Evidence for a 3p25 breakpoint hot spot region in thyroid tumors of follicular origin.

Epithelial tumors of the thyroid are cytogenetically well-investigated tumors. So far, the main cytogenetic subgroups, characterized by trisomy 7 and by rearrangements of either 19q13 or 2p21, respectively, have been described. Recently, we have been able to describe the involvement of a novel gene called THADA in benign thyroid lesions with 2p21 rearrangements. Other fusion genes found in thyroid lesions are RET/PTC and PAX8/PPAR(gamma). The latter occurs in follicular thyroid carcinomas with a t(2;3)(q13;p25). Here we present molecular-cytogenetic and cytogenetic investigations on a follicular thyroid adenoma with a t(2;20;3)(p21;q11.2; p25). In this case, an intronic sequence of PPAR(gamma) is fused to exon 28 of THADA. We used BAC clones containing the genomic sequence of PPARgamma for fluorescence in situ hybridization to confirm the localization of the breakpoint within intron 2 of PPAR(gamma) . Our findings suggest that the close surrounding of PPAR(gamma) is a breakpoint hot spot region, leading to recurrent alterations of this gene in thyroid tumors of follicular origin including carcinomas as well as adenomas with or without involvement of PAX8.

Delineation of a 150-kb breakpoint cluster in benign thyroid tumors with 19q13.4 aberrations.

Structural rearrangements involving the long arm of chromosome 19 characterize a cytogenetic subgroup of benign thyroid tumors and constitute one of the most frequent specific chromosome abnormalities in epithelial tumors. Recently, we have been able to narrow down the breakpoint region affected in two cell lines to a region covered by a single PAC clone. Close to that region a candidate gene has been identified which we tentatively referred to as RITA (Rearranged In Thyroid Adenomas) now named ZNF331 according to HUGO nomenclature. However, the results had been obtained on two cell lines only making it necessary to extend the studies to a larger number of tumors including primary material. Herein, we have used four further primary tumors showing translocations involving 19q13 for fluorescence in situ hybridization (FISH) mapping studies using a variety of molecular probes from a 470-kbp cosmid/BAC contig. Ten new STSs were characterized and physically mapped within an EcoRI restriction map. The results enabled us to define an approximately 150-kbp breakpoint cluster region of the 19q13 aberrations in benign thyroid tumors flanked by two newly established STS markers.

Chromosomal rearrangements leading to abnormal splicing within intron 4 of HMGIC?

Fusion of the high-mobility group protein gene HMGIC to other genes due to chromosomal rearrangements occurs in a variety of human benign tumors. In contrast to genes clearly derived from other chromosomes, some of the ectopic sequences fused to HMGIC have been assigned to chromosome 12 by CASH (chromosome assignment using somatic cell hybrids) analyses and thus can be assumed either to result from alternative splicing or to represent true ectopic sequences derived from other genes on chromosome 12. In an attempt to identify the ectopic sequences fused to this exon, we have sequenced the entire intron 4. Four of seven ectopic sequences previously described to be fused to exon 4 of HMGIC in different tumors were found to be located within intron 4 of the gene and thus are due to abnormal splicing. As for a mechanism explaining this observation, it can be suggested that breakpoints of chromosomal aberrations not directly disrupting HMGIC may induce small genomic alterations in their vicinity and thus facilitate abnormal splicing. The latter mechanism may underlie the development of part of the neoplasms characterized by 12q14--15 rearrangements.

Molecular cytogenetic investigations define a subgroup of thyroid adenomas with 2p21 breakpoints clustered to a region of less than 450 kb.

Structural rearrangements involving chromosome band 2p21 characterize a cytogenetic subgroup of benign thyroid tumors. To narrow down the breakpoints of these aberrations, we established two cell lines from benign thyroid tumors showing translocations involving 2p21. These two cell lines and one additional primary tumor were used for FISH-studies with 18 BAC clones. All breakpoints were mapped to a cluster of about 450 kb.

A 12q13 translocation involving the HMGI-C gene in richter transformation of a chronic lymphocytic leukemia.

We report a case of Richter transformation of a chronic lymphocytic leukemia with a 12q13 translocation involving the HMGI-C gene. Fluorescence in situ hybridization analysis with the use of two different cosmid pools spanning the entire HMGI-C region showed that the breakpoint on chromosome 12 was located in the HMGI-C gene, presumably within intron 3. In fact, the 3' region of HMGI-C had been translocated to a derivative chromosome 6. This translocation was not visible at the cytogenetic level. Immunohistochemical analysis performed on the bone marrow smear demonstrated the expression of the HMGI-C protein specifically in the blasts, suggesting that the aberrant expression of the HMGI-C gene might have an important role in the process of leukemogenesis.

A KRAB zinc finger protein gene is the potential target of 19q13 translocation in benign thyroid tumors.

In an attempt to identify the target gene of specific translocations involving chromosomal band 19q13 in benign follicular thyroid tumors, we have used two cell lines derived from benign thyroid tumors showing translocations with 19q13 breakpoints for fluorescence in situ hybridization mapping studies with cosmid and PAC clones located in a 400-kbp region. The breakpoints of the chromosome 19 abnormalities mapped within a 140-kb segment covered by a single PAC clone. Sequencing of part of this PAC clone allowed us to establish the cDNA sequence and the genomic structure of a candidate gene located in close vicinity to the breakpoints. The gene that we tentatively refer to as RITA (rearranged in thyroid adenomas) belongs to the KRAB zinc finger protein coding genes. From our results we have concluded that in the two cell lines investigated the breaks have occurred either within the 5' untranslated region of RITA or in its close 5' vicinity. By Northern blot analyses two transcripts of about 4.7 kbp and 5 kbp were detected in normal thyroid tissue as well as in other normal tissues tested. An additional 2.1-kbp transcript was found only in testicular tissue. In contrast to all normal tissues, both cell lines with 19q aberrations expressed larger transcripts of approximately 5.5 kbp and 6.2 kbp. From the close vicinity to the breakpoint region, the expression patterns of the gene, and its type, we consider RITA a strong candidate target gene of the specific 19q aberrations in benign thyroid tumors.

The expression pattern of the Hmgic gene during development.

The technique of RNA in situ hybridization to mouse embryo sections from different developmental stages was used to perform a detailed analysis of the expression pattern of the gene for the architectural chromatin factor Hmgic. At early stages of fetal development (day 9.5 post conceptionem), Hmgic is expressed at a high rate throughout the whole embryo. In the second half of development, the pattern of expression becomes more restricted. Expression is found in mesenchymal derivatives, which differentiate into cartilage or muscle, in epithelial cell layers of the lung, pancreas, submandibular gland, and vibrissae, and in some special parts of the central nervous system. The expression pattern of Hmgic was compared with the previously reported studies of Hmgiy gene expression, another member of the Hmgic protein family, and with the expression of histone H4, Hist4, which is representative of cellular proliferation stages. In some tissues the pattern of expression for both factors coincides, but in others the expression is different. Hmgic expression correlates throughout fetal development with high proliferative activity. In contrast, Hmgiy is expressed also in tissues with no proliferative activity, such as the cortical plate of the telencephalon and the spinal cord at late gestational stages.

Breakpoints of 19q13 translocations of benign thyroid tumors map within a 400 kilobase region.

Structural rearrangements involving the long arm of chromosome 19 characterize a cytogenetic subgroup of benign thyroid tumors. To localize the breakpoint of the 19q13 aberrations, we have established three cell lines derived from benign thyroid tumors showing translocations in this region. We have used these cell lines and four additional primary tumors with 19q13 abnormalities for fluorescence in situ hybridization (FISH) mapping studies with ten cosmid clones located between the molecular markers POLD1 and TNNT1. The breakpoints of all chromosome 19 abnormalities mapped within a 400 kb region.

Expression of HMGI-C, a member of the high mobility group protein family, in a subset of breast cancers: relationship to histologic grade.

The high-mobility-group (HMG) protein gene HMGI-C is apparently involved in the genesis of a variety of benign human solid tumors with rearrangements of chromosomal region 12q14-15 affecting the HMGI-C gene. So far, no expression of HMGI-C has been found in adult tissues, and no data are available on the expression of HMGI-C in primary human malignant tumors of epithelial origin. Therefore, we analysed the HMGI-C expression patterns in 44 breast cancer samples and 13 samples of nonmalignant adjacent tissue by hemi-nested reverse transcriptase-polymerase chain reaction for HMGI-C expression. There was no detectable expression of HMGI-C in any nonmalignant adjacent breast tissues analyzed. In contrast, we found expression in 20 of 44 breast cancer samples investigated. In invasive ductal tumors, expression was noted predominantly in tumors with high histologic grade: 17 of 21 breast cancer samples with histologic grade 3 but only three of 16 samples with histologic grades 1 or 2 showed expression of HMGI-C. In addition, all seven lobular breast cancer samples tested did not express HMGI-C. From these results, we concluded that HMGI-C expression may be of pathogenetic or prognostic importance in breast cancer.

A characteristic sequence of trisomies starting with trisomy 7 in benign thyroid tumors.

The cytogenetic results from a series of 113 thyroid hyperplasias and adenomas are reported; 15 showed clonal karyotypic alterations. In addition to a group showing translocations involving 19q13, another subset of lesions characterized by polysomies can be found. Based on our own cases belonging to this subset and a review of the cases reported in the literature, we conclude that the characteristic feature of this group is a sequence that always starts with trisomy 7, but that sometimes even leads to chromosome numbers in the hypertriploid range. This subset of thyroid tumors may be an example of a more common genetic pathway in human solid tumors.