XPO1 mutations identify early-stage CLL characterized by shorter time to first treatment and enhanced BCR signalling.

Here we evaluated the epigenomic and transcriptomic profile of XPO1 mutant chronic lymphocytic leukaemia (CLL) and their clinical phenotype. By ATAC-seq, chromatin regions that were more accessible in XPO1 mutated CLL were enriched of binding sites for transcription factors regulated by pathways emanating from the B-cell receptor (BCR), including NF-κB signalling, p38-JNK and RAS-RAF-MEK-ERK. XPO1 mutant CLL, consistent with the chromatin accessibility changes, were enriched with transcriptomic features associated with BCR and cytokine signalling. By combining epigenomic and transcriptomic data, MIR155HG, the host gene of miR-155, and MYB, the transcription factor that positively regulates MIR155HG, were upregulated by RNA-seq and their promoters were more accessible by ATAC-seq. To evaluate the clinical impact of XPO1 mutations, we investigated a total of 957 early-stage CLL subdivided into 3 independent cohorts (N = 276, N = 286 and N = 395). Next-generation sequencing analysis identified XPO1 mutations as a novel predictor of shorter time to first treatment (TTFT) in all cohorts. Notably, XPO1 mutations maintained their prognostic value independent of the immunoglobulin heavy chain variable status and early-stage prognostic models. These data suggest that XPO1 mutations, conceivably through increased miR-155 levels, may enhance BCR signalling leading to higher proliferation and shorter TTFT in early-stage CLL.

Do GWAS-Identified Risk Variants for Chronic Lymphocytic Leukemia Influence Overall Patient Survival and Disease Progression?

Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults worldwide. Although genome-wide association studies (GWAS) have uncovered the germline genetic component underlying CLL susceptibility, the potential use of GWAS-identified risk variants to predict disease progression and patient survival remains unexplored. Here, we evaluated whether 41 GWAS-identified risk variants for CLL could influence overall survival (OS) and disease progression, defined as time to first treatment (TTFT) in a cohort of 1039 CLL cases ascertained through the CRuCIAL consortium. Although this is the largest study assessing the effect of GWAS-identified susceptibility variants for CLL on OS, we only found a weak association of ten single nucleotide polymorphisms (SNPs) with OS (p < 0.05) that did not remain significant after correction for multiple testing. In line with these results, polygenic risk scores (PRSs) built with these SNPs in the CRuCIAL cohort showed a modest association with OS and a low capacity to predict patient survival, with an area under the receiver operating characteristic curve (AUROC) of 0.57. Similarly, seven SNPs were associated with TTFT (p < 0.05); however, these did not reach the multiple testing significance threshold, and the meta-analysis with previous published data did not confirm any of the associations. As expected, PRSs built with these SNPs showed reduced accuracy in prediction of disease progression (AUROC = 0.62). These results suggest that susceptibility variants for CLL do not impact overall survival and disease progression in CLL patients.

Prognostic Relevance of Multi-Antigenic Myeloma-Specific T-Cell Assay in Patients with Monoclonal Gammopathies.

Multiple Myeloma (MM) typically originates from underlying precursor conditions, known as Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM). Validated risk factors, related to the main features of the clonal plasma cells, are employed in the current prognostic models to assess long-term probabilities of progression to MM. In addition, new prognostic immunologic parameters, measuring protective MM-specific T-cell responses, could help to identify patients with shorter time-to-progression. In this report, we described a novel Multi-antigenic Myeloma-specific (MaMs) T-cell assay, based on ELISpot technology, providing simultaneous evaluation of T-cell responses towards ten different MM-associated antigens. When performed during long-term follow-up (mean 28 months) of 33 patients with either MGUS or SMM, such deca-antigenic myeloma-specific immunoassay allowed to significantly distinguish between stable vs. progressive disease (p < 0.001), independently from the Mayo Clinic risk category. Here, we report the first clinical experience showing that a wide (multi-antigen), standardized (irrespective to patients' HLA), MM-specific T-cell assay may routinely be applied, as a promising prognostic tool, during the follow-up of MGUS/SMM patients. Larger studies are needed to improve the antigenic panel and further explore the prognostic value of MaMs test in the risk assessment of patients with monoclonal gammopathies.

The dynamic functions of IRF4 in B cell malignancies.

The trajectory of B cell development goes through subsequent steps governed by complex genetic programs, strictly regulated by multiple transcription factors. Interferon regulatory factor 4 (IRF4) regulates key points from pre-B cell development and receptor editing to germinal center formation, class-switch recombination and plasma cell differentiation. The pleiotropic ability of IRF4 is mediated by its "kinetic control", allowing different IRF4 expression levels to activate distinct genetic programs due to modulation of IRF4 DNA-binding affinity. IRF4 is implicated in B cell malignancies, acting both as tumor suppressor and as tumor oncogene in different types of precursors and mature B cell neoplasia. Here, we summarize the complexity of IRF4 functions related to different DNA-binding affinity, multiple IRF4-specific target DNA motif, and interactions with transcriptional partners. Moreover, we describe the unique role of IRF4 in acute leukemias and B cell mature neoplasia, focusing on pathogenetic implications and possible therapeutic strategies in multiple myeloma and chronic lymphocytic leukemia.

Effects of the BTN162b2 mRNA COVID-19 vaccine in humoral and cellular immunity in patients with chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL), the most common leukemia in the western countries, is characterized by immunosuppression due to disease itself and cytotoxic treatments. Since the beginning of COVID-19 pandemic, patients with CLL appear to be a vulnerable population. In addition, phase III mRNA vaccine trials did not provide information about the efficacy in immunocomprised population. In CLL, the antibody-mediated response to SARS-CoV-2 vaccine is impaired. The goal of this study was to evaluate the effects of SARS-CoV-2 vaccination on humoral immune response and on cellular immunity in CLL patients. Humoral immune response to BNT162b2 messenger RNA COVID-19 vaccine was evaluated in 44 CLL patients comprising 20 treatment-naïve, 14 under treatment with ibrutinib and 10 in follow-up after completion of therapy. A positive serological response to SARS-CoV-2 vaccination with IgG titers higher than 13 UA/ml was detected in 54.6% of CLL patients with a higher response in patients who obtained remission after treatment. Reduced antibody response was detected in patients under ibrutinib treatment. T-cell response to overlapping pool of peptides representing the spike region was assessed in paired CLL samples collected before and after 1 month from the second dose of COVID-19 vaccine in treatment-naïve and ibrutinib-treated CLL patients using cytokine secretion assay. Both CD3+ CD4+ and CD3+ CD8+ T cells are able to mount a cellular response to spike peptides with secretion of IFNγ and TNFα before and after vaccination in both treatment naïve and ibrutinib-treated patients and this cellular immune response is independent by COVID-19 vaccination. Collectively, T cell response to spike peptides appeared more blunted in CLL patients under treatment with ibrutinib compared to untreated ones. Our study supports the need for optimization of vaccination strategy to achieve an adequate immune response keeping strict preventive measures by CLL patients against COVID-19.

Indoleamine 2, 3-Dioxygenase 1 Mediates Survival Signals in Chronic Lymphocytic Leukemia via Kynurenine/Aryl Hydrocarbon Receptor-Mediated MCL1 Modulation.

The indoleamine 2,3-dioxygenase 1 (IDO1) metabolic circuitry, comprising the first tryptophan (Trp) catabolite L-kynurenine (Kyn) and the aryl hydrocarbon receptor (AHR), has emerged as a mechanism of cancer immune evasion. Here, we investigated the functional role of the IDO1/Kyn/AHR axis in chronic lymphocytic leukemia (CLL). Our data show that CLL cells expressed an active form of the IDO1 enzyme and microenvironmental stimuli can positively modulate its expression. Interferon (IFN)-γ induces IDO1 expression through the Jak/STAT1 pathway and mediates Kyn production concomitantly with Trp consumption in CLL-conditioned media, while INCB018424 (ruxolitinib), a JAK1/2 inhibitor, impaired both effects. To characterize the involvement of IDO1 in leukemic cell maintenance, we overexpressed IDO1 by vector transfection measuring enhanced resistance to spontaneous apoptosis. IDO1 pro-survival influence was confirmed by treating CLL cells with Kyn, which mediated the increase of induced myeloid leukemia cell differentiation protein (MCL1). Conversely, AHR silencing or its blockade via CH-223191 improved the apoptosis of leukemic clones and mitigated MCL1 expression. Moreover, Kyn-treated CLL cells are less affected by the pro-apoptotic effect of ABT-199 (venetoclax), while CH-223191 showed synergistic/additive cytotoxicity with this drug. Lastly, targeting directly MCL1 in CLL cells with AMG-176, we abrogate the pro-survival effect of Kyn. In conclusion, our data identify IDO1/Kyn/AHR signaling as a new therapeutic target for CLL, describing for the first time its role in CLL pathobiology.

BTK Inhibitors Impair Platelet-Mediated Antifungal Activity.

In recent years, the introduction of new drugs targeting Bruton's tyrosine kinase (BTK) has allowed dramatic improvement in the prognosis of patients with chronic lymphocytic leukemia (CLL) and other B-cell neoplasms. Although these small molecules were initially considered less immunosuppressive than chemoimmunotherapy, an increasing number of reports have described the occurrence of unexpected opportunistic fungal infections, in particular invasive aspergillosis (IA). BTK represents a crucial molecule in several signaling pathways depending on different immune receptors. Based on a variety of specific off-target effects on innate immunity, namely on neutrophils, monocytes, pulmonary macrophages, and nurse-like cells, ibrutinib has been proposed as a new host factor for the definition of probable invasive pulmonary mold disease. The role of platelets in the control of fungal growth, through granule-dependent mechanisms, was described in vitro almost two decades ago and is, so far, neglected by experts in the field of clinical management of IA. In the present study, we confirm the antifungal role of platelets, and we show, for the first time, that the exposure to BTK inhibitors impairs several immune functions of platelets in response to Aspergillus fumigatus, i.e., the ability to adhere to conidia, activation (as indicated by reduced expression of P-selectin), and direct killing activity. In conclusion, our experimental data suggest that antiplatelet effects of BTK inhibitors may contribute to an increased risk for IA in CLL patients.

Role of Notch2 pathway in mature B cell malignancies.

In recent decades, the Notch pathway has been characterized as a key regulatory signaling of cell-fate decisions evolutionarily conserved in many organisms and different tissues during lifespan. At the same time, many studies suggest a link between alterations of this signaling and tumor genesis or progression. In lymphopoiesis, the Notch pathway plays a fundamental role in the correct differentiation of T and B cells, but its deregulated activity leads to leukemic onset and evolution. Notch and its ligands Delta/Jagged exhibit a pivotal role in the crosstalk between leukemic cells and their environment. This review is focused in particular on Notch2 receptor activity. Members of Notch2 pathway have been reported to be mutated in Chronic Lymphocytic Leukemia (CLL), Splenic Marginal Zone Lymphoma (SMZL) and Nodal Marginal Zone Lymphoma (NMZL). CLL is a B cell malignancy in which leukemic clones establish supportive crosstalk with non-malignant cells of the tumor microenvironment to grow, survive, and resist even the new generation of drugs. SMZL and NMZL are indolent B cell neoplasms distinguished by a distinct pattern of dissemination. In SMZL leukemic cells affect mainly the spleen, bone marrow, and peripheral blood, while NMZL has a leading nodal distribution. Since Notch2 is involved in the commitment of leukemic cells to the marginal zone as a major regulator of B cell physiological differentiation, it is predominantly affected by the molecular lesions found in both SMZL and NMZL. In light of these findings, a better understanding of the Notch receptor family pathogenic role, in particular Notch2, is desirable because it is still incomplete, not only in the physiological development of B lymphocytes but also in leukemia progression and resistance. Several therapeutic strategies capable of interfering with Notch signaling, such as monoclonal antibodies, enzyme or complex inhibitors, are being analyzed. To avoid the unwanted multiple "on target" toxicity encountered during the systemic inhibition of Notch signaling, the study of an appropriate pharmaceutical formulation is a pressing need. This is why, to date, there are still no Notch-targeted therapies approved. An accurate analysis of the Notch pathway could be useful to drive the discovery of new therapeutic targets and the development of more effective therapies.

Multiparametric Flow Cytometry for MRD Monitoring in Hematologic Malignancies: Clinical Applications and New Challenges.

Along with the evolution of immunophenotypic and molecular diagnostics, the assessment of Minimal Residual Disease (MRD) has progressively become a keystone in the clinical management of hematologic malignancies, enabling valuable post-therapy risk stratifications and guiding risk-adapted therapeutic approaches. However, specific prognostic values of MRD in different hematological settings, as well as its appropriate clinical uses (basically, when to measure it and how to deal with different MRD levels), still need further investigations, aiming to improve standardization and harmonization of MRD monitoring protocols and MRD-driven therapeutic strategies. Currently, MRD measurement in hematological neoplasms with bone marrow involvement is based on advanced highly sensitive methods, able to detect either specific genetic abnormalities (by PCR-based techniques and next-generation sequencing) or tumor-associated immunophenotypic profiles (by multiparametric flow cytometry, MFC). In this review, we focus on the growing clinical role for MFC-MRD diagnostics in hematological malignancies-from acute myeloid and lymphoblastic leukemias (AML, B-ALL and T-ALL) to chronic lymphocytic leukemia (CLL) and multiple myeloma (MM)-providing a comparative overview on technical aspects, clinical implications, advantages and pitfalls of MFC-MRD monitoring in different clinical settings.

Neoantigen-Specific T-Cell Immune Responses: The Paradigm of NPM1-Mutated Acute Myeloid Leukemia.

The C-terminal aminoacidic sequence from NPM1-mutated protein, absent in normal human tissues, may serve as a leukemia-specific antigen and can be considered an ideal target for NPM1-mutated acute myeloid leukemia (AML) immunotherapy. Different in silico instruments and in vitro/ex vivo immunological platforms have identified the most immunogenic epitopes from NPM1-mutated protein. Spontaneous development of endogenous NPM1-mutated-specific cytotoxic T cells has been observed in patients, potentially contributing to remission maintenance and prolonged survival. Genetically engineered T cells, namely CAR-T or TCR-transduced T cells, directed against NPM1-mutated peptides bound to HLA could prospectively represent a promising therapeutic approach. Although either adoptive or vaccine-based immunotherapies are unlikely to be highly effective in patients with full-blown leukemia, these strategies, potentially in combination with immune-checkpoint inhibitors, could be promising in maintaining remission or preemptively eradicating persistent measurable residual disease, mainly in patients ineligible for allogeneic hematopoietic stem cell transplant (HSCT). Alternatively, neoantigen-specific donor lymphocyte infusion derived from healthy donors and targeting NPM1-mutated protein to selectively elicit graft-versus-leukemia effect may represent an attractive option in subjects experiencing post-HSCT relapse. Future studies are warranted to further investigate dynamics of NPM1-mutated-specific immunity and explore whether novel individualized immunotherapies may have potential clinical utility in NPM1-mutated AML patients.

How to Improve Prognostication in Acute Myeloid Leukemia with CBFB-MYH11 Fusion Transcript: Focus on the Role of Molecular Measurable Residual Disease (MRD) Monitoring.

Acute myeloid leukemia (AML) carrying inv(16)/t(16;16), resulting in fusion transcript CBFB-MYH11, belongs to the favorable-risk category. However, even if most patients obtain morphological complete remission after induction, approximately 30% of cases eventually relapse. While well-established clinical features and concomitant cytogenetic/molecular lesions have been recognized to be relevant to predict prognosis at disease onset, the independent prognostic impact of measurable residual disease (MRD) monitoring by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR), mainly in predicting relapse, actually supersedes other prognostic factors. Although the ELN Working Party recently indicated that patients affected with CBFB-MYH11 AML should have MRD assessment at informative clinical timepoints, at least after two cycles of intensive chemotherapy and after the end of treatment, several controversies could be raised, especially on the frequency of subsequent serial monitoring, the most significant MRD thresholds (most commonly 0.1%) and on the best source to be analyzed, namely, bone marrow or peripheral blood samples. Moreover, persisting low-level MRD positivity at the end of treatment is relatively common and not predictive of relapse, provided that transcript levels remain stably below specific thresholds. Rising MRD levels suggestive of molecular relapse/progression should thus be confirmed in subsequent samples. Further prospective studies would be required to optimize post-remission monitoring and to define effective MRD-based therapeutic strategies.

Inflammatory Microenvironment and Specific T Cells in Myeloproliferative Neoplasms: Immunopathogenesis and Novel Immunotherapies.

The Philadelphia-negative myeloproliferative neoplasms (MPNs) are malignancies of the hematopoietic stem cell (HSC) arising as a consequence of clonal proliferation driven by somatically acquired driver mutations in discrete genes (JAK2, CALR, MPL). In recent years, along with the advances in molecular characterization, the role of immune dysregulation has been achieving increasing relevance in the pathogenesis and evolution of MPNs. In particular, a growing number of studies have shown that MPNs are often associated with detrimental cytokine milieu, expansion of the monocyte/macrophage compartment and myeloid-derived suppressor cells, as well as altered functions of T cells, dendritic cells and NK cells. Moreover, akin to solid tumors and other hematological malignancies, MPNs are able to evade T cell immune surveillance by engaging the PD-1/PD-L1 axis, whose pharmacological blockade with checkpoint inhibitors can successfully restore effective antitumor responses. A further interesting cue is provided by the recent discovery of the high immunogenic potential of JAK2V617F and CALR exon 9 mutations, that could be harnessed as intriguing targets for innovative adoptive immunotherapies. This review focuses on the recent insights in the immunological dysfunctions contributing to the pathogenesis of MPNs and outlines the potential impact of related immunotherapeutic approaches.

IRF4 modulates the response to BCR activation in chronic lymphocytic leukemia regulating IKAROS and SYK.

Interferon regulatory factor 4 (IRF4) is a transcriptional regulator of immune system development and function. Here, we investigated the role of IRF4 in controlling responsiveness to B-cell receptor (BCR) stimulation in chronic lymphocytic leukemia (CLL). We modulated IRF4 levels by transfecting CLL cells with an IRF4 vector or by silencing using small-interfering RNAs. Higher IRF4 levels attenuated BCR signaling by reducing AKT and ERK phosphorylation and calcium release. Conversely, IRF4 reduction improved the strength of the intracellular cascade activated by BCR engagement. Our results also indicated that IRF4 negatively regulates the expression of the spleen tyrosine kinase SYK, a crucial protein for propagation of BCR signaling, and the zinc finger DNA-binding protein IKAROS. We modulated IKAROS protein levels both by genetic manipulation and pharmacologically by treating CLL cells with lenalidomide and avadomide (IMIDs). IKAROS promoted BCR signaling by reducing the expression of inositol 5-phosphatase SHIP1. Lastly, IMIDs induced IRF4 expression, while down-regulating IKAROS and interfered with survival advantage mediated by BCR triggering, also in combination with ibrutinib. Overall, our findings elucidate the mechanism by which IRF4 tunes BCR signaling in CLL cells. Low IRF4 levels allow an efficient transmission of BCR signal throughout the accumulation of SYK and IKAROS.