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Rearrangements of chromosomes 1 and 10 generate transforming fusion genes derived from TRK and RET tyrosine kinases in papillary thyroid carcinomas
Abstracts
175
N-MYC REGULATION IN NEUROBLASTOMA.
Ngan Chinq Chenq, Huib Carom Alvin Chan, Mabel Beitsma, Peter van Sluis, Frank Speleman', Andries Westerveld, and Rogier Versteeg. Dept. of Human Genetics, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. "Dept. of Medical Genetics, University of Gent, Belgium.
Neuroblastoma is the most common extracranial solid turnout of childhood and comprises 8% of all childhood malignancies. A strong correlation between N-myc amplification and allelic loss of the lp36 chromosomal region was found. In 82 neuroblastomas, we found that 26 showed loss of lp36. All N-myc amplified tumors (16) belonged to this category. Therefore, the N-myc amplified tumours seem to be a subset of neuroblastomas with lp36 deletions. Deletion analysis in neuroblastoma cell lines showed that N-myc amplification is associated with large lp deletions, while in cell lines without N-myc amplification the deletions are smaller involving only the lp36 region. This indicates that lp harbours two distinctive neuroblastoma-associated genes: one distal gene with yet unknown function and a more proximal gene which is associated with N-myc amplification. To analyse this latter gene further, we performed cell fusion experiments. Two neuroblastoma cell lines, one with N-myc amplification and expression and the other without, were fused. Both cell lines have deletions of lp. The N-myc amplified line is deleted for 1p32->pter. The line without N-myc amplification has a deletion of lp36.1->pter. In the resulting hybrid cell lines the N-myc amplification is still present. Strikingly, the N-myc expression is totally switched off. This implicates that in the N-myc amplified line a factor controlling N-myc expression is
REARRANGEMENTS OF CHROMOSOMES 1 AND 10 GENERATE TRANSFORMING FUSION GENES DERIVED FROM TRK AND RET TYROSINE KINASES IN PAPILLARY THYROID CARCINOMAS. Marco A. Pierotti, Italia Bongarzone, Maria G. Borrello, Angela Greco, Gabriella Sozzi and Giuseppe Della Porta - lstituto Nazionale Tumori, Milan, Italy. In the last few years, we have reported an high frequency of tumorspecific rearrangements of the proto-oncogenes R E T and TRK, encoding tyrosine kinase membrane receptors, in human papillary thyroid carcinomas. By the combining use of cytogenetic and molecular approaches, we have determined that the oncogenic activation o f these genes is accomplished by the fusion of their tyrosine kinase domain with unlinked amino-terminal sequences following chromosomal rearrangements, mostly but not exclusively intrachromosomal, involving chromosome 10 and chromosome 1 in the case of RET and TRK, respectively. We have so far identified three different versions of RET (designated RET/ptcl, RET/ptc2 and RET/ptc3) and of TRK (named TRK-TI; TRK-T2 and TRKT3)-derived oncogenes. In particular, RET was found activated in 18 out of 52 cases of papillary thyroid carcinomas. In 10 of the 18 cases it resulted rearranged with an uncharacterized gene (H4/D10SI7) forming the transforming sequence RET/ptcl, in 2 cases with RI A regulatory subunit of pKA enzyme and in the last 6 cases with a novel gene designated ELE1. On the other end, TRK activation involved 3 case of rearrangement with tropomyosin gene (TPM3), 3 with TPR sequences and 1 with a newly identified gene designated TAG (Trk Activating Gene). A cytogenetic and molecular characterization of newly identified members (RET/ptc3 and TRK-T3) of the RET and TRK oncogene superfamily will be presented and the results discussed in a general model of oncogenesis mediated by the formation of fusion genes.
lost. The loss of control can apparently be complemented by the fusion partner with the short lp deletion. This suggests that a gene that can switch off N-myc expression is located proximal to lp36.1. Deletion of this gene could result in a certain level of N-
ThisworkispartiMlysupportedbyAIRCandCNRSpecialPr~ect "ACRO".
myc expression. Once the gene is expressed, amplification can further increase the expression, which could confer growth advantage to the cell.
A SINGLE MUTATION IN THE RET PROTO-ONCOGENE IS A S S O C I A T E D W I T H M U L T I P L E E N D O C R I N E N E O P L A S I A T Y P E 2B. Robert M.W. Hofstra',
Rudy M. Landsvater', Rein P. Stulp', Tineke Stelwagen', Giovanni R o m e o % Cornelis J.M. Lips" and Charles H.C.M. Buys v. "Dept. of Medical Genetics, U n i v e r s i t y of Groningen, Groningen, "Dept. of Internal Medicine and Pathology, Utrecht U n i v e r s i t y Hospital, Utrecht, The Netherlands, "Laboratorio di Genetica Molecolare, Instituto G. Gaslini, Genova, Italy. Multiple endocrine neoplasia type 2 (MEN 2) comprises three clinically distinct d o m i n a n t l y inherited cancer syndromes. MEN 2A patients develop m e d u l l a r y thyroid carcinoma (MTC) and phaeochromocytoma. MEN 2B patients show in addition ganglioneuromas of the gastro-intestinal tract and skeletal abnormalities. In familial MTC, only the thyroid is affected. Germline mutations of the R E T proto-oncogene have recently been reported in association with MEN 2A and familial MTC. All mutations occurred within codons specifying cysteine residues in the transition of the RET extracellular and transmembrane domains. We now show that in all (8) MEN 2B patients examined, a single mutation has been found in a different domain of the R E T proto-oncogene.
M U T A T I O N S IN T H E R E T P R O T O - O N C O G E N E IN SPORADIC MEDULLARY THYROID CARCINOMAS.
Tineke Stelwaqen', Robert M.W. Hofstra', Rein P. Stulp', Harm Nijveen', Giovanni Romeo A, Rudy M. Landsvater',Cornelis J.M. Lips" and Charles H.C.M. Buys','Dept. of Medical Genetics, University of Groningen, Groningen, "Dept. of Internal Medicine and Pathology, Utrecht University Hospital, Utrecht, The Netherlands, "Laboratorio di Genetica Molecolare, Instituto G. Gaslini, Genova, Italy. Medullary thyroid carcinoma (MTC) occur both in a sporadic and hereditary form. The hereditary MTCs are found in the MEN 2A and 2B syndromes and in familial MTC. For all these hereditary forms, mutations have been found in the R E T proto-oncogene. So far all MEN 2A associated mutations occurred within codons specifying cysteine residues in the 5' flanking region of transmembrane domain, while all MEN 2B patients examined showed a single mutation in a different domain of the R E T p r o t o - o n c o g e n e (see abstract Hofstra et al.). We looked for involment of the R E T proto-oncogene in the development of sporadic MTC by performing SSCP analysis for all 20 exons of the gene on 18 tumours. Using different SSCP conditions we could detect in several tumours the same mutations as have been found in patients of the MEN 2A and MEN 2B syndromes.
175
N-MYC REGULATION IN NEUROBLASTOMA.
Ngan Chinq Chenq, Huib Carom Alvin Chan, Mabel Beitsma, Peter van Sluis, Frank Speleman', Andries Westerveld, and Rogier Versteeg. Dept. of Human Genetics, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. "Dept. of Medical Genetics, University of Gent, Belgium.
Neuroblastoma is the most common extracranial solid turnout of childhood and comprises 8% of all childhood malignancies. A strong correlation between N-myc amplification and allelic loss of the lp36 chromosomal region was found. In 82 neuroblastomas, we found that 26 showed loss of lp36. All N-myc amplified tumors (16) belonged to this category. Therefore, the N-myc amplified tumours seem to be a subset of neuroblastomas with lp36 deletions. Deletion analysis in neuroblastoma cell lines showed that N-myc amplification is associated with large lp deletions, while in cell lines without N-myc amplification the deletions are smaller involving only the lp36 region. This indicates that lp harbours two distinctive neuroblastoma-associated genes: one distal gene with yet unknown function and a more proximal gene which is associated with N-myc amplification. To analyse this latter gene further, we performed cell fusion experiments. Two neuroblastoma cell lines, one with N-myc amplification and expression and the other without, were fused. Both cell lines have deletions of lp. The N-myc amplified line is deleted for 1p32->pter. The line without N-myc amplification has a deletion of lp36.1->pter. In the resulting hybrid cell lines the N-myc amplification is still present. Strikingly, the N-myc expression is totally switched off. This implicates that in the N-myc amplified line a factor controlling N-myc expression is
REARRANGEMENTS OF CHROMOSOMES 1 AND 10 GENERATE TRANSFORMING FUSION GENES DERIVED FROM TRK AND RET TYROSINE KINASES IN PAPILLARY THYROID CARCINOMAS. Marco A. Pierotti, Italia Bongarzone, Maria G. Borrello, Angela Greco, Gabriella Sozzi and Giuseppe Della Porta - lstituto Nazionale Tumori, Milan, Italy. In the last few years, we have reported an high frequency of tumorspecific rearrangements of the proto-oncogenes R E T and TRK, encoding tyrosine kinase membrane receptors, in human papillary thyroid carcinomas. By the combining use of cytogenetic and molecular approaches, we have determined that the oncogenic activation o f these genes is accomplished by the fusion of their tyrosine kinase domain with unlinked amino-terminal sequences following chromosomal rearrangements, mostly but not exclusively intrachromosomal, involving chromosome 10 and chromosome 1 in the case of RET and TRK, respectively. We have so far identified three different versions of RET (designated RET/ptcl, RET/ptc2 and RET/ptc3) and of TRK (named TRK-TI; TRK-T2 and TRKT3)-derived oncogenes. In particular, RET was found activated in 18 out of 52 cases of papillary thyroid carcinomas. In 10 of the 18 cases it resulted rearranged with an uncharacterized gene (H4/D10SI7) forming the transforming sequence RET/ptcl, in 2 cases with RI A regulatory subunit of pKA enzyme and in the last 6 cases with a novel gene designated ELE1. On the other end, TRK activation involved 3 case of rearrangement with tropomyosin gene (TPM3), 3 with TPR sequences and 1 with a newly identified gene designated TAG (Trk Activating Gene). A cytogenetic and molecular characterization of newly identified members (RET/ptc3 and TRK-T3) of the RET and TRK oncogene superfamily will be presented and the results discussed in a general model of oncogenesis mediated by the formation of fusion genes.
lost. The loss of control can apparently be complemented by the fusion partner with the short lp deletion. This suggests that a gene that can switch off N-myc expression is located proximal to lp36.1. Deletion of this gene could result in a certain level of N-
ThisworkispartiMlysupportedbyAIRCandCNRSpecialPr~ect "ACRO".
myc expression. Once the gene is expressed, amplification can further increase the expression, which could confer growth advantage to the cell.
A SINGLE MUTATION IN THE RET PROTO-ONCOGENE IS A S S O C I A T E D W I T H M U L T I P L E E N D O C R I N E N E O P L A S I A T Y P E 2B. Robert M.W. Hofstra',
Rudy M. Landsvater', Rein P. Stulp', Tineke Stelwagen', Giovanni R o m e o % Cornelis J.M. Lips" and Charles H.C.M. Buys v. "Dept. of Medical Genetics, U n i v e r s i t y of Groningen, Groningen, "Dept. of Internal Medicine and Pathology, Utrecht U n i v e r s i t y Hospital, Utrecht, The Netherlands, "Laboratorio di Genetica Molecolare, Instituto G. Gaslini, Genova, Italy. Multiple endocrine neoplasia type 2 (MEN 2) comprises three clinically distinct d o m i n a n t l y inherited cancer syndromes. MEN 2A patients develop m e d u l l a r y thyroid carcinoma (MTC) and phaeochromocytoma. MEN 2B patients show in addition ganglioneuromas of the gastro-intestinal tract and skeletal abnormalities. In familial MTC, only the thyroid is affected. Germline mutations of the R E T proto-oncogene have recently been reported in association with MEN 2A and familial MTC. All mutations occurred within codons specifying cysteine residues in the transition of the RET extracellular and transmembrane domains. We now show that in all (8) MEN 2B patients examined, a single mutation has been found in a different domain of the R E T proto-oncogene.
M U T A T I O N S IN T H E R E T P R O T O - O N C O G E N E IN SPORADIC MEDULLARY THYROID CARCINOMAS.
Tineke Stelwaqen', Robert M.W. Hofstra', Rein P. Stulp', Harm Nijveen', Giovanni Romeo A, Rudy M. Landsvater',Cornelis J.M. Lips" and Charles H.C.M. Buys','Dept. of Medical Genetics, University of Groningen, Groningen, "Dept. of Internal Medicine and Pathology, Utrecht University Hospital, Utrecht, The Netherlands, "Laboratorio di Genetica Molecolare, Instituto G. Gaslini, Genova, Italy. Medullary thyroid carcinoma (MTC) occur both in a sporadic and hereditary form. The hereditary MTCs are found in the MEN 2A and 2B syndromes and in familial MTC. For all these hereditary forms, mutations have been found in the R E T proto-oncogene. So far all MEN 2A associated mutations occurred within codons specifying cysteine residues in the 5' flanking region of transmembrane domain, while all MEN 2B patients examined showed a single mutation in a different domain of the R E T p r o t o - o n c o g e n e (see abstract Hofstra et al.). We looked for involment of the R E T proto-oncogene in the development of sporadic MTC by performing SSCP analysis for all 20 exons of the gene on 18 tumours. Using different SSCP conditions we could detect in several tumours the same mutations as have been found in patients of the MEN 2A and MEN 2B syndromes.