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

Abstract

BackgroundWe 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.MethodscirDNA 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.ResultscirDNA 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).ConclusionWe 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.

Liquid biopsy in ovarian cancer: recent advances on circulating tumor cells and circulating tumor DNA

“…They reported that LOH at two markers can predict tumor grade (p = 0.033) and FIGO stage (p = 0.004) in the LMWF cfDNA. Remarkably, a LOH at another marker can significantly predict patients OS (p = 0.030) in both HMWF and LMWF [13].…”

“…Recent studies aimed to detect copy number variations (CNV) [51] and to quantify specific LOH [13] or aberrant somatic chromosomal rearrangements [50] in ctDNA of ovarian cancer patients. Kuhlmann et al quantified cfDNA of 63 primary epithelial ovarian cancer patients before surgery and after chemotherapy.…”

“…Circulating tumor DNA (ctDNA) represents a small percentage of cfDNA that is shed in circulation by tumor cells and carries all these molecular alterations including tumor specific mutations, microsatellite instability (MI) [12], loss of heterozygosity (LOH) [13], and DNA methylation [14]. ctDNA is a very promising non-invasive diagnostic, prognostic and predictive tool, as it provides an easily accessible source of DNA derived from the tumor [15].…”

Monitoring tumor-derived cell-free DNA in patients with solid tumors: Clinical perspectives and research opportunities

“…In ovarian cancer patients were investigated also the MSI and the DNA integrity with promising results [97] and [98].…”

Circulating tumour DNA: a minimally invasive biomarker for tumour detection and stratification

“…However, the current study was limited by small sample size (20 patients) and retrospective study design. Other studies have also highlighted the role of ctDNA as a predictor of poor outcome and showed a consistent relationship between disease recurrence and reappearance of tumour-specific aberrations in circulating DNA in various cancer type (Diehl et al 2008;Kuhlmann et al 2012;Kinugasa et al 2015), also see Table 2. Moreover, studies have shown that the ability of ctDNA to predict recurrence were superior to standard biomarker (CEA) (Diehl et al 2008;Sato et al 2016).…”