[CANCER RESEARCH 53, 1889-1894. April 15. 1993] Altered Messenger RNA and Unique Mutational Profiles of p53 and Rb in Human Esophageal Carcinomas1 Ying Huang, Stephen J. Meltzer,2 Jing Yin, Yi Tong, Esther H. Chang, Shiv Srivastava, Tim McDaniel, Robert F. Boynton, and Zhi-Qiang Zou Departments of Microbiology and Immunology ¡Y.H.I. Medicine (Gastroenterology Division) /S. J. M.. J. Y.. Y. T.. T. M., R. F. BJ. Molecular and Cell Biology Graduate Program ¡S.J. M.. T. M.¡.Pathologv IS. J. M./. and Cancer Center [S. J. M.I, University of Mary/and and Veterans Affairs Hospitals, Baltimore 21201. and Department of Pathology, Uniformed Services University for the Health Sciences, Bethesda 20X14 [E. H. C.. S. S.. Z-Q. Z.], Maryland ABSTRACT Seventy-nine esophageal carcinoma patients were studied for genetic abnormalities in the p53 and Kb tumor suppressor genes. Single-strand conformation polymorphism analysis and DNA sequencing were used to detect p53 point mutations, Northern blotting was used to examine ab normal expression of />5.i and Kh, and polymerase chain reaction and Southern blotting were used to analyze alleile loss. Twenty-five cases were analyzed by DNA sequencing to detect mutations in p53. Fourteen samples contained mutations within exons S through 9 ot'/o.); seven had missense mutations giving rise to single amino acid substitutions. The remaining seven (50%) contained nonsense mutations leading to premature termi nation, five due to single base pair substitutions, and two that were the result of frameshift mutations. In other human tumors, p53 mutations are predominantly missense mutations, but our data as well as those from other groups show that nonsense mutations are common in human esoph ageal cancer. All but one of the constitutionally heterozygous samples containing mutations also manifested loss of the normal p53 alÃ-ele;the one exception without allelic loss contained a silent mutation, which should not have had any affect on the p53 protein product. In addition, Northern blotting analysis revealed abnormalities (altered transcript size or mRNA levels) in 5 of 7 cases involving p53 and in 2 of 7 cases analyzed for Kh. Thirty-four cases were informative for allelic loss studies of both p53 and Kb; of these, 25 (74%) lost heterozygosity ofp53, Rb, or both. When point mutations and mRNA expression abnormalities were also considered, 33 of 45 (73% ) tumors informative for allelic loss assays of both genes as well as for mRNA or point mutation studies showed one or more abnormalities in p53 or Kh. Our results strongly suggest that a unique profile of molec ular alterations involving p53 and Kh characterizes human esophageal cancer and that these specific genetic lesions are important in the devel opment and/or progression of most human esophageal carcinomas. INTRODUCTION A wealth of information now shows that the inactivation of tumor suppressor genes by various genetic mechanisms is implicated in the pathogenesis of most human tumors. Two such genes are p53 and Rb? both of which encode nuclear proteins that play a role in the regulation of normal cell growth. It has been shown that both p53 and Rb associate with the transforming proteins of DNA tumor viruses. For example, both Rb and p53 proteins associate with the simian virus 40 large T antigen (1,2). Genetic lesions in p53 occur at high frequencies in a wide variety of human tumors (3). Inactivation of p53 can occur through genetic mechanisms affecting one or both alÃ-eles.These mechanisms include allelic loss, point mutation, gene rearrangement, Received 10/23/92; accepted 2/10/93. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by Grant PDT-419 from the American Cancer Society; the Office of Research and Development (Medical Research Service). Department of Veterans Affairs; University of Maryland DRIF/GRA funds; NIH Grant CA45158 (E. H. C.); and Grant HV-0001 from the National Foundation for Cancer Research (E. H. C.). 2 To whom requests for reprints should be addressed, at Medicine/Gì. N3W62, Uni versity of Maryland Hospital, 22 South Greene Street. Baltimore, MD 21201. 1 The abbreviations used are; Rb. retinoblastoma; SSCP, single-strand conformation polymorphism; PCR, polymerase chain reaction; LOH, loss of heterozygosity. and altered gene expression (3). p53 mutations are the most frequently observed genetic lesions in human tumors; they tend to cluster within exons 5 through 9 (3). It has been proposed that p53 function can also be inactivated by mutation in a single alÃ-ele,showing a dominant negative effect (4). Quantitative alterations in p53 protein and mRNA expression have also been reported in a number of human cancers (5, 6). Abnormalities in the Rb gene are also common in many human malignancies. Alterations in Rb DNA structure or messenger RNA and protein expression have been described in small cell lung cancer (7), acute lymphoblastic (8) and chronic myelomonocytic (9) leukemias, testicular germ-cell tumors (10), breast cancers (11), and in tumors of the bladder or kidney (12) and prostate (13), among others. Esophageal carcinoma is one of the most common cancers world wide. Although the incidence of this cancer is relatively low in the United States, it is one of the leading causes of cancer death in some regions of Asia and Africa (14). Esophageal cancer occurs in two major forms: squamous cell carcinoma, which is associated with smoking and ethanol consumption (15); and adenocarcinoma, which occurs in patients with Barrett's esophagus (16). Esophageal cancer is also one of the most lethal cancers, with a 5-year survival rate as low as 5% (14). The molecular pathogenesis of this cancer is just begin ning to be understood. Ras family protooncogene mutations are very rare in esophageal cancers (17, 18); amplification of several protoon- cogenes is, in contrast, very common (19-22). The importance of tumor suppressor gene loci in human esophageal cancer has been demonstrated recently. Loss of heterozygosity involving chromosome 17p (22), the p53 gene locus (23), the Rb gene (24), and the APC/ MCC locus (25) has been reported. Point mutations in p53 were detected in 5 of 14 primary esophageal squamous cell carcinomas as well as in 2 of 4 squamous esophageal cancer cell lines in a recent study (18, 26). p53 mutations were also detected in 4 patients with Barrett's esophagus and in one with esophageal adenocarcinoma (27), as well as in a 25-year-old man with a Barrett's-associated cancer (28). A recent summary also described p53 mutations in esophageal cancers (3); transversions and chain-terminating mutations were very frequent in this group, although they were rare in other types of cancer. A study including tumors from a different geographic area by the same re search group described 5 premature termination or frameshift muta tions among a total of 21 (18). These studies suggested that esoph ageal cancer may have a unique mutational profile. We therefore examined genetic alterations involving lhep53 and Rb genes in a series of 79 human esophageal carcinomas. Abnormalities of p53 and Rb were found in the majority of tumor samples and included point mutations, microdeletions, allelic deletions of DNA, and abnormal size and/or quantity of mRNA. We also observed that allelic deletion of p53 occurs in virtually all cases that have p53 point mutation. Unlike other human tumors, in which p53 mutations are predominantly of the missense type (3), half of the mutations detected in the current study were nonsense mutations that created premature stop codons. Our findings strongly suggest a unique mutational profile in esophageal cancers and involvement of both p53 and Rb in the genesis and/or progression of most of these tumors. 1889 Research. on February 16, 2016. © 1993 American Association for Cancer cancerres.aacrjournals.org Downloaded from