Abstract
Detection of loss of heterozygosity (LOH) is usually performed on homogenised tumour specimens. In this type of analysis samples with a low percentage of tumour cells have to be excluded and possible intra-tumour heterogeneity is obscured. In this study we report the application of polymerase chain reaction (PCR)-driven LOH detection with in total 22 microsatellite markers for chromosome 1q, 3p, 3q, 4p, 6p, 6q, 11p, 11q, 17p, 17q, 18p, 18q, Xp and Xq on flow-sorted cells from fresh and paraffin-embedded ovarian tumour tissue. Titration experiments showed that LOH can be detected with as few as 100 cell equivalents of DNA. Clear examples of LOH could be detected in the sorted aneuploid fractions from one unilateral and two bilateral ovarian tumours from three patients. In two samples the sorted fraction was less than 10% of the total sample. The bilateral tumours from the same patient showed loss of identical alleles for one marker (case OV64) and two markers (case OV69), indicative of their monoclonal origin. Multiparameter flow cytometry using two different ovarian tumour markers (MOv18 and BMA180), an anti-cytokeratin monoclonal antibody (MAb) (M9), an anti-vimentin MAb (V9) and a MAb against the panepithelial antigen 17-1A on the fresh ascites cells of the fourth ovarian cancer patient was used to investigate possible intra-tumour heterogeneity. We showed the presence of at least three phenotypically different populations, of which the diploid, keratin-positive, vimentin-negative population showed a similar LOH pattern as the aneuploid population (DNA index = 1.7), indicative of its neoplastic origin. The same LOH pattern was shown in an omentum metastasis from this patient also having the same aneuploid DNA index of 1.7. The sharing of the same LOH pattern by the diploid and aneuploid tumour cell populations suggests that the observed allele loss events occurred before the development of aneuploidy. PCR on flow-sorted cells is thus an important tool to study clonal diversity in tumours.
Highlights
Study of loss of heterozygosity (LOH) is widely used to identify chromosomal locations of putative tumour-suppressor genes
Since it would be of interest to sample tumour cells on the basis of the expression of specific molecular features, we have investigated the possibility of performing molcular genetic (LOH) analysis on flow-sorted tumour cells
In this study 'we report the results from LOH analysis on isolated tumour cells from a total of four human ovarian carcinomas enriched by flow sorting on the basis of nuclear DNA content and/or cytoplasmic and surface antigen expression
Summary
Formalin-fixed, paraffin-embedded tissue blocks from three patients with ovarian carcinoma, operated on between 1982 and 1988, were retrieved from the archives of the Department of Pathology. Fresh ascites fluid and frozen tissue from an omentum metastasis from a patient with an ovarian carcinoma of the endometrioid type were included. The percentage of tumour cells in the solid tumours was estimated by visual examination of haematoxylin and eosin (H&E)-stained slides by an expenenced gynaecopathologist (G.J.F.). An established ovarian carcinoma cell line, OVCAR-3 (Hamilton et al, 1983), was used for determining the sensitivity of the polymerase chain reaction (PCR). Nuclei were isolated according to Hedley et al (1983) with minor modifications (Schueler et al, 1993) and stained with propidium iodide (PI) after RNAse treatment
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