NFKBIE mutations are selected by the tumor microenvironment and contribute to immune escape in chronic lymphocytic leukemia.

Loss-of-function mutations in NFKBIE, which encodes for the NF-κB inhibitor IκBε, are frequent in chronic lymphocytic leukemia (CLL) and certain other B-cell malignancies and have been associated with accelerated disease progression and inferior responses to chemotherapy. Using in vitro and in vivo murine models and primary patient samples, we now show that NFKBIE-mutated CLL cells are selected by microenvironmental signals that activate the NF-κB pathway and induce alterations within the tumor microenvironment that can allow for immune escape, including expansion of CD8+ T-cells with an exhausted phenotype and increased PD-L1 expression on the malignant B-cells. Consistent with the latter observations, we find increased expression of exhaustion markers on T-cells from patients with NFKBIE-mutated CLL. In addition, we show that NFKBIE-mutated murine CLL cells display selective resistance to ibrutinib and report inferior outcomes to ibrutinib treatment in NFKBIE-mutated CLL patients. These findings suggest that NFKBIE mutations can contribute to CLL progression through multiple mechanisms, including a bidirectional crosstalk with the microenvironment and reduced sensitivity to BTK inhibitor treatment.

Ibrutinib as first line therapy in chronic lymphocytic leukemia patients over 80 years old: A retrospective real-life multicenter Italian cohort.

Although chronic lymphocytic leukemia (CLL) predominantly affects the elderly, limited data exists about the outcomes of over 80-year-old patients, usually underrepresented in clinical trials. We conducted a multicenter study enrolling 79 consecutive CLL patients ≥80 years at the time of frontline therapy, all treated with ibrutinib. Nearly 48% of cases exhibited unmutated IGHV genes, 32% 17p deletion, and 39.2% TP53 mutations; 63.3% displayed a cumulative illness rating scale (CIRS) > 6. The overall response rate on ibrutinib, computed in 74/79 patients (5 patients excluded for early withdrawal), was 89.9%. After a median follow-up of 28.9 months, the median progression-free survival (PFS) and overall survival (OS) were 42.5 and 51.8 months, respectively. CIRS>6 and temporary discontinuation of ibrutinib lasting for 7-30 days were the only parameters associated with a significantly shorter PFS and were both relevant in predicting a shorter PFS compared to patients with CIRS≤6 and therapy discontinuation ≤7 days. The most common grade≥3 adverse events were infections (25.5%), neutropenia (10.1%), and anemia (2.5%). Eighteen patients (22.8%) experienced a cardiovascular event, including grade-2 atrial fibrillation (n = 9; 11%), grade-2 hypertension (n = 5; 6%), heart failure (n = 3; 3%), and acute coronary syndrome (n = 1; 1%). Mild bleeding events were observed in 27 patients (34.2%). Ibrutinib was permanently discontinued in 26 patients due to progressive disease (n = 11, including 5 Richter's syndromes), secondary malignancies (n = 6), infections (n = 3), cardiac failure (n = 3), severe bleeding (n = 2), and sudden death (n = 1). In conclusion, our analyses confirmed the overall effectiveness and favorable safety profile of the ibrutinib-single agent therapeutic approach in CLL patients ≥80 years.

ERBB4-Mediated Signaling Is a Mediator of Resistance to PI3K and BTK Inhibitors in B-cell Lymphoid Neoplasms.

BTK and PI3K inhibitors are among the drugs approved for the treatment of patients with lymphoid neoplasms. Although active, their ability to lead to long-lasting complete remission is rather limited, especially in the lymphoma setting. This indicates that tumor cells often develop resistance to the drugs. We started from a marginal zone lymphoma cell line, Karpas-1718, kept under prolonged exposure to the PI3Kδ inhibitor idelalisib until acquisition of resistance, or with no drug. Cells underwent transcriptome, miRNA and methylation profiling, whole-exome sequencing, and pharmacologic screening, which led to the identification of the overexpression of ERBB4 and its ligands HBEGF and NRG2 in the resistant cells. Cellular and genetic experiments demonstrated the involvement of this axis in blocking the antitumor activity of various BTK/PI3K inhibitors, currently used in the clinical setting. Addition of recombinant HBEGF induced resistance to BTK/PI3K inhibitors in parental cells and in additional lymphoma models. Combination with the ERBB inhibitor lapatinib was beneficial in resistant cells and in other lymphoma models already expressing the identified resistance factors. An epigenetic reprogramming sustained the expression of the resistance-related factors, and pretreatment with demethylating agents or EZH2 inhibitors overcame the resistance. Resistance factors were also shown to be expressed in clinical specimens. In conclusion, we showed that the overexpression of ERBB4 and its ligands represents a novel mechanism of resistance for lymphoma cells to bypass the antitumor activity of BTK and PI3K inhibitors and that targeted pharmacologic interventions can restore sensitivity to the small molecules.

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.

Lymphadenopathy as a predictor of progression during venetoclax treatment in chronic lymphocytic leukemia. A campus chronic lymphocytic leukemia study.

Clinical or biological parameters useful to predict progression during treatment in real-life setting with ibrutinib, idelalisib and venetoclax in relapsed/refractory chronic lymphocytic leukemia (CLL) are still debated. We conducted a multi-center retrospective study on CLL patients treated with ibrutinib and/or idelalisib who were switched to venetoclax for progression or due to adverse events to identify any clinical and/or biological parameters useful to predict progression during treatment with venetoclax. Of all the 128 evaluable patients, 81 had received ibrutinib prior to switching to venetoclax, 35 had received idelalisib and 12 both. When comparing the three subgroups, we did not notice any statistical difference in terms of clinical or biological features. No variable at baseline and at different time points during the follow-up (at 6, 12, 18 and 24 months) was found to predict progression nor to have significance for Progression Free Survival (PFS) in the ibrutinib group and in the idelalisib group and in subgroups according to the line of treatment. Analyzing the data of the venetoclax treatment, after a median follow up of 14.3 months, median PFS was not reached and estimated 3-year PFS was 54%. Of the 128 patients treated with venetoclax, 28 (22%) experienced progressive disease. At multivariate analysis for predictive factors for progression, lymph node diameter >56.5 mm before starting treatment emerged as an independent risk factor for progression. The lymph node predictive role for progression during venetoclax treatment could be a new parameter that deserves to be investigate in future studies.

Persistence of monoclonal B-cell expansion and intraclonal diversification despite virus eradication in patients affected by hepatitis C virus-associated lymphoproliferative disorders.

We investigated 23 hepatitis C virus (HCV)-infected patients with overt lymphoproliferative diseases (15 cases) or monoclonal B lymphocytosis (8 cases) treated with direct agent antiviral (DAAs) per clinical practice. DAA therapy yielded undetectable HCV-RNA, the complete response of cryoglobulinemia vasculitis and related signs, whilst the presence of B-cell clones (evaluated by flow cytometry, IGHV, and BCL2-IGH rearrangements), detected in 19/23 cases at baseline, was maintained (17/19). Similarly, IGHV intraclonal diversification, supporting an antigen-driven selection mechanism, was identified in B-cell clones at baseline and end of follow-up. DAA therapy alone, despite HCV eradication and good immunological responses, was less effective on the pathological B-cell clones.

BCR/Integrin Interaction in CLL: A Physiologic Remnant with Clinical Relevance.

CD49d, the alpha chain of the very late antigen-4 (VLA-4) integrin, has a negative prognostic impact in chronic lymphocytic leukemia treated with the Bruton's tyrosine kinase (BTK) inhibitors, ibrutinib and acalabrutinib. Despite BTK inhibition, VLA-4 remains inside-out activated via B-cell receptor, an activation dampened by phosphoinositide 3-kinase inhibitors. Evaluation of CD49d expression in patients starting BTK inhibitor therapy may improve their prognostic stratification. See related article by Alsadhan et al., p. 3612.

Targeted chitosan nanobubbles as a strategy to down-regulate microRNA-17 into B-cell lymphoma models.

Introduction: MicroRNAs represent interesting targets for new therapies because their altered expression influences tumor development and progression. miR-17 is a prototype of onco-miRNA, known to be overexpressed in B-cell non-Hodgkin lymphoma (B-NHL) with peculiar clinic-biological features. AntagomiR molecules have been largely studied to repress the regulatory functions of up-regulated onco-miRNAs, but their clinical use is mainly limited by their rapid degradation, kidney elimination and poor cellular uptake when injected as naked oligonucleotides. Methods: To overcome these problems, we exploited CD20 targeted chitosan nanobubbles (NBs) for a preferential and safe delivery of antagomiR17 to B-NHL cells. Results: Positively charged 400 nm-sized nanobubbles (NBs) represent a stable and effective nanoplatform for antagomiR encapsulation and specific release into B-NHL cells. NBs rapidly accumulated in tumor microenvironment, but only those conjugated with a targeting system (antiCD20 antibodies) were internalized into B-NHL cells, releasing antagomiR17 in the cytoplasm, both in vitro and in vivo. The result is the down-regulation of miR-17 level and the reduction in tumor burden in a human-mouse B-NHL model, without any documented side effects. Discussion: Anti-CD20 targeted NBs investigated in this study showed physico-chemical and stability properties suitable for antagomiR17 delivery in vivo and represent a useful nanoplatform to address B-cell malignancies or other cancers through the modification of their surface with specific targeting antibodies.

Measurable residual disease in chronic lymphocytic leukemia.

Measurable residual disease (MRD) is defined as the presence of residual cancer cells after treatment in patients with clinically undetectable disease, who would otherwise be considered in complete remission. It is a highly sensitive parameter which indicates the disease burden and predicts survival in this setting of patients. In recent years, MRD has gained a role in many hematological malignancies as a surrogate endpoint for clinical trials: undetectable MRD has been correlated to longer progression free survival (PFS) and overall survival (OS). New drugs and combinations have been developed with the aim to achieve MRD negativity, which would indicate favorable prognosis. Different methods to measure MRD have also been devised, which include flow cytometry, polymerase chain reaction (PCR) and next generation sequencing (NGS), with different sensitivity and accuracy in evaluating deep remission after treatment. In this review, we will analyze the current recommendations for the detection of MRD, with particular focus on its role in Chronic Lymphocytic Leukemia (CLL), as well as the different detection methods. Moreover, we will discuss the results of clinical trials and the role of MRD in new therapeutic schemes with inhibitors and monoclonal antibodies. MRD is not currently used in the clinical practice to evaluate response to treatment, due to technical and economical limitations, but it's gaining more and more interest in trials settings, especially since the introduction of venetoclax. The use of MRD in trials will likely be followed by a broader practical application in the future. The aim of this work is to provide a reader-friendly summary of the state of art in the field, as MRD will soon become an accessible tool to evaluate our patients, predict their survival and guide physician's therapeutic choices and preferences.

Hepatitis B virus-infection related cryoglobulinemic vasculitis. Clinical manifestations and the effect of antiviral therapy: A review of the literature.

Objective: Hepatitis B virus (HBV) infection causes chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Furthermore, about 20% of the patients develop extrahepatic manifestations such as cryoglobulinemic vasculitis (CV), polyarteritis nodosa, non-rheumatoid arthritis, glomerulonephritis and non-Hodgkin lymphoma. This review analyzed literature data on clinical manifestations of HBV-related CV and the impact of antiviral therapy with analoques nucleotide. Methods: A PubMed search was performed to select eligible studies in the literature, up to July 2022. Results: Some studies have analyzed clinical manifestations in HBV-related CV and have investigated the role of antiviral therapy with nucleotides analogues (NAs). Clinical manifestations of CV vary from mild to moderate (purpura, asthenia and arthralgias) to severe (leg ulcers, peripheral neuropathy, glomerulonephritis, and non-Hodking lymphoma). NAs therapy leads to suppression of HBV-DNA; therefore, it is capable of producing clinical response in the majority of patients with mild to moderate symptoms. Conclusion: Antiviral therapy with NAs is the first choice for HBV suppression and control of mild to moderate disease. In severe vasculitis (glomerulonephritis, progressive peripheral neuropathy and leg ulcers), rituximab alone or with plasma-exchange is always indicated in combination with antiviral therapy.

ERBB4-mediated signaling is a mediator of resistance to BTK and PI3K inhibitors in B cell lymphoid neoplasms.

BTK and PI3K inhibitors are among the drugs approved for the treatment of patients with lymphoid neoplasms. Although active, their ability to lead as single agents to long-lasting complete remission is rather limited especially in the lymphoma setting. This indicates that tumor cells often develop resistance to the drugs. Here, we show that the overexpression of ERBB4 and its ligands represents a modality for B cell neoplastic cells to bypass the anti-tumor activity of BTK and PI3K inhibitors and that targeted pharmacological interventions can restore sensitivity to the small molecules. We started from a marginal zone lymphoma (MZL) cell line, Karpas-1718, kept under prolonged exposure to the PI3Kδ inhibitor idelalisib until acquisition of resistance, or with no drug. Cells underwent transcriptome, miRNA and methylation profiling, whole exome sequencing, and pharmacological screening which led to the identification of the overexpression of ERBB4 and its ligands HBEGF and NRG2 in the resistant cells. Cellular and genetic experiments demonstrated the involvement of this axis in blocking the anti-tumor activity of various BTK and PI3K inhibitors, currently used in the clinical setting. Addition of recombinant HBEGF induced resistance to BTK and PI3K inhibitors in parental cells but also in additional lymphoma models. Combination with the ERBB inhibitor lapatinib was beneficial in resistant cells and in other lymphoma models already expressing the identified resistance factors. Multi-omics analysis underlined that an epigenetic reprogramming affected the expression of the resistance-related factors, and pretreatment with demethylating agents or EZH2 inhibitors overcame the resistance. Resistance factors were shown to be expressed in clinical samples, further extending the findings of the study. In conclusions, we identified a novel ERBB4-driven mechanism of resistance to BTK and PI3K inhibitors and treatments that appear to overcome it. Key points: A mechanism of secondary resistance to the PI3Kδ and BTK inhibitors in B cell neoplasms driven by secreted factors.Resistance can be reverted by targeting ERBB signaling.

Extrahepatic Manifestations of Chronic HBV Infection and the Role of Antiviral Therapy.

The hepatitis B virus (HBV) infection leads to chronic hepatitis, cirrhosis, and hepatocarcinoma. However, about 20% of patients experience extrahepatic manifestations such as polyarteritis nodosa, non-rheumatoid arthritis, non-Hodgkin lymphoma, cryoglobulinemic vasculitis, and glomerulonephritis. These influence the patient's morbidity, quality of life and mortality. The treatment of an HBV infection is based on nucleotide analogues (NAs) which are safe and effective for the suppression of HBV-DNA in almost 100% of cases. A few studies have shown that NAs induce a viral response and an improvement of extrahepatic diseases. There is a lack of a thorough analysis of the available treatments for extrahepatic HBV manifestations. In 90% to 100% of cases, the NAs stop the HBV replication, and they produce a clinical response in the majority of patients with mild to moderate extrahepatic signs/symptoms. Arthritis can definitely disappear after the HBV elimination and, in some cases, the HBV eradication following NAs therapy appears to improve the renal function in HBV-related nephropathies. Plasma exchange can be used in subjects who are suffering from the most aggressive forms of cryoglobulinemic vasculitis and glomerulonephritis, progressive peripheral neuropathy, and life-threatening cases, and this can be combined with glucocorticosteroids and antiviral agents. In selected refractory patients, the use of rituximab in conjunction with NAs therapy can be considered. The review provides an update on extrahepatic conditions that are linked to HBV and the impact of treating HBV with NAs.