Michael J. Groves

Somatic SF3B1 mutation in myelodysplasia with ring sideroblasts.

BACKGROUND Myelodysplastic syndromes are a diverse and common group of chronic hematologic cancers. The identification of new genetic lesions could facilitate new diagnostic and therapeutic strategies. METHODS We used massively parallel sequencing technology to identify somatically acquired point mutations across all protein-coding exons in the genome in 9 patients with low-grade myelodysplasia. Targeted resequencing of the gene encoding RNA splicing factor 3B, subunit 1 (SF3B1), was also performed in a cohort of 2087 patients with myeloid or other cancers. RESULTS We identified 64 point mutations in the 9 patients. Recurrent somatically acquired mutations were identified in SF3B1. Follow-up revealed SF3B1 mutations in 72 of 354 patients (20%) with myelodysplastic syndromes, with particularly high frequency among patients whose disease was characterized by ring sideroblasts (53 of 82 [65%]). The gene was also mutated in 1 to 5% of patients with a variety of other tumor types. The observed mutations were less deleterious than was expected on the basis of chance, suggesting that the mutated protein retains structural integrity with altered function. SF3B1 mutations were associated with down-regulation of key gene networks, including core mitochondrial pathways. Clinically, patients with SF3B1 mutations had fewer cytopenias and longer event-free survival than patients without SF3B1 mutations. CONCLUSIONS Mutations in SF3B1 implicate abnormalities of messenger RNA splicing in the pathogenesis of myelodysplastic syndromes. (Funded by the Wellcome Trust and others.).

Clinical significance of SF3B1 mutations in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms

In a previous study, we identified somatic mutations of SF3B1, a gene encoding a core component of RNA splicing machinery, in patients with myelodysplastic syndrome (MDS). Here, we define the clinical significance of these mutations in MDS and myelodysplastic/myeloproliferative neoplasms (MDS/MPN). The coding exons of SF3B1 were screened using massively parallel pyrosequencing in patients with MDS, MDS/MPN, or acute myeloid leukemia (AML) evolving from MDS. Somatic mutations of SF3B1 were found in 150 of 533 (28.1%) patients with MDS, 16 of 83 (19.3%) with MDS/MPN, and 2 of 38 (5.3%) with AML. There was a significant association of SF3B1 mutations with the presence of ring sideroblasts (P < .001) and of mutant allele burden with their proportion (P = .002). The mutant gene had a positive predictive value for ring sideroblasts of 97.7% (95% confidence interval, 93.5%-99.5%). In multivariate analysis including established risk factors, SF3B1 mutations were found to be independently associated with better overall survival (hazard ratio = 0.15, P = .025) and lower risk of evolution into AML (hazard ratio = 0.33, P = .049). The close association between SF3B1 mutations and disease phenotype with ring sideroblasts across MDS and MDS/MPN is consistent with a causal relationship. Furthermore, SF3B1 mutations are independent predictors of favorable clinical outcome, and their incorporation into stratification systems might improve risk assessment in MDS.

TP53 gene mutation is frequent in patients with acute myeloid leukemia and complex karyotype, and is associated with very poor prognosis

TP53 gene mutation is frequent in patients with acute myeloid leukemia and complex karyotype, and is associated with very poor prognosis

Δ160p53 is a novel N-terminal p53 isoform encoded by Δ133p53 transcript

p53 gene expresses several protein isoforms modulating p53‐mediated responses through regulation of gene expression. Here, we identify a novel p53 isoform, Δ160p53, lacking the first 159 residues. By knockdown experiments and site‐directed mutagenesis, we show that Δ160p53 is encoded by Δ133p53 transcript using ATG160 as translational initiation site. This hypothesis is supported by endogenous expression of Δ160p53 in U2OS, T47D and K562 cells, the latter ones carrying a premature stop codon that impairs p53 and Δ133p53 protein expression but not the one of Δ160p53. Overall, these results show that the Δ133p53 transcript generates two different p53 isoforms, Δ133p53 and Δ160p53.

Primary sciatic nerve lymphoma: a case report and review of the literature

A patient with primary B cell non-Hodgkin’s lymphoma of the sciatic nerve is described. He presented with neuropathic symptoms in the left leg, initially diagnosed as tarsal tunnel syndrome. Magnetic resonance imaging (MRI) identified the abnormality in the sciatic nerve. A fascicular biopsy of the sciatic nerve showed a diffuse large B cell non-Hodgkin’s lymphoma. The patient was treated with chemotherapy and rituximab (anti-CD20 monoclonal antibody). Four months later he was in remission, and remains so 48 months from presentation. Primary lymphoma of single peripheral nerves may be a unique subtype of extranodal lymphoma, which usually follows an aggressive course and has a variable response to current therapeutic strategies. MRI is useful, alongside electrophysiological studies, in patients with atypical peripheral nerve symptoms.

Absolute SILAC-Compatible Expression Strain Allows Sumo-2 Copy Number Determination in Clinical Samples

Quantitative mass spectrometry-based proteomics is a vital tool in modern life science research. In contrast to the popularity of approaches for relative protein quantitation, the widespread use of absolute quantitation has been hampered by inefficient and expensive production of labeled protein standards. To optimize production of isotopically labeled standards, we genetically modified a commonly employed protein expression Escherichia coli strain, BL21 (DE3), to construct an auxotroph for arginine and lysine. This bacterial strain allows low-cost, high-level expression of fully labeled proteins with no conversion of labeled arginine to proline. In combination with a fluorescence-based quantitation of standards and nontargeted LC–MS/MS analysis of unfractionated total cell lysates, this strain was used to determine the copy number of a post-translational modifier, small ubiquitin-like modifier (SUMO-2), in HeLa, human sperm, and chronic lymphocytic leukemia cells. By streamlining and improving the generation of labeled standards, this production system increases the breadth of absolute quantitation by mass spectrometry and will facilitate a far wider uptake of this important technique than previously possible.

Dysregulation of autophagy in chronic lymphocytic leukemia with the small-molecule Sirtuin inhibitor Tenovin-6

Tenovin-6 (Tnv-6) is a bioactive small molecule with anti-neoplastic activity. Inhibition of the Sirtuin class of protein deacetylases with activation of p53 function is associated with the pro-apoptotic effects of Tnv-6 in many tumors. Here, we demonstrate that in chronic lymphocytic leukemia (CLL) cells, Tnv-6 causes non-genotoxic cytotoxicity, without adversely affecting human clonogenic hematopoietic progenitors in vitro, or murine hematopoiesis. Mechanistically, exposure of CLL cells to Tnv-6 did not induce cellular apoptosis or p53-pathway activity. Transcriptomic profiling identified a gene program influenced by Tnv-6 that included autophagy-lysosomal pathway genes. The dysregulation of autophagy was confirmed by changes in cellular ultrastructure and increases in the autophagy-regulatory proteins LC3 (LC3-II) and p62/Sequestosome. Adding bafilomycin-A1, an autophagy inhibitor to Tnv-6 containing cultures did not cause synergistic accumulation of LC3-II, suggesting inhibition of late-stage autophagy by Tnv-6. Thus, in CLL, the cytotoxic effects of Tnv-6 result from dysregulation of protective autophagy pathways.

Molecular characterisation of a recurrent, semi-cryptic RUNX1 translocation t(7;21) in myelodysplastic syndrome and acute myeloid leukaemia

A proportion of cytogenetic abnormalities in myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML) may escape detection by high‐resolution genomic technologies, but can be identified by conventional cytogenetic and molecular analysis. Here, we report the detection of a reciprocal translocation t(7;21)(p22;q22) in the marrow of two adults with MDS and AML, using conventional cytogenetic analysis and fluorescence‐in situ‐hybridization (FISH). Reverse‐transcription polymerase chain reaction (RT‐PCR) and sequence analysis identified a fusion between RUNX1 and the gene encoding ubiquitin specific peptidase‐42 (USP42), with splice‐variants and variable break‐points within RUNX1. Combined cytomorphology and FISH studies in MDS marrow revealed abnormal RUNX1 signals within megakaryocytes, suggesting that the acquisition of t(7;21)(p22;q22) does not confer complete differentiation arrest and may represent an early genetic event in leukaemogenesis. Single nucleotide polymorphism‐arrays failed to detect additional sub‐microscopic genomic changes predisposing to or associated with t(7;21). Molecular analysis of 100 MDS and AML marrow specimens by RT‐PCR did not reveal new cases with the RUNX1‐USP42 fusion. Thus, our studies have identified t(7;21)(p22;q22) as a rare but recurrent abnormality in MDS/AML, with the existence of alternative spliced forms of the RUNX1‐USP42 transcript in different patients. Further studies are required to identify the potential contribution of these splice‐variants to disease heterogeneity.

Predicted costs of iron-chelators in myelodysplastic syndromes: A 10-year analysis based on actual prevalence and red cell transfusion rates†

Consideration of iron‐chelation (IC) in transfusion‐dependent patients is recommended in most clinical‐practice guidelines on myelodysplastic syndromes (MDS). The financial impact of IC on health‐care systems is predicted through economic modeling, but an analysis based on actual prevalence is lacking. Here, we have investigated the potential drug‐costs and need for IC in a cohort of 189 United Kingdom‐based MDS patients diagnosed from 2000 to 2010. Patients with low or intermediate‐1 IPSS scores were identified as eligible for IC if ≥24 red cell units (RCU) had been transfused over 12 consecutive months or the transfusion‐intensity averaged ≥2 RCU per month. Drug‐costs were calculated from the time patients qualified for IC until death or last follow‐up. In 159 patients with low/intermediate‐1 MDS, survival was superior with a low IPSS score (P = 0.014), age <70 years (P = 0.043), transfusion‐independence at diagnosis (P = 0.0056) and transfusion‐intensity of <2 RCU per month (P = 0.009). Reflecting the time elapsed since diagnosis, longer survival was observed with a cumulative red cell load of ≥75 U (P = 0.046). By logistic‐regression analysis, transfusion‐intensity independently predicted survival (P = 0.0035) in low and intermediate‐1 risk MDS patients. Forty‐one patients fulfilled criteria for consideration of IC. Of these, 6 patients died within 1 month; 35 patients survived for a median of 16 months (range 1–61). Had patients commenced IC, the anticipated drug‐costs alone would have been ∼

Factors influencing a second myeloid malignancy in patients with Philadelphia-negative -7 or del(7q) clones during tyrosine kinase inhibitor therapy for chronic myeloid leukemia

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