Assessment of a six gene panel for the molecular detection of circulating tumor cells in the blood of female cancer patients.
BACKGROUND
The presence of circulating tumor cells (CTC) in the peripheral blood of cancer patients has been described for various solid tumors and their clinical relevance has been shown. CTC detection based on the analysis of epithelial antigens might be hampered by the genetic heterogeneity of the primary tumor and loss of epithelial antigens. Therefore, we aimed to identify new gene markers for the PCR-based detection of CTC in female cancer patients.


METHODS
Gene expression of 38 cancer cell lines (breast, ovarian, cervical and endometrial) and of 10 peripheral blood mononuclear cell (PBMC) samples from healthy female donors was measured using microarray technology (Applied Biosystems). Differentially expressed genes were identified using the maxT test and the 50% one-sided trimmed maxT-test. Confirmatory RT-qPCR was performed for 380 gene targets using the AB TaqMan® Low Density Arrays. Then, 93 gene targets were analyzed using the same RT-qPCR platform in tumor tissues of 126 patients with primary breast, ovarian or endometrial cancer. Finally, blood samples from 26 healthy women and from 125 patients (primary breast, ovarian, cervical, or endometrial cancer, and advanced breast cancer) were analyzed following OncoQuick enrichment and RNA pre-amplification. Likewise, hMAM and EpCAM gene expression was analyzed in the blood of breast and ovarian cancer patients. For each gene, a cut-off threshold value was set at three standard deviations from the mean expression level of the healthy controls to identify potential markers for CTC detection.


RESULTS
Six genes were over-expressed in blood samples from 81% of patients with advanced and 29% of patients with primary breast cancer. EpCAM gene expression was detected in 19% and 5% of patients, respectively, whereas hMAM gene expression was observed in the advanced group (39%) only. Multimarker analysis using the new six gene panel positively identified 44% of the cervical, 64% of the endometrial and 19% of the ovarian cancer patients.


CONCLUSIONS
The panel of six genes was found superior to EpCAM and hMAM for the detection of circulating tumor cells in the blood of breast cancer, and they may serve as potential markers for CTC derived from endometrial, cervical, and ovarian cancers.
Department of Obstetrics and Gynecology, Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria. eva.obermayr@meduniwien.ac.at
Eva
E
Obermayr
Fatima
F
Sanchez-Cabo
Muy-Kheng M
MK
Tea
Christian F
CF
Singer
Michael
M
Krainer
Michael B
MB
Fischer
Jalid
J
Sehouli
Alexander
A
Reinthaller
Reinhard
R
Horvat
Georg
G
Heinze
Dan
D
Tong
Robert
R
Zeillinger
D000328:Adult; D000368:Aged; D000951:Antigens, Neoplasm / Q000235:genetics; D001317:Austria; D014408:Biomarkers, Tumor / Q000097:blood / Q000235:genetics*; D001943:Breast Neoplasms / Q000097:blood / Q000235:genetics* / Q000473:pathology; D016022:Case-Control Studies; D015815:Cell Adhesion Molecules / Q000235:genetics; D045744:Cell Line, Tumor; D016213:Cyclins / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D016889:Endometrial Neoplasms / Q000097:blood / Q000235:genetics / Q000473:pathology; D000071858:Epithelial Cell Adhesion Molecule; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D005833:Genital Neoplasms, Female / Q000097:blood / Q000235:genetics* / Q000473:pathology; D005858:Germany; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics; D008875:Middle Aged; D062806:Myelin and Lymphocyte-Associated Proteolipid Proteins; D009360:Neoplastic Cells, Circulating / Q000378:metabolism* / Q000473:pathology; D009419:Nerve Tissue Proteins / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D010051:Ovarian Neoplasms / Q000097:blood / Q000235:genetics / Q000473:pathology; D050480:Plasma Membrane Neurotransmitter Transport Proteins / Q000235:genetics; D011237:Predictive Value of Tests; D011379:Prognosis; D011510:Proteolipids / Q000235:genetics; D012333:RNA, Messenger / Q000097:blood*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics; D002583:Uterine Cervical Neoplasms / Q000097:blood / Q000235:genetics / Q000473:pathology; D033921:Vesicular Transport Proteins / Q000235:genetics
D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; C503688:CCNE2 protein, human; D015815:Cell Adhesion Molecules; D016213:Cyclins; D004268:DNA-Binding Proteins; C486403:EMP2 protein, human; C499379:EPCAM protein, human; D000071858:Epithelial Cell Adhesion Molecule; C438606:MAL2 protein, human; C430872:MAML1 protein, human; D008562:Membrane Glycoproteins; D062806:Myelin and Lymphocyte-Associated Proteolipid Proteins; D009419:Nerve Tissue Proteins; D050480:Plasma Membrane Neurotransmitter Transport Proteins; D011510:Proteolipids; D012333:RNA, Messenger; C094770:SLC6A8 protein, human; D014157:Transcription Factors; D033921:Vesicular Transport Proteins