Real life evaluation of AlphaMissense predictions in hematological malignancies.

High-throughput sequencing plays a pivotal role in hematological malignancy diagnostics, but interpreting missense mutations remains challenging. In this study, we used the newly available AlphaMissense database to assess the efficacy of machine learning to predict missense mutation effects and its impact to improve our ability to interpret them. Based on the analysis of 2073 variants from 686 patients analyzed for clinical purpose, we confirmed the very high accuracy of AlphaMissense predictions in a large real-life data set of missense mutations (AUC of ROC curve 0.95), and provided a comprehensive analysis of the discrepancies between AlphaMissense predictions and state of the art clinical interpretation.

Analysis of VH genes in marginal zone lymphoma reveals marked heterogeneity between splenic and nodal tumors and suggests the existence of clonal selection.

BACKGROUND AND OBJECTIVES: To clarify the relationship between splenic (SMZL) and nodal marginal zone (NMZL) lymphomas, we analyzed immunoglobulin variable heavy chain (VH) gene usage and mutation patterns in these tumors. DESIGN AND METHODS: VH genes were cloned and sequenced from 49 lymphoma samples (35 SMZL and 14 NMZL). RESULTS: A biased usage of VH gene was found with overrepresentation of VH1 in SMZL cases (13/35) and VH4 in NMZL cases (7/14). Evidence for antigen driven mutations was identified in 8 SMZL and 4 NMZL cases. Three cases out of 18 with clones analyzed from spleen and peripheral blood demonstrated intra-clonal diversity, with evidence of clonal selection in one case, indicating the possibility of antigen-driven clonal expansion. Eleven SMZL cases (31%) but only 2 NMZL (14%) cases were unmutated. No differences in clinical outcome and overall survival were found between the unmutated and mutated cases. INTERPRETATION AND CONCLUSIONS: The pattern of somatic mutation and the VH gene segment usage appear to differ between SMZL and NMZL, suggesting that these are distinct pathological entities. Moreover, a biased usage of certain sequences suggests that tumor cells in SMZL may be subjected to antigen selection.

Tumor necrosis factor-alpha mRNA stability in human peripheral blood cells after lipopolysaccharide stimulation.

TNF synthesis depends on many controls at transcriptional and post-transcriptional levels, including in particular mRNA stability and translational efficiency through the AU-rich elements (ARE) in the 3'untranslated region (3'UTR) of mRNA. We have previously reported that upon lipopolysaccharide (LPS) stimulation, TNF protein secreted by normal peripheral blood cells (PBC) from non-Hodgkin's lymphoma patients was slightly, but not significantly increased when compared to healthy control donors. In contrast, the relative amounts of TNF mRNA were significantly higher in lymphoma patients. Thus, the implication of TNF mRNA stability has been explored by investigating the decay rate of LPS-induced TNF mRNA and the expression of tristetraprolin (TTP), one of the factors involved in the destabilization of TNF mRNA. After LPS incubation, peak levels of TTP mRNA preceded those of TNF mRNA, supporting its implication in the control of TNF mRNA levels in human PBC. Furthermore, similar TTP expression in both groups correlated with an identical decay rate of TNF mRNA, which excludes this pathway for the higher LPS-induced TNF mRNA levels in PBC from lymphoma patients.

4-methylthio 2-oxobutanoate transaminase: a specific target for antiproliferative agents.

We have previously shown that the addition of 4-methylthio-2-oxobutanoate (MTOB) to cultures of methionine dependent neoplastic cells which lack endogenous MTOB restores their capacity to grow in the absence of exogenous methionine. Transition state inhibitors of the MTOB transaminase,responsible for the transamination of MTOB to methionine, had also been designed and selected for their capacity to inhibit the proliferation of methionine dependent neoplastic cells but not that of normal cells in culture. We now show that the transition state analogue : L-methionine ethyl esterpyridoxal(MEEP) with a structure corresponding to the oxo acid receptor covalently linked to pyridoxamine and the amine donor analogue: D-aspartate beta hydroxamate (D-AH) are efficient inhibitors of MTOB transaminase. [3H] MEEP uptake into transformed HeLa cells is similar to that in normal MRC5 cells, yet growth inhibition is seem in the transformed but not in the normal cells.MEEP irreversibly inhibits the activity of this enzyme when added to HeLa cells in culture but not that of the purified rat liver enzyme, probably due to pyridoxal phosphate already bound in the active site. On the contrary, D-AH is a noncompetitive reversible inhibitor of the purified rat liver enzyme in vitro and also inhibits intracellular HeLa MTOB transaminase. Furthermore, in HeLa cells both inhibitors induce DNA strand breaks typical of apoptotic cell death. These results provide evidence that MTOB transaminase is a potential target for antiproliferative agents which could selectively affect methionine-dependent neoplastic cells. The transition state intermediale : MEEP as an amine acceptor analogue was found to be 20 fold more effective than D-AH as the amine donor analogue in inducing apoptosis.

New Quantitative Method to Identify NPM1 Mutations in Acute Myeloid Leukaemia.

Somatic mutations in the NPM1 gene, which encodes for nucleophosmin, have been reported to be the most frequent genetic abnormalities found in acute myeloid leukaemia (AML). Their identification and quantification remain crucial for the patients' residual disease monitoring. We investigated a new method that could represent a novel reliable alternative to sequencing for its identification. This method was based on high-resolution melting analysis in order to detect mutated patients and on an allele-specific oligonucleotide real-time quantitative polymerase chain reaction (ASO-RQ-PCR) for the identification and quantification of the transcripts carrying NPM1 mutations (NPM1m). Few patients carrying known NPM1m enabled us to set up a table with the different primers' ΔCT values, identifying a profile for each mutation type. We then analysed a series of 337 AML patients' samples for NPM1 mutational status characterization and confirmed the ASO-RQ-PCR results by direct sequencing. We identified some mutations in 86 samples, and the results were fully correlated in 100% of the 36 sequenced samples. We also detected other rare NPM1m in two samples, that we confirmed by direct sequencing. This highly specific method provides a novel quick, useful, and costless tool, easy to use in routine practice.

High DNA methyltransferase DNMT3B levels: a poor prognostic marker in acute myeloid leukemia.

It has been recently shown that DNA methyl transferase overexpression is correlated with unfavourable prognosis in human malignancies while methylation deregulation remains a hallmark that defines acute myeloid leukemia (AML). The oncogenic transcription factor EVI1 is involved in methylation deregulation and its overexpression plays a major role for predicting an adverse outcome. Moreover, the identification of DNMT3A mutations in AML patients has recently been described as a poor prognostic indicator. In order to clarify relationship between these key actors in methylation mechanisms and their potential impact on patient outcomes, we analysed 195 de novo AML patients for the expression of DNMT3A, 3B (and its non-catalytic variant 3B(NC)) and their correlations with the outcome and the expression of other common prognostic genetic biomarkers (EVI1, NPM1, FLT3ITD/TKD and MLL) in adult AML. The overexpression of DNMT3B/3B(NC) is (i) significantly correlated with a shorter overall survival, and (ii) inversely significantly correlated with event-free survival and DNMT3A expression level. Moreover, multivariate analysis showed that a high expression level of DNMT3B/3B(NC) is statistically a significant independent poor prognostic indicator. This study represents the first report showing that the overexpression of DNMT3B/3B(NC) is an independent predictor of poor survival in AML. Its quantification should be implemented to the genetic profile used to stratify patients for therapeutical strategies and should be useful to identify patients who may benefit from therapy based on demethylating agents.

Interleukin-10 gene promoter polymorphisms influence the clinical outcome of diffuse large B-cell lymphoma.

The aim of the study was to investigate whether interleukin-10 (IL-10) genetic polymorphisms influence this cytokine production as well as the incidence and outcome of diffuse large B-cell lymphoma (DLBCL). The frequency of IL-10(-1082G) allele was found to be higher in 199 patients with DLBCL as compared with 112 control subjects (0.47 versus 0.39, P =.043). Increased serum levels of IL-10 were associated with adverse prognostic factors and poor DLBCL outcome. The frequencies of IL-10(-819T) and IL-10(-592A) alleles were lower in patients with elevated IL-10 serum levels (0.155 versus 0.32, P =.14). As compared with patients carrying the IL-10(-1082AA) genotype, patients with the IL-10(-1082G) allele (IL-10(-1082GG/GA) genotypes) had higher complete remission rate (78% [confidence interval (CI), 71%-85%] versus 65% [CI, 52%-78%], P =.07), 5-year freedom from progression (FFP) (60% [CI, 52%-68%] versus 40% [CI, 27%-53%], P =.013), and overall survival (OS) (63% [CI, 55%-71%] versus 33% [CI, 20%-45%], P =.0009). Among factors of the International Prognostic Index, IL-10(-1082G) allele remained an independent variable, predicting longer freedom from progression (FFP) (RR [relative risk] =.76, P =.00035) and OS (RR =.78, P =.0015). These results indicate that IL-10 production contributes to the clinical course of DLBCL and that this phenomenon involves a substantial genetic component.