Selection of potential markers for epithelial ovarian cancer with gene expression arrays and recursive descent partition analysis.
OBJECTIVE
Advanced-stage epithelial ovarian cancer has a poor prognosis with long-term survival in less than 30% of patients. When the disease is detected in stage I, more than 90% of patients can be cured by conventional therapy. Screening for early-stage disease with individual serum tumor markers, such as CA125, is limited by the fact that no single marker is up-regulated and shed in adequate amounts by all ovarian cancers. Consequently, use of multiple markers in combination might detect a larger fraction of early-stage ovarian cancers.


METHODS
To identify potential candidates for novel markers, we have used Affymetrix human genome arrays (U95 series) to analyze differences in gene expression of 41,441 known genes and expressed sequence tags between five pools of normal ovarian surface epithelial cells (OSE) and 42 epithelial ovarian cancers of different stages, grades, and histotypes. Recursive descent partition analysis (RDPA) was performed with 102 probe sets representing 86 genes that were up-regulated at least 3-fold in epithelial ovarian cancers when compared with normal OSE. In addition, a panel of 11 genes known to encode potential tumor markers [mucin 1, transmembrane (MUC1), mucin 16 (CA125), mesothelin, WAP four-disulfide core domain 2 (HE4), kallikrein 6, kallikrein 10, matrix metalloproteinase 2, prostasin, osteopontin, tetranectin, and inhibin] were similarly analyzed.


RESULTS
The 3-fold up-regulated genes were examined and four genes [Notch homologue 3 (NOTCH3), E2F transcription factor 3 (E2F3), GTPase activating protein (RACGAP1), and hematological and neurological expressed 1 (HN1)] distinguished all tumor samples from normal OSE. The 3-fold up-regulated genes were analyzed using RDPA, and the combination of elevated claudin 3 (CLDN3) and elevated vascular endothelial growth factor (VEGF) distinguished the cancers from normal OSE. The 11 known markers were analyzed using RDPA, and a combination of HE4, CA125, and MUC1 expression could distinguish tumor from normal specimens. Expression at the mRNA level in the candidate markers was examined via semiquantitative reverse transcription-PCR and was found to correlate well with the array data. Immunohistochemistry was performed to identify expression of the genes at the protein level in 158 ovarian cancers of different histotypes. A combination of CLDN3, CA125, and MUC1 stained 157 (99.4%) of 158 cancers, and all of the tumors were detected with a combination of CLDN3, CA125, MUC1, and VEGF.


CONCLUSIONS
Our data are consistent with the possibility that a limited number of markers in combination might identify >99% of epithelial ovarian cancers despite the heterogeneity of the disease.
Lu
Karen H
KH
Ovarian Cancer Research Laboratory, Department of Experimental Therapeutics, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
Patterson
Andrea P
AP
Wang
Lin
L
Marquez
Rebecca T
RT
Atkinson
Edward N
EN
Baggerly
Keith A
KA
Ramoth
Lance R
LR
Rosen
Daniel G
DG
Liu
Jinsong
J
Hellstrom
Ingegerd
I
Smith
David
D
Hartmann
Lynn
L
Fishman
David
D
Berchuck
Andrew
A
Schmandt
Rosemarie
R
Whitaker
Regina
R
Gershenson
David M
DM
Mills
Gordon B
GB
Bast
Robert C
RC
D014408:Biomarkers, Tumor; D045744:Cell Line, Tumor; D019070:Cell Lineage; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D005260:Female; D020558:GTP Phosphohydrolases / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry; D009369:Neoplasms / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D010051:Ovarian Neoplasms / Q000235:genetics* / Q000378:metabolism; D010053:Ovary / Q000378:metabolism; D011379:Prognosis; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016896:Treatment Outcome; D015854:Up-Regulation
D014408:Biomarkers, Tumor; D012333:RNA, Messenger; D012313:RNA; D020558:GTP Phosphohydrolases