Activity of venetoclax in patients with relapsed or refractory chronic lymphocytic leukaemia: analysis of the VENICE-1 multicentre, open-label, single-arm, phase 3b trial.

BACKGROUND: Most patients with chronic lymphocytic leukaemia progress after treatment or retreatment with targeted therapy or chemoimmunotherapy and have limited subsequent treatment options. Response levels to the single-agent venetoclax in the relapsed setting is unknown. We aimed to assess venetoclax activity in patients with or without previous B-cell receptor-associated kinase inhibitor (BCRi) treatment. METHODS: This multicentre, open-label, single-arm, phase 3b trial (VENICE-1) assessed activity and safety of venetoclax monotherapy in adults with relapsed or refractory chronic lymphocytic leukaemia, stratified by previous exposure to a BCRi. Eligible participants were aged 18 years or older with previously treated relapsed or refractory chronic lymphocytic leukaemia. Presence of del(17p) or TP53 aberrations and previous BCRi treatment were permitted. Patients received 5-week ramp-up to 400 mg of oral venetoclax once daily and were treated for up to 108 weeks, with 2 years follow-up after discontinuation, or optional extended access. The primary activity endpoint was complete remission rate (complete remission or complete remission with incomplete marrow recovery) in BCRi-naive patients. Analyses used the intent-to-treat (ie, all enrolled patients, which coincided with those who received at least one dose of venetoclax). This study was registered with ClinicalTrials.gov, NCT02756611, and is complete. FINDINGS: Between June 22, 2016, and March 11, 2022, we enrolled 258 patients with relapsed or refractory chronic lymphocytic leukaemia (180 [70%] were male; 252 [98%] were White; 191 were BCRi-naive and 67 were BCRi-pretreated). Median follow-up in the overall cohort was 49·5 months (IQR 47·2-54·1), 49·2 months (47·2-53·2) in the BCRi-naive group, and 49·7 months (47·4-54·3) in the BCRi-pretreated group. Of 191 BCRi-naive patients, 66 (35%; 95% CI 27·8-41·8) had complete remission or complete remission with incomplete marrow recovery. 18 (27%; 95% CI 16·8-39·1) of 67 patients in the BCRi-pretreated group had complete remission or complete remission with incomplete marrow recovery. Grade 3 or worse treatment-emergent adverse events were reported in 203 (79%) and serious adverse events were reported in 136 (53%) of 258 patients in the overall cohort. The most common treatment-emergent adverse event was neutropenia (96 [37%]) and the most common and serious adverse event was pneumonia (21 [8%]). There were 13 (5%) deaths reported due to adverse events; one of these deaths (autoimmune haemolytic anaemia) was possibly related to venetoclax. No new safety signals were identified. INTERPRETATION: These data demonstrate deep and durable responses with venetoclax monotherapy in patients with relapsed or refractory chronic lymphocytic leukaemia, including BCRi-pretreated patients, suggesting that venetoclax monotherapy is an effective strategy for treating BCRi-naive and BCRi-pretreated patients. FUNDING: AbbVie.

Collaborative cytometric inter-laboratory ring test for probiotics quantification.

Introduction: Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. From this definition, accurate enumeration of probiotic products is a necessity. Nonetheless, this definition does not specify the methods for assessing such viability. Colony forming units is the de facto gold standard for enumerating viable in probiotic products. The notion of microbial viability has been anchored in the concept of cultivability, which refers to a cell's capacity to replicate and form colonies on agar media. However, there is a growing consensus that the term "viability" should not be exclusively tied to the ability to cultivate cells. For example, bacterial cells can exist in a Viable But Non-Culturable (VBNC) state, characterized by the maintenance of characteristics such as membrane integrity, enzymatic activity, pH gradients, and elevated levels of rRNA, despite losing the ability to form colonies. Methods: Herein we present the results of a collaborative inter-laboratory ring test for cytometric bacterial quantification. Specifically, membrane integrity fluorescence flow cytometry (FFC) method and the newer impedance flow cytometry (IFC) method have been used. Both methods interrogate single cells in solution for the presence of intact membranes. FFC exploits fluorochromes that reflect the presence or absence of an intact membrane. IFC probes membrane integrity in a label-free approach by detecting membrane-induced hindrances to the propagation of electricity. Results: A performance ring-test and comparison design on the FFC method showed that the method is robust against the exchange of equipment, procedures, materials, and operators. After initial method optimization with assessments of rehydration medium, wake-up duration, and phase shift gating on the individual strains, the IFC method showed good agreement with the FFC results. Specifically, we tested 6 distinct species of probiotic bacteria (3 Lactobacillus and 3 Bifidobacterium strains) finding good agreement between FFC and IFC results in terms of total and live cells. Discussion: Together, these results demonstrate that flow cytometry is a reliable, precise, and user-friendly culture-independent method for bacterial enumeration.

High prognostic value of measurable residual disease detection by flow cytometry in chronic lymphocytic leukemia patients treated with front-line fludarabine, cyclophosphamide, and rituximab, followed by three years of rituximab maintenance.

It has been postulated that monitoring measurable residual disease (MRD) could be used as a surrogate marker of progression-free survival (PFS) in chronic lymphocytic leukemia (CLL) patients after treatment with immunochemotherapy regimens. In this study, we analyzed the outcome of 84 patients at 3 years of follow-up after first-line treatment with fludarabine, cyclophosphamide and rituximab (FCR) induction followed by 36 months of rituximab maintenance thearpy. MRD was assessed by a quantitative four-color flow cytometry panel with a sensitivity level of 10-4 Eighty out of 84 evaluable patients (95.2%) achieved at least a partial response or better at the end of induction. After clinical evaluation, 74 patients went into rituximab maintenance and the primary endpoint was assessed in the final analysis at 3 years of follow-up. Bone marrow (BM) MRD analysis was performed after the last planned induction course and every 6 months in cases with detectable residual disease during the 36 months of maintenance therapy. Thirty-seven patients (44%) did not have detectable residual disease in the BM prior to maintenance therapy. Interestingly, 29 patients with detectable residual disease in the BM after induction no longer had detectable disease in the BM following maintenance therapy. After a median followup of 6.30 years, the median overall survival (OS) and PFS had not been reached in patients with either undetectable or detectable residual disease in the BM, who had achieved a complete response at the time of starting maintenance therapy. Interestingly, univariate analysis showed that after rituximab maintenance OS was not affected by IGHV status (mutated vs unmutated OS: 85.7% alive at 7.2 years vs 79.6% alive at 7.3 years, respectively). As per protocol, 15 patients (17.8%), who achieved a complete response and undetectable peripheral blood and BM residual disease after four courses of induction, were allowed to stop fludarabine and cyclophosphamide and complete two additional courses of rituximab and continue with maintenance therapy for 18 cycles. Surprisingly, the outcome in this population was similar to that observed in patients who received the full six cycles of the induction regimen. These data show that, compared to historic controls, patients treated with FCR followed by rituximab maintenance have high-quality responses with fewer relapses and improved OS. The tolerability of this regime is favorable. Furthermore, attaining an early undetectable residual disease status could shorten the duration of chemoimmunotherapy, reducing toxicities and preventing long-term side effects. The analysis of BM MRD after fludarabine-based induction could be a powerful predictor of post-maintenance outcomes in patients with CLL undergoing rituximab maintenance and could be a valuable tool to identify patients at high risk of relapse, influencing further treatment strategies. This trial is registered with EudraCT n. 2007-002733-36 and ClinicalTrials.gov Identifier: NCT00545714.

Safety and efficacy of obinutuzumab with CHOP or bendamustine in previously untreated follicular lymphoma.

The GAUDI study assessed safety and preliminary efficacy of induction therapy with obinutuzumab plus chemotherapy, followed by maintenance therapy with obinutuzumab alone, in previously untreated patients with follicular lymphoma. Assignment to chemotherapy was decided on a per-center basis before the patients' enrollment. Patients (n=81) received four to six cycles of obinutuzumab plus bendamustine every 4 weeks or six to eight cycles of obinutuzumab plus CHOP every 3 weeks. Patients with an end-of-treatment response were eligible for obinutuzumab maintenance therapy every 3 months for 2 years or until disease progression. Induction treatment was completed by 90% of patients in the obinutuzumab plus bendamustine group and 95% in the obinutuzumab plus CHOP group, while maintenance was completed by 81% and 72% of patients, respectively. All patients experienced at least one adverse event during induction, most commonly infusion-related reactions (58%), the majority of which were grade 1/2. The most common hematologic adverse event was grade 3/4 neutropenia (36% during induction and 7% during maintenance). One treatment-related death occurred during the maintenance phase. At the end of induction, 94% of patients had achieved an overall response, with complete response based on computed tomography in 36%. The progression-free survival rate at 36 months was 90% in the obinutuzumab plus bendamustine group and 84% in the obinutuzumab plus CHOP group. These results demonstrate that induction therapy with obinutuzumab plus bendamustine or obinutuzumab plus CHOP, followed by obinutuzumab maintenance, is associated with tolerable safety and promising efficacy. This study is registered at ClinicalTrials.gov as NCT00825149.

Chronic lymphocytic leukemia: a clinical and molecular heterogenous disease.

The clinical heterogeneity that characterizes chronic lymphocytic leukemia (CLL), with survival times ranging from months to decades, reflects its biological diversity. Our understanding of the biology of CLL has helped us identify several markers of prognostic significance, by which CLL can be differentiated into several distinct diseases. The presence of specific chromosomal abnormalities is a prognostic indicator of disease progression and survival. Conventional cytogenetic analyses have revealed chromosomal aberrations in 40-50% of patients, but the detection of abnormalities is limited by the low mitotic activity of CLL cells. Metaphase analysis has recently undergone a "revival" because the metaphase yield has been improved by stimulation of CLL cells with alternative methods. Fluorescence in situ hybridization identifies chromosomal changes in approximately 80% of patients with CLL, and comparative genomic hybridization using high-density arrays (i.e., array comparative genomic hybridization [aCGH]) enables high-resolution genome-wide scanning for detecting copy number alterations in a single hybridization. The mutational status of the immunoglobulin heavy chain variable (IGHV) genes identifies two subsets of CLL with different outcomes. Unfortunately, the determination of IGHV mutation status may not be practical in all laboratories, and for this reason characteristics that are correlated with IGHV mutation status are needed-zeta-chain associated (TCR) protein kinase 70 kDa (ZAP-70) being that most commonly used currently in routine clinical practice. Whole genome sequencing has offered new insights into the mutational status of the disease, highlighting the role of several genes previously unrelated to CLL. Of these, NOTCH1 and SF3B1 are the most frequently mutated genes that predict poor prognosis. MicroRNA alterations are also involved in the initiation and progression of CLL, and the expression levels of some microRNAs correlate with previously established prognostic markers such as IGHV mutation status or ZAP-70. In addition, both global and gene-specific aberrant DNA methylation have been observed in CLL. Aberrant methylation has been described for genes that are specifically deregulated in CLL, such as BCL2, TCL1, and ZAP-70. Expanding knowledge of aberrant methylation profiles in CLL has a potential future impact on diagnosis, prognosis, and prediction of treatment response in CLL patients.

DNA methylation dynamics in blood after hematopoietic cell transplant.

Epigenetic deregulation is considered a common hallmark of cancer. Nevertheless, recent publications have demonstrated its association with a large array of human diseases. Here, we explore the DNA methylation dynamics in blood samples during hematopoietic cell transplant and how they are affected by pathophysiological events during transplant evolution. We analyzed global DNA methylation in a cohort of 47 patients with allogenic transplant up to 12 months post-transplant. Recipients stably maintained the donor's global methylation levels after transplant. Nonetheless, global methylation is affected by chimerism status. Methylation analysis of promoters revealed that methylation in more than 200 genes is altered 1 month post-transplant when compared with non-pathological methylation levels in the donor. This number decreased by 6 months post-transplant. Finally, we analyzed methylation in IFN-γ, FASL, IL-10, and PRF1 and found association with the severity of the acute graft-versus-host disease. Our results provide strong evidence that methylation changes in blood are linked to underlying physiological events and demonstrate that DNA methylation analysis is a viable strategy for the study of transplantation and for development of biomarkers.

The use of CD138 positively selected marrow samples increases the applicability of minimal residual disease assessment by PCR in patients with multiple myeloma.

We have evaluated the use of CD138+ positively selected bone marrow samples to identify a molecular target for minimal residual disease assessment by polymerase chain reaction (PCR) in 25 untreated patients with multiple myeloma. A fraction of each sample was used for CD138+ selection, and the rest served as a reference control. VDJH, DJH, and Kde gene rearrangements were tested for amplification according to the BIOMED-2 Concerted Action. PCR products were directly sequenced in an automated ABI 3130 DNA sequencer using Big-Dye terminators. Within the CD138+ selected group, VDJH rearrangements were detected in all cases (100 %), DJH in 16 (64 %), and Kde in 18 (72 %) cases; whereas in the control samples, VDJH, DJH, and Kde rearrangements were detected in 19 (76 %), 11 (44 %), and 12 (48 %) cases, respectively. After sequencing, 24 (96 %) cases within the CD138+ group had a PCR target for MRD detection compared with 15 (60 %) cases in the control group. We conclude that the use of CD138+ positively selected bone marrow samples increases the applicability of minimal residual disease studies by PCR in patients with multiple myeloma.

Common infectious agents and monoclonal B-cell lymphocytosis: a cross-sectional epidemiological study among healthy adults.

BACKGROUND: Risk factors associated with monoclonal B-cell lymphocytosis (MBL), a potential precursor of chronic lymphocytic leukaemia (CLL), remain unknown. METHODS: Using a cross-sectional study design, we investigated demographic, medical and behavioural risk factors associated with MBL. "Low-count" MBL (cases) were defined as individuals with very low median absolute count of clonal B-cells, identified from screening of healthy individuals and the remainder classified as controls. 452 individuals completed a questionnaire with their general practitioner, both blind to the MBL status of the subject. Odds ratios (OR) and 95% confidence interval (CI) for MBL were estimated by means of unconditional logistic regression adjusted for confounding factors. RESULTS: MBL were detected in 72/452 subjects (16%). Increasing age was strongly associated with MBL (P-trend<0.001). MBL was significantly less common among individuals vaccinated against pneumococcal or influenza (OR 0.49, 95% confidence interval (CI): 0.25 to 0.95; P-value=0.03 and OR: 0.52, 95% CI: 0.29 to 0.93, P-value=0.03, respectively). Albeit based on small numbers, cases were more likely to report infectious diseases among their children, respiratory disease among their siblings and personal history of pneumonia and meningitis. No other distinguishing epidemiological features were identified except for family history of cancer and an inverse relationship with diabetes treatment. All associations described above were retained after restricting the analysis to CLL-like MBL. CONCLUSION: Overall, these findings suggest that exposure to infectious agents leading to serious clinical manifestations in the patient or its surroundings may trigger immune events leading to MBL. This exploratory study provides initial insights and directions for future research related to MBL, a potential precursor of chronic lymphocytic leukaemia. Further work is warranted to confirm these findings.