Decreased apoptotic priming and loss of BCL-2 dependence are functional hallmarks of Richter's syndrome.

Richter's syndrome (RS) is the transformation of chronic lymphocytic leukemia (CLL) into a high-grade B-cell malignancy. Molecular and functional studies have pointed out that CLL cells are close to the apoptotic threshold and dependent on BCL-2 for survival. However, it remains undefined how evasion from apoptosis evolves during disease transformation. Here, we employed functional and static approaches to compare the regulation of mitochondrial apoptosis in CLL and RS. BH3 profiling of 17 CLL and 9 RS samples demonstrated that RS cells had reduced apoptotic priming and lower BCL-2 dependence than CLL cells. While a subset of RS was dependent on alternative anti-apoptotic proteins and was sensitive to specific BH3 mimetics, other RS cases harbored no specific anti-apoptotic addiction. Transcriptomics of paired CLL/RS samples revealed downregulation of pro-apoptotic sensitizers during disease transformation. Albeit expressed, effector and activator members were less likely to colocalize with mitochondria in RS compared to CLL. Electron microscopy highlighted reduced cristae width in RS mitochondria, a condition further promoting apoptosis resistance. Collectively, our data suggest that RS cells evolve multiple mechanisms that lower the apoptotic priming and shift the anti-apoptotic dependencies away from BCL-2, making direct targeting of mitochondrial apoptosis more challenging after disease transformation.

Extracellular Vesicles based STAT3 delivery as innovative therapeutic approach to restore STAT3 signaling deficiency.

Extracellular Vesicles (EVs) have been proposed as a promising tool for drug delivery because of their natural ability to cross biological barriers, protect their cargo, and target specific cells. Moreover, EVs are not recognized by the immune system as foreign, reducing the risk of an immune response and enhancing biocompatibility. Herein, we proposed an alternative therapeutic strategy to restore STAT3 signaling exploiting STAT3 loaded EVs. This approach could be useful in the treatment of Autosomal Dominant Hyper-IgE Syndrome (AD-HIES), a rare primary immunodeficiency and multisystem disorder due to the presence of mutations in STAT3 gene. These mutations alter the signal transduction of STAT3, thereby impeding Th17 CD4+ cell differentiation that leads to the failure of immune response. We set up a simple and versatile method in which EVs were loaded with fully functional STAT3 protein. Moreover, our method allows to follow the uptake of STAT3 loaded vesicles inside cells due to the presence of EGFP in the EGFP-STAT3 fusion protein construct. Taken together, the data presented in this study could provide the scientific background for the development of new therapeutic strategy aimed to restore STAT3 signaling in STAT3 misfunction associated diseases like AD-HIES. In the future, the administration of fully functional wild type STAT3 to CD4+ T cells of AD-HIES patients might compensate its loss of function and would be beneficial for these patients, lowering the risk of infections, the use of medications, and hospitalizations.

Outcomes of younger patients with mantle-cell lymphoma experiencing late relapse (>24 months): the LATE-POD study.

Patients with mantle cell lymphoma (MCL) who experience first relapse/refractoriness can be categorized into early or late progression-of-disease (POD) groups, with a threshold of 24 months from the initial MCL diagnosis. Bruton tyrosine kinase inhibitors (BTKi) are established standard treatment at first relapse, but their effectiveness as compared to chemoimmunotherapy (CIT) in late-POD patients remains unknown. In this international, observational cohort study, we evaluated outcomes amongst patients at first, late-POD beyond 24 months. Patients treated upfront with BTKi were excluded. The primary objective was progression-free survival from time of second-line therapy (PFS-2) of BTKi versus CIT. After accrual, all patients were prospectively followed-up. Overall, 385 late-POD patients were included from 10 countries. Their median age was 59 (range:19-70) years and 77% were males. Median follow-up from time of first relapse was 53 months (range:12-144). Overall, 114 patients had second-line BTKi, while 271 had CIT, consisting of rituximab-bendamustine (R-B, n=101), R-B and cytarabine (R-BAC, n=70), or other regimens (mostly cyclophosphamide-hydroxydaunorubicin-vincristine-prednisone-CHOP- or platinum-based, n=100). The two groups were balanced for clinicopathological features, and median time to first relapse (48 months for both). Overall, BTKi was associated with significantly prolonged median PFS-2 than CIT [not reached-NR vs 26 months, respectively, P=.0003], and overall survival [NR and 56 months, respectively, P=.03]. Multivariate analyses showed that BTKi was associated with lower risk of death than R-B and other regimens (hazard ratio-HR, 0.41 for R-B, 0.46 for others), but similar to R-BAC. These results may establish BTKi as the preferable second-line approach in BTKi-naïve MCL patients.

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.

B-cell receptor signaling activity identifies patients with mantle cell lymphoma at higher risk of progression.

Mantle cell lymphoma (MCL) is an incurable B-cell malignancy characterized by a high clinical variability. Therefore, there is a critical need to define parameters that identify high-risk patients for aggressive disease and therapy resistance. B-cell receptor (BCR) signaling is crucial for MCL initiation and progression and is a target for therapeutic intervention. We interrogated BCR signaling proteins (SYK, LCK, BTK, PLCγ2, p38, AKT, NF-κB p65, and STAT5) in 30 primary MCL samples using phospho-specific flow cytometry. Anti-IgM modulation induced heterogeneous BCR signaling responses among samples allowing the identification of two clusters with differential responses. The cluster with higher response was associated with shorter progression free survival (PFS) and overall survival (OS). Moreover, higher constitutive AKT activity was predictive of inferior response to the Bruton's tyrosine kinase inhibitor (BTKi) ibrutinib. Time-to-event analyses showed that MCL international prognostic index (MIPI) high-risk category and higher STAT5 response were predictors of shorter PFS and OS whilst MIPI high-risk category and high SYK response predicted shorter OS. In conclusion, we identified BCR signaling properties associated with poor clinical outcome and resistance to ibrutinib, thus highlighting the prognostic and predictive significance of BCR activity and advancing our understanding of signaling heterogeneity underlying clinical behavior of MCL.

Case Report: Rapid renal response to venetoclax monotherapy in a CLL patient with secondary membranous glomerulonephritis.

Membranous glomerulonephritis (MGN) is a rare extra-hematological autoimmune complication of chronic lymphocytic leukemia (CLL), clinically characterized by nephrotic-range proteinuria and, less frequently, renal failure. Because of the rarity of this condition, there is no standardized treatment. Chlorambucil and fludarabine-based regimens, possibly combined with rituximab, have been historically the most frequent therapeutic approaches, with renal response obtained in about two-third of the patients. However, responses are often transient and partial. Here we describe the first patient with rituximab-refractory, CLL-related MGN successfully treated with the Bcl-2 antagonist venetoclax. Nephrotic syndrome resolved as soon as three months after venetoclax initiation, with no unexpected toxicities. At the last follow-up, 17 months after venetoclax start, renal response persists, with proteinuria below 0.5 g/24 hours. This case suggests that targeted agents, particularly Bcl-2 antagonists, might be suitable options for patients with renal autoimmune disorders arising in the context of CLL.

An updated portrait of monocyte-macrophages in classical Hodgkin lymphoma.

Classical Hodgkin lymphoma (cHL) is a unique neoplastic ecosystem characterized by a heterogeneous immune infiltrate surrounding the rare malignant Hodgkin Reed-Sternberg cells. Though less abundant than T-cells, tumor-infiltrating macrophages play a pivotal role in supporting HRS survival through cell-to-cell and paracrine interactions. Traditional immunohistochemistry based upon the M1-M2 dichotomy yielded controversial results about the composition, functional role and prognostic impact of macrophages in cHL. More recent studies exploiting single-cell technologies and image analyses have highlighted the heterogeneity and the peculiar spatial arrangement of the macrophagic infiltrate, with the most immunosuppressive subpopulations lying in close proximity of HRS cells and the most tumor-hostile subsets kept far away from the neoplastic niches. High-throughput analysis of peripheral blood mononuclear cells in cHL patients have also identified a novel, potentially cytotoxic, subpopulation predicting better response to PD-1 blockade. This review examines the phenotypic profile, spatial localization and clinical impact of tumor-infiltrating macrophages and circulating monocytes in cHL, providing an up-do-date portrait of these innate immune cells with possible translational applications.

SARS-CoV-2 infection in patients with chronic lymphocytic leukemia: The Italian Hematology Alliance on COVID-19 cohort.

COVID-19, the disease caused by SARS-CoV-2, is still afflicting thousands of people across the globe. Few studies on COVID-19 in chronic lymphocytic leukemia (CLL) are available. Here, we analyzed data from the CLL cohort of the Italian Hematology Alliance on COVID-19 (NCT04352556), which included 256 CLL patients enrolled between 25 February 2020 and 1 February 2021. Median age was 70 years (range 38-94) with male preponderance (60.1%). Approximately half of patients (n = 127) had received at least one line of therapy for CLL, including 108 (83.7%) who were on active treatment at the time of COVID-19 or received their last therapy within 12 months. Most patients (230/256, 89.9%) were symptomatic at COVID-19 diagnosis and the majority required hospitalization (n = 176). Overall, after a median follow-up of 42 days (IQR 24-96), case fatality rate was 30.1%, and it was 37.5% and 24.4% in the first (25 February 2020-22 June 2020) and second wave (23 June 2020-1 February 2021), respectively (p = 0.03). At multivariate analysis, male sex (HR 1.82, 95% CI 1.03-3.24, p = 0.04), age over than 70 years (HR 2.23, 95% CI 1.23-4.05, p = 0.01), any treatment for CLL given in the last 12 months (HR 1.72, 95% CI 1.04-2.84, p = 0.04) and COVID-19 severity (severe: HR 5.66, 95% CI 2.62-12.33, p < 0.0001; critical: HR 15.99, 95% CI 6.93-36.90, p < 0.0001) were independently associated with poor survival. In summary, we report a dismal COVID-related outcome in a significant fraction of CLL patients, that can be nicely predicted by clinical parameters.

Real-world Outcomes of Relapsed/Refractory Diffuse Large B-cell Lymphoma Treated With Polatuzumab Vedotin-based Therapy.

After FDA and EMA approval of the regimen containing polatuzumab vedotin plus rituximab and bendamustine (PolaBR), eligible relapsed/refractory diffuse large B-cell lymphoma (DLBCL) patients in Italy were granted early access through a Named Patient Program. A multicentric observational retrospective study was conducted focusing on the effectiveness and safety of PolaBR in everyday clinical practice. Fifty-five patients were enrolled. There were 26 females (47.3%), 32 patients were primary refractory and 45 (81.8%) resulted refractory to their last therapy. The decision to add or not bendamustine was at physician's discretion. Thirty-six patients underwent PolaBR, and 19 PolaR. The 2 groups did not differ in most of baseline characteristics. The final overall response rate was 32.7% (18.2% complete response rate), with a best response rate of 49.1%. Median disease-free survival was reached at 12 months, median progression-free survival at 4.9 months and median overall survival at 9 months, respectively. Overall, 88 adverse events (AEs) were registered during treatment in 31 patients, 22 of grade ≥3. Eight cases of neuropathy occurred, all of grades 1-2 and all related to polatuzumab. The two groups of treatment did not differ for effectiveness endpoints but presented statistically significant difference in AEs occurrence, especially in hematological AEs, in AEs of grade equal or greater than 3 and in incidence of neuropathy. Our data add useful information on the effectiveness of Pola(B)R in the setting of heavily pretreated DLBCL and may also suggest a better tolerability in absence of bendamustine without compromise of efficacy.

Two Distinct Clinical Patterns of Ibrutinib-to-Venetoclax Transition in Relapsed Chronic Lymphocytic Leukemia Patients.

Patients with chronic lymphocytic leukemia (CLL) relapsing on ibrutinib are often treated with the Bcl-2 inhibitor venetoclax. However, the transition from one agent to another poses some clinical challenges due to disease flares sometimes occurring right after ibrutinib interruption. Here, we describe three clinical vignettes highlighting two distinct patterns of ibrutinib-to-venetoclax transition. While patients following the favorable pattern transited to venetoclax without experiencing disease flare, the one patient who took the unfavorable path showed rapid disease rebound, with large-cell transformation occurring one week after ibrutinib interruption. A high burden of BTK and PLCG2 mutations was found only in patients with the favorable transition pattern, suggesting that removing BTK inhibition might be particularly harmful if CLL cells are progressing through mechanisms external to the BTK axis.

Targeting CD19 in diffuse large B-cell lymphoma: An expert opinion paper.

The ubiquitous, early-stage expression, efficient internalization, limited off-target effects, and high disease specificity of CD19 make it an attractive therapeutic target. Currently available anti-CD19 therapies have demonstrated particular promise in patients with relapsed or refractory B-cell non-Hodgkin lymphoma. Selection of the most appropriate treatment strategy should be based on individual patient characteristics and the goal of therapy. However, evidence and knowledge about the sequencing of anti-CD19 therapies are limited. Here, we review the current evidence for CD19 as a target in diffuse large B-cell lymphoma and consider approaches to the use of anti-CD19 therapy.

The mitochondrial anti-apoptotic dependencies of hematologic malignancies: from disease biology to advances in precision medicine.

Mitochondria are critical organelles in the regulation of intrinsic apoptosis. As a general feature of blood cancers, different antiapoptotic members of the BCL-2 protein family localize at the outer mitochondrial membrane to sequester variable amounts of proapoptotic activators, and hence protect cancer cells from death induction. However, the impact of distinct anti-apoptotic members on apoptosis prevention, a concept termed anti-apoptotic dependence, differs remarkably across disease entities. Over the last two decades, several genetic and functional methodologies have been established to uncover the anti-apoptotic dependencies of the majority of blood cancers, inspiring the development of a new class of small molecules called BH3 mimetics. In this review, we highlight the rationale of targeting mitochondrial apoptosis in hematology, and provide a comprehensive map of the anti-apoptotic dependencies that are currently guiding novel therapeutic strategies. Cell-extrinsic and -intrinsic mechanisms conferring resistance to BH3 mimetics are also examined, with insights on potential strategies to overcome them. Finally, we discuss how the field of mitochondrial apoptosis might be complemented with other dimensions of precision medicine for more successful treatment of 'highly complex' hematologic malignancies.

Rituximab and Bendamustine (BR) Compared with Rituximab, Bendamustine, and Cytarabine (R-BAC) in Previously Untreated Elderly Patients with Mantle Cell Lymphoma.

BACKGROUND: Rituximab plus bendamustine (BR), and rituximab, bendamustine, and cytarabine (R-BAC) are well-known induction therapies in elderly patients with mantle cell lymphoma (MCL), according to clinical guidelines. However, a direct comparison between the two regimens has never been performed. METHODS: In this multicentre retrospective study, we compared the outcome of patients with newly diagnosed MCL, treated with BR or R-BAC. Primary endpoint was 2-year progression-free survival (PFS). Inclusion bias was assessed using a propensity score stratified by gender, age, MCL morphology, and MIPI score. RESULTS: After adjusting by propensity score, we identified 156 patients (53 BR, 103 R-BAC) with median age of 72 (53-90). Median follow-up was 46 months (range 12-133). R-BAC was administered in a 2-day schedule or with attenuated dose in 51% of patients. Patients treated with R-BAC achieved CR in 91% of cases, as compared with 60% for BR (p < 0.0001). The 2-year PFS was 87 ± 3% and 64 ± 7% for R-BAC and BR, respectively (p = 0.001). In terms of toxicity, R-BAC was associated with significantly more pronounced grade 3-4 thrombocytopenia than BR (50% vs. 17%). CONCLUSIONS: This study indicates that R-BAC, even when administered with judiciously attenuated doses, is associated with significantly prolonged 2-year PFS than BR in elderly patients with previously untreated MCL.

COVID-19 (SARS-CoV-2 infection) in lymphoma patients: A review.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection now has a global resonance and represents a major threat for several patient populations. Observations from initial case series suggested that cancer patients in general might have an unfavorable outcome following coronavirus disease 2019 (COVID-19), due to their underlying conditions and cytotoxic treatments. More recently, data regarding the incidence and clinical evolution of COVID-19 in lymphomas have been reported with the aim to identify those more frequently associated with severe complications and death. Patients with lymphoma appear particularly vulnerable to SARS-CoV-2 infection, only partly because of the detrimental effects of the anti-neoplastic regimens (chemotherapy, pathway inhibitors, monoclonal antibodies) on the immune system. Here, we systematically reviewed the current literature on COVID-19 in adult patients with lymphoma, with particular emphasis on disease course and prognostic factors. We also highlighted the potential differences in COVID-19 clinical picture according to lymphoma subtype, delivered treatment for the hematological disease and its relationship on how these patients have been managed thus far.

ONC212 is a Novel Mitocan Acting Synergistically with Glycolysis Inhibition in Pancreatic Cancer.

ONC212 is a fluorinated imipridone with preclinical efficacy against pancreatic and other malignancies. Although mitochondrial protease ClpP was identified as an ONC212-binding target, the mechanism leading to cancer cell death is incompletely understood. We investigated mitochondrial dysfunction and metabolic rewiring triggered by ONC212 in pancreatic cancer, a deadly malignancy with an urgent need for novel therapeutics. We found ClpP is expressed in pancreatic cancer cells and is required for ONC212 cytotoxicity. ClpX, the regulatory binding partner of ClpP, is suppressed upon ONC212 treatment. Immunoblotting and extracellular flux analysis showed ONC212 impairs oxidative phosphorylation (OXPHOS) with decrease in mitochondrial-derived ATP production. Although collapse of mitochondrial function is observed across ONC212-treated cell lines, only OXPHOS-dependent cells undergo apoptosis. Cells relying on glycolysis undergo growth arrest and upregulate glucose catabolism to prevent ERK1/2 inhibition and apoptosis. Glucose restriction or combination with glycolytic inhibitor 2-deoxy-D-glucose synergize with ONC212 and promote apoptosis in vitro and in vivo Thus, ONC212 is a novel mitocan targeting oxidative metabolism in pancreatic cancer, leading to different cellular outcomes based on divergent metabolic programs.

Activation of Protein Tyrosine Phosphatase Receptor Type γ Suppresses Mechanisms of Adhesion and Survival in Chronic Lymphocytic Leukemia Cells.

The regulatory role of protein tyrosine kinases in ß1- and ß2-integrin activation and in the survival of chronic lymphocytic leukemia (CLL) cells is well established. In contrast, the involvement of protein tyrosine phosphatases in CLL biology was less investigated. We show that selective activation of the protein tyrosine phosphatase receptor type γ (PTPRG) strongly suppresses integrin activation and survival in leukemic B cells isolated from patients with CLL. Activation of PTPRG specifically inhibits CXCR4- as well as BCR-induced triggering of LFA-1 and VLA-4 integrins and mediated rapid adhesion. Triggering of LFA-1 affinity is also prevented by PTPRG activity. Analysis of signaling mechanisms shows that activation of PTPRG blocks chemokine-induced triggering of JAK2 and Bruton's tyrosine kinase protein tyrosine kinases and of the small GTP-binding protein RhoA. Furthermore, activated PTPRG triggers rapid and robust caspase-3/7-mediated apoptosis in CLL cells in a manner quantitatively comparable to the Bruton's tyrosine kinase inhibitor ibrutinib. However, in contrast to ibrutinib, PTPRG-triggered apoptosis is insensitive to prosurvival signals generated by CXCR4 and BCR signaling. Importantly, PTPRG activation does not trigger apoptosis in healthy B lymphocytes. The data show that activated PTPRG inhibits, at once, the signaling pathways controlling adhesion and survival of CLL cells, thus emerging as a negative regulator of CLL pathogenesis. These findings suggest that pharmacological potentiation of PTPRG tyrosine-phosphatase enzymatic activity could represent a novel approach to CLL treatment.

Efficacy of R-COMP in comparison to R-CHOP in patients with DLBCL: A systematic review and single-arm metanalysis.

Doxorubicin represents the mainstay in the upfront treatment of diffuse large B-cell lymphoma (DLBCL) patients. However, its administration is sometimes hampered by the coexistence of former comorbidities/cardiac issues, especially in the elderly population. Liposome encapsulated drug delivery systems have been adopted to reduce the exposure of normal tissues to the drug, both in solid cancers and lymphomas. Despite claims for lower toxicity, the efficacy of non-pegylated liposome doxorubicin (NPLD) in DLBCL, as compared to standard doxorubicin, has never been established. We systematically reviewed relevant literature of NPLD in lymphoma treatment. Adjusting for age/comorbidities, our metanalysis revealed that the use of combinations including NPLD (R-COMP) were non-inferior in terms of response, overall and progression-free survival to the standard of care (R-CHOP) in overlapping series of DLBCL patients. R-COMP may represent a safe and active option for elderly patients with DLBCL, or for those with some extent of cardiac impairment at baseline.