Altered expression of key cell cycle regulators in renal cell carcinoma associated with Xp11.2 translocation.

Renal cell carcinoma (RCC) is a rare tumor in the pediatric population. Recently, a phenotypically and genetically distinct kidney carcinoma, mainly prevalent in children and associated with an Xp11.2 translocation or TFE3 gene fusion, has been described. It has been advanced that in this subtype of RCC, there is an accumulation of cyclin D1, cyclin D3, and p21 ((wafl/cip1)). The aim of the present study was to figure out in two pediatric RCC recently diagnosed in our department (one clear cell-type RCC and one TFE3-positive RCC) whether those features are indeed specific of the latter tumor or occur in pediatric RCC irrespective of the tumor type. The following immunostains were performed in both cases: Ki67, p16(ink4a), p21 ((wafl/cip1)), p27(kip1), p53, p63, mdm2, cyclin D1, cyclin D3, TFE3, CD10, vimentin, E-cadherin, and RCC-antigen. We observed in the TFE3-positive carcinoma an intense immunoreaction for p21 ((wafl/cip1)), cyclin D1, and cyclin D3, without expression for p53, p16, p27(kip1), and mdm2, whereas the immunoexpression profile observed in the classic RCC was similar to that of clear cell, adult-type RCC. Our study confirms that TFE3-positive RCC exhibits a deregulation of the cell cycle apparently unrelated to the young age of the patients.

MSH6 germline mutations in early-onset colorectal cancer patients without family history of the disease.

Germline MLH1 and MSH2 mutations are scarce in young colorectal cancer patients with negative family history of the disease. To evaluate the contribution of germline MSH6 mutations to early-onset colorectal cancer, we have analysed peripheral blood of 38 patients diagnosed with this disease before 45 years of age and who presented no family history of hereditary nonpolyposis colorectal cancer-related cancers. Blood samples from 108 healthy volunteers were analysed for those genetic alterations suspected to affect the function of MSH6. Of the seven (18.4%) MSH6 alterations found, we have identified three novel germline mutations, one 8 bp deletion leading to a truncated protein and two missense mutations resulting in the substitution of amino acids belonging to different polarity groups. High-frequency microsatellite instability was found in the patient with the MSH6 deletion, but not in the other 27 carcinomas analysed. No MLH1 promoter methylation was detected in tumour tissue. Our findings suggest that germline MSH6 mutations contribute to a subset of early-onset colorectal cancer. Further studies are warranted to understand the genetic and environmental factors responsible for the variable penetration of MSH6 germline mutations, as well as to identify other causes of early-onset colorectal cancer.

Cleft lip/palate and CDH1/E-cadherin mutations in families with hereditary diffuse gastric cancer.

We report the association of CDH1/E-cadherin mutations with cleft lip, with or without cleft palate (CLP), in two families with hereditary diffuse gastric cancer (HDGC). In each family, the CDH1 mutation was a splicing mutation generating aberrant transcripts with an in-frame deletion, removing the extracellular cadherin repeat domains involved in cell-cell adhesion. Such transcripts might encode mutant proteins with trans-dominant negative effects. We found that CDH1 is highly expressed at 4 and 5 weeks in the frontonasal prominence, and at 6 weeks in the lateral and medial nasal prominences of human embryos, and is therefore expressed during the critical stages of lip and palate development. These findings suggest that alteration of the E-cadherin pathway can contribute to human clefting.

Pathological exon skipping in an HNPCC proband with MLH1 splice acceptor site mutation.

One of the most commonly mutated mismatch repair genes in human nonpolyposis colorectal cancer (HNPCC) is MLH1. We identified a splice site mutation in MLH1 in a colorectal cancer proband (T-to-A at position -11 of intron 1 splice acceptor) and investigated its functional consequences by RT-PCR, using lymphocyte mRNA from the proband, two noncarrying siblings, and one unrelated individual. Subcloning of PCR products followed by sequencing of individual clones revealed increased transcript heterogeneity in the mutation carrier, attributable to the presence of a variety of mRNA forms lacking exon 2, or combinations of exons 2, 4, 6, 9, and 10. The full-length transcript subcloned from the mutation carrier was detected with a much reduced frequency, suggesting that only the wild-type allele produced functional MLH1 mRNA. The three noncarriers expressed some previously described transcripts and several novel variants, but none that lacked exon 2. The results are consistent with the hypothesis that this splice site mutation causes skipping of MLH1 exon 2 in a large proportion of mRNA transcripts derived from the mutated allele. Such an observation strengthens the case for identifying the mutation as pathogenic in this HNPCC family, which is of interest given the rarity of exon skipping defects resulting from splice acceptor site mutations outside the invariant AG dinucleotide.

Eleven novel APC mutations identified in Portuguese FAP families.

Germline mutations of the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis (FAP), an autosomal dominant predisposition to colorectal cancer. In the present study we screened all of the exons of the APC gene in individuals belonging to 85 Portuguese FAP families. We here report eleven novel mutations which are predominantly frameshifts or single base substitutions, resulting in premature stop codons. Hum Mutat 16:178, 2000.

Detection of prognostic significant translocations in childhood acute lymphoblastic leukaemia by one-step multiplex reverse transcription polymerase chain reaction.

The search for chromosomal translocations in de novo cases of childhood acute lymphoblastic leukaemia (ALL) is crucial for the selection of the appropriate therapeutic protocol. In this work, we describe a new method - one-step multiplex reverse transcription polymerase chain reaction (RT-PCR) - to screen for prognostic significant translocations in childhood ALL. Our approach involves a single PCR reaction for the simultaneous detection of the molecular rearrangements resulting from the t(9;22), t(12;21), t(4;11) and t(1;19), with a turnaround time of less than 24 h. This assay proved to be highly sensitive, specific, reproducible and easy to implement in a routine genetics laboratory.

Four novel MSH2 / MLH1 gene mutations in portuguese HNPCC families.

Hereditary non-polyposis colorectal cancer (HNPCC) is considered to be determined by germline mutations in the mismatch repair (MMR) genes, especially MSH2 and MLH1. While screening for mutations in these two genes in HNPCC portuguese families, 3 previously unreported MSH2 and 1 MLH1 mutations have been identified in families meeting strict Amsterdam criteria. Hum Mutat 15:116, 2000.

Significance of trisomy 7 and 12 in thyroid lesions with follicular differentiation: a cytogenetic and in situ hybridization study.

In a group of benign and malignant follicular thyroid lesions, previously analyzed by conventional cytogenetics, single- and double-target fluorescence in situ hybridization studies with pericentromeric probes specific for chromosomes 7 and 12 were performed, by using isolated nuclei from paraffin-embedded specimens of: 11 goiters, 21 adenomas, 9 follicular carcinomas, and adjacent normal thyroid tissue. Nonisotopic in situ hybridization with the same probes was used in 4-microm histologic sections of four follicular carcinomas. By fluorescence in situ hybridization analysis, the percentage of goiters, adenomas, and follicular carcinomas with gains of No. 7 was 18.2%, 52.4%, and 66.0%, respectively, and with gains of No. 12 was 9.0%, 42.9%, and 66.0%, respectively. The percentage of the same lesions (goiters, adenomas, carcinomas) exhibiting polysomies of No. 7 and No. 12, as assessed by cytogenetic analysis, was 5.0% and 0.0%, 20.0% and 20.0%, and 15.8% and 10.5%, respectively. Numerical alterations of these chromosomes were not observed in normal tissue. These findings reveal that gain of chromosomes 7 and 12 is a characteristic of the morphologically altered thyroid tissue; polysomies of chromosomes 7 and 12 are more frequent in thyroid lesions than it can be detected by conventional cytogenetic studies; the increasing frequency of polysomies of chromosomes 7 and 12 from hyperplastic lesions to benign and malignant tumors seem to substantiate the existence of a multistep pathway, ie, normal thyroid --> goiter --> adenoma --> follicular carcinoma in the pathogenesis of some thyroid neoplasms.

Cytogenetic and fluorescence in situ hybridization studies in a case of anaplastic thyroid carcinoma.

Cytogenetic analysis of a case of anaplastic thyroid carcinoma revealed multiple numerical and structural chromosomal changes, including a der(9) add(9)(p22)hsr(9)(p?). Fluorescence in situ hybridization (FISH) studies performed to identify the genetic nature of this derivative chromosome showed that both the additional material and the hsr region were composed of only chromosome 9 sequences and that the C-ABL oncogene was not one of the genes harbored at the hsr region. We suggest that amplification of gene(s) located at chromosome 9, other than the C-ABL, may play a significant role in anaplastic evolution of thyroid carcinomas.

Follicular thyroid carcinoma: chromosome analysis of 19 cases.

Short-term cultures of 19 follicular thyroid carcinomas were examined cytogenetically. Clonal chromosomal changes were detected in 12 tumors. Two follicular carcinomas had only numerical alterations: one with a hyperdiploid karyotype with trisomies/polysomies of chromosomes 7 and 12, similar to the karyotypes previously identified in a sub-group of benign thyroid lesions, and the other with monosomy 20. In the remaining ten cases several structural chromosome anomalies were found. Loss of the short arm of chromosome 3 was observed in one tumor. In two widely invasive and metastasizing follicular carcinomas there was a t(7;8)(p15;q24) as the sole abnormality in one case and a der(8)t(7;8)(p15;q24) together with other cytogenetic alterations in the other case. This finding suggests that t(7;8)(p15;q24) may be related to an aggressive behavior of follicular thyroid carcinomas.

Cytogenetic investigations of 340 thyroid hyperplasias and adenomas revealing correlations between cytogenetic findings and histology.

Cytogenetic analyses were performed on 340 follicular thyroid adenomas and goiters after short-term culture. Clonal chromosomal changes were found in 67 cases. Trisomy 7 as the sole abnormality or along with other trisomies was the most frequent type of aberration (19 cases). Other recurrent numerical changes were loss of chromosome 22 (4 cases) and the second X or the Y chromosome (5 cases). Translocations involving 19q13 (12 cases) were frequent structural chromosomal changes. Dicentric chromosomes or telomeric associations were frequent in goiters (12 cases). After a histopathologic classification of all cases, we have correlated the cytogenetic findings with the histology of the tumors. Only 8.4% of the goiters showed clonal abnormalities, whereas 44.9% of the adenomas revealed clonal abnormalities. Furthermore, simple clonal changes were predominantly found in goiters and complex changes in adenomas. The most impressive correlation was found in the group of lesions with trisomy 7. Although all but one lesion with one or two additional trisomies were goiters, those having three or more additional trisomies were all adenomas or adenomatous goiters.

[Cytogenetic changes in benign thyroid gland hyperplasias and adenomas correlated with histology]. / Zytogenetische Veränderungen bei benignen Schilddrüsenhyperplasien und Adenomen korrelieren mit der Histologie.

In order to elucidate cytogenetic changes associated with the development of benign growth of follicular epithelial cells of the thyroid, cytogenetic analyses were performed on 340 follicular thyroid adenomas and goiters after short-term culture. Clonal chromosome changes were found in 67 cases. Trisomy 7 as the sole abnormality or along with other trisomies was the most frequent type of aberration (19 cases). Other recurrent numerical changes were loss of chromosome 22 (4 cases) and the second X or the Y chromosome (5 cases). Translocations involving 19q13 (12 cases) were frequent structural chromosome changes. After a histopathological classification of all cases, we have correlated the cytogenetic findings with the histology of the tumors. Only 8.4% of the goiters showed clonal abnormalities whereas 44.9% of the adenomas revealed clonal abnormalities. Furthermore, simple clonal changes were predominantly found in goiters and complex changes in adenomas. The most impressive correlation was found in the group of lesions with trisomy 7: Whereas all but one lesion with one or two additional trisomies were goiters, those having three or more additional trisomies were all adenomas or adenomatous goiters.

Identification of two distinct regions of deletion at 6q in gastric carcinoma.

Loss of heterozygosity (LOH) affecting the long arm of chromosome 6 has been found repeatedly in human cancers. Recently, our group reported that del(6)(q21-22-->qter) was the most consistent structural cytogenetic abnormality in gastric carcinomas. To determine more precisely the deleted region, we studied 51 tumors with 9 polymorphic markers on this chromosome arm. LOH of one or more markers was found in 39% of the tumors. LOH at region 6q22.3 was detected in 50% of informative tumors and at 6q26-q27 in 37% of informative tumors. By comparative analysis of LOH regions, we identified two separate regions of overlapped deletions at 6q, one between 6q16.3-q21 and 6q22.3-q23.1, another distal to 6q23-q24. A comparison of clinicopathologic features of gastric carcinomas with and without LOH at 6q revealed statistically significant or suggestive differences between LOH and young age of the patients and proximal location of the tumors. The two informative early gastric carcinomas both showed LOH at 6q. The occurrence of LOH at 6q was similar in all histological types. We conclude that two distinct regions at 6q appear to be involved in the early stages of gastric carcinogenesis.

Detection of numerical alterations for chromosomes 7 and 12 in benign thyroid lesions by in situ hybridization. Histological implications.

Polysomies of chromosomes 7 and 12 have been frequently observed by conventional cytogenetics in a subgroup of thyroid follicular adenomas and in some cases of thyroid goiters. To further study possible cytogenetic similarities between these two types of thyroid lesions, we have used fluorescence in situ hybridization (FISH) to detect polysomies of chromosomes 7 and/or 12 in isolated nuclei from frozen and paraffin-embedded material of goiters and thyroid follicular adenomas and compared results with previous ones obtained by flow cytometry and conventional cytogenetics. With a set of two alpha-satellite DNA probes specific for the centromeric regions of chromosomes 7 and 12, used either separately (single-target fluorescence in situ hybridization) or simultaneously (double-target fluorescence in situ hybridization), we detected polysomies of chromosome 7 in 35.7% of the thyroid follicular adenomas and in 10.7% of the goiters. Polysomies of chromosome 12 were detected in 29.6% of the thyroid follicular adenomas and 6.7% of the goiters. The significantly higher frequency of adenomas with numerical alterations for chromosomes 7 and/or 12 supports the idea of a biological continuum and karyotypic evolution between both lesions. It is also noteworthy that polysomies of chromosomes 7 and/or 12 were observed only in lesions with an exclusive (or predominant) microfollicular histological component, as detected by enzymatic in situ hybridization on frozen sections.

Increasing levels of MYC and MET co-amplification during tumor progression of a case of gastric cancer.

The cytogenetic study of a nodal metastasis from a gastric carcinoma, after two passages in nude mice, revealed a large number of double minutes. Comparative genomic in situ hybridization (CGH) analysis using DNA extracted from this xenograft revealed the existence of three clear amplification units that originated from the chromosomal subregions 6q24-25, 7q31-32, and 8q24 in the xenograft DNA. Similar, though less prominent, CGH results were found with DNAs extracted from the primary tumor and its metastasis, implying that the same amplicons were also present, albeit less abundantly, in the DNAs of these neoplastic tissues. Southern analysis of the second-passage xenograft detected 18- and 10-fold amplification of MET (located at 7q31) and MYC (located at 8q24), respectively. The retrospective study of the first passage of the xenograft, as well as of the metastatic and primary tumors before xenografting, showed amplification levels of MET of, respectively, 12-, 9-, and 5-fold and MYC of, respectively, 8-, 7-, and 5-fold. Our results suggest that increased levels of co-amplification of MYC and MET correlate with enhanced growth potential in this case of gastric carcinoma.

Cytogenetic findings in 31 papillary thyroid carcinomas.

Chromosome studies performed on 31 papillary thyroid carcinomas (PTCs) revealed clonal numerical and structural abnormalities in 12 tumors. The numerical clonal aberrations found were trisomy 2, trisomy 7, and loss of the Y chromosome. A nonrandom telomeric association, tas(15;16)(p13;p13), was observed in one carcinoma. Structural alterations with a breakpoint at 10q11.2 were detected in two tumors. Other chromosomes involved in rearrangements were chromosomes 1, 2, 3, 5, 7, 9, 11, 12, and 14. The observation of clonal changes of chromosome 2 [i(2)(q10) and trisomy 2] in two tumors, which were both histologically classified as tall-cell PTC variants, suggests that gain of 2q may be important in the development of this morphological variant.