IGHV Mutation Research Articles

IGH 3'RR recombination uncovers a non-germinal center imprint and c-MYC-dependent IGH rearrangement in unmutated chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is an incurable indolent non-Hodgkin lymphoma characterized by tumor B cells that weakly express a B-cell receptor. The mutational status of the variable region (IGHV) within the immunoglobulin heavy chain (IGH) locus is an important prognosis indicator and raises the question of the CLL cell of origin. Mutated IGHV gene CLL are genetically imprinted by activation-induced cytidine deaminase (AID). AID is also required for IGH rearrangements: class switch recombination and recombination between switch Mu (Sμ) and the 3' regulatory region (3'RR) (Sμ-3'RRrec). The great majority of CLL B cells being unswitched led us to examine IGH rearrangement blockade in CLL. Our results separated CLL into two groups on the basis of Sμ-3'RRrec counts per sample: Sμ-3'RRrecHigh cases (mostly unmutated CLL) and Sμ-3'RRrecLow cases (mostly mutated CLL), but not based on the class switch recombination junction counts. Sμ-3'RRrec appeared to be ongoing in Sμ-3'RRrecHigh CLL cells and comparison of Sμ-3'RRrec junction structural features pointed to different B-cell origins for both groups. In accordance with IGHV mutational status and PIM1 mutation rate, Sμ-3'RRrecHigh CLL harbor a non-germinal center experienced B-cell imprint while Sμ-3'RRrecLow CLL are from AID-experienced B cells from a secondary lymphoid organ. In addition to the proposals already made concerning the CLL cell of origin, our study highlights that analysis of IGH recombinatory activity can identify CLL cases from different origins. Finally, on-going Sμ-3'RRrec in Sμ-3'RRrecHigh cells appeared to presumably be the consequence of high c-MYC expression, as c-MYC overexpression potentiated IGH rearrangements and Sμ-3'RRrec, even in the absence of AID for the latter.

Clinical and biological features and prognosis of patients with leukemic non-nodal mantle cell lymphoma

Objective: To investigate the clinical, biological and prognostic characteristics of leukemic non-nodal mantle cell lymphoma (nnMCL). Methods: The clinical data of 14 patients with nnMCL and 238 patients with classical mantle cell lymphoma (cMCL) in Blood Diseases Hospital, Chinese Academy of Medical Sciences from November 2000 to October 2020 were retrospectively analyzed. Among the 14 patients with nnMCL, there were 9 males and 5 females, with the age [M (Q1, Q3)] of 57.5 (52.3, 67.0) years. Among the 238 patients with cMCL, there were 187 males and 51 females, with the age of 58.0 (51.0, 65.3) years. The clinical and biological characteristics of the two groups were recorded and compared. Follow-up and efficacy evaluation were conducted by re-examination during hospital stay and telephone follow-up and so on. Results: The proportion of CD200 expression in nnMCL patients was 8/14, which was higher than that in cMCL patients [14.6% (19/130)] (P=0.001). The proportion of CD23 expression in nnMCL patients was 8/14, which was higher than that in cMCL patients [13.5% (23/171)] (P<0.001). The proportion of CD5 expression in nnMCL patients was 10/14, which was lower than that in cMCL patients [97.4% (184/189)] (P=0.001). The proportion of CD38 expression in nnMCL patients was 4/14, which was lower than that in cMCL patients [69.6% (112/161)] (P=0.005). The expression proportion of sex-determining region of Y chromosome-related high-mobility-group box 11 (SOX11) in nnMCL patients was 1/5, which was lower than that in cMCL patients [77.9% (60/77)] (P=0.014). The proportion of immunoglobulin heavy chain variable region (IGHV) mutations in nnMCL patients was 11/11, which was higher than that in cMCL patients [26.0% (13/50)] (P<0.001). As of April 11, 2021, the follow-up time for nnMCL and cMCL patients was 31 (8-89) months and 48 (0-195) months, respectively. Among the 14 nnMCL patients, 6 patients were still under observation, and 8 patients were treated. The overall response rate (ORR) was 8/8, including 4 patients with complete remission and 4 patients with partial response. The median overall survival and median progression-free survival were not reached in nnMCL patients. In the cMCL group, 50.0% (112/224) patients achieved a complete response, 24.6% (55/224) patients achieved a partial response, and ORR was 74.6% (167/224). There was no statistically significant difference in ORR between the two groups (P=0.205). Conclusions: nnMCL patients have an indolent progression, with higher expression rates of CD23 and CD200 and lower expression rates of SOX11, CD5 and CD38. Most patients have IGHV mutations, with a relatively good prognosis, and"watch and wait"approach is an optional treatment.

Characterization of the genetic and epigenetic landscape of B‐cell neoplasms with IG::BCL3‐translocation

Introduction: The BCL3 gene (19q13) encodes a transcriptional coactivator of the nuclear factor-kappaB (NF-κB) family. It is recurrently deregulated in B-cell neoplasms by a juxtaposition to regulatory elements of one of the immunoglobulin loci, mostly the immunoglobulin heavy chain (IGH) locus (14q32). Two distinct subsets of IG::BCL3 translocation-positive B-cell neoplasms, differing in the number of chromosomal aberrations, IGHV mutation status and histopathology, have been described [Martín-Subero et al., 2007]. In B-cell chronic lymphocytic leukaemia (CLL), the IG::BCL3 translocation has been associated with younger age at diagnosis, a more aggressive clinical course and an atypical tumour cell phenotype [Michaux et al., 1997; Au et al., 2002]. Methods: A total of 89 B-cell neoplasms with IG::BCL3 translocation detected by fluorescence in situ hybridisation (FISH), including predominately cases diagnosed as CLL, were studied. Breakpoints at the BCL3 and IGH loci were sequenced in a subset of samples using targeted capture and breakpoint-spanning PCR approaches. Chromosomal aberrations detected by FISH as well as the IGHV mutation status were analysed. In addition, DNA methylation and copy number aberration (CNA) analyses were performed for 48 samples with CLL using the 850K EPIC BeadChip array. Results: Targeted capture-based sequencing data showed that in the majority of cases BCL3 translocations are associated with aberrant class-switch recombination at the IGH locus, often involving IGHA segments (n = 4/10). From the 69 IG::BCL3-translocated cases with available IGHV mutation status, a total of 51 (74%) were classified as unmutated and 18 (26%) as mutated. This indicates an overrepresentation of cases with unmutated IGHV status among the IG::BCL3-translocated cases compared to the general CLL population. Based on EPIC BeadChip data (total n = 48) 25 (52%) IG::BCL3-translocated cases carried a trisomy 12, 11 cases (23%) a deletion 13q, 7 cases (15%) a deletion 17p and 3 cases (6%) a deletion 11q. FISH analysis of a partially overlapping set of neoplasms (n = 54) was in line with the EPIC BeadChip data and revealed 17 IG::BCL3-translocated cases (32%) with trisomy 12, 7 cases (13%) with deletion 17p and deletion 13q each, and 4 cases (7%) with deletion 11q. Thus, IG::BCL3-translocated cases showed a striking skewing towards the presence of trisomy 12 compared to the general CLL population (p = 0.011). In the DNA methylation analyses, the IG::BCL3-translocated CLLs segregated separately from other CLLs with and without IGH translocations. Conclusions: Our large multi-OMICs study of IG::BCL3-translocated B-cell neoplasms corroborates previous reports on the pathogenetic heterogeneity of these lymphomas and moreover provides evidence that IG::BCL3-translocated CLL might form a genetically and epigenetically distinct subtype of B-cell neoplasms distinct from common CLL. Keywords: Chronic Lymphocytic Leukemia (CLL), Genomics, Epigenomics, and Other -Omics No conflicts of interests pertinent to the abstract.

First dose infusion reaction during anti-CD20 monoclonal antibody therapy in chronic lymphocytic leukemia associates with high IP-10 levels amid cytokine release syndrome

Abstract Intravenous (IV) anti-CD20 monoclonal antibody (mAb) therapy for chronic lymphocytic leukemia (CLL) patients often produces a first dose infusion reaction (FDIR) within the first 2 h that requires careful monitoring to avoid serious complications and fatalities. To better understand FDIR, we studied blood samples collected from 37 treatment-naïve CLL patients in our clinical trial NCT03788291 undergoing IV treatment with 50 mg of anti-CD20 mAb (rituximab). We analyzed four time points: baseline (prior to infusion), 1 h later (during infusion), at the end of infusion (~2.5 h) and at 48 h. Infusion reactions (CTCAE (v5) grade ≥2 events) were managed by interrupting the infusion and with symptomatic treatment. 24 (65%) patients had a FDIR and all patients completed the infusion. Patient and CLL characteristics including measures of disease severity (Rai stage, IGHV mutation status, cytogenetic defects) did not correlate with FDIR. High levels of CLL cells and/or CD20 and their subsequent depletion resulting in decreased serum mAb and/or complement are thought to be risks for FDIR. However, measurements of these parameters did not correlate. Induction of cytokine release syndrome (CRS) may correlate with FDIR. However, CRS was observed in all samples after mAb infusion. The only significant associations with FDIR were higher levels of IP-10, IL-6, and IL-8, with IP-10 most significant. Anti-CD20 mAb activated cytotoxic effector cells, such as tissue-resident macrophages that ingest CLL cells via antibody-dependent cellular phagocytosis (ADCP), may be responsible for these events leading to FDIR. A mouse model of anti-CD20 mAb FDIR and in vitro ADCP cell systems will be useful to further understand the biology of FDIR. Supported by funding from Acerta/AstraZeneca, the Cadregari Foundation, a generous donation by Elizabeth Aaron, and the NIH NCI grant number R21CA267040.

Next-CLL, a New Next-Generation Sequencing–Based Method for Assessment of IGHV Gene Mutational Status in Chronic Lymphoid Leukemia

Current guidelines for patients with chronic lymphocytic leukemia (CLL) recommend mutation status determination of the clonotypic IGHV gene before treatment initiation to guide the choice of first-line therapy. Currently, commercially available next-generation sequencing (NGS) solutions have technical constraints, as they necessitate at least a 2×300 bp sequencing, which restricts their use for routine practice. The cost of the commercial kits also represents an important drawback. We present a new method called Next-CLL, a ready-to-use strategy to evaluate IGHV gene mutation status using any NGS device (including 2×150 bp sequencers) in routine diagnostic laboratories. The performance of Next-CLL was validated on genomic DNA and cDNA obtained from 80 patients with CLL at diagnosis. Next-CLL identified a productive clone in 100% of cases, whereas PCR with Sanger sequencing led to a 12.5% failure rate. Next-CLL had 100% concordance with the reference technique for IGHV gene identification and allowed assessment of the IGHV mutation status from the leader sequence, following international guidelines. Comparing a large retrospective series of samples, analyzed by using Sanger sequencing (n=773) or Next-CLL (n = 352), showed no bias in IGHV usage or mutational status, further validating our strategy in the real-life setting. Next-CLL represents a straightforward workflow for IGHV analysis in routine practice to assess clonal architecture and prognosis of patients with CLL.

OBINUTUZUMAB FOR THE TREATMENT OF CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS: SINGLE CENTER EXPERIENCE

Monoclonal antibodies (mAbs) targeting CD20 molecule on B lymphocyte are of great importance in the treatment of B-cell malignancies. In recent years a great effort has been put in developing novel mAbs that can provide greater efficacy than the well-known rituximab. A second class of mAbs obinutuzumab has been presented as a more powerful tool in the treatment of this group of patients. In this retrospective study 70 patients with symptomatic CLL were included. CLL patients were diagnosed and treated at the University Clinic for Hematology between January 2018 and January 2022. All patients were evaluated for traditional clinical and laboratory prognostic factors and newer prognostic factors including IGHV mutation status and CLL prognostic and predictive genetic abnormalities. Most of the patients treated with obinutuzumab had Binet B stage (56%). Mutational status of the immunoglobulin variable region heavy chain (IGHV) in most of CLL patients treated with obinutuzumab was unmated IGHV gene. The most frequently encountered adverse events were tumor lysis syndrome and leukopenia. The analysis of the initial results of the application of obinutuzumab-based therapy allows us to conclude that this therapeutical modality is not associated with severe adverse events that would limit the administration of therapy.

Accuracy of predicting IgHV mutation status in chronic lymphocytic leukemia using RNA expression profiling and machine learning

Background: Immunoglobulin heavy chain (IgHV) mutation status is a unique prognostic indicator for predicting clinical course and response to targeted therapy in chronic lymphocytic leukemia (CLL). Most of the tests for the IgHV mutation status require extensive and complicated genomic evaluation. We explored the potential of using RNA expression data generated from routine targeted RNA sequencing by Next Generation Sequencing (NGS) along with machine learning for the prediction of the IgHV mutation status.

An Unsupervised Machine Learning Method Stratifies Chronic Lymphocytic Leukemia Patients in Novel Categories with Different Risk of Early Treatment

Novel scoring systems have been developed in recent years to improve the accuracy of prognostication from historical clinical staging systems (Rai, Binet) for chronic lymphocytic leukemia (CLL). Most of them, however, rely on discretized and dichotomic values of the various biomarkers to infer prognosis. Here we analyzed the immunophenotypic and (immuno)genetic profiles in a wide CLL cohort by applying unsupervised machine learning methods elaborating prognostic factors as continuous variables, to identify novel relationships and interactions likely missed in conventional models. The study included 989 CLL patients with Rai stages 0-I-II (50, 36, 14% respectively), 420 (42.4%) treated, analyzed between 2003 and 2020. Treatment-free survival (TFS) was calculated from sampling (median TFS, 46 months). Median time of sampling from diagnosis was 7.2 months (~60% cases within 12 months). The studied laboratory-based markers were: CD20, FMC7, CD49d, CD49c, CD38, CD23, CD43, CD22, ZAP-70 expression by flow cytometry (reported as % of positive cells); del13, tris12, del11 and del17 cytogenetic abnormalities detected by FISH, reported as % of nuclei with abnormal signal; mutational status of TP53 by deep NGS, reported as % variant allele fraction (VAF); IGHV gene mutational status, reported as % mutations. By applying Cox proportional hazard model to estimate TFS on this pool of features, we selected the features with a fit p-value<0.05, i.e. del11, tris12, TP53 mutations, % IGHV mutation, expression of CD38 and CD49d. Then we grouped similar profiles with an unsupervised k-means algorithm, optimized by the Elbow method, to partition observations into 6 clusters (C1-C6). Clustering confidence for each patient was estimated through leave-one-out procedure and the average normalized score was 0.83 (0.85, 0.86, 0.79, 0.91, 0.73, 0.84, for C1 through C6, respectively). Centroid analysis was employed to evaluate which features mostly defined each cluster, as detailed below. C1 (n=220): all cases with <4.1% IGHV mutations, mostly (70%) IGHV-unmutated (UM, <2% cut-off), low CD49d expression (<30% of positive cells) in 90% of cases, low representation of other features (tris12, del11, CD38, TP53 mutations); C2 (n=210): high CD49d expression (99% of cases), equally balanced IGHV status (52% UM), low representation of other features; C3 (n=147): tri12 cases (100%; >10% of nuclei), concurrent high expression of CD49d (95%) and CD38 (67%), slightly enriched in UM IGHV cases (60%); C4 (n=303): cases heavily mutated in IGHV genes (mutation range 4.1-22.0%), low representation of all the other features; C5 (n=52): TP53 mutated cases with high mutation burden (VAF 35-97%), skewed towards UM IGHV (70%), irrelevant all the other features; C6 (n=57): highly clonal del11q cases (range 50-98%) mutually exclusive with TP53 mutations (2 mutated cases only), mostly UM IGHV (89%), low representation of all other features. Notably, in C1-C4, cases bearing TP53 mutations were present, although representing a minority (5-10%), mostly with low-VAF (median 6.7%, range 5-39%). Kaplan-Meier analysis revealed heterogeneous behaviors: C2, C3, C5, C6 presented a 50% TFS of 59, 25, 5, 9 months respectively, whereas TFS was not reached for C1 and C4 (Figure, A). Hierarchical agglomerative clustering identified 3 major risk classes (Figure, B). The high risk class (n=109) comprised C5 and C6 (TP53 mutations and del11q); the intermediate risk class (n=577) stratified on C1-2-3; the low risk class (n=303) was made by C4 only (CLL with highly mutated IGHV). The 50% TFS for high-intermediate-low risk was 7, 50 and not reached, respectively. In conclusion, we present here a novel machine-learning-driven, laboratory-based classification for predicting the risk of early treatment in CLL. Our approach identifies clusters at different risk with some novelties: i) a high IGHV mutational burden (i.e >4%) in the absence of other markers (e.g. CD49d) identifies patients with a particularly benign clinical course; ii) TP53 mutations and del11q associate with high risk of early treatment only if present in the vast majority of the CLL clone; iii) a IGHV status with low burden of mutations (i.e. <4%) along with CD49d expression or tris12 identifies patients at intermediate risk. These novel stratifications should be incorporated in risk algorithms for treatment prediction of CLL patients. Validation in additional independent cohorts is needed. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Genetic Markers and Front Line FCR/BR Vs. Rve, Gve and Give Treatment - Outcome Results from the CLL13/GAIA Trial

BACKGROUND: Genomic aberrations, IGHV mutation status and recurrent mutations in genes such as TP53 are established prognostic factors in CLL in the context of chemoimmunotherapy (CIT). For time limited treatment with venetoclax in combination with Rituximab (RVe), Obinutuzumab (GVe) and Obinutuzumab and Ibrutinib (GIVe) the role of these factors is less-well established. METHODS: The phase 3 GAIA/CLL13 trial compared 3 different time-limited venetoclax-based combinations against CIT in fit, treatment-naïve patients with CLL without del(17p) or TP53 mutations detected via Sanger sequencing. Patients were randomized to receive CIT (FCR for patients ≤65y; BR for patients >65y), RVe, GVe or GIVe. Co-primary endpoints were PFS and MRD in peripheral blood at month 15 via 4-colour flow. Genomic aberrations were assessed at enrolment by FISH, IGHV by sequencing with a threshold of 98% homology and mutations via amplicon-based targeted NGS for TP53, NOTCH1, SF3B1, MYD88, KRAS, NRAS, BRAF, EGR2, NFKBIE, RPS15, XPO1 and BIRC3 in 913 of 926 patients (98.6%) of the full trial cohort. RESULTS: The incidences of genomic aberrations based on the hierarchical model were del(11q) 17.5%, +12 16.2%, mono-allelic del(13q) 44.6% and normal karyotype 21.7%. IGHV was unmutated (U-IGHV) in 55.9% and mutated (M-IGHV) in 41.0% of patients (ITT). Subset#2 accounted for 50 patients equally distributed among U-IGHV and M-IGHV. The incidence of gene mutations was SF3B1 22.6%, NOTCH1 20.5% (exon 34 and 3'UTR), NFKBIE 13.6%, BIRC3 11.8%, BRAF/KRAS together 9.6%, XPO1 7.9%, EGR2 5.6%, RPS15 5.5% and MYD88 4.2%. Minor mutations with a variant allele fraction (VAF) of 1.5% to 10% were frequent in BIRC3 and NFKBIE (59.6% and 56.0% of all variants, respectively), while minor mutations in TP53 not previously detected by Sanger sequencing were rare (2.0%). Undetectable MRD (uMRD) was not associated with any genetic subgroup in the pooled analysis of GVe/RVe/GIVe. Single arm analysis revealed NOTCH1 mutation associated with lower rate of uMRD with GVe (74.4% vs 88.6%, p=0.02) and higher uMRD rate in +12 cases with RVe (73.5% vs. 52.2% in del(13q), p=0.03). With CIT a significantly lower uMRD rate was found in U-IGHV (42.0% vs. 66.3% in M-IGHV, p<0.01) and del(11q) (39.0% vs. 58.4% in del(13q), p=0.04) while mutated MYD88 associated with a higher uMRD rate (92.3% vs 50.2%, p=0.02). At a median follow-up of 38.8 months, there were 188 PFS events in the ITT population. For CIT U-IGHV (HR 4.47, p<0.01), del(11q) (HR 2.68, p<0.01) and mutated NOTCH1 with VAF>10% (HR 2.01, p=0.01) associated significantly with shorter PFS in univariate analysis. In the combined analysis for RVe/GVe/GIVe U-IGHV (HR 2.29, p<0.01), mutations in NOTCH1 (HR 1.76, p<0.01) and BRAF/RAS (HR 2.45, p<0.01) were associated with significantly shorter PFS and del(13q) (HR 0.67, p=0.03) with longer PFS. A separate analysis for each venetoclax arm identified mutated NOTCH1 (GVe: HR 2.2 p=0.04 and GIVe: HR 2.2, p=0.05), BIRC3 (GIVe: HR 3.6 p<0.01), BRAF/KRAS/NRAS (RVe: HR 3.6, p<0.01 and GIVe: HR 3.7, p<0.01) NFKBIE (GVe: HR 3.0, p<0.01), del(13q) (GVe: HR 0.4, p=0.01) and U-IGHV (GVe: HR 2.6, p=0.03 and GIVe: HR 2.7, p=0.03) as prognostic factors for PFS. Subset 2 was not associated with shorter PFS, also not among M-IGHV patients. In multivariable analysis including all clinical, laboratory and genetic markers significantly associated with PFS, treatment with GVe and GIVe were independent favorable factors (HR 0.42 and 0.33, respectively, both p<0.01) and U-IGHV (HR 2.43, p<0.01), and mutated NOTCH1 (HR 1.46, p=0.03) were confirmed as independent prognostic factors. In a separate multivariable analysis only for CIT, del(11q) (HR 1.89, p=0.03), U-IGHV (HR 3.08, p<0.01), and mutated NOTCH1 >10% VAF (HR 2.12, p=0.01) were verified as independent adverse genetic risk factors. For the combined RVe/GVe/GIVe group, mutated BRAF/RAS (HR 1.87, p=0.01), U-IGHV (HR 1.85, p<0.01), and mutated NOTCH1 (HR 1.54, p=0.04) were confirmed as independent adverse prognostic factors. CONCLUSIONS: Early results from CLL13/GAIA show association of gene mutations with lower rate for uMRD and shorter PFS after CIT or venetoclax-based therapy. IGHV and NOTCH1 mutation retain their prognostic role with both treatment types. Mutations in the RAF/RAS pathway (9.6% overall) were associated with shorter PFS with venetoclax-based therapy, while with CIT an impact on outcome is not apparent at present. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

An Ex Vivo Monocyte Stimulation Test Predicts Severity of COVID-19 Infection in Patients with Chronic Lymphocytic Leukemia (CLL)

Introduction The severity of coronavirus disease 2019 (COVID-19) is associated with the development of a cytokine storm driven by classical monocytes. Activation of the NLRP3 inflammasome mediates the production of IL-1β - a key pro-inflammatory cytokine that in excessive quantities causes tissue injury. Most CLL patients have an impaired humoral response to vaccination with subsequent higher risk of complications from COVID-19 infection. Currently, no pre-exposure test exists to assess the risk of a dysregulated immune response to COVID-19. We previously reported that open reading frame 8 (ORF8), a SARS-CoV-2 protein, when glycosylated and secreted into the blood, stimulates CD14+ monocytes and activates the NLRP3 pathway to produce IL-1β (Ruan et al, AAI 2022). We hypothesized that functional characteristics of CLL patients’ monocytes may predispose patients to develop severe COVID-19. First, we developed the ORF8 monocyte stimulation test (OMST), which measures monocyte reactivity via flow cytometry after stimulation of patient monocytes ex vivo with ORF8. Secondly, we applied the OMST to CLL patients to learn about its association with severe COVID-19 infection. Methods We identified patients from the Mayo Clinic CLL Database who were diagnosed with COVID-19 infection via a positive SARS-CoV-2 RT-PCR nasal swab between July 2020 to February 2022 and had a cryopreserved pre-COVID-19 peripheral blood mononuclear sample (PBMC) available. Our study was approved by the IRB, and samples were stored in the Mayo Clinic Predolin Biobank. For the OMST, samples were thawed and either stimulated with ORF8 or unstimulated (control) for 24 hours. The samples were harvested and intracellular cytokine/chemokine levels (IL-1β, IL-8, and CCL-2) in monocytes were analyzed by flow cytometry using the depicted gating strategy and formulas to calculate delta MFI (dMFI) and adjusted MFI (Figure 1). Clinical outcomes were abstracted by chart review. Severe infection was defined as hospitalization due to COVID-19 infection. Fisher's exact test, Wilcoxon rank sum test, and matched paired analysis were used to compare categorical, continuous, and paired variables, respectively. Receiver-operating curves (ROC) were used to calculate the area under the curve (AUC), sensitivity, specificity, and optimal cutoff levels for a reactive OMST. The optimal cutoff point was chosen as the point with the highest Youden index. Time to severe COVID-19 infection was calculated using the Kaplan-Meier method and associations of factors with severe COVID-19 were assessed using Cox proportional hazard model (CPHM). Results 60 patients were identified. The median age at COVID-19 diagnosis was 74 years (63-80). The median number of COVID-19 vaccinations before infection was 0 (range 0-3). The median time interval between vaccination and infection was 106 days (2-322). At diagnosis, 25 (42%) patients were receiving CLL-directed therapy and 22 (37%) were on a BTK-inhibitor. 22 patients had severe COVID-19 and 6 patients died. The OMST demonstrated that CD14+ monocytes had a significantly higher dMFI for IL-1β, IL-8, and CCL-2 after ORF8 stimulation compared to controls (495 vs. 158, p<0.0001), (298 vs. 121, p<0.001), and (385 vs. 171, p <0.0001) respectively. By ROC analysis, the AUC for IL-1β adjusted MFI was 0.68, 0.67 for IL-8 adjusted MFI, and 0.37 for CCL-2 adjusted MFI. A reactive OMST was defined by ROC as an IL-1β adjusted MFI >= 0.18 (sensitivity 67%, specificity 75%). The median time to hospitalization after COVID-19 infection for patients with a reactive OMST was 12 days (95% CI: 2 - not reached) versus not reached (NR) for patients with a non-reactive OMST [95% CI: NR-NR] with a hazard ratio of 7.7 (95% CI: 2.4-132, p=0.005) [Figure 2]. The following variables were not significant on univariate CPHM: age, gender, IGHV mutation, CLL-IPI risk, Charlson Comorbidity score, smoking status, number of mRNA COVID-19 vaccinations, receipt of monoclonal antibodies, or on therapy for CLL. Conclusions We have developed a novel assay, the OMST, that measures the reactivity of human monocytes to ORF8 and has significant value in determining the severity of COVID-19 infection in CLL patients. These findings are independent of patient age, sex, comorbidity score, prognostic models, and vaccination status. The OMST has the potential to assess monocyte reactivity and its implications in a broad range of clinical situations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Residual Disease Kinetics Among Patients with High-Risk Factors Treated with First-Line Fixed-Duration Ibrutinib Plus Venetoclax (Ibr+Ven) Versus Chlorambucil Plus Obinutuzumab (Clb+O): The Glow Study

Background: In the GLOW study (NCT03462719), fixed-duration Ibr+Ven demonstrated superior progression-free survival (PFS), deeper, better sustained undetectable minimal residual disease (uMRD) responses, and fewer patients (pts) requiring subsequent anti-cancer treatment versus Clb+O in older and/or comorbid pts with previously untreated chronic lymphocytic leukemia (CLL; Kater AP, et al. NEJM Evidence. 2022). Unmutated IGHV (uIGHV) and TP53 mutations are risk factors associated with worse outcomes for both chemoimmunotherapy and venetoclax plus anti-CD20 therapies for CLL. Here we investigate MRD kinetics and outcomes of Ibr+Ven in GLOW according to these risk factors. Methods: Pts aged ≥ 65 years or 18 to 64 years with cumulative illness rating scale score > 6 or creatinine clearance < 70 mL/min were enrolled and randomized 1:1 to Ibr+Ven (3 cycles of Ibr lead-in, followed by 12 cycles of Ibr+Ven) or 6 cycles of Clb+O, with a cycle defined as 28 days. Pts with del(17p) or known TP53 mutations at screening were excluded, and central evaluation of TP53 mutational status was performed during the study. The primary end point was PFS by independent review committee. Among pts with partial response or better, MRD in peripheral blood was evaluated using next-generation sequencing via clonoSEQ on-treatment and at 3-6 month intervals post-treatment. uMRD results are reported in peripheral blood (PB) at < 10-4 unless otherwise noted. Since the primary analysis, a post hoc retrospective reclassification of the IGHV status of baseline samples was conducted to reduce the number of unknowns. We used clonoSEQ data as part of CLL clonal testing (Adaptive Biotechnologies, Seattle, WA). Results: There were 106 pts randomized to Ibr+Ven and 105 to Clb+O. In the Ibr+Ven arm, uMRD rates increased from 46.2% after 6 cycles of the combination to 54.7% at 3 months after end of treatment (EOT+3), demonstrating that the majority of disease clearance in the PB occurred early, during the first 6 months of combination treatment. Post-treatment, 77.6% (45/58) of pts in the Ibr+Ven arm sustained their uMRD status from EOT+3 to EOT+18, compared with 12.2% (5/41) in the Clb+O arm. In the Ibr+Ven arm, 17/24 patients with intermediate MRD ≥ 10-4 to < 10-2 at EOT+3 did not worsen through EOT+18. Among pts with detectable MRD (≥ 10-4) at EOT+3, 6.5% (2/31) clinically progressed by EOT+18 in the Ibr+Ven arm versus 68.1% (32/47) in the Clb+O arm. After reclassification of baseline samples, uIGHV / mutated IGHV (mIGHV) / unknown status was 63.2%/30.2%/6.6% in the Ibr+Ven arm and 54.3%/33.3%/12.4% in the Clb+O arm. In the Ibr+Ven arm, rates of uMRD after 6 cycles of combination treatment and at EOT+3, respectively, were 52.2% and 59.7% in pts with uIGHV and 31.3% and 40.6% in pts with mIGHV (Fig A). On-treatment MRD kinetics differed by IGHV status in the Ibr+Ven arm, with uMRD achieved at a higher rate and earlier in pts with uIGHV. In the Ibr+Ven arm, uMRD was sustained post-treatment (from EOT+3 and EOT+18) in 80.0% (32/40) of pts with uIGHV and 76.9% (10/13) of pts with mIGHV. Among pts with detectable MRD (≥ 10-4) at EOT+3, 2/16 with uIGHV and 0/14 with mIGHV had clinically progressed by EOT+18 in the Ibr+Ven arm; corresponding rates were 83.3% (25/30) and 42.9% (6/14) in the Clb+O arm. Five of 7 pts with TP53-mutated CLL achieved uMRD at EOT+3 in the Ibr+Ven arm; TP53 variant allele frequencies ranged between 5.9% and 29.6% for these 5 pts and all maintained uMRD through EOT+18. With a median study follow-up of 34.1 months, PFS rates after IGHV reclassification were well sustained post-treatment regardless of IGHV status in the Ibr+Ven arm, while uIGHV pts in the Clb+O arm relapsed more quickly (Fig B). PFS, overall survival, and subgroup analyses with longer study follow-up will be presented. Conclusion: All-oral, once-daily, fixed-duration Ibr+Ven achieved uMRD responses that were better sustained than Clb+O during the first 18 months post-treatment. Notably, with Ibr+Ven, uMRD rates were higher and achieved earlier in pts with uIGHV CLL versus mIGHV CLL, while uMRD was similarly sustained post-treatment irrespective of IGHV status. Clinical progressions in the first 18 months post-treatment with Ibr+Ven were uncommon even among pts with detectable MRD ≥ 10-4. MRD kinetics and sustained responses demonstrate strong efficacy of the fixed-duration Ibr+Ven combination in older pts with high-risk genomic features. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Chronic Lymphocytic Leukemia Treatment: Single Center Experience

Background: Chronic lymphocytic leukemia (CLL) is relatively uncommon in the Middle East, with a younger age at diagnosis and different clinical characteristics compared to the West. There is little research on CLL in this region. This study aims to evaluate widely used prognostic markers and examine treatment outcomes of CLL patients in the Kuwait Cancer Control Center (KCCC), the only cancer center in Kuwait. Methods: This is a retrospective analysis of unselected 141 CLL patients receiving treatment in the KCCC. Diagnostic and treatment guidelines in our center were in accordance with the International Workshop on CLL criteria. Results: The median age of those patients at diagnosis was 58 years (IQR, 51-65), with 60% of patients under the age of 60. The male to female ratio in this group was 3.3:1. Median absolute lymphocyte count was 51x109/L (IQR, 51-65), and the median hemoglobin and platelet count were 11.8 g/dL (IQR, 10.1-13.8) and 170x109/L (IQR, 129-222). FISH analysis was performed in 118 patients, of which 9% (11) demonstrated del17p. Del13q and trisomy 21 were detected in 31% (36) and 26% (31) patients, respectively. IGHV mutational status was determined in 80 patients, 25% (20) were mutated, and 75% (60) were unmutated. When it comes to treatment, 47% (65) of patients were on FCR regimen, while 11% (15) and 12% (16) were receiving BR and CR, respectively. Approximately 15% (21) of patients in our study were on a BTKi. In 105 patients, the median progression-free survival (PFS) was 44.2 months [95% CI: 36-61.5], with a PFS rate of 48% and 32% at 25 and 50 months, respectively. In 121 patients with no history of progression or treatment in our study cohort, 41.5% and 21% progressed or received treatment at 25 and 50 months, respectively, with a median time to first progression or treatment around 37.6 months [95% CI: 29.1-49.5]. Examining 103 patients with prior history of progression or treatment, 34% and 13% progressed again or required a second course of treatment at 25 and 50 months, respectively, with a median time to second progression or treatment around 65.5 months [95% CI: 40.5-NA]. Those receiving chemoimmunotherapy exhibited a higher median PFS of 93 months [95% CI: 83-NA] compared to those receiving a BTKi with a median PFS of 42 months [95% CI: NA-NA]. However, the difference was not statistically significant (p=0.34). IGHV mutation status was significantly associated with OS (p=0.044) but not PFS (p=0.28). Those with unmutated IGHV had an inferior outcome compared to their mutated counterparts with OS rates at 50 months, around 15% and 25%, respectively, PFS rates were also lower in the unmutated compared to a mutated group, 21% vs. 31% at 50 months, respectively, and PFS medians around 49 [95% CI: 36.4-NA] and 94.5 [95% CI: 94.5-NA], respectively. Multivariate cox regression analysis on OS was not significant (p=0.160). None of the individual treatment regimens were significant predictors of OS compared to the reference (BR), except FCR. Treatment with FCR was associated with a hazard ratio of 0.11 (p = 0.011). Unmutated IGHV status, presence of del17p, and age greater than 60 were all associated with greater hazard ratios, but none were significant predictors of OS. Conclusion: Our CLL treatment group is younger. Unmutated IGHV was associated with a poor prognosis, consistent with the literature. However, treatment with BTKis was not associated with superior outcomes compared to CIT; however, this study is not powered to detect a difference between the two arms. Those receiving FCR had the best survival outcome compared to those receiving other therapies. Keywords: Chronic Lymphocytic Leukemia, Treatment Outcomes, Kuwait, Middle East Funding Agency: Kuwait Foundation for the Advancement of Science, Grant number: PR1713MM03 Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Chronic Lymphocytic Leukemia in African Ancestry Population Is Characterized By Increased Telomere Erosion

Background: Chronic lymphocytic leukemia (CLL), a neoplastic disease of relatively mature B-cells, is characterized by a variable clinical course. Although initial studies suggesting more aggressive disease phenotype and inferior clinical outcomes among CLL patients of African ancestry (AA) were published a decade ago, biological features contributing towards this augmented disease severity remain largely unknown. Telomere erosion in a cohort of CLL patients of European Ancestry (EA) has been previously associated with high-risk molecular markers including unmutated immunoglobulin heavy-chain variable region (u-IGHV) status, and genetic alterations such as del(17p) and del(11q) by FISH. Here we characterized telomere erosion in AA CLL patients compared to CLL patients of EA. Methods: We extracted DNA from CD5+/CD19+ cells peripheral blood mononuclear cells and utilized it to measure the average telomere length through quantitative-PCR (qPCR). Briefly, primers for telomere and the single copy gene (SCG) HBB were used for amplification of 20 ng of DNA in a total volume of 20 µL in triplicates using the KAPA SYBR® FAST optimized for LightCycler®480 and analyzed on a LightCycler®480. Absolute telomere lengths were then obtained by generating standard curves using synthetic oligonucleotide PCR standards for telomere and the SCG gene. The qPCR results were validated by terminal restriction fragment length (TRF) analysis and Southern hybridization (N=28; r=0.8531; p<0.0001) using the TeloTTAGGG Telomere Length Assay. TRF was used as the gold standard of telomere length quantification; however, the high quantity of genomic DNA required for the assay (2µg) limited its broad use. Five CLL samples with known telomere lengths through TRF analysis were included in every qPCR plate in order to perform a linear regression to account for variability between qPCR plates as described previously. Results: Telomere erosion was evaluated in a cohort of 133 AA (mean age at sample collection 62.7 years ±13.6) and 147 age-matched EA CLL patients (mean age at sample collection 61.6 years ±10.2). The AA samples were collected from 95 (71%) male patients and 71% presented u-IGHV, while EA samples were collected from 110 (77%) male patients and 66% u-IGHV. As expected, we observed shorter telomere length in u-IGHV CLL (median 3.7 kb; range 0.8-44.9) compared to mutated immunoglobulin heavy-chain variable region (m-IGHV) CLL (median 6.8 kb; range 1.8-44.7; p<0.0001) in the full cohort. AA CLL presented shorter telomere lengths (median 4.2 kb; range 0.6-13.1) when compared to EA CLL (median 4.7 kb; range 1.1-44.9; p=0.005; Figure 1A). We highlight that the differences in telomere length observed were restricted to the CD5+/CD19+ cells, with no difference observed in germline samples collected from AA and EA patients. When stratifying by IGHV mutation status, shorter telomere length was observed in u-IGHV when compared to m-IGHV among both AA CLL (median 3.5 kb and 6.3, respectively; p<0.0001) and EA CLL (median 3.9 kb and 7.5, respectively; p<0.0001; Figure 1B). The significant shorter telomere lengths in AA CLL remained significant (p=0.006) after accounting for IGHV status, age, sex, number of mutated CLL driver genes, and Rai stage in multivariate analyses. Conclusions: Our results demonstrated increased telomere erosion in AA CLL compared to EA CLL, even after accounting for other known prognostic factors. Telomere length has been described as an independent prognostic factor in multiple cancers. In CLL, telomere shortening can lead to a selective pressure for loss of function of DNA damage checkpoint genes, becoming permissive to further cell proliferation and telomere shortening. Future studies are needed to see if these findings contribute towards the severe clinical outcomes in AA CLL compared to EA CLL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Combined Ibrutinib and Venetoclax for First-Line Treatment of Patients with Chronic Lymphocytic Leukemia (CLL): 4-Year Follow-up Data

Background: Ibrutinib (IBR) and venetoclax (VEN) combination is an effective therapy for patients (pts) with CLL. We previously reported results of the first-line cohort of a phase II trial of combined IBR and VEN for high-risk pts with CLL (Jain, NEJM 2019, JAMA Oncology 2021). Here we report updated data for 120 pts (80 pts in the original published cohort and 40 pts in an expansion cohort) with a median follow-up of 4 years. Methods: Pts with previously untreated CLL meeting IWCLL treatment criteria were enrolled. All pts had at least one high-risk feature: del(17p), mutated TP53, del(11q), unmutated IGHV, or age ≥65 years. Pts received IBR 420 mg daily for 3 cycles followed by addition of VEN (weekly dose-escalation to 400mg daily). Combined therapy was given for 24 cycles (28 days/cycle). Pts with bone marrow (BM) undetectable MRD (U-MRD) (flow cytometry; sensitivity 10-4) at 24 cycles of combined therapy discontinued both VEN and IBR; MRD+ pts continued IBR. A trial amendment allowed an additional 12 cycles of combined VEN and IBR for pts who remained BM MRD+ after Cycle 24. Response assessments included BM and CT studies (2008 IWCLL criteria). U-MRD was defined as <0.01%; low MRD+ 0.01% to <1%; high MRD+ ≥1%. Progression-free survival (PFS) was assessed as the time from the start of study drug to CLL progression, Richter transformation, or death from any cause. Blood MRD was monitored every 6 months after active therapy. Results: Between August 2016 and February 2019, a total of 120 pts were enrolled. Baseline characteristics are shown in Table 1. The median follow-up is 48.8 months. Six pts came off study during 1st 3 cycles of IBR monotherapy; 114 pts initiated VEN. After 12 cycles of the combination, 62/120 (52%) achieved BM U-MRD remission; 43/120 (36%) were BM MRD-positive (low MRD+, n=35; high MRD+, n=8). After 24 cycles of the combination, 77/120 (64%) achieved BM U-MRD remission; 24/120 (20%) were BM MRD+ (low MRD+, n=23; high MRD+, n=1). Overall, 86/120 (72%) achieved BM U-MRD as the best response. We evaluated baseline pt and disease characteristics (age, gender, IGHV mutation status, CLL FISH panel, complex karyotype, TP53 mutation, del(17p)/TP53 mutation) and rate of bone marrow U-MRD at 6 months, 12 months and at any time during the combined IBR+VEN treatment. At 6 months of combined therapy, presence of TP53 mutation was associated with a lower rate of bone marrow U-MRD (p=0.03); however, none of the aforementioned characteristics, including TP53 mutation were associated with marrow U-MRD responses at 12 months or with best MRD response at any time during the combined therapy. There was a trend towards lower U-MRD rate among pts with mutated IGHV vs. unmutated IGHV at 12 month (p=0.09) and as best response (p=0.09). The 4-year PFS is 94.5% (95% CI, 90.3%-98.9%) and 4-year OS is 96.6% (95% CI, 93.3%-99.9%). The 4-year PFS by TP53 aberrant status is shown in Figure 1. The 4-year PFS for pts with del(17p)/TP53 mutation (n=27) is 90.9% and for pts without del(17p)/TP53 mutation (n=93) is 95.5%. None of the aforementioned pt/disease characteristics were associated with PFS. Of the 77 pts who were BM U-MRD at the end of cycle 24 of the combination, 73 discontinued all therapy, 4 pts continued IBR per treating physician discretion. Among these 77 pts, with a median time of 23.9 months post Cycle 24, 11 pts had recurrence of blood MRD (defined as MRD ≥0.01% in 2 consecutive visits). Of the 11 pts with MRD recurrence, 10 are being monitored without any active therapy for CLL and without clinical disease progression; 1 pt had disease progression after 22 months of treatment-free interval and is currently receiving acalabrutinib. There were 24 pts who were BM MRD+ at the end of cycle 24 of the combination (low MRD+, n=23; high MRD+, n=1). The only pt with high-MRD+ at end of cycle 24 was noted to have Richter transformation at that time. The remaining 23 pts (all low MRD+ in BM, range 0.01-0.95%) continued IBR monotherapy. With a recent trial amendment, MRD+ pts after Cycle 24 could get 12 additional cycles of VEN; 18/23 pts have resumed VEN. 11/18 (61%) pts achieved U-MRD remission during the third year of combined therapy. Conclusions: We report long term follow-up of combined IBR and VEN in first-line CLL (n=120) with a 4-year PFS of 94.5%. MRD responses and PFS were independent of baseline pt/disease characteristics. Continuation of combined therapy among marrow MRD+ pts during the second and the third year led to achievement of U-MRD in a subset of pts. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Mutational patterns along different evolution paths of follicular lymphoma.

Follicular lymphoma (FL) is an indolent disease, characterized by a median life expectancy of 18-20 years and by intermittent periods of relapse and remission. FL frequently transforms into the more aggressive diffuse large B cell lymphoma (t-FL). In previous studies, the analysis of immunoglobulin heavy chain variable region (IgHV) genes in sequential biopsies from the same patient revealed two different patterns of tumor clonal evolution: direct evolution, through acquisition of additional IgHV mutations over time, or divergent evolution, in which lymphoma clones from serial biopsies independently develop from a less-mutated common progenitor cell (CPC). Our goal in this study was to characterize the somatic hypermutation (SHM) patterns of IgHV genes in sequential FL samples from the same patients, and address the question of whether the mutation mechanisms (SHM targeting, DNA repair or both), or selection forces acting on the tumor clones, were different in FL samples compared to healthy control samples, or in late relapsed/transformed FL samples compared to earlier ones. Our analysis revealed differences in the distribution of mutations from each of the nucleotides when tumor and non-tumor clones were compared, while FL and transformed FL (t-FL) tumor clones displayed similar mutation distributions. Lineage tree measurements suggested that either initial clone affinity or selection thresholds were lower in FL samples compared to controls, but similar between FL and t-FL samples. Finally, we observed that both FL and t-FL tumor clones tend to accumulate larger numbers of potential N-glycosylation sites due to the introduction of new SHM. Taken together, these results suggest that transformation into t-FL, in contrast to initial FL development, is not associated with any major changes in DNA targeting or repair, or the selection threshold of the tumor clone.

Competing endogenous RNA networks related to prognosis in chronic lymphocytic leukemia: comprehensive analyses and construction of a novel risk score model

BackgroundChronic lymphocytic leukemia (CLL) is a heterogeneous B-cell malignancy that lacks specific biomarkers and drug targets. Competing endogenous RNAs (ceRNAs) play vital roles in oncogenesis and tumor progression by sponging microRNAs (miRNAs). Nevertheless, the regulatory mechanisms of survival-related ceRNA networks in CLL remain to be uncovered.MethodsWe included 865 de novo CLL patients to investigate RNA expression profiles and Illumina sequencing was performed on four CLL patients, two CLL cell lines and six healthy donors in our center. According to univariate Cox regression, LASSO regression as well as multivariate Cox regression analyses, we established a novel risk score model in CLL patients. Immune signatures were compared between the low- and high-risk groups with CIBERSORT and ESTIMATE program. Afterwards, we analyzed the relationship between differentially expressed miRNAs (DEmiRNAs) and IGHV mutational status, p53 mutation status and del17p. Based on the survival analyses and differentially expressed RNAs with targeting relationships, the lncRNA/circRNA-miRNA-mRNA ceRNA networks were constructed. In addition, the circRNA circ_0002078/miR-185-3p/TCF7L1 axis was verified and their interrelations were delineated by dual-luciferase reporter gene assay.ResultsTotally, 57 differentially expressed mRNAs (DEmRNAs) and 335 DEmiRNAs were identified between CLL patient specimens and normal B cells. A novel risk score model consisting of HTN3, IL3RA and NCK1 was established and validated. The concordance indexes of the model were 0.825, 0.719 and 0.773 in the training, test and total sets, respectively. The high-risk group was related to del(13q14) as well as shorter overall survival (OS). Moreover, we identified DEmiRNAs that related to cytogenetic abnormality of CLL patients, which revealed that miR-324-3p was associated with IGHV mutation, p53 mutation and del17p. The survival-related lncRNA/circRNA-miRNA-mRNA ceRNA networks were constructed to further facilitate the development of potential predictive biomarkers. Besides, the expression of circ_0002078 and TCF7L1 were significantly elevated and miR-185-3p was obviously decreased in CLL patients. Circ_0002078 regulated TCF7L1 expression by competing with TCF7L1 for miR-185-3p.ConclusionsThe comprehensive analyses of RNA expression profiles provide pioneering insights into the molecular mechanisms of CLL. The novel risk score model and survival-related ceRNA networks promote the development of prognostic biomarkers and potential therapeutic vulnerabilities for CLL.

Proteogenomics refines the molecular classification of chronic lymphocytic leukemia

Cancer heterogeneity at the proteome level may explain differences in therapy response and prognosis beyond the currently established genomic and transcriptomic-based diagnostics. The relevance of proteomics for disease classifications remains to be established in clinically heterogeneous cancer entities such as chronic lymphocytic leukemia (CLL). Here, we characterize the proteome and transcriptome alongside genetic and ex-vivo drug response profiling in a clinically annotated CLL discovery cohort (n = 68). Unsupervised clustering of the proteome data reveals six subgroups. Five of these proteomic groups are associated with genetic features, while one group is only detectable at the proteome level. This new group is characterized by accelerated disease progression, high spliceosomal protein abundances associated with aberrant splicing, and low B cell receptor signaling protein abundances (ASB-CLL). Classifiers developed to identify ASB-CLL based on its characteristic proteome or splicing signature in two independent cohorts (n = 165, n = 169) confirm that ASB-CLL comprises about 20% of CLL patients. The inferior overall survival in ASB-CLL is also independent of both TP53- and IGHV mutation status. Our multi-omics analysis refines the classification of CLL and highlights the potential of proteomics to improve cancer patient stratification beyond genetic and transcriptomic profiling.

CLL-137 SEQUOIA: Results of a Phase 3 Randomized Study of Zanubrutinib Versus Bendamustine + Rituximab (BR) in Patients With Treatment-Naïve (TN) Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma (CLL/ SLL)

The Bruton tyrosine kinase (BTK) inhibitor, zanubrutinib, was designed for high BTK specificity and minimal toxicity. SEQUOIA (NCT03336333) is a global, open-label, randomized phase 3 study in treatment-naïve patients with CLL/SLL without del(17p) who were unsuitable for fludarabine/cyclophosphamide/rituximab. Patients were randomized to receive zanubrutinib (160 mg twice daily) or bendamustine (day 1-2: 90 mg/m2) and rituximab (cycle 1: 375 mg/m2; cycles 2-6: 500 mg/m2); stratification factors were age (<65 years vs ≥65 years), Binet Stage, IGHV mutation, and geographic region. Primary endpoint was an independent review committee (IRC)-assessed progression-free survival (PFS). Secondary endpoints included investigator-assessed (INV) PFS, overall response rate (ORR), overall survival (OS), and safety. From October 31, 2017, to July 22, 2019, 479 patients were enrolled (zanubrutinib=241; BR=238). Baseline characteristics (zanubrutinib vs BR): median age, 70.0 years versus 70.0 years; unmutated IGHV, 53.4% versus 52.4%; del(11q), 17.8% versus 19.3%. With median follow-up of 26.2 months, PFS was significantly prolonged with zanubrutinib by IRC (HR 0.42; 2-sided P<.0001) and INV (HR 0.42; 2-sided P=.0001). Zanubrutinib treatment benefit occurred across age, Binet stage, bulky disease, del(11q) status, and unmutated IGHV (HR 0.24; 2-sided P<.0001), but not mutated IGHV (HR 0.67; 2-sided P=.1858). For zanubrutinib versus BR, 24-month PFS-IRC=85.5% versus 69.5%; ORR-IRC=94.6% versus 85.3%; complete response rate=6.6% versus 15.1%; ORR-INV=97.5% versus 88.7%; and 24-month OS=94.3% versus 94.6%. Select adverse event (AE) rates (zanubrutinib vs BR): atrial fibrillation (3.3% vs 2.6%), bleeding (45.0% vs 11.0%), hypertension (14.2% vs 10.6%), infection (62.1% vs 55.9%), and neutropenia (15.8% vs 56.8%). Treatment discontinuation due to AEs (zanubrutinib vs BR)=20 patients (8.3%) versus 31 patients (13.7%); AEs leading to death=11 patients (4.6%) versus 11 patients (4.8%). No sudden deaths occurred. In summary, zanubrutinib significantly improved PFS-IRC versus BR and was well tolerated, supporting the potential utility of frontline zanubrutinib in treatment-naïve CLL/SLL.

CLL-129 BRUIN CLL-313: A Phase 3 Open-Label, Randomized Study of Pirtobrutinib Versus Bendamustine Plus Rituximab in Untreated Patients With Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma (Trial in Progress)

Covalent Bruton's Tyrosine Kinase (BTK) inhibitors (BTKi) have transformed the management of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), but these treatments are not curative, and the majority of patients will require additional treatment. Covalent BTKi shares pharmacologic liabilities (e.g., low oral bioavailability, short half-life) that collectively may lead to suboptimal BTK target coverage, for example in rapidly proliferating tumors with high BTK protein turnover such as accelerating CLL/SLL, ultimately manifesting as acquired resistance in some patients. To address these limitations, pirtobrutinib, a highly selective, non-covalent BTKi that inhibits both wild type (WT) and C481-mutated BTK with equal low nM potency was developed. Determine whether pirtobrutinib is superior to bendamustine plus rituximab in treatment naïve CLL/SLL patients with retained 17p. BRUIN CLL-313 is a randomized, open-label, global phase 3 study comparing pirtobrutinib monotherapy versus bendamustine plus rituximab (BR) in treatment naïve CLL/SLL patients with retained 17p. Approximately 250 patients will be randomized 1:1. Randomization will be stratified by IGHV mutation status (mutated vs unmutated), and Rai stage (low/intermediate vs high). Patients in the BR arm are eligible to crossover to pirtobrutinib monotherapy if they experience progressive disease per iwCLL 2018 and confirmed by an independent review committee (IRC). Global; community hospitals, academic medical centers. Eligible patients are adults with confirmed diagnosis of CLL/SLL and who require therapy per iwCLL 2018 criteria. Key exclusion criteria include CNS involvement by CLL/SLL, Richter's transformation to DLBCL, prolymphocytic leukemia or Hodgkin lymphoma any time pre-enrollment, presence of 17p deletion, prior systemic therapy for CLL/SLL, and significant cardiovascular disease. Oral pirtobrutinib monotherapy versus bendamustine plus rituximab. The primary endpoint is progression-free survival (PFS) per iwCLL assessed by an independent review committee. Secondary endpoints include investigator-assessed PFS, overall survival (OS), overall response rate (ORR), duration of response (DoR), safety and tolerability, and patient-reported outcomes. The global study is currently enrolling patients (NCT05023980). Trial in Progress. Trial in Progress.

Diverse mutational landscapes in human lymphocytes

The lymphocyte genome is prone to many threats, including programmed mutation during differentiation1, antigen-driven proliferation and residency in diverse microenvironments. Here, after developing protocols for expansion of single-cell lymphocyte cultures, we sequenced whole genomes from 717 normal naive and memory B and T cells and haematopoietic stem cells. All lymphocyte subsets carried more point mutations and structural variants than haematopoietic stem cells, with higher burdens in memory cells than in naive cells, and with T cells accumulating mutations at a higher rate throughout life. Off-target effects of immunological diversification accounted for approximately half of the additional differentiation-associated mutations in lymphocytes. Memory B cells acquired, on average, 18 off-target mutations genome-wide for every on-target IGHV mutation during the germinal centre reaction. Structural variation was 16-fold higher in lymphocytes than in stem cells, with around 15% of deletions being attributable to off-target recombinase-activating gene activity. DNA damage from ultraviolet light exposure and other sporadic mutational processes generated hundreds to thousands of mutations in some memory cells. The mutation burden and signatures of normal B cells were broadly similar to those seen in many B-cell cancers, suggesting that malignant transformation of lymphocytes arises from the same mutational processes that are active across normal ontogeny. The mutational landscape of normal lymphocytes chronicles the off-target effects of programmed genome engineering during immunological diversification and the consequences of differentiation, proliferation and residency in diverse microenvironments.