IGHV-mutated Chronic Lymphocytic Leukemia Research Articles

Determining Clinical Practice Gaps for HCPs in Treating CLL Using CME-Based Surveys

Background: Targeted therapies have revolutionized the care of patients with chronic lymphocytic leukemia (CLL). In this context, new evidence continues to accumulate with the goal of optimizing the clinical use of these treatments as additional novel therapeutics continue to emerge. In this study, we sought to analyze current clinical practices of healthcare professionals (HCPs) regarding the care of patients with CLL, along with their awareness of the latest evidence on novel investigational therapies for these patients. Methods: Between March 2022 and June 2023, we conducted multiple live and online continuing medical education activities designed for experts to provide HCPs with evidence-based recommendations for the treatment of CLL. Baseline polling questions and surveys designed to assess key aspects of HCP knowledge, practice patterns, and confidence in this area were assessed across the activities. Results: Among 124 HCPs participating in educational activities between September 2022 and June 2023, 40% reported high confidence in their ability to plan optimal treatment strategies using BTK inhibitors for patients with CLL based on available evidence and guideline and expert recommendations. There was considerable discordance between HCP and expert treatment choices for various case scenarios in multiple educational activities; for example, for patient cases without del(17p)/ TP53-mutated/ IGHV-mutated CLL andbulky disease with diminished creatinine clearance, experts would recommend acalabrutinib with or without obinutuzumab for 77% of scenarios, while HCPs selected this treatment 35% of the time (Table). Among 57 HCPs who participated in educational programs from March 2022 to February 2023, 26% reported high confidence in their ability to recognize and manage AEs with BTK inhibitors in patients with CLL. Challenging AEs associated with the currently available BTK inhibitors also were self-identified by HCPs (Figure). One half of responding HCPs (52%) were able to identify characteristics that would qualify a patient for inpatient administration of the initial doses of venetoclax because of increased risk of tumor lysis syndrome (n = 48). Finally, among 213 HCPs participating in educational activities from September 2022 to June 2023, 43% were aware of recent data regarding the use of pirtobrutinib, an investigational noncovalent BTK inhibitor, for treating patients with CLL. Conclusion: Our data suggest that many HCPs are challenged to optimally incorporate contemporary treatment recommendations into the care of patients with CLL and struggle to maintain knowledge of investigational therapies that may affect the treatment landscape in the near future. Continuing educational activities designed to improve the knowledge, competence, and confidence of HCPs in the care of CLL would benefit patients with this disease. A more detailed data analysis of treatment trends and knowledge by geographic location and role on the care team will be presented.

Nucleosome repositioning in chronic lymphocytic leukemia.

The location of nucleosomes in the human genome determines the primary chromatin structure and regulates access to regulatory regions. However, genome-wide information on deregulated nucleosome occupancy and its implications in primary cancer cells is scarce. Here, we conducted a genome-wide comparison of high-resolution nucleosome maps in peripheral blood B cells from patients with chronic lymphocytic leukemia (CLL) and healthy individuals at single-base-pair resolution. Our investigation uncovered significant changes of nucleosome positioning in CLL. Globally, the spacing between nucleosomes-the nucleosome repeat length (NRL)-is shortened in CLL. This effect is stronger in the more aggressive IGHV-unmutated CLL subtype than in the IGHV-mutated CLL subtype. Changes in nucleosome occupancy at specific sites are linked to active chromatin remodeling and reduced DNA methylation. Nucleosomes lost or gained in CLL marks differential binding of 3D chromatin organizers such as CTCF as well as immune response-related transcription factors and delineated mechanisms of epigenetic deregulation. The principal component analysis of nucleosome occupancy in cancer-specific regions allowed the classification of samples between cancer subtypes and normal controls. Furthermore, patients could be better assigned to CLL subtypes according to differential nucleosome occupancy than based on DNA methylation or gene expression. Thus, nucleosome positioning constitutes a novel readout to dissect molecular mechanisms of disease progression and to stratify patients. Furthermore, we anticipate that the global nucleosome repositioning detected in our study, such as changes in the NRL, can be exploited for liquid biopsy applications based on cell-free DNA to stratify patients and monitor disease progression.

Integrating High-Throughput Dynamic BH3 Profiling and Molecular Phenotyping to Identify Therapeutic Vulnerabilities in CLL

Despite recent advances in chronic lymphocytic leukemia (CLL) therapy, such as the use of targeted agents including, Bruton's tyrosine kinase (BTK) inhibitor ibrutinib and the potent BCL-2 antagonist venetoclax, this disease remains incurable for most patients, who are refractory or become resistant to the novel agents. Thus, identifying new treatment regimens for CLL and building a precision medicine framework that can match CLL patients to the appropriate drugs are of high priority. To address this challenge, we optimized high-throughput dynamic BH3 profiling (HT-DBP), a functional assay that rapidly measures the initiation of apoptotic signaling after ex vivo exposure to drugs, for interrogation of CLL samples. The main advantages of this assay over other ex vivo assays are: (i) rapidity - under 24 hours, which is especially important in CLL, where cell viability substantially decreases after 24 hours; (ii) miniaturization - a very limited number of primary cells are required; and (iii) scalability - allowing to conduct hundreds of drug response tests in parallel on one 384-well plate. These features collectively maximize the information yield from a given patient sample. We recently identified prognostic genetic alterations and molecular subtypes of CLL, based on multi-omics profiling of >1100 CLL samples (Knisbacher et al., Nat Genet, in press). To determine if these molecular findings were associated with novel therapeutic vulnerabilities in CLL, we performed HT-DBP on 65 primary CLL samples previously characterized by exome, transcriptome and methylome profiling, using 42 FDA approved drugs that were selected for potential relevance to CLL biology. We isolated peripheral blood mononuclear cells (PBMCs) and cultured them in conditioned media derived from stroma cells to reduce spontaneous apoptosis. Target cells were treated with a drug for 20 hours followed by BH3 peptide exposure. Mitochondrial outer membrane permeabilization (MOMP) was then measured on digitonin-permeabilized cells in response to BH3-only synthetic peptides that mimic pro-apoptotic BCL-2 family proteins. Mitochondrial cytochrome c release was quantified as a measure of MOMP by flow cytometry, gating on CD19+ and CD5+ cells. This assay measures if the cell has moved closer to the threshold of apoptosis after drug treatment and thereby identifies drugs that enhance apoptosis priming. The peptides used in each experiment were derived from BIM or PUMA to measure increases in overall apoptotic priming, or BAD and MS1 peptides that identified BCL-2 and MCL-1 dependence, respectively. Our screen revealed differential drug-induced apoptotic priming for various drugs. As expected, venetoclax and ibrutinib were highly effective across CLL. Other drugs that demonstrated high priming included navitoclax (BCL-XL/BCL-2), nutlin-3 (MDM2), abexinostat (HDAC), gandotinib (JAK2), duvelisib (PI3K δ/γ), idelalisib (PI3Kδ) and cerdulatinib (SYK/JAK). The assay was robust, as indicated by an 0.92 median Pearson correlation across replicates. Additionally, the majority of drugs had greater effect on CLL samples than on healthy PBMCs (p<0.001, paired t-test), supporting their specificity. We focused our analysis on the differential drug effects among molecular subtypes of CLL. First, we found that IGHV-mutated CLLs (M-CLLs) became more primed to apoptosis than IGHV-unmutated CLLs (U-CLLs) across the panel of drugs (p<0.001, paired t-test) and significantly in response to fludarabine and umbralisib (FDR<0.1, t-test). Second, we compared drug-induced apoptotic priming among 8 CLL subtypes (i.e. RNA expression clusters (ECs); Knisbacher et al.). Notable among the many drug priming-EC relationships was that within M-CLL ECs, venetoclax was most effective in EC-m3 (high IL-10 expression) and least effective in EC-m2 (low IL-10 and enriched in trisomy 12). Interestingly, EC-m1, associated with high TFEC expression and poor outcome, was most sensitive to nutlin-3. For U-CLLs, EC-u1 was most sensitive to gandotinib and EC-u2 to navitoclax. EC-i, associated with the intermediate methylation subtype of CLL, was the most resistant EC to ibrutinib but was very sensitive to navitoclax, more than to any other drug. Altogether, we present a framework that links ex-vivo drug response with molecular features including expression subtypes to highlight new therapeutic opportunities in CLL.

Spatial organization and early signaling of the B-cell receptor in CLL.

Most chronic lymphocytic leukemia (CLL) clones express B-cell receptors (BcR) of both IgM/IgD isotypes; however, 5%–10% of CLL cases express isotype-switched immunoglobulin G (IgG). The early signaling and spatial patterning of the various BcRs at steady state and after activation are still fully unresolved. Herein, we show higher expression of the BcR signalosome elements and a more robust constitutive cell-intrinsic proximal BcR signaling in CLL with unmutated IGHV expressing IgM isotype (IgM U-CLL), compared with IGHV-mutated CLL (M-CLL) expressing either IgM or IgG isotypes. IgM in U-CLL is frequently located in the membrane plane in polarized patches, occasionally in caps, and sometimes inside the cells. Among M-CLL, IgM is scattered laterally in the membrane plane in a similar pattern as seen in normal B cells, whereas IgG is dispersed around the cell membrane in smaller clusters than in IgM U-CLL. Upon BcR engagement, both IgG and IgM expressing M-CLL showed attenuated signaling and only slight spatial reorganization dynamics of BcR microclusters and internalization, compared with the extensive reorganization and internalization of the BcR in IgM expressing U-CLL. The global gene signature of IgG M-CLL was closely related to that of IgM M-CLL rather than IgM U-CLL. Overall, we report fundamental differences in the basal composition, biochemical status, and spatial organization of the BcR in the three examined immunogenetic CLL subtypes that correlate with their clinical behavior. On the basis of our findings, IgG class-switched M-CLL likely represents the same disease as IgM M-CLL rather than a different biological and/or clinical entity.

Different Prognostic Impact of Recurrent Gene Mutations in IGHV-Mutated and IGHV-Unmutated Chronic Lymphocytic Leukemia: A Retrospective, Multi-Center Cohort Study By Eric, the European Research Initiative on CLL, in Harmony

Different Prognostic Impact of Recurrent Gene Mutations in IGHV-Mutated and IGHV-Unmutated Chronic Lymphocytic Leukemia: A Retrospective, Multi-Center Cohort Study By Eric, the European Research Initiative on CLL, in Harmony

Over-Expression of Activation-Induced Cytidine Deaminase in TCL1 Mice Leads to the Development of IGHV-Mutated CLL Clones That Associate with More Aggressive Disease

Introduction. Activation-induced cytidine deaminase (AID) mediates somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin genes (IG s), both of which are necessary for an effective immune response in normal B cells. An unwanted consequence of AID's role is its contribution to cellular transformation and tumor progression by aberrantly mutagenizing genes outside the IG locus (off-target mutations). In chronic lymphocytic leukemia (CLL), higher levels of IGHV clonal mutations in IGHV-mutated CLL (M-CLL) correlate strongly with better clinical outcomes, while, IGHV -unmutated (U-CLL) cases are associated with worse patient outcome. Paradoxically, U-CLL cells express significantly higher levels of AID than M- CLL cells.Eμ-T-cell leukemia-1 (TCL1) transgenic (Tg) mice are a valuable model of CLL. However, because SHM and CSR occur rarely in the leukemic clones of these animals, TCL1 Tg mice mimic only U-CLL and hence may not provide an understanding of the roles of SHM and CSR in disease evolution. To address this issue, we developed a TCL1 strain by interbreeding mice over-expressing AID in B lymphocytes (Eμ-TCL1xVκ-AID).Methods. B-cell clonal expansions were identified in spleen cells from 14 TCL1 and 23 TCL1xAID (DT) mice by amplifying cDNAs by PCR using consensus IGHV FR and isotype-specific primers for IgH chains and Vκ FR and Cκ for IgL chain genes. Ig sequences were compared to murine germline IGHV s and IGKV s, and IGHV-D-JH and IGKV-JK rearrangements by IMGT V-Quest. Six DT and 2 TCL1 tumors were transferred to SCID mice (5 mice/donor)Results.Clonal expansions: Monoclonal/oligoclonal expansions were detected in all TCL1 mice; these used only µ H and κ L chains. Similar expansions were detected in 21 of 23 DT mice; each animal bore an IgMκ+ clone and 5 also exhibited a concomitant IgGκ+ clone. Approximately 50% used VH1-55, VH11-2, or VH12-3.SHM: The IgM+ TCL1 clones had a mutation frequency of 0.05% for IGHV and 0.09% for IGKV . In contrast, the IGHV and IGKV mutation frequencies were considerably higher in DT mice: IGHV 0.47% in IgM+ and 3.0% in IgG+ clones, and IGKV 0.9% for IgM+ and IgG+ combined. These mutations localized more frequently in AID hotspots than coldspots at a ratio of ≥7:1.However, SHM did not affect all clones equally. Although the mutation frequency in VH12-3 and VH11-2 clones was higher (0.38%) than the TCL1 level (0.05%), it was considerably less than that found in the DT clones using other IGHV s (0.80%). In addition, no mutations were detected in VH1-55 clones from DT mice.IGHV gene use and SHM in clones that underwent CSR: Notably, in 2 of 5 instances the same IGHV-D-J rearrangement was found in IgMκ+ and IgGκ+ clones from the same mice. For the remaining 3, only the IgG+ version was detected. Also, within the IgG-only group, IGHV1-47 was used by 2 different clones and these were highly mutated (8.9%).Stereotyped IGHV-D-J and IGkV-Jk rearrangements: Among 32 IgM clones from DT mice, 4 expressed VH11-2 and Vκ14-126 and 8 expressed VH12-3 and Vκ4-91; these rearrangements were very similar to canonical anti-phosphatidylcholine-producing clones.DT mice develop more aggressive disease: we observed shorter median survival in DT mice comparing to TCL1 mice (279 days vs 356 days, P =0.0034)Transfer tumor development: Spleen cells from 6 CLL-bearing mice were transferred into SCID recipients, leading to a selected outgrowth of VH12-3/Vκ4-91 and VH11-2/Vκ14-126 clones. Survival in these mice inversely correlated with IGHV mutations (Figure).Summary and conclusions.Over-expression of AID in TCL1 mice leads to markedly increased SHM and CSR. However, SHM is not equivalent for all IGHV genes: certain IGHV s and IGK Vs are less susceptible to having major increases in the extent of SHM and the occurrence of CSR. This property resembles some human CLL IGHV s that rarely develop SHMs or undergo CSR despite the B-cell's ability to synthesize AID (e.g., many IGHV1-69+ clones in U-CLL patients). AID overexpression also led to IgG+ clones for which an IgM precursor was not found. This resembles those human stereotyped CLL clones that are only found as IgGs (e.g., stereotyped subsets 4 and 8). Finally, transfer of DT clones to SCID mice led to more IGHV and IGKV mutations and shorter survival opposite to that in M-CLL patients. Possibly this adverse effect might be a consequence of AID's aberrant action outside the IG loci which is facilitated in DT mice. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Genomic Complexity in Chronic Lymphocytic Leukemia Defined By Array-Based Analysis: Definitions, Associations with Other Biomarkers and Clinical Impact

Background: The advent of efficacious but also very expensive novel treatment regimens for high-risk chronic lymphocytic leukemia (CLL), has highlighted the need for accurate risk-stratification. Published evidence indicates that classic chromosome banding analysis (CBA) offers valuable complementary information to the routinely performed FISH analysis in CLL, potentially improving risk assessment. Indeed, recent studies identified complex karyotype (CK; defined as ≥3 abberations) as an independent predictive marker for refractoriness to ibrutinib and venetoclax, although for CLL, a precise evidence-based definition of genomic complexity (GC) is yet to be made. Array-based (CGH/SNP) analysis is capable of globally assessing the CLL genome, does not require in vitro mitogens, offers increased detection depth, and allows for easier standardization.Aims: 1. Determine a prognostic relevant definition of GC in CLL by array-based analysis. 2. Head-to-head comparison of the sensitivity of CBA versus array-based analysis.Methods: 1911 CLL and monoclonal B-cell lymphocytosis (MBL) patients (CLL=1895, 99% and MBL=16, 1%, respectively) were analyzed with CGH or SNP arrays mostly performed within the first year from diagnosis and before treatment administration (66% and 84%, respectively). For a subgroup (N=206) both CBA and array results were available (<1 year in between analyses). Main features of the studied cohort: median age: 60.7 years, males: 1252 (68%), Binet: A/B/C:623/324/141 (57%/30%/13%), IGHV-mutated CLL (M-CLL): 536/1082 (50%), del(13q): 1020/1910 (53%), del(11q): 353/1911 (19%), TP53 aberrations (TP53 abs, deletions and/or mutations of the TP53 gene): 205/1911 (11%). Recurrent chromosomal aberrations i.e. del(13)(q14), del(11)(q22.3) and del(17)(p13.1) defined by array were included in all analyses irrespective of size, while the cut-off for other aberrations are indicated.Results: First, analysis was performed using a size cut-off ≥5Mb according to Schoumans et al (Genes Chrom Cancer 2016). Applying the criteria ≥3 structural and/or numerical aberrations to define GC, 451/1911 cases (24%) displayed GC analyzed with CGH/SNP arrays. GC was significantly related (p<0.001) with advanced clinical stage (Binet B/C), IGHV-unmutated CLL (U-CLL), del(11q), del(13q) and TP53 abs. On univariate analysis, GC was associated with inferior OS (median: 10.0 vs 12.2 (p<0.001) years). GC cases were stratified into those with 3 (‘low-GC’, N=174, 39%), 4 (‘intermediate-GC’, N=103, 23%) and ≥5 (‘high-GC’, N=174, 39%) aberrations. High-GC cases differed significantly (p<0.05) from the other two subgroups, being enriched for U-CLL and TP53 abs (84% and 45%, respectively). The median OS was 5.8 years for the high-GC vs 10 years for the low- and 8.1 years for the intermediate-GC group (p<0.01). Only ‘high-GC’ remained significant (p<0.01) on multivariate analysis along with TP53 abs and IGHV status (see table).Lowering the array cut-off to 1Mb (N=353) resulted in detection of a higher number of aberrations (3.04 vs 2.34) compared to 5Mb cut-off (95% CI paired differences 0.475-0.924). Although a lower array cut-off resulted in upstaging of high risk GC (22 vs 12%), the median OS in the ‘high risk’ group was increased as compared to the cut-off of 5Mb (8.4 vs 5.5 years).Array-analysis (5Mb cut-off) showed a higher number of total detected aberrations as compared to CBA (2.35 vs 1.84, 95% CI paired differences 0.221-0.798). With array more patients were defined as GC with higher discriminatory power regarding survival (concordance index: 58 vs 55%) than with CBA. Prognostic impact of array vs CBA and of specific aberrations is currently being analyzed.Conclusion: According to this largest performed array-based analyses, current definitions for CK defined by the presence of ≥3 structural or numerical aberrations based on classical cytogenetic approaches may be suboptimal when applied for cases analyzed by array techniques. Instead, ≥5 aberrations detected by arrays were linked to high-risk disease, independent of clinical stage, IGHV and TP53 status. Since a lower array cut-off resulted in the detection of additional aberrations, without however been accompanied by the same dismal outcome as the 5Mb cut-off, further studies are warranted in order to define the actual prognostic relevance of smaller aberrations. [Display omitted] DisclosuresEldering:Roche: Research Funding; Gilead: Research Funding; Celgene: Research Funding. Ghia:Pharmacyclics LLC, an AbbVie Company: Consultancy; Janssen: Consultancy, Research Funding; Gilead: Consultancy, Research Funding, Speakers Bureau; Adaptive: Consultancy; AbbVie: Consultancy; Roche: Consultancy; Novartis: Research Funding. Stamatopoulos:Janssen Pharmaceuticals: Honoraria, Research Funding; Novartis SA: Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Strefford:Roche: Research Funding. Kater:Celgene: Consultancy, Research Funding; Johnson & Johnson: Research Funding; Abbvie: Research Funding.

The reference epigenome and regulatory chromatin landscape of chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a frequent hematological neoplasm in which underlying epigenetic alterations are only partially understood. Here, we analyze the reference epigenome of seven primary CLLs and the regulatory chromatin landscape of 107 primary cases in the context of normal B cell differentiation. We identify that the CLL chromatin landscape is largely influenced by distinct dynamics during normal B cell maturation. Beyond this, we define extensive catalogues of regulatory elements de novo reprogrammed in CLL as a whole and in its major clinico-biological subtypes classified by IGHV somatic hypermutation levels. We uncover that IGHV-unmutated CLLs harbor more active and open chromatin than IGHV-mutated cases. Furthermore, we show that de novo active regions in CLL are enriched for NFAT, FOX and TCF/LEF transcription factor family binding sites. Although most genetic alterations are not associated with consistent epigenetic profiles, CLLs with MYD88 mutations and trisomy 12 show distinct chromatin configurations. Furthermore, we observe that non-coding mutations in IGHV-mutated CLLs are enriched in H3K27ac-associated regulatory elements outside accessible chromatin. Overall, this study provides an integrative portrait of the CLL epigenome, identifies extensive networks of altered regulatory elements and sheds light on the relationship between the genetic and epigenetic architecture of the disease.

Presence of a Translocation Is Associated with Short Time to Treatment from Diagnosis in IGHV Mutated Chronic Lymphocytic Leukemia (CLL) Patients

Chronic Lymphocytic Leukemia (CLL) has a varied clinical course; some patients experience a long survival and others succumb to disease in a short time. Clinical factors correlated with either time to first treatment (TFT) and/or overall survival include Rai stage, IGHV somatic hypermutation status, fluorescence in situ hybridization (FISH) abnormalities, especially del(17p), karyotypic complexity and the presence of a cytogenetic translocation. Previous studies have included patients both at diagnosis and at various times throughout their diseases, and many included limited numbers of patients, precluding extensive analyses of relationships between the prognostic factors and their relative impact on clinical outcome. We sought to identify which factors determined within a short time of diagnosis (i.e., 1 year) were prognostic for TFT in untreated CLL patients.We identified 329 untreated CLL patients who had stimulated karyotypic and FISH analyses within 1 year of diagnosis seen at The Ohio State University (OSU). Patient characteristics and outcome were obtained from patient records. The studies were approved by the OSU IRB and were conducted according to the Declaration of Helsinki. A complex karyotype was defined as ≥ 3 unrelated aberrations by karyotype. Patient characteristics are given in Table 1. Translocations occurred in 87 (26.4%) patients: 38 balanced and 49 unbalanced translocations. Initial statistical analyses showed no large difference in TFT between balanced and unbalanced translations, so they were combined for final analyses. 144 patients (49 with and 95 without a translocation) had unmutated IGHV, and 144 patients (22 with and 122 without a translocation) had mutated IGHV. IGHV data were not available for 41 patients.TFT was calculated from date of diagnosis to date of first treatment. Untreated patients were censored at last known untreated date. Kaplan-Meier curves estimated TFT probability, and proportional hazard models were used to examine the association between potential risk factors and TFT. Using backward selection, variables with statistical significance when adjusting for all other covariates were included in the final model. To evaluate potential effect modifications, pairwise interactions among all the variables in the final model were examined and retained if statistically significant. Stata 14 (College Station TX) was used, and all tests were two-sided with statistical significance set at p<0.05.Median follow-up for censored patients was 30 months (range 0.03-102 months). Median TFT for the entire cohort was 47 months (95% confidence interval (CI) 40-63 months). In a univariable model, the following factors were significant: presence of a translocation (hazard risk (HR) 2.69, CI 1.91-3.78, p<0.001), Rai stage III/IV (HR 3.73, CI 2.32-5.99, p<0.001), complexity (HR 2.92, CI 1.98-4.31, p<0.001), unmutated IGHV (HR 3.54, CI 2.42-5.17, p<0.0001), del17p (HR 2.10, CI 1.31-3.37, p=0.002), del11q (HR 2.91,CI 1.92-4.40, p<0.001). In the multivariable model, there was significant effect modification of IGVH status on the relationship between translocation and TFT (p<0.001). In IGHV mutated patients, those with a translocation had over 5 times the risk of starting treatment relative to those without a translocation (HR 5.30, CI 2.76-10.17); however, in IGHV unmutated patients, a translocation did not significantly increase the risk of starting treatment (HR 1.32, CI 0.86-2.03). Independent of IGHV and translocation, Rai Stage (HR 2.07, CI 1.24-3.45, p=0.01) and del11q (HR 1.68, CI 1.09-2.60, p=0.02) were the only variables that remained statistically significant. Notably, once these variables were accounted for in the model, complexity did not provide additional significant prognostic information (p=0.12), perhaps due to its strong association with a translocation (p<0.001). In summary, the presence of a translocation in IGHV mutated patients appeared to negate the improved prognosis associated with mutated IGHV, but the presence of a translocation did not have an effect on TFT in high-risk IGHV unmutated patients (Figure 1). [Display omitted] [Display omitted] DisclosuresJones:Pharmacyclics, LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding. Andritsos:Hairy Cell Leukemia Foundation: Research Funding. Woyach:Morphosys: Research Funding; Acerta: Research Funding; Karyopharm: Research Funding. Awan:Pharmacyclics: Consultancy; Novartis Oncology: Consultancy; Innate Pharma: Research Funding.

Maintenance therapy in CLL: resolving the controversy

The standard of care for chronic lymphocytic leukaemia is fast changing. For younger patients, frontline chemoimmunotherapy with regimens such as fludarabine, cyclophosphamide, and rituximab 1 Thompson PA Tam CS O'Brien SM et al. Fludarabine, cyclophosphamide, and rituximab treatment achieves long-term disease-free survival in IGHV-mutated chronic lymphocytic leukemia. Blood. 2016; 127: 303-309 Crossref PubMed Scopus (381) Google Scholar , 2 Hallek M Fischer K Fingerle-Rowson G et al. Addition of rituximab to fludarabine and cyclophosphamide in patients with chronic lymphocytic leukaemia: a randomised, open-label, phase 3 trial. Lancet. 2010; 376: 1164-1174 Summary Full Text Full Text PDF PubMed Scopus (1545) Google Scholar are still appropriate standard-of-care, whereas kinase inhibitors such as ibrutinib are commonly used in older patients, and in those with relapsed disease. 3 Byrd JC Furman RR Coutre SE et al. Targeting BTK with ibrutinib in relapsed chronic lymphocytic leukemia. N Engl J Med. 2013; 369: 32-42 Crossref PubMed Scopus (1742) Google Scholar Attempts to intensify the regimen of fludarabine, cyclophosphamide, and rituximab with addition of drugs such as mitoxantrone 4 Faderl S Wierda W O'Brien S Ferrajoli A Lerner S Keating MJ Fludarabine, cyclophosphamide, mitoxantrone plus rituximab (FCM-R) in frontline CLL <70 years. Leuk Res. 2010; 34: 284-288 Summary Full Text Full Text PDF PubMed Scopus (39) Google Scholar resulted in additional toxicity and no improvement in therapeutic efficacy. Thus, current efforts are directed towards the use of less toxic drugs such as monoclonal antibodies and lenalidomide as post-induction therapy, commonly referred to as maintenance therapy. Rituximab maintenance versus observation alone in patients with chronic lymphocytic leukaemia who respond to first-line or second-line rituximab-containing chemoimmunotherapy: final results of the AGMT CLL-8a Mabtenance randomised trialRituximab maintenance therapy prolongs progression-free survival in patients achieving at least a PR to induction with rituximab plus chemotherapy, and the treatment is well tolerated overall. Although it is associated with an increase in infections, there is no excess in infection mortality, suggesting that remission maintenance with rituximab is an effective and safe option in the management of chronic lymphocytic leukaemia in early treatment phases. Full-Text PDF

Fludarabine, cyclophosphamide, and rituximab treatment achieves long-term disease-free survival in IGHV-mutated chronic lymphocytic leukemia

AbstractAccurate identification of patients likely to achieve long-progression-free survival (PFS) after chemoimmunotherapy is essential given the availability of less toxic alternatives, such as ibrutinib. Fludarabine, cyclophosphamide, and rituximab (FCR) achieved a high response rate, but continued relapses were seen in initial reports. We reviewed the original 300 patient phase 2 FCR study to identify long-term disease-free survivors. Minimal residual disease (MRD) was assessed posttreatment by a polymerase chain reaction-based ligase chain reaction assay (sensitivity 0.01%). At the median follow-up of 12.8 years, PFS was 30.9% (median PFS, 6.4 years). The 12.8-year PFS was 53.9% for patients with mutated immunoglobulin heavy chain variable (IGHV) gene (IGHV-M) and 8.7% for patients with unmutated IGHV (IGHV-UM). 50.7% of patients with IGHV-M achieved MRD-negativity posttreatment; of these, PFS was 79.8% at 12.8 years. A plateau was seen on the PFS curve in patients with IGHV-M, with no relapses beyond 10.4 years in 42 patients (total follow-up 105.4 patient-years). On multivariable analysis, IGHV-UM (hazard ratio, 3.37 [2.18-5.21]; P < .001) and del(17p) by conventional karyotyping (hazard ratio, 7.96 [1.02-61.92]; P = .048) were significantly associated with inferior PFS. Fifteen patients with IGHV-M had 4-color MRD flow cytometry (sensitivity 0.01%) performed in peripheral blood, at a median of 12.8 years posttreatment (range, 9.5-14.7). All were MRD-negative. The high rate of very long-term PFS in patients with IGHV-M after FCR argues for the continued use of chemoimmunotherapy in this patient subgroup outside clinical trials; alternative strategies may be preferred in patients with IGHV-UM, to limit long-term toxicity.

CLL with Mutated IGHV4-34 Antigen Receptors Is Clinically Heterogeneous: Antigen Receptor Stereotypy Makes the Difference

The IGHV4-34 gene is very frequent (~10%) in the B cell receptor immunoglobulin (BcR IG) gene repertoire of chronic lymphocytic leukemia (CLL). Over 30% of IGHV4-34 CLL cases can be assigned to different subsets with stereotyped BcR IG. The largest is subset #4 which represents ~1% of all CLL and ~10% of IGHV4-34 CLL and is considered a prototype for indolent disease. The BcR IG of a great majority (~85%) of IGHV4-34 CLL cases carry a significant load of somatic hypermutation (SHM), often with distinctive SHM patterns. This holds especially true for stereotyped subsets and is suggestive of particular modes of interactions with the selecting antigen(s). In detail, subsets #4 and #16, both involving IgG-switched cases (IgG-CLL), exhibit the greatest sequence similarity in SHM profiles, whereas they differ in this respect from IgM/D subsets #29 and #201. Prompted by these observations, here we explored the extent that these subset-biased SHM profiles in different IGHV4-34 stereotyped subsets were reflected in distinct demographics, clinical presentation, genomic aberrations and outcomes. Within a multi-institutional series of 20,331 CLL patients, 1790 (8.8%) expressed IGHV4-34 BcR IG. Following established bioinformatics approaches for the identification of BcR IG stereotypy, 573/1790 IGHV4-34 CLL cases (32%) were assigned to stereotyped subsets; of these, 340 cases (19% of all IGHV4-34 CLL and 60% of stereotyped IGHV4-34 cases) belonged to subsets #4, #16, #29 and #201, all concerning IGHV-mutated CLL (M-CLL). Clinicobiological information was available for 275/340 patients: #4, n=150; #16, n=44; #29, n=39; and #201, n=42. Comparisons between subsets revealed no differences in gender and age distribution. Interestingly, however, 36-43% of each subset cases were young for CLL (defined as patients aged ≤55 years), which is higher compared to general CLL cohorts, where young patients generally account for ~25% of cases. In contrast, significant differences were identified between subsets regarding: (i) disease stage at diagnosis, with >90% of IgG subsets #4 and #16 diagnosed at Binet stage A versus 83% in subset #201 and 74% in subset #29 (p=0.029); (ii) CD38 expression, ranging from 1% in subset #4 to 10% in subset #201 (p=0.013); (iii) the distribution of del(13q), peaking at a remarkable 92% in subset #29 versus only 37% in subset #16 (p<0.0001). Regarding other genomic aberrations, they were either absent (NOTCH1 mutations) or rare (SF3B1 mutations, trisomy 12, del(11q), TP53 aberrations due to either del(17p) and/or TP53 mutations). The sole exception concerned a high frequency (14%) of TP53 aberrations in subset #29 (p<0.05 compared with the other subsets), which is notable for M-CLL cases in general. Time to first treatment (TTFT) could be analyzed in 228 cases. IgG subsets #4 and #16 had significantly (p=0.036) longer TTFT (median TTFT: not yet reached) compared to the IgM/D subsets #29 and #201 (median TTFT: 11 and 12 years, respectively). In conclusion, we have identified distinct clinicobiological profiles for different stereotyped IGHV4-34 M-CLL subsets, highlighting subsets #4 and #16 as particularly indolent, which is important for both medical and social reasons, especially considering that a significant proportion of patients in these subsets are diagnosed at younger ages. Our findings support the notion that BcR IG stereotypy refines prognostication in CLL, superseding the crude immunogenetic distinction based on SHM load only. Additionally, the observed heterogeneity suggests that not all M-CLL are equal, prompting further research into the underlying biological background with the ultimate aim of tailored patient management. DisclosuresTausch:Gilead: Other: Travel support. Shanafelt:Glaxo-Smith_Kline: Research Funding; Genentech: Research Funding; Celgene: Research Funding; Polyphenon E Int'l: Research Funding; Hospira: Research Funding; Janssen: Research Funding; Pharmactckucs: Research Funding; Cephalon: Research Funding. Niemann:Gilead: Consultancy; Janssen: Consultancy; Roche: Consultancy; Novartis: Other: Travel grant. Langerak:InVivoScribe: Patents & Royalties: Licensing of IP and Patent on BIOMED-2-based methods for PCR-based Clonality Diagnostics.; DAKO: Patents & Royalties: Licensing of IP and Patent on Split-Signal FISH. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; Roche: Other: Lab services in the field of MRD diagnostics provided by Dept of Immunology, Erasmus MC (Rotterdam). Hallek:Celgene: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; AbbVie: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Roche: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Boehringher Ingelheim: Honoraria, Other: Speakers Bureau and/or Advisory Boards; Pharmacyclics: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Mundipharma: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Janssen: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Gilead: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding. Ghia:Janssen Pharmaceuticals: Research Funding.

The Nature of the Antigen Determines Leukemia Development and Behavior in the Eμ-TCL1 Transgenic Mouse Model of CLL

AbstractAbstract 181Studies conducted over the past decade have revealed a strong association between the mutational status of the immunoglobulin heavy-chain variable region (IGHV) genes and clinical course in patients with chronic lymphocytic leukemia (CLL). In patients with aggressive CLL, the leukemic cells typically express B cell receptors (BCRs) encoded by unmutated IGHV genes, whereas these genes are most often mutated in leukemic cells from patients with indolent disease. The mutational status of the IGHV genes reflects features of the antigen, such as antigen structure, form, presentation and affinity, indicating that the difference in the clinical course between IGHV-unmutated and IGHV-mutated CLL could be due to recognition of different types of antigens. In line with this possibility, recent studies have shown that IGHV-unmutated CLL (U-CLL) cells frequently express polyreactive BCRs that bind with low affinity to both microbial antigens and autoantigens translocated or exposed on apoptotic cells, whereas such reactivity is infrequent in IGHV-mutated CLL (M-CLL).To further explore the possibility that the clinical course in CLL is determined by the availability of particular types of antigenic stimuli, we investigated the impact of different antigen/BCR interactions on leukemia development and behavior in the Eμ-TCL1 transgenic mouse model of CLL. We initially established three cohorts of Eμ-TCL1 transgenic mice that expressed transgenic BCRs with different antigen specificity. Two of these cohorts expressed low-affinity unmutated transgenic BCRs reactive with the antigens phosphatidylcholine (PtC) and Sm (IgPtC and IgSm, respectively), whereas the third cohort expressed a high-affinity mutated transgenic BCR (IgHEL) specific for the antigen hen egg lysozyme (HEL). Of note, Sm is a ribonucleoprotein complex that is translocated to the surface of apoptotic cells and has been shown to be recognized by certain human U-CLL BCRs, whereas PtC is a cell membrane component that is exposed on senescent red blood cells and gut bacteria. Because no data are currently available regarding the reactivity of the M-CLL BCRs, we subdivided the cohort of Eμ-TCL1/IgHEL double transgenic mice into four additional cohorts. These included a cohort without antigen (Eμ-TCL1/IgHEL), a cohort in which HEL was provided as a foreign antigen (Eμ-TCL1/IgHEL double transgenic mice repetitively immunized with particles coated with HEL and CpG oligonucleotides), a cohort in which HEL was provided as a soluble autoantigen (Eμ-TCL1/IgHEL/sHEL triple transgenic mice) and a cohort in which HEL was provided as a membrane-bound autoantigen exposed on apoptotic cells (Eμ-TCL1/IgHEL/mHEL-KK triple transgenic mice). Each cohort consisted of 12–14 animals, of which at least 8 have been followed for >1 year.Animals from all cohorts developed CD5-positive B cell leukemias, but only in Eμ-TCL1/IgSm and Eμ-TCL1/IgPtC mice the leukemic cells expressed a transgenic BCR. In Eμ-TCL1/IgHEL mice the leukemias were always derived from the small percentage of B cells that express an endogenous BCR, whereas B cells that express the transgenic IgHEL BCR were never transformed. Interestingly, leukemia development and progression was more rapid in Eμ-TCL1/IgPtC than Eμ-TCL1/IgSm transgenic mice (7/14 at 6 months of age and 2/10 at 8 months of age, respectively). Since PtC is expressed as both a foreign- (gut flora) and self- (senescent red blood cells) antigen, we investigated whether suppression of gut flora will affect the growth of adoptively transferred Eμ-TCL1/IgPtC leukemias. Pretreatment of syngeneic recipient mice with a three-week course of broad-spectrum antibiotics significantly delayed leukemia growth, suggesting that PtC is more potent in driving the expansion of the leukemic clone when expressed as a foreign than self antigen.To summarize, these data demonstrate that U-CLL can be induced by both microbial antigens and autoantigens exposed on apoptotic cells, including autoantigens that are recognized by human CLL cells, such as Sm. In contrast, M-CLL can not be induced by chronic or repetitive antigen stimulation, regardless whether the antigen is provided as a foreign antigen, as a soluble autoantigen, or as a membrane-bound autoantigen exposed on apoptotic cells. Collectively, these data suggest that the mechanisms that drive U-CLL and M-CLL are different and indicate that only U-CLL is an antigen-driven disease. Disclosures:No relevant conflicts of interest to declare.

Enhanced levels of both the membrane-bound and soluble forms of IgM Fc receptor (FcμR) in patients with chronic lymphocytic leukemia

AbstractThe association of an IgM-Fc receptor (FcμR) with chronic lymphocytic leukemia (CLL) was suggested more than 30 years ago, but its authenticity has never been formally addressed. We examined the expression of the recently identified FcμR by B and T cells in CLL patients using receptor-specific monoclonal antibodies. CLL B cells (CD5+/CD19+) expressed much higher levels of FcμR on their cell surface than B cells from healthy donors. Such enhanced expression was more evident in immunoglobulin heavy chain variable region (IGHV)–mutated, CD38− or early Rai-stage CLL than in IGHV-unmutated, CD38+, or advanced Rai-stage CLL. Intriguingly, surface FcμR levels also were significantly elevated in the non-CLL B cells (CD5−/CD19+) and T cells (CD5+/CD19−), especially in IGHV-mutated CLL. CLL patients also had high serum titers of FcμR compared with healthy donors, and serum FcμR levels correlated significantly with circulating lymphocyte numbers but not with the IGHV mutation status or Rai stage. The serum FcμR was resolved as an ∼ 40-kDa protein, distinct from the cell surface FcμR of ∼ 60 kDa, and it was produced by both CLL B and non-CLL B cells. Mass spectrometric analysis revealed that the serum FcμR is a soluble form of the receptor encoded by an alternatively spliced FcμR transcript. These findings indicate enhanced levels of both membrane-bound and soluble forms of FcμR in CLL patients.

Importance of immunoglobulin heavy chain variable region mutational status in del(13q) chronic lymphocytic leukemia

Among prognostic factors for chronic lymphocytic leukemia (CLL), immunoglobulin heavy chain variable region (IGHV) mutation status and DNA analysis appear to be the most important. However, there is limited clinical outcome information for patients with the favorable-risk del(13q) and poor-risk unmutated IGHV. We retrospectively screened all patients with CLL at our institution between 2004 and June 2010 for del(13q) who also had an IGHV analysis. Unmutated IGHV was found in 38/79 patients; age, Rai stage, prior therapy, and time to evaluation were similar to those for patients with mutated IGHV. Unmutated patients were nearly four times more likely to harbor additional chromosomal aberrations compared to mutated patients (p < 0.001). During a median follow-up of 4.5 years, unmutated patients were more likely to demonstrate Rai stage progression (69% vs. 31%, log-rank p < 0.001) and to receive treatment (5-year cumulative probability of treatment: 65% vs. 32%, p < 0.001). Patients with unmutated CLL also had a shorter overall survival (5-year survival probability: 72% vs. 100%, p < 0.001). When limiting analysis to the 47 patients with del(13q) as a sole chromosomal abnormality, the 13 (28%) unmutated patients were more likely to demonstrate Rai progression (p < 0.001), to receive treatment (p = 0.02), and to have a shorter overall survival (p = 0.13) than the 34 mutated patients. These data suggest that del(13q) conveys an indolent course only in patients with IGHV-mutated CLL.