Chromosomal Rearrangements in Post-Chernobyl Papillary Thyroid Carcinomas: Evaluation by Spectral Karyotyping and Automated Interphase FISH

Structural genomic rearrangements are frequent findings in human cancers. Therefore, papillary thyroid carcinomas (PTCs) were investigated for chromosomal aberrations and rearrangements of the RET proto-oncogene. For this purpose, primary cultures from 23 PTC have been established and metaphase preparations were analysed by spectral karyotyping (SKY). In addition, interphase cell preparations of the same cases were investigated by fluorescence in situ hybridisation (FISH) for the presence of RET/PTC rearrangements using RET-specific DNA probes. SKY analysis of PTC revealed structural aberrations of chromosome 11 and several numerical aberrations with frequent loss of chromosomes 20, 21, and 22. FISH analysis for RET/PTC rearrangements showed prevalence of this rearrangement in 72% (16 out of 22) of cases. However, only subpopulations of tumour cells exhibited this rearrangement indicating genetic heterogeneity. The comparison of visual and automated scoring of FISH signals revealed concordant results in 19 out of 22 cases (87%) indicating reliable scoring results using the optimised scoring parameter for RET/PTC with the automated Metafer4 system. It can be concluded from this study that genomic rearrangements are frequent in PTC and therefore important events in thyroid carcinogenesis

Old and new immunophenotypic markers in multiple myeloma for discrimination of responding and relapsing patients: The importance of "normal" residual plasma cell analysis

Background Multiple myeloma is an incurable disease characterized by proliferation of clonal malignant plasma cells (CPCs), which can be immunophenotypically distinguished from polyclonal plasma cells (PPCs) by multiparameter flow cytometry (MFC). The utility of PPCs analysis in detecting prognostic and predictive information is still a matter of debate. Methods: we tested the ability of 11 MFC markers in detecting differences in the immunophenotype of CPCs and PPCs among patients in various disease stages; we verified if these markers could be associated with disease stage/response to therapy despite the role of clinical parameters. Results: significant changes in the expression of markers occurred both in CPCs and PPCs. CD58 on PPCs of responding patients was downregulated compared with PPC of relapsing group. Fraction of CD200 expressing PCs was lower in control subjects than in PPCs from MGUS and myeloma groups. CD11a levels of expression on both CPCs and PPCs showed an upregulation in newly diagnosed and relapsing patients versus PCs of controls; CD20 was less expressed on control PCs than on MGUS CPCs and PPCs. CD49d revealed to be advantageous in discrimination of PPCs from CPCs. In our multiple regression model, CD19 and CD49d on CPCs, and CD45, CD58 and CD56 on PPCs maintained their association with groups of patients independently of other prognostic variables. Conclusions: we provide a feasible start point to put in order ranges of expression on PPCs in healthy and myeloma subjects; we propose a new approach based on PPC analysis to monitor the stages of the disease

Integrative analysis of the microRNA-mRNA response to radiochemotherapy in primary head and neck squamous cell carcinoma cells

Background: Head and neck squamous cell carcinoma (HNSCC) is a very heterogeneous disease resulting in huge differences in the treatment response. New individualized therapy strategies including molecular targeting might help to improve treatment success. In order to identify potential targets, we developed a HNSCC radiochemotherapy cell culture model of primary HNSCC cells derived from two different patients (HN1957 and HN2092) and applied an integrative microRNA (miRNA) and mRNA analysis in order to gain information on the biological networks and processes of the cellular therapy response. We further identified potential target genes of four therapy-responsive miRNAs detected previously in the circulation of HNSCC patients by pathway enrichment analysis. Results: The two primary cell cultures differ in global copy number alterations and P53 mutational status, thus reflecting heterogeneity of HNSCC. However, they also share many copy number alterations and chromosomal rearrangements as well as deregulated therapy-responsive miRNAs and mRNAs. Accordingly, six common therapy-responsive pathways (direct P53 effectors, apoptotic execution phase, DNA damage/telomere stress induced senescence, cholesterol biosynthesis, unfolded protein response, dissolution of fibrin clot) were identified in both cell cultures based on deregulated mRNAs. However, inflammatory pathways represented an important part of the treatment response only in HN1957, pointing to differences in the treatment responses of the two primary cultures. Focused analysis of target genes of four therapy-responsive circulating miRNAs, identified in a previous study on HNSCC patients, revealed a major impact on the pathways direct P53 effectors, the E2F transcription factor network and pathways in cancer (mainly represented by the PTEN/AKT signaling pathway). Conclusions: The integrative analysis combining miRNA expression, mRNA expression and the related cellular pathways revealed that the majority of radiochemotherapy-responsive pathways in primary HNSCC cells are related to cell cycle, proliferation, cell death and stress response (including inflammation). Despite the heterogeneity of HNSCC, the two primary cell cultures exhibited strong similarities in the treatment response. The findings of our study suggest potential therapeutic targets in the E2F transcription factor network and the PTEN/AKT signaling pathway

Delineating Chromosomal Breakpoints in Radiation-Induced Papillary Thyroid Cancer

Recurrent translocations are well known hallmarks of many human solid tumors and hematological disorders, where patient- and breakpoint-specific information may facilitate prognostication and individualized therapy. In thyroid carcinomas, the proto-oncogenes RET and NTRK1 are often found to be activated through chromosomal rearrangements. However, many sporadic tumors and papillary thyroid carcinomas (PTCs) arising in patients with a history of exposure to elevated levels of ionizing irradiation do not carry these known abnormalities. We developed a rapid scheme to screen tumor cell metaphase spreads and identify candidate genes of tumorigenesis and neoplastic progression for subsequent functional studies. Using a series of overnight fluorescence in situ hybridization (FISH) experiments with pools comprised of bacterial artificial chromosome (BAC) clones, it now becomes possible to rapidly refine breakpoint maps and, within one week, progress from the low resolution Spectral Karyotyping (SKY) maps or Giemsa-banding (G-banding) karyotypes to fully integrated, high resolution physical maps including a list of candiate genes in the critical regions

Novel gene rearrangements in transformed breast cells identified by high-resolution breakpoint analysis of chromosomal aberrations

Chromosomal copy number alterations and chromosomal rearrangements are frequent mutations in human cancer. Unlike copy number alterations, little is known about the role and occurrence of chromosomal rearrangements in breast cancer. This may be due to the fact that chromosome-based breakpoint analysis is widely restricted to cultured cells. In order to identify gene rearrangements in breast cancer, we studied the chromosomal breakpoints in radiation-transformed epithelial breast cell lines using a high-resolution array-based approach using 1 Mb bacterial artificial chromosome (BAC) arrays. The breakpoints were further narrowed down by fluorescence in situ hybridisation (FISH) with clones from the 32 k BAC library. The analysis of the cell lines B42-11 and B42-16 revealed rearrangements of chromosomes 7, 8, 10 and 12. We identified the genes Has2, Grid1, Ret, Cpm, Tbx3, Tbx5, Tuba1a, Wnt1 and Arf3 within the breakpoint regions. Quantitative RT-PCR showed a deregulated expression of all of these candidate genes except for Tbx5 and Tbx3. This is the first study demonstrating gene rearrangements and their deregulated mRNA expression in radiation-transformed breast cells. Since the gene rearrangements occurred in the transformed and tumourigenic cell lines only, it is likely that these were generated in conjunction with malignant transformation of the epithelial breast cells and therefore might reflect early molecular events in breast carcinogenesis. Initial studies indicate that these gene alterations are also found in sporadic breast cancers. Endocrine-Related Cancer (2010) 17 87-98</p