Combined array comparative genomic hybridization and tissue microarray analysis suggest PAK1 at 11q13.5-q14 as a critical oncogene target in ovarian carcinoma

Abstract

Amplification of chromosomal regions leads to an increase of DNA copy numbers and expression of oncogenes in many human tumors. The identification of tumor-specific oncogene targets has potential diagnostic and therapeutic implications. To identify distinct spectra of oncogenic alterations in ovarian carcinoma, metaphase comparative genomic hybridization (mCGH), array CGH (aCGH), and ovarian tumor tissue microarrays were used in this study. Twenty-six primary ovarian carcinomas and three ovarian carcinoma cell lines were analyzed by mCGH. Frequent chromosomal overrepresentation was observed on 2q (31%), 3q (38%), 5p (38%), 8q (52%), 11q (21%), 12p (21%), 17q (21%), and 20q (52%). The role of oncogenes residing in gained chromosomal loci was determined by aCGH with 59 genetic loci commonly amplified in human tumors. DNA copy number gains were most frequently observed for PIK3CA on 3q (66%), PAK1 on 11q (59%), KRAS2 on 12p (55%), and STK15 on 20q (55%). The 11q13-q14 amplicon, represented by six oncogenes (CCND1, FGF4, FGF3, EMS1, GARP, and PAK1) revealed preferential gene copy number gains of PAK1, which is located at 11q13.5-q14. Amplification and protein expression status of both PAK1 and CCND1 were further examined by fluorescence in situ hybridization and immunohistochemistry using a tissue microarray consisting of 268 primary ovarian tumors. PAK1 copy number gains were observed in 30% of the ovarian carcinomas and PAK1 protein was expressed in 85% of the tumors. PAK1 gains were associated with high grade (P < 0.05). In contrast, CCND1 gene alterations and protein expression were less frequent (10.6% and 25%, respectively), suggesting that the critical oncogene target of amplicon 11q13-14 lies distal to CCND1. This study demonstrates that aCGH facilitates further characterization of oncogene candidates residing in amplicons defined by mCGH.

Figure 1.

A: The ovarian carcinoma TMA (overview) and two array elements (×40-fold, H&E staining) with a serous (B) and a mucinous (C) adenocarcinoma.

Figure 2.

Image of an AmpliOnc I microarray after hybridization of 59 genomic loci. Oncogenes CCND1, FGF4/FGF3, and EMS1 located on 11q13 are amplified.

Figure 3.

aCGH results of 26 primary ovarian carcinomas and three ovarian carcinoma cell lines.

Figure 4.

Comparison of aCGH and mCGH data obtained from tumor 4. aCGH (left): Amplified genes are represented in dark green, genes with gained copy numbers in green, and deleted genes in red columns. mCGH (right): Green and red bars indicate deleted or gained chromosomal regions (mean ratio + SD obtained from at least four chromosomes). Asterisk: Gene loss or gain not corresponding with mCGH profile.

Figure 5.

A: PAK1 amplification: tumor cells with clusters of red PAK1 signals and two green centromere 11 signals. B: CCND1 gain with four to six red CCND1 signals and two to four centromere 11 signals (green) in an ovarian carcinoma on the TMA. C: Cytoplasmic PAK1 protein expression in an ovarian carcinoma. D: Nuclear CCND1 protein expression in an ovarian carcinoma.