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1828
Gangliosides as Targets for Immunotherapy for
Pancreatic Adenocarcinoma
Kyo U. Chu, M.D.
Mepur H. Ravindranath, Ph.D.
Alexandra Gonzales, B.S.
Kevin Nishimoto, B.S.
Wai Yin Tam, B.S.
Daniel Soh, B.S.
Anton Bilchik, M.D., Ph.D.
Nonda Katopodis Ph.D.
Donald L. Morton, M.D.,
BACKGROUND. Pancreatic adenocarcinoma cells express gangliosides and sialyl
Presented in part at the American College of Surgeons Surgical Forum, Orlando, Florida, October
25–30, 1998, and the 90th Annual Meeting of the
American Association for Cancer Research, Philadelphia, Pennsylvania, April 10 –14, 1999.
Lewis (sLe) antigens. It is not known whether these carbohydrate antigens can be
targeted by immunotherapy. The authors measured the expression of GM2 and sLe
antigens on the surface of pancreatic carcinoma cells and the serum levels of total
gangliosides, GM2, and antiganglioside antibodies in patients with pancreatic
carcinoma.
METHODS. Cell surface GM2 and sLe antigens were measured by cell suspension
enzyme linked immunoadsorbent assay (ELISA) in four pancreatic carcinoma cell
lines. Sera from 20 pancreatic carcinoma patients and 20 age- and gender-matched
healthy volunteers were analyzed for antiganglioside and anti-sLe immunoglobulin
(Ig) M titers by ELISA. Serum levels of total gangliosides and GM2 also were
measured.
RESULTS. All cell lines expressed GM2 and sLe antigens. When compared with ageand gender-matched volunteers, patients had significantly higher serum levels of
total gangliosides (25.6 ⫾ 9.0 mg/dL vs. 15.6 ⫾ 2.7 mg/dL; P ⬍ 0.001), GM2 (0.278
⫾ 0.415 mg/dL vs. 0.013 ⫾ 0.018 mg/dL; P ⫽ 0.02), ELISA units of anti-GM2 IgM
antibody (368 ⫾ 95 vs. 155 ⫾ 25; P ⫽ 0.04) and anti-GD1b IgM antibody (351 ⫾ 91
vs. 138 ⫾ 26; P ⫽ 0.03), but not anti-sLex IgM (1389 ⫾ 345 vs. 1081 ⫾ 224; P ⫽ 0.46)
or anti-sLea IgM antibody (1097 ⫾ 253 vs. 1200 ⫾ 315; P ⫽ 0.80). Patients with
unresectable tumors had higher serum levels of total gangliosides compared with
patients with resectable tumors, and a serum level ⬎ 25 mg/dL was found to
correlate significantly with poor overall survival (P ⬍ 0.02).
CONCLUSIONS. Increased serum levels of total gangliosides and GM2 may reflect
shedding or release of gangliosides from the surface of tumor cells. Production of
IgM antibody against GM2 and GD1b indicates that these gangliosides are immunogenic antigens that may be potential targets for effective active immunotherapy.
Cancer 2000;88:1828 –36. © 2000 American Cancer Society.
The authors acknowledge support from NC1T32-CA 09689 and Rogovin-Davidow Foundation
(Los Angeles).
KEYORDS: pancreatic carcinoma, gangliosides, carbohydrate antigens, immunotherapy.
The authors thank Dr. Nobuo Hanoi for providing
the mouse monoclonal anti-GM2 antibody for this
investigation.
A
Roy E. Coats Research Laboratories and the Sonya
Valley Ghidossi Vaccine Laboratory of the John
Wayne Cancer Institute at Saint John’s Health
Center, Santa Monica, California.
Kyo U. Chu’s current address: Division of Surgical
Oncology, Department of Surgery, University of
Virginia Health System, Charlottesville, Virginia.
Address for reprints: Mepur H. Ravindranath,
Ph.D., Director, Laboratory of Glycolipid Immunotherapy, John Wayne Cancer Institute, 2200 Santa
Monica Boulevard, Santa Monica, CA 90404.
Received June 23, 1999; revision received December 3, 1999; accepted December 3, 1999.
© 2000 American Cancer Society
ccording to recent statistics from the American Cancer Society, in
the United States, the estimated number of new cases of invasive
pancreatic carcinoma and the estimated number of deaths due to
invasive pancreatic carcinoma each approached 30,000 during 1999.1
The most common type of invasive pancreatic carcinoma is ductal
adenocarcinoma, which is associated with a poor prognosis. Surgery
is the only potentially curative therapy, but complete resection is
possible in less than 10% of cases. Combined radiotherapy and chemotherapy have shown some efficacy after surgery.2–5 New chemotherapeutic agents appear to be promising,6 – 8 but as yet none has
produced a significant survival benefit. Overall 5-year survival rate
remains less than 10%, and overall median survival is less than 1 year.
Serum Gangliosides in Pancreatic Carcinoma/Chu et al.
Immunotherapeutic investigations for pancreatic
carcinoma have been hindered by the absence of information on immunogenic tumor-associated antigens and by the significant immunosuppression
caused by tumor-derived factors.9 –11 Pancreatic adenocarcinoma cells express glycoconjugate antigens
that are immunosuppressive. Among these glycoconjugates are gangliosides, the glycolipids containing
sialic acids.12 Because gangliosides are a target of active specific immunotherapy in patients with melanoma13 and colon carcinoma,14 we hypothesized that
they also might play a role in tumor immunity against
pancreatic adenocarcinoma. We examined the level of
expression of particular gangliosides on the surface of
pancreatic cell lines and the serum levels of gangliosides and antiganglioside antibodies in patients with
pancreatic adenocarcinoma.
PATIENTS AND METHODS
Study Population
The study population was drawn from patients treated
for pancreatic adenocarcinoma at the John Wayne
Cancer Institute between January 1991 and December
1997. The 20 patients chosen for this study were those
whose follow-up was accurately documented and
from whom sera had been preoperatively obtained
and cryopreserved. Charts for these patients were retrospectively reviewed, and clinical information was
entered into a computer database. Table 1 shows the
treatments received by these patients.
The control population was drawn from healthy
volunteers who donated blood at Saint John’s Health
Center during a 3-month interval (January through
March) in 1998. Twenty control subjects were selected
from this group by computer matching their age and
gender with those of the study patients. None of these
donors had an active medical problem or was taking
any medications. Informed consent was obtained
from the subjects to use their sera for the research
investigation. This study was approved by the joint
institutional review board of Saint John’s Health Center and John Wayne Cancer Institute (Protocol number: RavM-Cellsurf-1196).
Measurement of Serum Total Gangliosides
The most frequently used assay for measuring the
level of serum gangliosides (expressed as lipid-bound
sialic acid) is described by Katopodis et al.15 Approximately 30% of the sialic acids extracted with this
technique may be derived from sialoproteins, or proteolipids with sialic acid.16,17 However, some of these
sialic acid-containing proteins may include ganglioside binding transport proteins17 or immunoglobulins
complexed with gangliosides. For routine analysis, the
1829
TABLE 1
Patient Characteristics and Treatments Received
Patient
no.
Age (yrs)
Gender
Adjuvant treatment
1
71
Male
2
64
Male
3
4
5
7
6
8
9
10
11
12
13
14
15
16
17
18
19
20
87
59
65
70
54
48
68
58
68
47
52
63
56
58
78
63
66
42
Male
Female
Female
Male
Female
Female
Male
Female
Male
Male
Male
Male
Male
Male
Male
Male
Female
Female
Systemic chemotherapy (5-FU);
radiation
Systemic chemotherapy; hyperthermia;
radiation
None
Radiation
Regional chemotherapy; hyperthermia;
None
None
Systemic chemotherapy; hyperthermia;
Chemotherapy; radiation
None
Systemic chemotherapy; radiation
Systemic chemotherapy; hyperthermia
BCG/systemic chemotherapy
Chemotherapy; hyperthermia
None
Systemic chemotherapy; brachytherapy
Brachytherapy
None
None
Systemic chemotherapy; hyperthermia;
5-FU: 5-fluorouracil; BCG: bacillus Calmette–Guerin.
serum level of total gangliosides can be measured with
an assay that detects lipid-associated sialic acids (LASAs) (Dianon Systems, Inc., Stratford, CT). We checked
the reliability and reproducibility of this LASA assay by
a double blind analysis. In addition, the normal values
reported in this study were comparable to those reported by other investigators. Schutter et al.18 reported
that the sensitivity of serum ganglioside level as a
marker of various human cancers may vary from 30%
to 100% using a cutoff level of 20 mg/dL. The LASA
assay appears to have a particularly high positivity rate
in leukemia, Hodgkin disease, melanoma, sarcoma,
advanced ovarian carcinoma, and oropharyngeal tumors.
Briefly, for the LASA assay, 100 ␮L of serum was
extracted with 100 ␮L of a chloroform:methanol (2:1
v/v) mixture. After thorough vortexing and centrifuging of the mixture, 100 ␮L of the supernatant was
transferred to a clean test tube. The concentration of
sialic acid in the supernatant was determined using a
resorcinol method described by Svennerholm.19 The
resorcinol/HCl reagent was mixed with supernatant
and then heated at 100 °C for 15 minutes. After cooling
at room temperature, a butylacetate:n-butanol (85:15
volume/volume [v/v]) mixture was added, and the
organic layer containing the chromogen was read at
an absorbency of 580 nm. Using a standard curve
1830
CANCER April 15, 2000 / Volume 88 / Number 8
obtained with commercial N-acetylneuraminic acid
(NeuAc; Sigma, St. Louis, MO), the serum level of total
gangliosides was expressed as milligrams per deciliter.
Enzyme-Linked Immunosorbent Assay for Serum
Anticarbohydrate Antibody
Sera from 20 pancreatic carcinoma patients were
screened for immunoglobulin (Ig) G and IgM antibodies against GM1, GM2, GM3, GD1a, GD1b, GD2, GD3,
and GT1b by enzyme-linked immunosorbent assay
(ELISA). Serum titers of antiganglioside antibodies
were measured by a method described previously.20
Microtiter plates (Falcon Probind 3915) were coated
with an ethanolic suspension of gangliosides (3 nmol/
100 ␮L/well; Sigma) and dried in a vacuum desiccator
for 2 days. The plates were blocked with phosphatebuffered saline (PBS), 4% human serum albumin
(HSA), pH 7.2, for 90 minutes. Sera were diluted to
1:100 and incubated in a water bath for 30 minutes at
37 °C. Sera were further serially diluted up to 1:12,800,
overlaid on the plates, and incubated at 37 °C for 2
hours. The plates were manually washed five times
with washing buffer (PBS 0.1% HSA, 0.1% Tween 20).
Antihuman IgM and IgG antibodies coupled to peroxidase (Jackson Immunoresearch Laboratories, Inc.,
Pittsburgh, PA) were used as the second antibody at a
dilution of 1:5000, and the plates were incubated with
second antibody (100 ␮L/well) for 1 hour at 37 °C. The
plates were washed as before. The substrate, o-phenylenediamine dissolved in citrate–phosphate buffer
(pH 5.0) and hydrogen peroxide (4.2 ␮L/10 mL), was
added to the plates (100 ␮L/well), and the plates were
incubated for 45 minutes in the dark. Enzymatic oxidation was arrested with 6N H2SO4, and absorbency
was measured at 490 – 650 nm in a microplate reader
(Molecular Devices Corporation, Sunnyvale, CA). The
values were corrected against the background (wells
without antigen). The titer was determined at an absorbency of 0.1.
After identification of high anti-GM2 and GD1b
IgM levels in the 20 patients with pancreatic adenocarcinoma, sera from cancer and control groups were
analyzed for IgM antibodies against GM2, GD1b, sialyl
Lewisx (sLex), and sLea (the antigenic determinant of
CA 19-9). Serum titers of anti-sLe antibodies were
measured using the method described earlier.21 Microtiter plates (Falcon Probind 3915) were coated with
100 ␮L streptavidin (0.5 ␮g/well; Sigma) suspended in
PBS (pH 7.2) and incubated overnight at 4 °C. After
removing excess streptavidin, the plates were coated
overnight with biotinylated polyacrylamide sLe polymer (3.3 pmol of sLe; Glycotech, Rockville, MD) in
carbonate-bicarbonate buffer (pH 9.6). The plates
were blocked with 4% HSA PBS (pH 7.2) for 90 minutes
at 37 °C. The sera were serially diluted and tested on
the antigen-coated plates by ELISA. Pooled sera from
melanoma patients served as a positive control for
anti-GM2 and anti-GD1b antibodies, and pooled sera
from colon carcinoma patients served as a positive
control for anti-sLex and anti-sLea antibodies. Immunoglobulin M levels were expressed as ELISA units
(patient antibody titer/positive control titer ⫻100).
Measurement of Serum GM2
One hundred microliters of serum in polypropylene
Eppendorf tubes was admixed with 1 mL of chloroform:methanol (1:1), extensively vortexed, and centrifuged to determine the serum GM2 level. The supernatant was collected, dried over nitrogen gas, and
resuspended in 500 ␮L of ethanol. One hundred microliters of the ethanolic suspension was used to coat
the wells. After drying in a vacuum, the plates were
blocked with PBS 4% HSA. After removing the blocking agent, 100 ␮L of anti-GM2 IgM monoclonal antibody (KM696, 1.48 mg/ml affinity purified IgM; Kyowa
Hakko Kogyo Co., Tokyo, Japan) diluted to 1:500 was
added to the plates. The GM2 absorbency of patient
sera was compared with that of sera from the control
group of healthy volunteers.
Pancreatic Carcinoma Cell Lines
Pancreatic carcinoma cell lines (CRL-1687 [BxPC-3/
14223], CRL-1682 [AsPC-1/14736], CRL-1837 [SU-86-86/
15002], and CRL-1918 [CFPAC-1/11588]) were obtained
from American Type Culture Collection (Rockville, MD)
and cryopreserved. Cells were thawed22 and cultured in
RPMI-1640 medium (JRH Biosciences, Lenexa, KS) supplemented with 10% fetal bovine serum (Gemini Bioproducts, Calabassas, CA) and antibiotic antimycotic solution (Pen-Strep-Fungizone 5240-013; Gibco BRL,
Gaithersburg, MD). Lack of homogeneity of the cell population from fresh tumors, poor recovery of tumor cells
from individual tumors, and inability to grow the cells
under our routine culture conditions limited our investigations on the antigen expression to cell lines from
ATCC.
Quantitation of Cell Surface Antigens with Cell
Suspension ELISA
Quantitation of cell surface antigens was conducted
using methods described previously.22,23 Trypsin was
not used for harvesting the cells.15 All cells were
washed once with RPMI-4% HSA and then suspended
in cold RPMI-4% HSA. Cells were counted for viability
using 0.1% trypan blue. Suspensions of 0.5 ⫻ 106 cells
in microcentrifuge tubes containing 60 ␮L of solution
were treated with the secondary antibody only (background), with primary and secondary antibody (exper-
Serum Gangliosides in Pancreatic Carcinoma/Chu et al.
1831
TABLE 2
Serum Levels of Total Gangliosides and CA 19-9
Group
n
Total gangliosides
(mg/dL ⴞ SD)
Healthy donors
Patients with pancreatic carcinoma
Resectable tumor
Unresectable tumor
AJCC Stage II/III
AJCC Stage IV
20
20
5
15
10
10
15.6 ⫾ 2.7
26.0 ⫾ 9.0
19.1 ⫹ 4.9
28.3 ⫹ 9.0
22.7 ⫹ 7.6
29.3 ⫹ 9.5
P value
(two-tailed)
⬍ 0.001
⬍ 0.05
NS
CA 19-9
(mg/dL ⴞ SD)
10 ⫾ 8
33594 ⫾ 113547
362 ⫹ 570
44,671 ⫹ 130,275
1466 ⫹ 3794
65,721 ⫹ 157,829
P value
(two-tailed)
⬍ 0.001
NS
⬍ 0.05
SD: standard deviation; NS: not significant; AJCC: American Joint Committee on Cancer; NS: not significant.
imental), or with class-matched isotypes of the primary antibodies (negative control). The primary
antibodies (120 ␮L) used in this study were KM696
(IgM affinity purified, 1.23 mg/500 ␮L; Kyowa Hakko
Kogyo Co.; final dilution, 1:500) for GM2, KM 93 (IgM
affinity purified, 100 ␮L/␮L; Kamiya Biomed, Seattle,
WA; final dilution, 1:100) for sLex, and KM 231 (IgG1
affinity purified, 100 ␮L/␮L; Kamiya Biomed; final dilution, 1:100) for sLea. The fine specificity of these
antibodies has been well characterized.24
Localization of Immunogenic Epitopes in Glycoconjugates
by Enzyme-Treated Cell Suspension ELISA
Although gangliosides are glycolipids, the sugar
epitope of the gangliosides also may be found in glycoproteins.25 Therefore, the carrier molecules of GM2
and sLe can be proteins or lipids. Furthermore, gangliosides on the cell surface may be masked by transmembrane glycoproteins. To unmask gangliosides
and distinguish their sugar epitopes from the carbohydrate epitopes on proteins, we subjected pancreatic
carcinoma cells to a variety of enzymatic treatments.
To facilitate the access of enzymes to the peptide core
of cell surface glycoproteins, cells first were treated
with sialidase from Vibrio cholerae (Boehringer Mannheim, Indianapolis, IN) for 2 hours at 37 °C. N-linked
and O-linked oliogosaccharides then were cleaved
with N- and O-glycosidases (Boehringer Mannheim).
Pronase (Boehringer Mannheim) was used to cleave
the peptide core of cell surface proteins. The enzyme
activity was arrested with RPMI 4% HSA. Antibody
binding was assessed after washing viable cells three
times with RPMI-1640 4% HSA.
survival differences among patient subgroups. Overall
survival was defined as the period between the time of
diagnosis and the patient’s death or last follow-up
visit. Multivariate analysis using Cox proportional hazards regression model was performed to determine
the impact of significant factors on survival. Serum
levels of gangliosides, CA 19-9, anti-GM2 antibody,
and anti-GD1b antibody were analyzed as continuous
variables in multivariate analysis. All statistical analyses were two-tailed and performed with SAS software
(SAS Inc., Cary, NC). Results were considered significant if the probability value was less than 0.05.
RESULTS
Of the 20 patients with pancreatic adenocarcinoma, 5
underwent potentially curative resection, and 15 were
found to have unresectable disease either before or
during laparotomy. Ten patients had distant metastatic disease (American Joint Committee on Cancer
[AJCC] Stage IV26), nine had regional metastases (AJCC
Stage III), and one had localized disease (AJCC Stage
II). Median survival of the 20 patients was 10 months;
mean survival was 14.4 months.
Serum CA 19-9 Levels
Serum levels of CA 19-9 were elevated in 17 of 20
patients and were significantly (P ⬍ 0.001) higher in
the cancer group than the control group (Table 2).
Serum levels of CA 19-9 also were significantly higher
(P ⬍ 0.05) in Stage IV patients than Stage III patients.
CA 19-9 levels did not differ for patients with resectable versus unresectable disease.
Serum Total Ganglioside Levels
Statistical Analysis
Statistical analyses of serum levels of total gangliosides, GM2, and anticarbohydrate antibodies were
performed by Wilcoxon rank-sum test. Survival curves
were estimated by the Kaplan–Meier method. Univariate analysis by log rank test was used to determine
Serum levels of total gangliosides in the 20 healthy
volunteers ranged from 12.1 to 22.2 mg/dL; the mean
(15.6 ⫾ 2.7 mg/dL) was comparable to that of normal
individuals reported by other investigators.18 Serum
ganglioside levels were above the normal reference
range of 15–20 mg/dL in 16 of 20 patients and in 1 of
1832
CANCER April 15, 2000 / Volume 88 / Number 8
not significantly higher in the cancer group at any
stage of disease.
Serum GM2 Level
FIGURE 1. Kaplan–Meier curves for patients with pancreatic adenocarcinoma and serum ganglioside (SG) levels ⱖ 25 mg/dL versus ⬍ 25 mg/dL are
shown. Median overall survival was 14 months for patients with SG levels ⬍ 25
mg/dL and 10 months for patients with SG levels ⱖ 25 mg/dL (log rank test,
P ⫽ 0.03). SG: serum ganglioside.
20 normal volunteers. Serum ganglioside levels were
significantly higher in the cancer group than in the
control group (25.6 ⫾ 9.0 vs. 15.6 ⫾ 2.7 mg/dL; P
⬍ 0.001) (Table 2). Although patients with Stage IV
disease had a higher level of serum gangliosides than
did patients with Stage II/III disease, the difference
was not significant. However, serum ganglioside levels
were significantly higher in patients with unresectable
disease than in those with resectable disease (28.3 ⫾
9.0 vs. 19.1 ⫾ 4.9 mg/dL; P ⬍ 0.05). In addition, overall
survival was significantly better (P ⬍ 0.02) in patients
with low serum levels of total gangliosides (⬍ 25.00
mg/dL) than in those with high levels (ⱖ 25.00 mg/dL)
(Fig. 1). Multivariate analysis of prognostic factors including serum levels of gangliosides and CA 19-9,
AJCC stage, and patient gender showed that only serum gangliosides had a significant correlation with the
overall survival of patients with pancreatic adenocarcinoma (P ⬍ 0.05).
IgM Levels against Gangliosides and sLeX and sLeA
Sera from patients with cancer had high levels of antiGM2 and anti-GD1b IgM antibodies, very low levels of
IgM antibodies to other gangliosides, and very low
levels of IgG antibodies to all gangliosides (data not
shown). When compared with the control group, the
cancer group had higher levels of anti-GD1b IgM
(351 ⫾ 91 vs. 138 ⫾ 26; P ⫽ 0.03) and anti-GM2 IgM
(368 ⫾ 95 vs. 155 ⫾ 25; P ⫽0.04). However, anti-sLex
IgM (1389 ⫾ 345 vs. 1081 ⫾ 224; P ⫽0.46) and anti-sLea
IgM titers (1097 ⫾ 253 vs. 1200 ⫾ 315; P ⫽0.80) were
Because the cancer group had significantly higher
(P ⬍ 0.05) serum titers of anti-GM2 IgM antibodies, we
estimated serum GM2 levels by using an anti-GM2 IgM
mouse monoclonal antibody (KM696) to ethanolic supernatants of chloroform:methanol extracts. Box titration of varying concentrations of KM696 against varying concentrations of GM2 showed that the ELISA
absorbency was directly proportional to GM2 concentration (data not shown).
Serum GM2 absorbency ranged from 0 to 0.04
(mean ⫾ standard deviation [SD], 0.013 ⫾ 0.018) in the
control group of healthy volunteers. Serum GM2 absorbency was tested in 17 patients in the cancer group:
the GM2 absorbency was high in eight patients (range,
0.120 –1.414; mean ⫾ SD, 0.559 ⫾ 0.472). In the other
nine patients, the level of GM2 ranged from 0.013 to
0.086 (mean ⫾ SD, 0.028 ⫾ 0.023). However, overall,
the serum GM2 level was significantly higher in the
cancer group (0.278 ⫾ 0.415 vs. 0.013 ⫾ 0.018;
P ⫽ 0.02) than in the control group.
Density of GM2 on Pancreatic Carcinoma Cells
Because sera from 8 of 17 pancreatic carcinoma patients had significantly higher levels of GM2 than did
sera from control patients, we measured the density of
GM2 expression on the surface of cells from four pancreatic carcinoma lines. All pancreatic cell lines expressed GM2, but the level of expression was highest in
a cell line developed from a liver metastasis (Figure 2).
Expression of GM2 increased after the removal of Olinked or N-linked oligosaccharides associated with
cell surface proteins. Expression of GM2 was higher
after sialidase and O-glycosidase treatment or after
pronase treatment after deglycosylation (Figure 3).
Similarly, expression of GM2 was higher after N-glycosidase treatment (data not shown), suggesting that the
carbohydrate moiety of glycoproteins in pancreatic
carcinomas may be linked to serine and threonine
(O-linked) as well as to asparagine (N-linked). The
level of expression of GM2 may remain the same or
decrease slightly when deglycosylation is followed by
treatment with pronase. Interestingly cell surface expression of sLex was markedly reduced after deglycosylation (both N- and O-glycosidase), suggesting that
this antigen may be linked to serine/threonine as well
as to the asparagine in the protein moiety.
DISCUSSION
Our results document an apparent correlation between serum ganglioside levels and tumor burden in
Serum Gangliosides in Pancreatic Carcinoma/Chu et al.
1833
FIGURE 2.
Expression of GM2 on cells of
tumor cell lines derived from metastatic lesions
of the liver and pancreas and from ascites is
shown. The cell surface glycocalyx profile was
monitored by cell suspension ELISA by using
monospecific monoclonal antibodies, as described in Patients and Methods. ELISA: enzyme-linked immunosorbent assay.
pancreatic adenocarcinoma, similar to earlier reports.18 Levels of serum gangliosides are known to
increase with the stage of the disease and the extent of
metastasis in other malignancies.18 Our study population was too small to reveal a statistically significant
increase in the level of serum gangliosides among
AJCC Stage IV patients, but the level of serum gangliosides was significantly higher in patients with unresectable disease. Patients with unresectable disease
consistently had serum ganglioside levels greater than
25 mg/dL, and multivariate analysis identified serum
level of total gangliosides as the only factor that significantly influenced overall survival. These data suggest that patients with serum ganglioside levels less
than 25 mg/dL are more likely to be candidates for
complete surgical resection and have better survival.
Serum ganglioside level as a marker resembles
K-ras gene mutation in the plasma DNA of patients
with pancreatic carcinoma. K-ras mutation in codon
12 has been observed in pancreatic tissues of patients
with pancreatic adenocarcinoma and pancreatitis.27,28
In 9 of 16 patients with K-ras gene mutation-positive
tumors, an identical mutation was detected in the
plasma DNA.27 Such mutations in plasma DNA are not
common in patients with chronic pancreatitis
(⬍ 5%)28 or in healthy subjects.27 Plasma K-ras gene
mutation, similar to serum ganglioside level, reflected
tumor burden, failed to correlate with other clinicopathologic features (age, gender histologic type, mode
of invasion, and metastasis),27 correlated with poor
survival, and was identified as the only independent
prognostic factor.28 It would be worthwhile to investigate serum ganglioside level and K-ras gene mutation in plasma DNA as a combined-marker system to
evaluate tumor burden and resectability in patients
with pancreatic carcinoma. Because the K-ras gene
point mutation at codon 12 changes the amino acid
sequence (the normal Gly residue is substituted with
either a Val, Asp, or Cys residue), the K-ras products
are considered to be unique epitopes for T-cell (CD4⫹
and/or CD8⫹) recognition in cancer immunotherapy.29
Although serum levels of CA19-9 were higher in
patients with Stage IV disease, their tremendous variation concealed any significant elevation in patients
with unresectable disease, even though these patients
are more likely to have a larger tumor burden. This
variation makes CA 19-9 less reliable to determine
tumor burden. Most of the commercial antibodies for
CA 19-9 (121SLE [IgM], Neomarkers, Union City, CA;
1116-NS-19-9 [IgG1], Signet Laboratories, Dedham,
MA; M8073022 [IgG1], Fitzgerald, Concord, MA; ZYC09 [IgG1], Zymed, San Francisco, CA; MED-CLA 143,
Accurate Chemical and Scientific Corporation, Westbury, NY; NS19-9, International CIS, Cedex, France)
are directed against the sugar residues of sLea, which
is the antigenic determinant of CA 19-9.30 Sialyl Lewisa
is expressed in glycoproteins or glycolipids.30 –35 The
glycoproteins expressing sLea include mucin-type glycoproteins belonging to MUC family, such as MUC-1.
Tumor cells also may have more than one glycoprotein expressing the epitope of sLea. The heterogeneity
of pancreatic tumor cells expressing these sLea-associated antigens and the remarkable variability in the
carriers of sLea explain the variability observed in the
serum level of CA 19-9.
Although our pancreatic carcinoma patients had a
high level of IgM antibodies against GM2 and GD1b,
they did not have elevated levels of IgM antibodies
against sLea and sLex. This suggests that GM2 and
GD1b but not sLea or sLex are immunogenic antigens
in pancreatic carcinoma patients.
1834
CANCER April 15, 2000 / Volume 88 / Number 8
FIGURE 3. Expression of GM2 on four human pancreatic carcinoma (PC) cell
lines after deglycosylation. There was a noteworthy increase in the expression
of GM2 after deglycosylation or pronase treatment. The cell surface profile after
deglycosylation was monitored by enzyme-linked immunoadsorbent assay
(ELISA) cell suspension using monospecific monoclonal antibodies, as described in Patients and Methods. The vertical bars represent standard error of
five analyses. sLex: sialyl Lewisx.
The presence of anti-GM2 IgM antibody in patients with pancreatic carcinoma is noteworthy in
view of the observed correlation between elevated anti-GM2 IgM antibody titer and improved survival of
patients with melanoma.36 –38 Because anti-GM2 IgM
antibodies can fix complement and clear antigens
from the circulation, we assayed the levels of GM2 in
sera and tumor tissue. Eight of 17 patients in the
cancer group had significantly higher serum levels of
GM2 than did age- and gender-matched healthy volunteers. Our findings suggest that GM2 on pancreatic
carcinoma cells can be a potential target for immunotherapy. Deglycosylation significantly augments expression of GM2, possibly due to deletion of the oligosaccharide moiety of glycoproteins on the cell
surface. Interestingly, deglycosylation results in loss of
sLex expression and further facilitates access to GM2.
The use of factors such as cytokines and drugs to
down-regulate glycoprotein expression on the cell surface deserves further study.
The apparent inverse correlation between a serum
level of total gangliosides greater than 25 mg/dL and
survival may be related to increased tumor burden or
more advanced stage of disease. It also may reflect the
immunosuppressive effect of circulating gangliosides.
Gangliosides shed from a growing tumor39 can suppress expression of CD4 on human helper T lymphocytes,40 expression of major histocompatibility complex Class I and Class II genes,41 and mitogen-induced
lymphoblastic transformation. They can also induce
T-suppressor activity. Circulating gangliosides can reduce the number of macrophages and CD4⫹ and
CD8⫹ T lymphocytes,42 and they can inhibit interleukin-2– dependent lymphocyte proliferation43 by preventing the interaction of this cytokine with its cell
surface receptors. If ganglioside-induced immunosuppression is linked with poor prognosis, then clearance
of tumor-derived gangliosides from the circulation
may be of clinical benefit for patients with unresectable disease. The immunosuppressive role of GM2,
particularly its ability to mimic the receptor for interleukin-2, warrants its elimination from circulation.
The potential efficacy of anti-GM2 IgM antibody
for clearing the serum of immunosuppressive GM2
and for killing tumor cells by complement-mediated
cytotoxicity or apoptosis44 makes augmentation of anti-GM2 IgM antibodies a promising immunotherapeutic strategy. The level of anti-GM2 IgM can be augmented by passive administration of a murine antiGM2 IgM such as KM696; however, the development
of anti-mouse antibody responses45 limits the efficacy
of repeated administration. Nishinaka et al.46 developed a human anti-GM2 monoclonal antibody that
can cause complement-dependent cytotoxicity, but its
Serum Gangliosides in Pancreatic Carcinoma/Chu et al.
production is not cost-effective. Nakamura et al.44,47
constructed mouse/human chimeric anti-GM2 IgM
KM966 (mouse residue 33%; human residue 67%) and
humanized chimeric anti-GM2 IgM KM8969 (mouse
residue 10%; human residue 90%) with therapeutically
higher complement-mediated cytotoxicity, antibodydependent cytotoxicity, and apoptosis. However, Bator and Reading45 reported that anti-idiotypic and antiallotypic responses against human or humanized
monoclonal antibodies may negate any therapeutic
benefit of repeated administration. Suggested solutions to these problems may not be cost-effective for
treatment purposes.
An alternative to passive immunotherapy using
various anti-GM2 antibodies is active specific immunotherapy using purified GM2. Livingston48 augmented the immunogenicity of purified GM2 by conjugating it to hemocyanin, a xenogenic protein with a
molecular weight greater than 400 kilodaltons. However, the humoral and cellular immune responses induced by hemocyanin may prohibit effective use of
the vaccine. The poor immunogenicity of purified
GM2 can be improved by presenting this ganglioside
in the context of the cellular membrane.36 In human
melanoma, vaccination with tumor cells expressing
GM2 can augment serum levels of anti-GM2 IgM, and
elevated anti-GM2 IgM levels have been correlated
with improved survival.49 Therefore, use of a GM2based carcinoma cell vaccine may be a clinically effective strategy for patients with pancreatic carcinoma.
In summary, we have demonstrated that serum
gangliosides are elevated in patients with pancreatic
adenocarcinoma, suggesting their overexpression and
release by pancreatic adenocarcinoma cells. Our study
also shows that GM2 and GD1b are immunogenic antigens and thus potential targets for immunotherapy
in patients with pancreatic adenocarcinoma. In our
study, serum gangliosides were more reliable than
CA19-9 as an indicator of tumor burden. Serum ganglioside levels also may be useful for monitoring disease progression and response to surgery and postoperative adjuvant therapy.
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