Journal of Surgical Oncology 2008;97:451455

Clinical Significance of HLA Class I Heavy Chain Expression

in Patients With Gastric Cancer

YOSHITAKE UEDA, MD,1* KOICHI ISHIKAWA, MD,1 NORIO SHIRAISHI, MD,

1

SHIGEO YOKOYAMA, MD,2 AND SEIGO KITANO, MD1

1
Department of Gastroenterological Surgery, Oita University Faculty of Medicine, Oita, Japan
2
Department of Oncological Science (Pathology), Oita University Faculty of Medicine, Oita, Japan

Background: Little is known about the relation between HLA-I expression and the prognosis of patients with gastric cancer. The aim of this
retrospective study was to clarify the clinical significance of HLA-I heavy chain expression in gastric cancer.
Methods: The study subjects were 202 patients with gastric cancer who had undergone curative surgery. Tumors were examined for expression of
HLA-I heavy chain antigens by immunohistochemistry. We analyzed the association of HLA-I heavy chain expression with clinicopathological
parameters and patient prognosis.
Results: HLA-B/C expression showed association with deeper tumor invasion, higher incidence of lymph node metastasis, more advanced tumor
stage, and higher incidence of recurrence. Patients with positive HLA-B/C expression had shorter 5-year overall and 5-year disease-free survival
compared with patients whose tumors showed mixed and negative expression (P < 0.05 and 0.01, respectively). In multivariate analysis, although
HLA-B/C expression was not recognized as an independent prognostic factor, it was an independent factor in predicting peritoneal recurrence
after curative surgery in patients with gastric cancer [relative risk (RR): 9.924, P 0.003].
Conclusion: Expression of HLA-B/C heavy chain is associated with tumor progression, and it could be a significant predictor of peritoneal
recurrence after curative surgery in patients with gastric cancer.
J. Surg. Oncol. 2008;97:451455. 2008 Wiley-Liss, Inc.

KEY WORDS: gastric cancer; human leukocyte antigen class I molecule; immunohistochemistry

INTRODUCTION
Gastric cancer is one of the most common cancers and the second
leading cause of cancer-related deaths throughout the world [1].
Advances in diagnosis and treatment for early gastric cancer have
offered a better prognosis of patients, however, treatment of advanced
gastric cancer remains difficult and the prognosis of patients is still
poor. The establishment of other treatment modalities after surgery is
waited.
In the immune response against cancer cells, major histocompat-
ibility complex (MHC) molecules play an important role in presenting
antigens to T lymphocytes [24]. T lymphocytes recognize antigens by
the specific receptor, and this recognition activates T lymphocytes to
specifically kill the tumor cells. In addition, the difference between the
interaction of MHC molecules on tumor cells with T cells on one hand
and NK cells on the other, result either in T-cell activation and
specific cytotoxicity against the tumor cells or protection from the non-
specific NK-cell cytotoxicity mediated by inhibitory receptors for
MHC molecules [24]. One such molecule is the classical (class I)
human leukocyte antigen (HLA) [5]. HLA class I molecules (HLA-I) is
a complex molecule that consists of a heavy chain and light chain.
HLA-A, -B, and -C loci on chromosome 6 encode the heavy chain,
while b2 microglobulin (b2 m) on chromosome 15 encodes the light
chain [2]. Normal cells express six HLA-I alleles (2 HLA-A, 2 HLA-B,
and 2 HLA-C), and several major altered HLA-I down-regulation
phenotypes have been defined in different tumor tissues [6,7].
In recent years, reduced or loss of HLA-I expression has been
reported to be associated with tumor development or patients outcome
in breast cancer [810], malignant melanoma [11,12], laryngeal
carcinoma [13], small cell carcinoma of the lung [14], ovary carcinoma
[15], cervical carcinoma [16], and colorectal cancer [17]. However, the
relation of HLA-I, especially heavy chain of HLA-I, expression with
patients outcome in gastric cancer has not yet been investigated. In the
present study, we analyzed the correlation of HLA-I heavy chain
expression with various clinicopathological parameters and prognosis.
MATERIALS AND METHODS
Patients and Tumor Characteristics
The subjects of the study were 202 patients with gastric cancer who
had undergone curative surgery in the Department of Gastroenter-
ological Surgery, Oita University Hospital from 1991 to 2000. We
examined the clinicopathological findings of these patients by the
clinical and pathological records. The mean age of the patients
was 64.3 years (range: 3287 years) and 132 patients were men and
70 were women (Table I). Thirty-two patients received adjuvant
chemotherapy according to the histologic tumor stage. All patients had
been followed up by radiography, ultrasonography, and CT scan at
3-month intervals for the first year, every 6-month intervals for the
3-years, annually for the rest of 5-years. The follow-up period for the
survivors ranged from 0.5 to 12.0 years, with a median of 5.2 years. All
clinicopathological evaluations were based on the General rules for the
Gastric Cancer Study in Surgery and Pathology in Japan [18]. Informed
consent for this study was obtained from all patients.
*Correspondence to: Yoshitake Ueda, MD, Department of Gastroentero-
logical Surgery, Oita University Faculty of Medicine, 1-1 Idaigaoka,
Hasama-machi, Yufu, Oita 879-5593, Japan. Fax: 81-97-549-6039.
E-mail: yoshimd@med.oita-u.ac.jp
Received 26 August 2007; Accepted 21 December 2007
DOI 10.1002/jso.20985
Published online 4 February 2008 in Wiley InterScience
(www.interscience.wiley.com).

2008 Wiley-Liss, Inc.

452 Ueda et al.
TABLE I. Clinicopathological Features of Participating Patients According to HLA Typing

HLA-A HLA-B/C

Negative Mixed Positive P-value Negative Mixed Positive P-value

N 70 93 39 49 103 50
Patients
Age (years)
>65 41 43 27 <0.05 22 58 31 NS

65 29 50 12 27 45 19
Sex
Male 44 64 24 NS 31 68 33 NS
Female 26 29 15 18 35 17
Tumor
Depth of invasion
T1 (sm) 28 54 4 <0.01 16 67 3 <0.01
T2 (mp, ss)/T3 (se) 42 39 35 33 36 47
Differentiation
well/moderately 31 50 14 NS 21 56 18 NS
Poorly 39 43 25 28 47 32
LN metastasis
Negative 34 60 16 <0.05 23 68 19 <0.01
Positive 36 33 23 26 35 31
Vascular invasion
Negative 49 74 25 NS 38 80 30 <0.05
Positive 21 19 14 11 23 20
Lymphatic invasion
Negative 22 36 12 NS 20 38 12 NS
Positive 48 57 27 29 65 38
Stage
I/II 53 74 27 NS 36 87 31 <0.01
III/IV 17 19 12 13 16 19
Recurrences
No 52 74 26 NS 37 85 30 <0.01
Yes 18 19 13 12 18 20

NS, not significant.

Tumor Samples supernatant of culture medium as described previously [5], were

The resected tumor for the immunohistochemical analysis had been
used according to the guideline of Oita university hospital. Resected
tumors from each of the 202 patients were fixed in 10% formalin
solution and embedded in paraffin. Two tissue sections which represent
the tumor were cut at 4 mm from the paraffin-embedded tissue blocks
and used for immunohistochemical analysis.
Immunohistochemistry
Immunohistochemical staining was performed using two mono-
clonal antibodies, HC-A2 and the HC-10, which were provided by
Dr. J. J. Neefjes from the Netherlands Cancer Institute (Amsterdam,
The Netherlands). The monoclonal antibody HC-A2 reacts with HLA-
A locus heavy chains. The monoclonal antibody HC-10 has a
preference for HLA-B locus heavy chains, and also reacts with
HLA-C locus heavy chains [5]. The heavy chain possesses three
external domains designated a1, a2, and a3, a transmembrane domain,
and a cytoplasmic tail. The monoclonal antibody HC-A2 and HC-10
react with a3 and a1 domain of the b2 m-free HLA-I heavy chain,
respectively [19,20]. Each section was then deparaffinized and
rehydrated. Antigen retrieval was performed by placing the sample
in a microwave at 958C for 40 min, followed by cooling for 30 min to
room temperature. Endogenous peroxidase was blocked by incubation
in 0.03% hydrogen peroxidase in absolute methanol for 20 min at room
temperature and washed in Tris-buffered saline (TBS). The mono-
clonal antibodies, HC-A2 diluted 1:100, HC-10 diluted 1:200 from
incubated for 2 hr in a humidified chamber at room temperature.
The sections were rinsed in TBS and incubated for 30 min with
EnVision peroxidase mouse system (Dako Corporation, Carpinteria,
CA). After washing in TBS, the color reaction product was developed
with diaminobenzidine tetrahydrochloride (DAB, Sigma Chemical
Co., St. Louis, MO) as chromogen for 5 min. Finally, the sections were
counterstained with hematoxylin for 1 min, dehydrated in a series of
alcohols, cleared in xylene, and mounted with glass coverslips.
Evaluation of Immunohistochemistry
The specificity of immunostaining by HC-10 and HC-A2 mono-
clonal antibodies was confirmed by staining of positive control tissues,
that is, human kidney and salivary glands which had been known to be
constantly positive for HC-10 and HC-A2 [5]. Microscopic analysis
was carried out by two independent investigators who had no
knowledge of the clinical outcome of the patients. The tumors were
classified into three groups: positive (the percentage of stained cells in
the whole section was scored as >90%), mixed (1090%), and
negative (<10%; Fig. 1) [17].
Statistical Analysis
Statistical analysis was carried out using the Statistical Package for
Social Sciences (SPSS) software package. Comparison of various
clinicopathological parameters and HLA-I heavy chain expression
was evaluated by w2 test. Survival rates were calculated with the

Journal of Surgical Oncology


Fig. 1. a: Normal HLA-B/C expression in gastric cancer. The
expression of HLA-I heavy chain was observed in the cell membrane
and some cytoplasm of cancer cells by HC-10 antibody in all cancer
cells. b: Reduced HLA-B/C expression in gastric cancer. Some cancer
cells were stained by HC-10 antibody (arrow) and others were not
stained (arrowhead). c: Absent HLA-B/C expression in gastric cancer.
Note the lack of cancer cells stained by HC-10 antibody, but the
presence of positively stained infiltrating leukocytes.
KaplanMeier method, and the difference was evaluated by the log-
rank test. Patients prognosis and risk factors that influenced recurrence
were determined by multivariate analysis as using Coxs proportional
hazard model and logistic regression analysis, respectively. A P-value
of less than 0.05 was considered statistically significant.
RESULTS
Expression of HLA Class I Heavy
Chain in Gastric Cancer
Expression of HLA-I heavy chain including HLA-A and B/C was
observed in stroma, endothelium, and lymphocytes of normal gastric
tissues. In gastric cancer, the expression of HLA-I heavy chain had
Journal of Surgical Oncology
HLA-I Expression in Gastric Cancer 453
more intensity in the cell membrane than cytoplasm of cancer cells
(Fig. 1). Expression of HLA-A was positive in 39 cases (19%), mixed
in 93 cases (46%), and negative in 70 cases (35%) (Table I). Expression
of HLA-B/C was positive in 50 (25%), mixed in 103 (51%), and
negative in 49 (24%) (Table I). Both HLA-A and B/C expression were
positive in 29 (14%), mixed in 143 (71%), and negative in 30 (15%).
Relation of HLA Class I Heavy Chain Expression
With Clinicopathological Characteristics
Gastric cancers with positive expression of HLA-A were character-
ized by deeper cancer invasion (P < 0.01) and higher incidence of
lymph node metastasis (P < 0.05) than those with mixed and negative
expression (Table I). Gastric cancers with positive expression of HLA-
B/C were also characterized by deeper cancer invasion (P < 0.01),
higher incidence of lymph node metastasis (P < 0.01), more advanced
tumor stage (P < 0.01), and higher incidence of recurrences (P < 0.01)
than those with mixed and negative expression (Table I).
Analysis of Survival in Relation to HLA
Class I Heavy Chain Expression
Fifty patients developed recurrence or distant metastasis after
curative surgery. Of these patients, 18 had lymph node recurrence, 14
had peritoneal recurrence, and 8 had liver or lung metastasis. Forty-
four patients died of disease caused by cancer. The 5-year overall
survival and 5-year disease-free survival rates of patients with gastric
cancer were 78.2% and 75.3%, respectively. There was no relationship
between HLA-A expression and prognosis (data not shown). Both the
5-year overall survival and 5-year disease-free survival rates of patients
with positive expression HLA-B/C were significantly lower than those
of patients with mixed and negative expression (overall; 66% vs. 82%,
P < 0.05, disease-free; 60% vs. 80%, P < 0.01; Fig. 2a,b). In multi-
variate analysis, only tumor stage was an independent prognostic factor
[relative risk (RR): 5.769, P < 0.001; Table IIa]. The other factors such
as age, sex, depth of invasion, differentiation, lymph node metastasis,
vascular invasion, lymphatic invasion, HLA-A and B/C expression
were not recognized as independent prognostic factors. Lymph node
metastasis and tumor stage were independent risk factors for lymph
node recurrence (RR: 11.872, P 0.027 and RR: 3.202, P 0.041,
respectively; Table IIb). HLA-B/C expression was the most powerful
predictor of peritoneal recurrence (RR: 9.924, P 0.003) followed by
tumor stage (RR: 6.391, P 0.023; Table IIc).
DISCUSSION
In this study, we described the relationship of HLA-I heavy chain
expression with various clinicopathological parameters and prognosis
of patients with gastric cancer. Gastric cancer with positive expression
of HLA-A and B/C constituted 19% and 25% of all gastric cancer,
respectively. Gastric cancer with positive expression of HLA-B/C
exhibited deeper cancer invasion, higher incidence of lymph node
metastasis, more advanced tumor stage, and higher incidence of
recurrences than those with mixed and negative expression. Both the 5-
year overall and 5-year disease-free survival rates of patients with
positive expression of HLA-B/C were significantly lower than those
with mixed and negative expression. Although HLA-B/C expression
was not an independent prognostic factor, it was an independent
predictor of peritoneal recurrence after curative surgery in patients
with gastric cancer.
It is generally accepted that an anticancer immune reaction is
primarily mediated by cytotoxic T-lymphocytes (CTL). This process
requires the presence of HLA-I on the cancer cells. Many studies have
shown the relationship between HLA-I expression and prognosis of
454 Ueda et al.
TABLE II. Multivariate Analysis of Patients Prognosis and Risk Factors for Recurrence
RR
(a) Prognostic factors
Stage 5.769
(b) Risk factors for lymph node recurrence
LN metastasis 11.872
Stage 3.202
(c) Risk factors for peritoneal recurrence
HLA-B/C expression 9.924
Stage 6.391
RR, relative risk; CI, confidence interval.
patients with a variety of malignancies [817]. Most of these studies
showed that cancer patients with HLA-I expression had a better
prognosis than those with loss and reduced expression in breast cancer
[8], and malignant melanoma [11]. These studies suggested that
downregulation of HLA-I (i.e., mixed or negative HLA-I expression)
might reflect the escape of malignant cells from human immune
system. On the other hand, other studies reported that cancers with
downregulated HLA-I were correlated with early tumor stage and
better prognosis including breast cancer [9], uveal melanoma [12],
Fig. 2. Five-year overall and 5-year disease-free survival rates of
patients who underwent curative operation analyzed according to
HLA-I expression by the Kaplan-Meier method. Both 5-year overall
(a) and 5-year disease-free survival rates (b) of patients with positive
HLA-B/C expression were significantly lower than those with mixed
and negative expression (P < 0.05 and 0.01, respectively).
Journal of Surgical Oncology
95%CI P
2.28814.543 <0.001
1.321106.720 0.027
1.0519.753 0.041
2.16645.472 0.003
1.29031.668 0.023
non-small cell lung cancer [14], and colorectal cancer [17]. These
results reflect the controversy about the exact role of HLA-I expression
in malignancies.
With regard to gastric cancer, only a few studies investigated the
expression of HLA-I [2125]. Ferron et al. [21] analyzed the
expression of HLA-I and II antigens on gastric carcinomas and
autologous mucosa by cryopreserved tissue samples, and they reported
that the autologous mucosa lacked the expression of HLA antigens
before becoming malignant. Klein et al. [22] reported that reduced
HLA-I expression in gastric cancer, which correlated with deeper
cancer invasion. Their study has analyzed expression of the entire
HLA-I complex using polyclonal antihuman b2 microglobulin. HLA
class I antigen is a cell surface glycoprotein composed of heavy chain
and light chain. Shen et al. [23] reported that there was no relationship
between the down-regulation of HLA-I heavy chain and that of light
chain in gastric cancer. In our preliminary study, we also obtained
similar results about the relationship between the expression of HLA-
A, B/C and that of b2 microglobulin (data not shown). Therefore, in the
present study, we analyzed the expression of the HLA-A and B/C locus
of HLA-I heavy chains separately by using two monoclonal antibodies,
and showed that gastric cancer with positive expression of HLA-A and
B/C had deeper cancer invasion, higher incidence of lymph node
metastasis than those with mixed and negative expression. In the
future, we should clarify in detail the significance of expression pattern
of HLA-I heavy chain and light chain expression.
In the present study, we first showed the relationship between HLA-
I heavy chain expression and prognosis of patients with gastric cancer.
The relationship between HLA-1 expression and prognosis of patients
still remains controversial. Our study demonstrated that the prognosis
of gastric cancer patients with positive expression of HLA-I loci, HLA-
B/C of heavy chain is worse than that of patients with mixed and
negative expression. Menon et al. [17] also reported that patients
with positive HLA-I expression had a worse prognosis than those
with HLA-I-downregulated in colorectal cancer. Garcia-Lora et al. [3]
showed that HLA-I expression would activate T-lymphocytes, in
contrast, the same HLA-I would inhibit the NK cell by interacting with
NK inhibitory receptors. There was a possibility that positive HLA-I
expression worked on inhibiting NK cell activation more strongly than
on activating CTL in patients with colorectal cancer or gastric cancer.
In the present study, NK cell activities of patients with gastric cancer
were not examined. Further studies to clarify the association among
HLA-I heavy chain expression, NK cell activities and patients
prognosis are necessary.
Our results also suggest the feasibility of adjuvant chemotherapy for
gastric cancer with positive HLA-I heavy chain expression. Positive
HLA-I heavy chain expression causes anti-tumor T-cell-mediated
responses through CTL recognition. However, the prognosis for
patients with positive HLA-I heavy chain expression currently would
be poor. Therefore, in clinical settings, the chemotherapy that does not

involve the CTL, may be useful for this cancer. Ishikawa et al. [26]
reported that postoperative adjuvant chemotherapy with low-dose
cisplatin-5-fluorouracil was useful for patients with gastric cancer
because this chemotherapy activated NK cells. These chemotherapies
would be useful especially for the gastric cancer with positive HLA-I
heavy chain expression.
In conclusion, HLA-B/C of heavy chain expression is associated
with tumor progression, and it could be a significant predictor of
peritoneal recurrence after curative surgery in patients with gastric
cancer. Further studies are required to analyze the regulatory
mechanisms that control HLA-I heavy chain expression and the
significance of immune response through HLA-I in the gastric cancer.
ACKNOWLEDGMENTS
We thank Mrs. Hisaka and Miss Kawamura for the excellent
technical support and immunohistochemical staining.
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