal cell lines
used in these studies and their HLA class I alleles have been
reported previously (13
, 14)
.
Cell Culture.
One million PBMCs were incubated in 2 ml of the culture medium
containing 105 irradiated autologous tumor cells
in each well of a 24-well culture plate at 37°C in 5%
CO2. The culture medium consisted of RPMI 1640
with 10% heat-inactivated human AB serum, 0.1 mM MEM
nonessential amino acids solution, 100 IU/ml recombinant IL-2, 100
units/ml penicillin, 100 µg/ml streptomycin, 50 µg/ml gentamicin,
and 0.5 µg/ml fungizone. PBMCs stimulated with autologous tumor cells
were restimulated on day 7 of culture, followed by washing and
preparation for clinical use on day 14. PBMCs were also cultured with
IL-2 alone and were used as a control for the in vitro
analysis. Contamination in the cultured lymphocytes was checked by the
Department of Laboratory Medicine according to the guideline for
cellular therapy of our university. Endotoxin was not checked in this
study, although the cells were repeatedly washed before injection.
Assays.
The activated PBMCs were harvested after the second stimulation at day
14 of culture and were characterized for their phenotypes and
cytotoxicity. Autologous tumor cells were separated from the single
cell suspensions of biopsied tumor samples, pleural effusion, and
ascites as mentioned above. These autologous tumor cells were
cryopreserved in 90% human AB serum (Blood Center of Japanese Red
Cross) plus 10% DMSO (Sigma) at -178°C in liquid nitrogen for
restimulation and subsequent immunological assay. These cells were
thawed, cultured for several days, and used as stimulator or target
cells. A 6-h 51Cr-release assay was used to
measure
cytotoxicity of these activated PBMCs against autologous tumor cells or other tumor
cell lines by the methods previously reported (14)
. The
PBMCs were also measured for their IFN-
production in response to
various tumor cells by incubation of cells for 18 h with target
cells at an E:T ratio of 3:1. The amounts of IFN- in cell-free
supernatants were measured by an IFN- ELISA kit, and the limit of
sensitivity of ELISA was 5 pg/ml as reported previously
(14)
. The number of WBCs per mm3
was
counted by the Department of Laboratory Medicine. Heparinized blood was
applied for Ficoll-Hypaque solution and was centrifuged to obtain
PBMCs, as reported previously (14)
. Viability of PBMCs was
determined by a trypan blue dye-exclusion test. The surface phenotypes
of PBMCs were tested by the two-color immunostaining technique with
anti-CD3, -CD4, -CD8, and -CD16 monoclonal antibodies and FACScan flow
cytometry at 4-week intervals, as reported previously
(14)
. We used the term "CTL activity" in this study if
the activated PBMCs produced significantly higher levels of IFN-
production or percentage of cytotoxicity (P <
0.05 by a two-tailed Student’s t test) by
recognition of the autologous tumor cells compared with those in
response to the allogenic tumor cell lines.
Treatment Schedule.
The activated PBMCs were washed in PBS three times, resuspended in
5–10 ml of 0.9% saline, and administered by endoscopic intratumoral
injection or direct regional injection (Table 2)
. Biopsy for
preparation of the tumor cells was carried out before injection of the
activated PBMCs in cases 1–3 and 8. These injections were repeated at
least two times at 2-week intervals and for up to nine times until
disease progression or severe toxicity was seen. The first
clinical evaluation was performed 14 days after the second injection.
The clinical outcome was evaluated by the following methods:
esophagoscopy, esophagography and ultrasonography at 2-week intervals;
CT scan at 4-week intervals; and serum CEA at 4-week intervals. A CR
was evaluated as disappearance of all of the measurable tumor mass
without the appearance of new lesions, and a PR was defined as a
reduction of all of the measurable disease by 50% of the sum products
of the two greatest perpendicular diameters. A SR was defined as <50%
reduction in all of the measurable lesions. Adverse effects were
evaluated by history and physical examination and graded according to
the National Cancer Institute common toxicity scale.
Statistical Analysis.
Statistical analyses for lymphocyte phenotypic markers and IFN-
production were performed using the two-tailed Student’s t
test or the Fisher’s exact test.
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RESULTS
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Clinical Results.
Eleven patients with advanced (n = 4) or recurrent
(n = 7) esophageal cancer received the locoregional
cellular immunotherapy of the PBMC-activated autologous tumor cells and
IL-2 every 2 weeks for 2–9 times (mean, 5.6 times). The mean number of
administered cells per treatment was 0.8 x
109 cells (range, 0.5–2 x
109 cells). The patients’ profiles are
summarized in a Table 1, and the treatment schedule, CTL activity of
injected cells, adverse effects, and clinical response are summarized
in Table 2
. Six of the 11 patients developed a transient febrile
reaction (<38°C) within 72 h after the treatment. One patient
(case 1) developed grade III hypotension that required a Neo-Synephrine
pressure drip for recovery, and the other patient (case 11) had
transient grade II nausea and diarrhea. No adverse effect was observed
in the remaining three patients (Table 2)
. No toxicity was associated
with the intrapleural administration (cases 6, 7, and 10) of activated
cells. Transient grade II nausea and diarrhea were observed by the
intra-abdominal administration of the cells in case 11. The clinical
outcome was as follows: 1 CR, 3 PR, 2 SR, and 5 PD. Clinical findings
of the four cases that showed major tumor regression (1 CR and 3 PR)
are shown in the next paragraphs.
One patient (case 1) had a CR with no evidence of disease for >20
months after the last treatment. Esophagography of the lower esophagus
before treatment (Fig. 1
,part 1a) and after the third treatment (Fig. 1
, part
1b) showed a marked tumor regression for tumor size. Endoscopic
appearance before treatment (Fig. 1
, , part 2a), after the
first treatment (Fig. 1
, part 2, b and
c), and after the third treatment (Fig. 1
, part
2d) also clearly indicated a significant reduction in tumor size
followed by the complete disappearance of tumor. Moderately
differentiated SCC cells observed in the biopsied sample before
treatment (Fig. 1
, part 3a) were no longer observed in the
biopsied sample after the third treatment (Fig. 1
, part 3b).
Instead, the marked infiltration of mononuclear cells was seen in the
later sample. Similar histological changes were also observed in the
samples after the treatment in the other responders (data not shown).
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Fig. 1. Clinical findings of case 1. A barium
esophagogram of the lower esophagus in case 1 before treatment
(part 1a) showed a bulky irregular filling defect with
destruction of mucosal folds in double contrast and persistent severe
stenosis at the esophagogastric junction. Significant reduction in
tumor size and significant improvement in the narrowing of the lumen in
the same patient were observed after the third treatment (part
1b). The endoscopic appearance of the lower esophagus before
treatment (part 2a) showed typical appearance of an
exophytic polypoid carcinoma with a significant narrowing in the lumen
of the esophagus. Significant reduction in the tumor size and
improvement in the narrowing of the lumen were seen at 14 days after
the first and second treatments (part 2,
b and c, respectively). A feeding
tube was observed on the right side of esophagus in part
2c. Complete tumor regression was observed in the lower
esophagus after the third treatment (part 2d). Histology
of the tissues in the biopsied specimen before treatment showed
moderately differentiated SCC cells before treatment (part
3a), whereas the tumor cells were no longer observed and there
were some inflammatory changes after the third treatment (part
3b). H&E stain; x500.
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Three patients (cases 3, 5, and 9) had PRs. Esophagography before (Fig. 2
,part 1a) and after treatment (Fig. 2
, part 1b)
in case 3 demonstrated a marked reduction in tumor size. Similarly,
endoscopic appearance before (Fig. 2
, part 2, a-1
and a-2) and after treatment (Fig. 2
, part 2,
b-1 and b-2) also clearly showed a marked
reduction in tumor size. Marked infiltration of
CD3+CD8+ T cells was
observed in the biopsied sample of case 3 after treatment (Fig. 2
,
part 3, a–c). Injection of the activated cells
into metastatic tumors at the left supraclavicular LN resulted in
complete disappearance after the second treatment (data not shown).
Furthermore, the tumor size of liver metastases in the S2 (Fig. 3
a-1) and S4 (Fig. 3
a-2) areas of case 5 was
markedly reduced after the fourth treatment (Fig. 3
,
b-1 and b-2). This patient received regional
injections of the activated cells into metastatic LNs in the left
supraclavicular area but not into the liver. A significant decrease in
size of the metastatic tumor on the neck in the supraclavicular
LN was observed in case 9 after the seventh treatment (Fig. 4,a and b)
. The duration of PR was 11, 9, and 7
months in cases 3, 5, and 9, respectively. No additional or subsequent
treatment was given to these responders while they were in response.
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Fig. 2. Clinical findings of case 3. A barium
esophagogram in double contrast of the lower esophagus of patient 3
showed a bulky, irregular filling defect with destruction in the
mucosal folds and persistent severe stenosis in the middle thoracic
esophagus before treatment (part 1a). There was marked
regression in tumor size and a significant improvement in the narrowing
of the lumen after the sixth treatment (part 1b). The
endoscopic appearance of the lower esophagus showed typical appearance
of an exophytic polypoid carcinoma with a narrowing in the lumen of the
esophagus before treatment (part 2, a-1
and a-2). There was a significant improvement both in
tumor size and in the narrowing of lumen at day 12 after the seventh
treatment (part 2, b-1 and
b-2). Photomicrographs of the frozen tissue of the
biopsied specimen stained by anti-CD3 and anti-CD8 monoclonal
antibodies in case 3 before treatment (part 3a) and
after the third treatment (part 3, b and
c). There was significant infiltration of
CD3+ (part 3b) and CD8+
(part 3c) T cells in the specimen after treatment.
Immunohistochemical staining; x200.
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Fig. 3. A CT scan of the liver in case 5. There were
metastatic tumors in S2 (a-1, arrow) and
S4 area (b-2, arrow) before treatment.
There was marked tumor regression in the measurable mass of S2
(b-1, arrow) and S4 (b-2,
arrow) after the fourth treatment into the metastatic LN
located in the left supraclavicular LNs.
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Fig. 4. A CT scan of the neck in case 9. There
was marked tumor regression in the measurable mass of the left
supraclavicular LN after the seventh treatment (a,
before treatment; b, after treatment).
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In addition, the serum CEA levels largely decreased from 21.9 ng/ml
before treatment to 9.8 ng/ml after the fifth treatment in case 9, who
showed PR (Fig. 5)
. The levels also significantly decreased from 334 ng/ml before
treatment to 170 ng/ml after the fourth treatment in case 11, who
showed SR (Fig. 5)
. The decrease in the size of the
para-aortoarterial LN after the fifth treatment of case 11 as
measured by CT is shown in Fig. 6
.
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Fig. 5. Kinetic study of serum CEA levels in cases 9 and
11, according to the clinical course. The levels of serum CEA
significantly decreased in case 9 after the fifth treatment and case 11
after the fourth treatment of cellular therapy.
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Fig. 6. A CT scan of the abdomen in case 11. There was
marked tumor regression in the measurable mass of the
para-aortoarterial LN (arrow) after the fifth treatment
(a, before treatment; b, after
treatment).
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Laboratory Markers.
PBMCs stimulated with autologous tumor cells and IL-2 were measured for
their activity to produce IFN- in response to autologous tumor cells
and three allogeneic tumor cells (KE3,
A2/A24; TE10,
A24/A26; K562). PBMCs cultured with IL-2 alone
that were not used for the treatment were also measured for their
activity as the control. The PBMCs stimulated with autologous tumor
cells and IL-2 produced a significantly higher level of IFN- in
response to the autologous tumor cells than those in response to any of
the other three target cells (case 1) or to those in response to K562
target cells (cases 3, 5, 6, and 9; Fig. 7
a). The stimulated PBMCs of case 3 (HLA-A24/A26),
case 5 (HLA-A2/), and case 9 (HLA-A24/A11) also
produced significantly higher levels of IFN- by recognition of
allogeneic but HLA-A locus-matched tumor cells than those
produced by recognition of K562 target cells. Four (cases 1, 3, 5, and
9) of these five patients responded to the treatment, whereas the
remaining patient (case 6) had PD. There was no significant difference
among the levels of IFN- production by the stimulated PBMCs in
response to the four target cells tested in the remaining six patients
who had either SR (n = 2) or PD (n = 4;
data not shown). Similarly, there was no significant difference among
the levels of IFN- production by the control PBMCs by recognition of
the four target cells tested in any of the 11 patients. The results of
PBMCs of the responders are shown in Fig. 7
a.
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Fig. 7. CTL activity of the administered cells. In
a, PBMCs, stimulated with autologous tumor cells, and
IL-2 were measured on day 14 of culture for their activity to produce
IFN- in response to the autologous tumor cells or to the cells of
KE3 (A2/A24), TE10 (A24/A26) esophageal SCC cell line, and K562
erythroleukemia cell line. PBMCs cultured with IL-2 alone, taken as the
control, were also measured for their activity. Values represent the
mean of IFN- production of different effector cells
(n = 2–9). Each sample was measured in triplicate
determinants at an E:T ratio of 3:1. Background IFN- production
(50–100 pg/ml) was subtracted from the values. Two-tailed Student’s
t test was used for statistical analysis. In
b, PBMCs, stimulated with autologous tumor cells, and
IL-2 in cases 1 and 2 were measured for their cytotoxicity by a 6-h
51Cr-release assay against five different targets
[autologous tumor cells, KE3, TE10, K562, and MKN28 (A31/) gastric
adenocarcinoma cell line]. PBMCs cultured with IL-2 alone, taken as
the control, were also measured for cytotoxicity. Values represent the
mean of the percentage of specific lysis of the different effector
samples of the different experiments (n = 3 or
4). Each sample was measured in triplicate at an E:T ratio of
20:1. Bars, ±SD.
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These results suggested that the CTL activity was observed in the
stimulated PBMCs of 5 cases (cases 1, 3, 5, 6, and 9), whereas LAK cell
activity was observed both in the stimulated PBMCs of the remaining 6
cases and in the control PBMCs cultured with IL-2 alone in all of the
11 cases. A 6-h 51Cr-release assay was used in
cases 1 and 2, in which a relatively large number of cells were
available for the study (Fig. 7
b). The PBMCs of case 1
(HLA-A24/A26) showed significantly higher levels
of cytotoxicity against HLA-A24+
esophageal cancer cells than those against MKN28 (HLA-A31/)
stomach cancer cells or K562 target cells. In contrast, there was no
significant difference among the levels of cytotoxicity either by the
stimulated PBMCs in case 2 or by the control PBMCs in cases 1 and
2.
Eleven patients were divided into the three groups as follows:
responder group (n = 4, 1 CR + 3 PR; cases 1, 3, 5, and
9), SR group (n = 2; cases 8 and 11), and PD group
(n = 5; cases 2, 4, 6, 7, and 10) to find laboratory
markers useful for predicting the clinical response. Freshly isolated
PBMCs (n = 57 from 11 cases) consisted of 71 ±
4% CD3+ T cells, 44 ± 3%
CD4+ T cells, 18 ± 5%
CD8+ T cells, and 5 ± 2%
CD16+ NK cells (Table 3)
. The PBMCs stimulated with autologous tumor cells and IL-2
(n = 57 from 11 cases) consisted of 86 ± 5%
CD3+ T cells (P = 0.0007
versus freshly isolated PBMCs), 28 ± 3%
CD4+ T cells (P < 0.0001),
42 ± 4% CD8+ T cells (P <
0.0001), and 18 ± 4% CD16+ NK cells
(P = 0.0031). The percentages of surface markers of the
control PBMCs (n = 30 from 11 cases) cultured with IL-2
alone were similar to those of stimulated PBMCs, although the
percentages of CD8+ T cells and of
CD16+ NK cells in the stimulated PBMCs were
slightly higher than those in the control PBMCs. In the responder
group, the percentage of CD8+ T cells in the
stimulated PBMCs (55 ± 20%) was significantly higher than that
of freshly isolated PBMCs (22 ± 3%), whereas that in the
stimulated PBMCs of the SR group or the PD group was not significantly
different from that of fresh isolated PBMCs. The percentage of
CD16+ NK cells in stimulated PBMCs of the
responder group (17 ± 4%) and SR group (17 ± 5%)
was significantly higher than that in the freshly isolated PBMCs of the
responder group (7 ± 1%) and of the SR group (4 ± 2%),
respectively, whereas that in the stimulated PBMCs of the PD
group (15 ± 8%) was not significantly different from that of the
freshly isolated PBMCs (6 ± 3%).
The mean number of administrated lymphocytes per case was
8.2 x 109 cells in the responder group,
4.2 x 109 cells in the SR group, and
2.7 x 109 cells in the PD group. The mean
number of administrated lymphocytes per injection was 1.2 x
109 cells in the responder group, 0.7 x
109 cells in the SR group, and 0.7 x
109 cells in the PD group. A kinetic study showed
no significant difference in the mean number of WBCs, PBMCs, or
CD3+, CD4+, or
CD8+ cell counts that were measured before
treatment and after the second and fourth treatments in these three
groups (Fig. 8)
. In contrast, the mean number of CD16+
lymphocytes in the peripheral blood after the second and fourth
treatment in the responder group significantly increased compared with
that before treatment in the responder group (Fig. 8)
. The number
of CD16+ lymphocytes was also significantly
higher than those after the second and fourth treatments in the PD
group.
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Fig. 8. Kinetic study of cell numbers and their
phenotypes in peripheral blood. Number of WBCs; PBMCs in WBCs; and
CD3+, CD4+, and CD8+ cells in PBMCs
were measured before treatment and after the second and fourth
treatments. The 11 patients were divided into the three groups
as follows: responder group (n = 4, 1 CR + 3 PR);
SR group (n = 2); and PD group
(n = 5). *, #, two-tailed Student’s
t test was used for statistical analysis.
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DISCUSSION
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A number of clinical studies of adoptive immunotherapy using
mostly LAK cells and TILs have shown a significant response rate in
patients with melanoma, renal cell carcinoma, and some other cancers
(16, 17, 18, 19, 20, 21)
. However, there has been no report on the
clinical studies demonstrating the obvious tumor regression of advanced
esophageal cancers, to the extent that we searched in the
literature. This study showed that locoregional cellular
immunotherapy resulted in marked tumor regression in 4 of 11 patients
with advanced or recurrent esophageal cancer. Our report seems to be
the first report showing clear evidence for the potential of the
application of immunotherapy for esophageal cancer patients.
The adverse events in all of the 11 cases were moderate and tolerable.
There was no relationship between the number of administered cells and
adverse effects. Grade 1 fever was often observed and might have been
attributable in part to production of cytokines by the administered
lymphocytes (16)
. Other adverse effects included grade 3
hypotension in one case and grade 2 nausea and diarrhea in one other
case. These adverse effects may also have been attributable to cytokine
production from injected activated lymphocytes. These results suggest
that this regimen of cellular immunotherapy was safe.
We provided the activated lymphocytes by stimulation of PBMCs with
autologous tumor cells and IL-2 in vitro. These activated
lymphocytes consisted of 42% CD8+ and 28%
CD4+ cells. CTL activity was observed in these
PBMCs from 5 (cases 1, 3, 5, 6, and 9) of the 11 cases, and 4 of them
had significant tumor regression (1 CR and 3 PR), although the detailed
studies of CTL activity, including the
51Cr-release assay against various target cells
at different E:T ratios, were not carried out, mainly because of the
limited number of cells for the in vitro analyses. LAK cell
activity, instead of CTL activity, was observed in the PBMCs of the
remaining six cases, and none of them showed major tumor regression (2
SR and 4 PD). LAK cell activity was observed in the control PBMCs
cultured with IL-2 alone in all of the 11 patients tested. These
results suggested that CTLs but not LAK cells were needed to achieve
the tumor regression in this regimen of locoregional cellular
immunotherapy. Consequently, the CTL activity of administered cells
could be an appropriate laboratory marker to predict the clinical
response. This phenomenon was supported by the fact that the mean
percentage of CD8+ T cells (55 ± 20%) of
the stimulated PBMCs of the responder group was highest among those of
the different groups tested, and it was significantly higher than that
of fresh PBMCs. The other laboratory marker for prediction of clinical
response would be the percentage of CD16+ NK
cells in the peripheral blood, which significantly increased in PBMCs
of the responder group after treatment. These results suggest that both
CTLs at the tumor sites and NK cells in peripheral blood were needed
for tumor regression in this regimen. Therefore, both CTL activity in
the injected cells and percentage of CD16+ cells
in the peripheral blood might be useful laboratory markers for
monitoring the clinical response to this locoregional cellular
immunotherapy. These results, however, must be confirmed by a
large-scale clinical study. The magnitude of CTL activity of the
activated TILs was well correlated with clinical efficacy in metastatic
melanoma patients in the regimens of adoptive cellular therapy with
high-dose IL-2 (22)
. In contrast, no reliable laboratory
markers for PBMCs, including the percentage of
CD16+ cells, were found in the past decade,
regardless of numerous trials in the field of immunotherapy using LAK
cells, TILs, or cytokines.
It is of note that the injection of cells into the left supraclavicular
LN in case 5 resulted in both the disappearance of tumors at the
injection site and the decrease in tumor sizes at the uninjected site
(liver). Some element of systemic immunity might been achieved by local
therapy in this case, but this type of tumor regression was not
observed in any other cases tested. It is also of note that regional
treatment of pleural effusions and ascites was ineffective in 5 of the
11 patients. Subsequently, the application of this locoregional
immunotherapy to the control of cancer cells in pleural or abdominal
regions is not recommended. In addition, prior chemoradiotherapy is not
recommended for this immunotherapy. Three of five patients who received
no prior chemoradiotherapy responded to the immunotherapy, whereas only
one of six patients who received prior chemoradiotherapy responded to
the immunotherapy. This could be in part attributable to the number of
cells available for the treatment. The mean number of administered
cells per injection was 1.2 x 109 cells in
the responders, whereas it was 0.7 x 109
cells in the other groups. Prior chemoradiotherapy should suppress
T-cell proliferation in response to IL-2 and autologous tumor cells.
In conclusion, we have shown that this treatment regimen was safe and
resulted in tumor regression in 4 of 11 patients with advanced or
recurrent esophageal cancer. A large-scale clinical trial is
recommended to confirm the evidence from this Phase I clinical study.
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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 in part by Grants-in-Aid from the
Ministry of Education, Science, Sport and Culture, Japan, and Grant
H10–genome–003 from the Ministry of Health and Welfare,
Japan. 
2 To whom requests for reprints should be
addressed, at Department of Surgery, Kurume University School of
Medicine, 67 Asahi Machi, Kurume 830-0011, Japan. Phone:
81-942-31-7566; Fax: 81-942-34-0709; E-mail: hyamana{at}med.kurume-u.ac.jp
3 The abbreviations used are: SCC,
squamous cell carcinoma; PBMC, peripheral blood mononuclear cell; LN,
lymph node; HLA, human leukocyte antigen; IL, interleukin; CT, computed
tomography; CEA, carcinoembryonic antigen; TIL, tumor-infiltrating
lymphocyte; CR, complete response; PR, partial response; SR, stable
response; PD, progressive disease; LAK, lymphokine-activated killer;
NK, natural killer.
Received 5/ 8/00;
revised 7/ 7/00;
accepted 9/ 7/00.
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ABSTRACT
INTRODUCTION
