Identification of DNA copy number changes in microdissected serous ovarian cancer tissue using a cDNA microarray platform.
We have established a method for using a cDNA array platform in combination with degenerate oligonucleotide primer polymerase chain reaction (DOP-PCR) and taramide signal amplification (TSA) to identify DNA copy number abnormalities (CNA) in cancer cell lines and cancer cells procured with laser-based microdissection. To determine the sensitivity and specificity for detecting single-copy gain and loss, receiver-operator curve analysis was performed on hybridization signal ratios generated from non-DOP and DOP amplified female and male DNA using a 10,816-element cDNA microarray. A cutoff value of 1.12 and 1.07 average signal ratio for X-chromosomal genes versus autosomal genes provided a sensitivity and specificity of 50 and 79%, respectively, for non-DOP amplified DNA and a sensitivity and specificity of 50 and 72%, respectively, for DOP amplified DNA. We used this approach to identify DNA copy number abnormalities in the ovarian cancer cell line OVCA633, which has previously been shown to have 12p amplification. Transcription profiling of OVCA633 was also performed. Two amplified and overexpressed genes located on 12p11, KRAS2 and LRMP, were identified; these were validated with quantitative real-time PCR. Subsequently, the same approach was used to identify CNAs and gene expression alterations in 11 microdissected serous ovarian adenocarcinoma cases. Validated data revealed amplification and overexpression of ERBB3 and FOS and deletion and underexpression of KRT6 and APXL in more than 50% of the tissue samples. These results show the feasibility of using the cDNA array platform to identify changes in DNA and mRNA copy number simultaneously in microdissected tumor tissues.
Tsuda
Hiroshi
H
Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, Laboratory of Gynecologic Oncology, Brigham and Women's Hospital, Dana-Farber Cancer Institute, Harvard Medical School, BLI-447, 221 Longwood Avenue, Boston, MA 02115, USA.
Birrer
Michael J
MJ
Ito
Yoichi M
YM
Ohashi
Yasuo
Y
Lin
Ming
M
Lee
Cheng
C
Wong
Wing H
WH
Rao
Pulivarthi H
PH
Lau
Ching C
CC
Berkowitz
Ross S
RS
Wong
Kwong-Kwok
KK
Mok
Samuel C
SC
D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004273:DNA, Neoplasm / Q000032:analysis*; D005240:Feasibility Studies; D005260:Female; D005784:Gene Amplification; D017353:Gene Deletion; D018628:Gene Dosage; D015870:Gene Expression; D020869:Gene Expression Profiling; D015894:Genome, Human; D006801:Humans; D007834:Lasers; D008297:Male; D042282:Microdissection; D020411:Oligonucleotide Array Sequence Analysis; D010051:Ovarian Neoplasms / Q000235:genetics* / Q000378:metabolism*; D017698:Postmenopause; D012333:RNA, Messenger / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic
D018076:DNA, Complementary; D004273:DNA, Neoplasm; D012333:RNA, Messenger