Analysis of gene amplification and prognostic markers in ovarian cancer using comparative genomic hybridization for microarrays and immunohistochemical analysis for tissue microarrays.

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.

Microgenomics: Identification of new expression profiles via small and single-cell sample analyses.

BACKGROUND: Since the sequencing of the human genome has been finished, microgenomics has been booming, employing highly sophisticated, high-throughput platforms. But these mainly chip-based methods can only generate biologically relevant data if the samples investigated consist of homogeneous cell populations, in which no unwanted cells of different specificity and/or developmental stage obscure the results. METHODS: Different sampling methods have been routinely applied to overcome the problem presented by heterogeneous samples, e.g., global surveys, cell cultures, and microdissection. Various methods of laser-assisted microdissection, employing either positive or negative selection of tissue areas or even single cells, are available. RESULTS: These laser-assisted microdissection methods allow for fast and precise procurement of extremely small samples. Through subsequent application of recently developed methods of linear mRNA amplification in a pool of isolated total RNA, it has now become possible to perform complex high-throughput RNA expression profiling by microdissecting and processing even single-cell samples. CONCLUSIONS: Studies using the tools and methods of microgenomics have shed light on how those new approaches will eventually aid in the development of a new generation of diagnostics, e.g., leading to new patient-specific drugs tailored to the requirements assessed by assaying only a few biopsy cells.

Does endometriosis really have premalignant potential? A clonal analysis of laser-microdissected tissue.

Since 1925, epidemiological and histological evidence for an association between endometriosis and ovarian neoplasia has accumulated. Recently, publications assaying the clonality of a given cell population have implied endometriosis has premalignant properties. However, the human androgen receptor used as a marker in these studies is of highly questionable reliability due to the instability of its methylation pattern in nonmalignant cells and during the course of malignancy. Therefore, we decided to readdress the question of clonality of endometriotic foci by using an alternative assay based on a polymorphism of the phosphoglycerate kinase-1 gene. We overcame the limitation to using ovarian cysts (a problem encountered in other studies) by laser-microdissecting defined tissue fractions of interest. From the 13/29 informative patients, a total of 32 endometriotic samples from various sites was assayed. Only 2/32 samples from different patients bore monoclonal tissue. With one of those cases, we present the first direct evidence of the two morphological endometric compartments comprising a single biphasic developmental unit. Neither monoclonal patient was characterized by any outstanding clinical parameters, including neoplasia. Individual endometriotic foci from the only patient in this study with neoplasia was assayed as being polyclonal. Therefore, former studies stating endometriosis as premalignant have to be cautiously reinterpreted.