Journal of Surgical Oncology 2005;90:89–94

Expression of RCAS1 in Esophageal Squamous CellCarcinoma Is Associated With a Poor Prognosis

HIROYUKI KATO, MD, PhD,* MASANOBU NAKAJIMA, MD, PhD, NORIHIRO MASUDA, MD, PhD,AHMAD FARIED, MD, MAKOTO SOHDA, MD, YASUYUKI FUKAI, MD, TATSUYA MIYAZAKI, MD, PhD,

MINORU FUKUCHI, MD, PhD, KATSUHIKO TSUKADA, MD, PhD, AND HIROYUKI KUWANO, MD, PhD, FACS

Department of General Surgical Science (Surgery I), Gunma University,Graduate School of Medicine, Showa-machi, Maebashi, Gunma, Japan

Background: Receptor-binding cancer antigen expressed on SiSO cells (RCAS1) hasbeen reported to act as a ligand for a receptor present on normal peripherallymphocytes and to induce apoptotic cell death. We aimed to elucidate the significanceof RCAS1 expression in human esophageal squamous cell carcinomas (ESCC).Methods: RCAS1 expression was examined immunohistochemically in surgicallyresected esophageal carcinoma tissues from 114 patients. We also examined therelationships between RCAS1 expressions, the tumor Ki-67 indices (a marker ofproliferation), and the number of CD8þ tumor-infiltrating lymphocytes (TILs).Results: RCAS1 immunoreactivity was detected in the membranes and cytoplasm ofthe tumor cells. Of the 114 esophageal carcinomas, 39 (34.2%) were strongly positive forRCAS1 immunostaining on the membranes of the cancer cells, 41 (36.0%) were weaklypositive, and 34 (29.8%) were negative. A comparison of RCAS1 expression andclinicopathological characteristics in all 114 patients revealed significant associationsbetween RCAS1 expression and lymph node status (P< 0.05), and pathologic stage(P< 0.05). The survival rates of patients with RCAS1-negative tumors were significantlyhigher than those of patients with both RCAS1-weak positive tumors and RCAS1-strongpositive ones (log-rank P< 0.05). Multivariate analysis revealed that RCAS1 positivitywas an independent prognostic factor (P< 0.05). The relationship between RCAS1expression and the numbers of CD8þ T-cells in the primary tumors revealed that RCAS1-negative tumors tended to contain more of these cells than both RCAS1-weak positivetumors and RCAS1-strong positive ones (P¼ 0.2495).Conclusions: RCAS1 may play a significant role in tumor progression via an immuneescape mechanism; thus, RCAS1 expression could be used as a predictor of poorprognosis in patients with ESCC.J. Surg. Oncol. 2005;90:89–94. � 2005 Wiley-Liss, Inc.

KEY WORDS: esophageal carcinoma; immunohistochemistry; RCAS1;tumor-infiltrating lymphocytes; prognostic value

INTRODUCTION

Receptor-binding cancer antigen expressed on SiSocells (RCAS1), a type II membrane protein which isexpressed on the surfaces of human cancer cells, has beenreported to be expressed with high frequency in uterine,ovarian, and lung cancers [1–4]. It cannot be detectedimmunohistochemically in normal tissues, perhapsbecause it is usually expressed at very low levels or isrestricted to certain cell types. RCAS1 is a ligand for aputative receptor present on various human cell lines and

normal peripheral lymphocytes, such as T- , B- andnatural killer (NK) cells [5]. The expression of thisreceptor is enhanced by activation of these lymphocytes.RCAS1 acts to inhibit the growth of receptor-expressing

*Correspondence to: Hiroyuki Kato, MD, PhD, Department of GeneralSurgical Science (Surgery I), Gunma University, Graduate School ofMedicine, 3-39-22, Showa-machi, Maebashi, Gunma, 371-8511, Japan.Fax: þ81-27-220-8230. E-mail: hiroyuki@po.wind.ne.jp

Received 1 May 2004; Accepted 16 March 2005

DOI 10.1002/jso.20249

Published online in Wiley InterScience (www.interscience.wiley.com).

� 2005 Wiley-Liss, Inc.

cells and induce apoptotic cell death [5]. Indeed, over-expression of RCAS1 is known to inhibit the growth andinduce the apoptosis of immune cells [6].It has been suggested that tumor cells might evade

immune surveillance by expressing RCAS1. RCAS1 hasbeen reported to be expressed by uterine [1–3], ovarian[1], lung [4,7,8], esophageal [9,10], gastric [11,12],hepatocellular [13–15], pancreatic [16,17], colon [18,19],biliary tract [20–22], breast [23–26], skin [27], andprostatic carcinomas [28]. Expression of RCAS1 has alsobeen found to correlate with tumor progression and apoor prognosis in uterine and lung cancer [3,4,7–9]. Withrespect to esophageal carcinoma, Nakakubo et al. [9]demonstrated that RCAS1 expression was significantlyassociated with stage grouping and postoperative survi-val, indicating that it could act as a novel prognosticmarker for surgically resected esophageal squamouscell carcinoma (ESCC). On the other hand, Ikeguchiet al. [10] demonstrated that expression of the RCAS1gene and protein did not correlate with tumor progres-sion, lymph node metastasis, or patient prognosis inesophageal squamous cell carcinomas (ESCC). Thus, therole of RCAS1 in ESCC remains controversial.Tumor-infiltrating lymphocytes (TILs) are considered

to be a manifestation of the host immune response againstcancer cells [29]. TILs have been studied in various typesof carcinoma and have been reported to be prognosticfactors [30,31]. Inclusion of CD8þ T-cells among the TILpopulation appears to be important for the anticancerimmune response. Infiltration of CD8þ T-cells intocancer cell nests is a reliable prognostic indicator inhuman colorectal cancer [32] and esophageal cancer [33].In hepatocellular carcinoma, CD8þ TILs may play a rolein the occurrence of tumour cell apoptosis; however,CD8þ TILs may not be controlled by RCAS1 [15].In this study, we investigated the role of RCAS1

expression in ESCC more fully, using surgically resectedtissues from 114 patients with primary ESCC.We used animmunohistochemical approach to determine whetherthere was any correlation between RCAS1 expressionand the biological behavior of the individual tumors,including their proliferation as indicated by their Ki-67indices. In addition, we examined the relationship be-tween the expression of RCAS1 and the number of CD8þ

TILs present in the tumors, and investgated whether theexpression of RCAS1 protein by the tumors could controlCD8þ T-cell infiltration.

MATERIALS AND METHODS

Patient Characteristics and Surgical Specimens

We studied thoracic esophageal cancer specimensobtained from 114 patients (97 men and 17 women) whohad not received any preoperative treatment.

All of the patients underwent radical en bloc es-ophagectomy at the Department of General SurgicalScience (Surgery I), Gunma University, Graduate Schoolof Medicine, between 1990 and 2002. Their medianage was 62.2 years, with a range of 40–78 years. Tumorstages and disease grades were classified accordingto the sixth edition of the TNM classification of theInternational Union Against Cancer (UICC). All distantmetastases (M) occurred in lymph nodes. The meanfollow-up period for the entire patient group was41� 31.6 months, with a range of 6–82 months.The surgical specimens were fixed, embedded, stained

with hematoxylin and eosin, and examined microscopi-cally by two pathologists.

Immunohistochemical Detection of RCAS1

The resected specimens were fixed with 10% (v/v)formaldehyde and embedded in paraffin blocks. Immu-nohistochemical staining for RCAS1 was performed onsections using the standard avidin-biotin peroxidasecomplex (ABC) method. Sections 4 mm thick were cutfrom the paraffin blocks, deparaffinized and treated with a0.3% (v/v) H2O2—methanol solution for 30 min at roomtemperature to block endogenous peroxidase activity.After rehydration through a graded ethanol series, thesections were autoclaved in 1 mM ethylenediaminetetraacetic acid (EDTA) at 1208C for 2 min, then cooledto 308C. After rinsing in 0.1M phosphate-buffered saline(PBS; pH 7.4), nonspecific binding sites were blocked byincubation with 10% (v/v) normal rabbit serum for30 min. The sections were then incubated with theprimary antibody overnight at 48C. The primary antibodyraised against RCAS1 (anti-RCAS1 mouse IgM mono-clonal antibody, Medical & Biological Laboratories Co.,Ltd., Nagoya, Japan) was diluted 1:500. The primaryantibody against Ki-67 (MIB-1, Immunotech, Cedex 9,France) was diluted 1:50. The primary antibody againstCD8þ (clone C8/144B, Nichirei Corp., Tokyo, Japan)was diluted 1:60. Immunohistochemistry was performedusing an ABC system (Vectastain, Vector Laboratories,Inc., Burlingame, CA). Incubation with the secondaryantibody, biotinylated anti-rabbit IgG, was carried out for30 min. The chromogen utilized was 3,30—diaminoben-zidine tetrahydrochloride, applied as a 0.02% (w/v)solution containing 0.005% (v/v) H2O2 in 50 mMammonium acetate-citric acid buffer (pH 6.0). Thesections were counterstained lightly with hematoxylin.Negative controls were established by replacing theprimary antibody with PBS or normal rabbit serum.

Evaluation of RCAS1 Expression, Ki-67

Labeling Indices and CD8þ T-cells

Immunostaining for RCAS1 was graded as follows:negative (�) if fewer than 10% of the membranes of the

90 Kato et al.

cells at the invasive front of the tumor were stained in fiveconsecutive high-power fields, weak positive (þ) if stain-ing was present in 10%–50% of the cells, and strongpositive (þþ) if staining was present in more than 50% ofthe cells. The Ki-67 labeling indices were calculated asthe percentage nuclear staining in the cells at the invasivefront of the tumor in five consecutive high-poweredfields. The number of CD8þ T-cells was counted both inthe mesenchymal stroma and within the cancer cell nestsin five consecutive high power fields. The immunohisto-chemical evaluation was performed by two experiencedphysicians.

Statistical Analysis

The relationship between the expression of RCAS1and other parameters was determined using the w2

method and Fisher exact test. The relationship betweenthe expression of RCAS1 and the Ki-67 labeling indices,and the numbers of CD8þ T-cells was determined usingthe analysis of variance method. Survival rates werecalculated by the Kaplan–Meier method for analysis ofcensored data. The significance of differences in survivalwas analyzed by means of the log-rank test in a univariateanalysis. A Cox proportional hazards model based on riskratios was used to evaluate survival in univariate andmultivariate analyses.

RESULTS

RCAS1 Immunoreactivity and

Clinicopathologic Variables

Positive immunostaining for RCAS1 was detected bothon the membranes of the cancer cells and in theircytoplasm (Fig. 1). Of the 114 esophageal carcinomas,

39 (34.2%) were strongly positive for RCAS1 immu-nostaining on the membranes of the cancer cells, 41(36.0%) were weakly positive and 34 (29.8%) werenegative.

A comparison of RCAS1 expression and clinico-pathological characteristics in all 114 patients revealedsignificant associations between RCAS1 expressionand the lymph node status (P< 0.05), and pathologicstage (P< 0.05) of the esophageal cancers (Table I).The number of metastatic lymph nodes found duringsurgery was somewhat higher in patients with RCAS1-positive tumors (P¼ 0.0828), the average being 2.8 inthe RCAS1-strong positive patients, 2.2 in the RCAS1-weak positive ones and 1.2 in the RCAS1-negativeones.

Kaplan–Meier Survival and

Cox Proportional Hazards Analysis

The survival rates of patients with RCAS1-negativetumors were significantly higher than those of patientswith both RCAS1-weak positive tumors and RCAS1-strong positive tumors (log-rank P< 0.05; Fig. 2). The5-year survival rate of patients with RCAS1-negativetumors was 63%, while that of patients with RCAS1-weak positive tumors was 44%, and that of patients withRCAS1-strong positive tumors was 35%.

The Cox univariate regression analysis identifiedRCAS1 positivity (P¼ 0.0269), the T-category (P<0.0001) and the N-category (P< 0.0001) as significantprognostic predictors (Table II). The multivariate analysisrevealed that RCAS1 positivity was an independentprognostic factor (P¼ 0.0362, Table II).

RCAS1 Expression and Ki-67 Labeling Indices

The relationship between RCAS1 expression and theKi-67 labeling indices in the primary tumors revealed thatKi-67 labeling indices were not significantly higher inRCAS1-positive than in RCAS1-negative tumors (P¼0.6862). The Ki-67 labeling indices in the RCAS1-strongpositive, RCAS1-weak positive and RCAS1-negativetumors were 43.5� 15.8, 42.0� 15.5, and 40.3� 18.3,respectively.

RCAS1 Expression and Numbers of CD8þ T-cells

The relationship between RCAS1 expression and thenumbers of CD8þ T-cells in the primary tumors revealedthat RCAS1-negative tumors tended to contain more ofthese cells than RCAS1-positive tumors (P¼ 0.2495), theaverage being 34.2� 35.8/HPF (high power field) in theRCAS1-negative patients, 27.8� 24.4/HPF in the RCAS1-weak positive ones, and 23.8� 18.7/HPF in the RCAS1-strong positive ones.

Fig. 1. Positive immunostaining for RCAS1 on the membranes andin the cytoplasm of esophageal cancer cells. [Color figure can beviewed in the online issue, available at www.interscience.wiley.com.]

RCAS1 Expression in Esophageal Carcinoma 91

DISCUSSION

It has recently been reported that RCAS1 is a ligand fora receptor present on activated human T- and NK-cells,and that it acts to inhibit proliferation and induceapoptotic cell death [5]. A study using the TUNELmethod also demonstrated that apoptosis of TILs wasinduced around RCAS1-positive tumor cells [4].In the present study, RCAS1 was detected on the

membranes and in the cytoplasm of ESCC cells byimmunostaining. Of the 114 carcinomas studied, 80(70.2%) were positive for RCAS1 immunostaining. Acomparison of RCAS1 expression and clinicopathologi-cal characteristics in the 114 patients revealed significant

associations between RCAS1 expression and lymph nodestatus, pathologic stage and poorer prognosis. A multi-variate Cox analysis confirmed that RCAS1 positivitywas an independent prognostic factor. RCAS1 expressionwas also inversely associated with the number of CD8þ

T-cells in the primary tumor, but did not correlatewith tumor proliferation, as indicated by the tumorKi-67 indices. These results strongly suggest thatRCAS1 expression in ESCC is predictive of an un-favorable prognosis in patients who have undergonesurgical resection. Furthermore, RCAS1 may play asignificant role in tumor progression via an immuneescape mechanism, but does not reflect tumor prolifera-tive activity.

TABLE I. RCAS1 Expression and Clinicopathologic Characteristics in 114 Patients WithEsophageal Carcinomas

Parameters

Number of

patients

RCAS1 expression

P-valueNegative Weak positive Strong positive

Gender

Male 97 27 36 34 0.5389

Female 17 7 5 5

Age at surgery (years)

�61 52 14 21 17 0.6526

>61 62 20 20 22

Location

Upper 16 5 6 5 0.8534

Mid 71 20 24 27

Lower 27 9 11 7

Tumor status

T1 47 19 12 16 0.4094

T2 14 4 6 4

T3 46 9 20 17

T4 7 2 3 2

Lymph node status

N0 50 21 12 17 0.0186

N1 64 13 29 22

Metastatic status

M0 95 30 34 31 0.6040

M1 19 4 7 8

Pathologic stage

Stage I 33 16 8 9 0.0491

Stage II 35 10 14 11

Stage III 27 4 14 9

Stage IV 19 4 5 10

Histologic grade

G1 29 9 9 11 0.7404

G2 53 15 18 20

G3 32 10 14 8

Lymphatic invasion

Negative 34 15 12 7 0.0510

Positive 80 19 29 32

Blood vessel invasion

Negative 56 21 21 14 0.0831

Positive 58 13 20 25

G1: well differentiated carcinoma.

G2: moderately differentiated carcinoma.

G3: poorly differentiated carcinoma.

92 Kato et al.

Nakakubo et al. [9] demonstrated that a correlationbetween RCAS1 and the T-category of esophagealcarcinoma might be associated with tumour progression.In contrast, our results indicated that RCAS1 expressioncorrelated significantly with the lymph node status, butnot with the tumor status. This is consistent with previousreports on gastric and colon cancers, which found acorrelation between RCAS1 expression and lymph nodemetastasis [12,18].

The detection of RCAS1 expression in ESCC was alsocorrelated with a poor prognosis. Similar results havebeen reported for cancers of other organs, includinguterine [3], lung [4,7,8], gastric [12], pancreatic [16],colon [18], and prostatic cancer [28]. Furthermore,Hiraoka et al. [16] indicated that RCAS1 might be asignificant tumor marker for pancreatic adenocarcinomaand an unfavorable predictor of prognosis in patients whohave undergone surgical resection. During the presentstudy, we demonstrated that RCAS1 expression mightalso be an unfavorable prognostic indicator for patientswith esophageal carcinoma; this finding is consistent withresults reported by Nakakubo et al. [9].

Recent advances in molecular biology and immunol-ogy have led to the discovery and characterization of a

variety of molecules involved in the immune system [7].These molecules are currently being investigated withrespect to the development of new immune therapiesagainst cancer. Antigens expressed on tumor cells arerecognized by immune surveillance; tumor rejection thenensues because of the large amplification capacity ofimmune cells based on the clonal expansion principle.The immune response against tumors is mainly mediatedby four types of cells: NK-cells, lymphokine-activatedkiller (LAK) cells, cytotoxic T lymphocytes (CTL), andactivated macrophages [7]. The expression of RCAS1may be a mechanism by which cancer cells escape fromthe host immune system. In the present study, we suggestthat RCAS1 might play a significant role in tumorprogression via an immune escape mechanism, but doesnot reflect tumor proliferative activity. Conversely, Aokiet al. [14] demonstrated that, in hepatocellular carcinoma,RCAS1 was associated with increased proliferativeactivity, but not with metastasis. However, Okada et al.[18] demonstrated that the proportion of apoptotic TILswas significantly higher in RCAS1-positive colorectalcarcinomas than in RCAS1-negative tumors, suggestingthat over-expression of RCAS1 may negatively affect theprognosis of human colorectal carcinomas and thatRCAS1 may play a role in tumor immune privilege in vivo.

CD8þ T-cells can lyse tumor cells directly and destroylarge tumor masses in vivo. Immunohistochemical datahave demonstrated that infiltration of CD8þ T-cells intothe cancer cell nest is an important feature of theantitumor immune response [32,33]. Furthermore,patients with abundant CD8þ T-cells in either the cancercell nest or the mesenchymal stroma had a betterprognosis [33]. The numbers of CD8þ T-cells in thestroma and within the cancer cell nest itself werecorrelated, suggesting that accumulation of CD8þ T-cellsin the stroma is necessary for subsequent infiltration intothe cancer cell nest to attack the tumor. In the presentstudy, the number of CD8þ T-cells tended to be higher inRCAS1-negative than in RCAS1-positive tumors. How-ever, Ikeguchi et al. [15] demonstrated that although thedensity of CD8þ T-cells in tumors correlated positivelywith the occurrence of tumour cell apoptosis, it did not

TABLE II. Prognostic Factors in Cox Proportional Hazards Model

Variables

Univariate Multivariate

RR 95% CI P-value RR 95% CI P-value

RCAS1 (positive vs. negative) 2.124 1.090–4.141 0.0269 2.204 1.052–4.617 0.0362

pT (T2,3,4 vs. T1) 6.167 2.996–12.693 <0.0001 6.667 2.306–19.273 0.0005

pN (N1 vs. N0) 8.646 3.883–19.253 <0.0001 9.842 2.825–34.283 0.0003

pStage (2,3,4 vs. 1) 8.050 2.899–22.359 <0.0001 0.329 0.048–2.253 0.2574

Ki-67 (high vs. low) 2.227 1.271–3.905 0.0052 1.395 0.765–2.544 0.2768

RR: risk ratio.

CI: confidence interval.

Fig. 2. Kaplan–Meier survival curves for patients with esophagealcancers. The survival rates of patients with RCAS1-negative tumorswere significantly higher than those of patients with both RCAS1-weak positive tumors and RCAS1-strong positive tumors (log-rank P< 0.05). The 5-year survival rate of patients with RCAS1-negative tumors was 63%, while that of patients with RCAS1-weakpositive tumors was 44%, and that of patients with RCAS1-strongpositive tumors was 35%.

RCAS1 Expression in Esophageal Carcinoma 93

correlate with RCAS1 protein expression in hepatocel-lular carcinoma.In conclusion, we demonstrated that RCAS1 expres-

sion may play a significant role in tumor progression viaan immune escape mechanism, and that RCAS1 expres-sion could therefore be used as a predictor of poorprognosis in patients with ESCC. Further investigationsto clarify the role of RCAS1 in esophageal carcinoma arenow warranted.

ACKNOWLEDGMENTS

We thank A. Nakabayashi, H. Emura, M. Ohnuma,S. Ueno, T. Ogasawara, and Y. Saitoh for their excellentsecretarial assistance, and M. Ohno for her assistancewith the data management and biostatistical analysis.

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