Loss of heterozygosity proximal to the M6P/IGF2R locus is predictive for the presence of disseminated tumor cells in the bone marrow of ovarian cancer patients before and after chemotherapy

Abstract

Disseminated tumor cells (DTC) in the bone marrow (BM) are present in about 35% of ovarian cancers before surgery and after chemotherapy and are associated with worse prognosis. A molecular biomarker in the primary tumor predicting tumor cell spread would be highly desirable. The purpose of the study was to investigate loss of heterozygosity (LOH) in primary ovarian tumors at four ovarian cancer-relevant chromosomal loci involved in apoptosis, platinum sensitivity, or DNA-repair, to assess the prognostic value of LOH and to correlate LOH with DTC occurrence before surgery and after chemotherapy. Primary tumor DNA of 88 patients was analyzed for LOH at four polymorphic microsatellite markers using PCR-based fluorescence microsatellite analysis. BM aspirates were analyzed for DTC by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3. LOH at the entire marker set correlated with tumor grading (P = 0.0001) and histology (P = 0.004). LOH at marker D10S1765 correlated with FIGO stage (P = 0.046) and grading (P = 0.05), whereas LOH at D17S855 significantly associated with grading (P = 0.023) and histology (P = 0.012), respectively. DTC were detected in 49% of patients before surgery and in 50% of patients after chemotherapy. Interestingly, LOH proximal to D6S1581 significantly correlated with DTC presence before surgery (P = 0.05) and after chemotherapy (P = 0.022). Conclusively, our data suggest that allelic loss at D6S1581 (proximal to M6P/IGF2R locus) serves as a molecular biomarker for the presence of DTC in the BM before and after chemotherapy.

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

“…The ascertained prognostic impact of marker D6S1581 in cirDNA of ovarian cancer patients in our current study is of particular interest, as our present serum data corroborate our previous findings on ovarian tumor tissue showing that allelic loss at D6S1581 is a biomarker for tumor cell spread to the BM [5]. Thus, evidence is accumulating, that genomic region proximal to M6P/IGF2R locus seems to be functionally implicated for ovarian tumorigenesis and may serve as a blood-based biomarker for prognosis.…”

“…Significant associations of this marker with FIGO stage and grading coincide with our previous LOH investigation in primary ovarian tumor tissue [5]. D10S1765 is located at chromosomal band 10q23.3.…”

“…All sera of ovarian cancer patients, obtained before surgery and after chemotherapy, were tested for LOH at four ovarian cancer-relevant microsatellite markers, previously described by us in detail [5]. Before Surgery, 31/63 patients (49%) showed at least one LOH in one of the two fractions, whereas after chemotherapy in 24/58 patients (41%), at least one LOH was detectable.…”

“…DNA was selectively amplified by PCR reaction using primer pairs binding to microsatellite markers of interest, previously described by us in detail [5]. PCR conditions were: 10 ng of DNA were amplified in a 10-μl-reaction containing PCR Gold buffer (150 mM Tris–HCl, pH 8.0 and 500 mM KCl), 2.5 mM MgCl 2 (Applied Biosystems, Mannheim, Germany), 200 μM dNTPs (Roche, Mannheim, Germany), 0.2 μM of primer sets (Sigma, Taufkirchen, Germany) and 0.5 U AmpliTaq Gold DNA-Polymerase (Applied Biosystems).…”

“…We previously analyzed primary ovarian tumors for loss of heterozygosity (LOH) incidence at a panel of four microsatellite markers associated with ovarian cancer-relevant tumor suppressor genes involved in apoptosis, platinum sensitivity and DNA-repair, namely PTEN (marker D10S1765 ), BRCA1 (marker D13S218 ), BRCA2 (marker D17S855 ) and M6P/IGF2R ( D6S1581 ). We revealed that LOH incidence in the primary tumor at marker D6S1581 (directed proximal to M6P/IGF2R locus) is predictive for the presence of disseminated tumor cells (DTC) in the bone marrow (BM) before surgery and after chemotherapy [5]. However, primary tumor tissue is only available by resection, and it would be highly desirable to establish a blood-based biomarker which is suitable to monitor the course of disease.…”