Here we update our preliminary observations on array-comparative genome hybridization (array-CGH) analyses of rapidly dividing cells. In our array-CGH studies, we observed wave patterns in copy ...number determination on rapidly dividing cell systems, with population doubling times less than one day and high relative S phase fractions (1). DNA obtained from highly proliferative murine leukemic blasts and induced pluripotent stem (iPS) cells generated reproducible oscillation of data points around the baseline. Interestingly, after applying starvation conditions or direct G1 phase arrest during in vitro cultivation, genomic profiles smoothed to reliable copy number states, avoiding false positives and false negatives, respectively. Therefore, we strongly recommend G1 phase arrest prior to DNA extraction when highly proliferative cell systems are interrogated by array-CGH.
Genome-wide profiling of copy number alterations by array-based high resolution comparative genomic hybridization (array-CGH) is an important method to ensure the genomic integrity of cells in ...diverse conditions. We observed that the analysis of genomic profiles, in particular of fast-dividing murine leukemia cell lines, is challenging due to characteristic patterns oscillating around the array-CGH baseline. Here we show array-CGH data can be drastically improved by reducing proliferation rates of cultured cells using deprivation protocols or cell cycle inhibitors. Arresting cell cycle in the G1 phase leads to smoother genomic profiles, and hence to a more reliable detection of copy number alterations.
To gain more information about the molecular mechanisms leading to dedifferentiation of hepatocellular adenoma (HCA) and hepatocellular carcinoma (HCC), high-resolution array-based comparative ...genomic hybridization (array-CGH) was performed on 24 cases of HCC and 10 cases of HCA.
DNA chips containing 6251 individual bacterial artificial chromosome/plasmid artificial chromosome clones were used. They allowed for a genome-wide resolution of 1 Mb and an even higher resolution of up to 100 kb for chromosome regions recurrently involved in human tumors and for regions containing known tumor-suppressor genes and oncogenes.
Copy number changes on the genomic scale were found by array-based comparative genomic hybridization in all cases. In HCC, gains of chromosomal regions 1q (91.6%), and 8q (58.3%), and losses of 8p (54%) were found most frequently. Hierarchic cluster analysis branched all HCA from HCC. However, in 2 adenomas with a known history of glycogenosis type I and adenomatosis hepatis gains of 1q were found, too. The critically gained region was narrowed down to bands 1q22-23. Although no significant differences in the mean number of chromosomal aberrations were seen between adenomas and well-differentiated carcinomas (2.7 vs 4.6), a significant increase accompanied the dedifferentiation of HCC (14.1 in HCC-G2 and 16.3 in HCC-G2/3; P < .02). Dedifferentiation of HCC also was correlated closely to losses of 4q and 13q (P <.001 and <.005, respectively).
The increased chromosomal instability during dedifferentiation of HCC leads to an accumulation of structural chromosomal aberrations and losses and gains of defined chromosome regions.
Mantle cell lymphoma (MCL), a mature B-cell neoplasm, is genetically characterized by the translocation t(11;14)(q13;q32). However, secondary alterations are required for malignant transformation. ...The identification of inactivated tumor suppressor genes contributing to the development of MCL may lead to further elucidation of the biology of this disease and help to identify novel targets for therapy.
Whole genome microarray-based gene expression profiling on treated versus untreated MCL cell lines was used to identify genes induced by 5-aza-2'-deoxycytidine. The degree of promoter methylation and transcriptional silencing of selected genes was then proven in MCL cell lines and primary cases by methylation-specific polymerase chain reaction (PCR) techniques, real-time PCR and gene expression profiling.
After 5-aza-2'-deoxycytidine treatment, we identified more than 1000 upregulated genes, 16 of which were upregulated > or =3-fold. Most of them were not known to be silenced by methylation in MCL. A low expression of ING1, RUNX3 and BNIP3L was observed in three of the five the MCL cell lines. In addition, the expression of PARG1, which is located in the frequently deleted region 1p22.1, was substantially reduced and displayed at least partial promoter methylation in all investigated MCL cell lines as well as in 31 primary MCL cases.
In summary, we identified interesting novel candidate genes that probably contribute to the progression of MCL and suggest that PARG1 is a strong candidate tumor suppressor gene in MCL.
NGS‐based multiple gene panel resequencing in combination with a high resolution CGH‐array was used to identify genetic risk factors for hereditary breast and/or ovarian cancer in 237 high risk ...patients who were previously tested negative for pathogenic BRCA1/2 variants. All patients were screened for pathogenic variants in 94 different cancer predisposing genes. We identified 32 pathogenic variants in 14 different genes (ATM, BLM, BRCA1, CDH1, CHEK2, FANCG, FANCM, FH, HRAS, PALB2, PMS2, PTEN, RAD51C and NBN) in 30 patients (12.7%). Two pathogenic BRCA1 variants that were previously undetected due to less comprehensive and sensitive methods were found. Five pathogenic variants are novel, three of which occur in genes yet unrelated to hereditary breast and/or ovarian cancer (FANCG, FH and HRAS). In our cohort we discovered a remarkably high frequency of truncating variants in FANCM (2.1%), which has recently been suggested as a susceptibility gene for hereditary breast cancer. Two patients of our cohort carried two different pathogenic variants each and 10 other patients in whom a pathogenic variant was confirmed also harbored a variant of unknown significance in a breast and ovarian cancer susceptibility gene. We were able to identify pathogenic variants predisposing for tumor formation in 12.3% of BRCA1/2 negative breast and/or ovarian cancer patients.
What's new?
Risk for hereditary breast and ovarian cancer (HBOC) is mainly determined by BRCA1/2 mutations but in ~60% of cases the genetic predisposition remains unknown. The authors screened more than 200 women with BRCA1/2‐negative HBOC for new pathogenic variants using a combination of multi‐gene panel sequencing and comparative genomic hybridization. A remarkably high frequency of truncating variants in FANCM were discovered, a protein recently suggested as a susceptibility gene for hereditary breast cancer. The authors recommend that combined methods be used to identify new variants for HBOC risk assessment.
PAX5 is a member of the paired box (PAX) family of transcription factors involved in B‐cell development. PAX5P80R has recently been described as a distinct genetic B‐cell precursor (BCP) acute ...lymphoblastic leukemia (ALL) subtype with a favorable prognosis in adults. In contrast, an unfavorable outcome has been observed in children. Our aim was to determine the frequency of PAX5P80R in childhood BCP‐ALL treated according to the Associazione Italiana Ematologia ed Oncologia Pediatrica‐Berlin‐Frankfurt‐Muenster (AIEOP‐BFM) ALL 2000 protocol and to evaluate its clinical significance within this study cohort. The analyses included 1237 patients with ALL treated in the AIEOP‐BFM ALL 2000 trial with complete information for copy number variations (CNVs) of IKZF1, PAX5, ETV6, RB1, BTG1, EBF1, CDKN2A, CDKN2B, and ERG. A customized TaqMan genotyping assay was used to screen for PAX5P80R. Sanger sequencing was used to confirm PAX5P80R‐positive results as well as to screen for second variants in PAX5. Agilent CGH + SNP arrays (e‐Array design 85 320; Agilent Technologies) were performed in PAX5P80R‐positive patients to verify additional CNVs. Almost 2% (20/1028) of our BCP‐ALL cohort were PAX5P80R‐positive. White blood cell counts higher than 50 000/μl as well as male sex were significantly (P < .05) associated with PAX5P80R. Most of the PAX5P80R‐positive cases were 10 years of age or older. PAX5P80R‐positive samples were enriched for deletions affecting PAX5, IKZF1, CDKN2A, and CDKN2B. Compared to PAX5P80R‐wildtype BCP‐ALL, PAX5P80R‐positive patients showed a significantly reduced 5‐year overall survival (P = .042). Further studies should evaluate the interaction of PAX5P80R with other genetic aberrations to further stratify intermediate risk pediatric BCP‐ALL.
A complex karyotype, detected in myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML), is associated with a reduced median survival. The most frequent chromosomal aberrations in complex ...karyotypes are deletions of 5q and 17p harboring the tumor suppressor gene TP53. The unbalanced translocation der(5;17) involving chromosome 5q and 17p is a recurrent aberration in MDS/AML, resulting in TP53 loss. We analyzed the karyotypes of 178 patients with an unbalanced translocation der(5;17) using fluorescence R−/G‐banding analysis. Whenever possible, fluorescence in situ hybridization (FISH) (n = 138/141), multicolor FISH (n = 8), telomere length measurement (n = 9), targeted DNA sequencing (n = 13), array‐CGH (n = 7) and targeted RNA sequencing (n = 2) were conducted. The der(5;17) aberration was accompanied with loss of genetic material in 7q (53%), −7 (27%), gain of 21q (29%), +8 (17%) and − 18 (16%) and all analyzed patients (n = 13) showed a (likely) pathogenic variant inTP53. The der(5;17) cohort showed significantly shortened telomeres in comparison to the healthy age‐matched controls (P < .05), but there was no significant telomere shortening in comparison to MDS/AML patients with a complex karyotype without der(5;17). No fusion genes resulted from the unbalanced translocation. This study demonstrates that the unbalanced translocation der(5;17) is associated with a biallelic inactivation of TP53 due to a deletion of TP53 in one allele and a pathogenic variant of the second TP53 allele. Since the breakpoints are located within (near‐) heterochromatic regions, alterations of DNA methylation or histone modifications may be involved in the generation of der(5;17).
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