Abstract and Introduction
Abstract
To identify oncogene amplification involved in ovarian carcinogenesis, we studied 21 ovarian carcinomas and 5 serous borderline tumors using conventional comparative genomic hybridization (CGH) and CGH to a genomic DNA microarray. Immunohistochemical analysis of the proteins encoded by the genes that were amplified frequently (FGF3/4, FGFR1, CCNE1, PAK1, JUNB, and MDM2) was performed on a tissue microarray comprising 254 cases of ovarian neoplasms. Regarding histologic type, characteristic patterns of copy number changes were revealed. They correlated with histologic tumor type and with intratumoral heterogeneity. Gain of FGF3/4 and CCNE1 was found in all serous carcinomas. Endometrioid carcinomas most frequently showed gain of JUNB (83%), KRAS2 (67%), MYCN (50%), ESR (50%), and CCND2 (50%). Of the serous borderline tumors, 80% harbored amplification of FGFR1 and MDM2 and a 75% gain of PIK3CA. Only CCNE1 immunoreactivity was significantly correlated with CGH results (P < .05) and postoperative survival (P < .05). Microarray-based genomic analysis in combination with immunohistochemical analysis was found to be a powerful technique for identification of clinically relevant gene amplification in human ovarian cancer.
Introduction
DNA amplification is an important mechanism that allows cancer cells to increase expression of critical genes such as oncogenes involved in growth regulation and genes responsible for drug resistance. Therefore, the detection of gene amplification in tumors may be of diagnostic, prognostic, or therapeutic relevance for patient disease management.
Fixation of tumor tissues in formalin for routine diagnostics results in extensive cross-linking of nuclear proteins, formation of tight complexes between proteins and DNA, and fragmentation of DNA. It therefore is not surprising that it is almost impossible to extract intact DNA from fixed nuclei with compact chromatin. This is the reason that study of archival material was largely restricted to techniques such as fluorescence in situ hybridization and polymerase chain reaction, which can be performed with fragmented DNA. However, by these techniques, only one or a few genes can be analyzed in each experiment. Comparative genomic hybridization (CGH) has been used widely to screen for multiple chromosomal aberrations, including amplification and deletion, in various cancers.
Owing to the limited mapping resolution of CGH, genetic alterations comprising regions less than 20 megabases often may be overlooked. Therefore, array-based CGH was developed for more effective targeting of specific oncogenes. Most previous studies using CGH-based microarrays examined DNA extracted from cell lines and fresh frozen material. Daigo et al and Callagy et al found that CGH array works on formalin-fixed material by analyzing a few samples of archival breast carcinoma tissue by use of laser microdissection and degenerate oligonucleotide primed-polymerase chain reaction, supporting their general results of DNA analysis of 18 cancer cell lines.
In the present study, conventional CGH and a commercial CGH microarray analysis system were applied to a comprehensive series of formalin-fixed, paraffin-embedded samples of ovarian neoplasms. The microarray contained 59 target clones (P1, PAC, and BAC clones) representing 57 oncogenes that have been reported to be amplified in various tumors. To identify common oncogenes involved in ovarian carcinogenesis, 21 ovarian carcinomas and 5 serous ovarian borderline tumors were studied. We provide evidence that the microarray technique is suitable for this type of routinely fixed tissue material.
In addition, we studied the protein expression of the most frequently amplified genes by immunohistochemical analysis in these 21 tumors and in a large series of archived ovarian tumors. The aim was to identify genes and/or corresponding proteins in ovarian carcinomas that could be markers for diagnosis, targets of therapy, and a measure of prognosis.