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


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

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,
Fig. 1. a: Normal HLA-B/C expression in gastric cancer. The respectively; Table IIb). HLA-B/C expression was the most powerful
expression of HLA-I heavy chain was observed in the cell membrane
predictor of peritoneal recurrence (RR: 9.924, P 0.003) followed by
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 tumor stage (RR: 6.391, P 0.023; Table IIc).
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 DISCUSSION
presence of positively stained infiltrating leukocytes.
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,
KaplanMeier method, and the difference was evaluated by the log- respectively. Gastric cancer with positive expression of HLA-B/C
rank test. Patients prognosis and risk factors that influenced recurrence exhibited deeper cancer invasion, higher incidence of lymph node
were determined by multivariate analysis as using Coxs proportional metastasis, more advanced tumor stage, and higher incidence of
hazard model and logistic regression analysis, respectively. A P-value recurrences than those with mixed and negative expression. Both the 5-
of less than 0.05 was considered statistically significant. year overall and 5-year disease-free survival rates of patients with
positive expression of HLA-B/C were significantly lower than those
RESULTS with mixed and negative expression. Although HLA-B/C expression
was not an independent prognostic factor, it was an independent
Expression of HLA Class I Heavy predictor of peritoneal recurrence after curative surgery in patients
Chain in Gastric Cancer with gastric cancer.
It is generally accepted that an anticancer immune reaction is
Expression of HLA-I heavy chain including HLA-A and B/C was primarily mediated by cytotoxic T-lymphocytes (CTL). This process
observed in stroma, endothelium, and lymphocytes of normal gastric requires the presence of HLA-I on the cancer cells. Many studies have
tissues. In gastric cancer, the expression of HLA-I heavy chain had shown the relationship between HLA-I expression and prognosis of

Journal of Surgical Oncology


454 Ueda et al.
TABLE II. Multivariate Analysis of Patients Prognosis and Risk Factors for Recurrence

RR 95%CI P

(a) Prognostic factors


Stage 5.769 2.28814.543 <0.001
(b) Risk factors for lymph node recurrence
LN metastasis 11.872 1.321106.720 0.027
Stage 3.202 1.0519.753 0.041
(c) Risk factors for peritoneal recurrence
HLA-B/C expression 9.924 2.16645.472 0.003
Stage 6.391 1.29031.668 0.023

RR, relative risk; CI, confidence interval.

patients with a variety of malignancies [817]. Most of these studies non-small cell lung cancer [14], and colorectal cancer [17]. These
showed that cancer patients with HLA-I expression had a better results reflect the controversy about the exact role of HLA-I expression
prognosis than those with loss and reduced expression in breast cancer in malignancies.
[8], and malignant melanoma [11]. These studies suggested that With regard to gastric cancer, only a few studies investigated the
downregulation of HLA-I (i.e., mixed or negative HLA-I expression) expression of HLA-I [2125]. Ferron et al. [21] analyzed the
might reflect the escape of malignant cells from human immune expression of HLA-I and II antigens on gastric carcinomas and
system. On the other hand, other studies reported that cancers with autologous mucosa by cryopreserved tissue samples, and they reported
downregulated HLA-I were correlated with early tumor stage and that the autologous mucosa lacked the expression of HLA antigens
better prognosis including breast cancer [9], uveal melanoma [12], 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
Fig. 2. Five-year overall and 5-year disease-free survival rates of
patients who underwent curative operation analyzed according to gastric cancer with positive HLA-I heavy chain expression. Positive
HLA-I expression by the Kaplan-Meier method. Both 5-year overall HLA-I heavy chain expression causes anti-tumor T-cell-mediated
(a) and 5-year disease-free survival rates (b) of patients with positive responses through CTL recognition. However, the prognosis for
HLA-B/C expression were significantly lower than those with mixed patients with positive HLA-I heavy chain expression currently would
and negative expression (P < 0.05 and 0.01, respectively). be poor. Therefore, in clinical settings, the chemotherapy that does not

Journal of Surgical Oncology


HLA-I Expression in Gastric Cancer 455
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Journal of Surgical Oncology