A deep-sequencing study of chronic myeloid leukemia patients in blast crisis (BC-CML) detects mutations in 76.9% of cases

Grossmann, Vera; Kohlmann, Alexander; Zenger, Melanie; Schindela, Sonja; Eder, Christiane; Weissmann, Sandra; Schnittger, Susanne; Kern, Wolfgang; Müller, Martin C.; Hochhaus, Andreas; Haferlach, Torsten; Haferlach, Claudia

doi:10.1038/leu.2010.298

Cited by 159 publications

(175 citation statements)

References 7 publications

(15 reference statements)

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“…Using a modified PCR setup, amplicons with up to 77% GC content were successfully processed by deep sequencing. Moreover, miniaturization and automation of certain assay steps will allow a reasonable bundling of multiple markers per patient, 4,5 such that high-throughput laboratories will be in a position of offering panels of genes for an individualized approach to diagnose and monitor a patient's disease. For each comparison, the mean of the two relative variant frequencies from a single patient sample at the two centers (x axis) is plotted against the difference between the same two results (y axis).…”

Section: Discussionmentioning

confidence: 99%

“…1,2 It has become apparent that NGS platforms will have practical applications in clinical diagnostics and applications, such as detection of EGFR mutations in lung adenocarcinoma, 3 characterizing RAS and methylation pathway alterations in myeloproliferative diseases and myeloid leukemias, [4][5][6][7] or high-resolution, high-throughput human leukocyte antigen genotyping have been developed. 8,9 Thus far, limited data are available on the technical performance of amplicon deep sequencing in a clinical diagnostic setting.…”

Section: Introductionmentioning

confidence: 99%

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The Interlaboratory RObustness of Next-generation sequencing (IRON) study: a deep sequencing investigation of TET2, CBL and KRAS mutations by an international consortium involving 10 laboratories

et al. 2011

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Massively parallel pyrosequencing allows sensitive deep sequencing to detect molecular aberrations. Thus far, data are limited on the technical performance in a clinical diagnostic setting. Here, we investigated as an international consortium the robustness, precision and reproducibility of amplicon nextgeneration deep sequencing across 10 laboratories in eight countries. In a cohort of 18 chronic myelomonocytic leukemia patients, mutational analyses were performed on TET2, a frequently mutated gene in myeloproliferative neoplasms. Additionally, hotspot regions of CBL and KRAS were investigated. The study was executed using GS FLX sequencing instruments and the small volume 454 Life Sciences Titanium emulsion PCR setup. We report a high concordance in mutation detection across all laboratories, including a robust detection of novel variants, which were undetected by standard Sanger sequencing. The sensitivity to detect low-level variants present with as low as 1-2% frequency, compared with the 20% threshold for Sanger-based sequencing is increased. Together with the output of high-quality long reads and fast run time, we demonstrate the utility of deep sequencing in clinical applications. In conclusion, this multicenter analysis demonstrated that amplicon-based deep sequencing is technically feasible, achieves high concordance across multiple laboratories and allows a broad and in-depth molecular characterization of cancer specimens with high diagnostic sensitivity.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

The Interlaboratory RObustness of Next-generation sequencing (IRON) study: a deep sequencing investigation of TET2, CBL and KRAS mutations by an international consortium involving 10 laboratories

et al. 2011

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“…Then we derived an MDS GES by comparing the gene expression profiles of our 8 RARS to those of our 24 ) with high sensitivity and specificity (72% and 100%, respectively) and with a theoretical number of false positive of 5.…”

Section: Mds Gene Expression Signature Classifies Cmml Samplesmentioning

confidence: 99%

“…Could this similarity be the result of gene mutations common and specific to the two diseases? We 7,8,18 and others [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36] have previously studied several leukemogenic genes in CMML and RARS. However, several of those (e.g.…”

Section: Analysis Of Mutated Genes In Cmml and Mdsmentioning

confidence: 99%

Molecular similarity between myelodysplastic form of chronic myelomonocytic leukemia and refractory anemia with ring sideroblasts

Gelsi‐Boyer¹,

Cervera²,

Bertucci³

et al. 2012

Haematologica

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ABSTRACT(aCGH) and sequencing of candidate genes. 7,8 According to the French-American-British (FAB) 6 and WHO 3 classifications, the CMML series was made up of 31 MP and 22 MD cases (Online Supplementary Table S1) and the MDS panel 8 refractory anemia (RA) with ring sideroblasts (RARS), 13 RA with excess of blasts type 1 (RAEB1) and 11 RAEB type 2 (RAEB2). CMML and MDS cases selected for gene expression profiling were collected at the time of diagnosis or in therapeutic abstention; none had been treated. All patients signed an informed consent for research and the study was approved by our institutional review board ("Comité d'Orientation Scientifique" of the Institut PaoliCalmettes). CD34 enrichmentSamples were enriched in CD34-positive (CD34 + ) cells for 12 CMML and 32 MDS cases. Leukocytes were obtained after bone marrow red cell lysis and washing with PBS, and labeled with magnetic bead-conjugated anti-CD34 monoclonal antibody (AC34 MicroBead; Miltenyi Biotec, Auburn, CA, USA). CD34 + hematopoietic stem cell populations were then purified through a miniMACS magnetic cell separation column (Miltenyi Biotec). RNA/DNA extractionRNAs and DNAs were extracted from whole BM CMML samples. After BM aspiration, a red cell lysis was carried out, followed by rinses with PBS. Leukocytes were processed immediately or cryopreserved at -80°C at the sample bank of the Institute and processed later. DNA and RNA were extracted using Nucleobond RNA/DNA kit from Macherey-Nagel (Düren, Germany) as recommended by the supplier. RNA from CD34 + cells were similarly extracted using Nucleobond RNA/DNA kit from Macherey-Nagel. Sequencing of 18 candidate genesMutations in ASXL1 (exon 12), CBL (exons 8, 9), DNMT3A (exons 15-23), EZH2 (all exons), FLT3 (exons 14, 15, 20), IDH1/2 (exons 4), JAK2 (exon 14), NF1 (exons 1-50), N/KRAS (exons 1, 2), PTPN11 (exons 3, 11) ), RUNX1 (exons 1-8), SF3B1 (exons 12-16), SUZ12 (exons 14-16), SRSF2 (exon 2), TET2 (exons 3-11), U2AF35/U2AF1 (exons 2, 6), and ZRSR2 (exons 1-11) were analyzed using BM DNA as previously described. 7-11 Gene expression profilingGene expression profiles of 39 CMML (out of the 53) and 32 MDS (all from CD34 + cells) mRNAs were established. Among the 39 CMML cases, 37 were studied as BM (10 of these were also studied as CD34 + ) and 2 as CD34 + RNAs. In other words, 10 CMML samples were studied as both CD34 + and whole BM RNAs, and 2 as CD34 + only (12 CD34 + in total). RNA quality and purity were assessed with Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, USA). Preparation of cRNA, hybridizations onto Affymetrix U133 Plus 2.0 human oligonucleotide microarrays, washes and detection were carried out as recommended by the supplier and as previously described. Data were analyzed by the Robust Multichip Average (RMA) method in R using Bioconductor and associated package, as previously described.12 Before analysis, a first filtering process removed from the data set the probe sets with low and poorly measured expression as defined by an expression value inferior to ...

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“…Based on the DNA sequencing, several somatic gene mutations in patients with CML-BC has been revealed. These gene including RUNX1, ASXL1, IKZF1, WT1, TET2, IDH1, IDH2, NRAS, KRAS, CBL, CBLB, TP53, and GATA2 (Zhang et al, 2008;Grossmann et al, 2011;Makishima et al, 2011).…”

Section: Dear Editormentioning

confidence: 99%

Absence of EZH2 Gene Mutation in Chronic Myeloid Leukemia Patients in Blast Crisis

Chen¹,

Yao²,

Wu³

et al. 2013

Asian Pacific Journal of Cancer Prevention

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A deep-sequencing study of chronic myeloid leukemia patients in blast crisis (BC-CML) detects mutations in 76.9% of cases

Cited by 159 publications

References 7 publications

The Interlaboratory RObustness of Next-generation sequencing (IRON) study: a deep sequencing investigation of TET2, CBL and KRAS mutations by an international consortium involving 10 laboratories

The Interlaboratory RObustness of Next-generation sequencing (IRON) study: a deep sequencing investigation of TET2, CBL and KRAS mutations by an international consortium involving 10 laboratories

Molecular similarity between myelodysplastic form of chronic myelomonocytic leukemia and refractory anemia with ring sideroblasts

Absence of EZH2 Gene Mutation in Chronic Myeloid Leukemia Patients in Blast Crisis

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