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BMC Cancer. 2012; 12: 325.
Published online Jul 31, 2012. doi:  10.1186/1471-2407-12-325
PMCID: PMC3488536
LOH at 6q and 10q in fractionated circulating DNA of ovarian cancer patients is predictive for tumor cell spread and overall survival
Jan Dominik Kuhlmann,corresponding author1 Heidi Schwarzenbach,2 Pauline Wimberger,1 Micaela Poetsch,3 Rainer Kimmig,1 and Sabine Kasimir-Bauer1
1Department of Gynecology and Obstetrics, University Hospital of Essen, Hufelandstrasse 55, Essen, D-45122, Germany
2Institute of Tumor Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
3Institute of Legal Medicine, University Hospital of Essen, Essen, Germany
corresponding authorCorresponding author.
Jan Dominik Kuhlmann: jandominik.kuhlmann/at/uk-essen.de; Heidi Schwarzenbach: hschwarz/at/uke.uni-hamburg.de; Pauline Wimberger: pauline.wimberger/at/uk-essen.de; Micaela Poetsch: micaela.poetsch/at/uk-essen.de; Rainer Kimmig: rainer.kimmig/at/uk-essen.de; Sabine Kasimir-Bauer: sabine.kasimir-bauer/at/uk-essen.de
Received March 30, 2012; Accepted July 18, 2012.
Abstract
Background
We recently showed that LOH proximal to M6P/IGF2R locus (D6S1581) in primary ovarian tumors is predictive for the presence of disseminated tumor cells (DTC) in the bone marrow (BM). For therapy-monitoring, it would be highly desirable to establish a blood-based biomarker. Therefore, we quantified circulating DNA (cirDNA) in sera of 63 ovarian cancer patients before surgery and after chemotherapy, measured incidence of LOH at four cancer-relevant chromosomal loci, correlated LOH with tumor cell spread to the BM and evaluated prognostic significance of LOH.
Methods
cirDNA was fractionated into high- and low molecular-weight fraction (HMWF, LMWF) for LOH-profiling, utilizing PCR-based fluorescence microsatellite analysis. BM aspirates were analyzed for DTC by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3.
Results
cirDNA levels in the HMWF before surgery were predictive for residual tumor load (p = 0.017). After chemotherapy, we observed a significant decline of cirDNA in the LMWF (p = 0.0001) but not in the HMWF. LOH was prevalently detected in the LMWF with an overall frequency of 67%, only moderately ablating after chemotherapy (45%). Before surgery, LOH in the LMWF at marker D10S1765 and D13S218 significantly correlated with tumor grading and FIGO stage (p = 0.033, p = 0.004, respectively). In both combined fractions, LOH at D6S1581 additionally associated with overall survival (OS) (p = 0.030). Moreover, solely LOH at D10S1765 in LMWF after therapy correlated with DTC in BM after therapy (p = 0.017).
Conclusion
We demonstrate the applicability and necessity of DNA-fractionation prior to analyzing circulating LOH and identify LOH at D10S1765 and D6S1581 as novel blood-based biomarkers for ovarian cancer, being relevant for therapy-monitoring.
Keywords: Circulating DNA, Loss of heterozygosity, Disseminated tumor cells, DNA-fractionation, Tumor suppressor genes
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