Characterization of gene mutations and copy number changes in acute myeloid leukemia using a rapid target enrichment protocol

Prognostic stratification is critical for making therapeutic decisions and maximizing survival of patients with acute myeloid leukemia. Advances in the genomics of acute myeloid leukemia have identified several recurrent gene mutations whose prognostic impact is being deciphered. We used HaloPlex target enrichment and Illumina-based next generation sequencing to study 24 recurrently mutated genes in 42 samples of acute myeloid leukemia with a normal karyotype. Read depth varied between and within genes for the same sample, but was predictable and highly consistent across samples. Consequently, we were able to detect copy number changes, such as an interstitial deletion of BCOR, three MLL partial tandem duplications, and a novel KRAS amplification. With regards to coding mutations, we identified likely oncogenic variants in 41 of 42 samples. NPM1 mutations were the most frequent, followed by FLT3, DNMT3A and TET2. NPM1 and FLT3 indels were reported with good efficiency. We also showed that DNMT3A mutations can persist post-chemotherapy and in 2 cases studied at diagnosis and relapse, we were able to delineate the dynamics of tumor evolution and give insights into order of acquisition of variants. HaloPlex is a quick and reliable target enrichment method that can aid diagnosis and prognostic stratification of acute myeloid leukemia patients.This project was funded by the Wellcome Trust. NB is a fellow of the European Hematology Association and was supported by the Academy of Medical Sciences. EP is a European Hematology Association Advanced Research Fellow. GV is a Wellcome Trust Senior Fellow in Clinical Science. IV is funded by Spanish Ministerio de Economía y Competitividad subprograma Ramón y Cajal

JAK2 V617F hematopoietic clones are present several years prior to MPN diagnosis and follow different expansion kinetics.

TO THE EDITOR: The JAK2 V617F mutation is the most common somatic mutation in the classical myeloproliferative neoplasms (MPNs), present in >95% of cases of polycythemia vera (PV) and ∼50% of essential thrombocythemia (ET) and myelofibrosis (MF).1⇓⇓-4 It is usually the sole identifiable driver mutation in MPNs5 and was recently also identified as a driver of age-related clonal hemopoiesis in healthy individuals.6⇓⇓-9 In order to investigate the preclinical clonal evolution of MPNs, we identified 12 individuals with a JAK2 V617F mutant MPN, who 4.6 to 15.2 years previously (median 10.2 years) had also donated blood to register with the Cyprus Bone Marrow Donor Registry at the Karaiskakio FoundationThis work was supported by the Wellcome Trust Sanger Institute (WT098051). T. McKerrell is funded by a Wellcome Trust Clinician Scientist Fellowship (100678/Z/12/Z). G.S.V. is funded by a Wellcome Trust Senior Fellowship in Clinical Science (WT095663MA), and work in his laboratory is also funded by Cancer Research UK, Bloodwise, the Kay Kendall Leukaemia Fund, and Celgene. I.V. is supported by the Spanish Ministerio de Economía y Competitividad, Programa Ramón y Cajal

Development and validation of a comprehensive genomic diagnostic tool for myeloid malignancies.

The diagnosis of hematologic malignancies relies on multidisciplinary workflows involving morphology, flow cytometry, cytogenetic, and molecular genetic analyses. Advances in cancer genomics have identified numerous recurrent mutations with clear prognostic and/or therapeutic significance to different cancers. In myeloid malignancies, there is a clinical imperative to test for such mutations in mainstream diagnosis; however, progress toward this has been slow and piecemeal. Here we describe Karyogene, an integrated targeted resequencing/analytical platform that detects nucleotide substitutions, insertions/deletions, chromosomal translocations, copy number abnormalities, and zygosity changes in a single assay. We validate the approach against 62 acute myeloid leukemia, 50 myelodysplastic syndrome, and 40 blood DNA samples from individuals without evidence of clonal blood disorders. We demonstrate robust detection of sequence changes in 49 genes, including difficult-to-detect mutations such as FLT3 internal-tandem and mixed-lineage leukemia (MLL) partial-tandem duplications, and clinically significant chromosomal rearrangements including MLL translocations to known and unknown partners, identifying the novel fusion gene MLL-DIAPH2 in the process. Additionally, we identify most significant chromosomal gains and losses, and several copy neutral loss-of-heterozygosity mutations at a genome-wide level, including previously unreported changes such as homozygosity for DNMT3A R882 mutations. Karyogene represents a dependable genomic diagnosis platform for translational research and for the clinical management of myeloid malignancies, which can be readily adapted for use in other cancers

Induction of discrete apoptotic pathways by bromo-substituted indirubin derivatives in invasive breast cancer cells

Indirubin derivatives gained interest in recent years for their anticancer and antimetastatic properties. The objective of the present study was to evaluate and compare the anticancer properties of the two novel bromo-substituted derivatives 6-bromoindirubin-3'-oxime (6BIO) and 7-bromoindirubin-3'-oxime (7BIO) in five different breast cancer cell lines. Cell viability assays identified that 6BIO and 7BIO are most effective in preventing the proliferation of the MDA-MB-231-TXSA breast cancer cell line from a total of five breast cancer cell lined examined. In addition it was found that the two compounds induce apoptosis via different mechanisms. 6BIO induces caspase-dependent programmed cell death through the intrinsic (mitochondrial) caspase-9 pathway. 7BIO up-regulates p21 and promotes G(2)/M cell cycle arrest which is subsequently followed by the activation of two different apoptotic pathways: (a) a pathway that involves the upregulation of DR4/DR5 and activation of caspase-8 and (b) a caspase independent pathway. In conclusion, this study provides important insights regarding the molecular pathways leading to cell cycle arrest and apoptosis by two indirubin derivatives that can find clinical applications in targeted cancer therapeutics.Katerina A. Nicolaou, Vasilis Liapis, Andreas Evdokiou, Constantina Constantinou, Prokopios Magiatis, Alex L. Skaltsounis, Laura Koumas, Paul A. Costeas and Andreas I. Constantino

Induction of discrete apoptotic pathways by bromo-substituted indirubin derivatives in invasive breast cancer cells

Indirubin derivatives gained interest in recent years for their anticancer and antimetastatic properties. The objective of the present study was to evaluate and compare the anticancer properties of the two novel bromo-substituted derivatives 6-bromoindirubin-3'-oxime (6BIO) and 7-bromoindirubin-3'-oxime (7BIO) in five different breast cancer cell lines. Cell viability assays identified that 6BIO and 7BIO are most effective in preventing the proliferation of the MDA-MB-231-TXSA breast cancer cell line from a total of five breast cancer cell lined examined. In addition it was found that the two compounds induce apoptosis via different mechanisms. 6BIO induces caspase-dependent programmed cell death through the intrinsic (mitochondrial) caspase-9 pathway. 7BIO up-regulates p21 and promotes G2/M cell cycle arrest which is subsequently followed by the activation of two different apoptotic pathways: (a) a pathway that involves the upregulation of DR4/DR5 and activation of caspase-8 and (b) a caspase independent pathway. In conclusion, this study provides important insights regarding the molecular pathways leading to cell cycle arrest and apoptosis by two indirubin derivatives that can find clinical applications in targeted cancer therapeutics. © 2012 Elsevier Inc

Combined effect of glutamine at position 70 of HLA-DRB1 and alanine at position 57 of HLA-DQB1 in type 1 diabetes: An epitope analysis

<div><p>The contribution of specific HLA Class II alleles in type 1 diabetes is determined by polymorphic amino acid epitopes that direct antigen binding therefore, along with conventional allele frequency analysis, epitope analysis can provide important insights into disease susceptibility. We analyzed the highly heterogeneous Cypriot population for the HLA class II loci of T1DM patients and controls and we report for the first time their allele frequencies. Within our patient cohort we identified a subgroup that did not carry the DRB1*03:01-DQA1*05:01-DQB1*02:01 and DRB1*04:xx-DQA1*03:01-DQB1*03:02 risk haplotypes but a novel recombinant one, DRB1*04:XX-DQA1*03:01-DQB1*02:01 designated DR4-DQ2.3. Through epitope analysis we identified established susceptibility (DQB1 A⁵⁷, DRB1 H¹³) and resistance (DQB1 D⁵⁷) residues as well as other novel susceptibility residues DRB1 Q⁷⁰, DQB1 L²⁶ and resistance residues DRB1 D⁷⁰, R⁷⁰ and DQB1 Y⁴⁷. Prevalence of susceptibility epitopes was higher in patients and was not exclusively a result of linkage disequilibrium. Residues DRB1 Q⁷⁰, DQB1 L²⁶ and A⁵⁷ and a 10 amino acid epitope of DQA1 were the most significant in discriminating risk alleles. An extended haplotype containing these epitopes was carried by 92% of our patient cohort. Sharing of susceptibility epitopes could also explain the absence of risk haplotypes in patients. Finally, many significantly associated epitopes were non-pocket residues suggesting that critical immune functions may exist spanning further from the binding pockets.</p></div