Molecular detection of tumour DNA in serum and peritoneal fluid from ovarian cancer patients

Abstract

We have analysed DNA extracted from the serum and peritoneal fluid of 20 ovarian cancer patients for the presence of tumour-specific genetic alterations. The 20 patients included six with stage Ia disease. Using six polymorphic microsatellite loci we were able to detect novel alleles or loss of heterozygosity in 17/20 serum samples and 12/19 peritoneal fluid samples. Tumour-specific abnormalities were detected in the serum of all but one of the stage Ia cases. Half of the occurrences of loss of heterozygosity identified in primary tumour material were detectable in the serum samples. Novel alleles indicative of microsatellite instability were found in 3/6 patients with stage Ia disease but in only 1/14 of patients with more advanced disease. One of the eight patients in the control group displayed abnormalities in her serum DNA. The ease with which tumour-specific alterations were detected in serum and peritoneal samples from ovarian cancer patients, using a panel of only six polymorphic microsatellite markers on four chromosomes, suggests that molecular detection methods could prove useful in the staging, monitoring and screening of this disease. © 1999 Cancer Research Campaign

The Acquisition of hMLH1 Methylation in Plasma DNA after Chemotherapy Predicts Poor Survival for Ovarian Cancer Patients

“…This is most likely to be due to a proportion of patients not having detectable tumor DNA in plasma at relapse and is consistent with the 73% sensitivity of detecting the same genetic change in plasma DNA as present in a MS-S, microsatellite stable; MSI-L, low microsatellite instability (a shift in one locus of at least five loci examined); MSI-H, high microsatellite instability (a shift in more than one locus); Pre-chemo, before chemotherapy. the tumor reported previously for ovarian cancer at presentation (8). The observation that three of the relapse plasma samples that had apparently lost MLH1 methylation had very low DNA yields (Ͻ100 ng/ml plasma) further supports this interpretation.…”

“…The choice of microsatellite loci used reflects those used in previous colorectal cancer and ovarian analysis (8,18). PCR was carried out using primers (synthesized by Oswel DNA Service, Southampton, United Kingdom) specific for six polymorphic DNA microsatellite loci (Mfd15CA, D2S123, P53, D5S346, D18S69, and D18S58).…”

“…DNA can be detected in plasma from cancer patients with the same characteristic changes, including CpG island methylation, found in the corresponding tumor (7). In the case of ovarian cancer, such changes have been detected with high specificity and have been suggested as a diagnostic tool (4,8). DNA methylation is particularly suited for such analysis of plasma DNA because sensitive methylation-specific PCR (MSP)-based assays require only small amounts of DNA, and methylation of genes frequently aberrantly methylated in tumors is rarely observed in normal tissue, including peripheral blood mononuclear cell (PBMC) DNA that may be present with tumor DNA in plasma (9).…”

Tumor Cell-Specific BRCA1 and RASSF1A Hypermethylation in Serum, Plasma, and Peritoneal Fluid from Ovarian Cancer Patients

“…Molecular detection of loss of heterozygosity or new alleles by microsatellite analysis has been reported in 17 of 20 (85%) serum and 12 of 19 (63%) peritoneal fluid DNA from ovarian cancer patients (43) and by digital single nucleotide polymorphism analysis in 19 of 20 (95%) ascitic fluids from ovarian carcinoma patients (44). Successful detection of p53 point mutation in matched peritoneal fluid from three patients has also been demonstrated (45); however, p53 is not mutated in the majority of ovarian tumors (6).…”

A change in microsatellite instability caused by cisplatin-based chemotherapy of ovarian cancer

“…Four loci (BAT25, BAT26, D2S123, and D5S346) were recommended for analysing MSI, and 2 loci (BAT40 and D18S58) were recommended as alternative loci as published in the International Guidelines for the Evaluation of MSI in Colorectal Cancer (Boland et al, 1998). We selected 4 more microsatellite loci including D3S1612 (hMLH1) (Hass et al, 1999), D11S904 (Hickey et al, 1999) (chromosome 11p), D17S795 (Pieretti et al, 1995) (chromosome 17q), and TP53 (p53) for the assay of MSI. The forward primer was 5 ′ -end-labelled with [γ 32 P]-ATP (Amersham Pharmasia Biotech, Tokyo, Japan) using T 4 -polynucleotide kinase (Promega, Madison, WI), and was used together with an unlabelled reverse primer.…”