CANCER RESEARCH55, 5617-5620. December I. 9951 ABSTRACT Since the Chernobyl nuclear reactor accident, a striking increase of thyroid carcinoma has been reported in children exposed to radiation in Belarus Because ofits unprecedented scale and its emotional implications, this finding has raised concern and called the attention of the scientific community to this major health problem. Although epidemiologically documented, a direct correlation between thyroid cancer and radiation exposure has not been definitely proven at the molecular level. On the assumption that ionizing radiation could cause specific and common cancer-associated genetic lesions, an analysis of oncogene activation and/or tumor suppressor gene inactivation would help to define radiation induced thyroid carcinomas. Therefore, we have analyzed by different molecular approaches, in cluding Southern blotting, DNA transfection assay on NIH-3T3 cells, and reverse transcription-PCR analysis, six papillary carcinomas from chil dren living in the region of Belarus at the time of the Chernobyl nuclear accident to identify tumor-specific gene rearrangements of the proto oncogenes RET and TRK, previously found activated in a tumor type specific manner in papillary thyroid carcinoma. Using Southern blot analysis in four cases, we could detect specific rearranged bands indicat ing an oncogenic activation of RET that in three cases resulted in rear ranged sequences provided by the same activating gene. Moreover, the DNA of the last three cases showed a biological activity in transforming NIH-3T3 cells after the DNA-mediated transfection assay, and the respec tive NIH-3T3 transfectants were found to express the oncogenic fusion transcripts. These results support the possibility that RET oncogemc activation could represent a major genetic lesion associated with thyroid carcinoma in children exposed to the Chernobyl nuclear accident. INTRODUCTION The greatly increased number of thyroid carcinomas reported in children who were living in southern Belarus at the time of the Chernobyl nuclear accident has aroused a number of concerns regard ing both a true increase in the incidence of thyroid cancer and its link to the disaster ( 1—3). Relevant answers to these questions could be provided by an analysis of oncogene activation in these thyroid tumors because of the possibility that radiation may lead with high frequency to specific genetic lesions. Potential candidates for such a role are the oncogenes derived from the activation of the tyrosine kinase receptor genes RET and TRK, which are found rearranged with significant frequency in papillary thyroid carcinomas (4). In particu lar, the human RET proto-oncogene was discovered following its oncogenic activation by gene rearrangement that occurred in vitro during transfection experiments (5). Subsequently, through the same technique, specific oncogenic RET rearrangements occurring at the Received 9/19/95; accepted 9/19/95. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I This work was supported by Consiglio Nazionale delle Ricerche “ACRO Project,― Associazione Italiana Ricerche sul Cancro, and JSP4 EEC Chernobyl Projects COSU CT93-0052. 2 To whom requests for reprints should be addressed. Phone: 2-2390236; Fax: 2-2390764. somatic level in tumor cells have been identified and characterized in papillary thyroid carcinoma (4, 6). So far, three different oncogenic versions of RET have been characterized and designated as RET! PTC.3 In this oncogene family the tyrosine kinase domain of RET, localized to chromosome lOql 1.2, is juxtaposed to sequences derived from different genes. In RET/PTCJ and RET/PTC3 oncogenes, the genes (H4/DJOSJ7O and ELE]) providing the promoter and the 5' end of the chimeric transforming sequences were localized to the same chromosome 10: 10q21 and lOql 1.2, respectively, thus indicat ing that intrachromosomal rearrangements (inversions) led to the fusion of the two different genes (7, 8). Differently, RETIPTC2 is formed by the RET rearrangement with the R12 gene which maps to chromosome l7q23 (9). The involvement of the RET proto-oncogene in radiation-induced thyroid cancer has been suggested by the following observations: RET proto-oncogene activation has been documented in a PTC from a patient who had experienced previous accidental exposure to a high dose of radiation (10). In addition, in vitro irradiation of human undifferentiated thyroid carcinoma cells has been shown to cause RET gene rearrangements (1 1). Finally, the activation by gene rearrange ment of RET has been demonstrated to be highly specific for the papillary type of thyroid carcinoma that is more frequently associated with radiation exposure (12), with a frequency which varies signifi cantly depending on the geographic origin of the tumor specimens (13).In thisstudy, wehaveinvestigated thepossibility of anonco genic activation of RET and/or TRK proto-oncogenes in a sample of six PTCs from children living in the region of Belarus at the moment of the Chernobyl nuclear accident. MATERIALS AND METHODS Patients and Tumors. Table 1 gives a description of the characteristics of the six patients. Microscopically, all of the tumor specimens examined on frozen sections were consistent with a diagnosis of papillary carcinoma. In two cases (patients 2 and 6), the tumor component represented <20% of the sample and in patient 3 an extensive fibrosis was present. Southern Blot Analysis and Transfection Assay. Ten @gDNA were digested with EcoRl, blotted, and hybridized with the following 32Pmolecular probes: probe 1, 1-kb BglII-BamHI fragment specific for the 6.7-kb EcoRI fragment of proto-RET(7); probe 2, 1.8-kb BamHI-BamHI fragment related to the 5' end of proto-RET (7); probe 3, 0.8-kb EcoRI cDNA fragment encom passing the ELEllRETbreakpoint (15); and probe 4, 0.8-kb fragment related to the 5‘ end of the RI2 intron where the two previously characterized oncogenic rearrangements of R12 occurred (9). The transfection assay on NIH-3T3 cells was performed as previously described (14) using 16—40 @gof high molecular weight DNA. RNA Amplification and Oligonucleotide Primers. RT-PCR analysis was performed with 2 @tgRNA from each sample that was reverse transcribed and amplified as previously described (15). PCR primers were synthesized on a DNA synthesizer (Applied Biosystems, Foster, CA), and the sequences were as 3 The abbreviations used are: PTC, papillary thyroid carcinoma; RI, reverse transcrip tion. 5617 Oncogenic Rearrangements of the RET Proto-Oncogene in Papillary Thyroid Carcinomas from Children Exposed to the Chernobyl Nuclear Accident1 Laura Fugazzola, Silvana Pilotti, Aldo Pinchera, Tatiana V. Vorontsova, Piera Mondellini, Italia Bongarzone, Angela Greco, Larisa Astakhova, Marta G. Butti, Eugene P. Demidchik, Furio Pacini, and Marco A. Pierotti2 Istituto di Endocrinologia. Universita ‘ di Pisa. Vie dei Tirreno 64. 56018 Tirrenia Pisa IL F., A. P.. F. P.]; Divisione di Anatomia Patologica e Citologia (S. P.] and Divisione di Oncoiogia Sperimentale A [P. M., 1. B., A. G., M. G. B.. M. A. P.]. Istituto Nazionaie Tunwri, Via G. Venezian 1, 20133 Miiano, Italy; and Institute of Radiation Medicine IT. V. V. L A.] and Oncology Center of Thyroid fE. P. D.]. Minsk. Byeiorussia Research. on September 30, 2021. © 1995 American Association for Cancer cancerres.aacrjournals.org Downloaded from