Molecular cytogenetic analysis consistently identifies translocations involving chromosomes 1, 2 and 15 in five embryonal rhabdomyosarcoma cell lines and a PAX-FOXO1A fusion gene negative alveolar rhabdomyosarcoma cell line

Abstract

Rhabdomyosarcoma in children is a “small round blue cell tumour” that displays skeletal muscle differentiation. Two main histological variants are recognised, alveolar (ARMS) and embryonal (ERMS) rhabdomyosarcoma. Whereas consistent chromosome translocations characteristic of ARMS have been reported, no such cytogenetic abnormality has yet been described in ERMS. We have used multiple colour chromosome painting to obtain composite karyotypes for five ERMS cell lines and one PAX-FOXO1A fusion gene negative ARMS. The cell lines were assessed by spectral karyotyping (SKY), tailored multi-fluorophore fluorescence in situ hybridisation (M-FISH) using series of seven colour paint sets generated to examine specific abnormalities, and comparative genomic hybridisation (CGH). This approach enabled us to obtain karyotypes of the cell lines in greater detail than previously possible. Several recurring cytogenetic abnormalities were demonstrated, including translocations involving chromosomes 1 and 15 and chromosomes 2 and 15, in 4/6 and 2/6 cell lines respectively. All six cell lines demonstrated abnormalities of chromosome 15. Translocations between chromosomes 1 and 15 have previously been recorded in two primary cases of ERMS by conventional cytogenetics. Analysis of the translocation breakpoints may suggest mechanisms of ERMS tumourigenesis and may enable the development of novel approaches to the clinical management of this tumour.

Paint‐assisted microdissection‐FISH: Rapid and simple mapping of translocation breakpoints in the embryonal rhabdomyosarcoma cell line RD

“…Seven rearrangements originally reported by us in RD (13) were not confirmed by PAM-FISH (Table 1, rows I, L, M, N, O, Q, S). We conclude that these rearrangements were either misidentified in our earlier study; are present infrequently in RD metaphases; or have been lost from the cell line during passage in vitro.…”

“…2). Table 1 provides a complete listing of the translocations mapped in RD by PAM-FISH, in comparison to the data previously obtained by SKY, M-FISH and CGH (13). In brief, as a result of PAM-FISH analysis 18 structural rearrangements were confirmed in RD, rather than the 20 that we originally reported.…”

“…For selected rearrangements, we used bacterial artificial chromosome FISH (BAC-FISH) to test the accuracy of the breakpoint mapping information produced by PAM-FISH. We compared the cytogenetic data from our PAM-FISH study with our previously published description of the RD karyotype, which we obtained using SKY, M-FISH and CGH (13). We conclude that PAM-FISH is a more accurate and reliable means to derive high-resolution cytogenetic data than any other whole chromosome painting methodology.…”

“…Whole chromosome-specific paints labelled with Biotin 16-dUTP (Roche, Basel, Switzerland) were produced using standard protocols (4,7), with modifications (8). Specific paints were selected to enable localisation of each abnormal chromosome identified in RD in our previous study (13). Details of the forward paints used are given in the second column of Table 1.…”

“…Following optimisation of protocols for PAM-FISH, we used the technique in an attempt to map all 20 chromosomal rearrangements previously described by us using SKY, M-FISH and CGH in the ERMS cell line RD (13). We analysed a minimum of 10 reverse painted chromosome homologues per experiment, in order to derive consensus breakpoint locations.…”