Molecular dissection of the chromosome band 7q21 amplicon in gastroesophageal junction adenocarcinomas identifies cyclin-dependent kinase 6 at both genomic and protein expression levels.

Amplification of chromosome band 7q21 has been frequently detected in various types of cancer including gastroesophageal junction (GEJ) adenocarcinomas. At present, no gene has been disclosed that can explain this frequent amplification of 7q21 in GEJ carcinomas. Therefore, a detailed genomic analysis of the 7q21 region was performed on a selected series of GEJ adenocarcinomas, i.e., 14 primary adenocarcinomas and 10 cell lines, by array comparative genomic hybridization (aCGH) with a 7q11.22-q31.2 contig array. A distinct peak of amplification was identified at 92.1 Mb in 7q21.2, precisely comprising cyclin-dependent kinase 6 (CDK6), a gene involved in cell cycle regulation. A smaller peak was seen at 116.2 Mb in 7q31.2, the locus of the MET proto-oncogene. No distinct peak was detected for the hepatocyte growth factor (HGF) at 81.3 Mb in 7q21.11. An immunoprofile of HGF, CDK6 and MET revealed a strong correlation between aCGH and immunohistochemical protein expression for CDK6 (P = 0.002). Furthermore, immunohistochemistry did not show expression of CDK6 in Barrett's dysplasia and carcinoma in situ, correlating expression of CDK6 with a malignant phenotype. We conclude that high-resolution genomic analysis and immunoprofiling identify CDK6 as the main candidate target for the recurrent amplification of 7q21 in GEJ adenocarcinomas.

High-resolution array comparative genomic hybridization of chromosome 8q: evaluation of putative progression markers for gastroesophageal junction adenocarcinomas.

Amplification of 8q is frequently found in gastroesophageal junction (GEJ) cancer. It is usually detected in high-grade, high-stage GEJ adenocarcinomas. Moreover, it has been implicated in tumor progression in other cancer types. In this study, a detailed genomic analysis of 8q was performed on a series of GEJ adenocarcinomas, including 22 primary adenocarcinomas, 13 cell lines and two xenografts, by array comparative genomic hybridization (aCGH) with a whole chromosome 8q contig array. Of the 37 specimens, 21 originated from the esophagus and 16 were derived from the gastric cardia. Commonly overrepresented regions were identified at distal 8q, i.e. 124-125 Mb (8q24.13), at 127-128 Mb (8q24.21), and at 141-142 Mb (8q24.3). From these regions six genes were selected with putative relevance to cancer: ANXA13, MTSS1, FAM84B (alias NSE2), MYC, C8orf17 (alias MOST-1) and PTK2 (alias FAK). In addition, the gene EXT1 was selected since it was found in a specific amplification in cell line SK-GT-5. Quantitative RT-PCR analysis of these seven genes was subsequently performed on a panel of 24 gastroesophageal samples, including 13 cell lines, two xenografts and nine normal stomach controls. Significant overexpression was found for MYC and EXT1 in GEJ adenocarcinoma cell lines and xenografts compared to normal controls. Expression of the genes MTSS1, FAM84B and C8orf17 was found to be significantly decreased in this set of cell lines and xenografts. We conclude that, firstly, there are other genes than MYC involved in the 8q amplification in GEJ cancer. Secondly, the differential expression of these genes contributes to unravel the biology of GEJ adenocarcinomas.

Evaluation of oncogene amplification in intact and truncated cell nuclei of gastro-esophageal cancer cell lines by DNA in situ hybridisation.

Adenocarcinoma arising around the gastro-esophageal junction (GEJ) is a highly malignant form of cancer. Its incidence is rising sharply. The study of oncogenes in these carcinomas may give information concerning treatment and prognosis. In the present study, the fluorescence in situ hybridisation (FISH) technique was optimised for genetic characterisation of oncogenes in archival cancer specimens. Three cell lines derived from GEJ adenocarcinomas were investigated, i.e. JROECL 19, JROECL 33 and OACM5.1C, both in fresh and paraffin-embedded preparations. Furthermore, paraffin-embedded material of three xenografts was studied, i.e. JROECL 19, JROECL 33, and OACM4.1X. We focussed on the oncogenes MYC and HER2/neu, since they are frequently involved in intestinal cancers. Firstly, our results indicate that it is feasible to detect oncogene-specific probes with the FISH technique in formalin-fixed, paraffin-embedded material. Secondly, it appeared that the optimal section thickness for analysis was 2 microm. This thickness resulted in minimal nuclear overlap, which facilitates counting of FISH spots. Due to the truncation phenomenon, however, the sensitivity of the technique is less than FISH on intact nuclei. Importantly, (high level) oncogene amplifications were easily recognised in 2 microm thick sections. Finally, counting of the individual copy number of the MYC and HER2/neu oncogenes was feasible enabling an arbitrary assessment of low- and high-level amplification. In conclusion, FISH is an accurate technique for detecting amplification of oncogenes in paraffin-embedded patient material.

Genomic alterations in malignant transformation of Barrett's esophagus.

The incidence of adenocarcinoma in Barrett's esophagus has been increasing rapidly over the past decades. Neoplastic progression is characterized by three well-defined premalignant stages: metaplasia, low-grade dysplasia, and high-grade dysplasia. A genome-wide overview, based on comparative genomic hybridization, was performed, evaluating 30 Barrett's adenocarcinomas and 25 adjacent precursors, i.e., 6 metaplasias, 9 low-grade dysplasias, and 10 high-grade dysplasias. The frequency of losses and gains significantly increased in the subsequent stages of malignant transformation. Losses of 5q21-q23, 9p21, 17p12-13.1, 18q21, and Y were revealed in low-grade dysplasias. This was followed by loss of 7q33-q35 and gains of 7p12-p15, 7q21-q22, and 17q21 in high-grade dysplasias along with high-level amplification (HLA) of 7q21 and 17q21. In the invasive cancers, additional losses of 3p14-p21, 4p, 4q, 8p21, 13q14-q31, 14q24.3-q31, 16q21-q22, and 22q as well as gains of 3q25-q27, 8q23-24.1, 12p11.2-12, 15q22-q24, and 20q11.2-q13.1 were distinguished along with HLAs of 8p12-p22 and 20q11.2-q13.1. Approximately one-third of the alterations in the dysplasias were also found in the adjacent adenocarcinomas, illustrating that multiple clonal lineages can be present in Barrett's esophagus. Novel findings include loss on 7q, gain on 12p, and the observation of several HLAs in high-grade dysplasias. Furthermore, loss of 7q33-q35 was found to represent a significant distinction between low-grade and high-grade dysplasia (P = 0.01), whereas loss of 16q21-q22 and gain of 20q11.2-q13.1 were disclosed to significantly discriminate between high-grade dysplasia and adenocarcinoma (P = 0.02 and P = 0.03, respectively). This inventory of genetic aberrations increases our understanding of malignant transformation in Barrett's esophagus and might provide useful biomarkers for disease progression.

Involvement of cancer-activating genes on chromosomes 7 and 8 in esophageal (Barrett's) and gastric cardia adenocarcinoma.

UNLABELLED: The incidence of adenocarcinomas of the distal esophagus (Barrett's esophagus) and proximal stomach (gastric cardia) has increased rapidly over the past decades. In contrast to this dramatic increase, genetic knowledge is sparse. MATERIALS AND METHODS: We investigated genomic amplification on chromosomes 7 and 8 by comparative genomic hybridization (CGH) and protein expression of relevant oncogenes (EGFR, HGF, MET, CTSB, MYC) by immunohistochemistry (IHC) in 22 esophageal and 22 gastric cardia carcinomas. RESULTS: The CGH and IHC patterns were very similar for the two cancer locations. IHC showed positive immunostaining in 93% of the adenocarcinomas for at least one of the investigated genes, whereas CGH disclosed genomic gains on chromosome 7 and/or 8 in 80%. CONCLUSION: Cancer-activating genes on chromosomes 7 and 8 are frequently involved in gastro-esophageal junction adenocarcinomas. Moreover, the similarities in chromosomal changes and protein expression patterns strongly suggest that esophageal and gastric cardia adenocarcinomas have a shared etiology. This is in agreement with studies addressing gastroesophageal reflux disease and intestinal metaplasia at these locations.

Expression of the neurofibromatosis type 2 gene in human tissues.

The neurofibromatosis Type 2 tumor suppressor gene is implicated in the hereditary tumor syndrome NF2, hallmarked by bilateral vestibular schwannomas, meningiomas, and ocular non-neoplastic features. The gene product has characteristics of a membrane cytoskeleton-linking protein but the mechanism of tumor suppression by the NF2 protein remains to be elucidated. The NF2 gene is widely expressed in mouse and rat tissues. In humans, most of the expression data have accumulated through Northern blot analysis, RT-PCR and, more recently, Western blot analysis, providing information on whole tissues and organs rather than on specific cell types. We report here an extensive survey of NF2 gene expression in human tissues using a combination of mRNA in situ hybridization (mRNA ISH) and immunohistochemistry (IH) with a panel of monoclonal antibodies (MAbs) supplemented by tissue immunoprecipitation experiments with affinity-purified polyclonal antibodies. Expression was observed in many different cell types, most of which appear functionally normal in individuals affected by NF2. Surprisingly, expression could not be consistently documented in Schwann cells and arachnoidal cells by IH or by mRNA ISH in formalin-fixed tissue. However, consistent immunostaining of Schwann cells was seen in frozen sections. (J Histochem Cytochem 47:1471-1479, 1999)

Effect of bone decalcification procedures on DNA in situ hybridization and comparative genomic hybridization. EDTA is highly preferable to a routinely used acid decalcifier.

Decalcification is routinely performed for histological studies of bone-containing tissue. Although DNA in situ hybridization (ISH) and comparative genomic hybridization (CGH) have been successfully employed on archival material, little has been reported on the use of these techniques on archival decalcified bony material. In this study we compared the effects of two commonly used decalcifiers, i.e. , one proprietary, acid-based agent (RDO) and one chelating agent (EDTA), in relation to subsequent DNA ISH and CGH to bony tissues (two normal vertebrae, six prostate tumor bone metastases with one sample decalcified by both EDTA and RDO). We found that RDO-decalcified tissue was not suited for DNA ISH in tissue sections with centromere-specific probes, whereas we were able to adequately determine the chromosomal status of EDTA-decalcified material of both control and tumor material. Gel electrophoresis revealed that no DNA could be successfully retrieved from RDO-treated material. Moreover, in contrast to RDO-decalcified tumor material, we detected several chromosomal imbalances in the EDTA-decalcified tumor tissue by CGH analysis. Furthermore, it was possible to determine the DNA ploidy status of EDTA- but not of RDO-decalcified material by DNA flow cytometry. Decalcification of bony samples by EDTA is highly recommended for application in DNA ISH and CGH techniques.

Loss of heterozygosity on chromosome 9 and loss of chromosome 9 copy number are separate events in the pathogenesis of transitional cell carcinoma of the bladder.

The most frequent genetic aberration found in transitional cell carcinoma (TCC) of the bladder involves chromosome 9. Loss of heterozygosity (LOH) analyses show deletions of both chromosome 9p and 9q, while in situ hybridization studies suggest a significant percentage of tumours with monosomy 9. To investigate the types of chromosome 9 losses that occur in bladder cancer, we have studied 40 tumours with different techniques such as in situ hybridization (ISH), flow cytometry and LOH analysis. LOH for one or more markers was found in 43% of the tumours. This percentage does not differ from previous reports. With ISH, complete monosomy for chromosome 9 was observed in only 1 of the 40 tumours. Four other tumours had monosomic subpopulations, representing 23-40% of the cells. In 18 cases, an underrepresentation of the chromosome 9 centromere relative to chromosome 6 or to the ploidy of the tumour was observed, including the cases with monosomy. In 5 of these 18 cases, the relative loss could not be confirmed by LOH. In addition, when LOH and a relative underrepresentation were observed in the same tumour, the extent of LOH as measured by the intensity of allele loss, was often not related to the extent of underrepresentation. We therefore conclude that complete monosomy of chromosome 9 is rare in TCCs of the bladder and that a relative loss of centromere signal may not be related to a loss compatible with inactivation of a tumour suppressor gene. LOH was found in TCCs of all stages and grades. Our results suggest that loss of tumour suppressor genes on chromosome 9 is an early event in the pathogenesis of bladder cancer.

Cytogenetic analysis of Barrett's mucosa and adenocarcinoma of the distal esophagus and cardia.

We performed flow cytometry and cytogenetic analysis of 37 adenocarcinomas of the distal esophagus and cardia, of which 22 arose in Barrett's mucosa. Two of eight analyzed specimens of Barrett's mucosa had clonal chromosomal abnormalities. In 19 cases clonal chromosomal abnormalities were found in tumor tissue. The complex pattern of cytogenetic changes did not differ among the adenocarcinomas arisen in Barrett's esophagus, and those in the distal esophagus without Barrett's mucosa or cardia. Abnormal karyotypes with multiple and complex rearrangements were seen in 11 cases and with single or a few numeric changes in eight. Losses of chromosomes 4, 18, 21, and Y were the most frequent numeric changes. Loss of the Y chromosome was observed in eight of 26 tumors of males (31%). Gains of chromosomes 14 and 20 were also frequent numeric changes. Structural abnormalities were observed in 13 of the abnormal karyotypes (68%). The chromosome arms most frequently rearranged were 1p, 3q, 11p and 22p. The chromosome arm most frequently contributing to losses was 1p, with the shortest region of overlap being 1p22-33. The chromosome arms most often involved in gains were 11p and 22p, and i(3q) was the isochromosome that was most frequently identified.

Interphase in situ hybridization to disaggregated and intact tissue specimens of prostatic adenocarcinomas.

A comparative study was performed of interphase in situ hybridization (ISH) to deparaffinized 4-microns tissue sections and nuclear suspensions from eight prostatic adenocarcinomas, as well as one normal prostatic control. Whole nuclear suspensions were derived from the same tumor areas to evaluate differences of ISH to truncated versus whole nuclei. DNA probes specific for the centromeres of chromosome 1, 7, 8, 10, and Y were used for detection of numerical chromosomal changes and aneuploidy. In six adenocarcinomas chromosome aberrations (+7, +8, -8, -10, -Y) were seen. However, ISH to sections revealed focal aberrations (-10, -Y) in four cases that could not be distinguished in the suspensions. Chromosomal alterations occurring in larger tumor areas were also detected in the nuclear suspensions. Chromosome copy number changes, especially gains, were better discriminated in the nuclear suspensions. The rate of ISH aneuploidy seen in nuclear suspensions corresponded with that observed in the tissue sections (P < 0.01). Ploidy patterns as assessed by ISH to sections and nuclear suspensions were in concordance with DNA flow cytometry (both P < 0.001). We conclude that both section and suspension ISH were able to accurately detect aneuploidy and numerical chromosomal aberrations occurring in larger histological areas. However, section ISH was also capable of revealing (small) focal cytogenetic abnormalities, due to a precise analysis of only target cells. Focal abnormalities were not detected by suspension ISH, probably due to an admixture of non-aberrant tumor cells and stromal elements.

Interphase cytogenetics of prostatic adenocarcinoma and precursor lesions: analysis of 25 radical prostatectomies and 17 adjacent prostatic intraepithelial neoplasias.

Twenty-five radical prostatectomy specimens were screened for the presence of numerical chromosome changes within the adenocarcinoma as well as 17 adjacent prostatic intraepithelial neoplasias (PIN) by means of interphase in situ hybridization (ISH) to routinely processed tissue sections. To this end a defined alfoid repetitive DNA probe set was used, specific for the centromeres of chromosomes 1, 7, 8, 10, 15, and Y. The cytogenetic information was correlated with histopathological and clinical features as well as with DNA ploidy. Numerical aberrations of at least one chromosome were shown in 13 of 25 cases (52%). Alterations of chromosome 8 and loss of the Y chromosome were the most frequent findings (both 20%), followed by loss of chromosomes 15 (16%) and 10 (12%). Gain of chromosome 7 was seen in 8% of cases. No aberrations of chromosomes 7, 8, 10, and 15 were found in the adjacent PIN lesions, whereas loss of the Y chromosome in both PIN and tumor occurred in two cases. Also, (low level) aneuploidy was observed in 76% of these PIN lesions. Ploidy of the carcinomas as assessed by ISH correlated well with ploidy measured by DNA flow cytometry (FCM; P < 0.02). Due to the more specific correspondence between ISH and tumor pathology, pathologic grade correlated with ISH aneuploidy (P < 0.05), whereas FCM ploidy did not. Furthermore, genetic heterogeneity within a tumor was seen, as judged by the focal appearance of chromosomal aberrations. Chromosomal alterations occurred in all grades and stages, although loss of chromosome 10, gain of chromosome 7, and aberrations of chromosome 8 tended to predominate in more advanced cancers.