Venetoclax resistance in acute lymphoblastic leukemia is characterized by increased mitochondrial activity and can be overcome by co-targeting oxidative phosphorylation.

Deregulated apoptosis signaling is characteristic for many cancers and contributes to leukemogenesis and treatment failure in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Apoptosis is controlled by different pro- and anti-apoptotic molecules. Inhibition of anti-apoptotic molecules like B-cell lymphoma 2 (BCL-2) has been developed as therapeutic strategy. Venetoclax (VEN), a selective BCL-2 inhibitor has shown clinical activity in different lymphoid malignancies and is currently evaluated in first clinical trials in BCP-ALL. However, insensitivity to VEN has been described constituting a major clinical concern. Here, we addressed and modeled VEN-resistance in BCP-ALL, investigated the underlying mechanisms in cell lines and patient-derived xenograft (PDX) samples and identified potential strategies to overcome VEN-insensitivity. Leukemia lines with VEN-specific resistance were generated in vitro and further characterized using RNA-seq analysis. Interestingly, gene sets annotated to the citric/tricarboxylic acid cycle and the respiratory electron transport chain were significantly enriched and upregulated, indicating increased mitochondrial metabolism in VEN-resistant ALL. Metabolic profiling showed sustained high mitochondrial metabolism in VEN-resistant lines as compared to control lines. Accordingly, primary PDX-ALL samples with intrinsic VEN-insensitivity showed higher oxygen consumption and ATP production rates, further highlighting that increased mitochondrial activity is a characteristic feature of VEN-resistant ALL. VEN-resistant PDX-ALL showed significant higher mitochondrial DNA content and differed in mitochondria morphology with significantly larger and elongated structures, further corroborating our finding of augmented mitochondrial metabolism upon VEN-resistance. Using Oligomycin, an inhibitor of the complex V/ATPase subunit, we found synergistic activity and apoptosis induction in VEN-resistant BCP-ALL cell lines and PDX samples, demonstrating that acquired and intrinsic VEN-insensitivity can be overcome by co-targeting BCL-2 and the OxPhos pathway. These findings of reprogrammed, high mitochondrial metabolism in VEN-resistance and synergistic activity upon co-targeting BCL-2 and oxidative phosphorylation strongly suggest further preclinical and potential clinical evaluation in VEN-resistant BCP-ALL.

Genetic alterations in chronic lymphocytic leukemia and plasma cell neoplasms - a practical guide to WHO HAEM5.

The 5th edition of the World Health Organization Classification of Haematolymphoid Tumours (WHO-HAEM5) provides a revised classification of lymphoid malignancies including chronic lymphocytic leukemia (CLL) and plasma cell myeloma/multiple myeloma (PCM/MM). For both diseases the descriptions of precursor states such as monoclonal B-cell lymphocytosis and monoclonal gammopathy of uncertain significance (MGUS) have been updated including a better risk stratification model. New insights on mutational landscapes and branching evolutionary pattern were embedded as diagnostic and prognostic factors, accompanied by a revised structure for the chapter of plasma cell neoplasms. Thus, the WHO-HAEM5 leads to practical improvements of biological and clinical relevance for pathologists, clinicians, geneticists and scientists in the field of lymphoid malignancies. The present review gives an overview on the landscape of genetic alterations in CLL and plasma cell neoplasms with a focus on their impact on classification and treatment.

First-line venetoclax combinations versus chemoimmunotherapy in fit patients with chronic lymphocytic leukaemia (GAIA/CLL13): 4-year follow-up from a multicentre, open-label, randomised, phase 3 trial.

BACKGROUND: In the primary analysis report of the GAIA/CLL13 trial, we found that venetoclax-obinutuzumab and venetoclax-obinutuzumab-ibrutinib improved undetectable measurable residual disease (MRD) rates and progression-free survival compared with chemoimmunotherapy in patients with previously untreated chronic lymphocytic leukaemia. However, to our knowledge, no data on direct comparisons of different venetoclax-based combinations are available. METHODS: GAIA/CLL13 is an open-label, randomised, phase 3 study conducted at 159 sites in ten countries in Europe and the Middle East. Eligible patients were aged 18 years or older, with a life expectancy of at least 6 months, an Eastern Cooperative Oncology group performance status of 0-2, a cumulative illness rating scale score of 6 or lower or a single score of 4 or lower, and no TP53 aberrations. Patients were randomly assigned (1:1:1:1), with a computer-generated list stratified by age, Binet stage, and regional study group, to either chemoimmunotherapy, venetoclax-rituximab, venetoclax-obinutuzumab, or venetoclax-obinutuzumab-ibrutinib. All treatments were administered in 28-day cycles. Patients in the chemoimmunotherapy group received six cycles of treatment, with patients older than 65 years receiving intravenous bendamustine (90 mg/m2, days 1-2), whereas patients aged 65 years or younger received intravenous fludarabine (25 mg/m2, days 1-3) and intravenous cyclophosphamide (250 mg/m2, days 1-3). Intravenous rituximab (375 mg/m2, day 1 of cycle 1; 500 mg/m2, day 1 of cycles 2-6) was added to chemotherapy. In the experimental groups, patients received daily venetoclax (400 mg orally) for ten cycles after a 5-week ramp-up phase starting on day 22 of cycle 1. In the venetoclax-rituximab group, intravenous rituximab (375 mg/m2, day 1 of cycle 1; 500 mg/m2, day 1 of cycles 2-6) was added. In the obinutuzumab-containing groups, obinutuzumab was added (cycle 1: 100 mg on day 1, 900 mg on day 2, and 1000 mg on days 8 and 15; cycles 2-6: 1000 mg on day 1). In the venetoclax-obinutuzumab-ibrutinib group, daily ibrutinib (420 mg orally, from day 1 of cycle 1) was added until undetectable MRD was reached in two consecutive measurements (3 months apart) or until cycle 36. The planned treatment duration was six cycles in the chemoimmunotherapy group, 12 cycles in the venetoclax-rituximab and the venetoclax-obinutuzumab group and between 12 and 36 cycles in the venetoclax-obinutuzumab-ibrutinib group. Coprimary endpoints were the undetectable MRD rate in peripheral blood at month 15 for the comparison of venetoclax-obinutuzumab versus standard chemoimmunotherapy and investigator-assessed progression-free survival for the comparison of venetoclax-obinutuzumab-ibrutinib versus standard chemoimmunotherapy, both analysed in the intention-to-treat population (ie, all patients randomly assigned to treatment) with a split α of 0·025 for each coprimary endpoint. Both coprimary endpoints have been reported elsewhere. Here we report a post-hoc exploratory analysis of updated progression-free survival results after a 4-year follow-up of our study population. Safety analyses included all patients who received at least one dose of study treatment. This study is registered with ClinicalTrials.gov, NCT02950051, recruitment is complete, and all patients are off study treatment. FINDINGS: Between Dec 13, 2016, and Oct 13, 2019, 1080 patients were screened and 926 were randomly assigned to treatment (chemoimmunotherapy group n=229; venetoclax-rituximab group n=237; venetoclax-obinutuzumab group n=229; and venetoclax-obinutuzumab-ibrutinib group n=231); mean age 60·8 years (SD 10·2), 259 (28%) of 926 patients were female, and 667 (72%) were male (data on race and ethnicity are not reported). At data cutoff for this exploratory follow-up analysis (Jan 31, 2023; median follow-up 50·7 months [IQR 44·6-57·9]), patients in the venetoclax-obinutuzumab group had significantly longer progression-free survival than those in the chemoimmunotherapy group (hazard ratio [HR] 0·47 [97·5% CI 0·32-0·69], p<0·0001) and the venetoclax-rituximab group (0·57 [0·38-0·84], p=0·0011). The venetoclax-obinutuzumab-ibrutinib group also had a significantly longer progression-free survival than the chemoimmunotherapy group (0·30 [0·19-0·47]; p<0·0001) and the venetoclax-rituximab group (0·38 [0·24-0·59]; p<0·0001). There was no difference in progression-free survival between the venetoclax-obinutuzumab-ibrutinib and venetoclax-obinutuzumab groups (0·63 [0·39-1·02]; p=0·031), and the proportional hazards assumption was not met for the comparison between the venetoclax-rituximab group versus the chemoimmunotherapy group (log-rank p=0·10). The estimated 4-year progression-free survival rate was 85·5% (97·5% CI 79·9-91·1; 37 [16%] events) in the venetoclax-obinutuzumab-ibrutinib group, 81·8% (75·8-87·8; 55 [24%] events) in the venetoclax-obinutuzumab group, 70·1% (63·0-77·3; 84 [35%] events) in the venetoclax-rituximab group, and 62·0% (54·4-69·7; 90 [39%] events) in the chemoimmunotherapy group. The most common grade 3 or worse treatment-related adverse event was neutropenia (114 [53%] of 216 patients in the chemoimmunotherapy group, 109 [46%] of 237 in the venetoclax-rituximab group, 127 [56%] of 228 in the venetoclax-obinutuzumab group, and 112 [48%] of 231 in the venetoclax-obinutuzumab-ibrutinib group). Deaths determined to be associated with study treatment by the investigator occurred in three (1%) patients in the chemoimmunotherapy group (n=1 due to each of sepsis, metastatic squamous cell carcinoma, and Richter's syndrome), none in the venetoclax-rituximab and venetoclax-obinutuzumab groups, and four (2%) in the venetoclax-obinutuzumab-ibrutinib group (n=1 due to each of acute myeloid leukaemia, fungal encephalitis, small-cell lung cancer, and toxic leukoencephalopathy). INTERPRETATION: With more than 4 years of follow-up, venetoclax-obinutuzumab and venetoclax-obinutuzumab-ibrutinib significantly extended progression-free survival compared with both chemoimmunotherapy and venetoclax-rituximab in previously untreated, fit patients with chronic lymphocytic leukaemia, thereby supporting their use and further evaluation in this patient group, while still considering the higher toxicities observed with the triple combination. FUNDING: AbbVie, Janssen, and F Hoffmann-La Roche.

ERIC recommendations for TP53 mutation analysis in chronic lymphocytic leukemia-2024 update.

In chronic lymphocytic leukemia (CLL), analysis of TP53 aberrations (deletion and/or mutation) is a crucial part of treatment decision-making algorithms. Technological and treatment advances have resulted in the need for an update of the last recommendations for TP53 analysis in CLL, published by ERIC, the European Research Initiative on CLL, in 2018. Based on the current knowledge of the relevance of low-burden TP53-mutated clones, a specific variant allele frequency (VAF) cut-off for reporting TP53 mutations is no longer recommended, but instead, the need for thorough method validation by the reporting laboratory is emphasized. The result of TP53 analyses should always be interpreted within the context of available laboratory and clinical information, treatment indication, and therapeutic options. Methodological aspects of introducing next-generation sequencing (NGS) in routine practice are discussed with a focus on reliable detection of low-burden clones. Furthermore, potential interpretation challenges are presented, and a simplified algorithm for the classification of TP53 variants in CLL is provided, representing a consensus based on previously published guidelines. Finally, the reporting requirements are highlighted, including a template for clinical reports of TP53 aberrations. These recommendations are intended to assist diagnosticians in the correct assessment of TP53 mutation status, but also physicians in the appropriate understanding of the lab reports, thus decreasing the risk of misinterpretation and incorrect management of patients in routine practice whilst also leading to improved stratification of patients with CLL in clinical trials.

Fludarabine, cyclophosphamide, and rituximab as first-line treatment in patients with chronic lymphocytic leukemia: A long-term analysis of the German CLL Study Group (GCLLSG) registry.

Long-term data of chronic lymphocytic leukemia (CLL) patients with favorable risk who were treated with fludarabine, cyclophosphamide, and rituximab (FCR) within clinical trials show good efficacy. We here report long-term data collected within the GCLLSG registry. Altogether, 417 CLL patients who received first-line treatment with FCR were analyzed, of which 293 (70.3%) were treated outside of clinical trials. The median observation time from first-line was 95.8 (interquartile range 58.7-126.8) months. Focusing on data of 194 (46.5%) patients who received FCR first-line treatment after 2013 (start of data collection within GCLLSG registry), responses were documented in 85% of the patients, non-responses in 15%, and for 3.6% the assessment was missing. Median event-free survival (EFS, time until disease progression, subsequent treatment, or death) was 60.2 months with a 5-year EFS-rate of 50.6%. Patients with higher-risk disease, characterized by unmutated IGHV (N = 78), had a median EFS of 45.4 months with a 5-year EFS rate of 36.3%, while the median EFS was 77.5 months with a 5-year EFS rate of 60.3% in patients with mutated IGHV (N = 40). Median overall survival was not reached with a 5-year survival rate of 92.7%. In summary, first-line FCR was associated with long EFS, especially in patients exhibiting a mutated IGHV status.

Acalabrutinib, venetoclax and obinutuzumab in relapsed CLL: Final efficacy and ctDNA analysis of the CLL2-BAAG trial.

The phase 2 CLL2-BAAG trial tested the measurable residual disease (MRD)-guided triple combination of acalabrutinib, venetoclax and obinutuzumab after an optional bendamustine debulking in 45 patients with relapsed/refractory CLL (one patient was excluded from the analysis due to a violation of exclusion criteria). MRD was measured by flow cytometry (FCM, undetectable MRD <10-4) in peripheral blood (PB) and circulating tumor DNA (ctDNA) by digital droplet PCR (ddPCR) of variable-diversity-joining (VDJ) rearrangements and CLL-related mutations in plasma. MRD recurrence was defined as detectable ctDNA and/or MRD ≥10-4 after achieving both uMRD/undetectable ctDNA. The median number of previous treatments was 1 (range 1-4), 18 patients (40%) had received a BTK inhibitor (BTKi) and/or venetoclax prior to inclusion, 14/44 (31.8%) had TP53 aberrations, 34 (75.6%) had unmutated IGHV. With a median observation time of 36.3 months and all patients off treatment for a median of 21.9 months, uMRD <10-4 in PB was achieved in 42/45 patients (93.3%) at any time point, including 17/18 (94.4%) previously exposed to venetoclax/BTKi and 13/14 (92.9%) with TP53 aberrations. The estimated three-year progression-free and overall survival rates were 85.0% and 93.8%. Overall 585 paired FCM/ctDNA samples were analyzed and 18 MRD recurrences (5 with and 13 without clinical progression) occurred after the end of treatment. Twelve were first detected by ctDNA, three by FCM and three synchronously. Patients with earlier detection by ctDNA appeared to have genetically higher risk disease. In conclusion, time-limited MRD-guided acalabrutinib, venetoclax and obinutuzumab achieved deep remissions in almost all patients with relapsed/refractory CLL. The addition of ctDNA-based analyses to FCM MRD assessment seems to improve early detection of relapses. ClinicalTrials.gov Identifier: NCT03787264.

Reassessing the chronic lymphocytic leukemia International Prognostic Index in the era of targeted therapies.

ABSTRACT: We evaluated the chronic lymphocytic leukemia International Prognostic Index (CLL-IPI) in patients with CLL treated first line with targeted drugs (n = 991) or chemoimmunotherapy (n = 1256). With a median observation time of 40.5 months, the 3-year progression-free survival (PFS) rates for targeted drug-treated patients varied by CLL-IPI risk group: 96.5% (low), 87.6% (intermediate), 82.4% (high), and 78.7% (very high). Differences between consecutive CLL-IPI risk groups were observed for intermediate vs low and high vs intermediate, but not very high vs high. CLL-IPI factors ß2-microglobulin, immunoglobulin heavy variable (IGHV) status, and TP53 status each retained prognostic value for PFS. The 3-year overall survival (OS) rates by CLL-IPI risk groups were 100%, 96%, 93.9%, and 89.4%, respectively, with no differences between consecutive risk groups. Age, Binet stage, ß2-microglobulin, and TP53 status each retained prognostic value for OS. In chemoimmunotherapy patients (median observation time, 66.9 months), 3-year PFS rates for CLL-IPI risk groups were 78.1%, 51.4%, 40.1%, and 16.5%, respectively; corresponding 3-year OS rates were 97.4%, 93.1%, 81.8%, and 57.3%. In a matched-pair analysis, PFS differences in targeted therapies (n = 812) vs chemoimmunotherapy (n = 812) across all risk groups and OS differences in all but patients at low risk were demonstrated. The CLL-IPI maintains its prognostic value in predicting PFS outcomes with targeted drugs, but its impact in predicting survival appears diminished. Targeted therapies showed enhanced outcomes over chemoimmunotherapy, highlighting their effectiveness across various risk groups. Our findings support ongoing assessment of prognostic tools in CLL treatment evolution. These trials were registered at www.ClinicalTrials.gov as #NCT02345863, #NCT02401503, #NCT02689141, #NCT02445131, #NCT02758665, #NCT02950051, #NCT02242942, #NCT00262782, #NCT00281918, and #NCT01010061.

Six-year follow-up and subgroup analyses of a phase 2 trial of venetoclax for del(17p) chronic lymphocytic leukemia.

ABSTRACT: Chromosome 17p deletion (del[17p]) is associated with poor prognosis in patients with chronic lymphocytic leukemia (CLL). Venetoclax is approved for treatment of previously untreated and relapsed/refractory (R/R) CLL, including patients with del(17p), based on the open-label, multicenter, phase 2 M13-982 trial (NCT01889186). Here, we detail the 6-year follow-up analysis for M13-982. A total of 158 patients with previously untreated (n = 5) or R/R (n = 153) del(17p) CLL received 400 mg venetoclax daily after initial ramp-up until progressive disease. After a median follow-up of 70 months, the best objective response rate (ORR) was 77% (21% complete remission [CR] and 49% partial remission [PR]), with a median duration of response (DOR) of 39.3 months (95% confidence interval [CI], 31.1-50.5). The median progression-free survival (PFS) was 28.2 months (95% CI, 23.4-37.6), and median overall survival (OS) was 62.5 months (95% CI, 51.7-not reached), with 16% of patients remaining on treatment after 6 years. Multivariable analysis did not identify statistically significant correlation between patient subgroups defined by clinical or laboratory variables and ORR or PFS. The most common grade ≥3 adverse events were neutropenia (42%), infections (33%), anemia (16%), and thrombocytopenia (16%). Post hoc comparative analyses of PFS and OS from treatment initiation, from a 24-month landmark, and by minimal residual disease status were performed between patients with del(17p) in the M13-982 and MURANO studies in the interest of understanding these data in another context. These long-term data show the continued benefits of venetoclax in patients with del(17p) CLL. The trial was registered at www.clinicaltrials.gov as #NCT01889186.

Tislelizumab plus zanubrutinib for Richter transformation: the phase 2 RT1 trial.

In patients with chronic lymphocytic leukemia, Richter transformation (RT) reflects the development of an aggressive lymphoma that is associated with poor response to chemotherapy and short survival. We initiated an international, investigator-initiated, prospective, open-label phase 2 study in which patients with RT received a combination of the PD-1 inhibitor tislelizumab plus the BTK inhibitor zanubrutinib for 12 cycles. Patients responding to treatment underwent maintenance treatment with both agents. The primary end point was overall response rate after six cycles. Of 59 enrolled patients, 48 patients received at least two cycles of treatment and comprised the analysis population according to the study protocol. The median observation time was 13.9 months, the median age was 67 (range 45-82) years. Ten patients (20.8%) had received previous RT-directed therapy. In total, 28 out of 48 patients responded to induction therapy with an overall response rate of 58.3% (95% confidence interval (CI) 43.2-72.4), including 9 (18.8%) complete reponse and 19 (39.6%) partial response, meeting the study's primary end point by rejecting the predefined null hypothesis of 40% (P = 0.008). Secondary end points included duration of response, progression-free survival and overall survival. The median duration of response was not reached, the median progression-free survival was 10.0 months (95% CI 3.8-16.3). Median overall survival was not reached with a 12-month overall survival rate of 74.7% (95% CI 58.4-91.0). The most common adverse events were infections (18.0%), gastrointestinal disorders (13.0%) and hematological toxicities (11.4%). These data suggest that combined checkpoint and BTK inhibition by tislelizumab plus zanubrutinib is an effective and well-tolerated treatment strategy for patients with RT. ClinicalTrials.gov Identifier: NCT04271956 .

Final analysis of the CLL2-GIVe trial: obinutuzumab, ibrutinib, and venetoclax for untreated CLL with del(17p)/TP53mut.

The final analysis of the open-label, multicenter phase 2 CLL2-GIVe trial shows response and tolerability of the triple combination of obinutuzumab, ibrutinib, and venetoclax (GIVe regimen) in 41 previously untreated patients with high-risk chronic lymphocytic leukemia (CLL) with del(17p) and/or TP53 mutation. Induction consisted of 6 cycles of GIVe; venetoclax and ibrutinib were continued up to cycle 12 as consolidation. Ibrutinib was given until cycle 15 or up to cycle 36 in patients not achieving a complete response and with detectable minimal residual disease. The primary end point was the complete remission rate at cycle 15, which was achieved at 58.5% (95% CI, 42.1-73.7; P < .001). The last patient reached the end of the study in January 2022. After a median observation time of 38.4 months (range, 3.7-44.9), the 36-month progression-free survival was 79.9%, and the 36-month overall survival was 92.6%. Only 6 patients continued ibrutinib maintenance. Adverse events of concern were neutropenia (48.8%, grade ≥3) and infections (19.5%, grade ≥3). Cardiovascular toxicity grade 3 occurred as atrial fibrillation at a rate of 2.4% between cycles 1 and 12, as well as hypertension (4.9%) between cycles 1 and 6. The incidence of adverse events of any grade and grade ≥3 was highest during induction and decreased over time. Progressive disease was observed in 7 patients between cycles 27 and 42. In conclusion, the CLL2-GIVe regimen is a promising fixed-duration, first-line treatment for patients with high-risk CLL with a manageable safety profile.

First-Line Venetoclax Combinations in Chronic Lymphocytic Leukemia.

BACKGROUND: Randomized trials of venetoclax plus anti-CD20 antibodies as first-line treatment in fit patients (i.e., those with a low burden of coexisting conditions) with advanced chronic lymphocytic leukemia (CLL) have been lacking. METHODS: In a phase 3, open-label trial, we randomly assigned, in a 1:1:1:1 ratio, fit patients with CLL who did not have TP53 aberrations to receive six cycles of chemoimmunotherapy (fludarabine-cyclophosphamide-rituximab or bendamustine-rituximab) or 12 cycles of venetoclax-rituximab, venetoclax-obinutuzumab, or venetoclax-obinutuzumab-ibrutinib. Ibrutinib was discontinued after two consecutive measurements of undetectable minimal residual disease or could be extended. The primary end points were undetectable minimal residual disease (sensitivity, <10-4 [i.e., <1 CLL cell in 10,000 leukocytes]) as assessed by flow cytometry in peripheral blood at month 15 and progression-free survival. RESULTS: A total of 926 patients were assigned to one of the four treatment regimens (229 to chemoimmunotherapy, 237 to venetoclax-rituximab, 229 to venetoclax-obinutuzumab, and 231 to venetoclax-obinutuzumab-ibrutinib). At month 15, the percentage of patients with undetectable minimal residual disease was significantly higher in the venetoclax-obinutuzumab group (86.5%; 97.5% confidence interval [CI], 80.6 to 91.1) and the venetoclax-obinutuzumab-ibrutinib group (92.2%; 97.5% CI, 87.3 to 95.7) than in the chemoimmunotherapy group (52.0%; 97.5% CI, 44.4 to 59.5; P<0.001 for both comparisons), but it was not significantly higher in the venetoclax-rituximab group (57.0%; 97.5% CI, 49.5 to 64.2; P = 0.32). Three-year progression-free survival was 90.5% in the venetoclax-obinutuzumab-ibrutinib group and 75.5% in the chemoimmunotherapy group (hazard ratio for disease progression or death, 0.32; 97.5% CI, 0.19 to 0.54; P<0.001). Progression-free survival at 3 years was also higher with venetoclax-obinutuzumab (87.7%; hazard ratio for disease progression or death, 0.42; 97.5% CI, 0.26 to 0.68; P<0.001), but not with venetoclax-rituximab (80.8%; hazard ratio, 0.79; 97.5% CI, 0.53 to 1.18; P = 0.18). Grade 3 and grade 4 infections were more common with chemoimmunotherapy (18.5%) and venetoclax-obinutuzumab-ibrutinib (21.2%) than with venetoclax-rituximab (10.5%) or venetoclax-obinutuzumab (13.2%). CONCLUSIONS: Venetoclax-obinutuzumab with or without ibrutinib was superior to chemoimmunotherapy as first-line treatment in fit patients with CLL. (Funded by AbbVie and others; GAIA-CLL13 ClinicalTrials.gov number, NCT02950051; EudraCT number, 2015-004936-36.).

High karyotypic complexity is an independent prognostic factor in patients with CLL treated with venetoclax combinations.

Complex karyotypes have been associated with inferior outcomes in chronic lymphocytic leukemia (CLL) treated with chemoimmunotherapy (CIT), whereas their prognostic impact in the context of venetoclax-based treatments is still debated. In this prospective analysis on karyotype complexity in CLL, we evaluated the impact of complex (≥3 chromosomal aberrations [CAs], CKTs) and highly complex karyotypes (≥5 CAs; hCKTs) as well as specific aberrations in previously untreated patients without TP53 aberrations undergoing either CIT or time-limited venetoclax-based therapies in the phase 3 GAIA/CLL13 trial. Karyotype analyses were available for 895 of 926 patients (96.7%), of whom 153 (17%) had a CKT and 43 (5%) hCKT. In the CIT arm, CKT was associated with shorter progression-free survival (PFS) (hazard ratio [HR] 2.58; 95% confidence interval [95% CI], 1.54-4.32; P < .001) and overall survival (HR, 3.25; 95% CI, 1.03-10.26; P = .044). In the pooled venetoclax arms, a multivariable analysis identified hCKTs (HR, 1.96; 95% CI, 1.03-3.72; P = .041), but not CKTs, as independent adverse prognosticators for PFS. The presence of translocations (unbalanced and/or balanced) was also independently associated with shorter PFSs in the venetoclax arms. CIT led to the acquisition of additional CAs (mean CAs, 2.0-3.4; from baseline to CLL progression), whereas karyotype complexity remained stable after venetoclax-based treatments (2.0, both time points). This analysis establishes highly complex karyotypes and translocations as adverse prognostic factors in the context of venetoclax-based combination treatments. The findings of this study support the incorporation of karyotyping into the standard diagnostic workup of CLL, because it identifies patients at high risk of poor treatment outcomes and thereby improves prognostication. This trial was registered at www.clinicaltrials.gov as #NCT02950051.

Transcriptomic profiles and 5-year results from the randomized CLL14 study of venetoclax plus obinutuzumab versus chlorambucil plus obinutuzumab in chronic lymphocytic leukemia.

Data on long-term outcomes and biological drivers associated with depth of remission after BCL2 inhibition by venetoclax in the treatment of chronic lymphocytic leukemia (CLL) are limited. In this open-label parallel-group phase-3 study, 432 patients with previously untreated CLL were randomized (1:1) to receive either 1-year venetoclax-obinutuzumab (Ven-Obi, 216 patients) or chlorambucil-Obi (Clb-Obi, 216 patients) therapy (NCT02242942). The primary endpoint was investigator-assessed progression-free survival (PFS); secondary endpoints included minimal residual disease (MRD) and overall survival. RNA sequencing of CD19-enriched blood was conducted for exploratory post-hoc analyses. After a median follow-up of 65.4 months, PFS is significantly superior for Ven-Obi compared to Clb-Obi (Hazard ratio [HR] 0.35 [95% CI 0.26-0.46], p < 0.0001). At 5 years after randomization, the estimated PFS rate is 62.6% after Ven-Obi and 27.0% after Clb-Obi. In both arms, MRD status at the end of therapy is associated with longer PFS. MRD + ( ≥ 10-4) status is associated with increased expression of multi-drug resistance gene ABCB1 (MDR1), whereas MRD6 (< 10-6) is associated with BCL2L11 (BIM) expression. Inflammatory response pathways are enriched in MRD+ patient solely in the Ven-Obi arm. These data indicate sustained long-term efficacy of fixed-duration Ven-Obi in patients with previously untreated CLL. The distinct transcriptomic profile of MRD+ status suggests possible biological vulnerabilities.

Evolutionary history of transformation from chronic lymphocytic leukemia to Richter syndrome.

Richter syndrome (RS) arising from chronic lymphocytic leukemia (CLL) exemplifies an aggressive malignancy that develops from an indolent neoplasm. To decipher the genetics underlying this transformation, we computationally deconvoluted admixtures of CLL and RS cells from 52 patients with RS, evaluating paired CLL-RS whole-exome sequencing data. We discovered RS-specific somatic driver mutations (including IRF2BP2, SRSF1, B2M, DNMT3A and CCND3), recurrent copy-number alterations beyond del(9p21)(CDKN2A/B), whole-genome duplication and chromothripsis, which were confirmed in 45 independent RS cases and in an external set of RS whole genomes. Through unsupervised clustering, clonally related RS was largely distinct from diffuse large B cell lymphoma. We distinguished pathways that were dysregulated in RS versus CLL, and detected clonal evolution of transformation at single-cell resolution, identifying intermediate cell states. Our study defines distinct molecular subtypes of RS and highlights cell-free DNA analysis as a potential tool for early diagnosis and monitoring.

Molecular characterization of Richter syndrome identifies de novo diffuse large B-cell lymphomas with poor prognosis.

Richter syndrome (RS) is the transformation of chronic lymphocytic leukemia (CLL) into aggressive lymphoma, most commonly diffuse large B-cell lymphoma (DLBCL). We characterize 58 primary human RS samples by genome-wide DNA methylation and whole-transcriptome profiling. Our comprehensive approach determines RS DNA methylation profile and unravels a CLL epigenetic imprint, allowing CLL-RS clonal relationship assessment without the need of the initial CLL tumor DNA. DNA methylation- and transcriptomic-based classifiers were developed, and testing on landmark DLBCL datasets identifies a poor-prognosis, activated B-cell-like DLBCL subset in 111/1772 samples. The classification robustly identifies phenotypes very similar to RS with a specific genomic profile, accounting for 4.3-8.3% of de novo DLBCLs. In this work, RS multi-omics characterization determines oncogenic mechanisms, establishes a surrogate marker for CLL-RS clonal relationship, and provides a clinically relevant classifier for a subset of primary "RS-type DLBCL" with unfavorable prognosis.

In Vivo Modeling of CLL Transformation to Richter Syndrome Reveals Convergent Evolutionary Paths and Therapeutic Vulnerabilities.

Transformation to aggressive disease histologies generates formidable clinical challenges across cancers, but biological insights remain few. We modeled the genetic heterogeneity of chronic lymphocytic leukemia (CLL) through multiplexed in vivo CRISPR-Cas9 B-cell editing of recurrent CLL loss-of-function drivers in mice and recapitulated the process of transformation from indolent CLL into large cell lymphoma [i.e., Richter syndrome (RS)]. Evolutionary trajectories of 64 mice carrying diverse combinatorial gene assortments revealed coselection of mutations in Trp53, Mga, and Chd2 and the dual impact of clonal Mga/Chd2 mutations on E2F/MYC and interferon signaling dysregulation. Comparative human and murine RS analyses demonstrated tonic PI3K signaling as a key feature of transformed disease, with constitutive activation of the AKT and S6 kinases, downmodulation of the PTEN phosphatase, and convergent activation of MYC/PI3K transcriptional programs underlying enhanced sensitivity to MYC/mTOR/PI3K inhibition. This robust experimental system presents a unique framework to study lymphoid biology and therapy. SIGNIFICANCE: Mouse models reflective of the genetic complexity and heterogeneity of human tumors remain few, including those able to recapitulate transformation to aggressive disease histologies. Herein, we model CLL transformation into RS through multiplexed in vivo gene editing, providing key insight into the pathophysiology and therapeutic vulnerabilities of transformed disease. This article is highlighted in the In This Issue feature, p. 101.