Open Access Research article

LOH at 6q and 10q in fractionated circulating DNA of ovarian cancer patients is predictive for tumor cell spread and overall survival

Jan Dominik Kuhlmann1*, Heidi Schwarzenbach2, Pauline Wimberger1, Micaela Poetsch3, Rainer Kimmig1 and Sabine Kasimir-Bauer1

Author Affiliations

1 Department of Gynecology and Obstetrics, University Hospital of Essen, Hufelandstrasse 55, Essen, D-45122, Germany

2 Institute of Tumor Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

3 Institute of Legal Medicine, University Hospital of Essen, Essen, Germany

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BMC Cancer 2012, 12:325  doi:10.1186/1471-2407-12-325

Published: 31 July 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