Molecular definition of a narrow interval at 7q22.1 associated with myelodysplasia

Chromosome 7 translocations, deletions, or monosomy are associated with myelodysplasia (MDS) and acute myeloid leukemia both in children and adults. These chromosomal anomalies represent one of the most common cytogenetic abnormalities associated with these diseases and usually herald a poor prognosis. In this study two cosmid DNA probes that mapped to 7q22.1 and were known to be separated by approximately 500 kb were identified to flank the proximal inversion breakpoint in a patient carrying a constitutional inversion (7q22.1-34) associated with MDS. A yeast artificial chromosome (YAC) clone that encompassed the two cosmids was identified and shown to span the breakpoint. Fluorescence in situ hybridization was then used to analyze six additional patients with myelodysplasia and chromosomal rearrangements of the 7q22 region (three patients had translocations and three carried deletions). The breakpoint in one of the patients was found to be contained within the same YAC clone that spanned the inversion breakpoint. Moreover, this same interval was determined to be absent in all three patients with chromosomal deletions. These results suggest that this segment of DNA on chromosome 7q22.1 may contain specific gene(s) that have a significant role in myeloid malignancies.link_to_OA_fulltex

The morphology of CLL revisited: the clinical significance of prolymphocytes and correlations with prognostic/molecular markers in the LRF CLL4 trial.

Historically, an increase in the percentage and number of circulating prolymphocytes in chronic lymphocytic leukaemia (CLL) has been associated with strong expression of surface immunoglobulin, trisomy 12 and a poor outcome. This study re-examines the biological and clinical significance of increased peripheral blood prolymphocytes in 508 patients at entry into the randomized UK Leukaemia Research Fund CLL4 trial. It also investigates the associations between increased prolymphocytes and a comprehensive array of biomarkers. 270 patients (53%) had <5% prolymphocytes, 167 (33%) had 5-9%, 60 (12%) had 10-14% and 11 (2%) had ≥15% prolymphocytes. We show that a higher proportion of prolymphocytes (≥10%) was independently associated with NOTCH1 mutations (P = 0·006), absence of 13q deletion (P = 0·001), high CD38 expression (P = 0·02) and unmutated IGHV genes (P = 0·01). Deaths due to Richter syndrome were significantly more common amongst patients who had ≥10% vs <10% prolymphocytes (13% vs 2%) respectively (P < 0·0001). ≥10% prolymphocytes was also associated with a shorter progression-free survival (Hazard ratio [HR] 1·50 [95% confidence interval [CI]: 1·16-1·93], P = 0·002) and overall survival (HR 1·99 [95% CI: 1·53-2·59], P < 0·0001). Our data support the routine examination of blood films in CLL and suggest that a finding of an increased proportion of prolymphocytes may be a trigger for further evaluation of clinical and laboratory features of progressive disease

Multi-label classification using ensembles of pruned sets

This paper presents a Pruned Sets method (PS) for multi-label classification. It is centred on the concept of treating sets of labels as single labels. This allows the classification process to inherently take into account correlations between labels. By pruning these sets, PS focuses only on the most important correlations, which reduces complexity and improves accuracy. By combining pruned sets in an ensemble scheme (EPS), new label sets can be formed to adapt to irregular or complex data. The results from experimental evaluation on a variety of multi-label datasets show that [E]PS can achieve better performance and train much faster than other multi-label methods

The SF3B1 inhibitor spliceostatin A (SSA) elicits apoptosis in chronic lymphocytic leukemia cells through downregulation of Mcl-1

The pro-survival Bcl-2 family member Mcl-1 is expressed in chronic lymphocytic leukemia (CLL), with high expression correlated with progressive disease. The spliceosome inhibitor spliceostatin A (SSA), is known to regulate Mcl-1 and so here we assessed the ability of SSA to elicit apoptosis in CLL. SSA induced apoptosis of CLL cells at low nanomolar concentrations in a dose- and time-dependent manner, but independently of SF3B1 mutational status, IGHV status and CD38 or ZAP70 expression. However, normal B and T cells were less sensitive than CLL cells (P=0.006 and P&lt;0.001, respectively). SSA altered the splicing of anti-apoptotic MCL-1L to MCL-1s in CLL cells coincident with induction of apoptosis. Overexpression studies in Ramos cells suggested Mcl-1 was important for SSA-induced killing since its expression inversely correlated with apoptosis (P=0.001). IL4 and CD40L, present in patient lymph nodes, are known to protect tumor cells from apoptosis and significantly inhibited SSA, ABT-263 and ABT-199 induced killing following administration to CLL cells (P=0.008). However, by combining SSA with the Bcl-2/Bcl-xL antagonists ABT-263 or ABT-199, we were able to overcome this pro-survival effect. We conclude that SSA combined with Bcl-2/Bcl-xL antagonists may have therapeutic utility for CL

Telomere length predicts progression and overall survival in chronic lymphocytic leukemia: data from the UK LRF CLL4 trial

Telomere erosion and fusion play an important role in the pathology of many common human malignancies including CLL.1,2 Previous studies in CLL have shown that short telomeres defined on the basis of the median value or receiver operating characteristic (ROC) analysis are associated with unmutated IGHV genes, poor risk genomic abnormalities, genomic complexity and high expression of CD38, CD49d, and ZAP70 whereas long telomeres are associated with increasing IGHV mutational load, isolated deletion of 13q and low CD49d expression. In addition, in predominantly diagnostic or mixed patient cohorts, telomere length (TL) predicts time to first treatment and/or overall survival (OS) in multivariate analyses of models incorporating established biomarkers. 3-7 However uncertainties about the most clinically relevant measure of telomere length, the optimal choice of assay, the need for assay standardisation and the lack of published data on the prognostic value of TL in patients entered into randomised trials have hindered the implementation of TL measurement into routine clinical practice. We have attempted to address these issues by measuring telomere length using monochrome multiplex Q-PCR (MMQ-PCR) in 384 patients at randomisation into the UK LRF CLL4 phase 3 chemotherapy trial (Table S1), of whom 111 samples were also screened by single telomer

Genetic variation at MECOM, TERT, JAK2 and HBS1L-MYB predisposes to myeloproliferative neoplasms

Clonal proliferation in myeloproliferative neoplasms (MPN) is driven by somatic mutations in JAK2, CALR or MPL, but the contribution of inherited factors is poorly characterized. Using a three-stage genome-wide association study of 3,437 MPN cases and 10,083 controls, we identify two SNPs with genome-wide significance in JAK2V617F-negative MPN: rs12339666 (JAK2; meta-analysis P=1.27 × 10−10) and rs2201862 (MECOM; meta-analysis P=1.96 × 10−9). Two additional SNPs, rs2736100 (TERT) and rs9376092 (HBS1L/MYB), achieve genome-wide significance when including JAK2V617F-positive cases. rs9376092 has a stronger effect in JAK2V617F-negative cases with CALR and/or MPL mutations (Breslow–Day P=4.5 × 10−7), whereas in JAK2V617F-positive cases rs9376092 associates with essential thrombocythemia (ET) rather than polycythemia vera (allelic χ2 P=7.3 × 10−7). Reduced MYB expression, previously linked to development of an ET-like disease in model systems, associates with rs9376092 in normal myeloid cells. These findings demonstrate that multiple germline variants predispose to MPN and link constitutional differences in MYB expression to disease phenotype

Proteomics Profiling of CLL Versus Healthy B-cells Identifies Putative Therapeutic Targets and a Subtype-independent Signature of Spliceosome Dysregulation

Chronic lymphocytic leukemia (CLL) is a heterogeneous B-cell cancer exhibiting a wide spectrum of disease courses and treatment responses. Molecular characterization of RNA and DNA from CLL cases has led to the identification of important driver mutations and disease subtypes, but the precise mechanisms of disease progression remain elusive. To further our understanding of CLL biology we performed isobaric labeling and mass spectrometry proteomics on 14 CLL samples, comparing them with B-cells from healthy donors (HDB). Of 8694 identified proteins, ∼6000 were relatively quantitated between all samples (q<0.01). A clear CLL signature, independent of subtype, of 544 significantly overexpressed proteins relative to HDB was identified, highlighting established hallmarks of CLL (e.g. CD5, BCL2, ROR1 and CD23 overexpression). Previously unrecognized surface markers demonstrated overexpression (e.g. CKAP4, PIGR, TMCC3 and CD75) and three of these (LAX1, CLEC17A and ATP2B4) were implicated in B-cell receptor signaling, which plays an important role in CLL pathogenesis. Several other proteins (e.g. Wee1, HMOX1/2, HDAC7 and INPP5F) were identified with significant overexpression that also represent potential targets. Western blotting confirmed overexpression of a selection of these proteins in an independent cohort. mRNA processing machinery were broadly upregulated across the CLL samples. Spliceosome components demonstrated consistent overexpression (p = 1.3 × 10⁻¹²) suggesting dysregulation in CLL, independent of SF3B1 mutations. This study highlights the potential of proteomics in the identification of putative CLL therapeutic targets and reveals a subtype-independent protein expression signature in CLL

Targeting the Ataxia Telangiectasia Mutated-null Phenotype in Chronic Lymphocytic Leukemia with Pro-oxidants

Inactivation of the Ataxia Telangiectasia Mutated gene in chronic lymphocytic leukemia results in resistance to p53-dependent apoptosis and inferior responses to treatment with DNA damaging agents. Hence, p53-independent strategies are required to target Ataxia Telangiectasia Mutated-deficient chronic lymphocytic leukemia. As Ataxia Telangiectasia Mutated has been implicated in redox homeostasis, we investigated the effect of the Ataxia Telangiectasia Mutated-null chronic lymphocytic leukemia genotype on cellular responses to oxidative stress with a view to therapeutic targeting. We found that in comparison to Ataxia Telangiectasia Mutated-wild type chronic lymphocytic leukemia, pro-oxidant treatment of Ataxia Telangiectasia Mutated-null cells led to reduced binding of NF-E2 p45-related factor-2 to antioxidant response elements and thus decreased expression of target genes. Furthermore, Ataxia Telangiectasia Mutated-null chronic lymphocytic leukemia cells contained lower levels of antioxidants and elevated mitochondrial reactive oxygen species. Consequently, Ataxia Telangiectasia Mutated-null chronic lymphocytic leukemia, but not tumours with 11q deletion or TP53 mutations, exhibited differentially increased sensitivity to pro-oxidants both in vitro and in vivo. We found that cell death was mediated by a p53- and caspase-independent mechanism associated with apoptosis inducing factor activity. Together, these data suggest that defective redox-homeostasis represents an attractive therapeutic target for Ataxia Telangiectasia Mutated-null chronic lymphocytic leukemia

KLF2 mutation is the most frequent somatic change in splenic marginal zone lymphoma and identifies a subset with distinct genotype.

To characterise the genetics of splenic marginal zone lymphoma (SMZL), we performed whole exome sequencing of 16 cases and identified novel recurrent inactivating mutations in Kruppel-like factor 2 (KLF2), a gene whose deficiency was previously shown to cause splenic marginal zone hyperplasia in mice. KLF2 mutation was found in 40 (42%) of 96 SMZLs, but rarely in other B-cell lymphomas. The majority of KLF2 mutations were frameshift indels or nonsense changes, with missense mutations clustered in the C-terminal zinc finger domains. Functional assays showed that these mutations inactivated the ability of KLF2 to suppress NF-κB activation by TLR, BCR, BAFFR and TNFR signalling. Further extensive investigations revealed common and distinct genetic changes between SMZL with and without KLF2 mutation. IGHV1-2 rearrangement and 7q deletion were primarily seen in SMZL with KLF2 mutation, while MYD88 and TP53 mutations were nearly exclusively found in those without KLF2 mutation. NOTCH2, TRAF3, TNFAIP3 and CARD11 mutations were observed in SMZL both with and without KLF2 mutation. Taken together, KLF2 mutation is the most common genetic change in SMZL and identifies a subset with a distinct genotype characterised by multi-genetic changes. These different genetic changes may deregulate various signalling pathways and generate cooperative oncogenic properties, thereby contributing to lymphomagenesis.The research was supported by grants from Leukaemia & Lymphoma Research, U.K., Addenbrooke’s Charitable Trust. SM is a PhD student supported by MRC and Department of Pathology, University of Cambridge. LEI is a PhD student supported by the Pathological Society of UK & Ireland. NB is a fellow of the European Hematology Association and was supported by a starter grant from the Academy of Medical Sciences