A novel polymerase chain reaction (PCR) technique has been combined with chromosome flow sorting to characterise two lymphoblastoid cell lines and one medullary thyroid carcinoma cell line carrying translocations close to the locus for multiple endocrine neoplasia type 2A (MEN 2A). Five hundred copies of the derivative chromosome(s) were flow sorted from each cell line and amplified by degenerate oligonucleotide-primed-polymerase chain reaction (DOP-PCR). This generated pools of DNA sequences corresponding to the abnormal chromosomes, which were then used as probes in fluorescence in situ hybridisation (FISH) experiments on normal metaphase cells. The resultant chromosome paints revealed the portions of the normal chromosomes related to those involved in the translocations. By this technique, translocation breakpoints in bands p15, q11.2, and q21 of chromosome 10 were defined in the above cell lines, in two cases refining previous cytogenetic data. This study shows that flow sorting of aberrant chromosomes and chromosome painting can be used as a rapid aid to cytogenetic analysis, particularly in cases of difficult karyotypes, such as tumours. Furthermore, the DOP-PCR technique described here will have applications to other areas of genome analysis, such as cloning of new markers; its design will allow a general and representative amplification to occur from any starting DNA in any species.

Medullary thyroid carcinoma (MTC) occurs sporadically or as part of the inherited cancer syndrome multiple endocrine neoplasia (MEN) type 2. In MEN 2A, germline missense mutations are found in one of five cysteine codons within exons 10 and 11 in the extracellular domain of the RET protooncogene. In MEN 2B, germline mutations occur in codon 918 (exon 16) within the catalytic core of the tyrosine kinase domain. To determine if RET mutations similar to those in MEN 2A and 2B play a role in the pathogenesis of sporadic MTC, we analysed 71 sporadic tumours comprising 68 primary tumours and three cell lines, for mutations in RET exons 10, 11, and 16. We found that 23% of sporadic MTC had RET codon 918 mutations, while only 3% had exon 10 mutations, and none had mutations in exon 11. We found no exon 16 mutations in MTC from 14 MEN 2A cases. Thus, exon 10 and 11 mutations, commonly found in familial MTC and MEN 2A, rarely occur in sporadic MTC; somatic mutation of RET codon 918 appears to play a role in the tumourigenesis of a significant minority of sporadic MTC but not MEN 2A tumours. In addition to their biological interest, these findings may have some clinical application in determining whether a patient presenting with isolated MTC is truly sporadic or is part of an inherited cancer syndrome.

Thirteen male newborns of a family spanning five generations revealed a syndrome consisting in elevated birth weight and length, a disproportionately large head with coarse, distinctive facies, short neck, slight obesity, and broad, short hands and feet. The affected who reached adulthood attained heights of about 2 m; their unusual facial and general appearance and the clumsiness of all their motions, remarkable during infancy and childhood, had become somewhat less conspicuous. In all but one affected individual, intellectual development was normal. In two index cases neither clinical nor laboratory evaluations revealed a basic defect. X-linked recessive inheritance is most probable.

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Hereditary and sporadic medullary thyroid carcinoma (MTC) are closely associated with RET proto-oncogene mutations. However, the role of additional changes in the tumor genomes remains unclear. Our objective was the identification of chromosomal regions involved in MTC tumorigenesis and to assess their significance by using MTC-derived cell lines. We used array-CGH (comparative genomic hybridization) to map chromosomal imbalances in 52 primary tumors and ten metastases. Eleven tumors (11/52, 21%) were hereditary and 41 (41/52, 79%) were sporadic. Among the latter, 15 tumors (15/41, 37%) harbored RET mutations. Furthermore, we characterized five MTC cell lines in detail and evaluated the tumorigenicity by severe combined immunodeficiency (SCID)-mouse experiments. Most MTCs had only few copy number changes, and losses of chromosomes 1p, 4q, 19p and 22q were observed most frequently. The number of chromosomal aberrations increased in metastases. Twenty-three percent (12/52) of the primary tumors did not even show any chromosomal gains and losses. We injected three cell lines (two of these were without chromosomal changes and pathogenic RET mutations) into immune deficient SCID mice, and in each case, we observed rapid tumor growth at the injection sites. Our data suggest that MTCs-in contrast to most other tumor entities-do not acquire a multitude of genomic imbalances. SCID mouse experiments performed with chromosomally normal cell lines and without RET mutations suggest that presently unknown submicroscopic genomic changes are sufficient in MTC tumorigenesis.Human medullary thyroid carcinoma (MTC) is a calcitoninproducing tumor originating from the parafollicular C-cells. It constitutes about 5-8% of all thyroid cancers and may occur sporadic (70-80% of cases) or hereditary (20-30%). 1Hereditary MTC is inherited in an autosomal-dominant pattern, either as familial MTC (FMTC) without other endocrinopathies or as part of multiple endocrine neoplasia syndrome type 2A (MEN-2A), or in multiple endocrine neoplasia type 2B (MEN-2B). 2As the disease may develop over many years without clinical signs, lymph node metastasis are documented in more than 50% of patients, and distant metastasis are found in at least in one quarter of patients at the time of diagnosis. With 5-and 10-year survival reported at 86% and 78%, respectively, prognosis is less favorable than for follicular cellderived carcinomas. Patients with distant metastases have only a 35% survival after 5 years. MTC, both in patients and in preclinical models, is resistant to chemotherapy and radiation therapy. Surgical removal of all malignant tissue is the only potentially curative treatment in localized disease.1 Management guidelines for treatment of MTC were recently published by the American Thyroid Association. 3MTC is closely associated with mutations in the REarranged during Transfection (RET) proto-oncogene in chromosome 10q11.2. The hereditary variants MEN-2/FMTC are associated with activating germline point mutations in the RET proto-oncogene and near...