Allelic imbalance of 14q32 in esophageal carcinoma

Cancer Genetics and Cytogenetics 135 (2002) 177–181

Allelic imbalance of 14q32 in esophageal carcinoma Yuji Iharaa,*, Yuji Katob, Tadashi Bandoa, Fuminori Yamagishia, Tetsuji Minamimuraa, Takashi Sakamotoa, Kazuhiro Tsukadaa, Masaharu Isobeb a The Second Department of Surgery, Toyama Medical and Pharmaceutical University School of Medicine, 2630 Sugitani Toyama 930-0194, Japan Molecular and Cellular Biology Laboratory, Department of Materials and Biosystem Engineering, Faculty of Engineering, Toyama University, 3190 Gofuku Toyama 930-8555, Japan Received 3 April 2001; received in revised form 4 December 2001; accepted 6 December 2001

b

Abstract

It has been demonstrated that the accumulation of alterations in several oncogenes and tumor suppressor genes plays a role in the initiation and progression of esophageal carcinoma. However, to our knowledge, very few studies have described the molecular genetic changes of chromosome arm 14q in esophageal carcinoma. In this study, we examined 35 primary esophageal carcinomas for allelic imbalance on 14q32. Analysis of four polymorphic microsatellite markers identified 13 (37%) tumors exhibiting allelic imbalance on 14q32 in at least one locus. In particular, the allelic imbalance of the D14S267 marker that has been reported in various tumors as having a high frequency of deletion was observed in 10 of 26 informative cases (38.5%). The commonly deleted regions were delineated by markers D14S65 and D14S250 on 14q32. In regard to the macroscopic features of tumor, the 14q allelic imbalance rate of protruding type tumors was higher than that of the ulcerative type. These data suggest that potential suppressor loci on 14q32 are related to the development and progression of esophageal carcinoma. © 2002 Elsevier Science Inc. All rights reserved.

1. Introduction Recent developments in the early detection of esophageal carcinoma and progress in therapeutic modalities have shown good results [1]. However, patients whose disease was diagnosed at advanced stages have shown poor prognoses. To improve on the survival rates associated with this disease, understanding of the molecular genetic alterations causing esophageal carcinoma is important. Accumulation of multiple deletions and activation of oncogenes, Cyclin D1, INT-2, and C-ERBB, have been noted in cases of esophageal carcinoma [2,3]. Furthermore, highly frequent deletions have been observed on chromosome arms 3p, 5q, 9p, 13q, and 17p by allelotype analysis [4,5]. These results suggested that the inactivation of multiple tumor suppressor genes is associated with the initiation and progression of esophageal carcinoma. However, these results do not conclusively determine the genetic pathway of esophageal carcinoma. Several studies have shown 14q allelic loss in several tumors, including neuroblastoma, colorectal carcinoma, blad-

der cancer, meningioma, ovarian cancer, renal cell carcinoma, and head and neck squamous cell carcinoma [6–12]. Earlier, we also were able to determine the frequency of loss of heterozygosity (LOH) and to define a minimal region of LOH contributing to the eventual localization of potential tumor suppressor gene at 14q32 in colorectal carcinoma [13]. These observations suggest the existence of a candidate tumor suppressor gene for many types of human neoplasms in this region. Thus, we considered that the LOH on 14q32 also may participate in genetic changes in esophageal carcinoma. The purpose of this study was to identify the frequency of allelic imbalance and to determine a minimal region on 14q32 in esophageal carcinoma. We analyzed the frequency of allelic imbalance and determined the smallest minimal region by using fluorescent polymerase chain reaction (PCR) [14,15] and a PCR–single-strand conformation polymorphism (SSCP) [16,17] methods using four highly microsatellite markers in 35 esophageal carcinomas. 2. Materials and Methods 2.1. Tissue samples

* Corresponding author. Tel.: 0235-24-8872; fax: 0235-24-8872. E-mail address: [email protected] (Y. Ihara).

Thirty-five esophageal carcinomas and corresponding normal mucosae samples were obtained from 24 freshly fro-

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Y. Ihara et al. / Cancer Genetics and Cytogenetics 135 (2002) 177–181

zen surgically resected specimens and 11 endoscopic biopsies at the Second Department of Surgery/Toyama Medical and Pharmaceutical University School of Medicine. The specimens were frozen and stored at 80C until DNA extraction. The tumors used in this study were primary site cancers that had not been previously treated with chemoradiative therapy and were representative of macroscopic feature, lymphnode metastasis, stage, histologic subtype, and clinical outcome based one the Japanese Society for Esophageal Disease [18]. Tumors were 33 squamous cell carcinomas, one adenoid cystic carcinoma, and one basaloid squamous carcinoma as determined histologically. The two cases were obtained from double carcinomas in same patients. 2.2. DNA extraction The samples were digested in proteinase K, followed by phenol-chloroform extraction and ethanol precipitation as described previously [19]. 2.3. Allelic imbalance analysis Allelic imbalance on 14q32 was assessed using the four microsatellite (dinucleotide repeat) markers from the Genethon genetic linkage map [20] and are shown in Table 2. We conducted PCR in a total volume of 5 l containing 50–100 ng genomic DNA, 20 mM Tris-HCl (pH 8.0), 100 mM KCl, 1.5 mM MgCl2, each deoxynucleotide triphosphage at 2.5 mM, each primer at 0.25 M, and 0.5 units of

Taq polymerase (Nippon Gene Co., Ltd., Toyama, Japan). One primer per pair was labeled fluorescently with IR770 (Takara Co., Ltd., Japan) Twenty to 25 PCR cycles were performed with denaturation of 95C for 60 seconds, annealing at 55–62C for 40 seconds, and extension at 72C for 120 seconds with a final extension step of 72C for 4 minutes. Amplification products were electrophoresed at 30 W in 6% urea-polyacrylamide gel for 14 hours using an automated DNA sequencer (Li-Cor Model 4000, Li-Cor Co., Ltd, USA). The imbalance factor, defined as the ratio of allelic intensity in a tumor sample relative to the ratio of allelic intensity in normal DNA, was calculated using the peak height data generated by the NIH image analysis software (National Institutes of Health, Bethesda, MD, USA). 2.4. SSCP analysis The SSCP analysis was performed essentially as described by Mitsumori et al. [17]. After heating at 95C for 5 minutes, the PCR products were electrophoresed in a 15% polyacrylamide gel for 3 hours, and the resulting DNA bands were photographed after staining with CYBER Green I nucleic acid stain (Molecular Probes, Eugene, OR, USA). 2.5. Statistical analysis Statistical significance of difference was evaluated by Fisher’s exact test. The survival rate was determined using the Kaplan–Meier method, and statistical significance was evaluated by the log-rank test (P0.05 was considered significant).

Fig. 1. Examples of allelic imbalance data in tumors used to define minimal regions of deletion. (A) Tumor 35 demonstrates retention of both alleles at D14S250, and allelic imbalance at D14S62, D14S65, and D14S267. (B) Tumor 7 demonstrates retention of both alleles at D14S65 and D14S250 and allelic imbalance at D14S267 and D14S250. (C) Tumor 27 demonstrates retention of both alleles at D14S65, and allelic imbalance at D14S62, D14S267, and D14S250. Abbreviations: T, tumor DNA; N, normal DNA.

Y. Ihara et al. / Cancer Genetics and Cytogenetics 135 (2002) 177–181 Table 1 LOH on 14q32 in 35 esophageal carcinomas Microsatellite markers Informative cases (%) LOH (%)

D14S62

D14S65

D14S267

D14S250

20 (57.1) 8 (40)

26 (74.3) 8 (30.7)

26 (74.3) 10 (38.5)

27 (77.1) 9 (33.3)

3. Results DNA from 35 esophageal carcinomas were examined for allelic imbalance using PCR method using four microsatellite markers on chromosome 14q32. Fig. 1 shows the results of typical bands in examined samples. Correct evidence of allelic imbalance at one or more 14q32 markers was observed in 13 of 35 (37%) primary esophageal carcinomas. Table 2 Deletion map of 14q32 in esophageal carcinoma Microsatellite markers Tumor no.

D14S62

D14S65

D14S267

D14S250

1 2 3 4 12 13 14 16 17 18 19 20 22 23 26 28 29 30 31 33 34 21 6 7 27 35 5 8 9 10 11 15 24 25 32

RH RH RH NI NI NI NI RH NI RH RH NI RH RH RH NI NI NI RH RH NI NI RH NI LOH LOH LOH LOH NI NI NI LOH LOH LOH LOH

RH RH RH NI RH RH NI RH RH RH NI RH RH RH RH RH RH RH NI NI NI RH NI RH RH LOH LOH NI LOH LOH LOH LOH LOH LOH NI

NI NI RH RH RH NI RH RH RH RH NI NI RH RH RH RH NI NI RH RH RH RH LOH LOH LOH LOH LOH LOH LOH NI LOH LOH LOH LOH NI

RH RH RH NI RH RH RH RH RH RH RH NI RH RH RH NI RH RH NI NI NI RH LOH RH LOH RH NI LOH LOH LOH LOH LOH LOH NI LOH

A summary of results for four microsatellite markers on chromosome 14q32 are presented. Minimal area of deletion is between loci D14S65 and D14S250. Abbreviations: NI, not informative; RH, retention of heterozygosity.

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The highest rates of allelic imbalance were 38.5% (10 of 26 informative tumors) at D14S267 and 40% (8 of 20) at D14S62 (Table 1). Of the 12 tumors with allelic imbalance at one or more markers, nine displayed complete allelic imbalance at all markers that we examined. The remaining three tumors with partial allelic imbalance were used to define minimal regions of deletion. Tumors 7 and 27 demonstrated a centromeric border at D14S65, and tumors 7 and 35 defined a telomeric border at D14S250. Only tumor 7 demonstrated the borders at both D14S65 and D14S250. Therefore, the common deletion region on 14q32 is likely between loci D14S65 and D14S250 (Table 2). There was no correlation between allelic imbalance and no allelic imbalance cases with regard to sex, age, depth of tumor invasion, extent of lymph node metastasis, and stage. However, when the macroscopic features of all tumors were divided to protruding, ulcerative types and others, the 14q32 allelic imbalance rate (54%) of the protruding type was higher than that of the ulcerative type (31%) with statistical significance (Table 3). Fourteen patients who had survived in the 6 months since diagnosis were eligible for outcome analysis. They were treated with chemotherapy, irradiation, and/or surgery. The follow-up range was from 12 to 101 months, with a mean of 32 months. The survival rate of the D14S62 allelic imbalance group tended to be lower than that of the no allelic imbalance group (Fig. 2). 4. Discussion Recently, we reported a highly frequent deletion on 14q32 in colorectal carcinoma and suggested the existence of a putative tumor suppressor gene(s) in this region. ThereTable 3 Relation between clinicopathologic findings and LOH on 14q32 LOH on 14q32 Retained

Lost

2 14 6

7 4 2

10 4

6 4

1 1 1 7 4

2 1 2 4 1

a

Macroscopic features Protruding type*,** Ulcerative type*,** Others** Lymph node metastasisb,c Positive Negative Histologic stageb,c 0 I II III IV

Abbreviation: LOH, loss of heterozygosity. *P  0.0090. **P  0.0044 (Fisher’s exact test). a All tumors were divided into three types: protruding, ulcerative, and other. b According to classification of the Japanese Society for esophageal disease. c Not significant.

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Fig. 2. Survival curves for patients who had survived in the 6 months from diagnosis with the D14S62 allelic imbalance and no allelic imbalance groups. The survival rate of the allelic imbalance group tends to be lower than that of the no allelic imbalance group (P0.05 log-rank test).

fore, we analyzed the allelic imbalance on 14q32 in esophageal carcinoma and demonstrated that more than one-third of all esophageal carcinomas examined in this study had sustained allelic imbalances. Until our study, no evidence has reported the high frequency of 14q allelic imbalance in esophageal carcinoma. In a reported allelotype of esophageal carcinoma, restriction fragment length polymorphism (RFLP) analysis revealed LOH on chromosome 14q in 14–15% of the examined tumors [4,20]. These studies used D14S13 RFLP marker at 14q32 locus in their analysis. However, our results revealed 30.7–42.3% allelic imbalance on chromosome 14q32 by using five microsatellite markers in this tumor. The discrepancy between the frequency found in the previous studies and those found in ours is probably attributable to the differing the cytogenetic locations of the markers. It may be that the primers we used were located in more centromeric side on 14q32 than the primer used by these studies. Highly frequent allelic imbalance on chromosome 14q has been reported previously and correlated with statistical significance to a more advanced stage or poor prognosis in bladder, ovarian, and head and neck squamous cell carcinomas [8,10,12]. In this study, although tumors of all grades, stages, histologic subtypes, and depths of invasion were examined, we were unable to notice any clincopathologic correlates with 14q deletions except that the macroscopic features of the tumors seem to be affected by deletions on this chromosome. In the present report, we identified the small deletion between D14S65 and D14S250, approximately 8 cM spanning 14q32. This region overlaps in ovarian, colorectal, bladder, head and neck squamous cell carcinomas, and Barrett esophageal adenocarcinomas [8,12,13,21] and is consistent with the same loci that is considered the potential tumor suppressor gene. Recently, van Dekken et al. used the comparative genomic hybridization method to reveal that loss of 14q31q-32.1 occurred significantly more frequently in Barrett esophageal adenocarcinoma than in gastric cardia

cancers and suggested that this is a result of Barrett transformed esophageal tissue specificity [21]. It is interesting that these genetic changes on the same loci occurred in different histologies of esophageal carcinoma. This genetic abnormality on 14q32 may play an important role in the carcinogenesis of esophageal neoplasms as well as in other solid tumors. Thus, our observations might begin to characterize of 14q32 allelic imbalance in the genetic progression of this tumor. In our study, the number of cases examined was rather low; however, many cases might reveal the clincopathologic significance of allelic imbalance in esophageal carcinoma, especially regarding its prognosis or biological behavior. Our characterization of the putative tumor suppressor locus will help in isolating a candidate gene associated with esophageal carcinoma and other solid tumors. Acknowledgments This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology and by Joint-Research for Regional Intensive from Japan Science and Technology Corporation.

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