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The ras gene family consists of 3 closely related genes

ras Gene Mutations and Expression of Ras Signal Transduction Mediators in Gastric Adenocarcinomas Jinyoung Yoo, MD, PhD; Sonya Y. Park; Robert A. Robinson, MD, PhD; Seok Jin Kang, MD, PhD; Woong Shick
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ras Gene Mutations and Expression of Ras Signal Transduction Mediators in Gastric Adenocarcinomas Jinyoung Yoo, MD, PhD; Sonya Y. Park; Robert A. Robinson, MD, PhD; Seok Jin Kang, MD, PhD; Woong Shick Ahn, MD, PhD; Chang Suk Kang, MD, PhD Objective. To investigate ras gene alteration in human gastric adenocarcinomas and its potential relationship to ras signal transduction mediators. Design. Genomic DNA from 10 gastric tumors were analyzed by sequencing of polymerase chain reaction amplified products for the presence of ras mutations. All the samples were further investigated with the use of immunohistochemical analysis for ERK1 and ERK2. Setting. Tertiary care teaching hospital. Patients. Seventy patients from a Korean population and 3 from a Midwestern US population composed of white Americans and African Americans. Results. Fifteen tumors (1%) were positive for either H-ras or K-ras mutation: 9 (13%) of 70 Korean patients and 6 (18%) of 3 US patients. Seven (78%) of the 9 mutated tumors from Korean patients and all 6 (100%) from the US patients were intestinal-type lesions. Either ERK1 and/or ERK2 was overexpressed in 68 samples (65%). No association was established between ras mutations and overexpression of ERK1/2. However, the correlation between ERK1/2 and progression (early vs late) was statistically significant (P.007). Conclusions. These data suggest that ras mutations are uncommon in gastric adenocarcinomas and that differing racial and/or geographic mechanisms may not underlie ras gene alteration. Most ras mutations were, however, observed in the group of intestinal-type samples, supporting the different genetic mechanisms of carcinogenesis between the intestinal- and diffuse-type tumors. It is noteworthy that enhanced ERK1/2 activity could be one of the characteristics of tumor invasiveness in gastric cancers. (Arch Pathol Lab Med. 2002;126: ) The ras gene family consists of 3 closely related genes (H-ras, K-ras, and N-ras), which have similar structures and encode for a p21 ras. These p21 ras proteins, known to play an important role in the regulation of normal signal transduction, bind guanosine triphosphate and guanosine diphosphate with high affinity and possess guanosine triphosphatase activity, a process that is necessary to shift the ras proteins to an inactive state. Activation by amino acid substitution occurs as a consequence of mutation nearly exclusively at codons 12, 13, or 61 of each ras and creates a functional reduction of intrinsic guanosine triphosphatase activity of the proteins, leading to increased cell groh. 1,2 The ras gene alterations have been detected in a variety of human malignancies, most frequently in pancreatic adenocarcinomas. 3 Data regarding ras activation in stomach cancers were, however, limited and controversial. The incidence of ras mutations varied among popula- Accepted for publication April 2, From the Departments of Pathology (Drs Yoo, S. J. Kang, and C. S. Kang and Ms Park) and Obstetrics and Gynecology (Dr Ahn), St Vincent s Hospital, Catholic University, Suwon, Kyungkido, South Korea, and Department of Pathology, University of Iowa, Iowa City (Dr Robinson). Presented at the American Association for Cancer Research 93rd Annual Meeting, San Francisco, Calif, April 6 10, Reprints: Seok Jin Kang, MD, PhD, Department of Pathology, St Vincent s Hospital, Catholic University, Suwon, Kyungkido, South Korea ( tions; no mutations were observed in samples studied in China and South Africa, whereas the mutations were detected in 28% of the cases from a US population. 6 Japanese and Italian groups revealed mutation rates of 3% and 9%, respectively. 7 9 Furthermore, high rates of mutated ras genes in gastric adenomas but a low incidence in gastric adenocarcinomas were described. 10 Mitogen-activated protein kinase (MAPK) is a serinethreonine kinase that transfers extracellular signals from the cytoplasm to the nucleus. Extracellular signal-regulated kinases (ERK1 and ERK2), an MAPK subfamily, are crucial components of signaling pathways, and their activation has been shown to induce cell proliferation. 11 The ERK cascade is considered a major downstream target of ras and may be constitutively active in cancer cells with activating ras mutations. To evaluate the frequency of ras gene alterations in gastric adenocarcinomas and to determine whether there were racial or geographic factors relevant to the mutations, we investigated 10 stomach cancer samples obtained from different population groups, using the polymerase chain reaction (PCR) technique with the direct automated sequencing method. In addition, since ras is an important link in extracellular stimuli transduction in the cell and it signals the cell by means of numerous mediators, including MAPKs, further studies were performed by the application of immunohistochemical analysis for ERK1 and ERK2 to explore the biological effects of the mutated ras and its potential relationship with signal transduction mediators and other clinicopathologic parameters Arch Pathol Lab Med Vol 126, September 2002 ras and ERK1/2 in Gastric Cancer Yoo et al MATERIALS AND METHODS One hundred four patients with gastric adenocarcinoma were included in this study. Of these, 70 were Korean patients who were surgically treated at the Catholic University St Vincent s Hospital, Suwon, Kyungkido, South Korea, between 1999 and The remaining 3 samples were obtained from white American and African American patients in the Midwestern United States who underwent resections or biopsies of their tumor at the University of Iowa Hospitals and Clinics. None of the patients had received therapy before surgery. Only those with newly incident, primary lesions were selected. Histologic reviews were performed by one of the authors (J.Y.) to confirm the diagnosis and reevaluate the histologic subtype according to Lauren s classification. The tumors located in the gastroesophageal junction and cardia were determined to be proximal, whereas the tumors present in the body and antrum were considered distal. DNA Extraction Microdissection and DNA extraction were performed from 5 paraffin sections (10 m thick). Using the hematoxylin-eosin stained section as a guide, precisely identified tumor tissue was obtained with care by use of a needle to ensure that more than 75% of the recovered cells were tumor cells as opposed to unremarkable connective tissue elements, necrotic debris, and inflammatory or hemorrhagic cell populations. The procured tissue was suspended in 100 L of lysis buffer (50mM Tris, 1mM EDTA, 0.5% Tween 20, containing proteinase K) and incubated at 55 C overnight. After phenol-chloroform extraction, genomic DNA was precipitated with ethanol. Quantitation was performed by UV absorption. As a control for H-ras mutation, T2 bladder carcinoma cells, knowntohaveh-ras codon 12 mutation, were used. The positive control for K-ras mutation was the human colon carcinoma cell line SW80, which has a known homozygous K-ras mutation at codon 12. Normal tissues were also obtained from surrounding sites of 10 tumor specimens as a negative control for the analysis. By the application of the PCR technique on a PTC-100 thermal cycler (MJ Research, Watertown, Mass), we amplified a sequence spanning 121 base pairs across codons 12 and 13 of the H-ras gene and a sequence spanning 219 base pairs across codon 61 of the H-ras gene by using the primers 5 -CTG-AGG-AGC-GAT- GAC-GGA-ATA-TAA-GC-3 (sense) (Genosys, Woodlands, Tex) and 5 -CTC-TAT-AGT-GGG-GTC-GTA-TTC-GTC-CA-3 (antisense) and 5 -TGA-GCC-CTG-TCC-TCC-TGC-AGG-ATT-C-3 (sense) and 5 -GCC-AGC-CTC-ACG-GGG-TTC-ACC-TGT-A-3 (antisense), respectively. The primers used for K-ras gene analysis were 5 -ATG-ACT-GAA-TAT-AAA-CTT-GTG-GTA-3 (sense) (Genosys) and 5 -AC-CTC-TAT-TGT-TGG-ATC-A-3 (antisense), which flank codons 12/13, and 5 -TTC-CTA-CAG-GAA-GCA- AGT-AG-3 (sense) and 5 -CAC-AAA-GAA-AGC-CCT-CCC-CA- 3 (antisense), which flank codon 61. The PCR reaction mixture (100 L) contained approximately 0.5 g of genomic DNA in 20mM Tris hydrochloride (ph 8.), 50mM potassium chloride, 2.0mM magnesium chloride, 0.2mM each of deoxyribonucleoside triphosphate, 0.2 M of each primer, and 5 U of Platinum Taq DNA polymerase (Life Technologies, Gaithersburg, Md). Templates were initially denatured for 5 minutes at 9 C, followed by 35 cycles of PCR with incubations of 1 minute at 9 C, 1 minute at 55 C, and 1 minute at 72 C. The reaction was incubated at 72 C for 10 minutes on the last cycle. Negative controls without DNA templates were run to exclude the possibility of contamination of reagents. The PCR reactions were checked for the appropriately amplified DNA fragments on an ethidium bromide stained agarose gel using UV light transillumination. DNA Sequencing The PCR product of each sample was purified with a QIAquick PCR purification kit (Qiagen, Valencia, Calif) and then sequenced with a 373A DNA sequencer (Applied Biosystems, Foster City, Calif) using dye-primer conditions recommended by the manufacturer. Both strands were sequenced for each DNA analyzed, and genomic DNA from control samples was sequenced in parallel to confirm the mutations. Immunohistochemical Analysis Immunohistochemical staining was performed by a sensitive peroxidase-streptavidin method with a LASB kit (Dako Co, Ltd, Kyoto, Japan). Monoclonal antibodies against ERK1 and ERK2 (Santa Cruz Biotechnology, Inc, Santa Cruz, Calif) were purchased and applied after antigen retrieval in the citrate buffer. Briefly, tissue sections m thick were deparaffinized in xylene and rehydrated. Endogenous peroxidase was blocked by soaking in 3% hydrogen peroxide at 5 C for minutes. The slides were placed in a Coplin jar containing citrate buffer (2.1 g/l, ph 6.0) and heated to 120 C in an autoclave for 15 minutes to unmask the antigen. They were treated with protein-blocking reagent before the incubation at 5 C for 1 hour with primary antibodies at a 1:100 dilution. After extensive washing, the sections were incubated at 5 C for 10 minutes with biotinylated anti-mouse immunoglobulin antibodies (Dako) at a 1:20 dilution and subsequently with streptavidin-biotin peroxidase complexes at a 1:25 dilution. The staining was visualized by using aminoethylcarbonate as the final chromogen. The nuclei were counterstained with hematoxylin. Cases were considered positive if more than 10% of cells showed distinct cytoplasmic and/or nuclear immunostaining. Statistical Analysis The proportions of samples expressing the mutant ras genes were computed and compared with ERK expression status and selected clinicopathologic parameters, using the Fisher exact test and Pearson correlation coefficient. P.05 was considered statistically significant. RESULTS The first exon of H-ras and K-ras genes was examined in 10 gastric tumors by the application of an automated DNA sequencing of PCR-amplified ras sequences. In a control study using 2 cell lines, each had the expected mutation for H-ras and K-ras genes, respectively. Data from mutation-containing cases are shown in Table 1, along with ERK expression status. Fifteen tumors (1%) were positive for either H-ras or K-ras mutation: 9 (13%) of 70 cases from Korean patients and 6 (18%) of 3 cases from US patients. All 5 H-ras mutations, found in Korean and 1 US patient, were G-to-T transversion mutations in the second base of codon 12 (coding for valine instead of the normal glycine residue) (Figure 1). Of the samples with K-ras activation (5 in each population group), there were GGC-to-AGC transition mutations at codon 13 (replacing glycine by serine) in 7 (5 of which were tumors from US patients), CAA-to-CGA mutations at codon 61 (substituting arginine for glutamine) in 2, and a CAA-to- CTA mutation at codon 61 (substituting leucine for glutamine) in 1 (Figure 2). No mutations were observed at H- ras 13 or 61 or K-ras 12. The tumors we analyzed included 71 cases of the intestinal type and 33 cases of the diffuse type. All but 2 of the identified mutations were in the group of intestinal lesions: 7 (78%) of the 9 mutated tumors from Korean patients and all 6 (100%) from US patients. As to the vertical location of the tumor in the stomach, we found 5 proximal tumors (17%) in 29 patients and 10 distal tumors (13%) in 75 patients harboring the mutation. The ras mutations established no correlation with clinicopathologic characteristics, including the patient s age, sex, tumor location, size, differentiation, subtype, and prognosis. ERK1 was expressed in 66 samples (62%): 5 tumors Arch Pathol Lab Med Vol 126, September 2002 ras and ERK1/2 in Gastric Cancer Yoo et al 1097 Case No. K-7 K-11 K-1 K-21 K- K-51 K-57 K-61 K-73 US-3 US-13 US-25 US-27 US-30 US-36 Tumor Table 1. Depth of Invasion pt pt List of Mutation-Containing Cases in Each Ethnic Group* Subtype Diffuse Diffuse Mutation H-ras K-ras ERK1/ERK2 61/CTA/Leu 61/CGA/Arg 61/CGA/Arg 13/GGC AGC/Gly Ser * K indicates Korean patients; US, American patients;, wild type;, primary tumor involving lamina propria or submucosa;, primary tumor involving muscularis propria or subserosa;, primary tumor penetrating serosa without invasion of adjacent structures; pt, primary tumor invading adjacent structures; plus sign, positive; and minus sign, negative. Heterozygous mutation. Table 2. Penetration ras mutation Negative Positive ERK1/2 Negative Positive Correlation Among the ras Mutation, ERK1/2 Expression, Tumor, Subtype, and Penetration in Gastric Tumors ras Mutation Negative Positive P Value ERK1/2 Negative Positive P Value P Value Subtype Diffuse P Value (6%) from Korean patients and 21 (62%) from US patients (Figure 3, A). Overexpression of ERK2 was detected in 2 cases (0%): 36 (51%) from a Korean population and 6 (18%) from a US population (Figure 3, B). The frequency of either ERK1 and/or ERK2 abnormality was 65% (68/ 10): 73% (11/15) with and 6% (57/89) without ras mutations (Table 2). No association was found between the ras mutations and ERK1/2 immunoreactivity (P.9). However, the correlation between ERK1/2 expression and progression (early vs late) was statistically significant (P.007); 23 (51%) of 5 early lesions and 5 (76%) of 59 advanced cancers were positive for ERK1/2. COMMENT Although recent molecular studies have revealed various genetic changes in stomach cancer, the essential genetic features are yet to be determined. Occasional small series of gastric cancers have described the relatively low incidence of ras gene alterations. However, the studies are limited and there exists the possibility of ethnic variability in the overall mutation rates. The activated ras genes have rarely been detected in Asian patients,,7,8 although gastric carcinomas are known to occur more frequently in these populations. In contrast, the incidence of mutated ras genes in non-asian patients from the United States, in which carcinogens and etiologic factors differ from those in Asia, was reported to be as high as 28%. 6 In this study, all the specimens were subjected to the same analytic procedures in one laboratory under consistent conditions using a direct DNA sequencing of PCRamplified ras sequences. We found mutations in the ras gene in 15 (1%) of 10 patients. It may be that the activation of ras genes by point mutations is not directly involved in the development of gastric cancer but it does not exclude the involvement of ras by other mechanisms. Additional studies of ras gene alterations along with other related oncogens and mitogens should lead to a more detailed understanding of the tumorigenesis in gastric tumors. There were no major differences in the frequency of ras gene alterations between Korean and US patients, suggesting that geographic or racial factors may not play a role in the induction of these tumors. It will be of interest to investigate the frequencies of ras gene activation in sam Arch Pathol Lab Med Vol 126, September 2002 ras and ERK1/2 in Gastric Cancer Yoo et al Figure 1. Demonstration of H-ras mutation in the second base of codon 12 (GGC GTC) of the sense strand, as indicated by a C-to-A transversion in the antisense strand (case K-11). Figure 2. Demonstration of heterozygous mutation in the first position of codon 13 (GGC AGC) of the sense strand of K-ras, as indicated by the presence of both C and T in the antisense strand of sequence (case US-30). ples collected from multiple ethnic groups, including emigrants from high-risk to low-risk areas and vice versa. The discrepancies in the ras mutation rates among studies may be due to definable factors such as the nature of the specimens, differences in the analytic methods used, the members of the ras gene family and their codons investigated, and the histologic subtypes (intestinal vs diffuse), location (proximal vs distal), and progression (early vs late) of the tumors included. It has been suggested that the mechanism of carcinogenesis in stomach cancer might be different between intestinal- and diffuse-type lesions and between proximal and distal tumors. 5,7,16,17,19 -type tumors, which grow mostly in glandular formation, are accompanied and presumably preceded by intestinal metaplasia and dysplasia, whereas diffuse-type tumors, which show individual cell groh with loose cell-to-cell adhesion, are not preceded by precancerous lesions and may originate directly from undifferentiated cells in the germinative zones of gastric glands. Recently, the -catenin gene, essential for cell-to-cell adhesion, has been reported to mutate only in intestinal-type gastric cancers, 20 suggesting that -catenin gene alterations may be associated with unstable adhesion molecules during the progression of intestinal-type tumors. Miki et al 21 detected K-ras mutations in the intestinal-type lesions only. In the present study, of the 15 samples containing a mutated ras gene, all but 2 (87%) were of intestinal-type carcinoma. Along with other observations of dissimilar patterns of increased p21 ras expression in each type of tumor, 22 the results support the notion of different mechanisms in the tumorigenesis of intestinal and diffuse types of gastric carcinomas. With regard to the vertical location of tumor, Lee et al 19 identified a significantly higher frequency of K-ras mutations in proximally located tumors (37.5% in the upperthird lesions vs 13.8% in the middle and 3% in the lower parts of the stomach). We, however, failed to demonstrate the particular association of ras mutations with tumor location (17% in proximal lesions vs 13% in distal tumors), which is in accordance with findings described by Nanus et al. 17 Therefore, the possibility of tumor location related activated ras genes in stomach cancers cannot be considered at the present time. ERKs are downstream factors of the ras-erk cascade, and they are now considered to be key molecules in signaling processes. 11,23 26 As shown in our study and a study by Kiyokawa et al, 27 overexpression of ERK was frequent in gastric cancers. ERK1/2 expression was detected in 68 (65%) of our samples, 57 (8%) of which, however, harbored no ras mutation, suggesting that ERK activation does not indicate the presence of ras mutations and other non-ras pathways may exist. A recent investigation on MAPK activity in advanced colon cancers demonstrated similar results. No mutated ras was found in 75% of the samples with MAPK activation. 28 It is thus possible that there may be other crucial mechanisms controlled by the specific up-regulators and down-regulators of ERKs in tumors, either ras dependent or ras independent. Despite a rapidly expanding literature on the biochem- Figure 3. Immunohistochemical analysis of ERK1 (A) and ERK2 (B). Strong expression is present in the cytoplasm and nucleus of most tumor cells (case K-73) (original magnification 200). Arch Pathol Lab Med Vol 126, September 2002 ras and ERK1/2 in Gastric Cancer Yoo et al 1099 ical and physiologic properties of ERKs, few data were available regarding their relation to clini
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