The BCR-ABL1 fusion gene defines the subgroup of acute lymphoblastic leukemia (ALL) with the worst clinical prognosis. To identify oncogenic lesions that combine with BCR-ABL1 to cause ALL, we used Affymetrix Genome-Wide Human SNP arrays (250K NspI and SNP 6.0), fluorescence in situ hybridization, and genomic polymerase chain reaction to study 106 cases of adult BCR-ABL1-positive ALL. The most frequent somatic copy number alteration was a focal deletion on 7p12 of IKZF1, which encodes the transcription factor Ikaros and was identified in 80 (75%) of 106 patients. Different patterns of deletions occurred, but the most frequent were those characterized by a loss of exons 4 through 7 (⌬4-7) and by removal of exons 2 through 7 (⌬2-7). A variable number of nucleotides (patient specific) were inserted at the conjunction and maintained with fidelity at the time of relapse. The extent of the ⌬4-7 deletion correlated with the expression of a dominant-negative isoform with cytoplasmic localization and oncogenic activity, whereas the ⌬2-7 deletion resulted in a transcript lacking the translation start site. The IKZF1 deletion also was identified in the progression of chronic myeloid leukemia to lymphoid blast crisis (66%) but never in myeloid blast crisis or chronicphase chronic myeloid leukemia or in patients with acute myeloid leukemia. Known DNA sequences and structural features were mapped along the breakpoint cluster regions, including heptamer recombination signal sequences recognized by RAG enzymes during V(D)J recombination, suggesting that IKZF1 deletions could arise from aberrant RAGmediated recombination. (Blood. 2009;
BackgroundThe assessment of TP53 mutational status is becoming a routine clinical practice for chronic lymphocytic leukemia patients (CLL). A broad spectrum of molecular techniques has been employed so far, including both direct Sanger sequencing and next generation sequencing. Oxford Nanopore Technologies recently released the MinION an USB-interfaced sequencer. In this paper we report our experience, with the MinION technology for the detection of the TP53 gene mutation in CLL patients.Twelve CLL patients at diagnosis were included in this study. All except one patient showed the TP53 gene deletion in Fluorescence in situ hybridization experiments.Patients were investigated for TP53 mutation by Sanger and by MinION sequencing.Analysis by Sanger was performed according with the IARC protocol.Analysis by MinION was performed adopting a strategy based on long template PCR, read error correction, and post variant calling filtering.ResultsDue to the high error rate of nanopore technology, sequence data were both used directly and before correction with two different in silico methods: ALEC and nanocorrect. A mean error rate of 15 % was detected before correction that was reduced to 4-5 % after correction.Analysis by Sanger sequencing was able to detect four patients mutated for TP53. MinION analysis detected one more mutated patient previously not detected from Sanger.ConclusionIn our hands, the Nanopore technology shows correlation with Sanger sequencing but more sensitive, manageable and less expensive, and therefore has proven to be a useful tool for TP53 gene mutation detection.Electronic supplementary materialThe online version of this article (doi:10.1186/s13000-016-0550-y) contains supplementary material, which is available to authorized users.
Purpose: The 9p21 locus, encoding three important tumor suppressors (p16/CDKN2A, p14/ARF, and p15/CDKN2B), is a major target of inactivation in the pathogenesis of many human tumors.Patients and Methods: To explore, at high resolution, the frequency and size of alterations affecting this locus in adult BCR-ABL1-positive acute lymphoblastic leukemia (ALL) and to investigate their prognostic value, 112 patients (101 de novo and 11 relapsed cases) were analyzed by genome-wide single-nucleotide polymorphism arrays and gene candidate deep exon sequencing. Paired diagnosis-relapse samples were further available and analyzed for 19 (19%) cases.Results: CDKN2A/ARF and CDKN2B genomic alterations were identified in 29% and 25% of newly diagnosed patients, respectively. Deletions were monoallelic in 72% of cases, and in 43% of them, the minimal overlapping region of the lost area spanned only the CDKN2A/B gene locus. An analysis conducted at relapse showed an increase in the detection rate of CDKN2A/ARF loss (47%) compared with the time of diagnosis (P ¼ 0.06). Point mutations within the 9p21 locus were found at very low levels, with only a nonsynonymous substitution in the exon 2 of CDKN2A. Of note, deletions of CDKN2A/B were significantly associated with poor outcomes in terms of overall survival (P ¼ 0.0206), disease free-survival (P ¼ 0.0010), and cumulative incidence of relapse (P ¼ 0.0014).Conclusions: Inactivation of the 9p21 locus by genomic deletion is a frequent event in BCR-ABL1-positive ALL. Deletions are frequently acquired during leukemia progression and are a poor prognostic marker of long-term outcomes. Clin Cancer Res; 17(23); 7413-23. Ó2011 AACR.
We report a customized gene panel assay based on multiplex long-PCR followed by third generation sequencing on nanopore technology (MinION), designed to analyze five frequently mutated genes in chronic lymphocytic leukemia (CLL): TP53, NOTCH1, BIRC3, SF3B1 and MYD88. For this purpose, 12 patients were selected according to specific cytogenetic and molecular features significantly associated with their mutational status. In addition, simultaneous analysis of the targets genes was performed by molecular assays or Sanger Sequencing. Data analysis included mapping to the GRCh37 human reference genome, variant calling and annotation, and average sequencing depth/error rate analysis. The sequencing depth resulted on average higher for smaller amplicons, and the final breadth of coverage of the panel was 94.1%. The error rate was about 6% and 2% for insertions/deletions and single nucleotide variants, respectively. Our gene panel allows analysis of the prognostically relevant genes in CLL, with two PCRs per patient. This strategy offers an easy and affordable workflow, although further advances are required to improve the accuracy of the technology and its use in the clinical field. Nevertheless, the rapid and constant development of nanopore technology, in terms of chemistry advances, more accurate basecallers and analysis software, offers promise for a wide use of MinION in the future.
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