MethScore as a new comprehensive DNA methylation-based value refining the prognosis in acute myeloid leukemia.

BACKGROUND: Changes in DNA methylation are common events in the pathogenesis of acute myeloid leukemia (AML) and have been repeatedly reported as associated with prognosis. However, studies integrating these numerous and potentially prognostically relevant DNA methylation changes are lacking. Therefore, we aimed for an overall evaluation of these epigenetic aberrations to provide a comprehensive NGS-based approach of DNA methylation assessment for AML prognostication. RESULTS: We designed a sequencing panel targeting 239 regions (approx. 573 kb of total size) described in the literature as having a prognostic impact or being associated with AML pathogenesis. Diagnostic whole-blood DNA samples of adult AML patients divided into a training (n = 128) and a testing cohort (n = 50) were examined. The libraries were prepared using SeqCap Epi Enrichments System (Roche) and sequenced on MiSeq instrument (Illumina). Altogether, 1935 CpGs affecting the survival (p < 0.05) were revealed in the training cohort. A summarizing value MethScore was then calculated from these significant CpGs. Patients with lower MethScore had markedly longer overall survival (OS) and event-free survival (EFS) than those with higher MethScore (p < 0.001). The predictive ability of MethScore was verified on the independent testing cohort for OS (p = 0.01). Moreover, the proof-of-principle validation was performed using the TCGA dataset. CONCLUSIONS: We showed that comprehensive NGS-based approach of DNA methylation assessment revealed a robust epigenetic signature relevant to AML outcome. We called this signature MethScore and showed it might serve as a strong prognostic marker able to refine survival probability of AML patients.

ABC transporters are predictors of treatment failure in acute myeloid leukaemia.

INTRODUCTION: To date, no chemoresistance predictors are included in acute myeloid leukaemia (AML) prognostic scoring systems to distinguish responding and refractory AML patients prior to chemotherapy. ABC transporters have been described as altering AML chemosensitivity; however, a relevant study investigating their role at various molecular levels was lacking. METHODS: Gene expression, genetic variants, methylation and activity of ABCA2, ABCA5, ABCB1, ABCB6, ABCC1, ABCC3 and ABCG2 were analysed in AML blasts and healthy myeloblasts. Differences between responding and refractory AML in a cohort of 113 patients treated with 3 + 7 induction therapy were explored. RESULTS: ABCC3 variant rs2301837 (p = 0.049), ABCG2 variant rs11736552 (p = 0.044), higher ABCA2 (p = 0.021), ABCC1 (p = 0.017), and ABCG2 expression (p = 0.023) and a higher number of concurrently overexpressed transporters (p = 0.002) were predictive of treatment failure by multivariate analysis. Expression of ABCA5 (p = 0.003), ABCB6 (p = 0.001) and ABCC3 (p < 0.0001) increased significantly after chemotherapy. Higher ABCG2 promoter methylation correlated with lower ABCG2 expression (p = 0.0001). ABCC1 was identified as the most active transporter in AML blasts by functional analysis. CONCLUSIONS: ABC transporters, especially ABCC1 seem to contribute substantially to AML chemoresistance. A detailed understanding of chemoresistance mechanisms and the clinical implications of chemosensitivity predictors may lead to alternative therapeutic approaches for AML patients with unveiled chemoresistance signatures.

A validation study of potential prognostic DNA methylation biomarkers in patients with acute myeloid leukemia using a custom DNA methylation sequencing panel.

BACKGROUND: Multiple studies have reported the prognostic impact of DNA methylation changes in acute myeloid leukemia (AML). However, these epigenetic markers have not been thoroughly validated and therefore are still not considered in clinical practice. Hence, we aimed to independently verify results of selected studies describing the relationship between DNA methylation of specific genes and their prognostic potential in predicting overall survival (OS) and event-free survival (EFS). RESULTS: Fourteen studies (published 2011-2019) comprising of 27 genes were subjected to validation by a custom NGS-based sequencing panel in 178 newly diagnosed non-M3 AML patients treated by 3 + 7 induction regimen. The results were considered as successfully validated, if both the log-rank test and multivariate Cox regression analysis had a p-value ≤ 0.05. The predictive role of DNA methylation was confirmed for three studies comprising of four genes: CEBPA (OS: p = 0.02; EFS: p = 0.03), PBX3 (EFS: p = 0.01), LZTS2 (OS: p = 0.05; EFS: p = 0.0003), and NR6A1 (OS: p = 0.004; EFS: p = 0.0003). For all of these genes, higher methylation was an indicator of longer survival. Concurrent higher methylation of both LZTS2 and NR6A1 was highly significant for survival in cytogenetically normal (CN) AML group (OS: p < 0.0001; EFS: p < 0.0001) as well as for the whole AML cohort (OS: p = 0.01; EFS < 0.0001). In contrast, for two studies reporting the poor prognostic effect of higher GPX3 and DLX4 methylation, we found the exact opposite, again linking higher GPX3 (OS: p = 0.006; EFS: p < 0.0001) and DLX4 (OS: p = 0.03; EFS = 0.03) methylation to a favorable treatment outcome. Individual gene significance levels refer to the outcomes of multivariate Cox regression analysis. CONCLUSIONS: Out of twenty-seven genes subjected to DNA methylation validation, a prognostic role was observed for six genes. Therefore, independent validation studies are necessary to reveal truly prognostic DNA methylation changes and to enable the introduction of these promising epigenetic markers into clinical practice.

WT1 Expression in Peripheral Blood at Diagnosis and During the Course of Early Consolidation Treatment Correlates With Survival in Patients With Intermediate and Poor-Risk Acute Myeloid Leukemia.

BACKGROUND: Up to 55% of non-APL acute myeloid leukemias (AML) lack a molecular target suitable for standardized disease monitoring. We aimed to evaluate the prognostic significance of WT1 gene expression at early stages of intensive treatment. PATIENTS AND METHODS: A total of 106 consecutive patients with intermediate and high-risk AML who had WT1 expression at diagnosis >500 copies/104ABL and who achieved remission after 1 to 2 cycles of induction treatment were analyzed. WT1 expression was measured in peripheral blood using a standardized European LeukemiaNet method. Overexpression was defined as >50 copies/104ABL. The median follow-up was 30 months. RESULTS: Patients with normal versus high WT1 expression after 2 cycles of chemotherapy had overall survival (OS) at 3 years of 66% versus 41% (P = .01); event-free survival (EFS) 45% versus 22% (P = .01). Prognostic significance of WT1 expression after 2 cycles of treatment was maintained in the group of patients treated with chemotherapy alone without hematopoietic stem cell transplantation in first line treatment (OS 70% vs. 36%, P = .02; EFS 35% vs. 0%, P = .03). Significant prognostic factors for EFS on multivariate analysis were the achievement of molecular remission (<50 copies of WT1) at any time during treatment (hazard ratio [HR] 0.47, P = .04) and increased WT1 expression after 2 cycles of chemotherapy (HR 2.0, P = .03). CONCLUSION: Increased WT1 expression after 2 cycles of chemotherapy is a negative prognostic factor for survival. WT1 remains a valuable molecular marker in AML without any leukemia-specific mutation, especially if next generation sequencing and/or digital polymerase chain reaction are not routinely available.

DNA Methylation Validation Methods: a Coherent Review with Practical Comparison.

Here, we present a practical overview of four commonly used validation methods for DNA methylation assessment: methylation specific restriction endonucleases (MSRE) analysis, pyrosequencing, methylation specific high-resolution DNA melting (MS-HRM) and quantitative methylation specific polymerase chain reaction (qMSP). Using these methods, we measured DNA methylation levels of three loci in human genome among which one was highly methylated, one intermediately methylated and one unmethylated. We compared the methods in terms of primer design demands, methods' feasibility, accuracy, time and money consumption, and usability for clinical diagnostics. Pyrosequencing and MS-HRM proved to be the most convenient methods. Using pyrosequencing, it is possible to analyze every CpG in a chosen region. The price of the instrument may represent the main limitation of this methodology. MS-HRM is a simple PCR-based method. The measurement was quick, cheap and very accurate. MSRE analysis is based on a methylation specific digestion of DNA. It does not require a bisulfite conversion of DNA as the other methods. MSRE analysis was very easy to perform, however, it was not suitable for intermediately methylated regions and it was also quite expensive. qMSP is a qPCR-based method that uses primers designed specifically for methylated and unmethylated alleles of a chosen region. This was the least accurate method and also the primer design and optimization of PCR conditions were highly demanding.

Nuclear transport of nicotinamide phosphoribosyltransferase is cell cycle-dependent in mammalian cells, and its inhibition slows cell growth.

Nicotinamide phosphoribosyltransferase (NAMPT) is located in both the nucleus and cytoplasm and has multiple biological functions including catalyzing the rate-limiting step in NAD synthesis. Moreover, up-regulated NAMPT expression has been observed in many cancers. However, the determinants and regulation of NAMPT's nuclear transport are not known. Here, we constructed a GFP-NAMPT fusion protein to study NAMPT's subcellular trafficking. We observed that in unsynchronized 3T3-L1 preadipocytes, 25% of cells had higher GFP-NAMPT fluorescence in the cytoplasm, and 62% had higher GFP-NAMPT fluorescence in the nucleus. In HepG2 hepatocytes, 6% of cells had higher GFP-NAMPT fluorescence in the cytoplasm, and 84% had higher GFP-NAMPT fluorescence in the nucleus. In both 3T3-L1 and HepG2 cells, GFP-NAMPT was excluded from the nucleus immediately after mitosis and migrated back into it as the cell cycle progressed. In HepG2 cells, endogenous, untagged NAMPT displayed similar changes with the cell cycle, and in nonmitotic cells, GFP-NAMPT accumulated in the nucleus. Similarly, genotoxic, oxidative, or dicarbonyl stress also caused nuclear NAMPT localization. These interventions also increased poly(ADP-ribosyl) polymerase and sirtuin activity, suggesting an increased cellular demand for NAD. We identified a nuclear localization signal in NAMPT and amino acid substitution in this sequence (424RSKK to ASGA), which did not affect its enzymatic activity, blocked nuclear NAMPT transport, slowed cell growth, and increased histone H3 acetylation. These results suggest that NAMPT is transported into the nucleus where it presumably increases NAD synthesis required for cell proliferation. We conclude that specific inhibition of NAMPT transport into the nucleus might be a potential avenue for managing cancer.

DNA methylation and hydroxymethylation patterns in acute myeloid leukemia patients with mutations in DNMT3A and IDH1/2 and their combinations.

BACKGROUND: Aberrant epigenetic patterns are a hallmark of acute myeloid leukemia (AML). Mutations in profound epigenetic regulators DNMT3A and IDH1/2 often occur concurrently in AML. OBJECTIVES: The aim was to analyze DNA methylation, hydroxymethylation and mRNA expression profiles in AML with mutations in DNMT3A and IDH1/2 (individually and in combinations). METHODS: Infinium MethylationEPIC BeadChip (Illumina) covering 850,000 CpGs was utilized. The validation of hydroxy-/methylation data was done by pyrosequencing. HumanHT-12 v4 Expression BeadChip (Illumina) was used for expression examination. RESULTS: Hierarchical clustering analysis of DNA hydroxy-/methylation data revealed clusters corresponding to DNMT3A and IDH1/2 mutations and CD34+ healthy controls. Samples with concurrent presence of DNMT3A and IDH1/2 mutations displayed mixed DNA hydroxy-/methylation profile with preferential clustering to healthy controls. Numbers and levels of DNA hydroxymethylation were low. Uniformly hypermethylated loci in AML patients with IDH1/2 mutations were enriched for immune response and apoptosis related genes, among which hypermethylation of granzyme B (GZMB) was found to be associated with inferior overall survival of AML patients (P= 0.035). CONCLUSIONS: Distinct molecular background results in specific DNA hydroxy-/methylation profiles in AML. Site-specific DNA hydroxymethylation changes are much less frequent in AML pathogenesis compared to DNA methylation. Methylation levels of enhancer located upstream GZMB gene might contribute to AML prognostication models.