Correlation between genomic alterations assessed by array comparative genomic hybridization, prognostically informative histologic subtype, stage, and patient survival in gastric cancer

It is difficult to evaluate the prognostic value of histologic criteria in gastric cancer because of the high variability of morphologic patterns. Recently, histologic subtypes of low, intermediate, or high malignant potential have been identified, providing the basis for a prognostically informative grading system. Because array comparative genomic hybridization systems allow systematic analysis of chromosome alterations, which may be prognostically and pathogenetically informative, we applied high-resolution genome-wide array comparative genomic hybridization to archival material from 81 gastric cancer cases followed for a median of 150 months after surgery. The DNA extracted from paraffin sections gave useful results in 49 tumors, 18 of which were of low-grade, 24 of intermediate, and 7 of high-grade histotypes. Based on the number of chromosome aberrations and the presence/absence of amplifications, 3 tumor clusters of increasing genomic lesion severity were constructed, which proved to correlate significantly with histologic grade and stage as well as with patient survival. Further investigation documented the lower number and severity of genomic alterations in tumors with microsatellite DNA instability and high CD8-rich lymphoid response; the close association of 8p23.1 amplification with cardial cancer; the frequent amplification of genes involved in cell renewal (CDC6, HER2, GRB7, IGFBP4) at 17q12-q21.1, with close histochemical correlation with HER2 membranous expression; and more sporadic amplification of chromosome regions harboring important oncogenes like MYC, KRAS, NRAS, CRKL, CCNE1, or ZNF217. We conclude that genome-wide array comparative genomic hybridization of gastric cancer contributes prognostically relevant information providing a genetic background for histologic grading.     

Chromosomal imbalances in gastric cancer. Correlation with histologic subtypes and tumor progression

DNA copy number changes were analyzed by comparative genomic hybridization (CGH) in 38 gastric carcinomas and correlated with tumor histologic type and progression. Gains of copy numbers were observed in all tumors, affecting all chromosomes except chromosome 16. The average number of copy number gains was 7 (range, 1-13), most frequently located on chromosomes 11, 12, 15, 17, and 20 in 45% to 97% of tumors. High-level amplifications were found on chromosomes 12, 15, 17, and 20; the latter was affected most frequently (66%). Loss of DNA copy numbers was detected in 14 tumors affecting 7 chromosomes. No statistically significant differences in the frequency and pattern of chromosomal imbalances were observed in tumor histologic type (Lauren classification) and grade of differentiation, as well as the prognostic parameters depth of invasion (pT) and lymph node involvement (pN). Our results indicate that in gastric cancer there is no specific recurrent pattern of DNA aberrations to be correlated with tumor histologic type or stage. However, CGH analysis could reveal new, recurrent genetic changes in gastric cancer affecting chromosomes sites that harbor genes known to participate in tumorigenesis and progression of several human malignant neoplasms.     

Genetic alterations of gastric cancer: comparative genomic hybridization and fluorescence In situ hybridization studies

Genetic changes leading to the development of gastric cancers are still in dispute. In the following study, we used comparative genomic hybridization (CGH) to screen for DNA copy number changes along all chromosomes in 37 gastric carcinomas, and fluorescence in situ hybridization (FISH) with the C-MYC and TP53 probes in 14 cases for comparison. The aim of this study was to identify those chromosome regions that contain genes important for the development of gastric carcinomas and to identify genetic markers associated with tumor progression. The most often involved gains were 2q, 7pq, 8pq, 13q, 17q, 18q, and 20pq. The most commonly deleted regions were 17p. The pattern of genetic changes was different depending on the existence of nodal metastasis and histologic types. Gains in 8q and losses in 17p were the most common features of the CGH changes. However, only 3 among the available 10 cases (30%) showed an amplification of the C-MYC gene by FISH. Allelic loss of TP53 was found in 2 of 4 cases (50%). This difference might be due to another rearrangement of these 2 genes which cannot be detected by FISH, or other possible genes in that area may be involved in the tumorigenesis and nodal metastasis of gastric carcinomas.     

DNA copy number aberrations in intestinal-type gastric cancer revealed by array-based comparative genomic hybridization

Genomic instability can be divided into 2 categories: chromosomal instability (CIN) and microsatellite instability (MSI). CIN has been linked to aneuploidy and chromosomal aberrations, and high-level loss of heterozygosity (LOH-H) has been suggested to be an indicator of CIN. High-level MSI (MSI-H), which results from nonfunctional mismatch repair, has previously been suggested to be mutually exclusive with CIN. Four MSI-H and three LOH-H primary gastric tumors of intestinal histology were used for copy number analysis by array-based comparative genomic hybridization (aCGH) with 13,000 cDNA targets. The MSI-H group showed fewer gains (0-12, average 4.5) and losses (0-10, average 2.5) per tumor as compared to the LOH-H group (9-15 gains, average 11.6 and 1-6 losses, average 4). Two MSI-H tumors did not show any copy number changes and one showed only gains of whole chromosomes. The most common alterations were gains of 20q (5/7 samples), 1q, 8, and 10p (3/7 samples) and losses of 1p and 5p (3/7 samples). The minimal amplified regions in 1q and 20q were localized to 1q21.1 approximately q21.2, 1q21.3, 20q11.2, 20q13.12, and 20q13.3 approximately qter. No copy number change was found to be specific for MSI-H or LOH-H. The results suggest that the LOH-H phenotype revealed by microsatellite analysis predicts reliably copy number abnormalities on aCGH and that a subset of MSI-H and all LOH-H tumors share the CIN phenotype.     

Genetic differences in endocrine pancreatic tumor subtypes detected by comparative genomic hybridization

The molecular pathogenesis as well as histogenesis of endocrine pancreatic tumors (EPTs) is not well understood, and the clinical behavior of EPTs is difficult to predict using current morphological criteria. Thus, more accurate markers of risk and better understanding of tumor initiation and progression are needed to allow a precise classification of EPTs. We have studied 44 benign and malignant EPTs by comparative genomic hybridization to correlate the overall number of genetic alterations with clinical and histopathological parameters and to identify chromosomal regions which might harbor genes involved in EPT pathogenesis and progression. Aberrations were found in 36 EPTs, and chromosomal losses (mean, 5.3) were slightly more frequent than gains (mean, 4. 6). The most frequent losses involved Y (45% of male EPTs), 6q (39%), 11q (36%), 3p, 3q, 11p (each 30%), 6p (27%), and 10q and Xq (each 25%), whereas most common gains included 7q (43%), 17q (41%), 5q and 14q (each 32%), 7p, 9q, 17p, 20q (each 27%), and 12q and Xp (each 25%). A correlation was found between the total number of genetic changes per tumor and both tumor size and disease stage. In particular, losses of 3p and 6 and gains of 14q and Xq were found to be associated with metastatic disease. Furthermore, characteristic patterns of genetic changes were found in the various EPT subtypes, eg, 6q loss in malignant insulinomas, indicating that these groups might evolve along genetically different pathways. The highlighted genetic aberrations, including the newly found involvement of 6q losses and sex chromosome alterations, should stimulate the further analysis of these chromosomal regions, which may lead to the discovery of novel genes important in the tumorigenesis and evolution of EPTs.     

Analysis by comparative genomic hybridization of gastric cancer with peritoneal dissemination and/or positive peritoneal cytology

Peritoneal metastasis is an important prognostic factor in cases of gastric cancer. Although studies on comparative genomic hybridization (CGH) in gastric cancer have been reported, there are few reports on the peritoneal metastasis (P) and peritoneal cytology (CY) factors in this cancer. In this study, we analyzed the chromosomal changes in the primary tumor with a combination of laser microdissection analysis and CGH in an attempt to detect the unknown abnormal chromosomal regions. We analyzed 34 primary tumors, including 13 primary tumors with peritoneal metastasis (P1) and/or positive peritoneal cytology (CY1) using a combination of laser microdissection and CGH. The minimal overlapping regions in gains were assigned to 5p14 (46.2%), 7q21.3 (61.5%), 7q31 (46.2%), 7q36 (46.2%), 8q23 (53.8%), 15q26 (46.2%), 20q12 (61.5%), 20q13.1 (53.8%), and 20q13.2 (53.8%) in primary tumors with P1 and/or CY1. The minimal regions of losses that occurred most frequently were 4q34-q35 (23.1%) and 22q11.2 (23.1%). There were significant differences in the minimal regions of 5p14 (P=0.033), 7q21.3 (P < 0.0001), 7q31 (P=0.013), 7q36 (P=0.033), and 22q11.2 (P=0.048) between primary tumors with and without P1 and/or CY1. In this study, gain/amplification of 5p14, 7q21.3, 7q31, and 7q36, and loss of 22q11.2 were significant in gastric cancer cases with peritoneal dissemination and/or positive peritoneal cytology.     

三种比较基因组杂交技术及其在肿瘤研究中的应用

比较基因组杂交技术作为一种新型细胞一分子遗传学技术,已成为近年广泛应用的重要技术,其主要应用于实体瘤领域的研究中.高分辨比较基因组杂交在传统的比较基因组杂交基础上提高了分辨率,具有耗费低、分辨率较高、诊断效率高等特点而具有较好的临床应用前景.而微阵列比较基因组杂交不仅使分辨率提高,甚至可以确定肿瘤相关基因并提供精确的定位,该技术在肿瘤的临床研究及临床应用上具有重要意义.本文则对上述方法及其在肿瘤研究中的应用作一综述.     

乳腺癌中比较基因组杂交研究的进展

近年来,随着比较基因组杂交（comparative genomic hybridization,CGH）技术在研究肿瘤相关染色体异常方面的应用,已发现乳腺癌发生、发展不同时期存在非随机性染色体基因组DNA拷贝数改变,而不同类型的肿瘤在基因组异常方面又存在很大的异质性;利用芯片-CGH技术的高分辨率还能鉴别肿瘤的不同亚型。[第一段]     

Intratumoral genomic heterogeneity in advanced head and neck cancer detected by comparative genomic hybridization

Little is known about the extent of intratumoral genetic heterogeneity in head and neck squamous cell carcinoma (HNSCC). We therefore examined 79 stage III and IV primary HNSCCs (P) and matched lymph node metastases (M) for over- and underrepresentation of specific chromosome regions by comparative genomic hybridization (CGH). The overall ratio of gains and losses was higher in metastases than in primary tumors (4/1 vs. 2.5/1). Gains of 3q (78.1% P vs. 87.5% M) and 11q (78.1% P vs. 62.5% M) and deletions of 3p (43.8% P vs. 34.4% M) and 9p (31.3% P vs. 15.6% M) were most frequently detected. The highest rate of intratumoral discordance was observed for primary tumors and corresponding metastases (32.8%) compared with matched pairs of two metastases (26.5%) and of two anatomically distinct sides of one primary tumor (24.3%). Furthermore, the discordance rate was dependent on the primary tumor site (oral cavity 49.2%, oropharynx 31%, hypopharynx 30.3%, and larynx 27.3%). In some tumors, the extent of genomic discordance argues against a monoclonal origin. In conclusion, we found a high individual variation of intratumoral genomic heterogeneity depending on the localization and selection of matched pairs. These findings are of specific importance in view of establishing prognostic markers.     

Array comparative genomic hybridization analysis revealed four genomic prognostic biomarkers for primary gastric cancers

Unlike the case with some other solid tumors, whole genome array screening has not revealed prognostic genetic aberrations in primary gastric cancer. Comparative genomic hybridization (CGH) using bacterial artificial chromosome (BAC) arrays for 56 primary gastric cancers resulted in identification of four prognostic loci in this study: 6q21 (harboring FOXO3A; previously FKHRL1), 9q32 (UGCG), 17q21.1∼q21.2 (CASC3), and 17q21.32 (HOXB3 through HOXB9). If any one of these four loci was deleted, the prognosis of the patient was significantly worse (P = 0.0019). This was true even for advanced tumors (stage IIIA, IIB, or IV, n = 39) (P = 0.0113), whereas only 1 of the 17 patients with less advanced tumors (stage IA, IB, or II; n = 17) died of disease after surgery. Multivariate analysis according to the status of four BACs or pathological stage based on the Japanese Classification of Gastric Carcinoma (stages IA, IB, and II vs. stages IIIA, IIIB, and IV) demonstrated that the BAC clone status was also an independent prognostic factor (P = 0.006). These findings may help predict which patients with malignant potential need more intensive therapy, or may point to new therapeutic approaches especially for advanced tumors. The parameter here termed the integrated genomic prognostic biomarker may therefore be of clinical utility as a prognostic biomarker.     

Correlation of comparative genomic hybridization results of 100 archival uveal melanomas with patient survival

Comparative genomic hybridization (CGH) was used to elucidate DNA sequence copy number imbalances in 100 archival formalin-fixed, paraffin-embedded (FFPE) uveal melanoma cases. Of these 100 cases, 51 were from patients who survived >or=9 years post diagnosis without evidence of metastasis; the remaining 49 patients died from metastatic disease. Viable probe was generated from 82 of the 100 cases, allowing correlation of CGH findings with survival for all but 18 cases. Copy number imbalances revealed by CGH were tested for univariate prognostic significance. The most powerful predictor of a poor prognosis was gain of 18q11.2, which was subsequently compared with other significant chromosomal regions, as well as histologic and clinical factors, in a multivariate analysis. There was also evidence of differential X chromosome involvement in the survival correlations between male and female cases, which may be of significance to prognosis. This large-scale CGH analysis of archival material is intended to direct further gene-specific study of malignancy in uveal melanoma.     

比较基因组杂交应用于结直肠癌检测中的质量控制

比较基因组杂交（comparative genomic hybridization,CGH）是在染色体荧光原位杂交（fluorescence in situ hybridization,FISH）基础上发展起来的一种新的分子细胞遗传学技术,与其他肿瘤细胞遗传学方法相比,主要的优点是无需制备肿瘤细胞的中期分裂象,对肿瘤标本没有限制,只要能得到少量基因组DNA的标本如新鲜或冻存的或石蜡包埋的癌组织、癌细胞系均可用来分析,且只经一次实验便可得到整个基因组的扩增和丢失的情况。与FISH相比,它克服了需制备染色体特异区域的探针且仅能检测个别染色体或部分位点的局限性,它是一种全方位检测法,一次实验即可检测出待测标本整个基因组DNA拷贝数增减。     

Genetic alterations in primary gastric carcinomas correlated with clinicopathological variables by array comparative genomic hybridization

Genetic alterations have been recognized as an important event in the carcinogenesis of gastric cancer (GC). We conducted high resolution bacterial artificial chromosome array-comparative genomic hybridization, to elucidate in more detail the genomic alterations, and to establish a pattern of DNA copy number changes with distinct clinical variables in GC. Our results showed some correlations between novel amplified or deleted regions and clinical status. Copy-number gains were frequently detected at 1p, 5p, 7q, 8q, 11p, 16p, 20p and 20q, and losses at 1p, 2q, 4q, 5q, 7q, 9p, 14q, and 18q. Losses at 4q23, 9p23, 14q31.1, or 18q21.1 as well as a gain at 20q12 were correlated with tumor-node-metastasis tumor stage. Losses at 9p23 or 14q31.1 were associated with lymph node status. Metastasis was determined to be related to losses at 4q23 or 4q28.2, as well as losses at 4q15.2, 4q21.21, 4q 28.2, or 14q31.1, with differentiation. One of the notable aspects of this study was that the losses at 4q or 14q could be employed in the evaluation of the metastatic status of GC. Our results should provide a potential resource for the molecular cytogenetic events in GC, and should also provide clues in the hunt for genes associated with GC.     

Chromosomal aberrations in prostate cancer xenografts detected by comparative genomic hybridization

A major problem in studying prostate cancer has been the lack of model systems because of the difficulties in growing prostate cancer cells in vitro. Recently, however, several human prostate cancer xenografts, grown in immune-deficient mice, have been established. Here, we characterized 13 such xenografts (LuCaP 23.8, 23.12, 35, 41, 49, 58, 69, 70, 73, LAPC-4AD, LAPC-4AI, LAPC-9AD, and LAPC-9AI) as well as one prostate cancer cell line (22Rv1) derived from a xenograft for chromosomal alterations by comparative genomic hybridization and a modification of multicolor fluorescence in situ hybridization. On average, the xenografts contained 13 (range 5-28) aberrations, 5 (1-13) gains, and 8 (1-15) losses, per case. The chromosome arms that most often contained losses were 2q, 5q, 6q, 8p, 13q, and 18q, and gains were 7q, 8q, and Xq. The same regions were previously shown to be often altered in advanced prostate carcinomas in patients. The androgen-dependent and corresponding androgen-independent sublines of LAPC-4 and LAPC-9 shared all genetic alterations, suggesting that the transition of the growth from androgen dependency to independence does not involve major chromosomal aberrations in these two models. In conclusion, the identified genetic aberrations lay the groundwork for further detailed genetic analyses of these xenografts.     

Gains, losses, and amplifications of genomic materials in primary gastric cancers analyzed by comparative genomic hybridization

By means of comparative genomic hybridization (CGH), we screened 58 primary gastric cancers for changes in copy number of DNA sequences. We detected frequent losses on Ip32-33 (21%), 3p21-23 (22%), 5q14-22 (36%), 6q16 (26%), 9p21-24 (22%), 16q (21%), 17p13 (48%), 18q11-21(33%), and 19(40%). Gains were most often noted at I p36 (22%), 8p22-23 (24%), 8q23-24 (29%), 11q12-13 (24%), 16p(21%), 20p (38%), 20q (45%), Xp21-22(38%), and Xq21-23 (43%), with high-level amplifications at 6p21(2%),7q31(10%), 8p22-23(5%), 8q23-24 (7%), 11q13(4%), 12p12-13(4%), 17q21(2%), 19q12-13(2%), and 20q13(2%). High-level amplification at 8p22-23 has never been reported in any other cancer type and its frequency was as high as that reported for the MYC, MET, and KRAS genes. We narrowed down the smallest common amplicon to 8p23.1 by reverse-painting FISH to prophase chromosomes. Southern blot analysis using one EST marker (D38736) clearly demonstrated that amplification of this exon-like sequence had occurred in all three tumors in which amplifications at 8p22-23 had been detected by CGH. Our data provide evidence for several, previously undescribed, genomic aberrations that are characteristic of gastric cancers.     

Recurrent chromosome changes in 62 primary gastric carcinomas detected by comparative genomic hybridization

Comparative genomic hybridization (CGH) has been applied to detect recurrent chromosome alterations in 62 primary gastric carcinomas. Several nonrandom chromosomal changes, including gains of 8q (31 cases, 50%), 20q (29 cases, 47%) with a minimum gain region at 20q11. 2-q12, 13q (21 cases, 34%) with a minimum gain region at 13q22, and 3q (19 cases, 31%) were commonly observed. The regions most frequently lost included: 19p (23 cases, 37%), 17p (21 cases, 33%), and 1p (14 cases, 23%). High copy number gain (DNA sequence amplification) was detected in 6 cases. Amplification of 8q23-q24.2 and 20q11.2-q12 were observed in 3 cases. Gain of 20q and loss of 19p were confirmed by fluorescence in situ hybridization using corresponding bacterial artificial chromosomes (BAC) clones from those regions. The gain and loss of chromosomal regions identified in this study provide candidate regions involved in gastric tumorigenesis.     

比较基因组杂交技术的应用

肿瘤发生和发育畸形与染色体的不平衡有关,包括染色体的扩增和缺失.常规的比较基因组杂交技术(conventional comparative genomic hybridization)由于能在一次试验中掌握整个基因组DNA拷贝数的变化并可以将此改变准确的定位于染色体,因而在探讨肿瘤的发生和生长发育畸形机理的研究方面得到了广泛的应用.在此基础上发展起来的微阵列比较基因组杂交技术(microarray CGH/ array-based CGH),为精确、定量地研究人类基因组微缺失和微扩增及其定位提供了有力工具.本文综述了CGH和CGHa 技术的原理及应用.     

Mapping of chromosomal gains and losses in prostate cancer by comparative genomic hybridization

Comparative genomic hybridization (CGH) allows detection of chromosomal imbalances in whole genomes in a comprehensive manner. With this approach, ten cases of prostate cancer (seven primary tumors and three metastases) were analyzed. Frequent chromosomal gains detected by CGH involved chromosome arms 7q, 8q, 9q, and 16p, and chromosomes 20 and 22, as well as frequent losses of chromosome arms 16q and 18q, in at least three of the ten cases. Overrepresentation of chromosome arm 9q has not been described in published reports. The CGH data were compared with results of a loss of heterozygosity (LOH) study, in which complete allelotyping was performed in the same prostate tumors with 74 different polymorphic markers. In general, a high concordance between the CGH and LOH results was observed (92%). Tumors revealing discrepancies by CGH and LOH analysis were investigated further by interphase cytogenetics, and the resulting picture regarding the genomic alterations is discussed in detail.     

Challenges in array comparative genomic hybridization for the analysis of cancer samples

PurposeTo address some of the challenges facing the incorporation of array comparative genomic hybridization technology as a clinical tool, including archived tumor tissue, tumor heterogeneity, DNA quality and quantity, and array comparative genomic hybridization platform selection and performance.MethodsExperiments were designed to assess the impact of DNA source (e.g., archival material), quantity, and amplification on array comparative genomic hybridization results. Two microdissection methods were used to isolate tumor cells to minimize heterogeneity. These data and other data sets were used in a further performance comparison of two commonly used array comparative genomic hybridization platforms: bacterial artificial chromosome (Roswell Park Cancer Institute) and oligonucleotide (Agilent Technologies, Santa Clara, CA).ResultsArray comparative genomic hybridization data from as few as 100 formalin-fixed, paraffin-embedded cells isolated by laser capture microdissection and amplified were remarkably similar to array comparative genomic hybridization copy number alterations detected in the bulk (unamplified) population. Manual microdissection from frozen sections provided a rapid and inexpensive means to isolate tumor from adjacent DNA for amplification and array comparative genomic hybridization. Whole genome amplification introduced no appreciable allele bias on array comparative genomic hybridization. The array comparative genomic hybridization results provided by the bacterial artificial chromosome and Agilent platforms were concordant in general, but bacterial artificial chromosome array comparative genomic hybridization showed far fewer outliers and overall less technical noise, which could adversely affect the statistical interpretation of the data.ConclusionsThis study demonstrates that copy number alterations can be robustly and reproducibly detected by array comparative genomic hybridization in DNA isolated from challenging tumor types and sources, including archival materials, low DNA yield, and heterogeneous tissues. Furthermore, bacterial artificial chromosome array comparative genomic hybridization offers the advantage over the Agilent oligonucleotide platform of presenting fewer outliers, which could affect data interpretation.     

Detection of genetic alterations in advanced prostate cancer by comparative genomic hybridization

In this study, we examined nine cases of advanced Japanese prostate cancer by comparative genomic hybridization (CGH) to detect chromosomal imbalances across the entire genome and to identify several new regions likely to contain genes important to the development and progression of this disease. These cases had been previously examined for numerical chromosomal aberrations by fluorescence in situ hybridization (FISH). By CGH, the following regions were found to be over-represented (gains), with fluorescence ratio values higher than the threshold: 4p, 6p, 8q, 11q, 12q, 15q, 16p, 17q, 20, and 21 (>4 cases); underrepresentation (losses) involved: 1q, 4q, 5q, 6q, 13q, 14q, and 22 (>4 cases). The shortest regions of overlap (SRO) of gains were noted at 8q24.1 through q24.3, 12q23, and 17q23 through q24 (>5 cases). The SRO of losses were seen at 5q14 through q21, 6q16.1 through q21, 13q21.3 through q22, and 14q21 (>5 cases). Notably, the gain of chromosomes 8 and 12 by CGH was in agreement with the FISH data, suggesting that the gain of chromosomes 8 and 12 may play an important role in prostate carcinogenesis. The genes on the SRO regions were also discussed in relation to oncogenes and bone metastases.