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.

Prognostication in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in Western countries. CLL is a highly heterogeneous disease: some patients may never require therapy and others relapse several times after different therapeutic strategies. Therefore, in CLL, prognostic markers are essential to capture high-risk patients for different clinical endpoints including early treatment requirement, early progression after BTK or BCL2 inhibitors and Richter transformation. In early stage CLL, different biological and clinical biomarkers have been identified to predict time to treatment requirement that could be used to identify the most appropriate population for early intervention clinical trial. However, at the moment, the standard of care for early stage CLL remains watch & wait since no survival benefit has been identified in clinical trials with chemoimmunotherapy and with BTK inhibitors. In patients requiring treatment TP53 disruptions identify high-risk patients who benefit the most from long-term continuous therapy with BTKi. On the opposite side of the spectrum, IGHV mutated patients devoid of TP53 disruption benefit the most from fixed-duration therapy with venetoclax-obinutuzumab. In between, the highly heterogenous subgroup of patients with IGHV unmutated genes represents the group in which further efforts are needed to identify additional prognostic biomarkers aimed at selecting patients who can benefit from fixed-duration and patients who can benefit from long term BTKi therapy. In the context of the aggressive transformation of CLL, namely Richter syndrome, the clonal relationship to the CLL counterpart represents the strongest prognostic biomarker. Clonally related Richter syndrome still represents an unmet clinical need which requires further efforts to identify new therapeutic strategies.

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.

High BECN1 Expression Negatively Correlates with BCL2 Expression and Predicts Better Prognosis in Diffuse Large B-Cell Lymphoma: Role of Autophagy.

Diffuse large B-cell lymphoma (DLBCL) is characterized by high molecular and clinical heterogeneity. Autophagy, a lysosome-driven catabolic process devoted to macromolecular turnover, is fundamental in maintaining normal hematopoietic stem cells and progenitors homeostasis, and its dysregulation plays a critical role in the initiation and progression of hematological malignancies. One main regulator of autophagy is BECLIN-1, which may interact alternatively with either BCL-2, thus allowing apoptosis, or PI3KC3, thus promoting autophagy. The altered expression of BCL2 and BECN1 correlates with lymphoma outcomes, but whether this is associated with dysregulated cross-talk between autophagy and apoptosis remains to be elucidated. Analysis of the TCGA database revealed that BCL2 and BECN1 mRNA expression were inversely correlated in DLBCL patients. In representative DLBCL cell lines exposed to doxorubicin, the cells highly expressing BCL-2 were resistant, while the ones highly expressing BECLIN-1 were sensitive, and this correlated with low and high autophagy flux, respectively. Venetoclax targeting of BCL-2 increased while the spautin-1-mediated inhibition of BECLIN-1-dependent autophagy reversed doxorubicin sensitivity in the former and in the latter, respectively. By interrogating the TCGA DLBCL dataset, we found that BCL2 and BECN1 acted as negative and positive prognostic markers for DLBCL, respectively. The differentially expressed gene analysis in the respective cohorts revealed that BCL2 positively correlated with oncogenic pathways (e.g., glucose transport, HIF1A signaling, JAK-STAT signaling, PI3K-AKT-mTOR pathway) and negatively correlated with autophagy-related transcripts, while BECN1 showed the opposite trend. Notably, patients with high BECN1 expression displayed longer survival. Our data reveal, for the first time, that the modulation of BECLIN-1-dependent autophagy influences the prognosis of DLBCL patients and provide a mechanistic explanation supporting the therapeutic use of drugs that, by stimulating autophagy, can sensitize lymphoma cells to chemotherapy.

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.

Liquid biopsy in hematological malignancies: current and future applications.

The assessment of the cancer mutational profile is crucial for patient management, stratification, and therapeutic decisions. At present, in hematological malignancies with a solid mass, such as lymphomas, tumor genomic profiling is generally performed on the tissue biopsy, but the tumor may harbor genetic lesions that are unique to other anatomical compartments. The analysis of circulating tumor DNA (ctDNA) on the liquid biopsy is an emerging approach that allows genotyping and monitoring of the disease during therapy and follow-up. This review presents the different methods for ctDNA analysis and describes the application of liquid biopsy in different hematological malignancies. In diffuse large B-cell lymphoma (DLBCL) and Hodgkin lymphoma (HL), ctDNA analysis on the liquid biopsy recapitulates the mutational profile of the tissue biopsy and can identify mutations otherwise absent on the tissue biopsy. In addition, changes in the ctDNA amount after one or two courses of chemotherapy significantly predict patient outcomes. ctDNA analysis has also been tested in myeloid neoplasms with promising results. In addition to mutational analysis, liquid biopsy also carries potential future applications of ctDNA, including the analysis of ctDNA fragmentation and epigenetic patterns. On these grounds, several clinical trials aiming at incorporating ctDNA analysis for treatment tailoring are currently ongoing in hematological malignancies.

Long-term follow-up of rituximab plus bendamustine and cytarabine in older patients with newly diagnosed MCL.

The combination of rituximab, bendamustine, and low-dose cytarabine (R-BAC) has been studied in a phase 2 prospective multicenter study from Fondazione Italiana Linfomi (RBAC500). In 57 previously untreated elderly patients with mantle cell lymphoma (MCL), R-BAC was associated with a complete remission rate of 91% and 2-year progression-free survival (PFS) of 81% (95% confidence interval [CI], 68-89). Here, we report the long-term survival outcomes, late toxicities, and results of minimal residual disease (MRD) evaluation. After a median follow-up of 86 months (range, 57-107 months), the median overall survival (OS) and PFS were not reached. The 7-year PFS and OS rates were 55% (95% CI, 41-67), and 63% (95% CI, 49-74), respectively. Patients who responded (n = 53) had a 7-year PFS of 59% (95% CI, 44-71), with no relapse or progression registered after the sixth year. In the multivariate analysis, blastoid/pleomorphic morphology was the strongest adverse predictive factor for PFS (P = .04). Patients with an end of treatment negative MRD had better, but not significant, outcomes for both PFS and OS than patients with MRD-positive (P = 0.148 and P = 0.162, respectively). There was no signal of late toxicity or an increase in secondary malignancies during the prolonged follow-up. In conclusion, R-BAC, which was not followed by maintenance therapy, showed sustained efficacy over time in older patients with MCL. Survival outcomes compare favorably with those of other immunochemotherapy regimens (with or without maintenance), including combinations of BTK inhibitors upfront. This study was registered with EudraCT as 2011-005739-23 and at www.clinicaltrials.gov as #NCT01662050.

The immunomodulatory molecule TIGIT is expressed by chronic lymphocytic leukemia cells and contributes to anergy.

T-cell immunoreceptor with Ig and ITIM domains (TIGIT) is an inhibitory checkpoint receptor that negatively regulates Tcell responses. CD226 competes with TIGIT for binding to the CD155 ligand, delivering a positive signal to the T cell. Here we studied the expression of TIGIT and CD226 in a cohort of 115 patients with chronic lymphocytic leukemia (CLL) and report expression of TIGIT and CD226 by leukemic cells. By devising a TIGIT/CD226 ratio, we showed that CLL cells favoring TIGIT over CD226 are typical of a more indolent disease, while those favoring CD226 are characterized by a shorter time to first treatment and shorter progression-free survival after first treatment. TIGIT expression was inversely correlated to the B-cell receptor (BCR) signaling capacity, as determined by studying BTK phosphorylation, cell proliferation and interleukin- 10 production. In CLL cells treated with ibrutinib, in which surface IgM and BCR signaling capacity are temporarily increased, TIGIT expression was downmodulated, in line with data indicating transient recovery from anergy. Lastly, cells from patients with Richter syndrome were characterized by high levels of CD226, with low to undetectable TIGIT, in keeping with their high proliferative drive. Together, these data suggest that TIGIT contributes to CLL anergy by downregulating BCR signaling, identifying novel and actionable molecular circuits regulating anergy and modulating CLL cell functions.

Different prognostic impact of recurrent gene mutations in chronic lymphocytic leukemia depending on IGHV gene somatic hypermutation status: a study by ERIC in HARMONY.

Recent evidence suggests that the prognostic impact of gene mutations in patients with chronic lymphocytic leukemia (CLL) may differ depending on the immunoglobulin heavy variable (IGHV) gene somatic hypermutation (SHM) status. In this study, we assessed the impact of nine recurrently mutated genes (BIRC3, EGR2, MYD88, NFKBIE, NOTCH1, POT1, SF3B1, TP53, and XPO1) in pre-treatment samples from 4580 patients with CLL, using time-to-first-treatment (TTFT) as the primary end-point in relation to IGHV gene SHM status. Mutations were detected in 1588 (34.7%) patients at frequencies ranging from 2.3-9.8% with mutations in NOTCH1 being the most frequent. In both univariate and multivariate analyses, mutations in all genes except MYD88 were associated with a significantly shorter TTFT. In multivariate analysis of Binet stage A patients, performed separately for IGHV-mutated (M-CLL) and unmutated CLL (U-CLL), a different spectrum of gene alterations independently predicted short TTFT within the two subgroups. While SF3B1 and XPO1 mutations were independent prognostic variables in both U-CLL and M-CLL, TP53, BIRC3 and EGR2 aberrations were significant predictors only in U-CLL, and NOTCH1 and NFKBIE only in M-CLL. Our findings underscore the need for a compartmentalized approach to identify high-risk patients, particularly among M-CLL patients, with potential implications for stratified management.

High reproducibility of spleen stiffness measurement by vibration-controlled transient elastography with a spleen-dedicated module.

Spleen stiffness measurement (SSM) by vibration-controlled transient elastography (VCTE) is a noninvasive technique for estimating portal hypertension in patients with chronic liver disease (CLD), with its reproducibility yet to be established and its feasibility still unknown beyond CLD. We have studied 420 participants from two tertiary referral centers for liver diseases (Novara, Milan): 297 patients with CLD (32% with cirrhosis) of different etiology (Group A), 63 Philadelphia-negative myeloproliferative neoplasms (Group B), and 60 heathy volunteers (Group C). All underwent SSM by VCTE with a spleen-dedicated module (SSM@100 Hz) and liver stiffness measurement (LSM), blindly performed by 2 different operators. In total, 1680 VCTE examinations for SSM were performed (1000 in Novara, 680 in Milan), with an overall 3.2% failure rate. Median SSM was 26.5 kPa (interquartile range [IQR] 20.0-42.3) in Group A, 26.3 kPa (IQR 22.3-33.6) in Group B, and 16.1 kPa (IQR 14.6-18.7) in Group C. In Group A, the median LSM was 6.8 kPa (IQR 4.9-11.3) in Novara and 8.3 kPa (IQR 7.1-10.8) in Milan, the proportion of patients with cirrhosis being 34% in Novara and 31% in Milan. The Group A interobserver agreement ICC was 0.90 (0.88-0.92), significantly lower in the absence of splenomegaly (ICC 0.87 vs. 0.91) and in absence of cirrhosis (ICC 0.84 vs. 0.90); overweight slightly, but not significantly reduced the interobserveragreement. The intra-observer agreement ICC ranged from 0.91 to 0.96 for the four operators. The Group B interobserver agreement ICC was 0.90 (0.83-0.94). In conclusion, SSM measured by the new spleen-dedicated VCTE module is a feasible, reliable, and highly reproducible tool in patients with CLD and hematological disorders, and in healthy volunteers.

Detection of early seeding of Richter transformation in chronic lymphocytic leukemia.

Richter transformation (RT) is a paradigmatic evolution of chronic lymphocytic leukemia (CLL) into a very aggressive large B cell lymphoma conferring a dismal prognosis. The mechanisms driving RT remain largely unknown. We characterized the whole genome, epigenome and transcriptome, combined with single-cell DNA/RNA-sequencing analyses and functional experiments, of 19 cases of CLL developing RT. Studying 54 longitudinal samples covering up to 19 years of disease course, we uncovered minute subclones carrying genomic, immunogenetic and transcriptomic features of RT cells already at CLL diagnosis, which were dormant for up to 19 years before transformation. We also identified new driver alterations, discovered a new mutational signature (SBS-RT), recognized an oxidative phosphorylation (OXPHOS)high-B cell receptor (BCR)low-signaling transcriptional axis in RT and showed that OXPHOS inhibition reduces the proliferation of RT cells. These findings demonstrate the early seeding of subclones driving advanced stages of cancer evolution and uncover potential therapeutic targets for RT.

Minimal residual disease-driven treatment intensification with sequential addition of ibrutinib to venetoclax in R/R CLL.

Undetectable measurable residual disease (uMRD) is achievable in patients with chronic lymphocytic leukemia (CLL) with the BCL2-inhibitor venetoclax alone or combined with the Bruton's tyrosine kinase inhibitor ibrutinib. This phase 2, multicenter, MRD-driven study was designed to discontinue treatment upon reaching uMRD4 (<10-4) in patients with relapsed/refractory CLL receiving venetoclax monotherapy or after the addition of ibrutinib. Primary end point of the study was proportion of uMRD4 with venetoclax ± ibrutinib. Secondary end points were overall response rate, partial response, complete response, progression-free survival, duration of response, overall survival, and safety of venetoclax ± ibrutinib. Patients with uMRD4 at Cycle 12 Day 1 discontinued venetoclax. MRD+ patients added ibrutinib and continued both drugs up to Cycle 24 Day 28/uMRD4/progression/toxicity. After Cycle 24 Day 28, MRD+ patients continued ibrutinib. Thirty-eight patients (29% with TP53 aberrations; 79% with unmutated IGHV) started venetoclax. Overall response rate with venetoclax was 36 (95%) of 38 patients (20 complete; 16 partial response). Seventeen patients (45%) with uMRD4 at Cycle 12 Day 1 discontinued venetoclax. Nineteen (55%) MRD+ subjects added ibrutinib. After a median of 7 months (range, 3-10 months) of combined treatment, 16 (84%) of 19 achieved uMRD4, thus stopping both drugs. Two MRD+ patients at Cycle 24 Day 28 continued ibrutinib until progression/toxicity. After a median follow-up of 36.5 months, median progression-free survival was not reached; 10 patients progressed (4 restarted venetoclax, 3 without treatment need, 2 developed Richter transformation, and 1 dropped out). Seven (22%) of 32 patients remain uMRD4 after 3 years of follow-up. Neutropenia was the most frequent grade 3 to 4 adverse event; no grade 5 events occurred on study. This sequential MRD-guided approach led to uMRD4 in 33 (87%) of 38 patients, with venetoclax monotherapy or combined with ibrutinib, delivering treatment combination only in a fraction, and ultimately identifying the few patients benefiting from continuous therapy. This trial was registered at www.clinicaltrials.gov as # NCT04754035.

Clonally unrelated Richter syndrome are truly de novo diffuse large B-cell lymphomas with a mutational profile reminiscent of clonally related Richter syndrome.

Richter syndrome (RS) is mostly due to the direct transformation of the chronic lymphocytic leukaemia (CLL) clone, as documented by the same immunoglobulin heavy-chain variable region (IGHV) rearrangement in both CLL and RS cells. In rare cases characterized by a better outcome, the RS clone harbours a different IGHV rearrangement compared to the CLL phase. We investigated the CLL phase of clonally unrelated RS to test whether the RS clone was already identifiable prior to clinicopathologic transformation, albeit undetectable by conventional approaches. CLL cells of eight patients with unrelated RS were subjected to an ultra-deep next-generation sequencing (NGS) approach with a sensitivity of 10-6 . In 7/8 cases, the RS rearrangement was not identified in the CLL phase. In one case, the RS clone was identified at a very low frequency in the CLL phase, conceivably due to the concomitance of CLL sampling and RS diagnosis. Targeted resequencing revealed that clonally unrelated RS carries genetic lesions primarily affecting the TP53, MYC, ATM and NOTCH1 genes. Conversely, mutations frequently involved in de novo diffuse large B-cell lymphoma (DLBCL) without a history of CLL were absent. These results suggest that clonally unrelated RS is a truly de novo lymphoma with a mutational profile reminiscent, at least in part, of clonally related RS.