Optical Genome Mapping as a Tool to Unveil New Molecular Findings in Hematological Patients with Complex Chromosomal Rearrangements.

Standard cytogenetic techniques (chromosomal banding analysis-CBA, and fluorescence in situ hybridization-FISH) show limits in characterizing complex chromosomal rearrangements and structural variants arising from two or more chromosomal breaks. In this study, we applied optical genome mapping (OGM) to fully characterize two cases of complex chromosomal rearrangements at high resolution. In case 1, an acute myeloid leukemia (AML) patient showing chromothripsis, OGM analysis was fully concordant with classic cytogenetic techniques and helped to better refine chromosomal breakpoints. The OGM results of case 2, a patient with non-Hodgkin lymphoma, were only partially in agreement with previous cytogenetic analyses and helped to better define clonal heterogeneity, overcoming the bias related to clonal selection due to cell culture of cytogenetic techniques. In both cases, OGM analysis led to the identification of molecular markers, helping to define the pathogenesis, classification, and prognosis of the analyzed patients. Despite extensive efforts to study hematologic diseases, standard cytogenetic methods display unsurmountable limits, while OGM is a tool that has the power to overcome these limitations and provide a cytogenetic analysis at higher resolution. As OGM also shows limits in defining regions of a repetitive nature, combining OGM with CBA to obtain a complete cytogenetic characterization would be desirable.

TNFRSF13B gene mutation in familial acute myeloid leukemia: A new piece in the complex scenario of hereditary predisposition?

TNFRSF13B mutations are widely associated with common variable immunodeficiency. TNFRSF13B was recently counted among relevant genes associated with childhood-onset of hematological malignancies; nonetheless, its role in acute myeloid leukemia (AML) remains unexplored. We report the study of a family with two cases of AML, sharing a germline TNFRSF13B mutation favoring the formation of a more stable complex with its ligand TNFSF13: a positive regulator of AML-initiating cells. Our data turn the spotlight onto the TNFRSF13B role in AML onset, inserting a new fragment into the complex scenario of a hereditary predisposition to myeloid neoplasms.

Case report: biallelic DNMT3A mutations in acute myeloid leukemia.

DNMT3A gene mutations, detected in 20-25% of de novo acute myeloid leukemia (AML) patients, are typically heterozygous. Biallelic variants are uncommon, affecting ~3% of cases and identifying a worse prognosis. Indeed, two concomitant DNMT3A mutations were recently associated with shorter event-free survival and overall survival in AML. We present an AML case bearing an unusual DNMT3A molecular status, strongly affecting its function and strangely impacting the global genomic methylation profile. A 56-year-old Caucasian male with a diagnosis of AML not otherwise specified (NOS) presented a complex DNMT3A molecular profile consisting of four different somatic variants mapping on different alleles (in trans). 3D modelling analysis predicted the effect of the DNMT3A mutational status, showing that all the investigated mutations decreased or abolished DNMT3A activity. Although unexpected, DNMT3A's severe loss of function resulted in a global genomic hypermethylation in genes generally involved in cell differentiation. The mechanisms through which DNMT3A contributes to AML remain elusive. We present a unique AML case bearing multiple biallelic DNMT3A variants abolishing its activity and resulting in an unexpected global hypermethylation. The unusual DNMT3A behavior described requires a reflection on its role in AML development and persistence, highlighting the heterogeneity of its deregulation.

Feasibility of Optical Genome Mapping in Cytogenetic Diagnostics of Hematological Neoplasms: A New Way to Look at DNA.

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.

The Role of NLRP3, a Star of Excellence in Myeloproliferative Neoplasms.

Nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) is the most widely investigated inflammasome member whose overactivation can be a driver of several carcinomas. It is activated in response to different signals and plays an important role in metabolic disorders and inflammatory and autoimmune diseases. NLRP3 belongs to the pattern recognition receptors (PRRs) family, expressed in numerous immune cells, and it plays its primary function in myeloid cells. NLRP3 has a crucial role in myeloproliferative neoplasms (MPNs), considered to be the diseases best studied in the inflammasome context. The investigation of the NLRP3 inflammasome complex is a new horizon to explore, and inhibiting IL-1ß or NLRP3 could be a helpful cancer-related therapeutic strategy to improve the existing protocols.

Clonal hematopoiesis onset in chronic myeloid leukemia patients developing an adverse cardiovascular event.

Life expectation of chronic myeloid leukemia patients in the tyrosine kinase inhibitors era is almost equal to that of healthy subjects. On the other hand, their long-term management must take into account a higher risk of adverse events, at least partly related to the treatment. Various studies reported a higher incidence of cardiovascular events in these patients. Clonal hematopoiesis is broadly considered a major independent risk factor for cardiovascular events. Of note, the underlying physiopathological mechanisms connect clonal hematopoiesis with a global proinflammatory status, triggering a vicious circle in which the somatic mutations and inflammation feed each other. All this considered, we investigated the occurrence of clonal hematopoiesis in chronic myeloid leukemia patients developing a cardiovascular event under tyrosine kinase inhibitor therapy.

IKAROS in Acute Leukemia: A Positive Influencer or a Mean Hater?

One key process that controls leukemogenesis is the regulation of oncogenic gene expression by transcription factors acting as tumor suppressors. Understanding this intricate mechanism is crucial to elucidating leukemia pathophysiology and discovering new targeted treatments. In this review, we make a brief overview of the physiological role of IKAROS and the molecular pathway that contributes to acute leukemia pathogenesis through IKZF1 gene lesions. IKAROS is a zinc finger transcription factor of the Krüppel family that acts as the main character during hematopoiesis and leukemogenesis. It can activate or repress tumor suppressors or oncogenes, regulating the survival and proliferation of leukemic cells. More than 70% of Ph+ and Ph-like cases of acute lymphoblastic leukemia exhibit IKZF1 gene variants, which are linked to worse treatment outcomes in both childhood and adult B-cell precursor acute lymphoblastic leukemia. In the last few years, much evidence supporting IKAROS involvement in myeloid differentiation has been reported, suggesting that loss of IKZF1 might also be a determinant of oncogenesis in acute myeloid leukemia. Considering the complicated "social" network that IKAROS manages in hematopoietic cells, we aim to focus on its involvement and the numerous alterations of molecular pathways it can support in acute leukemias.

IRF4 Gene Expression on the Trail of Molecular Response: Looking at Chronic Myeloid Leukemia from Another Perspective.

INTRODUCTION: Interferon regulatory factor 4 (IRF4) is a transcriptional factor with a key role in the modulation of inflammation and immune surveillance. The IRF4 gene is downregulated in Philadelphia-negative myeloproliferative neoplasms, and its expression is associated with prognosis and response to treatment. METHODS: We evaluated the IRF4 expression kinetics during tyrosine kinase inhibitor (TKI) treatment in a cohort of 116 chronic myeloid leukemia (CML) patients to elucidate its role in the disease course. RESULTS: A relationship between the IRF4 expression and the disease burden was observed at various disease stages. A correlation analysis between the International Scale (IS) and IRF4 values confirmed this close association. A significant increase is detected after 3 months of TKI treatment. Patients achieving an early molecular response (EMR) had higher IRF4 values at both diagnosis and after 3 months of therapy as compared to those failing the EMR target. Patients achieving treatment-free remission did not show IRF4 fluctuations during monitoring, while a decreased IRF4 expression emerged at the time of molecular relapse. CONCLUSION: Our data seem to confirm the relevance of IRF4 in the pathogenesis of CML, suggesting a pivotal role at the disease onset and a predictive value during the CML course.

JAK2 V617f in chronic myeloid leukemia: driving force or passive bystander?

OBJECTIVES: BCR-ABL1 and JAK2 V617F coexistence in myeloproliferative neoplasms has been described as concomitant or sequential events. Despite this, we present a unique case of chronic myeloid leukemia (CML) not referable to either of the known scenarios. METHODS: BCR-ABL1 molecular monitoring was performed by real-time quantitative PCR (RQ-PCR). At the time of molecular relapse, a targeted next-generation sequencing analysis with a customized panel of 26 genes commonly mutated in myeloid diseases was performed. To investigate the kinetics of the JAK2 variant and its association with the BCR-ABL1 rearrangement, RQ-PCR was performed at different time points during the patient's follow-up. RESULTS: While negative at CML diagnosis, the JAK2 mutation was first detected 9 years later (VAF: 7.2%). The mutational burden of JAK2 remained stable in multiple determinations, with minor fluctuations independent of BCR-ABL1 kinetics. At the last available time point, the patient was in deep molecular response (MR4), the JAK2 mutational burden was 7%, and no clinical-laboratory findings of Ph-MPN were detectable. DISCUSSION: In the presented case, the JAK2variantoccurring during the course of the disease seems to stay in the shadows of CML, just as a bystander. The impact of this event (that may be considered suggestive of clonal hematopoiesis of indeterminate potential) on the disease outcome, even if seemingly irrelevant, has still to be explored.

Genome characterization and CRISPR-Cas9 editing of a human neocentromere.

The maintenance of genome integrity is ensured by proper chromosome inheritance during mitotic and meiotic cell divisions. The chromosomal counterpart responsible for chromosome segregation to daughter cells is the centromere, at which the spindle apparatus attaches through the kinetochore. Although all mammalian centromeres are primarily composed of megabase-long repetitive sequences, satellite-free human neocentromeres have been described. Neocentromeres and evolutionary new centromeres have revolutionized traditional knowledge about centromeres. Over the past 20 years, insights have been gained into their organization, but in spite of these advancements, the mechanisms underlying their formation and evolution are still unclear. Today, through modern and increasingly accessible genome editing and long-read sequencing techniques, research in this area is undergoing a sudden acceleration. In this article, we describe the primary sequence of a previously described human chromosome 3 neocentromere and observe its possible evolution and repair results after a chromosome breakage induced through CRISPR-Cas9 technologies. Our data represent an exciting advancement in the field of centromere/neocentromere evolution and chromosome stability.

Identification and Characterisation of pST1023 A Mosaic, Multidrug-Resistant and Mobilisable IncR Plasmid.

We report the identification and characterisation of a mosaic, multidrug-resistant and mobilisable IncR plasmid (pST1023) detected in Salmonella ST1023, a monophasic variant 4,[5],12:i: strain of widespread pandemic lineage, reported as a Southern European clone. pST1023 contains exogenous DNA regions, principally gained from pSLT-derivatives and IncI1 plasmids. Acquisition from IncI1 included oriT and nikAB and these conferred the ability to be mobilisable in the presence of a helper plasmid, as we demonstrated with the conjugative plasmids pST1007-1D (IncFII) or pVC1035 (IncC). A sul3-associated class 1 integron, conferring resistance to aminoglycosides, chloramphenicol and trimethoprim-sulphonamides, was also embedded in the acquired IncI1 DNA segment. pST1023 also harboured an additional site-specific recombination system (rfsF/rsdB) and IS elements of the IS1, IS5 (IS903 group) and IS6 families. Four of the six IS26 elements present constituted two pseudo-compound-transposons, named PCT-sil and PCT-Tn10 (identified here for the first time). The study further highlighted the mosaic genetic architecture and the clinical importance of IncR plasmids. Moreover, it provides the first experimental data on the ability of IncR plasmids to be mobilised and their potential role in the horizontal spread of antimicrobial-resistant genes.

The genomic analysis brings a new piece to the molecular jigsaw of idiopathic erythrocytosis.

Erythrocytosis is a clinical condition characterized by increased red cell mass, hemoglobin, and hematocrit values. A significant fraction of patients is described as having idiopathic erythrocytosis. We have previously demonstrated an association between erythrocytosis and the JAK2 GGCC_46/1 haplotype and CALR rs1049481_G allele. In the present study, we investigated genomic and clinical features of 80 erythrocytosis patients with the aim to provide useful information in clinical practice. Patients with idiopathic erythrocytosis could have a genomic germline background, eventually associated with somatic variants. Through association analysis, we show that male patients presenting with idiopathic erythrocytosis, and normal EPO levels could be the best candidates for the search for the JAK2 GGCC_46/1 haplotype and CALR rs1049481_G allele. Further studies are needed to confirm these findings and to depict detailed genomic and phenotypical characteristics of these patients.

Exploring the Potential of Eltrombopag: Room for More?

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.

Clonal hematopoiesis in clinical practice: walking a tightrope.

The understanding of clonal hematopoiesis (CH) and its features is rapidly evolving in step with the spread of sequencing techniques. Indeed, CH detection is now an emerging aspect in clinical practice. The awareness of CH intersects with consolidated diagnostic paths, thus exposing 'grey zone' circumstances under the magnifying lens of clinicians. The interpretation of genomic data poses, in some cases, a true clinical challenge, sometimes further complicating the route to diagnosis. The line separating different entities is thin. The present work aims to review some of these challenging situations to help clinicians keep their balance along this tightrope.

Clonal Hematopoiesis at the Crossroads of Inflammatory Bowel Diseases and Hematological Malignancies: A Biological Link?

Inflammatory bowel diseases (IBDs) are a group of chronic conditions of the gastrointestinal tract in which nationwide studies have revealed a higher risk of hematological malignancies (HMs). Clonal hematopoiesis (CH) is a premalignant condition defined by the presence of an acquired somatic mutation characterized by a variant allele frequency (VAF) of ≥2%, in a gene frequently associated with HMs. A growing body of evidence suggests a correlation between inflammation and CH; its occurrence in the context of IBD has been previously demonstrated. With the aim to assess CH possible co-occurrence in patients with an IBD associated with HMs, we performed a targeted next-generation sequencing analysis in a cohort of thirteen patients who were referred to our center with IBD associated with HMs. Eleven (85%) patients showed one or more mutations in CH-associated genes; DNMT3A was the most frequently mutated gene, followed by ASXL1 and JAK2. These results may suggest that the mechanisms at the basis of the inflammatory environment could potentially select for the growth of hematopoietic clones harboring specific mutations. In this context, CH emergence may be boosted by the proinflammatory IBD environment, thus acting as a biological link between IBD and the HM onset. If these data are confirmed, IBD patients screened and positive for CH should undergo a hematologic follow-up to assess the risk of developing HM. Future study will clarify the relationship between these conditions.

Second Cancer Onset in Myeloproliferative Neoplasms: What, When, Why?

The risk of developing a solid cancer is a major issue arising in the disease course of a myeloproliferative neoplasm (MPN). Although the connection between the two diseases has been widely described, the backstage of this complex scenario has still to be explored. Several cellular and molecular mechanisms have been suggested to link the two tumors. Sometimes the MPN is considered to trigger a second cancer but at other times both diseases seem to depend on the same source. Increasing knowledge in recent years has revealed emerging pathways, supporting older, more consolidated theories, but there are still many unresolved issues. Our work aims to present the biological face of the complex clinical scenario in MPN patients developing a second cancer, focusing on the main cellular and molecular pathways linking the two diseases.

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.