The VLA-4 integrin is constitutively active in circulating chronic lymphocytic leukemia cells via BCR autonomous signaling: a novel anchor-independent mechanism exploiting soluble blood-borne ligands.

In chronic lymphocytic leukemia (CLL), survival of neoplastic cells depends on microenvironmental signals at lymphoid sites where the crosstalk between the integrin VLA-4 (CD49d/CD29), expressed in ~40% of CLL, and the B-cell receptor (BCR) occurs. Here, BCR engagement inside-out activates VLA-4, thus enhancing VLA-4-mediated adhesion of CLL cells, which in turn obtain pro-survival signals from the surrounding microenvironment. We report that the BCR is also able to effectively inside-out activate the VLA-4 integrin in circulating CD49d-expressing CLL cells through an autonomous antigen-independent BCR signaling. As a consequence, circulating CLL cells exhibiting activated VLA-4 express markers of BCR pathway activation (phospho-BTK and phospho-PLC-γ2) along with higher levels of phospho-ERK and phospho-AKT indicating parallel activation of downstream pathways. Moreover, circulating CLL cells expressing activated VLA-4 bind soluble blood-borne VCAM-1 leading to increased VLA-4-dependent actin polymerization/re-organization and ERK phosphorylation. Finally, evidence is provided that ibrutinib treatment, by affecting autonomous BCR signaling, impairs the constitutive VLA-4 activation eventually decreasing soluble VCAM-1 binding and reducing downstream ERK phosphorylation by circulating CLL cells. This study describes a novel anchor-independent mechanism occurring in circulating CLL cells involving the BCR and the VLA-4 integrin, which help to unravel the peculiar biological and clinical features of CD49d+ CLL.

Early reappearance of intraclonal proliferative subpopulations in ibrutinib-resistant chronic lymphocytic leukemia.

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib represents an effective strategy for treatment of chronic lymphocytic leukemia (CLL), nevertheless about 30% of patients eventually undergo disease progression. Here we investigated by flow cytometry the long-term modulation of the CLL CXCR4dim/CD5bright proliferative fraction (PF), its correlation with therapeutic outcome and emergence of ibrutinib resistance. By longitudinal tracking, the PF, initially suppressed by ibrutinib, reappeared upon early disease progression, without association with lymphocyte count or serum beta-2-microglobulin. Somatic mutations of BTK/PLCG2, detected in 57% of progressing cases, were significantly enriched in PF with a 3-fold greater allele frequency than the non-PF fraction, suggesting a BTK/PLCG2-mutated reservoir resident within the proliferative compartments. PF increase was also present in BTK/PLCG2-unmutated cases at progression, indicating that PF evaluation could represent a marker of CLL progression under ibrutinib. Furthermore, we evidence different transcriptomic profiles of PF at progression in cases with or without BTK/PLCG2 mutations, suggestive of a reactivation of B-cell receptor signaling or the emergence of bypass signaling through MYC and/or Toll-Like-Receptor-9. Clinically, longitudinal monitoring of the CXCR4dim/CD5bright PF by flow cytometry may provide a simple tool helping to intercept CLL progression under ibrutinib therapy.

End of induction [18F]FDG PET is prognostic for progression-free survival and overall survival in follicular lymphoma patients enrolled in the FOLL12 trial.

PURPOSE: To evaluate the reliability of the Deauville score (DS) in therapy response assessment and to define the prognostic value of the metabolic response of end of induction (EOI) [18F]FDG PET (PET) in follicular lymphoma patients. METHODS: Adult patients with untreated grade 1-3a FL/ stage II-IV enrolled in the multicentre, prospective, phase III FOLL12 trial (NCT02063685) were randomized to receive standard immunochemotherapy followed by rituximab maintenance (standard arm) versus standard immunochemotherapy followed by response-adapted post-induction management (experimental arm). Baseline and EOI PET were mandatory for the study. All PET scans were centralized on the WIDEN® platform and classified according to DS in a blind independent central review. DS1-3 was considered negative (CMR), whereas DS4-5 was considered positive (not CMR). The primary endpoint was PFS. The main secondary endpoint was overall survival (OS). RESULTS: Overall, 807 follicular lymphoma patients-52% women, 89% stage III-IV disease, 40% with a high-risk FLIPI-2 score (3-5)-were enrolled in the study; 729 (90.4%) baseline and EOI PET were available for the analysis. EOI PET was positive (DS4-5) in 88/729 (12.1%) cases. Overall inter-reviewer agreement on PET pos/neg result was 0.92, while agreement on positive and negative cases was 0.77 and 0.94, respectively. The median follow-up was 69 months; 247 events were registered in the 5-yr follow-up, with a 5-yr PFS of 67% (95%CI: 63%-70%). The 5-yr PFS rate for PET neg (DS1-3) and PET pos (DS4-5) patients was 71% (95%CI: 67%-75%) and 36% (95%CI: 25%-46%), respectively, with HR 3.49 (95%CI: 2.57-4.72). Five-year PFS was worse as DS increased, with 74% (70%-78%), 58% (48%-67%; HR 1.71; p = 0.001)] and 36% (25%-46%; HR 3.88; p < 0.001) in DS1-2, DS3 and DS4-5, respectively. EOI PET maintained its prognostic value in both the standard and experimental arms. In the whole population, 5-yr OS was 94% (95%CI: 92%-96%), with 96% (95%CI: 94-97) and 82% (95%CI: 72%-89%) in EOI PET negative (DS1-3) and positive (DS4-5), respectively (HR 4.48; p < 0.001). When DS was associated with FLIPI-2, patients with DS3 or DS1-2 with high FLIPI-2 (3-5) experienced worse OS than patients with DS1-2 and low FLIPI-2 (1-2) (p = 0.003). CONCLUSION: This study shows that DS is a reliable prognostic tool to evaluate EOI PET in follicular lymphoma patients, with prognostic value maintained both in the standard and experimental arms, making metabolic imaging a robust tool to assess response in FL. Moreover, although preliminary, this study provides further information on DS3 patients, who are considered as CMR but show a less favourable PFS than DS1-2 patients.

Innovative Combinations, Cellular Therapies and Bispecific Antibodies for Chronic Lymphocytic Leukemia: A Narrative Review.

In the last few years, several agents targeting molecules that sustain the survival and the proliferation of chronic lymphocytic leukemia (CLL) cells have become clinically available. Most of these drugs target surface proteins, such as CD19 or CD20, via monoclonal or bispecific monoclonal antibodies (BsAbs), CAR T cells, intracellular proteins like BTK by using covalent or non-covalent inhibitors or BCL2 with first or second generation BH3-mimetics. Since the management of CLL is evolving quickly, in this review we highlighted the most important innovative treatments including novel double and triple combination therapies, CAR T cells and BsAbs for CLL. Recently, a large number of studies on novel combinations and newer strategic options for CLL therapy have been published or presented at international conferences, which were summarized and linked together. Although the management of treatment with a single continuous agent is easier, the emergence of protein mutations, long-term toxicities and costs are important concerns that favor the use of a fixed duration therapy. In the future, a measurable residual disease (MRD)-guided treatment cessation and MRD-based re-initiation of targeted therapy seems to be a more feasible approach, allowing identification of the patients who might benefit from continuous therapy or who might need a consolidation with BsAbs or CAR T cells to clear the neoplastic clone.

Monocyte-to-platelets ratio (MPR) at diagnosis is associated with inferior progression-free survival in patients with mantle cell lymphoma: a multi-center real-life survey.

Mantle cell lymphoma (MCL) pathogenesis is strongly related to the role of the tumor immune microenvironment (TIME) in which MCL cells proliferate. TIME cells can produce growth signals influencing MCL cells' survival and exert an antitumoral immune response suppression. The activity of TIME cells might be mirrored by some ratios of peripheral blood cell subpopulations, such as the monocyte-to-platelet ratio (MPR). We reviewed the clinical features of 165 consecutive MCL patients newly diagnosed and not eligible for autologous stem cell transplantation (both for age or comorbidities) who accessed two Italian Centers between 2006 and 2020. MPR was calculated using data obtained from the complete blood cell count at diagnosis before any cytotoxic treatment and correlated with PFS. Univariate analysis showed that MPR ≥ 3 was associated with inferior PFS (p = 0.02). Multivariate analysis confirmed that MPR ≥ 3, LDH > 2.5 ULN, and bone marrow involvement were significant independent variables in predicting PFS. For these reasons, MPR ≥ 3 seems the most promising prognostic factor in patients with MCL, and it could be considered a variable in new predictive models.

Real-World Outcome of Treatment with Single-Agent Ibrutinib in Italian Patients with Chronic Lymphocytic Leukemia: Final Results of the EVIdeNCE Study.

Real-world data in clinical practice are needed to confirm the efficacy and safety that ibrutinib has demonstrated in clinical trials of patients with chronic lymphocytic leukemia (CLL). We described the real-world persistence rate, patterns of use, and clinical outcomes in 309 patients with CLL receiving single-agent ibrutinib in first line (1L, n = 118), 2L (n = 127) and ≥3L (n = 64) in the prospective, real-world, Italian EVIdeNCE study. After a median follow-up of 23.9 months, 29.8% of patients discontinued ibrutinib (1L: 24.6%, 2L: 29.9%, ≥3L: 39.1%), mainly owing to adverse events (AEs)/toxicity (14.2%). The most common AEs leading to discontinuation were infections (1L, ≥3L) and cardiac events (2L). The 2-year retention rate was 70.2% in the whole cohort (1L: 75.4%, 2L: 70.1%, ≥3L: 60.9%). The 2-year PFS and OS were, respectively, 85.4% and 91.7% in 1L, 80.0% and 86.2% in 2L, and 70.1% and 80.0% in ≥3L. Cardiovascular conditions did not impact patients' clinical outcomes. The most common AEs were infections (30.7%), bleeding (12.9%), fatigue (10.0%), and neutropenia (9.7%), while grade 3-4 atrial fibrillation occurred in 3.9% of patients. No new safety signals were detected. These results strongly support ibrutinib as a valuable treatment option for CLL.

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.

Baseline IgM Amounts Can Identify Patients with Poor Outcomes: Results from a Real-Life Single-Center Study on Classical Hodgkin Lymphoma.

Hodgkin Lymphoma (HL) is characterized by an inflammatory background in which the reactive myeloid cells may exert an immune-suppressive effect related to the progression of the disease. Immunoglobulin M is the first antibody isotype produced during an immune response, which also plays an immunoregulatory role. Therefore, we investigated if, as a surrogate of defective B cell function, it could have any clinical impact on prognosis. In this retrospective, observational, single-center study, we evaluated 212 newly diagnosed HL patients, including 132 advanced-stage. A 50 mg/dL level of IgM at baseline resulted in 84.1% sensitivity and 45.5% specificity for predicting a complete response in the whole cohort (area under curve (AUC) = 0.62, p = 0.013). In multivariate analysis, baseline IgM ≤ 50 mg/dL and the presence of a large nodal mass (<7 cm) were independent variables able to predict the clinical outcome, while, after two cycles of treatment, IgM ≤ 50 mg/dL at baseline and PET-2 status were independent predictors of PFS. The amount of IgM at diagnosis is a valuable prognostic factor much earlier than PET-2, and it can also provide information for PET-2-negative patients. This can help to identify different HL classes at risk of treatment failure at baseline.

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.

Patients' preferences for chronic lymphocytic leukemia treatment: The CHOICE study.

Chronic lymphocytic leukemia (CLL) therapies differ in efficacy, side effects, route, frequency, and duration of administration. We assessed patient preferences for treatment attributes and evaluated associations with disease stage, treatment line, and socio-demographic characteristics in a cross sectional, observational study conducted at 16 Italian hematology centers. Study visits occurred between February and July 2020; 401 adult patients with CLL (201 Watch and Wait (W&W), 200 treated) participated in a discrete choice experiment (DCE), composed of 8 choices between pairs of treatment profiles with different levels of 5 attributes of currently available CLL treatments (length of response, route and duration of administration, risk of side effects including diarrhea, infections, or organ damage). Health-related quality of life was assessed with the EQ-5D-5L, EORTC QLQ-C30 and QLQ CLL-16. Previously treated patients had longer disease duration (7 vs. 5 years), higher prevalence of serious comorbidities (45.5% vs. 36.2%) and high-risk molecular markers (unmutated IGHV 55.6% vs. 17.1%; TP53 mutation 15.2% vs. 4.0%). Health-related quality of life scores were similar between groups. In the DCE, W&W patients rated "possible occurrence of infections" highest (relative importance [RI] = 36.2%), followed by "treatment and relevant duration" (RI = 28.0%) and "progression-free survival (PFS)" (RI = 16.9%). Previously treated patients rated "treatment and relevant duration" highest (RI = 33.3%), followed by "possible occurrence of infections" (RI = 28.8%), "possible occurrence of organ damage" (RI = 19.4%), and "PFS" (RI = 9.8%). Concern over infection was rated highest overall; unexpectedly PFS was not among the most important criteria in either group, suggesting that the first COVID-19 pandemic wave may have influenced patient preferences and concerns about CLL therapy options.

Efficacy and safety of tixagevimab-cilgavimab versus SARS-CoV-2 breakthrough infection in the hematological conditions.

BACKGROUND: Managing SARS-CoV-2 infection in frail and immunosuppressed patients still represents an open challenge, but, starting from the phase 3 PROVENT study, prophylaxis with tixagevimab-cilgavimab has improved the approach in this category of patients, guaranteeing a better outcome and inferior mortality. Real-life data in a heterogeneous cohort are few. METHODS: The aim of this study is to evaluate the benefit of prophylaxis with tixagevimab-cilgavimab in a cohort of 202 patients affected by different hematological diseases (lymphoproliferative, myeloproliferative, autoimmune, patients recently receiving a bone marrow transplant), active (with ongoing treatment), or in watch-and-wait strategy, followed in our center, during a median follow-up of 249 (45-325) days. RESULTS: An incidence of 44 breakthrough infections (21.8%) is reported, with no treatment-related adverse effects. Age ≥70 years, ongoing treatment (above all with monoclonal antibodies), baseline lymphoproliferative disorders, and prior virus exposure are identified as risk factors related to subsequent infection (p < 0.05). Moreover, the incidence is higher in low/nonresponse to prior vaccination (p = .002). Patients treated with tixagevimab-cilgavimab had a mild course of the infection and a reduction of the duration compared with preprophylaxis infection (11 vs. 15 days, p < .001). The concurrent treatment with anti-CD20 monoclonal antibodies and B-non-Hodgkin lymphoma still confers a higher duration of infection despite prophylaxis. No deaths attributable to the infection occurred. CONCLUSION: Prophylaxis treatment seems to be a valid and safe strategy, although not preventing breakthrough infection, but the severe complications associated with the infection and the possible delays in administering lifesaving therapies from long positivity.

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.

Coexisting conditions and concomitant medications do not affect venetoclax management and survival in chronic lymphocytic leukemia.

Background: The question of which parameters may be informative on venetoclax outcome in chronic lymphocytic leukemia (CLL) is still unclear. Furthermore, the choice to treat with venetoclax can be challenging in patients with baseline characteristics or comorbidities that may potentially favor some specific adverse events. Objectives: This study was aimed to evaluate whether age, fitness status, patients'/disease characteristics, or concomitant medications may predict outcomes in CLL patients receiving venetoclax. Design: Retrospective observational study. Methods: Impact of age, presence of Cumulative Illness Rating Scale (CIRS) >6 or severe organ impairment (CIRS3+), Eastern Cooperative Oncology Group-Performance Status (ECOG-PS), renal function, and concomitant medications were retrospectively analyzed on treatment management (definitive discontinuation due to toxicity, discontinuation due to toxicity, Tox-DTD; permanent dose reduction, PDR) and survival [progression free survival (PFS), event free survival (EFS), overall survival (OS)] in unselected patients receiving venetoclax monotherapy in common practice. Results: A total of 221 relapsed/refractory patients were included. Tox-DTD and PDR were reported in 5.9% and 21.7%, respectively, and were not influenced by any fitness parameter, age, number or type of concomitant medication, baseline neutropenia, or impaired renal function. None of these factors were associated with tumor lysis syndrome (TLS) development. Age and coexisting conditions had no influence on PFS and EFS. At univariate analysis, OS was significantly shorter only in patients with ECOG-PS >1 (p < 0.0001) and elderly (⩾65 years) with CIRS >6 (p = 0.014) or CIRS3+ (p = 0.031). ECOG-PS >1 retained an independent role only for EFS and OS. While Tox-DTD affected all survival outcomes, no differences in PFS were reported among patients permanently reducing dose or interrupting venetoclax for > 7 days. Conclusion: Clinical outcome with venetoclax is not influenced by comorbidities, patients' clinical characteristics, or concomitant medications. Differently from other targeted therapies, this demonstrates that, except ECOG-PS, none of the parameters generally considered for treatment choice, including baseline neutropenia or impaired renal function, should rule the decision process with this agent. Anyway, if clinically needed, a correct drug management does not compromise treatment efficacy and may avoid toxicity-driven discontinuations. Plain Language Summary: Chapter 1: Why was this study done? Chapter 2: Which are the main findings of the study? Chapter 3: How these findings may impact on clinical practice? Coexisting conditions and concomitant medications do not affect venetoclax management and survival in chronic lymphocytic leukemia • The question of which parameters may be informative on venetoclax outcome in chronic lymphocytic leukemia is still unclear. Furthermore, the choice to treat with venetoclax can be challenging in patients with baseline characteristics or comorbidities that may potentially favor some specific adverse events (e.g. compromised renal function or baseline neutropenia).• In our large series of patients treated outside of clinical trials, we demonstrated that neither age, fitness, comorbidities nor concomitant medications impact on venetoclax management and survival. Importantly, patients presenting with baseline neutropenia or impaired renal function did not have a higher rate of dose reductions or toxicity-driven discontinuations, thus further underlining that venetoclax may be safely administered even in those categories with no preclusions.• Differently from other targeted agents, our data demonstrate that none of the baseline factors commonly considered in treatment decision process retains a role with venetoclax. Finally, permanent dose reductions and temporary interruptions did not adversely impact PFS suggesting that, if clinically needed, a correct drug management should be adopted with no risk of compromising venetoclax efficacy.

Previous therapy with immune checkpoint inhibitor as a cause of hypothyroidism, myositis, and renal insufficiency in a candidate for allogeneic hematopoietic transplantation.

Treatment of neoplastic diseases resistant to conventional chemotherapies is still an open challenge. The increasing development of chemical molecules or monoclonal antibodies able to recognize precise molecular targets of cancer disease has played an increasingly important role in treating patients suffering from solid or hematological tumors, and constitutes the basis of so-called 'targeted therapy'. Immunotherapy has become a cornerstone for treating refractory or relapsed cancer disease patients after standard chemotherapies. Immune checkpoint (including PD-1) inhibitors are essential drugs that significantly improve the therapeutic possibilities for neoplastic patients. Still, foreseeable or unpredictable adverse effects can potentially arise during or after the end of therapy. Specifically, toxicity involving several organs is capable of delaying or preventing the continuation of programmed treatment, as described in this case, where we will discuss the possibility of toxicity affecting various organs (kidney, muscle tissue, and thyroid) attributed to nivolumab and which resulted in temporary ineligibility for allogeneic transplantation.