(From Abstract) In 1986, an accident at the nuclear power plant in Chernobyl, USSR, led to the release of high levels of radioisotopes. Ten years later, the incidence of childhood papillary thyroid cancer (chPTC) near Chernobyl had risen by 2 orders of magnitude, most likely as a consequence of increased exposure to ionizing radiation. The routes and mechanisms by which radiation generated these additional thyroid cancers remain mysterious. Eight years ago, we began to archive thyroid tumor tissues from patients who underwent thyroidectomy near the site of the power plant, among them 214 specimens from children. Several tumors from this collection aberrantly expressed ret tyrosine kinase transcripts due to a ret/PTC1 or ret/PTC3 chromosomal rearrangements on chromosome 10. However, many other chPTC tumors have phenotypes not attributable to aberrant ret expression. Moreover, even within a ret-positive chPTC tumor, not all cells express ret or contain a rearranged chromosome 10. We hypothesize that these other classes of tumors may inappropriately express a different oncogene or have lost function of a tumor suppressor as a result of chromosomal rearrangements and that knowledge of the kind of genetic alterations leading to chPTC may facilitate the early detection and staging of tumors as well as provide guidance for therapeutic intervention. To test this hypothesis, we propose to map the sites of chromosomal breakpoints in 38 cases of radiation-induced chPTC and identify genes with abnormal pattern of expression. We will localize the breakpoints in radiation-induced tumors for which we have metaphase spreads using G-banding results and Spectral Karyotyping. With the breakpoints grossly determined, we will define the targets for positional cloning and prepare breakpoint-spanning YAC contigs and high-resolution physical maps based on co-linear BAC clones. Control groups will be comprised of children who developed thyroid cancer without prior radiation exposure as well as tumors in adult patients who underwent radiotherapy as children or adults. Interphase cell preparations from the control groups will be studied with comparative genomic hybridization and probes specific for candidate loci to detect translocations as well as gene amplifications and deletions. We can then identify genes at the chPTC-specific breakpoints by exon-trapping, direct selection, and DNA sequencing. We will prepare stable cell lines carrying the oncogenic rearrangements for further characterization and distribution. Finally, we will examine the levels and localization of mRNAs with Northern and in situ hybridization analysis.
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