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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.

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.

Abstract 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.

Abstract 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.

Abstract 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.

Abstract 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.

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.

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-1β or NLRP3 could be a helpful cancer-related therapeutic strategy to improve the existing protocols.

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.

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.

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.

Abstract Background Alu repeats, belonging to the Short Interspersed Repetitive Elements (SINEs) class, contain about 25% of CpG sites in the human genome. Alu sequences lie in gene-rich regions, so their methylation is an important transcriptional regulation mechanism. Aberrant Alu methylation has been associated with tumor aggressiveness, and also previously discussed in hematological malignancies, by applying different approaches. Moreover, today different techniques designed to measure global DNA methylation are focused on the methylation level of specific repeat elements. In this work we propose a new method of investigating Alu differential methylation, based on droplet digital PCR (ddPCR) technology. Methods Forty-six patients with hematological neoplasms were included in the study: 30 patients affected by chronic lymphocytic leukemia, 7 patients with myelodysplastic syndromes at intermediate/high risk, according with the International Prognostic Scoring System, and 9 patients with myelomonocytic leukemia. Ten healthy donors were included as controls. Acute promyelocytic leukemia-derived NB4 cell line, either untreated or treated with decitabine (DEC) hypomethylating agent, was also analyzed. DNA samples were investigated for Alu methylation level by digestion of genomic DNA with isoschizomers with differential sensitivity to DNA methylation, followed by ddPCR. Results Using ddPCR, a significant decrease of the global Alu methylation level in DNA extracted from NB4 cells treated with DEC, as compared to untreated cells, was observed. Moreover, comparing the global Alu methylation levels at diagnosis and after azacytidine (AZA) treatment in MDS patients, a statistically significant decrease of Alu sequences methylation after therapy as compared to diagnosis was evident. We also observed a significant decrease of the Alu methylation level in CLL patients compared to HD, and, finally, for CMML patients, a decrease of Alu sequences methylation was observed in patients harboring the SRSF2 hotspot gene mutation c.284C>D. Conclusions In our work, we propose a method to investigate Alu differential methylation based on ddPCR technology. This assay introduces ddPCR as a more sensitive and immediate technique for Alu methylation analysis. To date, this is the first application of ddPCR to study DNA repetitive elements. This approach may be useful to profile patients affected by hematologic malignancies for diagnostic/prognostic purpose.

Acute myeloid leukemia (AML) is a haematological neoplasm resulting from the accumulation of genetic and epigenetic alterations. Patients’ prognoses vary with AML genetic heterogeneity, which hampers successful treatments. Single-cell approaches have provided new insights of the clonal architecture of AML, revealing the mutational history from diagnosis, during treatment and to relapse. In this review, we imagine single-cell technologies as the Ariadne’s thread that will guide us out of the AML maze, provide a precise identikit of the leukemic cell at single-cell resolution and explore genomic, transcriptomic, epigenetic and proteomic levels.

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.

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.

Micro RNAs (miRNAs) are a class of small non-coding RNAs that have a crucial role in cellular processes such as differentiation, proliferation, migration, and apoptosis. miRNAs may act as oncogenes or tumor suppressors; therefore, they prevent or promote tumorigenesis, and abnormal expression has been reported in many malignancies. The role of miRNA in leukemia pathogenesis is still emerging, but several studies have suggested using miRNA expression profiles as biomarkers for diagnosis, prognosis, and response to therapy in leukemia. In this review, the role of miRNAs most frequently involved in leukemia pathogenesis is discussed, focusing on the class of circulating miRNAs, consisting of cell-free RNA molecules detected in several body fluids. Circulating miRNAs could represent new potential non-invasive diagnostic and prognostic biomarkers of leukemia that are easy to isolate and characterize. The dysregulation of some miRNAs involved in both myeloid and lymphoid leukemia, such as miR-155, miR-29, let-7, and miR-15a/miR-16-1 clusters is discussed, showing their possible employment as therapeutic targets.

Retinoic acid receptor γ (RARγ) belongs to the nuclear receptor superfamily and shares 90% homology with retinoic acid receptor α (RARα) and retinoic acid receptor β (RARβ). RARA rearrangements are well-known to be involved in acute promyelocytic leukemia (APL), but RARG rearrangements can also resemble this kind of leukemia. In this review we trace the role of RARγ, considering both its physiological and oncogenic contribution; from 2011 to date, nine cases of patients harboring RARG fusions have been reported. These patients showed typical APL features, including the clinical presentation, coagulation abnormalities and morphological features of bone marrow (BM), but are not responsive to APL standard therapy. We stress the urgent need for a better comprehension of the critical role of RARG dysregulation in the leukemogenesis process, since optimum therapy strategies have not yet been established.

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.

TP53 dysregulation plays a pivotal role in the molecular pathogenesis of myelodysplastic syndromes (MDS), identifying a subgroup of patients with peculiar features. In this review we report the recent biological and clinical findings of TP53-mutated MDS, focusing on the molecular pathways activation and on its impact on the cellular physiology. In MDS, TP53 mutational status is deeply associated with del(5q) syndrome and its dysregulation impacts on cell cycle, DNA repair and apoptosis inducing chromosomal instability and the clonal evolution of disease. TP53 defects influence adversely the MDS clinical outcome and the treatment response rate, thus new therapeutic approaches are being developed for these patients. TP53 allelic state characterization and the mutational burden evaluation can therefore predict prognosis and identify the subgroup of patients eligible for targeted therapy. For these reasons, in the era of precision medicine, the MDS diagnostic workup cannot do without the complete assessment of TP53 mutational profile.

The digital polymerase chain reaction (dPCR) is considered to be the third-generation polymerase chain reaction (PCR), as it yields direct, absolute and precise measures of target sequences. dPCR has proven particularly useful for the accurate detection and quantification of low-abundance nucleic acids, highlighting its advantages in cancer diagnosis and in predicting recurrence and monitoring minimal residual disease, mostly coupled with next generation sequencing. In the last few years, a series of studies have employed dPCR for the analysis of hematologic malignancies. In this review, we will summarize these findings, attempting to focus on the potential future perspectives of the application of this promising technology.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer and accounts for about a quarter of adult acute leukemias, and features different outcomes depending on the age of onset. Improvements in ALL genomic analysis achieved thanks to the implementation of next-generation sequencing (NGS) have led to the recent discovery of several novel molecular entities and to a deeper understanding of the existing ones. The purpose of our review is to report the most recent discoveries obtained by NGS studies for ALL diagnosis, risk stratification, and treatment planning. We also report the first efforts at NGS use for minimal residual disease (MRD) assessment, and early studies on the application of third generation sequencing in cancer research. Lastly, we consider the need for the integration of NGS analyses in clinical practice for genomic patients profiling from the personalized medicine perspective.

The family of retinoic acid receptors (RARs: RARα, -β, and -γ) has remarkable pleiotropy characteristics, since the retinoic acid/RARs pathway is involved in numerous biological processes not only during embryonic development, but also in the postnatal phase and during adulthood. In this review, we trace the roles of RA/RARs signaling in the immune system (where this pathway has both an immunosuppressive role or is involved in the inflammatory response), in hematopoiesis (enhancing hematopoietic stem cell self-renewal, progenitor cells differentiation or maintaining the bone marrow microenvironment homeostasis), and in bone remodeling (where this pathway seems to have controversial effects on bone formation or osteoclast activation). Moreover, in this review is shown the involvement of RAR genes in multiple chromosomal rearrangements generating different fusion genes in hematological neoplasms, with a particular focus on acute promyelocytic leukemia and its variant subtypes. The effect of different RARs fusion proteins on leukemic transformation, on patients’ outcome, and on therapy response is also discussed.

The germline JAK2 haplotype known as “GGCC or 46/1 haplotype” (haplotypeGGCC_46/1) consists of a combination of single nucleotide polymorphisms (SNPs) mapping in a region of about 250 kb, extending from the JAK2 intron 10 to the Insulin-like 4 (INLS4) gene. Four main SNPs (rs3780367, rs10974944, rs12343867, and rs1159782) generating a “GGCC” combination are more frequently indicated to represent the JAK2 haplotype. These SNPs are inherited together and are frequently associated with the onset of myeloproliferative neoplasms (MPN) positive for both JAK2 V617 and exon 12 mutations. The association between the JAK2 haplotypeGGCC_46/1 and mutations in other genes, such as thrombopoietin receptor (MPL) and calreticulin (CALR), or the association with triple negative MPN, is still controversial. This review provides an overview of the frequency and the role of the JAK2 haplotypeGGCC_46/1 in the pathogenesis of different myeloid neoplasms and describes the hypothetical mechanisms at the basis of the association with JAK2 gene mutations. Moreover, possible clinical implications are discussed, as different papers reported contrasting data about the correlation between the JAK2 haplotypeGGCC_46/1 and blood cell count, survival, or disease progression.