Nanopore Sequencing in Blood Diseases: A Wide Range of Opportunities

The molecular pathogenesis of hematological diseases is often driven by genetic and epigenetic alterations. Next-generation sequencing has considerably increased our genomic knowledge of these disorders becoming ever more widespread in clinical practice. In 2012 Oxford Nanopore Technologies (ONT) released the MinION, the first long-read nanopore-based sequencer, overcoming the main limits of short-reads sequences generation. In the last years, several nanopore sequencing approaches have been performed in various “-omic” sciences; this review focuses on the challenge to introduce ONT devices in the hematological field, showing advantages, disadvantages and future perspectives of this technology in the precision medicine era

Can the new and old drugs exert an immunomodulatory effect in acute myeloid leukemia?

Acute myeloid leukemia (AML) is considered an immune-suppressive neoplasm capable of evading immune surveillance through cellular and environmental players. Increasing knowledge of the immune system (IS) status at diagnosis seems to suggest ever more attention of the crosstalk between the leukemic clone and its immunologic counterpart. During the last years, the advent of novel immunotherapeutic strategies has revealed the importance of immune dysregulation and suppression for leukemia fitness. Considering all these premises, we reviewed the “off-target” effects on the IS of different drugs used in the treatment of AML, focusing on the main advantages of this interaction. The data reported support the idea that a successful therapeutic strategy should consider tailored approaches for performing leukemia eradication by both direct blasts killing and the engagement of the IS

Inside the biology of early T-cell precursor acute lymphoblastic leukemia: the perfect trick

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) is a rare, distinct subtype of T-ALL characterized by genomic instability, a dismal prognosis and refractoriness to standard chemotherapy. Since its first description in 2009, the expanding knowledge of its intricate biology has led to the definition of a stem cell leukemia with a combined lymphoid-myeloid potential: the perfect trick. Several studies in the last decade aimed to better characterize this new disease, but it was recognized as a distinct entity only in 2016. We review current insights into the biology of ETP-ALL and discuss the pathogenesis, genomic features and their impact on the clinical course in the precision medicine era today

FLT3 mutational analysis in acute myeloid leukemia: Advantages and pitfalls with different approaches

FMS-like tyrosine kinase 3 (FLT3) is one of the most closely studied genes in blood diseases. Numerous methods have been adopted for analyses, mainly in acute myeloid leukemia (AML) diagnostic work-up. According to international recommendations, the current gold standard approach allows FLT3 canonical mutations to be investigated, providing the main information for risk assessment and treatment choice. However, the technological improvements of the last decade have permitted “black side” gene exploration, revealing numerous hidden aspects of its role in leukemogenesis. The advent of the next-generation sequencing era emphasizes lights and shadows of FLT3 conventional mutational analysis, highlighting the need for a more comprehensive study of the gene. However, more extensive analysis is opening new, unexplored questions whose impact on clinical outcomes is still unknown. The present work is focused on the main topics regarding FLT3 mutational analysis in AML, debating the strengths and weaknesses of the current gold standard approach. The rights and wrongs of NGS introduction in clinical practice will be discussed, showing that a more extensive knowledge of FLT3 mutational status could lead to reconsidering its role in AML management

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

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

IRF4 expression is low in Philadelphia negative myeloproliferative neoplasms and is associated with a worse prognosis

Interferon regulatory factor 4 (IRF4) is involved in the pathogenesis of various hematologic malignancies. Its expression has been related to the negative regulation of myeloid-derived suppressor cells (MDSCs) and the polarization of anti-inflammatory M2 macrophages, thereby altering immunosurveillance and inflammatory mechanisms. An abnormal inflammatory status in the bone marrow microenvironment of myeloproliferative neoplasms (MPNs) has recently been demonstrated; moreover, in chronic myeloid leukemia a downregulated expression of IRF4 has been found. In this context, we evaluated the IRF4 expression in 119 newly diagnosed consecutive Philadelphia negative MPNs (Ph- MPNs), showing a low expression among the MPNs phenotypes with a more significant decrease in primary myelofibrosis patients. Lower IRF4 levels were associated with JAK2 + and triple negatives cases carrying the worst prognosis. Furthermore, the IRF4 levels were related to leukemic transformation and a shorter leukemia-free survival; moreover, the risk of myelofibrosis transformation in polycythemia vera and essential thrombocythemia patients was more frequent in cases with lower IRF4 levels. Overall, our study demonstrates an IRF4 dysregulated expression in MPNs patients and its association with a worse prognosis. Further studies could validate these data, to improve our knowledge of the MPNs pathogenesis and confirm the IRF4 role as a new prognostic factor

Valutazione in vitro dell'affaticamento di strumenti rotanti in nichel-titanio

Riassunto Obiettivo L'obiettivo di questo lavoro e quello di valutare, in vitro, la fatica ciclica di ProFile, K3 e Twisted File, strumenti rotanti in nichel-titanio, in canali radicolari simulati aventi angoli di curvatura di 45°, 60° e 90°. Materiali e metodi Per effettuare questo studio sono stati utilizzati: 18 file 25/.06 ProFile; 18 file 25/.06 K3; 18 file 25/.06 Twisted File. La resistenza a fatica ciclica degli strumenti rotanti Ni-Ti e stata testata ad angolazioni di 45°, 60° e 90° mediante un modello sperimentale creato specificatamente. Risultati e conclusioni Dall'analisi dei dati e emersa una buona resistenza alla fatica ciclica per quanto concerne le sistematiche ProFile e Twisted File, di contro, la sistematica K3 e risultata essere inferiore alle altre per tutte le curvature prese in esame. I Twisted File all'angolazione di 90° hanno mostrato una resistenza a fatica ciclica nettamente superiore rispetto ai ProFile e ai K3

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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