MOLECULAR-GENETIC ANALYSIS OF FLOW-SORTED OVARIAN TUMOR-CELLS - IMPROVED DETECTION OF LOSS OF HETEROZYGOSITY

Detection of loss of heterozygosity (LOH) is usually performed on homo genised tumour specimens. In this type of analysis samples with a low percentage of tumour cells have to be excluded and possible intra-tumo ur 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 Iq, 3p, 3q, 4p, 6p, 6q, 11p, 11q, 17p, 17q, 18p, 18q, Xp and Xq on flow-sorted cells from fresh and paraffin-embedded ovarian rumour 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 fracti ons from one unilateral and two bilateral ovarian tumours from three p atients. In two samples the sorted fraction was less than 10% of the t otal sample. The bilateral rumours from the same patient showed loss o f identical alleles for one marker (case OV64) and two markers (case O V69), indicative of their monoclonal origin. Multiparameter flow cytom etry 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 a scites cells of the fourth ovarian cancer patient was used to investig ate 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 i ts neoplastic origin. The same LOH pattern was shown in an omentum met astasis 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 cell s is thus an important tool to study clonal diversity in tumours.