Epitranscriptomics in normal and malignant hematopoiesis

Epitranscriptomics analyze the biochemical modifications borne by RNA and their downstream influence. From this point of view, epitranscriptomics represent a new layer for the control of genetic information and can affect a variety of molecular processes including the cell cycle and the differentiation. In physiological conditions, hematopoiesis is a tightly regulated process that produces differentiated blood cells starting from hematopoietic stem cells. Alteration of this process can occur at different levels in the pathway that leads from the genetic information to the phenotypic manifestation producing malignant hematopoiesis. This review focuses on the role of epitranscriptomic events that are known to be implicated in normal and malignant hematopoiesis, opening a new pathophysiological and therapeutic scenario. Moreover, an evolutionary vision of this mechanism will be provided

A NOVEL FUSION 5'AFF3/3'BCL2 ORIGINATED FROM A t(2;18)(Q11.2-Q21.33) TRANSLOCATION IN FOLLICULAR LYMPHOMA

Follicular lymphoma is the second most frequent type of non-Hodgkin's lymphoma in adults. The basic molecular defect consists of the t(14;18)(q32;q21) translocation, juxtaposing the B-cell lymphoma protein 2 gene BCL2 to the immunoglobulin heavy chain locus IGH@, and leading to the antiapoptotic BCL2 protein overproduction. Variations in the t(14;18) are rare and can be classified into two categories: (i) simple variants, involving chromosomes 18 and 2, or 22, in which the fusion partner of BCL2 is the light-chain IGK@ or IGL@; (ii) complex variant translocations occurring among chromosomes 14, 18 and other chromosomes. We report a follicular lymphoma case showing BCL2 overexpression, detected by immunohistochemistry and real-time quantitative PCR, consequently to the formation of a novel fusion gene between the 5' of the lymphoid nuclear transcriptional activator gene AFF3 at 2q11.2, and the 3' of BCL2. This case shows evidence, for the first time, of BCL2 overexpression consequently to the fusion of BCL2 to a non-IG partner locus

BCR-ABL1 doubling-times and halving-times may predict CML response to tyrosine kinase inhibitors

In Chronic Myeloid Leukemia (CML), successful treatment requires accurate molecular monitoring to evaluate disease response and provide timely interventions for patients failing to achieve the desired outcomes. We wanted to determine whether measuring BCR-ABL1 mRNA doubling-times (DTs) could distinguish inconsequential rises in the oncogene’s expression from resistance to tyrosine kinase inhibitors (TKIs). Thus, we retrospectively examined BCR-ABL1 evolution in 305 chronic-phase CML patients receiving imatinib mesylate (IM) as a first line treatment. Patients were subdivided in two groups: those with a confirmed rise in BCR-ABL1 transcripts without MR3.0 loss and those failing IM. We found that the DTs of the former patients were significantly longer than those of patients developing IM resistance (57.80 vs. 41.45 days, p = 0.0114). Interestingly, the DT values of individuals failing second-generation (2G) TKIs after developing IM resistance were considerably shorter than those observed at the time of IM failure (27.20 vs. 41.45 days; p = 0.0035). We next wanted to establish if decreases in BCR-ABL1 transcripts would identify subjects likely to obtain deep molecular responses. We therefore analyzed the BCR-ABL1 halving-times (HTs) of a different cohort comprising 174 individuals receiving IM in first line and observed that, regardless of the time point selected for our analyses (6, 12, or 18 months), HTs were significantly shorter in subjects achieving superior molecular responses (p = 0.002 at 6 months; p < 0.001 at 12 months; p = 0.0099 at 18 months). Moreover, 50 patients receiving 2G TKIs as first line therapy and obtaining an MR3.0 (after 6 months; p = 0.003) or an MR4.0 (after 12 months; p = 0.019) displayed significantly shorter HTs than individuals lacking these molecular responses. Our findings suggest that BCR-ABL1 DTs and HTs are reliable tools to, respectively, identify subjects in MR3.0 that are failing their assigned TKI or to recognize patients likely to achieve deep molecular responses that should be considered for treatment discontinuation

Clinical implications of discordant early molecular responses in CML patients treated with imatinib

A reduction in BCR-ABL1/ABL1IS transcript levels to <10% after 3 months or <1% after 6 months of tyrosine kinase inhibitor therapy are associated with superior clinical outcomes in chronic myeloid leukemia (CML) patients. In this study, we investigated the reliability of multiple BCR-ABL1 thresholds in predicting treatment outcomes for 184 subjects diagnosed with CML and treated with standard-dose imatinib mesylate (IM). With a median follow-up of 61 months, patients with concordant BCR-ABL1/ABL1IS transcripts below the defined thresholds (10% at 3 months and 1% at 6 months) displayed significantly superior rates of event-free survival (86.1% vs. 26.6%) and deep molecular response (≥ MR4; 71.5% vs. 16.1%) compared to individuals with BCR-ABL1/ABL1IS levels above these defined thresholds. We then analyzed the outcomes of subjects displaying discordant molecular transcripts at 3-and 6-month time points. Among these patients, those with BCR-ABL1/ABL1IS values >10% at 3 months but <1% at 6 months fared significantly better than individuals with BCR-ABL1/ABL1IS <10% at 3 months but >1% at 6 months (event-free survival 68.2% vs. 32.7%; p < 0.001). Likewise, subjects with BCR-ABL1/ABL1IS at 3 months >10% but <1% at 6 months showed a higher cumulative incidence of MR4 compared to patients with BCR-ABL1/ABL1IS <10% at 3 months but >1% at 6 months (75% vs. 18.2%; p < 0.001). Finally, lower BCR-ABL1/GUSIS transcripts at diagnosis were associated with BCR-ABL1/ABL1IS values <1% at 6 months (p < 0.001). Our data suggest that when assessing early molecular responses to therapy, the 6-month BCR-ABL1/ABL1IS level displays a superior prognostic value compared to the 3-month measurement in patients with discordant oncogenic transcripts at these two pivotal time points

Height estimates for Killing graphs

The paper aims at proving global height estimates for Killing graphs defined over a complete manifold with nonempty boundary. To this end, we first point out how the geometric analysis on a Killing graph is naturally related to a weighted manifold structure, where the weight is defined in terms of the length of the Killing vector field. According to this viewpoint, we introduce some potential theory on weighted manifolds with boundary and we prove a weighted volume estimate for intrinsic balls on the Killing graph. Finally, using these tools, we provide the desired estimate for the weighted height in the assumption that the Killing graph has constant weighted mean curvature and the weighted geometry of the ambient space is suitably controlled.Comment: 26 pages. Final version. To appear on Journal of Geometric Analysi

Absolute quantification of the pretreatment PML-RARA transcript defines the relapse risk in acute promyelocytic leukemia.

In this study we performed absolute quantification of the PML-RARA transcript by droplet digital polymerase chain reaction (ddPCR) in 76 newly diagnosed acute promyelocytic leukemia (APL) cases to verify the prognostic impact of the PML-RARA initial molecular burden. ddPCR analysis revealed that the amount of PML-RARA transcript at diagnosis in the group of patients who relapsed was higher than in that with continuous complete remission (CCR) (272 vs 89.2 PML-RARA copies/ng, p = 0.0004, respectively). Receiver operating characteristic analysis detected the optimal PML-RARA concentration threshold as 209.6 PML-RARA/ng (AUC 0.78; p < 0.0001) for discriminating between outcomes (CCR versus relapse). Among the 67 APL cases who achieved complete remission after the induction treatment, those with > 209.6 PML-RARA/ng had a worse relapse-free survival (p = 0.0006). At 5-year follow-up, patients with > 209.6 PML-RARA/ng had a cumulative incidence of relapse of 50.3% whereas 7.5% of the patients with suffered a relapse (p < 0.0001). Multivariate analysis identified the amount of PML-RARA before induction treatment as the sole independent prognostic factor for APL relapse. Our results show that the pretreatment PML-RARA molecular burden could therefore be used to improve risk stratification in order to develop more individualized treatment regimens for high-risk APL cases

ADAMTS2 gene dysregulation in T/myeloid mixed phenotype acute leukemia.

Background: Mixed phenotype acute leukemias (MPAL) include acute leukemias with blasts that express antigens of more than one lineage, with no clear evidence of myeloid or lymphoid lineage differentiation. T/myeloid (T/My) MPAL not otherwise specified (NOS) is a rare leukemia that expresses both T and myeloid antigens, accounting for less than 1% of all leukemias but 89% of T/My MPAL. From a molecular point of view, very limited data are available on T/My MPAL NOS. Case presentation: In this report we describe a T/My MPAL NOS case with a complex rearrangement involving chromosomes 5 and 14, resulting in overexpression of the ADAM metallopeptidase with thrombospondin type 1 motif, 2 (ADAMTS2) gene due to its juxtaposition to the T cell receptor delta (TRD) gene segment. Conclusion: Detailed molecular cytogenetic characterization of the complex rearrangement in the reported T/My MPAL case allowed us to observe ADAMTS2 gene overexpression, identifying a molecular marker that may be useful for monitoring minimal residual disease. To our knowledge, this is the first evidence of gene dysregulation due to a chromosomal rearrangement in T/My MPAL NOS. Keywords: Mixed phenotype acute leukemia, ADAMTS2, TRD, Complex chromosomal rearrangement, Promoter swapping, Gene dysregulatio

Nanopore sequencing approach for immunoglobulin gene analysis in chronic lymphocytic leukemia

The evaluation of the somatic hypermutation of the clonotypic immunoglobulin heavy variable gene has become essential in the therapeutic management in chronic lymphocytic leukemia patients. European Research Initiative on Chronic Lymphocytic Leukemia promotes good practices and standardized approaches to this assay but often they are labor-intensive, technically complex, with limited in scalability. The use of next-generation sequencing in this analysis has been widely tested, showing comparable accuracy and distinct advantages. However, the adoption of the next generation sequencing requires a high sample number (run batching) to be economically convenient, which could lead to a longer turnaround time. Here we present data from nanopore sequencing for the somatic hypermutation evaluation compared to the standard method. Our results show that nanopore sequencing is suitable for immunoglobulin heavy variable gene mutational analysis in terms of sensitivity, accuracy, simplicity of analysis and is less time-consuming. Moreover, our work showed that the development of an appropriate data analysis pipeline could lower the nanopore sequencing error rate attitude

RUNX1 gene alterations characterized by allelic preference in adult acute myeloid leukemia

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Molecular-biology-driven treatment for metastatic colorectal cancer

Background: Metastatic CRC (mCRC) is a molecular heterogeneous disease. The aim of this review is to give an overview of molecular-driven treatment of mCRC patients. Methods: A review of clinical trials, retrospective studies and case reports was performed regarding molecular biomarkers with therapeutic implications. Results: RAS wild-type status was confirmed as being crucial for anti-epidermal growth factor receptor (EGFR) monoclonal antibodies and for rechallenge strategy. Antiangiogenic therapies improve survival in first- and second-line settings, irrespective of RAS status, while tyrosine kinase inhibitors (TKIs) remain promising in refractory mCRC. Promising results emerged from anti-HER2 drugs trials in HER2-positive mCRC. Target inhibitors were successful for BRAFV600E mutant mCRC patients, while immunotherapy was successful for microsatellite instability-high/defective mismatch repair (MSI-H/dMMR) or DNA polymerase epsilon catalytic subunit (POLE-1) mutant patients. Data are still lacking on NTRK, RET, MGMT, and TGF-β, which require further research. Conclusion: Several molecular biomarkers have been identified for the tailored treatment of mCRC patients and multiple efforts are currently ongoing to increase the therapeutic options. In the era of precision medicine, molecular-biology-driven treatment is the key to impro patient selection and patient outcomes. Further research and large phase III trials are required to ameliorate the therapeutic management of these patients