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Vol. 4, 6/9-627, Marc/i 1998 Clinical Cancer Research 619
Active Immunotherapy with Ultraviolet B-irradiated Autologous
Whole Melanoma Cells plus DETOX in Patients with
Metastatic Melanoma1
Omar Eton,2 Dmitri D. Kharkevitch,
Mary Ann Gianan, Merrick L Ross, Kyogo Itoh,
Michael W. Pride, Cherrie Donawho,
Antonio C. Buzaid, Paul F. Mansfield,
Jeffrey E. Lee, Sewa S. Legha, Carl Plager,
Nicholas E. Papadopoulos, Agop Y. Bedikian,
Robert S. Benjamin, and Charles M. Balch
Departments of Melanoma/Sarcoma Medical Oncology [0. E..M.A.G.,A.C.B.,S.S.L.,C.P..N.E.P..A.Y.B.,R.S.B.].Surgical Oncology [D. D. K., M. I. R.. P. F. M., J. E. L.], and
Immunology [M. W. P.. C. D.]. The University of Texas M. D.
Anderson Cancer Center, Houston, Texas 77030: Department of
Immunology. Kurume University School of Medicine. Kurume,
Fukuoka 830, Japan [K. I.]: and City of Hope National MedicalCenter and Beckman Research Institute, Duane, California 91010
[C.M.B.]
ABSTRACT
Our objective was to determine the clinical activity,toxicity, and immunological effects of active immunotherapy
using UVB-irradiated (UVR) autologous tumor (AT) cells
plus adjuvant DETOX in metastatic melanoma patients.
Eligibility included nonanergic patients fully recovered after
resection of 5 or more grams of metastatic melanoma. Treat-
ment consisted of intradermal injections of iO� UVR-AT
plus 0.25 ml of DETOX every 2 weeks x 6, then monthly.
Peripheral blood mononuclear cells (PBMCs) were har-vested for cytotoxicity assays, and skin testing was per-
formed for delayed-type hypersensitivity (DTH) determina-tions before the first, fourth, seventh, and subsequent
treatments. Forty-two patients were treated, 18 in the adju-
vant setting and 24 with measurable disease. Among thelatter group, there were two durable responses in soft-tissue
sites and in a bone metastasis. Treatment was well tolerated.
Thirty-five patients were assessable for immunological pa-
rameters; 10 of these patients, including the 2 responders,demonstrated early induction of PBMC cytotoxicity against
AT cells that persisted up to 10 months on treatment beforefalling to background levels. In five of seven patients, the
Received 6/12/97: revised 12/5/97: accepted 12/15/97.
The costs of publication of this article were defrayed in part by thepayment 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.
I This work was supported in part by NIH Grant RR-551 1.
2 To whom requests for reprints should be addressed, at The Universityof Texas, M. D. Anderson Cancer Center, 1515 Holcombe Boulevard,
Box #77, Houston, TX 77030. Phone: (713) 794-4633: Fax: (713) 794-1934.
fall-off heralded progressive disease. Late induction of aweak DTH reaction to AT cells was observed in eight pa.
tients. Active immunotherapy with UVR-AT + DETOX hadmodest but definite clinical activity in advanced melanoma.The induction of both PBMC cytotoxicity and DTH reactiv-ity to AT cells supported a specific systemic immune effect of
treatment, although the former more closely followed dis-ease course in this study.
INTRODUCTION
Malignant cells must elude any host immune surveillance
to grow and overcome the host. Hewitt et a!. ( I ) have shown that
spontaneous tumors in aging mice do not induce a detectable
host immune response. Nevertheless, the hypothesis that those
same cells that escape immune surveillance in vivo might be
manipulated in vitro to become more immunogenic is a basis for
tumor immunotherapy. Methybcholanthrene and UVB-induced
tumors, when excised and reimplanted in mice, have resulted in
varying degrees of specific immune protection (2-5). In pa-
tients, tumor burden, immunosebection (6), tolerance induction
(7-I 0), and biophysical barriers ( 1 1 ) all present challenges to
developing effective antitumor immunotherapy programs.
Efforts to circumvent these obstacles in the 1990s are
focused on identifying specific factors that either drive or inhibit
antitumor immunity (12). However, while the relative impor-
tance of these factors continues to evolve almost on a daily
basis, cruder vaccine preparations from the patients’ tumors
continue to have a role in providing a plethora of characterized
and as yet unidentified targets. These vaccines have resulted in
objective response rates under 20%, including responses in
visceral organ metastases consisting of billions of tumor cells
(13-15). Such clinical activity supports the continued develop-
ment of active immunotherapy in the multidisciplinary antican-
cer armamentarium. Viable autobogous whole tumor cells tested
in melanoma vaccine preparations have some advantages: (a)
the relative safety of intradermab injection of autobogous rather
than ablogeneic material; (b) the potential for inducing as yet
uncharacterized immune responses to altered self-antigens in
common with the patient’s tumor; and (c) the diminution of
some artifacts of vaccine preparation. Disadvantages include:
(a) the difficulty in obtaining and preparing individualized vac-
cines; (b) the limited source material; and (c) the inability to
control for intervaccine variation important in clinical trials.
At M. D. Anderson Cancer Center, Hostetler et a!. (16-18)
demonstrated that UVR3 (280-320 nm) of the weakly immu-
3 The abbreviations used are: UVR, UVB irradiation; AV, antigenicvariant; PBMC, peripheral blood mononuclear cell; DTH, delayed-type
hypersensitivity; AT, autologous tumor.
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620 Active Immunotherapy: Autologous Melanoma Cells, DETOX
nogenic melanoma cell line K1735P (C3H/HeN; MTV) re-
sulted in the generation of a high frequency of highly immuno-
genic AVs. Immunizing syngeneic mice with AV clones
induced protective immunity in in vivo challenge assays against
the immunizing AV clone and the untreated parental tumor line
but not against unrelated tumors (16-18). The parental tumor
did not induce such protective immunity. These AV clones grew
in immunosuppressed mice but not in immunocompetent mice,
and they induced cytotoxic/helper I-lymphocyte activity that
cross-reacted with parental tumor cells (18).
Based on these data, it was hypothesized that the poly-
cbonab population of UVR-parental cells might provide a mul-
titude of immunogenic AVs for active immunotherapy without
requiring individual AV clones to be isolated. Umezu et al. (19)
developed conditions for UVR of human melanoma and renal
cell carcinoma cells to optimize inducing cytotoxic activity of
PBMCs against irradiated- and nonirradiated parental cells. Us-
ing in vivo immunization/challenge and DTH experiments, Pride
et a!. (20, 2 1) demonstrated that UVR of the K! 735-M2 cell line
greatly increased its immunogenicity. Pride et a!. (21) also
found that UVR not only up-regulated expression of MHC
classes I and II molecules but also induced expression of murine
B7.l on Kl735-M2 melanoma.
Mycobacteriab preparations are useful adjuvants in active
immunotherapy (22). In melanoma, Bacillus Calmette-Gu#{233}rin
induces responses in up to 90% of locally injected and 15% of
adjacent lesions but has had no effect on survival in Phase III
trials and can cause disseminated infection. DETOX (Ribi Im-
munoChem Research, Inc. , Hamilton, MI) is a novel adjuvant
available in a lyophilized oil droplet emulsion consisting of both
cell wall cytoskeleton from Mycobacterium phlei and mono-
phosphoryl lipid A from Salmonella minnesota R595. DETOX
can augment I cell and humoral immunity. In the murine
experiments by Pride et a!. (20), DETOX was effective in
significantly augmenting immunoprotection and DTH. In pa-
tients, DETOX has been used only as an adjuvant, and dosing
has been empiric because its effects have been more host than
dose rebated (23, 24).
We undertook the present study to determine the clinical
activity and toxicity of active immunotherapy with UVR-AT
cells + DETOX in patients with metastatic melanoma. We used
serial monitoring of PBMC cytotoxicity and DTH reactions
against AT cells with controls to detect and track any evidence
of a specific systemic immune response to local intradermal
therapy during the course of vaccine treatment.
PATIENTS AND METHODS
Patients. Patients > 15 years with metastatic melanoma
pathologically confirmed at M. D. Anderson Cancer Center and
from whom more than S g of nonnecrotic tumor had been
resected as a part of routine management were candidates for
treatment. Patients were enrolled only after full postoperative
recovery and with: a Karnofsky performance status >70%; life
expectancy >3 months; DIH reaction positive to foreign anti-
gens (see below); results of complete blood counts, serum
chemistry studies, immunogbobulins (IgG, IgA, and 1gM), com-
plement (C3, C4, and CHSO), and I-cell subsets in the clinically
normal range; and signed informed consent forms. Patients were
required to avoid immunomodubatory agents (e.g., corticoste-
roids, nonsteroid anti-inflammatory agents, antihistamines, es-
trogens, high doses of vitamins, and concurrent specific antitu-
mon therapy). All sites of clinically detectable disease were
objectively evaluated at baseline using computed tomography,
magnetic resonance imaging, sonography, photographs, and, for
s.c. disease, multiple investigator assessment.
Vaccine Preparation. Fresh tumor was collected at the
time of surgery from the frozen section laboratory and frag-
mented by slicing. Aliquots of 106 cells each were frozen for
immunoassays. To maximize yield of viable tumor cells for
vaccine preparation, the bulk of tumor was dissociated using
colbagenase type I (2 mg/mb) and type IV DNase (0.4 mg/ml;
Sigma Chemical Co., St. Louis, MO; Ref. 25). These enzymes
can alter the immunogenicity of the resulting cell preparation
(26-29). In control flow cytometry experiments, Itoh et a!. (26)
did not find enough significant alteration to obviate the use of
the enzymes; furthermore, incubation of the tumor cells for 24 h
before vaccine administration, as described below, resulted in
reexpression of specific suppressed antigens (26, 29).
The dissociated cells were washed in sterile HBSS with
gentamicin and resuspended in equal volumes of HBSS and
chilled 10% DMSO + 4% human serum albumin. Aliquots
containing 1.5-2 x i0� viable tumor cells were stored under
liquid nitrogen. For some specimens, an aliquot of fresh tumor
was also put into culture to establish a tumor cell line.
Twenty-four h before patient arrival, an abiquot of tumor
cells was thawed in a 37#{176}Cwater bath, washed twice, resus-
pended in 5 ml of PBS in a 10-cm Petni dish, and exposed to
UVR (2.2 J/m2/s) for 30 s using an FS4O sunlamp (Westing-
house Electric Corp., Bboomfield, NJ; Refs. 18 and 19). Incu-
bation in macrophage-/serum-free medium (Life Technologies,
Inc.) for 24 h resulted in >80% cell viability. Cell suspensions
were checked for bacterial contamination and for Mycoplasma
contamination using the Gene-Probe rapid detection system
(Gene Probe, Inc., San Diego, CA). After incubation, cells were
detached by scraper, washed, resuspended in 0.75 ml of PBS,
and X-irradiated (20,000 cGy) to prevent propagation in vivo
and insure sterility (30, 31). Concurrently, DETOX (each vial
stored at 2-7#{176}Cconsisted of 500 p.g of cell wall cytoskeleton, 50
p�g of monophosphoryl lipid A, 2% squalene, and 0.4% Tween
80 in 2X normal saline) was reconstituted by repeated aspiration
with 0.5 ml of sterile water. In a total volume of 1 ml, approx-
imateby 10� viable replication-deficient UVR autologous mela-
noma cells in PBS were mixed with 0.25 ml of DETOX just
before each treatment (24).
Treatment Plan. Active immunotherapy was adminis-
tered at M. D. Anderson Cancer Center by intradermal injection
into the anteromedial deltoid and femoral regions near intact
lymph node basins, every 2 weeks X 6 and then every 4-6
weeks. Treatment ended with progression of disease (25%
growth) or exhausted supply of vaccine material. Response was
assessed initially at 6 weeks and then at 8-12-week intervals
during long-term follow-up. Toxicity was graded according to
National Cancer Institute guidelines.
Cytotoxicity Assays. For uniformity, all cytotoxicity as-
says were performed by a single investigator (D. K.) throughout
the study. At baseline and before the fourth, seventh, and sub-
sequent vaccinations, PBMCs were isolated by Ficoll-Paque
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Clinical Cancer Research 621
Tab le I Pretreatme nt patient and tumor characteristics
n % of total Indicator lesions Adjuvant setting
All patients 42Age (yr): median, 52: range, 27-81
Male 24 57Prior treatment
None 15 36
Biotherapy 14 33
Chemotherapy ± biotherapy 21 50
Regional perfusion 2 5
Karnofsky performance status
90% 36 86
80% 6 14Normal serum level of LDH” 38 90Normal serum level of albumin 40 95
Tumor stage and sites
All patients 42 24 18
AJCC stage III N2 (regional) 6 1 5
AJCC stage IV (disseminated) 36 23 (55%) 13 (3 1%)
Soft-tissue only 16 8 8
Visceral metastases 20 15 5
One visceral organ 12 8 4Lung alone 7 5 2Brain 6 5 1Liver 4 3 1
Spleen 4 2 2Adrenal 2 1 1
Gastrointestinal 2 0 2
C, LDH, lactate dehydrogenase: AJCC, American Joint Committee on Cancer.
gradient (Pharmacia AB, Uppsala, Sweden) from 30 ml of fresh
heparinized peripheral blood at room temperature (22). These
PBMCs were assayed fresh and were also cryopreserved until
sufficient specimens could be obtained over 2 or more months
of treatment to perform a single experiment, thereby minimizing
interassay variability. Four-h � ‘Cr-release assays were used to
detect cytotoxic activity, as described previously (32). Cryopre-
served, uncultured autobogous and albogeneic melanoma cells,
renal cell carcinoma cells, cultured tumor cell lines, and natural
killer-sensitive tumor line K562 were used as the targets in these
assays. Frozen aliquots of uncultured tumor cells were thawed
on the day of the assay; adherent cell lines were trypsinized.
Target cells were labeled with 50 p.Ci/million cells of
Na25tCrO4 (Amersham Corp., Arlington Heights, IL) at 37#{176}C
for 1 h. After washing twice, labeled cell suspensions were
passed through a cotton column to remove cell aggregates and
debris. Labeled target cells at a concentration of 5000 cells/well
in triplicate were added to effector lymphocytes at each of three
E:T ratios, 40: 1, 20: 1, and 10: 1, in 96-well, round-bottomed
plates. The plates were incubated for 4 h at 37#{176}C,after which
100 p.1 of supematant/well was harvested, and radioactivity was
measured in a gamma counter. In all experiments performed, the
spontaneous release did not exceed 20% of the maximum
release. Cytotoxicity assays were also performed with ad-
herent melanoma cell targets in 96-well, flat-bottomed plates.
The percentage of specific lysis was calculated as: (Xa x��)/
(Xmax x��) X 100, where Xa �5 the mean � ‘ Cr release at a
specified E:I ratio; x� is the mean spontaneous 51Cr release (no
effector cells); Xmax �5 the maximum 5tCr release by target cells
treated with 0. 1 N HC1. Positive cytotoxicity as a result of
treatment was defined as > 10% specific lysis and at beast 2-fold
higher specific bysis than at baseline.
Antibody-blocking Experiments. To confirm and char-
acterize any positive cytotoxicity results detected, blocking ex-
periments were performed using antibodies to CD3, CD4, CD8,
DR. DP, DQ (Becton Dickinson, Sunnyvale, CA), and class I
MHC (W6/32; DAKO, Copenhagen, Denmark). Monoclonal
antibodies were mixed with � ‘Cr-labeled AT targets in a volume
of 50 p.1 each for 1 h at 4#{176}Cto try to abrogate lysis. Effector cells
were added at a 40: 1 E:T ratio, and cytotoxicity was measured
as described above. Controls were murine IgG and medium
alone.
DTH Assays. Intradermal skin testing on the vobar
surface of the forearm was performed at baseline against:
foreign recall antigens (Merieux Skintest Multitest Device:
tetanus, diphtheria, strep, tuberculosis, Candida, trichophy-
ton, and proteus); l0� X-irradiated AT, and i05 autobogous
PBMCs (negative control). AT and PBMC skin tests were
repeated before the fourth, seventh, and subsequent vaccina-
tions. A positive DTH reaction consisted of induration over 2
mm in diameter at 48 h. Hypoergy to foreign antigens using
the Merieux skin-test device was defined as two or fewer
positive DTH reactions or sum of induration diameters under
1 cm. In this study, patients were not asked to undergo punch
biopsy of skin test sites to detect subclinical evidence of a
DTH reaction.
Statistical Considerations. Complete or partial re-
sponse required disappearance or >50% decrease, respec-
tively, in the sum of the products of largest diameters of all
indicator lesions lasting at beast 1 month. Stable disease
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�- -.�
622 Active Immunotherapy: Autologous Melanoma Cells, DETOX
A
B
Fig. 1 Clinical responses to active immunotherapy using autologous UVR-melanoma cells + DETOX. A, partial response in bone 6 months from
the start of treatment; this patient also had a complete response in s.c. nodules. B, single view of some of several dozen in-transit lower-extremity
metastases which, after an initial flare at 10 weeks, disappeared over a period of 17 months from the start of treatment; surgery to harvest more cells
for vaccine preparation resulted in this patient’s formal response being characterized as mixed to vaccine and complete to vaccine plus surgery.
required less than 25% change in the sum of the products of
diameters lasting at least 8 weeks. Survival from start of
treatment was plotted by the method of Kaplan-Meier and
evaluated using the bog-rank test. PBMC cytotoxicity and
DTH reaction to AT were assessable only after six or more
treatments. A modification of Simon’s design for Phase II
clinical trials was used; assuming a 10% significance level
and 90% power, I 2 patients were enrolled in stage I and 23
additional patients in stage II (32). Induction of PBMC
cytotoxicity in 6 of 35 patients would support further inves-
tigation, assuming that response rates of 30 and 10%, respec-
tively, would and would not be of interest. Enrollment would
have terminated early if none of 12 patients achieved a
clinical response or if 6 of 9 patients treated in an adjuvant
setting had progressive disease within 3 months.
RESULTS
Between September 1992 and May 1995, 42 patients re-
ceived a median of eight treatments (range, 3-21) with UVR-
autobogous melanoma cells plus DETOX. Pretreatment charac-
teristics are summarized in Table I . All patients were assessable
for clinical response and toxicity and 35 for biological response.
Seven patients had disease progression before completing the
first six treatments.
Clinical Responses. Among 24 patients with indicator
lesions, two had major responses and six showed stabilization in
soft-tissue metastases. One 39-year-old woman treated previ-
ously with chemo/biotherapy underwent resection of painful s.c.
nodules for vaccine preparation. Her indicator lesions consisted
of nine s.c. nodules, each 1 cm or smaller, and a large sympto-
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40 A
I!11 12
.�‘ 30
#{149}0
� 20
0
a� 10
Before 0Vaccine #: I
Months: �
40
�‘ 30U
� 20
0
a� 10
Before 0 -
Vaccine #: IMonths: 0
4 7 8 9 10
3 6 9
B
In�jI
4
Clinical Cancer Research 623
IlLI12
- 40:1 I- 20:1 I
10:1]
Li � Icil � I7 9 10 11 12 14 15 1617 18 LAK
3 6 9 12 15
Fig. 2 Mean PBMC cytotoxicity over time against autologous tumor atthree E:T ratios for each of the two responders (A and B) shown in Fig.
I. L4K, bymphokine-activated killer activity (positive control).
matic lytic bone metastasis that had progressed radiographically
1 year after radiation therapy. Four days after her first treatment,
she noted soreness at all tumor sites. Three skin nodules initially
enlarged by less than 20% with minimal inflammatory changes,
three remained stable, and three regressed; however, by the
seventh treatment (at 4 months), all s.c. lesions had completely
regressed as had her bone pain, with subsequent confirmation of
major radiological response (Fig. 1A). Nine months after her last
treatment, she developed new s.c. nodules and recurrence in
bone (time to progression, 21 months). From a negative base-
line, her unstimubated PBMCs demonstrated persistently posi-
tive cytotoxicity against AT cells (Fig. 2A). She had an unusu-
ally large DTh reaction (4 cm with 10 cm of surrounding
inflammation) at the first vaccine intradermal injection site,
smaller with subsequent treatments. No DTH reaction to the i0�
AT control was detected during 1 year of treatment.
The second responder was a 63-year-old man with exten-
sive, progressing, lower-extremity, in-transit nodules, despite
regional and systemic chemo/biotherapy and radiation therapy.
Resected symptomatic lesions were used for vaccine prepara-
tion. Innumerable in-transit nodules, 0.5-2 cm in caliber, were
used as indicator lesions. By 10 weeks, most lesions had flat-
tened or remained stable; however, a minority appeared more
prominent, although by <20%. Because of early resection of
these lesions to make more vaccine, this was strictly a “mixed”
response. However, after 19 treatments for >20 months, to-
gether with surgery, he ultimately achieved a complete response
lasting 5 months (Fig. 1B). From a negative baseline and ex-
tending through the 16th treatment, his unstimulated PBMCs
demonstrated cytotoxicity against AT cells (Fig. 2B). Fall-off in
cytotoxicity coincided with progressive soft-tissue disease. He
93 consistently had a large DTH reaction (3 cm) to vaccine but
never to the AT control.
Survival. The median survival of the stage IV patients
was 16 months from start of treatment, 20 months from initial
diagnosis of stage IV disease. Review of pretreatment prognos-
tic factors revealed a high frequency of favorable factors (nor-
mal serum levels of lactate dehydogenase; Refs. 33 and 34),
metastases in soft tissue sites alone, or metastases in one visceral
LAK organ such as lung (33), in this study.
Toxicity. Grade I toxicities were limited to local irrita-
tion at the vaccine injection site and in 10% of patients. with
malaise lasting 24-48 h. There was no incidence of skin ulcer-
ation or other grades I1-IV toxicities. Palpable residual granu-
bomas at the injection sites measuring <2 cm became less
apparent over weeks to months. In one-half of the patients,
routine evaluation by an ophthalmologist yielded no abnormal-
ities attributable to treatment. Five of 15 patients (33%) purified
protein derivative negative at baseline converted to purified
protein derivative positive (asymptomatic) using the Merieux
skin-test device, possibly a result of repeated exposure to DE-
lOX (or to the anergy panel).
Detection of PBMC Cytotoxicity against Autologous
Tumor. During the course of treatment, 10 of 35 assessable
patients had an early rise in mean PBMC cytotoxicity against
AT cells within 6 weeks, all from a negative baseline of under
10% specific lysis (Table 2). Lytic activity remained detectable
up to 10 months, later falling back to background bevels in 7 of
the 10 patients. In five patients, the fall-off coincided with
progression of disease within 3 months. For the three patients
without fall-off, autologous material was insufficient to continue
bong-term treatment and monitoring.
Table 3 illustrates the individual spectrum of specificity for
PBMC cytotoxicity. The selective rather than general positivity
in the cytotoxicity assay results against a panel of allogeneic
tumor cells, and K562 does not support a non-specific effect of
vaccine preparation, such as the use of collagenase. In four
patients, the level of lysis of K562 cells was minimal, indicating
that the cytotoxicity was not mediated by natural killer cells.
Table 4 shows that lysis could be abrogated by anti-CD3,
anti-class I, and anti-CD8 but not by anti-class II and anti-CD4
antibodies, indicating that the observed cytolytic activity was
restricted by class I and was mediated by CD8 + I cells.
Univariate analysis indicated that patients with stage IV
disease and positive cytotoxicity had both a prolonged median
time to progression and overall survival duration compared with
those with negative cytotoxicity (Table 5 and Fig. 3). Although
time bias was minimized because PBMC cytotoxicity was al-
ways detected early, the positive cytotoxicity group also had a
lower disease burden, confounding the results of the analysis.
DTH. Among 42 nonanergic patients, 21 (50%) were
hypoergic to foreign recall antigens at baseline, a finding that
did not correlate with any pre- or posttreatment factors. Baseline
hypoergy was evident in 4 of the 10 patients with positive
PBMC cytotoxicity results (Table 5).
Among the 35 assessable patients, 8 (23%) developed a bate
DIH reaction to iO� AT cells (and no reaction to PBMC
control); indurations under I cm first appeared after a median of
8 courses (4-15 courses). Neither responder and only three
patients with positive PBMC cytotoxicity also had a DTH
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Patient
Table 2 Ten patients with specific lysis > 10% and rising over 2 X baseline during the course of treatment
E:T 15” 16”16” 17b
2 02 32 3
4 0
2 03 0
9 0
8 0
0 3
Table 3 SpecifIcity of positive PBMC cytotoxicity in four patients
Cytotoxicity against targets
624 Active Immunotherapy: Autologous Melanoma Cells, DETOX
I” 4” 7”
0” 1b 4/’
40:1 8 14 21
20:1 4 14 21
10:1 12 3 19
2 40:1 4 15 1420:1 5 13 17
10:1 4 10 3
3 40:1 6 22 23
20:1 0 15 23
10:1 0 10 15
4 40:1 0 10 0
20:1 1 9 1
10:1 0 9 0
5 40:1 2 6 10
20:1 5 0 5
10:1 1 0 0
6 40:1 4 24 18
20:1 0 17 3
10:1 0 8 0
7 40:1 8 9 12
20:1 5 10 8
10:1 0 0 3
8 40:1 0 1 2
20:1 I 0 1
10:1 0 0 29 40:1 2 19 24
20:1 0 7 18
10:1 0 1 6
10 40:1 1 0 0
20:1 1 3 1
10:1 0 3 0
‘I Vaccine number.
I, Months.
9” ioa6”
7b
49
8” 11”
_�t48 31
28 3429 22
15 20 10
5 17 6
0 5 8
28 30 31 4717 23 23 15
8 2 5 15
6 21 24
5 23 12
5 15 2
15 15 10 13
5 10 9 4
I 7 3 0
38 46 15 1
21 18 8 0
11 13 8 0
11 16 12 8
0 6 14 1
0 0 5 3
16 20 16 23
6 14 11 12
2 7 1 2
25 13 26 3619 4 17 19
3 4 3 4
27 30 28 823 33 17 0
9 10 5 0
12” 13” 14”
11” 12” 14”
27 55 5122 47 8
15 22 6
9 2 5
6 I 3
5 0 1
16 10 5
19 7 4
I 1 4
30 23 15
17 11 10
2 0 8
7 3 0
1 3 2
0 2 1
12 13 5
2 1 3
0 0 6
18 28
12 16
8 7
2717
50
0
0
0 3
0 0
0 0
Patient Autologous Allogeneic Renal cell NK”-sensitive
no. melanoma melanoma carcinoma K562
I + + - -
4 + - - -
10 + ± ± ±
11 + + - -
“ NK, natural killer.
reaction to AT cells. Skin testing using UVR-AT was not
performed. Contaminants in preparation could have resulted in
a high frequency of false-positive DTH reactions to both AT and
PBMC control skin tests; this was not observed. No tests, such
as punch biopsy, were done to evaluate for false-negative
results.
The size of the DTH induration to the vaccine itself (UVR-
AT + DETOX), measuring primarily response to adjuvant, was
above the average and median values in 7 of the 10 patients with
positive cytotoxicity, including the two responders and the three
patients with a DTH reaction to AT. No consistent trend in the
size of the DTH reaction to vaccine was observed in individuals
or in population groups over time. Biopsy and staining of a
1-month-old indurated injection site in one patient revealed
heavy infiltration by histiocytes and I-cells but not by B cells
(data not shown).
DISCUSSION
Among 24 patients with indicator lesions, intradermal ac-
tive immunotherapy with UVR-AT + DETOX resulted in two
major responses in soft-tissue sites and in a bone metastasis,
supporting clinically meaningful systemic activity, albeit in
<20% of patients with 95% confidence. This is no different
from the results reported in other vaccine trials and does not
support using UVR alone to enhance the clinical activity of
autologous whole-cell vaccines plus adjuvant. As in other such
trials, the overall survival data were encouraging but likely
reflect selection bias. The present American Joint Committee on
Cancer criteria for stage IV disease only distinguish between
soft-tissue and visceral metastases in patients having a median
overall of survival of 8 months. Newer, more effective stratifi-
cation parameters for clinical trials using pretreatment serum
levels of lactate dehydogenase and albumin (33-34) and the
presence of visceral disease in zero-to-one organ versus more-
than-one organ (33) have identified prognostic groups with
median survivals of over 12 months versus <6 months. Until a
regimen with a high response rate is found, stratification might
help determine which active immunotherapy trials warrant con-
firmation of “encouraging” survival data in a Phase III trial
setting.
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Table 5 Characteristics of patients with positive and negative
detection of PBMC cytotoxicity against autologous tumor
0.8
0.6zC
C
Q.0.4
0.2
Clinical Cancer Research 625
4 M. Pride, unpublished data.
Table 4 Antibody inhibitio n of PBMC cyto toxicity against aut ologous melanoma
Patient Media
no. alone IgG anti-CD3 anti-CD8 anti-class I anti-CD4 anti-DR anti-DP
1 50” 56 14 13 12 54 55 52
4 47 53 0 1 1 45 46 48
11 46 43 7 4 6 42 41 45
a Percentage of cytotoxicity.
PBMC c
Positive
ytotoxicity
Negative
Patients (n = 35) 10 25
Male 8 14
Median age (yr) 53 52
Prior treatmentNone 50% 36%
Biotherapy alone 10% 8%
Chemotherapy ± biotherapy 40% 56%
Tumor stage and site(s):AJCC� stage III I 5
AJCC stage IV 9 (90%) 20 (80%)
Adjuvant 6 6Soft-tissue only or one visceral organ 8 13
Two or more visceral organs 1 1
Major objective response 2 0Median time to progression (mo) 18 3
Median survival (mo)
All patients 28+ 16Stage IV patients 28+ 14
DTH
Hypoergy to foreign antigens 40% 68%
Positive reaction to 10� AT 30% 20%
Larger than average reaction to vaccine 70%
a AJCC, American Joint Committee on Cancer.
38%
With regard to laboratory tests that might also guide such
determination, none to-date has proven reliable, in part because
of the dearth of correbatable clinical responses and in part
because of the inability to demonstrate reproducible dose-
response relationships in a heterogeneous patient population.
Posttreatment laboratory results cannot be stratified prospec-
tively in Phase I or II studies because of time bias and other
factors that complicate any analysis of such data (35).
The serial detection of PBMC cytotoxicity against AT in
10 of 35 assessable patients (Tables 2 and 5) supported induc-
tion of a specific systemic immune response to intradermal
treatment, a primary end point of this study. There are no data
to permit a comparison of the frequency of induction of positive
cytotoxicity with that using a non-UVR-AT + DETOX vaccine
because no patient received such treatment in this pilot study.
Among the stage IV patients, almost uniformly with favorable
clinical pretreatment prognostic features, detection of PBMC
cytotoxicity against AT cells correlated significantly with sur-
vival (P = 0.009; Fig. 3). Different patterns of specificity were
observed, suggesting that the positive results were not likely to
be an artifact of the assays themselves (Table 3). Also, MHC
class I-restricted CD8+ I cells and not natural killer cells were
the primary mediators of this activity in the patients tested
I : � � H�1 �,
4---+ -�
PBMC Cytotoxicity Total Dead
Positive 9 3
Negative 20 12 P a 0.009
0 12 24 36 48
MONThS
Fig. 3 Kaplan-Meier survival curves for 29 assessable patients with
stage IV melanoma and similar pretreatment prognostic features dis-
tinguished by the presence or lack of detectable PBMC cytotoxicity
against autologous tumor.
(Table 4). The fall-off of detection of PBMC cytotoxicity con-
current with tumor progression in some patients suggested in-
duction of tolerance. It must be noted, however, that the periph-
eral blood is only one pharmacological compartment (36), and
this study did not account for efficient trafficking of cytotoxic I
cells to metastatic deposits and any resulting immunoselection
(37).
The induction of a DTH reaction to AT cells was not
observed in clinical responders. Subclinical reactivity could
have been evaluated further by punch biopsy of skin test sites;
this would require an informed consent from the patients. The
prolonged time to first clinically apparent DTH reaction to AT
cells (median of 8 courses) introduced a substantial “time bias”
precluding developing this test for prognostic purposes (35).
Among all DTH assays performed, only above average DTH
reaction to vaccine, largely a response to the adjuvant DETOX,
correlated favorably with the clinical course of disease. The
study was too small to evaluate the effect of concurrent detec-
tion of both PBMC cytotoxicity and DTH reactivity to AT cells,
observed in only three patients.
Demonstration of UVR-induced expression of B7. 1 on
human melanoma, as reported recently on murine melanoma
(21) as well as on CT-26 (colon carcinoma) and HCA (hepato-
cellular carcinoma) cell lines,4 could support using UVR to
study the antigen-presenting capability of tumor cells without
resorting to gene transfection. Besides the ongoing character-
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626 Active Immunotherapy: Autologous Melanoma Cells. DETOX
ization of the positive cellular immune responses as presented
above and in cytokine elaboration experiments, future efforts
will be geared to testing cytokines that drive TH’ immune
responses (25) or that recruit dendritic cells or other professional
antigen-presenting cells to the vaccine injection site (15).
Suto and Srivastava (38) has been able to extract from
autobogous tumor cells heat shock protein-96, a chaperon that
complexes with the cells’ unique repertoire of soluble immuno-
genic peptides (HSPPC-96). He has shown that HSPPC-96 can
elicit antigen-specific CILs. Treatment with HSPPC-96 could
provide an array of autobogous tumor-specific peptide targets for
CIL activation without the need to characterize each peptide or
to exclude patients based on human leukocyte antigen pheno-
type. This may prove to be a far more exploitable and versatile
strategy for immunotherapy than trying to convert an autologous
tumor cell that has already eluded host immune surveillance into
a better antigen-presenting cell. Autologous tumor HSPPC-96
could be used in the future to prime dendritic cells activated or
expanded in vitro with cytokines such as granubocyte/macro-
phage colony-stimulating factor, interleukin 4, or flt3 bigand.
No active immunotherapy program will be optimized, how-
ever, until methods are developed in individual patients to
circumvent immunoselection (6), local secretion of tolerizing
molecules by tumors (7-10), and biophysicab barriers that make
metastases sanctuary sites from the immune system ( I I).
ACKNOWLEDGMENTS
We thank Dr. Margaret Kripke for helpful suggestions and thought-
ful review of the manuscript.
REFERENCES
1. Hewitt, H., Blake, E., and Walder, A. A critique of the evidence for
active host defense against cancer based on personal studies of 27
murine tumors ofspontaneous origin. Br. J. Cancer, 33: 241-259, 1976.
2. Gross, L. Intradermal immunization of C3H mice against a sarcomathat originated in an animal of the same line. Cancer Res.. 3: 326-333,
1943.
3. Prehn, R. T., and Main, J. M. Immunity to methylcholanthrene-
induced sarcomas. J. Natl. Cancer Inst., 18: 769-778, 1957.
4. Klein, G., Sjogren. H.. Klein, E.. and Hellstrom, H. E. Demonstration
of resistance against methylcholanthrene induced sarcomas in the pri-
mary autochthonous host. Cancer Res., 20: 1561-1572. 1960.
5. Kripke, M. L. Antigenicity of murine skin tumors induced by ultra-violet light. J. Natl. Cancer Inst., 53: 1333-1336, 1974.
6. Jager, E., Ringhoffer, M.. Altmannsberger, M., Arand, M., Karbach,J., Jager, D., Ocsch, F., and Knuth, A. Immunoselection in i’iio: loss of
MHC class I and melanocyte differentiation antigen expression in met-
astatic melanoma. Abstracts 521 and M16, Cancer Vaccines, Cancer
Research Institute (New York). 1996.
7. Suzuki, T., Tahara, H., Narula, S., Moore, K. W., Robbins, P. D., and
Lotze, M. T. Viral interleukin 10 (IL-b), the human herpes virus 4
cellular IL-b homologue, induces local anergy to allogeneic and syn-
geneic tumors. J. Exp. Med., 182: 477-486, 1995.
8. Mizoguchi, H., O’Shea, J. J., Longo, D. L., Loeffler, C. M., McVicar,
D. W., and Ochoa, A. C. Alterations in signal transduction molecules in
T lymphocytes from tumor-bearing mice. Science (Washington DC).258: 1795-1798, 1992.
9. Hahne, M., Rimoldi, D., Schroter, M., Romero, P., Schreier, M.,
French, L. E., Schneider, P., Bornand, T., Fontana. A., Lienard, D..
Cerottini, J., and Tschopp, J. Melanoma cell expression of Fas (Apol/
CD95) ligand: implications for tumor immune escape. Science (Wash-ington DC), 274: 1363-1366. 1996.
10. Wiertz, E. J., Tortorella, D., Bogyo, M., Yu, J., Mothes, W., Jones,
T. R.. Rapoport. T. A., and Ploegh, H. L. Sec6l-mediated transfer of a
membrane protein from the endoplasmic reticulum to the proteasome for
destruction. Nature (Lond.), 384: 432-438, 1996.
11. Jam, R. K. Barriers to drug delivery in solid tumors. Sci. Am., 271:
58-65. 1994.
12. Cheson, B. D., Phillips. P. H., and Sznol, M. Trials using tumor
vaccines. Oncology (Basel), 11: 81-90, 1997.
13. Berd, D.. Maguire, H. C., Jr., McCue, P., and Mastrangelo, M. J.
Treatment of metastatic melanoma with an autologous tumor cell vac-
cine: clinical and immunologic results in 64 patients. J. Clin. Oncol., 8:
1858-1867, 1990.
14. Morton, D. L., Foshag, L. J.. Hoon, D. S., Nizze, J. A., Wanek,
L. A.. Chang. C., Davtyan, D. 0.. Gupta. R. K.. Elashoff, R., and Irie,
R. F. Prolongation of survival in metastatic melanoma after activespecific immunotherapy with a new polyvalent melanoma vaccine. Ann.
Surg.. 216: 463-482, 1992.
15. Leong, S. P. L., Enders-Zohr. P., Zhou, Y. M., Allen, R. W., Jr..
Saagebiel, A. B., Glassberg, A. B., and Hayes, F. A. Active specific
immunotherapy with GM-CSF as an adjuvant to autologous melanoma
vaccine in metastatic melanoma. Proc. Am. Soc. Clin. Oncol. Annu.
Meet., 15: 437. 1996.
16. Hostetler, L. W., Ananthaswamy, H. N., and Kripke. M. L. Gener-
ation of tumor-specific transplantation antigens by UV radiation can
occur independently of neoplastic transformation. J. Immunol.. /37:
2721-2725. 1986.
I 7. Hostetler, L. W., and Kripke. M. L. Proportion of antigenic variantsinduced by iii vitro UV irradiation differs in clones derived from a single
tumor. J. Immunol., /40: 666-670, 1988.
18. Hostetler. L. W., Romerdahl, C. A., and Kripke. M. L. Specificity
of antigens on UV radiation-induced antigenic tumor cell variants meas-
ured in vitro and in vivo. Cancer Res., 49: 1207-1213, 1989.
19. Umezu, Y., Augustus. L. B.. Seito. D.. Hayakawa, K.. Ross, M. I.,
Eton. 0.. Swanson, D. A., and Itoh, K. Increase in the ability of human
cancer cells to induce cytotoxic T lymphocytes by ultraviolet irradiation.
Cancer Immunol. Immunother., 37: 392-399, 1993.
20. Pride, M. L., Donawho, C. K., and Kripke, M. L. Active specific
immunotherapy using UVB-irradiated whole melanoma cell vaccine.
Abstract 52 1 1, 9th International Congress of Immunology (San Fran-
cisco), 1995.
21. Pride, M. L., Donawho, C. K.. and Kripke, M. L. UVB irradiation
of tumor cells enhances the immunogenicity of melanoma cell vaccines
by augmenting cell surface expression of MHC class I and II and B7-l.
Proc. Am. Assoc. Cancer Res., 38: 630, 1997.
22. Shinomiya. Y.. Harada, M.. Kurosawa. S., Okamoto. T.. Terao, H..
Matsuzaki, G., Shirakusa, T., and Nomoto, K. Anti-metastatic activity
induced by the in vito activation of purified protein derivative (PPD)-
recognizing ThI type CD4+ T cells. Immunobiology, /93: 439-455,
1995.
23. Schultz, N., Oratz, R., Chen, D., Zeleniuch-Jacquotte. A., Abeles,
G., and Bystryn. J. C. Effect of DETOX as an adjuvant for melanoma
vaccine. Vaccine, /3: 503-508, 1995.
24. Mitchell, M. S., Harel, W., Kempf� R. A.. Hu, E., Kan-Mitchell, J.,
Boswell, W. D.. Dean, G.. and Stevenson, L. Active-specific immuno-
therapy for melanoma. J. Clin. Oncol.. 8: 856-869, 1990.
25. Itoh, K., Platsoucas, C. D., and Balch, C. M. Autologous tumor-
specific cytotoxic T lymphocytes in the infiltrates of human metastaticmelanoma. Activation by interleukin 2 and autologous tumor cells, and
involvement oftheTcell receptor. J. Exp. Med., /68: 1419-1441, 1988.
26. Itoh, K., Tilden, A. B., and Balch, C. M. Interleukin-2 activation of
cytotoxic T lymphocytes infiltrating into human metastatic melanomas.Cancer Res., 46: 301 1-3017, 1986.
27. Mulder, W. M., Koenen, H., van de Muysenberg. A. J.. Bloemena,
E.. Wagstaff. J.. and Scheper. R. J. Reduced expression of distinct T-cell
CD molecules by collagenase/DNase treatment. Cancer Immunol. Im-
munother., 38: 253-258, 1994.
Research. on March 21, 2021. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Clinical Cancer Research 627
28. Abuzakouk, M., Feighery. C.. and O’Farrelly. C. Collagenase and
dispase enzymes disrupt lymphocyte surface molecules. J. Immunol.
Methods, 194: 21 1-216. 1996.
29. Berd, D., Herlyn, M., Koprowski, H., and Mastrangelo. M. J. Flowcytometric determination of the frequency and heterogeneity of expres-
sion of human melanoma-associated antigens. Cancer Res., 49: 6840-
6844. 1989.
30. Bystryn. J. C., Jacobson, S., Harris, M. N., Roses, D. F., Speyer. J. L..
and Levin, M. Preparation and characterization of a human polyvalent
melanoma antigen vaccine. J. Biol. Response Modif.. 5: 21 1-224, 1986.
3 1 . Mather, J. P.. and Sato, G. H. The growth of mouse melanoma cellsin hormone supplemented serum-free medium. Exp. Cell Res., /20:
199-200. 1979.
32. Simon. R. Optimal two-stage designs for Phase II clinical trials.Control. Clin. Trials, 10: 1-10, 1989.
33. Eton, 0., Legha. S. S.. Moon. T. E.. Papadopoulos. N. E.. Plager,C., Burgess. A. M., Bedikian, A. Y.. Buzaid, A. C., Ring. S.. Dong. Q..Glassman. A. B., Balch, C. M.. and Benjamin, R. S. Prognostic factors
for survival of patients treated systemically for disseminated melanoma.J. Clin. Oncol., in press. 1998.
34. Sirott, M. N., Bajorin, D. F., Wong, G. Y., Tao, Y., Chapman, P. B.,
Templeton. M. A., and Houghton, A. N. Prognostic factors in patients
with metastatic malignant melanoma: a multivariate analysis. Cancer
(Phila.), 72: 3091-3098, 1993.
35. Anderson, J. R., Crowley, J. J., and Propert, K. J. Interpretation of
survival data in clinical trials. Oncology, 5: 104-1 14, 1991.
36. Zhu, H., Melder, R. J., Baxter, L. T., and Jam, R. K. Physiologically
based kinetic model of effector cell biodistribution in mammals: impli-
cations for adoptive immunotherapy. Cancer Res.. 56: 3771-3781,
1996.
37. Economou, J. S., Belldegrun, A. S., Glaspy. J.. Toloza, E. M.,Figlin. R.. Hobbs, J.. Meldon. N., Kaboo, R., Tso, C. L.. Miller, A., La.
R.. McBride, W., and Moen, R. C. In vito trafficking of adoptively
transferred interleukin-2 expanded tumor-infiltrating lymphocytes andperipheral blood lymphocytes. Results of a double gene marking trial.
J. Clin. Invest., 97: 515-521, 1996.
38. Suto. R.. and Srivastava, P. K. A mechanism for the specific
immunogenicity of heat shock protein-chaperoned peptides. Science(Washington DC), 269: 1585-1588, 1995.
Research. on March 21, 2021. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
1998;4:619-627. Clin Cancer Res O Eton, D D Kharkevitch, M A Gianan, et al. melanoma.whole melanoma cells plus DETOX in patients with metastatic Active immunotherapy with ultraviolet B-irradiated autologous
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