KRAS Mutations Predict Progression of Preneoplastic Gastric Lesions 1 Changning Gong, Robertino Mera, Juan C. Bravo, Bernardo Ruiz, Rafael Diaz-Escamilla, Elizabeth T. H. Fontham, Pelayo Correa, and Jay D. Hunt 2 Departments of Biochemistry and Molecular Biology [C. G., J. D. H.], Pathology [R. M., J. C. B., B. R., R. D. E., E. T. H. F., P. C.], and Public Health and Preventive Medicine [E. T. H. F.], and the Stanley S. Scott Cancer Center [R. M., E. T. H. F., P. C., J. D. H.], Louisiana State University Medical Center, New Orleans, Louisiana 70112 Abstract Eight hundred sixty-three subjects with atrophic gastritis were recruited to participate in an ongoing chemoprevention trial in Narin ˜ o, Colombia. The participants were randomly assigned to intervention therapies, which included treatment to eradicate Helicobacter pylori infection followed by daily dietary supplementation with antioxidant micronutrients in a 2  2  2 factorial design. A series of biopsies of gastric mucosa were obtained according to a specified protocol from designated locations in the stomach for each participant at baseline (before intervention therapy) and at year three. A systematic sample of 160 participants was selected from each of the eight treatment combinations. DNA was isolated from each of these biopsies (n  320), and the first exon of KRAS was amplified using PCR. Mutations in the KRAS gene were detected using denaturing gradient gel electrophoresis and confirmed by sequence analysis. Of all baseline biopsies, 14.4% (23 of 160) contained KRAS mutations. Among those participants with atrophic gastritis without metaplasia, 19.4% (6 of 25) contained KRAS mutations, indicating that mutation of this important gene is likely an early event in the etiology of gastric carcinoma. An important association was found between the presence of KRAS mutations in baseline biopsies and the progression of preneoplastic lesions. Only 14.6% (20 of 137) of participants without baseline KRAS mutations progressed from atrophic gastritis to intestinal metaplasia or from small intestinal metaplasia to colonic metaplasia; however, 39.1% (9 of 23) with baseline KRAS mutations progressed to a more advanced lesion after 3 years [univariate odds ratio (OR), 3.76 (P  0.05); multivariate OR adjusted for treatment, 3.74 (P  0.04)]. In addition, the specificity of the KRAS mutation predicted progression. For those participants with G3 T transversions at position 1 of codon 12 (GGT3 TGT), 19.4% (5 of 17) progressed (univariate OR, 2.4); however, 60.0% (3 of 5) of participants with G3 A transitions at position 1 of codon 12 (GGT3 AGT) progressed (univariate OR, 8.7; P  0.004 using  2 test). Introduction Gastric cancer is one of the most frequent cancers in the world (1). The 5-year survival rate in the United States is only 21% for all histological stages (2). According to the Lauren classifica- tion (3), there are two major types of gastric carcinomas: intestinal and diffuse. The most frequent gastric malignancy is the intestinal type, which is often preceded by sequential steps of precancerous changes (Fig. 1), including atrophic gastritis, intestinal metaplasia (type I, complete, or small intestinal meta- plasia; and type III, incomplete, or colonic metaplasia), and dysplasia (reviewed in Ref. 4). These progressive stages, which usually proceed over decades, have been defined as a sequence of histopathological events that confer an increasing risk of malignant transformation. The conversion of normal epithelial cells to cancer cells requires the accumulation of multiple genetic abnormalities. Interestingly, the two types of gastric carcinoma have some common genetic components but differ in some important aberrations. Abnormal expression and ampli- fication of the MET gene, inactivation of the p53 tumor sup- pressor gene, abnormal transcription of CD44, and loss of telomeres are common events in both types (5–7). Reduction or loss of cadherins and catenins and KSAM gene amplification are unique to the diffuse type of gastric cancer (8). KRAS muta- tions, ERBB2 gene amplification, LOH 3 and mutations of the APC gene, LOH of BCL2 gene, and LOH of the DCC locus are preferentially associated with the intestinal type (8). However, because few studies have been done on preneoplastic lesions, the significance of genetic events in gastric carcinogenesis remains unclear. Mutations of KRAS are detected in many types of human malignancies and are associated with the development and progression of human cancer (9). The KRAS gene encodes a M r 21,000 membrane-associated protein (p21 RAS ) with intrinsic GTPase activity involved in cellular signal transduction. Point mutations of KRAS at specific codons lead to activated onco- protein (GTP-RAS) with reduced GTPase activity (9). KRAS codons 12, 13, and 61 are the most frequently detected mutation “hot spots” in human cancers. The frequency of mutated KRAS varies greatly among different tumor types. KRAS mutations are found in 10% of intestinal type gastric carcinomas but are rarely detected in the diffuse type (8). Very few studies have been done on premalignant gastric mucosa, and the significance Received 6/25/98; revised 11/13/98; accepted 11/17/98. 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 This study was supported in part by Program Project Grant P01-CA28842 from the National Cancer Institute. 2 To whom requests for reprints should be addressed, at Louisiana State Univer- sity Medical Center, Department of Biochemistry and Molecular Biology, 1901 Perdido Street, P7-2, New Orleans, LA 70112. Phone: (504) 568-4734; Fax: (504) 599-1014; E-mail: jhunt@lsumc.edu. 3 The abbreviations used are: LOH, loss of heterozygosity; DGGE, denaturing gradient gel electrophoresis; OR, odds ratio. 167 Vol. 8, 167–171, February 1999 Cancer Epidemiology, Biomarkers & Prevention on July 10, 2021. © 1999 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from