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Abstract Richter transformation (RT) is an aggressive lymphoma occurring in patients with chronic lymphocytic leukaemia. Here we investigated the anti-CD3/anti-CD19 T-cell-engager blinatumomab after R-CHOP (i.e. rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) in patients with untreated RT of diffuse large B-cell lymphoma histology (NCT03931642). In this multicentre phase 2 study, patients without complete response (CR) after two cycles of R-CHOP were eligible to receive an 8-week blinatumomab induction via continuous vein infusion with stepwise dosing until 112 μg/day. The primary endpoint was the CR rate after blinatumomab induction and secondary endpoint included safety, response duration, progression-free and overall survival. Thirty-nine patients started the first cycle of R-CHOP, 25 of whom received blinatumomab. After blinatumomab induction, five (20%) patients achieved CR, four (16%) achieved partial response, and six (24%) were stable. Considering the entire strategy, the overall response rate in the full-analysis-set was 46% (n = 18), with CR in 14 (36%) patients. The most common treatment-emergent adverse events of all grades in the blinatumomab-safety-set included fever (36%), anaemia (24%), and lymphopaenia (24%). Cytokine release syndrome (grade 1/2) was observed in 16% and neurotoxicity in 20% of patients. Blinatumomab demonstrated encouraging anti-tumour activity (the trial met its primary endpoint) and acceptable toxicity in patients with RT.

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Abstract Chimeric antigen receptor T cells (CAR T cells) can induce prolonged remission in a substantial subset of patients with relapse/refractory lymphoma. However, little is known about patients' life after CAR T‐cell therapy. We prospectively assessed the multidimensional recovery of lymphoma patients in remission, before leukapheresis, before CAR T‐cell infusion, and 3, 6, and 12 months thereafter. Validated tools were used to measure lymphoma‐related and global health‐related quality of life (HRQoL; Functional Assessment of Cancer Therapy‐Lymphoma [FACT‐Lym] and EQ‐5D‐5L), cognitive complaint (FACT‐Cognition), fatigue (FACIT‐Fatigue subscale), psychological status (Hospital Anxiety and Depression Scale, Post‐Traumatic Check List Scale), and sexuality (Relationship and Sexuality Scale). Beyond 12 months of remission, we also surveyed physical, professional, sexual, and general life status. At 3, 6, and 12 months, 53, 35, and 23 patients were evaluable, respectively. Improvement in lymphoma‐related HRQoL was clinically relevant at 3, 6, and 12 months with a mean change from baseline of 10.9 (95% confidence interval [CI]: 5.8; 16.1), 12.2 (95% CI: 4.2; 20.1), and 11.72 (95% CI: 2.06; 21.38), respectively. Improvement in global HRQoL, fatigue, and anxiety was clinically relevant, but 20%–40% of patients experienced persistent fatigue, psychological distress, and cognitive complaints over time. Beyond 12 months after CAR T cells, 81.8% of 22 evaluable patients were satisfied with their daily life. Physical activity, professional, sexual, and global well‐being had returned to prediagnosis levels in nearly half of the patients. We found an improvement in HRQoL after CAR T‐cell therapy including anxiety, depression, sexual satisfaction, and general well‐being. However, not all patients recover a “normal life.” Further research is needed to determine which patients are at risk of quality‐of‐life impairment to improve recovery after CAR T‐cell infusion.

Chimeric antigen receptor (CAR) T-cell therapy has transformed the care of patients with relapsed/refractory B-cell derived hematologic malignancies. To date, six CAR T-cell therapies, targeting either CD19 or B-cell maturation antigen, have received regulatory approval. Along with the promising survival benefit, CAR T-cell therapy is associated with potentially lifethreatening adverse events (AE), including cytokine release syndrome and immune effector cellassociated neurotoxicity syndrome. While clinical trials evaluating CAR T-cell therapy consistently report the incidence of these AE, most trials do not collect health-related quality of life (HRQoL) data. As such, the impact of CAR T-cell therapy process and related AE on the physical and psychological well-being of patients remains uncertain. HRQoL and other patientreported outcome (PRO) assessments in patients with relapsed or refractory hematologic malignancies are of utmost importance, as individuals may have unmet needs and a high demand for tolerable therapy if a cure is not obtained. In addition, it is important to standardize methods of data collection to better assess the impact of CAR T-cell therapy on quality of life, optimize patient care and costs, and enable comparison between different studies. We conducted a literature search up to June 2023 to identify the HRQoL tools used in clinical trials and in realworld studies investigating CAR T-cell therapy in patients with lymphomas or leukemias. In the present comprehensive review, we summarize the most commonly used CAR T-cell specific and non-specific HRQoL tools and discuss how the use of HRQoL and other PRO tools may be optimized.

Background Follicular lymphoma (FL), the most common indolent non-Hodgkin’s Lymphoma, is a heterogeneous disease and a paradigm of the contribution of immune tumor microenvironment to disease onset, progression, and therapy resistance. Patient-derived models are scarce and fail to reproduce immune phenotypes and therapeutic responses.Methods To capture disease heterogeneity and microenvironment cues, we developed a patient-derived lymphoma spheroid (FL-PDLS) model culturing FL cells from lymph nodes (LN) with an optimized cytokine cocktail that mimics LN stimuli and maintains tumor cell viability.Results FL-PDLS, mainly composed of tumor B cells (60% on average) and autologous T cells (13% CD4 and 3% CD8 on average, respectively), rapidly organizes into patient-specific three-dimensional (3D) structures of three different morphotypes according to 3D imaging analysis. RNAseq analysis indicates that FL-PDLS reproduces FL hallmarks with the overexpression of cell cycle, BCR, or mTOR signaling related gene sets. FL-PDLS also recapitulates the exhausted immune phenotype typical of FL-LN, including expression of BTLA, TIGIT, PD-1, TIM-3, CD39 and CD73 on CD3+ T cells. These features render FL-PDLS an amenable system for immunotherapy testing. With this aim, we demonstrate that the combination of obinutuzumab (anti-CD20) and nivolumab (anti-PD1) reduces tumor load in a significant proportion of FL-PDLS. Interestingly, B cell depletion inversely correlates with the percentage of CD8+ cells positive for PD-1 and TIM-3.Conclusions In summary, FL-PDLS is a robust patient-derived 3D system that can be used as a tool to mimic FL pathology and to test novel immunotherapeutic approaches in a context of personalized medicine.

Summary: Monocyte-derived macrophages help maintain tissue homeostasis and defend the organism against pathogens. In tumors, recent studies have uncovered complex macrophage populations, including tumor-associated macrophages, which support tumorigenesis through cancer hallmarks such as immunosuppression, angiogenesis, or matrix remodeling. In the case of chronic lymphocytic leukemia, these macrophages are known as nurse-like cells (NLCs) and they protect leukemic cells from spontaneous apoptosis, contributing to their chemoresistance. We propose an agent-based model of monocyte differentiation into NLCs upon contact with leukemic B cells in vitro. We performed patient-specific model optimization using cultures of peripheral blood mononuclear cells from patients. Using our model, we were able to reproduce the temporal survival dynamics of cancer cells in a patient-specific manner and to identify patient groups related to distinct macrophage phenotypes. Our results show a potentially important role of phagocytosis in the polarization process of NLCs and in promoting cancer cells’ enhanced survival.

The Bruton tyrosine kinase (BTK) inhibitor ibrutinib is widely used for treatment of patients with relapsed/refractory or treatment-naïve chronic lymphocytic leukemia (CLL). A prominent effect of ibrutinib is to disrupt the retention of CLL cells from supportive lymphoid tissues, by altering BTK-dependent adhesion and migration. To further explore the mechanism of action of ibrutinib and its potential impact on non-leukemic cells, we quantified multiple motility and adhesion parameters of human primary CLL cells and non-leukemic lymphoid cells. In vitro, ibrutinib affected CCL19-, CXCL12- and CXCL13-evoked migration behavior of CLL cells and non-neoplastic lymphocytes, by reducing both motility speed and directionality. De-phosphorylation of BTK induced by ibrutinib in CLL cells was associated with defective polarization over fibronectin and inability to assemble the immunological synapse upon B-cell receptor engagement. In patients’ samples collected during a 6-month monitoring of therapy, chemokine-evoked migration was repressed in CLL cells and marginally reduced in T cells. This was accompanied by profound modulation of the expression of chemokine receptors and adhesion molecules. Remarkably, the relative expression of the receptors governing lymph node entry (CCR7) versus exit (S1PR1) stood out as a reliable predictive marker of the clinically relevant treatment-induced lymphocytosis. Together, our data reveal a multifaceted modulation of motility and adhesive properties of ibrutinib on both CLL leukemic cell and T-cell populations and point to intrinsic differences in CLL recirculation properties as an underlying cause for variability in treatment response.

Abstract The presence of numerous mast cells (MCs) mixed with tumor cells in the bone marrow (BM) is a hallmark of the diagnosis of Waldenström's macroglobulinemia (WM). MCs have been shown to support lymphoplasmacytic cell growth, but there is thus far no demonstration of the prognostic impact of BM MC density in WM. We investigated BM MC density by sensitive and specific digital quantification, allowing the analysis of a large area infiltrated by BM tumor cells. A total of 65 WM patients were investigated, including 54 at diagnosis and 11 at relapse. Tryptase and CD20 immunohistochemisty staining was performed on contiguous sections of deparaffinized BM trephine biopsies. After numerization of each section, the BM surface area was manually marked out, excluding the bone framework and adipocytes to limit the analyses to only hematopoietic tissue. MCs were assessed using a digital tool previously used to quantify immune‐cell infiltrates on tumor‐tissue sections. Deep next‐generation sequencing and allele‐specific PCR were used to explore the MYD88 and CXCR4 mutational status. MC density was heterogeneous among the WM patients. An optimal MC density threshold (> 56 MC.mm–2) was defined according to ROC curve analysis of overall survival. A higher MC density (> 56 MC.mm–2) was associated with greater BM involvement by WM lymphoplasmacytic cells and less hepatosplenic involvement (p = 0.023). Furthermore, MC density significantly correlated with a higher ISSWM score (p = 0.0003) in symptomatic patients. Patients with a higher MC density showed shorter median OS (56.5 months vs. nonreached, p = 0.0004), even in multivariate analysis after controlling for other predictive variables, such as age, ISSWM score, and CXCR4 mutational status. In conclusion, MC density can be accurately measured in WM patients using a specific digital tool on well‐outlined hematopoietic tissue surfaces. High MC density is associated with aggressive features and a poor clinical outcome, emphasizing the need for further investigation of the involvement of MCs in the pathophysiology of WM.

Abstract Background Age-adjusted lymphoma incidence rates continue to rise in France since the early 80’s, although rates have slowed since 2010 and vary across subtypes. Recent improvements in patient survival in major lymphoma subtypes at population level raise new questions about patient outcomes (i.e. quality of life, long-term sequelae). Epidemiological studies have investigated factors related to lymphoma risk, but few have addressed the extent to which socioeconomic status, social institutional context (i.e. healthcare system), social relationships, environmental context (exposures), individual behaviours (lifestyle) or genetic determinants influence lymphoma outcomes, especially in the general population. Moreover, the knowledge of the disease behaviour mainly obtained from clinical trials data is partly biased because of patient selection. Methods The REALYSA (“REal world dAta in LYmphoma and Survival in Adults”) study is a real-life multicentric cohort set up in French areas covered by population-based cancer registries to study the prognostic value of epidemiological, clinical and biological factors with a prospective 9-year follow-up. We aim to include 6000 patients over 4 to 5 years. Adult patients without lymphoma history and newly diagnosed with one of the following 7 lymphoma subtypes (diffuse large B-cell, follicular, marginal zone, mantle cell, Burkitt, Hodgkin, mature T-cell) are invited to participate during a medical consultation with their hematologist. Exclusion criteria are: having already received anti-lymphoma treatment (except pre-phase) and having a documented HIV infection. Patients are treated according to the standard practice in their center. Clinical data, including treatment received, are extracted from patients’ medical records. Patients’ risk factors exposures and other epidemiological data are obtained at baseline by filling out a questionnaire during an interview led by a clinical research assistant. Biological samples are collected at baseline and during treatment. A virtual tumor biobank is constituted for baseline tumor samples. Follow-up data, both clinical and epidemiological, are collected every 6 months in the first 3 years and every year thereafter. Discussion This cohort constitutes an innovative platform for clinical, biological, epidemiological and socio-economic research projects and provides an opportunity to improve knowledge on factors associated to outcome of lymphoma patients in real life. Trial registration 2018-A01332–53, ClinicalTrials.gov identifier: NCT03869619 .

Abstract Venetoclax with high‐dose methotrexate and rituximab seem effective and safe to treat central nervous system involvement of chronic lymphocytic leukemia.

Abstract Background Previous studies have suggested that lymphoma survivors commonly display altered Health-Related Quality of Life (HRQoL). Because these were predominantly cross-sectional studies, the dynamic of events as well as the factors which influence HRQoL remain to be determined. Methods We conducted a prospective study on a cohort of 204 Hodgkin and non-Hodgkin lymphoma survivors who remained disease-free 2 years after undergoing chemotherapy (referred to the M0-M12-M24 periods). Results We found that although Physical and Mental Component Scores (PCS and MCS) of HRQoL significantly improved from M0 to M24 in the vast majority of patients (favorable group), approximately 20% of patients displayed severe alterations in HRQoL (global SF-36 scores

Follicular lymphoma (FL) is the most common indolent lymphoma. Despite the clear benefit of CD20-based therapy, a subset of FL patients still progress to aggressive lymphoma. Thus, identifying early biomarkers that incorporate positron emission tomography metrics could be helpful to identify patients with a high risk of treatment failure with rituximab. We retrospectively included a total of 132 untreated FL patients separated into training and validation cohorts. Optimal threshold of baseline whole-body maximum standardized uptake (SUVmax) was first determined in the training cohort (n=48) to predict progression-free survival (PFS). The PET results were investigated along with the tumor and immune microenvironment, which were determined by immunochemistry and transcriptome studies involving gene set enrichment analyses and immune cell deconvolution, together with the tumor mutation profile. We report that baseline SUVmax>14.5 was associated with poorer PFS than baseline SUVmax≤14.5 (hazard ratio =0.28; P=0.00046). Neither immune T-cell infiltration nor immune checkpoint expression were associated with baseline PET metrics. By contrast, FL samples with Ki-67 staining ≥10% showed enrichment of cell cycle/DNA genes (P=0.013) and significantly higher SUVmax values (P=0.007). Despite similar oncogenic pathway alterations in both SUVmax groups of FL samples, four out of five cases harboring the infrequent FOXO1 transcription factor mutation were seen in FL patients with SUVmax>14.5. Thus, high baseline SUVmax reflects FL tumor proliferation and, together with Ki-67 proliferative index, can be used to identify patients at risk of early relapse with rituximab chemotherapy.

Abstract. As a result of significant recent developments, the management of patients with chronic lymphocytic leukemia (CLL) is changing, and new therapeutic options will continue to emerge in the near future. The recommendations of the French Innovative Leukemia Organization (FILO-CLL) group presented here are intended to provide practical recommendations for physicians taking care of CLL patients, taking into account the availability of both biological tests and therapies in daily practice in France at the time of publication. This text details the documented information and guidelines on diagnosis, indications for treatment, infectious complications and therapeutic strategies in frontline and relapsed CLL as well as in particular conditions such as autoimmune cytopenia or Richter syndrome.

Complex karyotype (CK) identified by chromosome-banding analysis (CBA) has shown prognostic value in chronic lymphocytic leukemia (CLL). Genomic arrays offer high-resolution genome-wide detection of copy-number alterations (CNAs) and could therefore be well equipped to detect the presence of a CK. Current knowledge on genomic arrays in CLL is based on outcomes of single center studies, in which different cutoffs for CNA calling were used. To further determine the clinical utility of genomic arrays for CNA assessment in CLL diagnostics, we retrospectively analyzed 2293 arrays from 13 diagnostic laboratories according to established standards. CNAs were found outside regions captured by CLL FISH probes in 34% of patients, and several of them including gains of 8q, deletions of 9p and 18p (p

While efficient at treating B-cell malignancies, Bruton tyrosine kinase (BTK) inhibitors are consistently reported to increase the risk of bleeding. Analyzing platelet aggregation response to collagen in platelet-rich plasma allowed us to identify two groups in the healthy population characterized by low or high sensitivity to ibrutinib in vitro. Inhibition of drug efflux pumps induced a shift from ibrutinib low-sensitive platelets to high-sensitive ones. At a clinically relevant dose, acalabrutinib, a second-generation BTK inhibitor, did not affect maximal collagen-induced platelet aggregation in the ibrutinib low-sensitive group but did inhibit aggregation in a small fraction of the ibrutinib high-sensitive group. Consistently, acalabrutinib delayed aggregation, particularly in the ibrutinib high-sensitive group. In chronic lymphocytic leukemia patients, acalabrutinib inhibited maximal platelet aggregation only in the ibrutinib high-sensitive group. Acalabrutinib inhibited collagen-induced tyrosine-753 phosphorylation of phospholipase Cγ2 in both groups, but, in contrast to ibrutinib, did not affect Src-family kinases. Acalabrutinib affected thrombus growth under flow only in the ibrutinib high-sensitive group and potentiated the effect of cyclooxygenase and P2Y12 receptor blockers in both groups. Since the better profile of acalabrutinib was observed mainly in the ibrutinib low-sensitive group, replacement therapy in patients may not systematically reduce the risk of bleeding.

CD8+ T cells are frontline defenders against cancer and primary targets of current immunotherapies. In CLL, specific functional alterations have been described in circulating CD8+ T cells, yet a global view of the CD8+ T cell compartment phenotype and of its real impact on disease progression is presently elusive. We developed a multidimensional statistical analysis of CD8+ T cell phenotypic marker expression based on whole blood multi-color flow-cytometry. The analysis comprises both unsupervised statistics (hClust and PCA) and supervised classification methods (Random forest, Adaboost algorithm, Decision tree learning and logistic regression) and allows to cluster patients by comparing multiple phenotypic markers expressed by CD8+ T cells. Our results reveal a global CD8+ T cell phenotypic signature in CLL patients that is significantly modified when compared to healthy donors. We also uncover a CD8+ T cell signature characteristic of patients evolving toward therapy within 6 months after phenotyping. The unbiased, not predetermined and multimodal approach highlights a prominent role of the memory compartment in the prognostic signature. The analysis also reveals that imbalance of the central/effector memory compartment in CD8+ T cells can occur irrespectively of the elapsed time after diagnosis. Taken together our results indicate that, in CLL patients, CD8+ T cell phenotype is imprinted by disease clinical progression and reveal that CD8+ T cell memory compartment alteration is not only a hallmark of CLL disease but also a signature of disease evolution toward the need for therapy.

Follicular lymphoma (FL) is a common non Hodgkin’s lymphoma subtype in which immune escape mechanisms are implicated in resistance to chemo-immunotherapy. Although molecular studies point to qualitative and quantitative deregulation of immune checkpoints, in depth cellular analysis of FL immune escape is lacking. Here, by functional assays and in silico analyses we show that a subset of FL patients displays a ‘high’ immune escape phenotype. These FL cases are characterized by abundant infiltration of PD1+ CD16+ TCRVγ9Vδ2 γδ T lymphocytes. In a 3D co-culture assay (MALC), γδ T cells mediate both direct and indirect (ADCC in the presence of anti-CD20 mAbs) cytolytic activity against FL cell aggregates. Importantly, PD-1, which is expressed by most FL-infiltrating γδ T lymphocytes with ADCC capacity, impairs these functions. In conclusion, we identify a PD1-regulated γδ T cell cytolytic immune component in FL. Our data provide a treatment rational by PD-1 blockade aimed at boosting γδ T cell anti-tumor functions in FL.

Therapeutic drug monitoring of ibrutinib is based on the area under the curve of concentration vs. time (AUCIBRU) instead of trough concentration (Cmin,ss) because of a limited accumulation in plasma. Our objective was to identify a limited sampling strategy (LSS) to estimate AUCIBRU associated with Bayesian estimation. The actual AUCIBRU of 85 patients was determined by the Bayesian analysis of the full pharmacokinetic profile of ibrutinib concentrations (pre-dose T0 and 0.5, 1, 2, 4 and 6 h post-dose) and experimental AUCIBRU were derived considering combinations of one to four sampling times. The T0–1–2–4 design was the most accurate LSS (root-mean-square error RMSE = 11.0%), and three-point strategies removing the 1 h or 2 h points (RMSE = 22.7% and 14.5%, respectively) also showed good accuracy. The correlation between the actual AUCIBRU and Cmin,ss was poor (r2 = 0.25). The joint analysis of dihydrodiol-ibrutinib metabolite concentrations did not improve the predictive performance of AUCIBRU. These results were confirmed in a prospective validation cohort (n = 27 patients). At least three samples, within the pre-dose and 4 h post-dose period, are necessary to estimate ibrutinib exposure accurately.

Alteration in the DNA replication, repair or recombination processes is a highly relevant mechanism of genomic instability. Despite genomic aberrations manifested in hematologic malignancies, such a defect as a source of biomarkers has been underexplored. Here, we investigated the prognostic value of expression of 82 genes involved in DNA replication-repair-recombination in a series of 99 patients with chronic lymphocytic leukemia without detectable 17p deletion or TP53 mutation. We found that expression of the POLN gene, encoding the specialized DNA polymerase ν (Pol ν) correlates with time to relapse after first-line therapy with fludarabine. Moreover, we found that POLN was the only gene up-regulated in primary patients’ lymphocytes when exposed in vitro to proliferative and pro-survival stimuli. By using two cell lines that were sequentially established from the same patient during the course of the disease and Pol ν knockout mouse embryonic fibroblasts, we reveal that high relative POLN expression is important for DNA synthesis and cell survival upon fludarabine treatment. These findings suggest that Pol ν could influence therapeutic resistance in chronic lymphocytic leukemia. (Patients’ samples were obtained from the CLL 2007 FMP clinical trial registered at: clinicaltrials.gov identifer: 00564512).

Findings among a cohort of 26 patients who had hematologic malignancies and hepatitis E virus (HEV) infection support that HEV can induce chronic hepatitis. However, a 3-month course of ribavirin can induce a rapid viral clearance, reducing the risk for chronic hepatitis and enabling continuation of cytotoxic treatments for underlying malignancies.

Cell survival mediated by integrin engagement has been implicated in cell adhesion-mediated drug resistance. We have recently demonstrated that the activation of glycogen synthase kinase 3 beta (GSK3beta) is a new pathway supporting the chemoresistance of leukemic cells adhered to fibronectin.We show here that in conditions of serum starvation, the fibronectin receptor alpha(5)beta(1) integrin, but not alpha(4)beta(1), induced activation of GSK3beta through Ser-9 dephosphorylation in adherent U937 cells. The GSK3beta-dependent survival pathway occurred in adherent leukemic cells from patients but not in the HL-60 and KG1 cell lines. In adhesion, activated GSK3beta was found in the cytosol/plasma membrane compartment and was co-immunoprecipitated with alpha(5) integrin, the phosphatase PP2A and the scaffolding protein RACK1. PP2A and its regulatory subunit B' regulated the Ser-9 phosphorylation of GSK3beta. In adherent leukemic cells, alpha(5)beta(1) integrin but not alpha(4)beta(1) upregulated the resistance to TNFalpha-induced apoptosis. Both extrinsic and intrinsic apoptotic pathways were under the control of alpha(5)beta(1) and GSK3beta.Our data show that, upon serum starvation, alpha(5)beta(1) integrin engagement could regulate specific pro-survival functions through the activation of GSK3beta.