Refinement of Regions with Allelic Loss on Chromosome 18P11.2 and 18Q12.2 in Esophageal Squamous Cell Carcinoma1

Vol. 6, 3565–3569, September 2000 Clinical Cancer Research 3565

Refinement of Regions with Allelic Loss on Chromosome 18p11.2 and 18q12.2 in Esophageal Squamous Cell Carcinoma1

Jayaprakash D. Karkera,2 Saleh Ayache,2 more frequently in African-Americans than in Caucasians, Rodman J. Ransome, Jr., Marvin A. Jackson, whereas adenocarcinoma is more prevalent among Caucasians Ahmed F. Elsayem, Rajagopalan Sridhar, (2). Tobacco and alcohol consumption represents major envi- ronmental risk factors for this malignancy (3), but the molecular Sevilla D. Detera-Wadleigh, and events leading to esophageal cancer and the genetic components 3 Robert G. Wadleigh that are mutated at the inception and course of the neoplasm are Medical Oncology Section, Department of Veterans Affairs Medical largely unknown. Unraveling the genetic factors could provide Center, Washington, DC 20422 [J. D. K., S. A., R. G. W.]; targets for the development of more effective therapeutic agents. Departments of Pathology [R. J. R., M. A. J.], Medicine [A. F. E.], A variety of tumor suppressor genes have been implicated and Radiation Therapy and Cancer Center [R. S.], Howard University, Washington, DC 20060; and National Institute of Mental Health, in esophageal cancer (4, 5). Chromosomal abnormalities and NIH, Bethesda, MD 20892 [S. D. D.] LOH4 in which random isolated markers are used have been associated with esophageal tumors (6, 7). Analysis of chromosome 18 has focused mainly on 18q21, where two known tumor ABSTRACT suppressor genes, DCC (8) and DPC4 (9) have been mapped. Esophageal cancer ranks among the 10 most common However, only infrequent deletions of DCC and DPC4 have cancers worldwide and is almost invariably fatal. The de- been found (7, 10). Our previous effort to screen chromosome tailed genetic repertoire involved in esophageal carcinogen- 18 using microsatellite markers that span the entire length of the esis has not been defined. We have shown previously that the chromosome detected a broad peak of allelic deletion spanning esophageal squamous cell carcinoma genome exhibits a fre- the p and q sides of the centromere (11). Within this peak, quent loss of heterozygosity (LOH) in the pericentromeric D18S542 on 18p and D18S978 on 18q displayed LOH frequen- region of chromosome 18. To construct a fine deletion map, cies of 38 and 72%, respectively. These findings suggest the we screened 76 new samples composed of microdissected involvement of tumor suppressor genes located on 18p and 18q esophageal squamous cell carcinoma and matched morpho- in esophageal carcinogenesis. To obtain a global representation logically normal epithelial cells using closely spaced mark- of the sites that are lost, gained, and amplified in ESC, we ers. Maximal LOH frequency (54%) was displayed by conducted CGH (12). CGH identified multiple chromosomal D18S542 on 18p11.2. The pattern of LOH in selected pa- imbalances, including some on chromosome 18. Chromosomal tients indicated that the short region of overlap extends 3 cM losses detected in this study overlap with those previously on either side of D18S542. On the long arm of chromosome reported in other squamous cell neoplasms (13, 14). 18, the highest frequency of allelic loss (42%) was detected In the present study, we typed 11 new polymorphic mark- by D18S978 on 18q12.2-q21.1. This analysis revealed a short ers which flanked D18S53 and D18S978. These markers includ- region of overlap of ϳ0.8 cM. These findings further impli- ing D18S53 and D18S978 were used to screen for LOH with the cate unreported tumor suppressor genes encoded by 18p11.2 use of 76 different DNA templates derived from microdissected and 18q12.2 in esophageal squamous cell carcinogenesis and ESC cells. Here, we report additional evidence for allelic loss on they indicate a refinement of their map location. 18p and 18q and a narrowing of these deleted regions reported in our previous study (11). INTRODUCTION Squamous cell and adenocarcinoma comprise the two most common cell types of esophageal cancer (1). Squamous cell MATERIALS AND METHODS carcinoma is common in all populations examined, and it occurs Patients and Samples. The present study includes archival tissue from 76 patients with ESC from the Department of Pathology, Howard University. There were 57 male and 19 female patients; age range was from 40 to 88 years (mean, 62.8 Received 1/27/00; revised 6/29/00; accepted 7/6/00. years). There were 73 African-Americans and 3 Caucasians. The costs of publication of this article were defrayed in part by the Eleven tumors (14%) were located in the upper esophagus, 40 payment of page charges. This article must therefore be hereby marked (53%) were in the middle esophagus, and 25 (33%) were in the advertisement in accordance with 18 U.S.C. Section 1734 solely to lower esophagus. The American Joint Committee on Cancer indicate this fact. 1 Supported by grants from the Elsa U. Pardee Foundation and the Robert Leet and Clara Guthrie Patterson Trust. 2 Contributed equally to this work. 3 To whom requests for reprints should be addressed, at Medical Oncology Section, Department of Veterans Affairs Medical Center, 50 4 The abbreviations used are: LOH, loss of heterozygosity; ESC, esoph- Irving Street NW, Washington, DC 20422. Phone: (202) 745-8482; ageal squamous cell carcinoma; CGH, comparative genomic hybridiza- Fax: (202) 745-8131; E-mail: WADLEIGH.ROBERT_Gϩ_@ tion; MMRF, Marshfield Medical Research Foundation; LDB, Genetic WASHINGTON.VA.GOV. Location Database.

Fig. 1 Allelic loss on chromosome 18 in esophageal cancer detected by D18S542 and D18S978. Lanes N and T, normal and tumor banding patterns, respectively.

staging showed 7 stage I, 9 stage IIA, 16 stage IIB, 24 stage III, and 20 stage IV. Microdissection and Genomic DNA Extraction. A to- tal of 76 new cases of formalin-fixed, paraffin-embedded archi- Fig. 2 Chromosomal pattern of LOH in selected cases. F, LOH; E,no LOH; R, noninformative site. Genetic distances shown are based on the val samples of ESC that had been reviewed by pathologists LDB (17). (R. J. R. and M. A. J.) were used to microdissect morphologically malignant and normal cells. One 5-␮m section was cut and stained with H&E and used as a guide for microdissection. Areas containing tumor and normal cells were microdissected a 6% sequencing gel. Autoradiography was done either at room Ϫ from six de-paraffinized 5-␮m sections with a sterile 25-gauge temperature or at 70°C with an intensifying screen. LOH was needle under a microscope (Stereostar, A.O. Scientific). Micro- defined as either the absence of one allele or a decrease in dissected cells were collected in an Eppendorf tube and resus- intensity of one allele by at least 50%. Gels were read by two pended in 30 ␮l of lysis buffer [10 mM Tris-HCl (pH 8), 25 mM reviewers (J. D. K. and R. G. W.) and checked by a third re- EDTA (pH 8), and 0.5% SDS] containing 1 mg/ml proteinase K, viewer (S. D. D-W.) when reading differences occurred. Read- incubated overnight at 55oC (15). DNA was isolated as de- ing discrepancies were resolved by repeating the experiments. scribed previously (16). PCR-LOH Analysis. PCR amplification was conducted RESULTS by using microsatellite markers that map to 18p (D18S542, To refine the deletion map of the regions flanking the D18S1158, D18S1150, D18S1116, D18S453) and to 18q centromere, we typed 76 new cases of archival squamous esoph- (D18S67, D18S451, D18S978, D18S65, D18S455, D18S1094, ageal tumor tissues using closely spaced markers on 18p11.2 D18S460). D18S1104 mapped to 18p11-18q12. These markers and 18q12-q21. LOH analysis was performed with DNA de- were purchased from Research Genetics (Huntsville, AL). Each rived from microdissected tumor cells and corresponding mor- PCR was performed in a 10-␮l reaction mixture containing 1 ␮l phologically normal epithelial cells from each individual case. of genomic DNA from microdissected cells, dNTP mix (1.25 The success rate of PCR amplification from these templates for mM dGTP, dATP, dTTP, dCTP), 10 mM Tris-HCl (pH 8.6), 1.5 LOH analysis was ϳ90%. Ј 32 mM MgCl2, one 5 - P-end-labeled primer, and 0.5 unit of We used 13 highly informative genetic markers mapping to AmpliTaq (Perkin-Elmer). PCR was performed with an initial 18p11.2 and 18q12.1-q21.1, with heterozygosity ranging from denaturation at 94°C for 4 min followed by 40 cycles of dena- 65% (D18S1094) to 83% (D18S453). In the samples studied turation at 94°C for 30 s, annealing at 55°C for 30 s, extension here, the number of informative cases was highest with at 72°C for 1 min, and a final extension at 72°C for 5 min. The D18S978 (89%) and lowest with D18S1116 (57%). amplicons were denatured at 95°C for 5 min in 10 ␮lof On 18p11.2, we scanned a ϳ6-cM region using five mic- sequence loading buffer, and electrophoresis was conducted on rosatellite markers based on the genetic maps of the Center for

Fig. 3 Percentage of LOH detected by chromosome 18 markers in informative cases. The markers used, number of cases, and percentage of LOH per informative case are indicated.

Medical Research of the MMRF (www.marshmed.org/genet- DISCUSSION ics/) and the LDB (cedar.genetics.soton.ac.uk/public_html/) Recently, we found a broad region of LOH on chromosome (17). D18S542 displayed the highest frequency of LOH (54%; 18 encompassing both sides of the centromere, from 18p11.2 to Figs. 1–3). Note that D18S542 and D18S53 amplify the same 18q12-q21.1 (11). In that study, typing was done on template locus (http://gdbwww.gdb.org/). The flanking marker DNA derived from ESC biopsies. Because LOH could be D18S1116 displayed a 20% LOH frequency. D18S453, 2.25 cM masked by the presence of normal cells, which could lead to centromeric of D18S542 by MMRF and 3.172 cM by LDB false negatives, we decided to isolate ESC cells separately from detected LOH in 8 of 65 informative cases (Fig. 3). The fre- morphologically normal epithelial cells by microdissection and quency of allelic deletions detected by markers telomeric of to prepare DNA representing each genome. To permit a nar- D18S1116 decreased considerably. rowing of the critical region, we examined a relatively large We searched for the shortest commonly deleted region number of cases and increased the marker density by selecting from among the LOH patterns displayed by the 76 cases. In Fig. markers that closely flanked D18S53 and D18S978, markers that 2, Patient 50 exhibited LOH at both D18S542 and D18S1116, displayed the highest frequency of allelic loss in our prior study but not in markers flanking these loci. This patient did not show (11). There were 11 such new markers. Our findings indicate LOH anywhere else on the chromosome. In the MMRF map, D18S542 and D18S1116 are at the same genetic location that LOH on 18p11.2 and 18q12.2-q21 is frequent in ESC cells whereas in the LDB map (17) the distance between these mark- as displayed by D18S542 and D18S978, respectively. However, ers is ϳ1 cM. D18S1158 is 2.3 cM telomeric of D18S1116 and lower LOH frequencies using the same markers in the present D18S453 is 2.3 cM centromeric of D18S542. Based on this study compared with our previous study (11) probably indicate analysis the shortest region of overlap extends #3 cM on either a more accurate representation of the actual LOH frequencies side of D18S542 or D18S1116. because of the increased sample size. Seven microsatellite markers mapping to18q12-q21 and Prior studies have implicated 18p in other tumors. In spanning ϳ6 cM were used for LOH screening. In this region, esophageal adenocarcinoma, LOH was detected by D18S53 D18S978 exhibited the highest frequency of LOH (42%; Fig. 3). (18). Recently, Tran et al. (19) reported a novel region on 18p, This contrasts with our prior study where a smaller number of D18S452, that was frequently deleted in tumors of the lung, biopsies were used and the LOH frequency was higher in brain, and breast. These, coupled with our present findings, D18S978 than in D18S542 (11). In the present study, the allelic increase the evidence for an unknown tumor suppressor gene on deletions extended to ϳ0.9 cM telomeric of D18S978 (Figs. 1 the short arm of chromosome 18. Two genes that might qualify and 2). The LOH frequencies are shown in Fig. 3. In 25% of the as positional and functional candidates are ZFP161 (20), a cases, allelic deletions occurred at both the 18p11.21 and 18q12- c-myc repressor, and PTPR1 (21), a tyrosine phosphatase q21 regions (data not shown). receptor. In this panel of samples, the shortest region of overlap on Our initial LOH study suggested the existence of another 18q12-q21 was smaller than found on 18p11.2. Cases 09 and 18 unreported tumor suppressor gene for ESC on 18q12-q21 (11). displayed patterns that permitted narrowing of the location of In squamous cell carcinoma of the head and neck, D18S39, a the potential tumor suppressor gene to ϳ0.8 cM (Fig. 2). marker located on 18q12 detected a high percentage of LOH

(22). Similarly, loss of 18q is reported to be a prognostic factor 5. Montesano, R., Hollstein, M., and Hainaut, P. Genetic alterations in for poor patient survival with head and neck cancer (23) or with esophageal cancer and their relevance to etiology and pathogenesis: a cohesive-type gastric cancer (24). Also, Frank et al. (25) re- review. Int. J. Cancer, 69: 225–235, 1996. ported loss of 18q in metastatic and locally recurrent squamous 6. Hu, N., Roth, M. J., Polymeropolous, M., Tang, Z. Z., Emmert-Buck, M. R., Wang, Q. H., Goldstein, A. M., Feng, S. S., Dawsey, S. M., Ding, cell tumors, but not in primary tumors from the same patients. T., Zhuang, Z. P., Han, X. Y., Ried, T., Griffen, C., and Taylor, P. R. Data from the present study on a larger number of samples Identification of novel regions of allelic loss from a genomewide scan of suggest that the critical region of allelic loss on 18q is proximal esophageal squamous-cell carcinoma in a high-risk Chinese population. to DCC. 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Jayaprakash D. Karkera, Saleh Ayache, Rodman J. Ransome, Jr., et al.

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