Explore the vast range of titles available.

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.

We report a customized gene panel assay based on multiplex long-PCR followed by third generation sequencing on nanopore technology (MinION), designed to analyze five frequently mutated genes in chronic lymphocytic leukemia (CLL): TP53, NOTCH1, BIRC3, SF3B1 and MYD88. For this purpose, 12 patients were selected according to specific cytogenetic and molecular features significantly associated with their mutational status. In addition, simultaneous analysis of the targets genes was performed by molecular assays or Sanger Sequencing. Data analysis included mapping to the GRCh37 human reference genome, variant calling and annotation, and average sequencing depth/error rate analysis. The sequencing depth resulted on average higher for smaller amplicons, and the final breadth of coverage of the panel was 94.1%. The error rate was about 6% and 2% for insertions/deletions and single nucleotide variants, respectively. Our gene panel allows analysis of the prognostically relevant genes in CLL, with two PCRs per patient. This strategy offers an easy and affordable workflow, although further advances are required to improve the accuracy of the technology and its use in the clinical field. Nevertheless, the rapid and constant development of nanopore technology, in terms of chemistry advances, more accurate basecallers and analysis software, offers promise for a wide use of MinION in the future.

Since its introduction in clinical practice, eltrombopag (ELT) has demonstrated efficacy in heterogeneous clinical contexts, encompassing both benign and malignant diseases, thus leading researchers to make a more in-depth study of its mechanism of action. As a result, a growing body of evidence demonstrates that ELT displays many effects ranging from native thrombopoietin agonism to immunomodulation, anti-inflammatory, and metabolic properties. These features collectively explain ELT effectiveness in a broad spectrum of indications; moreover, they suggest that ELT could be effective in different, challenging clinical scenarios. We reviewed the extended ELT mechanism of action in various diseases, with the aim of further exploring its full potential and hypothesize new, fascinating indications.

Optical genome mapping (OGM) is a new genome-wide technology that can reveal both structural genomic variations (SVs) and copy number variations (CNVs) in a single assay. OGM was initially employed to perform genome assembly and genome research, but it is now more widely used to study chromosome aberrations in genetic disorders and in human cancer. One of the most useful OGM applications is in hematological malignancies, where chromosomal rearrangements are frequent and conventional cytogenetic analysis alone is insufficient, necessitating further confirmation using ancillary techniques such as fluorescence in situ hybridization, chromosomal microarrays, or multiple ligation-dependent probe amplification. The first studies tested OGM efficiency and sensitivity for SV and CNV detection, comparing heterogeneous groups of lymphoid and myeloid hematological sample data with those obtained using standard cytogenetic diagnostic tests. Most of the work based on this innovative technology was focused on myelodysplastic syndromes (MDSs), acute myeloid leukemia (AML), and acute lymphoblastic leukemia (ALL), whereas little attention was paid to chronic lymphocytic leukemia (CLL) or multiple myeloma (MM), and none was paid to lymphomas. The studies showed that OGM can now be considered as a highly reliable method, concordant with standard cytogenetic techniques but able to detect novel clinically significant SVs, thus allowing better patient classification, prognostic stratification, and therapeutic choices in hematological malignancies.

Acute myeloid leukemia (AML) clinical settings cannot do without molecular testing to confirm or rule out predictive biomarkers for prognostic stratification, in order to initiate or withhold targeted therapy. Next generation sequencing offers the advantage of the simultaneous investigation of numerous genes, but these methods remain expensive and time consuming. In this context, we present a nanopore-based assay for rapid (24 h) sequencing of six genes (NPM1, FLT3, CEBPA, TP53, IDH1 and IDH2) that are recurrently mutated in AML. The study included 22 AML patients at diagnosis; all data were compared with the results of S5 sequencing, and discordant variants were validated by Sanger sequencing. Nanopore approach showed substantial advantages in terms of speed and low cost. Furthermore, the ability to generate long reads allows a more accurate detection of longer FLT3 internal tandem duplications and phasing double CEBPA mutations. In conclusion, we propose a cheap, rapid workflow that can potentially enable all basic molecular biology laboratories to perform detailed targeted gene sequencing analysis in AML patients, in order to define their prognosis and the appropriate treatment.

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.

Background The assessment of TP53 mutational status is becoming a routine clinical practice for chronic lymphocytic leukemia patients (CLL). A broad spectrum of molecular techniques has been employed so far, including both direct Sanger sequencing and next generation sequencing. Oxford Nanopore Technologies recently released the MinION an USB-interfaced sequencer. In this paper we report our experience, with the MinION technology for the detection of the TP53 gene mutation in CLL patients. Twelve CLL patients at diagnosis were included in this study. All except one patient showed the TP53 gene deletion in Fluorescence in situ hybridization experiments. Patients were investigated for TP53 mutation by Sanger and by MinION sequencing. Analysis by Sanger was performed according with the IARC protocol. Analysis by MinION was performed adopting a strategy based on long template PCR, read error correction, and post variant calling filtering. Results Due to the high error rate of nanopore technology, sequence data were both used directly and before correction with two different in silico methods: ALEC and nanocorrect. A mean error rate of 15 % was detected before correction that was reduced to 4-5 % after correction. Analysis by Sanger sequencing was able to detect four patients mutated for TP53. MinION analysis detected one more mutated patient previously not detected from Sanger. Conclusion In our hands, the Nanopore technology shows correlation with Sanger sequencing but more sensitive, manageable and less expensive, and therefore has proven to be a useful tool for TP53 gene mutation detection.

Background Splenomegaly is an event occurring in a variable range between 10–40% of de novo acute myeloid leukemia (AML), recently linked to poorer prognosis. Studies in murine models have shown that loss of the additional sex combs-like 1 (ASXL1) gene function leads to a significantly enlarged spleen volume, due to an increased infiltration of myeloid cells into the spleen. Methods In 58 de novo AML patients presenting with splenomegaly at diagnosis, we evaluated the occurrence of ASXL1 somatic mutations, deepened the molecular profile and conducted high-throughput RNA sequencing, with the aim of unveiling possible peculiar aspects of this rare clinical scenario. Results ASXL1 mutations ( ASXL1 mut) were detected in 23/58 (40%) patients, being the most frequently mutated gene, followed by TET2 and NRAS . ASXL1 mut cases were significantly older than ASXL1 wt (71 vs 64 years old, p = 0.003), showed a significantly higher white blood cells count (31,970/uL vs 17,810/uL, p = 0.044) and a higher platelet count (177,700/uL vs 67,700/uL, p = 0.0006). In contrast, the median bone marrow blasts percentage was lower in the ASXL1 mut subset compared to ASXL1 wt (36.4% vs 72,1%, p = 0.002). Comparing the gene expression profile of the ASXL1 mut and ASXL1 wt groups, we found the upregulation of PCDHB2 and LURAP1L/LURAP1L-AS1 (all involved in mechanisms of cellular interaction and migration) genes in the former group, unveiling a role in the splenic infiltration of ASXL1 mut leukemic cells. Conclusions Overall, our data paves the way for further studies of an AML subgroup with a distinctive phenotype, whose prompt identification could improve patient management and therapeutic decision making.

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.