Abstract
Fluorescence in situ hybridisation (FISH) has been used increasingly for gene mapping and ordering probes on interphase and metaphase preparations. The association of consistent chromosomal aberrations with certain malignancies allows the possibility of using interphase cytogenetics as a diagnostic tool. In small round cell tumours of children accurate diagnosis may be difficult using existing methods. We have therefore evaluated the diagnostic potential of this technique when applied to the characteristic t(11;22) found in Ewing's sarcoma and peripheral neuroectodermal tumour (ES and PNET). Interphase nuclei were prepared from normal human foreskin fibroblasts (HFF), two Ewing's sarcoma cell lines and several fresh tumour biopsies. DNA probes each side of the breakpoint at 22q12 were labelled with biotin and digoxygenin, hybridised to chromosomes in interphase and detected in different colours. Measurements between pairs of signals arising from each copy of chromosome 22 were taken and statistical analysis performed. There was a highly significant difference (P < 0.0001) between the two populations of measurements obtained (from nuclei with and without the t(11;22)). Studying four tumours and one further ES line (blind) it was found that median values from 30 nuclei could correctly identify which samples contained the t(11;22). This application of interphase cytogenetics contributes a reliable, accurate and conceptually simple diagnostic test for ES and PNET. It may now be applied to other tumours with characteristic translocations, amplifications or deletions when suitable probes are available. This approach is likely to become a routine in clinical diagnosis.
Highlights
There was a highly significant difference (P
Sample preparation Interphase nuclei were prepared from normal human foreskin fibroblasts (HFF), a human Ewing's sarcoma cell line RD-ES, and several fresh tumour biopsies
A total of 130 pairs of measurements were taken from normal HFF nuclei and 224 pairs from the RD-ES cell line
Summary
Sample preparation Interphase nuclei were prepared from normal human foreskin fibroblasts (HFF), a human Ewing's sarcoma cell line RD-ES (from American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, 20852-1776 USA), and several fresh tumour biopsies (from Great Ormond Street Hospital, London).The cell line was cultured in RPMI 1640, with 15% foetal calf serum (FCS) (Gibco) and 1% glutamine at 37°C, with 5% CO2. In order to assess whether the HFF ratios differed from the RD-ES ratios, a two sample rank test called the Mann-Whitney test was used (Altman, 1991) This test assumes that the ratios observed in the HFF and RD-ES nuclei are random samples taken from two populations characterised by similar variation but different medians. In order to determine how many pairs of measurements should be carried out in practice in the diagnosis of tumours with the t(I 1;22) we have used a computer-intensive method known as 'bootstrap' (Efron & Tibshirani, 1986). This method allowed us to simulate the populations of HFF and RD-ES ratios from which the original observations were
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