Incidence of chromosome numerical changes in multiple myeloma: fluorescence in situ hybridization analysis using 15 chromosome-specific probes

Fue el primer trabajo precursor de los estudios con FISH en pacientes con Mieloma múltiple continuando la exitosa línea de investigación en mieloma del grupo de Salamanca, y por añadidura, del Grupo español de mieloma. La metodología FISH es hoy en día la principal herramienta de pronóstico en mieloma múltiple en todo el mundo.[EN]The presence of complex karotypes with frequent numerical and structural abnormalities has been reported in 20 to 50% of multiple myeloma (MM) patients. This variability is mainly due to the difficulty of conventional cytogenetics to obtain tumor metaphases representative of all possible neoplastic clones in MM. To gain insight into the real incidence of numerical chromosome changes in MM we have studied by fluorescence in situ hybridization technique 15 different human chromosomes, 1, 3, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, X, and Y, in a series of 52 MM patients. In all cases, the DNA index assessed by a propidium iodide/CD38 double-staining technique with flow cytometry was simultaneously investigated for correlation, with fluorescence in situ hybridization results. Additional aims of this study were 1) to analyze whether the abnormalities detected were common to all plasma cells or were present in only a subpopulation of tumor cells, 2) to explore changes caused by disease progression, and 3) to establish possible associations among the altered chromosomes. Although the overall incidence of numerical abnormalities was 67%, this frequency increased to 80% in the 41 cases in which 7 or more chromosomes were analyzed. Trisomies were significantly more common than monosomies (84% versus 16%). Chromosomes 9 and 15 were the most frequently altered (52% and 48% of cases, respectively), with all of their abnormalities corresponding to trisomies. The most frequent losses involved chromosomes 13 (26%) and X in females (32%). Other common numerical changes corresponded to chromosomes 1 (39%), 11 (37%), 6 (32%), 3 (31%), 18 (29%), 7 (28%), and 17 (22%). By contrast, chromosomes 8(13%), 10(8%), and 12(3%) were rarely altered. DNA aneuploidy by flow cytometry was detected in 67% of patients, and a high degree of correlation was observed between the DNA index obtained by flow cytometry and the chromosome index derived from fluorescence in situ hybridization studies, calculated according to two mathematical formulas (coefficient of correlation of 0.82 and 0.91 when at least 7 or 12 chromosomes were considered, respectively). The frequency of numeric chromosome aberrations was higher in those patients with progressive disease and, interestingly, trisomy of chromosome 8 was exclusively detected in this latter group of patients. Our study shows that, with the exception of chromosome 8, a possible marker of clonal evolution, the numeric chromosome changes are present in nearly all malignant plasma cells (r > 0.84). Finally, frequent associations between chromosomal aberrations were observed (ie, chromosomes 6, 7, 9, and 17; 7 and 15; and 11 and 17). By excluding them, it was found that two triple combinations of chromosome-specific probes, chromosomes 1 and 9 together with either chromosome 13 or 15, could be a useful marker for detection of residual disease, as it permits the identification of most MM patients displaying numerical changes.University Hosptial of Salamanca Universidad de SalamancaHospital Universitario de Salamanc

Cancer-associated mutations in chromatin remodeler hSNF5 promote chromosomal instability by compromising the mitotic checkpoint

The hSNF5 subunit of human SWI/SNF ATP-dependent chromatin remodeling complexes is a tumor suppressor that is inactivated in malignant rhabdoid tumors (MRTs). Here, we report that loss of hSNF5 function in MRT-derived cells leads to polyploidization and chromosomal instability. Re-expression of hSNF5 restored the coupling between cell cycle progression and ploidy checkpoints. In contrast, cancer-associated hSNF5 mutants harboring specific single amino acid substitutions exacerbated poly- and aneuploidization, due to abrogated chromosome segregation. We found that hSNF5 activates the mitotic checkpoint through the p16(INK4a)-cyclinD/CDK4-pRb-E2F pathway. These results establish that poly- and aneuploidy of tumor cells can result from mutations in a chromatin remodeler

A novel t(6;14)(q25∼q27;q32) in acute myelocytic leukemia involves the BCL11B gene

Cytogenetic studies in a patient with acute myelocytic leukemia (AML) revealed as the sole karyotypic alteration a half-cryptic rearrangement, identified with 48-color combined binary ratio-labeled fluorescence in situ hybridization (pq-COBRA-FISH) as a reciprocal t(6;14)(q?;q?). The breakpoints were later assigned on the basis of G-banding to t(6;14)(q25∼q26;q32). FISH experiments using genomic probes showed that the breakpoint on 14q32.2 was within bacterial artificial chromosome RP11-782I5 and revealed BCL11B as the only candidate gene in the region. BCL11B is a homolog to BCL11A (2p13), a highly conserved gene implicated in mouse and human leukemias. To our knowledge, this is the first report implicating BCL11B in hematological malignancies. Because of lack of material, the translocation partner remains unknown

A Novel Strategy for Human Papillomavirus Detection and Genotyping with SybrGreen and Molecular Beacon Polymerase Chain Reaction

Human papillomaviruses (HPVs) play an important role in the pathogenesis of cervical cancer. For identification of the large number of different HPV types found in (pre)malignant lesions, a robust methodology is needed that combines general HPV detection with HPV genotyping. We have developed for formaldehyde-fixed samples a strategy that, in a homogenous, real-time fluorescence polymerase chain reaction (PCR)-based assay, accomplishes general HPV detection by SybrGreen reporting of HPV-DNA amplicons, and genotyping of seven prevalent HPV types (HPV-6, -11, -16, -18, -31, -33, -45) by real-time molecular beacon PCR. The false-positive rate of the HPV SybrGreen-PCR was 4%, making it well suited as a prescreening, general HPV detection technology. The type specificity of the seven selected HPV molecular beacons was 100% and double infections were readily identified. The multiplexing capacity of the HPV molecular beacon PCR was analyzed and up to three differently labeled molecular beacons could be used in one PCR reaction without observing cross talk. The inherent quantitation capacities of real-time fluorescence PCR allowed the determination of average HPV copy number per cell. We conclude that the HPV SybrGreen-PCR in combination with the HPV molecular beacon PCR provides a robust, sensitive, and quantitative general HPV detection and genotyping methodology