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Home , Polysomy, Trisomy 8

ANTICANCER RESEARCH 24: 155-160 (2004)

Numerical Aberrations of 8 in Gastric Cancer Detected by Fluorescence In Situ Hybridization

ANNA D. PANANI, ANGELIKI D. FERTI, ALEXANDROS AVGERINOS and SATIRIOS A. RAPTIS

The Second Department of Internal Medicine Propaedeutic, Research Unit, Athens University, Evangelismos Hospital, Athens, Greece

Abstract. Background: Limited data are available on the X, 8, 9 and 19, del (7q), i(8q), as well as complex genetic events underlying gastric cancer. Studying a few cases abnormalities involving 1, 3, 6, 7, 11, 13 and by conventional cytogenetic techniques, we previously reported 17 (2-7). However the detection of recurrent genetic that might be frequently involved. The aim of changes by conventional cytogenetic studies is particularly our study was to evaluate the numerical aberrations of problematic because of the complex nature of the chromosome 8 in gastric cancer using fluorescence in situ chromosomal aberrations and the difficulty in preparing hybridization (FISH). Materials and Methods: FISH, with an metaphase spread of adequate quality and quantity. a-satellite DNA probe specific for chromosome 8, was applied Studying a few cases by conventional staining techniques, we to 37 primary gastric tumors directly processed for cytogenetic found that numerical aberrations of chromosome 8 are study. Results: Numerical aberrations of chromosome 8 were frequent in gastric cancer (3). Furthermore, we described in observed in 23 out of 37 tumors (62.16%). Trisomy was one gastric cancer case with very long survival an i(8q) as detected in 16 cases (43.24%), in 4 cases (10.81%) the sole anomaly (5). Xiao et al. (4) have also observed and in 3 cases (8.10%). No correlation was found in a case with minimal chromosomal changes, between 8 and the histopathologic characteristics of suggesting that this abnormality might be a non-random the tumors. Conclusion: An increase in the number of event in gastric tumorigenesis. chromosome 8 may frequently occur in gastric cancer. Molecular cytogenetic techniques have proven valuable Advanced and more aggressive gastric tumors did not harbor in the study of primary solid tumors. By these techniques a polysomy 8. Further studies at molecular and clinical level must complex pattern of gains and losses of many chromosomes be carried out to identify the alterations reflected by was observed in gastric cancer. Thus DNA copy number polysomy 8 and possibly to facilitate the detection of specific gains were reported to be frequent on 20q, 17q, 8q and 1p, tumors subtypes. whereas loss of 4q, 5q, 3p, 18q and 17p genetic material seemed to be common (8-13). Gastric cancer is of major importance world-wide. Statistical The fluorescence in situ hybridization (FISH) technique data on mortality rates identified that gastric with centromeric specific DNA probes allows the rapid represents the second most common cause of cancer-related detection of numerical aberrations in interphase nuclei of death in the world (1). Unfortunately there is not clear tumor cells. FISH studies have shown numerical aberrations agreement on the genetic changes present in this type of 7, 9, 17 and Y to be common in gastric cancer. However, a malignancy. Conventional cytogenetic studies of gastric few studies only using FISH techniques were focused on cancer have shown simple abnormalities, such as polysomy chromosome 8 (14-17). Genetic changes involved in gastric tumorigenesis or progression and their clinical significance for different subtypes of gastric cancer have not been clearly defined. Correspondence to: Anna D. Panani, Second Department of This might partly be due to the fact that most of the studies Internal Medicine Propaedeutic, Research Unit, Evangelismos focused on the analysis of selected or chromosomal Hospital, Athens University, 45 Ipsilandou St, Athens 106 76, regions, while studies simultaneously comparing various Greece. Tel: +30210 7259307, Fax: +30210 7259307, e-mail: clinicopathological parameters and genetic alterations [email protected] remain limited (11-13, 18). Key Words: Fluorescence in situ hybridization, chromosome 8 The aim of this study was to evaluate, by FISH technique, aberrations, gastric cancer, interphase . the numerical aberrations of chromosome 8 in gastric

0250-7005/2004 $2.00+.40 155 ANTICANCER RESEARCH 24: 155-160 (2004)

Table I. Histopathological characteristics of gastric adenocarcinomas (n=37).

Histopathological characteristics Total number of patients (%)

Histological type (WHO) Well-differentiated 1 (2.70%) Moderate-differentiated 19 (51.35%) Poorly-differentiated 14 (37.83%) Mucinus 3 (8.1%) Histological type (Lauren’s classification) Intestinal 25 (67.56%) Diffuse 12 (32.43%) Lymphatic vessel invasion Negative 25 (67.56%) Positive 12 (32.43%) Venous invasion Negative 21 (56.75%) Positive 16 (43.24%) Stages of tumor invasion Stage I 8 (21.62%) Stage II 10 (27.02%) Stage III 15 (40.54%) Stage IV 4 (10.81%)

cancer. Correlation of these abnormalities with certain histopathological characteristics, representing prognostic parameters in this type of malignancy, was also estimated.

Materials and Methods

Thirty-seven patients with gastric cancer, who underwent gastrectomy, were included in this study. None of the patients had ever received or radiation prior to surgery. Tissue specimens were collected from fresh surgically resected tumors and a routine histopathological examination followed. The tumors were classified histopathologically according to the World Health Organization guidelines (19) and Lauren (20). A small portion of each resected tumor was directly processed for cytogenetic study as described elsewhere (8). FISH was applied on recently made slides from the methanol/acetic acid-fixed cells of all patients. An a-satellite DNA probe D8Z2 specific for chromosome 8 (chromosome region 8p11.1-q11.1) was used. The slide was washed in 2xSaline Sodium Citrate (SSC) buffer and dehydrated in a sequence of 70%, 80% and 95% ethanol. Both slide and hybridization solution containing the chromosome 8 a-satellite DNA probe were prewarmed at 37ÆC for 5 minutes. Next, 10 Ìl of the hybridization Figure 1. Copy number of chromosome 8 (red spots) detected by FISH in mixture was added to the slide under a glass cover-slip. Denaturation gastric cancer cells from different cases. was performed at 75ÆC on a heating plate for 2 minutes. In situ hybridization was carried out for 1 hour at 37ÆC in a humidified lightproof chamber. Post hybridization washings were done and the nuclei were counterstained with DAPI antifade. The hybridization of the probe with the cellular DNA site was visualized by fluorescence lymphocyte nuclei were used as a control. Approximately 97% of microscopy NICON E 600 with a triple filter DAPI/FITC/TEXAS control lymphocyte nuclei showed two signals for probe specific for RED. Positive chromosome signals appeared as red spots in the chromosome 8. A case was counted as aberrant if more than 10% of nuclei. A minimum of 200 cells from each slide were evaluated for cell nuclei showed loss or gain of signals for chromosome 8. each case. Signals were scored using the criteria of Hopman et al. For statistical evaluation the Chi-square test and Kruskal-Wallis (21). To avoid misinterpretation due to technical error, normal test were used.

156 Panani et al: Chromosome 8 in Gastric Cancer

Table II. Copy number of chromosome 8 and histopathological genetic alterations by conventional cytogenetics was parameters in gastric cancer cases (n=37). hampered by the complexity of the chromosomal abnormalities and the difficulty in preparing adequate Histopathological Disomy Monosomy polysomy parameters n (%) n (%) n (%) metaphase spreads. Molecular cytogenetic techniques have been valuable in solving some of these problems. Differentiation Abnormalities of chromosome 8 are a frequent finding in Well 1 (100%) gastric cancer. Whether they result in loss or excess of Moderately 6 (31.57%) 1 (5.26%) 12 (63.15%) chromosome 8 material is still in dispute (4, 10, 12, 22, 23). Poorly 6 (42.85%) 1 (7.14%) 7 (50%) Mucinus 1 (33.33%) 1 (33.33%) 1 (33.33%) In the present study we evaluated the numerical Histological type aberrations of chromosome 8 in a total of 37 gastric Intestinal 10 (40%) 1 (4%) 14 (56%) adenocarcinomas. We used the FISH technique, which is Diffuse 4 (33.33%) 2 (16.66%) 6 (50%) considered a valuable method for the detection of numerical Lymphatic vessel chromosomal aberrations. In most specimens (54.05%) an Invasion Negative 9 (36%) 3 (12%) 13 (52%) increase in the copy number of chromosome 8 was detected, Positive 5 (41.66%) - 7 (58.33%) while loss of this chromosome was found in only 8.10% of Venous invasion the tumors. Han et al. (15), by FISH studies of paraffin- Negative 8 (38.09%) 3 (14.28%) 10 (47.61%) embedded tumor cells of 18 gastric adenocarcinomas, Positive 6 (37.5%) - 10 (62.5%) detected polysomy 8 in 5 cases (27.8%) and monosomy in Stages of tumor invasion Stage I 2 (25%) 1 (12.5%) 5 (62.5%) one case (5.5%). In another FISH study (16), with Stage II 2 (20%) 2 (20%) 6 (60%) centromeric probes specific for chromosomes 7, 8, 11, 17 Stage III 8 (53.33%) - 7 (46.66%) and Y on deparaffinized tissue sections from 40 gastric Stage IV 2 (50%) - 2 (50%) tumors, polysomy 8 was found in 62.5% of the cases. Also, comparative genomic hybridization (CGH) studies have shown gains on 8q material in approximately 18-55% of the cases studied (11, 13, 18). Results Numerical abnormalities involving chromosome 8, in which c- is located, have been suggested as a mechanism Histopathological characteristics of resected gastric tumors accounting for the increase of c-myc copy number. are shown in Table I. A representative example of FISH Amplification leading to activation of c-myc have been analysis is shown in Figure 1. Numerical aberrations of observed in various solid tumors including gastric cancer chromosome 8 were observed in 23 out of 37 tumors (24-27). However, other investigators did not find (62.16%). The vast majority of these aberrations involved concordance of c-myc overexpression with amplification in an increase in the number of chromosome 8. Trisomy 8 was gastric cancer (28). The possibility that numerical detected in 16 cases (43.24%) and tetrasomy in 4 cases aberrations of chromosome 8 might reflect alterations of (10.81%). One copy of chromosome 8 was observed only in other genes implicated in genesis and progression of gastric 3 cases (8.10%). Table II shows the numerical aberrations cancer could not be excluded. Our study has not focused on of chromosome 8 in association with the histopathological the c-myc alterations reflected by polysomy 8. characteristics of resected gastric tumors. Statistical analysis Aberrations in certain chromosome copy numbers may be using the Chi-square test showed that there was no related to tumor aggressiveness and many studies have association of chromosome 8 copy number with the investigated the relationship between numerical chromosomal histological type according to WHO or Lauren’s abnormalities and clinicopathological parameters of various classification and the lymphatic vessel or venous invasion cancers (29, 30). As far as we know there are no previous p>0.1. Also Kruskal-Wallis statistical test did not show any reports on the association between chromosome 8 numerical correlation between chromosome 8 copy number and the aberrations detected by FISH and the clinicopathological stage of tumor invasion, p=0.14. characteristics of gastric tumors. However a few studies by CGH have been reported on the association between excess Discussion on 8q material and the clinicopathological characteristics of gastric cancers with different results. Koo et al. (12) reported Cytogenetic studies focused on the evaluation of recurrent amplifications on the 8q material predominantly in lymphnode chromosomal aberrations in tumor cells are of great metastatic of diffuse type gastric adenocarcinomas. Also Wu et importance, providing entry points for molecular studies to al. (11) by CGH studies showed gains on 8q to be higher in identify genes involved in the pathogenesis of human advanced gastric cancer of intestinal type. On the other hand, cancer. However, in solid tumors, the detection of recurrent Wu et al. (13), in a series of 62 gastric adenocarcinomas

157 ANTICANCER RESEARCH 24: 155-160 (2004) studied by CGH, did not find any correlation between excess 7 Panani AD, Ferti A, Malliaros S and Raptis S: Cytogenetic on 8q material and the clinicopathological characteristics of study of 11 gastric adenocarcinomas. Cancer Genet Cytogenet the disease. 81: 169-172, 1995. Although the number of cases we studied was not so 8 Stamouli MI, Ferti AD, Panani AD, Raftakis J, Consoli C, Raptis SA and Young BD: Application of multiplex fluorescence large, a correlation of chromosome 8 numerical aberrations in situ hybridization in the cytogenetic analysis of primary gastric with certain histopathological characteristics representing carcinoma. Cancer Genet Cytogenet 135: 23-27, 2002. prognostic factors in gastric cancer was evaluated. Our 9 Kokkola A, Monni O, Puolakkainen P, Larramendy ML, results did not reveal any association of chromosome 8 copy Victorzon M, Nording S, Haapiainen R, Kivilaakso E and number with the histological type, the tumor aggressiveness Knuutila S: 17q 12-21 amplification, a novel recurrent genetic and the tumor invasion. Thus, despite the notion that change in intestinal type of gastric carcinoma. Genes tumors accumulate more genetic abnormalities as they Chromosomes Cancer 20: 38-43, 1997. 10 Sakakura C, Mori T, Sakabe T, Ariyama Y, Shinomiya T, Date progress, in the present study advanced gastric cancer did K, Hagiwara A, Yamaguchi T, Takayashi T, Nakamura Y, Abe not harbor polysomy 8. Our findings are in agreement with T and Inazawa J: Gains, losses and amplifications of genomic those of Wu et al. (13) regarding correlation between excess materials in primary gastric cancers analyzed by comparative on 8q material by CGH studies and the clinicopathological genomic hybridization. Genes Chromosomes Cancer 24: 299- characteristics of gastric tumors. 305, 1999. Gastric cancer is a genetically heterogeneous disease 11 Wu MS, Chang MC, Huang SP, Tseng CC, Sheu JC, Lin YM, proceeding via different pathways of tumorigenesis and/or Shun CT, Lin MT and Lin JT: Correlation of histologic subtypes and replication error phenotype with comparative progression, but the possible genetic causes of this genomic hybridization in gastric cancer. Genes Chromosomes heterogeneity have not been thoroughly investigated. Cancer 30: 80-86, 2001. Stocks et al. (18), by CGH studies of junctional and distal 12 Koo SH, Kwon KC, Shin SY, Jeon YM, Park JW, Kim SH and gastric tumors, showed that DNA aberrations may Noh SM: Genetic alterations of gastric cancer: Comparative distinguish distinct tumor subtypes among histologically genomic hybridization and fluorescence in situ hybridization identical tumors. studies. Cancer Genet Cytogenet 117: 97-103, 2000. It is clear from all the above that further studies at 13 Wu CW, Chen GD, Faun CSJ, Lee AFY, Chi CW, Liu JM, Weier U and Chen JY: Clinical implications of chromosomal molecular and clinical level must be carried out to identify abnormalities in gastric adenocarcinomas. Genes Chromosomes the gene alterations reflected by polysomy 8 and to Cancer 35: 219-231, 2002. contribute to the classification of this disease. 14 Dekken HV, Pizzolo JG, Kelsen DP and Melaled MR: Targeted cytogenetic analysis of gastric tumors by in situ Acknowledgements hybridization with a set of chromosome-specific DNA probes. Cancer 66: 491-497, 1990. The technical assistance of Mrs France Stamatelli is gratefully 15 Han K, Oh EJ, Kim SY, Kim YG, Lee YK, Kang CS, Kim BK, aknowledged. Kim WL, Shim SI and Kim SM: Chromosomal numerical aberrations in gastric carcinoma: analysis of 18 cases using in References situ hybridization. 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Carey FA and Kernohan NM: Chromosomal imbalances in Cancer Genet Cytogenet 24: 63-73, 1987. gastric and esophageal adenocarcinoma: specific comparative 4 Xiao S, Geng JS, Feng XL, Liu QZ and Li P: Cytogenetic genomic hybridization detected abnormalities segregate with studies of eight primary gastric cancers. Cancer Genet junctional adenocarcinomas. Genes Chromosome Cancer 32: Cytogenet 58: 79-84, 1992. 50-58, 2001. 5 Panani AD, Ferti A, Malliaros S and Raptis S: Gastric cancer 19 Oota K and Sobin LH: Histological typing of gastric and with an i(8q) and long survival. Cancer Genet Cytogenet 58: esophageal tumors. In: International Histological Classification 214-215, 1992. of Tumors. No 18, WHO, Genova, 1997. 6 Seruca R, Castedo S, Correia C, Gomes P, Carneiro F, Soares 20 Lauren P: The two histological main types of gastric carcinoma: P, De Jong B and Sobrinho-Simoes M: Cytogenetic findings in diffuse and so-called intestinal type carcinoma. An attempt at eleven gastric . 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21 Hopman AHN, Ramaekers FCS, Raap AK, Beck JLM, Devilee 27 Takahashi Y, Shintaku K, Ishii Y, Asai S, Ishikawa K and Fujii P, Ploeg M and Vooijs GP: In situ hybridization as a tool to M: Analysis of MYC and chromosome 8 copy number changes study numerical aberrations in solid bladder tumors. in gastrointestinal cancer by dual-color fluorescence in situ Histochemistry 89: 307-316, 1988. hybridization. Cancer Genet Cytogenet 107: 61-64, 1998. 22 Castedo S, Correia C, David L and Sobrinbo-Simoes M: 28 Nakata B, Onoda N, Chung YS et al: Correlation between 8q. A recurrent change in gastric carcinoma. malignancy of gastric cancer and c-myc DNA amplification or Cancer Genet Cytogenet 54: 137-38, 1991. overexpression of c-myc protein. Gan To Kayaku Ryoho 22: 23 Baffa R, Santoro R, Bullrich F, Mandes B, Ishii H and Eroce 176-179, 1995. CM: Definition and refinement of chromosome 8p regions of 29 Wada Y, Igawa M, Shiina H, Shigeno K, Yokogi H, Urakami loss of heterozygosity in gastric cancer. Clin Cancer Res 6: S, Yoneda I and Maruyma R: Comparison of chromosomal 1372-77, 2000. aberrations detected by fluorescence in situ hybridization with 24 Watson PH, Safneck JR, Le K, Dubik D and Shiu RP: clinical parameters, DNA and Ki 67 expression in renal Relationship of c-myc amplification to progression of breast cell carcinoma. Br J Cancer 7: 2003-2007, 1998. cancer from in situ to invasive tumor and lymph node 30 Tsukamoto F, Miyoshi Y, Egawa C, Kasugai T, Inazawa J and metastasis. J Natl Cancer Inst 85: 902-907, 1993. Nogushi S: Clinopathologic study of breast carcinoma with 25 Suzuki S, Tenjin T, Watanabe H, Matsushima S, Shibuya T and chromosomal aneusomy detected by fluorescence in situ Tanaka S: Low level c-myc gene amplification in gastric cancer hybridization. Cancer 93: 165-170, 2001. detected by dual-color fluorescence in situ hybridization analysis. J Surg Oncol 66: 173-178, 1997. 26 Kozma L, Kiss I, Hajdu J, Szentkereszty Z, Szakall S and Ember I: C-myc amplification and cluster analysis in human Received August 29, 2003 gastric carcinoma. Anticancer Res 21: 707-710, 2001. Accepted October 20, 2003

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