Early Stage Chronic Lymphocytic Leukemia Research Articles

Immunoglobulin light chain mutational status refines IGHV prognostic value in identifying chronic lymphocytic leukemia patients with early treatment requirement.

The mutational status of immunoglobulin (IG) light chain genes in chronic lymphocytic leukemia (CLL) and its clinical impact have not been extensively studied. To assess their prognostic significance, the IG light chain gene repertoire in CLL patients has been evaluated using a training-validation approach. In the training cohort (N = 573 CLL), 92.5% showed productive IG light chain genes rearrangements, with IGKV4-1 (20.5%) and IGLV3-21 (19.0%) being the most common. A 99.0% somatic hypermutation cut-off was identified as the best predictor for time to first treatment (TTFT) in 414 Binet A CLL patients of the training cohort. Patients with unmutated (UM) light chain genes displayed a 10-year treatment free probability of 32.4% versus 73.2% for those with mutated (M) genes (p < 0.0001). Importantly, UM light chain genes maintained an independent association with a shorter TTFT when adjusted for the IPS-E prognostic model variables, that also includes IGHV mutational status. The validation cohort of 343 Rai 0 patients confirmed these findings, with UM light chain genes predicting a 7-year treatment free probability of 42.0% versus 73.7% for M genes (p < 0.0001). These results indicate that the mutational status of the light chain genes is an independent predictor of shorter TTFT in early-stage CLL patients.

Ibrutinib in Early-Stage Chronic Lymphocytic Leukemia: The Randomized, Placebo-Controlled, Double-Blind, Phase III CLL12 Trial.

The CLL12 trial reassesses the watch-and-wait consensus for early-stage chronic lymphocytic leukemia (CLL) in the context of targeted therapies. The German CLL Study Group conducted a randomized, double-blind, placebo-controlled phase III trial with 363 patients with asymptomatic, treatment-naïve Binet stage A CLL at increased risk of progression to receive ibrutinib (n = 182) at a daily dose of 420 mg or placebo (n = 181). Additionally, 152 low-risk patients were allocated to the watch-and-wait group. The final analysis included event-free survival, progression-free survival, time to next treatment, overall survival, and safety assessments. Ibrutinib significantly delayed progression to symptomatic disease (P < .001; hazard ratio, 0.276 [95% CI, 0.188 to 0.407]), but no survival benefit was observed with 26 death cases (P = .562) at a median observation time of 69.3 months. Five-year survival rates were excellent: 93.3% (95% CI, 89.3 to 97.3) in the ibrutinib group, 93.6% (95% CI, 89.5 to 97.7) in the placebo group, and 97.9% (95% CI, 95.6 to 100) in the watch-and-wait cohort. Estimated 10-year survival rates from diagnosis were 86.5% (95% CI, 78.7 to 94.3, placebo), 89.8% (95% CI, 83.3 to 96.3, ibrutinib), and 95.3% (95% CI, 91.1 to 99.4, watch and wait). In the ibrutinib group, one of 12 deaths was CLL-associated, compared with four of 14 fatal cases of CLL progression or Richter transformation in the placebo group. Adverse and serious adverse events occurred in 99.4% and 60% of both treatment groups, respectively. The safety profile indicated increased cardiovascular toxicity in the ibrutinib group. Ibrutinib treatment in early-stage CLL delayed disease progression compared with placebo. However, with the given observation time and few deaths, no survival benefit was demonstrated. In the era of targeted therapies, watch and wait remains the standard of care irrespective of risk factors.

Unmutated IGHV at diagnosis in patients with early stage CLL independently predicts for shorter follow-up time to first treatment (TTFT)

The mutational status of the IGHV gene is routinely assessed in patients with chronic lymphocytic leukaemia (CLL), since it is both prognostic of clinical outcome and predictive of response to treatment. This study evaluates the IGHV mutational status, assessed in newly diagnosed CLL patients, as a stand-alone predictor of time to first treatment (TTFT). We analysed the data of 236 CLL patients, diagnosed at our centre between January 2004 and September 2020, with a minimum follow-up period of 3.0 years, Binet A-B and Rai 0-II stages. IGHV was unmutated in 38.1 % and mutated in 61.9 % of cases. The univariate analysis showed a statistically significant difference (p < 0.001) in TTFT based on unmutated (85.2 % at 14 years, 95 % CI = 63.3–94.5 %) or mutated (41.3 % at 14 years, 95 % CI = 29.5–51.8 %) and the need for treatment at 1, 3 and 5 years was of 20.0 % vs 4.1 % (p < 0.001), 42.7 % vs 11.4 % (p < 0.001) and 55.8 % vs 20.0 % (p < 0.001) in unmutated and mutated IGHV patients, respectively. Multivariate analysis confirmed that unmutated IGHV status negatively affects TTFT (p < 0.001), in addition to high-risk genomic aberration (p = 0.025), Rai stage I (p = 0.007) and II (p-value < 0.001). The difference in TTFT based on unmutated or mutated IGHV status remains statistically significant also when considering the subgroups by the genomic aberrations and Rai stages. Our findings suggest that, with the single analysis of the IGHV mutational status at CLL diagnosis, along with clinical and laboratory data, and without karyotype and TP53 data, clinicians will have prognostic and predictive indications for the first clinical treatment and appropriate follow-up of patients.

#2991 Two cases of chronic lymphocytic leukemia associated immunotactoid glomerulopathy: variable clinical presentation, close hematological collaboration

Abstract Background and Aims Immunotactoid glomerulopathy (ITG) is a very rare disease, observable in less than 0.1% of native kidney biopsy samples. It is most often associated with hematological diseases. We confirmed ITG in two cases among our 850 kidney biopsy patients in the past 9 years. In both patients, chronic lymphocytic leukemia (CLL) was the underlying cause. Method Case 1, a 56-year-old man was admitted due to severely increased proteinuria (2 g/day) with preserved renal function, while case 2, a 57-year-old-man was referred with rapidly decreasing eGFR (20 ml/min/1.73m2) with nephrotic syndrome (serum albumin level was 24 g/l). In case 1, no previous history of hematological disease was known, in case 2, CLL - that did not require treatment - was diagnosed half a year before admission. He still had no kidney involvement even 3 months after the CLL diagnosis. We performed kidney biopsy. Light microscopy revealed mesangial proliferative-atypical membranous pattern in case 1, and membranoproliferative pattern in case 2. Immunofluorescence microscopy showed IgG1-lambda deposition, electron microscopy confirmed the organised deposits, microtubular structures with a diameter of 44 nanometers in both cases. Neither monoclonal gammopathy nor systemic immune disease was confirmed. In case 2, low titer ANA positivity, low C3 and high anti-C1q antibody titer was detected, lupus nephritis was ruled out. Previously, lymphocyte count was slightly increased in case 1, CLL was confirmed after kidney biopsy. The immunoglobulin heavy chain variable region gene somatic hypermutation status was borderline in case 1, while in the other case it was unmutated. Fluorescein in situ hybridisation revealed trisomy 12 in both cases. In case 1, chemotherapy was not used due to Rai stage 0, and with appropriate antihypertensive treatment, we could preserve kidney function, and proteinuria was persistently low (below 1 g/day). In case 2, due to Rai stage 1, and progressive renal and lymph node involvement, immunochemotherapy was started (obinutuzumab-venetoclax). Three months later eGFR stabilized at 27 ml/min/1.73 m2, proteinuria decreased below 1 g/day, the C3 level normalized. Conclusion ITG can develop in the early stages of CLL, but it can also indicate CLL progression. The light microscopy histological pattern and, in parallel, the clinical appearance can be of varying severity. Immunochemotherapy resulting reduction of CLL tumor burden and cessation of complement damage, both play a role of improving renal status. Our cases also draw attention to the need for close collaboration with hematologists.

Vitamin D supplement for patients with early-stage chronic lymphocytic leukemia is associated with a longer time to first treatment

AbstractLow levels of vitamin D are associated with a shorter time to first treatment (TTFT) and inferior overall survival in patients with chronic lymphocytic leukemia (CLL). But whether vitamin D supplement affects the clinical course of patients with CLL, remains an open question. In this study, we aimed to retrospectively explore the clinical benefit of vitamin D supplement or one of its analogs, on TTFT and treatment-free survival (TFS) in a large cohort of patients with asymptomatic CLL, who were under watch-and-wait approach. Among the 3474 patients included in the study, 931 patients (26.8%) received either vitamin D supplement or its analog, for a minimum of 6 months. We found that vitamin D supplement was statistically significant for longer TTFT in the young cohort (age ≤65) and was associated with a longer TFS for all ages (P = .004). Among non–vitamin-D users, the median TFS was found to be 84 months, whereas among vitamin D supplement users the median TFS extended to 169 months. In conclusion, our long-term retrospective study demonstrates that the administration of vitamin D to patients with CLL in a watch-and-wait active surveillance is significantly associated with a longer TFS (in any age) and a longer TTFT among young patients (age ≤65). A prospective clinical trial is needed to validate results.

Predicting Stage Progression in Binet Stage a Chronic Lymphocytic Leukemia.

The variable clinical course of chronic lymphocytic leukemia (CLL) and the lack of consensus on follow-up and treatment strategies have necessitated a prognostic model for identifying high-risk patients at the time of diagnosis. We involved a retrospective analysis of demographic and clinical characteristics of 212 patients diagnosed with Binet stage A CLL and thus eligible for risk stratification by both the International Prognostic Score for Early-stage CLL (IPS-E) and the alternative IPS-E (AIPS-E). We evaluated the applicability of these prognostic indices in our young, Middle Eastern cohort (median age 59 at diagnosis). During the study period with a median follow-up of 3.5 years, 67 patients (32 %) experienced progression to first treatment and cumulative incidence of treatment was 13 % at 1 year and 28 % at 3 years after diagnosis. Sixty-nine (51 % of the 136 with a known value) patients harbored an unmutated immunoglobulin heavy chain gene (IGHV) and 21 (10 %) an 11q or 17p deletion with 11 % lacking FISH results. For each early-stage CLL prognostic index, more patients were identified as high-risk for disease progression (51 % of 124 patients evaluable for IPS-E; 42 % of 109 patients evaluable for AIPS-E) than intermediate-risk and low-risk. Multivariable models involving the IPS-E and AIPS-E components revealed that unmutated IGHV and elevated absolute lymphocyte count were significant predictors of earlier treatment requirement. Both prognostic scores were discriminative of time to first treatment (log-rank p < 0.001; c-statistics of 0.74 for IPS-E and 0.69 for AIPS-E). Although clarity on clinical behavior with regard to initiation of treatment remains elusive, IPS-E and AIPS-E are valuable tools for identifying high-risk patients.

Prognostication in chronic lymphocytic leukemia

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

IGLV3-21 R110 Is a Prognostic Marker for Early Stage CLL Patients Under Ibrutinib Treatment or Watch & Wait: Results from the Double-Blind, Randomized, Placebo-Controlled GCLLSG CLL12 Trial

INTRODUCTION: IGLV3-21 R110 is a point mutation that can confer the ability for autonomous signaling to B cell receptors (BCR) using light chains encoded by the allele IGLV3-21 ∗ 01 or IGLV3-21 ∗ 04. Chronic lymphocytic leukemia (CLL) patients with IGLV3-21 R110 have a shorter time to first treatment and shorter overall survival, independent of their IGHV mutational status. However, outcomes of CLL patients using IGLV3-21 R110 have not been evaluated in clinical trials of BTK inhibitors. METHODS: The CLL12 trial was a prospective, double-blind, randomized phase 3 trial that compared ibrutinib (ibr, n=182) vs. placebo (pcb, n=181) in previously untreated Binet A CLL patients with intermediate to very high risk, while low risk patients were assigned to watch &amp; wait (w&amp;w, n=152). The primary endpoint of the study was event-free survival (EFS) (event defined as death, symptomatic progression or initiation of CLL treatment), while overall survival (OS) was a secondary endpoint. The IGLV3-21 locus was sequenced via amplicon-based targeted next generation sequencing (tNGS) in 514 of 515 samples collected at enrolment. IGLV rearrangements and point mutations were detected with ScanIndel as well as IgCaller and the results of the genetic analyses were correlated with outcome. RESULTS: IGHV was unmutated (U-IGHV) in 148 (28.3%) patients (w&amp;w n=8, pcb n=70, ibr n=70) and 10 patients belonged to stereotyped B cell receptor (BCR) subset 2 (w&amp;w n=2, pcb n=1, ibr n=7). IGLV3-21 rearrangement was found in 34 of 514 patients (6.6%) and was more frequent in the intermediate/high risk groups (pcb n=16, ibr n=13) than in the low risk group (w&amp;w n=5). Twenty of 34 patients with IGLV3-21 rearrangement had an IGLV3-21 R110 mutation (w&amp;w n=3/5, pcb n=10/16, ibr n=7/13). For 5 patients with IGLV3-21 (3 pcb, 2 ibr) the mutational status for IGLV3-21 R110 could not be determined by tNGS. In the total cohort, IGLV3-21 R110 often co-occurred with mutations in SF3B1 (odds ratio 8.54, p &amp;lt; 0.001), but not with mutations in TP53 or any of the other frequently mutated genes in CLL. Conversely, SF3B1 mutations were not enriched in subset 2 patients (p = 0.165). Also, there was no association of IGLV3-21 R110 or IGLV3-21 usage in general with IGHV mutational status. At a median follow-up of 69.3 months, there were 166 events for EFS and 32 for OS. CLL with IGLV3-21 had a shorter EFS in the pcb (HR 1.93, CI95% 1.05-3.55, p=0.033) and ibr (HR 3.71, CI95% 1.62-8.46, p=0.002) arm (Figure 1A). The estimated 5-year EFS rate was 51.0% without and 21.4% with IGLV3-21 rearrangementin the pcb group vs. 80.7% and 44.8% in the ibr group, respectively. Similarly, IGLV3-21 R110 associated with shorter EFS with ibr (HR 3.30, CI95% 1.17-9.31, p=0.024) while no impact was observed in the pcb group (HR 1.51, CI95% 0.66-3.46, p=0.332, Figure 1B). Patients with IGLV3-21 R110 mutation and ibr treatment had an estimated 5-year EFS rate similar to that of patients in the pcb group without IGLV3-21 R110 (50.0% vs. 49.8%). In low risk patients with w&amp;w, both IGLV3-21 rearrangement and IGLV3-21 R110 were adverse markers for EFS (HR 4.99, CI95% 1.52-16.41, p=0.008 and HR 17.0, CI95% 4.99-58.13, p&amp;lt;0.001, respectively). For the full trial, we observed a prognostic impact of IGLV3-21 in the M-IGHV (HR 3.87, CI95% 2.14-6.99, p&amp;lt;0.001) but not in the U-IGHV (HR 1.61, CI95% 0.74-3.54, p=0.233) group. Multivariable analysis for the treatment arms including the IGLV3-21 R110 status and other variables associated significantly with EFS in univariate analyses (treatment arm, serum β2-microglobulin, IGHV status, del(17p), del(11q), +(12), mutations in XPO1, NOTCH1, SF3B1 and NFKBIE) revealed presence of IGLV3-21 R110 (HR 2.76, CI95% 1.32-5.80, p = 0.007) as an independent prognostic factor. OS was excellent in all genetic subgroups and in all arms of the trial. The estimated 5-year OS rate was between 92.9% and 95.8% for any of the 4 subgroups defined jointly by IGHV status and presence of IGLV3-21 R110. CONCLUSIONS: IGLV3-21 R110 was identified as an independent prognostic factor for shorter EFS in early stage CLL with intermediate/high risk score and was associated with reduced ibrutinib efficacy in the CLL12 trial.

Extended Follow up of a Phase 2 Study of Early Intervention with Lenalidomide in Patients with High-Risk Chronic Lymphocytic Leukemia

CONCLUSIONS Lenalidomide (LEN) is an effective therapy for chronic lymphocytic leukemia (CLL) and has immunomodulatory effects that were hypothesized to improve immune function in patients (pts) with untreated early stage CLL. A NCI/CTEP sponsored phase 2 study (NCT01351896) randomized asymptomatic pts with high-risk CLL to receive LEN either concurrent with or delayed from (3 months after 1st dose) two doses of 13-valent protein-conjugated pneumococcal vaccine (PCV13). The primary endpoint was the proportion of pts who achieve an antibody response to the 2nd PCV13 vaccine and was not significantly different between groups (Thangavadivel et al., Clinical Cancer Research 2020). Despite this, many pts experienced effective CLL disease control with LEN. Here we report long-term results from this trial to examine the impact of LEN early treatment on CLL disease course, complicating infections (INF), and second neoplasms (SN). Pts ≥18 and &amp;lt;80 years-old who had CLL with a high-risk feature, defined as FISH positive for del(17)(p13.1) and/or del(11)(q22.3), complex karyotype (≥3 abnormalities), or unmutated IGHV status, no prior treatment, and no iwCLL 2008 criteria for treatment were eligible. LEN was started at 2.5mg daily and increased to 5mg as tolerated. Data on INF and SN was determined from study adverse events and graded (gr) using CTCAE v4.0 and 5.0. Overall survival (OS) and progression-free survival (PFS) were calculated from the start of LEN and discontinuation of LEN, and analyzed using the Kaplan-Meier method. Time to next therapy (TTNT) was calculated from start or discontinuation of LEN to the initiation of next therapy treating death without new therapy as a competing risk. Data cut for analysis was 6/12/2023. A total of 49 pts were enrolled between 11/2011 and 7/2014. The median age was 59 (range 40-70) years, 69% were men, and 96% were Caucasian. In terms of CLL features, 94% (46/49) were IGHV unmutated, 47% (23/49) had complex karyotype, 14% (7/49) had del(17)(p13.1), and 31% (15/49) had del(11q)(q22.2). The median follow-up from LEN start was 4.6 (range 0.4-11.2) years and median time on LEN treatment was 3.7 (range 0.02-10.1) years with 2 pts remaining on LEN at 9.7 and 10.1 years. The median PFS after starting LEN was 5.7 (95% CI: 3.2-6.4) years with an estimated 8-year PFS of 25.9% (95% CI: 13.9%-39.6%) (Figure). Eleven pts had progressive disease (PD) at the time of LEN discontinuation and their median time from first dose of LEN to PD was 3.7 (range 0.3-6.0) years. For those who discontinued without PD, the median PFS from discontinuation was 1.5 (95% CI: 0.6-2.6) years. For those who initiated a subsequent therapy, the median TTNT from starting LEN was 4.4 (range 0.3-9.1) years and from discontinuing LEN was 1.1 (range 0.1-7.8) years. There were 7 deaths, and the median OS was not reached with an estimated 8-year OS of 73.9% (95% CI: 51.6%-87.2%). There were 217 episodes of INF that occurred in 88% (43/49) of pts. Patients experienced multiple INF: 24 (49%) pts had 1-3 INFs and 19 (39%) had ≥3 INFs with 5 of those having &amp;gt;10 INFs. The most frequent INFs were upper respiratory (n=71), sinus (n=37), skin and soft tissue (n=27), and lower respiratory (n=22) (Table). INFs were gr 1-2 in 95% of cases. Gr ≥3 INF were experienced by 18% (9/49) of patients with 10 events. There were 9 gr 3 INFs (lower respiratory tract n=4, kidney n=1, urinary tract n=1, appendix n=1, joint n=1, meninges n=1) and 1 gr 5 COVID-19 INF which occurred in a pt who discontinued LEN &amp;gt;2 years prior. The rate of gr ≥3 INF was 3.8 (95% CI: 1.8-7.0) per 100-person-years. SN were experienced by 8 pts who had 19 SNs. The most common SN were non-melanoma skin cancers (n=15). Other SN were lung adenocarcinoma (n=2 in 1 pt), melanoma in situ (n=1), and squamous cell carcinoma in a parotid lymph node from a skin SCC (n=1). The rate SN was 7.2 (95% CI 4.3-11.3) per 100-person-years. There were no cases of acute leukemia. In this population of pts with CLL with high-risk disease features predictive of need for earlier treatment, LEN resulted in a prolonged time to subsequent therapy, even in cases of LEN discontinuation. The incidence of gr ≥3 INFs and SN were as expected for this population supporting that LEN does not increase risk for these complications.

Whole Genome Classification of Pre-Malignant B-Cells Disorder of the Oxplored Study

Background. Chronic lymphocytic leukaemia (CLL) is always preceded by preclinical clonal B-cell disorders such as monoclonal B-cell lymphocytosis (MBL). Pre-malignant clonal B-cells are identifiable in asymptomatic individuals, a minority of whom will progress to symptomatic CLL. While some people remain at pre-cancerous or asymptomatic early stage CLL, others experience clinically aggressive disease. There is currently no method to effectively stratify individuals at higher risk of developing symptomatic CLL. Despite recent advances in treatment options available, the disease remains incurable. Therefore, we need to expand our knowledge and improve current classification methods. Here, we propose a molecular classifier using whole genome sequencing (WGS). This classifier was obtained by performing genomic characterization in pre-malignancy / early stages of the disease and comparing the genomic subgroups with those of symptomatic CLL. Methods. Individuals with newly diagnosed monoclonal B-cell lymphocytosis and early asymptomatic chronic lymphocytic leukaemia were enrolled in the OXPLORED clinical trial (Oxford Pre-cancerous Lymphoproliferative Disorders: Analysis and Interception study). We performed WGS in 400 samples from 200 individuals (sorted CD19+ B-cells as tumour, and matched salivary DNA as germline) to detect somatic alterations. We derived immunoglobulin heavy chain variable (IGHV) mutational status, stereotype and an additional 186 genomic features as previously described (Robbe*, Ridout* et al. Nature Genetics 2022) including known and recently discovered candidate drivers of CLL, recurrent structural variants, mutational signatures, genomic complexity measures, mutational burden, and pathway alterations. All the data was compiled into a matrix to extract meaningful sets of features to cluster patients' genomes using non-negative matrix factorization (NMF). Genomic findings were compared to the largest CLL whole genome cohort of symptomatic patients requiring frontline treatment (n=443). Results. The IGHV mutational status was found unmutated in 31% of individuals and hypermutated in 69% (excluding missing data for 6 genomes). Next, we considered mutations and CNAs in 58 common CLL drivers and 34 regions with recurrent CNAs. Although numbers were lower than in frontline CLL, most individuals presented at least one driver (86%) or more (57%) (by comparison, the CLL frontline cohort presented at least one CLL driver in 98% of patients). Genomic complexity, defined as 4 or more CNAs was detected in 12%, and complex genome defined as the presence of both CN gains and CN losses in 22%, indicating that newly diagnosed monoclonal B-cell lymphocytosis and early asymptomatic CLL present a relatively high degree of genomic complexity similar to what was observed in frontline CLL. The most common driver was del13q (60%), including as a sole driver in a third of these genomes. Other recurrent alterations were IGLL5 mutations (20%), trisomy 12 (9%), and mutations in MYD88 (8%), CREBBP (5%), NOTCH1 (5%), and SPEN (5%). Noncoding elements were also found mutated, including 30 promoters (including BIRC3 in 7%), five UTRs and eight enhancers, including BCL6 and PAX5 enhancers in 18% and 3.4% of individuals, respectively. When comparing this cohort with CLL genomes, we found that alterations in the main cancer pathways were depleted in OXPLORED, including known drivers such as ATM, NOTCH1, SF3B1. Finally, moving away from single-alteration grouping, we clustered the 643 genomes (443 CLL + 200 OXPLORED) using 186 genomic features by applying NMF. Importantly, we found distinct genomic profiles in the early disease cohort, including a subgroup like symptomatic CLL at frontline and another distinct subgroup, hinting towards identifying a genomic subgroup at higher risk of progressing to CLL. Conclusion: The comprehensive characterisation of genomic events that determine progression to malignancy are essential to understand the mechanisms of disease progression. Our study opens new horizons towards identifying individuals at higher risk and might pinpoint to individuals who need early intervention with curative intent. Therefore, we propose to analyse newly generated whole-genome sequencing (WGS) data to establish genomic subgroups in early CLL / pre-malignancy stages.

Genetic markers and ibrutinib vs placebo treatment in early stage chronic lymphocytic leukemia -results from the GCLLSG CLL12 trial

Background: Watch and wait is standard of care in patients with early stage chronic lymphocytic leukemia (CLL). In the CLL12 trial, 515 stage Binet-A patients were risk stratified and randomized 1:1 to ibrutinib (ibr, n=182) or placebo (pcb, n=181) when assigned to (very) high or intermediate risk, while low risk patients were only observed (w&amp;w, n=152). Treatment with ibr was associated with longer progression-free survival (PFS) (event defined as progression or death) and event-free survival (EFS) (event defined as death, symptomatic progression or initiation of CLL treatment) but not overall survival (OS) when compared to pcb (Langerbeins et al. EHA 2023). However, it is unclear if specific subgroups defined by genetic markers may derive particular benefit from early ibr treatment. Methods: Genetic markers were analyzed in all 515 patients at study entry. Genomic aberrations were assessed by FISH, IGHV mutational status by sequencing with a threshold of 98% homology and gene mutations via targeted NGS for TP53, FBXW7, NOTCH1, SF3B1, NRAS, KRAS, BRAF, BIRC3, MYD88, NFKBIE, EGR2 and XPO1. Results: The prevalence of genomic aberrations and gene mutations is shown in the table 1. At a median follow up of 69.3 months, there were 166 events for EFS and 32 for OS respectively. EFS was shorter with del(17p) (compared to del(13q): Hazard ratio (HR) 2.89, 95%CI 1.14-7.37, p&amp;lt;0.05), del(11q) (HR 4.39, 95%CI 2.48-7.76, p&amp;lt;0.001); +(12) (HR 1.78, 95%CI 1.01-3.13, p&amp;lt;0.05) in the pcb arm, while with ibr only del(17p) (HR 5.07, 95%CI 1.70-15.17 p&amp;lt;0.01) was associated with shorter EFS. IGHV mutation status had an impact on EFS with pcb (U-IGHV vs. M-IGHV, HR 4.44, 95%CI 2.88-6.85, p&amp;lt;0.001) but not with ibr (HR 1.52, 95%CI 0.81-2.85, p=0.19). In the pcb arm, mutations in NOTCH1 (HR 2.26, 95%CI 1.35-3.76, p&amp;lt;0.01), NRAS/KRAS/BRAF, (HR 5.38, 95%CI 1.92-15.03, p&amp;lt;0.01) and NFKBIE (HR 4.12, 95%CI 1.97-8.64, p&amp;lt;0.001) but not TP53 (HR 1.24, 95%CI 0.57-2.68, p=0.59) were significantly associated with a shorter EFS. In contrast, with ibr treatment, only TP53 (HR 2.88, 95%CI 1.31-6.32, p&amp;lt;0.01) and NFKBIE (HR 2.45, 95%CI 1.03-5.87, p=0.04) were associated with shorter EFS. Notably, in the full trial population (i.e. including the w&amp;w cohort) TP53 mutations were associated with shorter EFS only when combined with U-IGHV (HR 2.37, 95%CI 1.22-4.63, p&amp;lt;0.05) but not with M-IGHV (HR 0.53, 95%CI 0.26-1.10. p=0.09). In the w&amp;w cohort high risk markers were rare and only +(12) (compared to del(13q)(HR 4.66, 95%CI 1.4-15.51, p&amp;lt;0.05) and mutations in TP53 (HR 4.88, 95%CI 1.49-16.04, p&amp;lt;0.01), SF3B1 (HR 5.01, 95%CI 1.53-16.45, p&amp;lt;0.01), BIRC3 (HR 3.43, 95%CI 1.05-11.23, p&amp;lt;0.05) and NFKBIE (HR 12.58, 95%CI 3.68-42.99, p&amp;lt;0.001) were significantly associated with shorter EFS. Significant EFS benefit from ibr as compared to pcb with HR&amp;lt;0.2 was observed in subgroups with U-IGHV (HR 0.18, 95%CI 0.1-0.3, p&amp;lt;0.001); del(11q) (HR 0.10, 95%CI 0.04-0.29, p&amp;lt;0.001) and cases with mutations in NOTCH1 (HR 0.18, 95%CI 0.06-0.55, p&amp;lt;0.01) and NFKBIE (HR 0.15, 95%CI 0.04-0.53, p&amp;lt;0.01), while no significant benefit was observed with del(17p) (HR 0.68, 95%CI 0.16-2.85, p=0.59) and mutated TP53 (HR 0.69, 95%CI 0.25-1.92, p=0.48, figure 1). Regarding OS none of the subgroups defined by genetic markers derived significant benefit from ibr as compared to pcb. In multivariable analysis for EFS including treatment arms, clinical disease characteristics and genetic markers as variables, ibr treatment was an independent favorable factor (HR 0.25, 95%CI 0.16-0.37, p&amp;lt;0.001), while U-IGHV (HR 3.38, 95%CI 2.29-4.98 p&amp;lt;0.001), del(17p) (HR 3.53, 95%CI 1.76-7.06, p&amp;lt;0.001), +(12) (HR 1.58, 95%CI 1.01-2.45 p&amp;lt;0.05) and mutated NFKBIE (HR 2.22, 95%CI 1.23-4.01, p&amp;lt;0.01) were independent adverse prognostic factors. Conclusion: In addition to the well-known prognostic factors, also mutated SF3B1, NFKBIE and BIRC3 are associated with shorter EFS in low risk early stage CLL. While some genetic subgroups derived particular EFS benefit from ibr, patients with high-risk TP53 aberration and/or del(17p) did not. In summary, the CLL12 trial fails to demonstrate survival superiority by early ibr treatment, including genetic subgroups and w&amp;w remains the standard of care for all patients with Binet stage A CLL.

ZMYM3 Mutations Cooperate with NOTCH1 Alterations, Reduce Histone H4 Acetylation and Promote Apoptosis Evasion in Chronic Lymphocytic Leukemia

Introduction: Recent next-generation sequencing (NGS) studies have identified up to 200 recurrently mutated genes in chronic lymphocytic leukemia (CLL), the vast majority of which appear mutated in &amp;lt;5% of CLL cases. Among the long list of CLL candidate driver genes is ZMYM3, a gene mutated in 2-4% of CLL patients. Since the impact of ZMYM3 mutations is unknown, we aimed to identify their clinical and biological consequences in CLL. Methods: 487 CLL patients were analyzed by targeted NGS using a customized panel of 54 CLL-related genes to characterize the mutational profile of ZMYM3 mutated patients ( ZMYM3MUT) and the clinical impact of these variants. In addition, we introduced ZMYM3 truncating mutations into the CLL-derived HG3 cell line with two genetic backgrounds: wild-type (WT), and NOTCH1 mutated cells ( NOTCH1MUT), to generate cells with either single ZMYM3MUT, NOTCH1MUT or combined ZMYM3MUTNOTCH1MUT. In these CLL models, we evaluated the impact of these alterations in gene expression, apoptosis, growth and DNA damage response (DDR). Results: A total of 32 ZMYM3 variants were identified in 30 CLL patients, most of which were loss-of-function mutations (24/32; 75%). These variants were distributed throughout the protein sequence, with a particular recurrent frameshift mutation occurring in 6 patients (p.P48fs) that was subsequently reproduced in our CLL model. Attending to their mutational profile, we identified that 70% (21/30) of ZMYM3MUT patients harbored mutations in the NOTCH pathway, either in NOTCH1 (60%) or in negative regulators such as MED12, FBXW7 and SPEN, suggesting a cooperation between ZMYM3 dysfunction and NOTCH1 signaling mutations in CLL evolution. Interestingly, ZMYM3MUT patients exhibited significantly shorter time to first treatment (TFT) than ZMYM3WT cases (median: 35 vs 52 months; p=0.010). Furthermore, ZMYM3 mutations shortened TFT of early stage CLL patients, including Binet A (48 vs 108 months, p=0.002) or Rai 0/1 (48 vs 91 months, p=0.016), as well as in low-risk cytogenetics del(13q) patients (median: 45 vs 93 months; p=0.033). We next addressed the biological implications of ZMYM3 mutations and their co-occurrence with NOTCH1 mutations in HG3 cells. First, we performed RNA-sequencing and analyzed the number of differentially expressed genes (DEGs) (|fold change|&amp;gt;2, adjusted p-value&amp;lt;0.05). Comparing with WT cells, we detected 155 DEGs in ZMYM3MUT and 83 DEGs in NOTCH1MUT, whereas ZMYM3MUTNOTCH1MUT cells showed 690 DEGs,indicating that the combination of both mutations profoundly dysregulate gene expression. Immune regulation, apoptosis and DNA damage response were some of the biological processes dysregulated by ZMYM3 mutations (p&amp;lt;0.05). Furthermore, considering the suggested role of ZMYM3 in chromatin remodeling (Puente et al, Nature. 2015) and that ZMYM3MUT cells showed altered expression of histone acetylation related genes (p&amp;lt;0.05), we identified that ZMYM3 mutations reduced global acetylation levels of histone H4 (p&amp;lt;0.05), which may underlie the ZMYM3-related changes in gene expression. To further define the functional effects of ZMYM3 mutations, we assessed in vitro their impact in the main dysregulated pathways identified by RNA-seq. First, we identified that ZMYM3 mutations impair DNA damage response, as reflected by the difficulties to arrest cell cycle in G2/M phase after irradiation. Moreover, we detected more γH2AX foci (p&amp;lt;0.05) in ZMYM3MUTcells, indicating persistent DNA damage; and fewer RAD51 and BRCA1 foci than WT cells (p&amp;lt;0.001) after irradiation, suggesting defective DNA damage response. In parallel, we identified that ZMYM3 mutations promote apoptosis evasion, especially in the context of NOTCH1 mutations (p&amp;lt;0.001), which led to enhanced growth of ZMYM3MUTNOTCH1MUT cells (p&amp;lt;0.05). Of note, ZMYM3MUT cells showed down-expression of caspases (-8, -7 and -3) and slightly higher levels of anti-apoptotic proteins (BCL2, MCL1 and BCL-XL), which may explain this apoptosis resistance. Conclusions: Mutations in ZMYM3 are mainly loss-of-function, associate with NOTCH1 signaling mutations and shorten TFT in CLL patients, suggesting that ZMYM3 mutational status may be a useful marker in the management of early stage CLL patients. Moreover, ZMYM3 mutations reduce histone H4 acetylation and cooperate with NOTCH1 mutations to dysregulate gene-expression, leading to impair DNA damage response and apoptosis evasion.

XPO1 mutations identify early-stage CLL characterized by shorter time to first treatment and enhanced BCR signalling.

Here we evaluated the epigenomic and transcriptomic profile of XPO1 mutant chronic lymphocytic leukaemia (CLL) and their clinical phenotype. By ATAC-seq, chromatin regions that were more accessible in XPO1 mutated CLL were enriched of binding sites for transcription factors regulated by pathways emanating from the B-cell receptor (BCR), including NF-κB signalling, p38-JNK and RAS-RAF-MEK-ERK. XPO1 mutant CLL, consistent with the chromatin accessibility changes, were enriched with transcriptomic features associated with BCR and cytokine signalling. By combining epigenomic and transcriptomic data, MIR155HG, the host gene of miR-155, and MYB, the transcription factor that positively regulates MIR155HG, were upregulated by RNA-seq and their promoters were more accessible by ATAC-seq. To evaluate the clinical impact of XPO1 mutations, we investigated a total of 957 early-stage CLL subdivided into 3 independent cohorts (N = 276, N = 286 and N = 395). Next-generation sequencing analysis identified XPO1 mutations as a novel predictor of shorter time to first treatment (TTFT) in all cohorts. Notably, XPO1 mutations maintained their prognostic value independent of the immunoglobulin heavy chain variable status and early-stage prognostic models. These data suggest that XPO1 mutations, conceivably through increased miR-155 levels, may enhance BCR signalling leading to higher proliferation and shorter TTFT in early-stage CLL.

Increased Perforin- and IL-21-Expressing NK Cells in Patients with Early-Stage Chronic Lymphocytic Leukemia

Objective: CD5+CD19+ cells have a low proliferation capacity and elevated expression of anti-apoptotic protein BCL-2, mostly in the G0/ G1 cell phase and accumulate in the pathogenesis of chronic lymphocytic leukemia (CLL). Natural killer (NK) cells have the ability to kill the intracellular pathogen-infected or cancer cells and secrete cytotoxic enzymes. This study evaluated the frequency, expression of cytotoxic enzymes, and intracellular cytokine levels of NK cells in CLL patients. Materials and Methods: In this study, peripheral blood mononuclear cells were isolated from peripheral blood samples of CLL patients (n=29) and healthy controls (n=16) by density gradient centrifugation method. The frequency of the total NK cells and the intracellular levels of perforin, granzyme, interferon (IFN)-γ, interleukin (IL)-21, IL-4 and IL-17 in NK cells were investigated. Results: Elevated total NK cell frequency were found in the CLL patients compared to the healthy controls, and negatively correlated with CD5+CD19+ malign B cell frequency. Increased perforin expression was observed in patients’ total NK cells. Additionally, increased levels of IL-17 and IL-21 in total NK cell of CLL patients were obtained compared to healthy subjects. Conclusion: The findings suggest that in the early stage of CLL, increased total NK cell frequency, and elevated perforin and IL-21 levels in NK cells might have a protective impact against the progression of the disease for CLL patients.

A gene expression assay based on chronic lymphocytic leukemia activation in the microenvironment to predict progression

AbstractSeveral gene expression profiles with a strong correlation with patient outcomes have been previously described in chronic lymphocytic leukemia (CLL), although their applicability as biomarkers in clinical practice has been particularly limited. Here we describe the training and validation of a gene expression signature for predicting early progression in patients with CLL based on the analysis of 200 genes related to microenvironment signaling on the NanoString platform. In the training cohort (n = 154), the CLL15 assay containing a 15-gene signature was associated with the time to first treatment (TtFT) (hazard ratio [HR], 2.83; 95% CI, 2.17-3.68; P < .001). The prognostic value of the CLL15 score (HR, 1.71; 95% CI, 1.15-2.52; P = .007) was further confirmed in an external independent validation cohort (n = 112). Notably, the CLL15 score improved the prognostic capacity over IGHV mutational status and the International Prognostic Score for asymptomatic early-stage (IPS-E) CLL. In multivariate analysis, the CLL15 score (HR, 1.83; 95% CI, 1.32-2.56; P < .001) and the IPS-E CLL (HR, 2.23; 95% CI, 1.59-3.12; P < .001) were independently associated with TtFT. The newly developed and validated CLL15 assay successfully translated previous gene signatures such as the microenvironment signaling into a new gene expression–based assay with prognostic implications in CLL.

Serum B-Cell maturation antigen is an independent prognostic marker in previously untreated chronic lymphocytic leukemia

B-Cell maturation antigen (BCMA) is a cell membrane receptor expressed on mature B lymphocytes, with elevated serum levels found among patients with B-cell malignancies, including chronic lymphocytic leukemia (CLL). Serum BCMA (sBCMA) levels were measured in 331 untreated, newly diagnosed CLL patients using an enzyme-linked immunosorbent assay with a polyclonal anti-BCMA antibody. Elevated sBCMA levels were found among patients with CLL compared with age- and sex-matched healthy controls and those with more active CLL based on prognostic factors. The relationships between sBCMA, time to first treatment (TTFT), overall survival (OS) and multiple prognostic factors were compared using Mann-Whitney and Kruskal-Wallis tests. The median sBCMA level in the CLL cohort (48.6 ng/mL) was significantly higher (p < 0.001) compared with those of age- and sex-matched healthy subjects (n=100; 37.8 ng/mL). Serum BCMA correlated with TTFT(hazard ratio [HR]=2.9, 95% confidence interval 2.0-4.2, p < 0.001) and OS (HR=2.5, 95% confidence interval: 1.5-4.0, p < 0.001). Multiple models were used to test the predictive effects of sBCMA, sex, CLL International Prognostic Index (CLL-IPI) and International Prognostic Score for early-stage CLL (IPS-E) on TTFT and OS. The addition of sBCMA to CLL-IPI and IPS-E improved their prognostic ability to predict TTFT and OS. Thus, sBCMA is a new promising prognostic biomarker for CLL.

Identifying patients with chronic lymphocytic leukemia without need of treatment: End of endless watch and wait?

Early-stage chronic lymphocytic leukemia (CLL) challenges specialized management and follow-up. We developed and validated a prognostic index to identify newly diagnosed patients without need of treatment (CLL-WONT) by a training/validation approach using data on 4708 patients. Composite scores derived from weighted hazards by multivariable analysis defined CLL-WONT risk groups. Age (>65years: 1 point), Binet stage (B: 2 points), lactate dehydrogenase (LDH) (>205U/L: 1 point), absolute lymphocyte count (15-30×109 /L: 1 point; >30×109 /L; 2 points), β2-microglobulin (>4mg/L: 1 point), IGHV mutation status (unmutated: 1 point), and 11q or 17p deletion (1 point) were independently associated with shorter time to first treatment (TTFT). Low-risk patients demonstrated 5-year TTFT of 2% by internal validation, but 7-19% by external validation. Including all patients with complete scores, the 5-year TTFT was 10% for the 756 (39%) CLL-WONT low-risk patients, and the 704 (37%) patients who were both CLL-WONT and CLL-IPI low risk demonstrated even lower 5-year TTFT (8%). We have adopted the CLL-WONT at an institution covering 1800000 individuals to allow patients with both low-risk CLL-WONT and CLL-IPI to be managed by primary healthcare providers, thereby prioritizing specialized hematology services for patients in dire need.

The Effect of Dietary Intervention With High-Oleocanthal and Oleacein Olive Oil in Patients With Early-Stage Chronic Lymphocytic Leukemia: A Pilot Randomized Trial.

AimOleocanthal and oleacein (OC/OL) have important in vitro and in vivo antitumor properties; however, there is no data about their anticancer activity in humans. The aim of this pilot study was to test if patients at early stage of chronic lymphocytic leukemia (CLL) could adhere to and tolerate an intervention with high OC/OL extra virgin olive oil (EVOO) and if this intervention could lead to any changes in markers related to the disease.MethodsA pilot dietary intervention (DI) was made in patients with CLL in Rai stages 0–II who did not follow any treatment (NCT04215367). In the first intervention (DI1), 20 CLL patients were included in a blind randomized study and were separated into two groups. One group (A) of 10 patients consumed 40 ml/day of high OC/OL-EVOO (416 mg/Kg OC and 284 mg/kg OL) for 3 months. A second group (B) of 10 patients consumed 40 ml/day of low OC/OL (82 mg/kg OC and 33 mg/kg OL) for 3 months. After a washout period of 9–12 months, a second intervention (DI2) only with High OC/OL-EVOO for 6 months was performed with 22 randomly selected patients (16 from the DI1 (8 from each group) and 6 new). Hematological, biochemical, and apoptotic markers were analyzed in the serum of the patients. In addition, cellular proliferation and apoptosis markers were studied in isolated proteins from peripheral blood mononuclear cells.ResultsThe results of the DI1 showed beneficial effects on hematological and apoptotic markers only with High OC/OL-EVOO. During the DI2, a decrease in the white blood cell and lymphocyte count was observed (p ≤0.05), comparing 3 months before the intervention and 6 months after it. After 3 and 6 months of DI2, an increase (p ≤0.05) was observed in the apoptotic markers ccK18 and Apo1-Fas, and also in the cell cycle negative regulator p21, and also a decrease in the antiapoptotic protein Survivin, and in the cellular proliferation marker Cyclin D.ConclusionsThis is the first clinical trial with High OC/OL-EVOO that indicates that it could be a promising dietary feature for the improvement of CLL inducing the apoptosis of their cancer cells and improving the metabolism of the patients.Clinical Trial Registration https://clinicaltrials.gov/ct2/show/NCT04215367, identifier: NCT04215367.

Ibrutinib Treatment in Early Stage CLL Prolongs Time to Active Disease.

Ibrutinib treatment extended time to active disease in early stage chronic lymphocytic leukemia.

The CLL12 trial: ibrutinib vs placebo in treatment-naïve, early-stage chronic lymphocytic leukemia

AbstractObservation is the current standard of care for patients with early-stage asymptomatic chronic lymphocytic leukemia (CLL), as chemotherapy-based interventions have failed to prolong survival. We hypothesized that early intervention with ibrutinib would be well tolerated and lead to superior disease control in a subgroup of early-stage patients with CLL. The phase 3, double-blind, placebo-controlled CLL12 trial randomly assigned asymptomatic, treatment-naïve Binet stage A CLL patients at increased risk of progression in a 1:1 ratio to receive ibrutinib (n = 182) or placebo (n = 181) at a dose of 420 mg daily. At a median follow-up of 31 months, the study met its primary endpoint by significantly improving event-free survival in the ibrutinib group (median, not reached vs 47.8 months; hazard ratio = 0.25; 95% confidence interval = 0.14-0.43, P < .0001). Compared with placebo, ibrutinib did not increase overall toxicity, yielding similar incidence and severity of adverse events (AEs). The most common serious AEs were atrial fibrillation, pneumonia, and rash in the ibrutinib group, and basal cell carcinoma, pneumonia, and myocardial infarction in the placebo group. Ibrutinib-associated risk for bleeding (33.5%) was decreased by prohibiting the use of oral anticoagulants through an amendment of the study protocol and by avoiding CYP3A4 drug–drug interactions. Ibrutinib confirms efficacy in CLL patients at an early stage with an increased risk of progression. However, the results do not justify changing the current standard of “watch and wait.” This trial was registered at www.clinicaltrials.gov as #NCT02863718.