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Proc. Natl. Acad. Sci. USA Vol. 89, pp. 3385-3388, April 1992 Medical Sciences Loss of heterozygosity involving the APC and MCC genetic loci occurs in the majority of human esophageal (tumor suppressor /polymerase chain reaction/Barrett /squamous carcnoma/alelic deletion) ROBERT F. BOYNTON*, PATRICIA L. BLOUNTt, JING YIN*, VICTORIA L. BROWN*, YING HUANG4, Yi TONG*, TIM MCDANIEL*§, CARNELL NEWKIRK$, JAMES H. RESAU1, WENDY H. RASKINDt, RODGER C. HAGGITTII, BRIAN J. REIDt, AND STEPHEN J. MELTZER*§¶**tt Departments of *Medicine (GI Division), 1Pathology, tMicrobiology and Immunology, ** Center, and §Graduate Program in Molecular and Cell Biology, University of Maryland School of Medicine and Veterans Affairs Hospital, Baltimore, MD 21201; and Departments of tMedicine and IlPathology, University of Washington, Seattle, WA 98195 Communicated by Donald D. Brown, January 6, 1991

ABSTRACT The tumor suppressor geneAPC was recently mosome 17p encoding tumor p53 (designated TP53), identified, and the cDNA was cloned from 5q21. point mutations in TP53, and overexpression of p53 protein Point mutations affecting APC are seen in the hereditary have all been reported in esophageal (24-26). syndrome familial adenomatous polyposis, and point mutations LOH of the retinoblastoma gene, RB, also has been reported in APC and a closely linked gene, MCC, as well as loss of in recent studies of esophageal cancer (27). Therefore, it is heterozygosity involving chromosome 5q have been reported in likely that abnormalities at several genetic loci are involved sporadic colon cancer. To our knowledge, loss ofheterozygosity in the pathogenesis of this disease. involving APC or MCC or both has not yet been described in To our knowledge, LOH of 5q21 has not been evaluated in any other human cancer besides cancer. We used the previous studies ofesophageal cancer (28); and LOH directly polymerase chain reaction and DNA content flow cytometric involving APC or MCC has only been reported in colorectal nuclear sorting to examine 30 primary human esophageal and lung (12). We undertook this study to deter- cancers for loss of heterozygosity ofAPC or MCC or both. Loss ofone allele was detected in 77% of26 informative cases. These mine the prevalence of APC-LOH and MCC-LOH in esoph- data suggest that loss of heterozygosity of regions on Sq ageal carcinoma. DNA extracted from aneuploid nuclei pu- including the APC and MCC genetic loci is involved in the rified by flow cytometry (29) from 30 human esophageal development and/or progression of most human esophageal cancers (12 squamous cell carcinomas and 1i adenocarcino- cancers. They imply that inactivation ofAPC, MCC, and/or a mas) was examined for LOH at the APC and MCC loci by linked gene on chromosome Sq plays a role in the pathogenesis using the polymerase chain reaction (PCR). of some cancers of the upper , as well as in colon cancer and familial adenomatous polyposis. MATERIALS AND METHODS Recently, a known as APC or DP2.5 Tissue Specimens and DNA Extractions. After informed was identified, cloned, and sequenced (1-4). Previous cyto- consent was obtained from the patient, extra tissue samples genetic studies (5) and linkage analyses (6-8) had already were obtained during diagnostic endoscopic or after localized the site of this gene to 5q21. APC is mutated in the . Matching normal tissue was simultaneously col- germ-line DNA of patients with familial adenomatous poly- lected from normal gastric mucosa in all patients. Tumor type posis (or FAP) (2-4). Another gene from this region, MCC, ( versus ) was ver- has also been implicated in the pathogenesis of sporadic ified by histological examination. Aneuploid tumor nuclei (9). Somatic mutations of APC or allelic were separated from contaminating diploid stromal cell nu- deletions of Sq or both have also been described in sporadic clei by flow cytometric sorting on the basis of DNA content colorectal cancers (2, 10, 11). Moreover, allelic deletion (29). These samples were obtained from involving MCC occurs in 20-25% of lung cancers (12). specimens at the time of surgery, placed in minimal essential Previous studies therefore suggest that APC functions as a medium (GIBCO) containing 10% (vol/vol) dimethyl sulfox- tumor-suppressor gene in colon and possibly lung cancer, ide and 5% (vol/vol) fetal calf serum on ice, and frozen according to the model proposed by Knudson (13). immediately at -700C until processed. Nuclei were isolated Esophageal cancer accounts for 10,000 deaths annually in from tumor samples and stained with 4,6-diamidino-2- the United States and ranks among the leading causes of phenylindole (Accurate Chemicals, Westbury, NY) by the cancer death worldwide (14). There are two histologic types single-step detergent method (29). Nuclei were then sorted of esophageal cancer: squamous cell carcinoma, associated according to DNA content on a Coulter Epics cell sorter, and with excessive ethanol and tobacco use (14); and adenocar- high molecular weight DNA was obtained from the aneuploid cinoma, associated with Barrett's esophagus (15). Various nuclear preparation as described (26). Each sorted nuclear molecular genetic abnormalities have been reported in esoph- preparation contained approximately 50,000 nuclei for DNA ageal cancer. These include amplification of the protoonco- extraction. DNA was extracted by using a modification ofthe MYC, INT2, HSTJ, and the epidermal growth factor method of Goelz et al. (30) as described (24). Each total receptor gene, EGFR (formerly ERBB) (16-19). Investiga- nuclear genomic DNA preparation was dissolved in 150 ,A of tions of RAS family protooncogenes in esophageal cancer mM mM EDTA. revealed infrequent or absent RAS mutations (20-23). Loss 10 Tris, pH 8.0/1 of heterozygosity (LOH) at loci including the gene on chro- Abbreviations: LOH, loss ofheterozygosity; PCR, polymerase chain reaction. The publication costs of this article were defrayed in part by page charge ttTo whom reprint requests should be addressed at: Department- of payment. This article must therefore be hereby marked "advertisement" Medicine/GI, University of Maryland Hospital, Room N3W62, 22 in accordance with 18 U.S.C. §1734 solely to indicate this fact. South Greene Street, Baltimore, MD 21201.

3385 Downloaded by guest on October 1, 2021 3386 Medical Sciences: Boynton et al. Proc. Natl. Acad. Sci. USA 89 (1992)

PCR. For APC, primers flanking a Rsa I restriction frag- Table 1. Esophageal tumors analyzed for LOH ment length polymorphism within exon 11 were synthesized. APC exon MCC exon The upstream primer was 5'-GGACTACAGGCCATTGCA- Case no. 11 10 GAA-3'; the downstream primer was 5'-GGCTACATCTC- 424TS SQ CA + uncut Homo lower CAAAAGTCAA-3'. The following ingredients were mixed 485TS SQ CA - + upper with H20 in a total volume of 100 pl: 10-20 1.l ofeach nuclear 489TS SQ CA Homo cut Homo lower genomic DNA preparation, 40 pmol of each primer, 10 A1 of 499TS SQ CA Homo cut reaction buffer, 2 units of Taq DNA polymerase (Promega), 533TS SQ CA - and 75 AtM of each deoxynucleotide triphosphate. Amplifi- 547TS SQ CA Homo uncut ND cation was carried out with an Ericomp programmable cyclic 934TS SQ CA Homo uncut + upper reactor (Ericomp, San Diego). One cycle of 5 min at 95TC, 1 E14TS SQ CA Homo uncut + lower min at 590C, and 1 min at 720C was followed by 34 cycles of E30TS SQ CA + uncut + lower 30 sec at 95TC, 1 min at 590C, and 1 min at 72TC. The E87A1/A2 SQ CA + cut + upper 133-base-pair (bp) PCR products were digested with 1 pl of ElOlA SQ CA Homo uncut + upper Rsa I (New England Biolabs) and electrophoresed on 7% S9 SQ CA + uncut Homo upper polyacrylamide gels. PCR product contamination was pre- 429TS AD CA Homo cut + lower vented by stringent measures described previously (24). 415TS AD CA Homo cut For MCC, a variable insertion region within exon 10 was 504TS AD CA - - amplified by using the primers 5'-TACGAATCCAATGC- 939TS AD CA + cut Homo lower CACA-3' (upstream) and 5 '-CTGAAGTAGCTC- S1 AD CA + cut + upper CAAACA-3' (downstream). This PCR amplified a product S2 AD CA + cut Homo lower either 79 or 93 bp long, depending upon the absence or S3 AD CA + uncut + upper presence of the insertion. Conditions were as for APC, S4 AD CA + cut + upper except that only 29 cycles of amplification were performed. S5 AD CA + cut + upper At the MCC locus, a tendency to form the heterodimer was S6 AD CA + uncut Homo lower seen in heterozygotes, with a resulting third band migrating S7 AD CA + uncut + lower above the 93-bp band. This tendency could be minimized by S8 AD CA + uncut + upper decreasing the number of PCR cycles to 29. No restriction Slo AD CA Homo cut Homo lower enzyme digestion was necessary. Sil AD CA Homo uncut + upper S12 AD CA Homo cut Homo lower S13 AD CA - ND RESULTS S14 AD CA - Homo upper Normal DNA from heterozygous patients showed three S15 AD CA + uncut Homo upper bands at the APC exon 11 locus: a 133-bp "uncut" band from -SQ CA, squamous cell carcinoma; AD CA, adenocarcinoma; the allele lacking the Rsa I restriction enzyme site and two homo, constitutionally homozygous patient; -, negative for LOH; "cut" bands (85 bp and 48 bp) representing the allele that ND, not done; + uncut, positive for loss of the uncut allele ofAPC; contained the site. APC-LOH was demonstrated when the + cut, positive for loss of the cut allele of APC; homo cut, tumor DNA showed loss of either the single uncut band or of uninformative patient homozygous for the cut allele of APC; homo the cut bands. Several examples ofAPC-LOH are illustrated uncut, uninformative patient homozygous for the uncut allele of APC; + upper, positive for loss of the upper (93 bp) band (allele I) in Fig. 1. Individual cases are displayed in Table 1 and of MCC; + lower, positive for loss of the lower (79 bp) band (allele summarized in Table 2. At the APC exon 11 locus, 19 of 30 II) of MCC; homo upper, uninformative patient homozygous for the patients were informative or heterozygous; 14 ofthe 19 (74%) upper allele of MCC; homo lower, uninformative patient homozy- demonstrated LOH. Heterozygous patients showed three gous for the lower allele of MCC. bands at the MCC exon 10 locus: a lower 79-bp band representing allele I, an upper 93-bp band representing allele heterodimer band formed only at very high PCR product II, and a more slowly migrating band representing the pre- concentrations. At the MCC exon 10 locus, 18 of 28 individ- sumed heterodimer ofthese two alleles. This uppermost band uals assayed were informative; 14 of the 18 patients (78%) was seen only in heterozygotes and disappeared when LOH demonstrated LOH. Examples ofLOH at the MCC locus are occurred. It was verified as a heterodimer by reamplifying a illustrated in Fig. 2. 1-pl aliquot of the PCR product in a new 100-,l reaction'tube Twenty-six of 30 patient tumor DNAs were informative at for two PCR cycles: the uppermost band (but not the lower one or both loci; 20 of the 26 (77%) showed LOH. Sixteen of two bands) then disappeared, suggesting that this presumed 18 were informative at APC or MCC or both; 12 of the 16 (75%) demonstrated LOH. Ten of 12 squamous cancers were informative at one or both loci; 8 of 133 the 10 (80%) demonstrated LOH. LOH was observed in two separate aneuploid populations from one tumor (E87). Agree- 85 ment between data for APC and MCC was seen in 11 of 12 cases informative at both loci; one squamous cancer (485TS) 418 showed MCC-LOH but not APC-LOH. In addition, LOH at several other chromosomal loci occurred in only 10%o to 40%6 FIG. 1. LOH at exon 11 of APC detected with PCR. Lanes: N, Table 2. APC/MCC-LOH in esophageal cancers normal tissue; T, tumor tissue. All eight patients in this figure are constitutionally heterozygous or informative. Each normal DNA Informative/total LOH/informative lane is immediately followed by its corresponding tumor DNA. In the Adenocarcinoma 16/18 12/16 (75%) first case, there is no difference between normal and tumor DNAs; Squamous carcinoma 10/12 8/10 (80%) this result is negative for LOH. In each ofthe remaining seven cases, there is loss of either the 133-bp allele lacking the Rsa I restriction Total carcinoma 26/30 20/26 (77%) site or the allele containing the site, represented by bands at 48 and Numbers given refer to patients informative at APC, MCC, or both 85 bp. loci. Downloaded by guest on October 1, 2021 Medical Sciences: Boynton et al. Proc. NatL. Acad. Sci. USA 89 (1992) 3387

A B C progression (or both). They add APC/MCC to TP53 (24-26) N T N T N T and RB (27) as turhor-suppressor gene sites that are fre- quently abnormal in this type of tumor. They enlarge the potential role ofthe APC/MCC loci in human tumors. Future investigations should also address the possibility that other dimer. tumor-suppressor genes are altered in esophageal cancer. 93bp - The continued use of PCR to detect LOH should facilitate 79bp -_ these analyses, particularly when aneuploid nuclei prepared from small endoscopic biopsy specimens are used as sub- strates. We thank Bert Vogelstein and Kenneth W. Iinzler for sequence information ait the restriction fragment length polymorphisms used in this study; Jordan Denner, Richard Milanich, and Thomas Bauer for aid in preparing figures; and Carrie Zimmerman and Robert W. Lash for oligonucleotide synthesis. This work was supported by the FIG. 2. LOH at eXon 10 of MCC detected by PCR. Lanes: N, Department of Veterans Affairs, American Cancer Society Grants normal tissue; T, tumor tissue. A variably present 14-bp insertion PDT-419 and PDT-316C, the Crohn's and Colitis Foundation of results in alleles of 79 or 93 bp with approximately equal frequency. America, Inc., National Institutes of Health Grant P01-DK32971, Heterozygotes also possess a third band above these two bands, and a Graduate Research Assistantship/Special Research Initiative presumed to be due to annealing of one PCR strand from each of the Support award from the University of Maryland Designated Re- two different alleles. LOH is evident as absence ofthis third band and search Initiative Fund. either the 79-bp or the 93-bp allele in the tumors of constitutionally heterozygous individuals. 1. Kinzler, K. W., Nilbert, M. C., Su, L.-K., Vogelstein, B., Bryan, T. M., Levy, D. B., Smith, K. J., Preisinger, A. C., of these same aneuploid tumor cell populations (unpublished Hedge, P., McKechnie, D., Finniear, R., Markham, A., Grof- data). fen, J., Boguski, M. S., Altschul, S. F., Horiee, A., Ando, H., Miyoshi, Y., Miki, Y., Nishisho, I. & Nakamura, Y. (1991) Science 253, 661-664. DISCUSSION 2. Nishisho, I., Nakamura, Y., Miyoshi, Y., Miki, Y., Ando, H., The preceding results show that deletions of one allele of Horii, A., Koyama, K., Utsunomiya, J., Baba, S., Hedge, P., APC or MCC or both occur in 77% of human esophageal Markham, A., Krush, A. J., Petersen, G., Hamilton, S. R., A. cancers. This is the most frequent molecular abnormality Nilbert, M. C., Levy, D. B., Bryan, T. M., Preisinger, C., Smith, K. J., Su, L.-K., Kinzler, K. W. & Vogelstein, B. (1991) detected in esophageal cancer, with the exception of allelic Science 253, 665-669. deletion of 17p reported in 12 of 13 patients with esophigeal 3. Groden, J., Thliveris, A., Samowitz, W., Carlson, M., Gelbert, adenocarcinoma (26). The prevalence of APC/MCC LOH L., Albertsen, H., Joslyn, G., Stevens, J., Spirio, L., Robert- was approximately equal in squamous cancers and adeno- son, M., Sargeant, L., Krapcho, K., Wolff, E., Burt, R., carcinomas. These data also suggest that APC or MCC or Hughes, J. P., Warrington, J., McPherson, J., Wasmuth, J., Le both may be involved in the pathogenesis of a broader Paslier, D., Abderrahim, H., Cohen, D., Leppert, M. & White, spectrum of human tumors than familial adenomatous poly- R. (1991) Cell 66, 589-600. 4. Joslyn, G., Carlson, M., Thliveris, A., Albertsen, H., Gelbert, posis and colon cancer. L., Samowitz, W., Groden, J., Stevens, J., Spirio, L., Rob- Abnormalities of chromosome Sq have been studied in ertson, M., Sargeant, L., Krapcho, K., Wolff, E., Burt, R., other cancers at the cytogenetic level (31-34) and by South- Hughes, J. P., Warrington, J., McPherson, J., Wasmuth, J., Le ern blotting (35-43). In some tumors, such as ovarian and Paslier, D., Abderrahim, H., Cohen, D., Leppert, M. & White, germinal cancers, no LOH was detected (35, 36); in others, R. (1991) Cell 66, 601-613. LOH was detected in 9-60% (37-41). The highest incidence 5. Herrera, L., Kakati, S., Gibas, L., Pietrzak, E. & Sandberg, of LOH on chromosome 5q (5q35-ter) was observed in A. A. (1986) Am. J. Med. Genet. 25, 473-476. 6. Bodmer, W. F., Bailey, C. J., Bodmer, J., Bussey, H. J. R., well-differentiated, but not in poorly differentiated, gastric Ellis, A., Gorman, P., Lucibello, F. C., Murday, V. A., Rider, adenocarcinomas (40). This study did not specify whether the S. H., Scambler, P., Sheer, D., Solomon, E. & Spurr, N. K. gastric cancers were located in the cardia or more distally in (1987) Nature (London) 328, 614-616. the . Cancers of the gastric cardia, but not distal 7. Leppert, M., Dobbs, M., Scambler, P., O'Connell, P., Naka- gastric cancers, show anatomic, epidemiologic, and patho- mura, Y., Stauffer, D., Woodward, S., Burt, R., Hughes, J., logic similarities to esophageal adenocarcinoma (44-46). Gardner, E., Lathrop, M., Wasmuth, J., Lalouel, J.-M. & Therefore, it will be important to determine the involvement White, R. (1987) Science 238, 1411-1413. of 5q allelic deletions in both types of gastric cancer. 8. Nakamura, Y., Lathrop, M., Leppert, M., Dobbs, M., Was- It is possible that neither APC nor MCC are targets of muth, J., Wolff, E., Carlson, M., Fujimoto, E., Krapcho, K., Sears, T., Woodward, S., Hughes, J., Burt, R., Gardner, E., allelic deletion in these cancers or that inactivation of other Lalouel, J.-M. & White, R. (1988) Am. J. Hum. Genet. 43, genes is important at this locus. However, the high percent- 638-644. age of tumors showing LOH (77%), when compared with the 9. Kinzler, K. W., Nilbert, M. C., Vogelstein, B., Bryan, T. M., published "background" LOH rate of 15-20o (28) and our Levy, D. B., Smith, K. J., Preisinger, A. C., Hamilton, S. R., own unpublished data at other chromosomal loci, strongly Hedge, P., Markham, A., Carlson, M., Joslyn, G., Groden, J., implies that APC/MCC-LOH is not a random event in White, R., Miki, Y., Miyoshi, Y., Nishisho, I. & Nakamura, Y. esophageal cancer. The DNA polymorphisms within APC (1991) Science 251, 1366-1370. and MCC that we used to measure LOH may be linked to 10. Solomon, E., Voss, R., Hall, V., Bodmer, W. F., Jass, J. R., H. another gene on Sq that is involved in esophageal cancer. Jeffreys, A. J., Lucibello, F. C., Patel, I. & Rider, S. (1987) Nature 616-619. cancers mutations (London) 328, Future studies in esophageal of point 11. Vogelstein, B., Fearon, E. R., Hamilton, S. R., Kern, S. E., involving APC/MCC and mapping studies using banks of Preisinger, A. 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