Unmutated IGHV Genes Research Articles

Complex evaluation of serum immunoglobulin levels in patients with chronic lymphocytic leukemia: Significant increase in IgA after first-line chemoimmunotherapy.

The impact of chemoimmunotherapy (CIT) on immunoglobulin (Ig) quantities in patients with chronic lymphocytic leukemia (CLL) has not been extensively studied. We analyzed Ig levels in 45 stable patients with indolent CLL (without indication for treatment) and 87 patients with progressive disease before first-line treatment. Fifty-five patients were evaluated again after the treatment with CIT. We observed significantly lower levels of all Ig classes and subclasses in patients with progressive disease compared to patients with indolent disease. After treatment, median IgA increased from 0.59 g/L to 0.74 g/L (p = 0.0031). In stable patients, lower IgA2 was associated with shorter time to first treatment, although it did not reach statistical significance (p = 0.056). Shorter overall survival was observed in patients with progressive disease and lower IgG2 (p = 0.043). Surprisingly, among the patients with progressive CLL, unmutated IGHV genes were associated with higher levels of IgG, IgG1 and IgM, while TP53 mutation and/or 17p deletion were associated with higher levels of IgA and IgA1. CIT may lead to increase in IgA levels. Hypogammaglobulinemia is more common in patients with progressive CLL and unmutated IGHV or TP53 dysfunction.

Long-term follow up of the combination of ofatumumab, high-dose methylprednisolone, and lenalidomide for untreated chronic lymphocytic leukemia with biomarker analysis

BackgroundAdvancements in frontline therapy and chemotherapy-sparing treatments in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) have altered the treatment algorithms of this disease. We present a frontline alternative for treatment- naïve (TN) CLL/SLL patients. MethodsThis was a single-center, phase 2 study of high-dose methylprednisolone (HDMP) and ofatumumab with lenalidomide and ofatumumab consolidative therapy for all comers with TN CLL/SLL. Treatment was continued until disease progression or intolerable side effects. Patients were assessed for response per iwCLL 2008 criteria after completing cycles 3 and 12. ResultsForty-five patients were enrolled (median age, 62.6 years). High-risk features included del17p (18%), Del11q (22%), and unmutated IGHV gene (76%). Median treatment duration was 32·2 (2·7-75·9) months. Thirty-six patients discontinued treatment due to disease progression (22%), adverse events (40%), allogeneic hematopoietic cell transplantation (allo-HCT) (7%), consent withdrawal (4%), and secondary malignancies (7%). The best overall and complete response rates were 96& and 29% respectively. At median follow-up of 61·7 (5·6-84·9) months, 9 patients remained on treatment. Median progression-free survival was 54·4 (2·9-77·6) months. Three patients underwent allo-HCT after a median of 3 (3-4) treatment cycles. Treatment was well tolerated, with a grade 3/4 infusion reaction in one patient. The most common grade 3/4 hematological adverse event was neutropenia (69%). Four patients had grade 3/4 infections. No grade 3/4 tumor flares, tumor lysis syndrome, or thrombosis were observed. ConclusionThe combination of ofatumumab, HDMP, and lenalidomide was effective and relatively well tolerated in treatment-naive CLL/SLL. Its role in the frontline setting remains unclear given the current available and effective treatment options. FundingThe funders had no role in the study.

CO139 Acalabrutinib (AKA) Versus Venetoclax With Obinutuzumab (VEN+OBI) in Patients With Untreated Chronic Lymphocytic Leukemia (CLL) and Unmutated IGHV Genes – Indirect Comparison

CO139 Acalabrutinib (AKA) Versus Venetoclax With Obinutuzumab (VEN+OBI) in Patients With Untreated Chronic Lymphocytic Leukemia (CLL) and Unmutated IGHV Genes – Indirect Comparison

Telomere Length and DNA Methylation Epitype Both Provide Independent Prognostic Information in CLL Patients; Data from the UK CLL4, Arctic and Admire Clinical Trials

The presence of TP53 aberrations ( TP53ab) and/or unmutated IGHV genes (U-CLL) helps select initial treatment for CLL patients (Hallek 2018 & Walewska 2022). However, many other biomarkers identified in recent years have failed to impact clinical decisions due to their coexistence with poor-risk indicators and uncertainty in their predictive abilities, often without suitable validation in long-term Phase II/III trials with comprehensive molecular analysis. We evaluated the clinical significance of two biomarkers, DNA methylation-based epitype (DME) and telomere length (TL), in (immuno-)chemotherapy clinical trials using samples from UKCLL4 (n=304) and ARCTIC/ADMIRE (ARC/ADM) (n=215). To our knowledge, DME and TL have not been assessed in a single study. Therefore, we utilized previously published DME (Wojdacz, 2019) and TL data (Strefford, 2015 & Norris, 2019), supplemented with new MMQ-PCR data (n=60). TL cut-offs of short (TL-S, <2.92kb), intermediate (TL-I, 2.92-3.57kb), and long (TL-L, >3.57kb) and DME classifications of n-CLL (naive B-cell-like), i-CLL (intermediate B-cell-like) and m-CLL (memory B-cell-like) were employed. 73% of m-CLL and 50% of n-CLL patients harboured TL-L and TL-S, respectively (p<0.001, Figure 1). Additionally, n-CLL and TL-S were associated with poor-risk indicators, such as TP53ab and del(11q) (p<0.05, Figure 1). We then assessed the impact of 10 clinico-biological features, including DME, TL, age and treatment arm, on progression-free survival (PFS) and overall survival (OS) in CLL4 and ARC/ADM cohorts using univariate analysis (UA). The n-CLL group exhibited the shortest PFS and OS in the ARC/ADM cohort (PFS hazard ratio (HR):4.47, 95% confidence interval (CI):2.56-7.82 & OS HR:3.95, CI:1.66-9.35, p<0.01), greater than the presence of TP53ab (PFS HR:4.39, CI:2.69-7.17 & OS HR:3.77, CI:1.94-7.33, p<0.001). In CLL4, whilst TP53ab was the strongest predictor of PFS and OS (PFS HR:3.61, CI:2.39-5.44 & OS HR: 3.66, CI:2.4-5.57, p<0.001), both n-CLL (PFS HR:1.96, CI:1.32-2.9 & OS HR:2.8, CI:1.81-4.34, p<0.001) and TL-S (PFS HR:2.36, CI:1.7-3.29 & OS HR:2.66, CI:1.87-3.76, p<0.001) were in the top five predictors of PFS and OS along with U-CLL and biallelic ATM inactivation ( biATM). Next, we performed Kaplan-Meier subgroup analysis, investigating DME in TL subgroups and vice versa. Examination of both biomarkers in the opposing subgroups in the ARC/ADM cohort, showed that TL could further stratify the i-CLL subgroup, with TL-L predicting longer PFS (median:6.12 years) compared to TL-S (HR:5.78, CI:2.34-14.33, median:3.8 years, p<0.001) or TL-I (HR:3.29, CI:1.4-7.76, median:4.35 years, p<0.01). As this pairwise analysis suggested that DME and TL may differentially contribute to outcome, we performed a multivariate cox regression, whilst controlling for confounding variables such as TP53ab and U-CLL. Covariates that were significant in UA were included in a stepwise backwards elimination process until a final model was reached. The CLL4 models were based on 246 subjects with 221 PFS and 205 OS events, ARC/ADM models were based on 138 and 176 subjects with 86 and 45 events for PFS and OS, respectively. For PFS models, TL-S emerged as significant (CLL4 HR:2.14, CI:1.39-3.3, p<0.001 & ARC/ADM HR:2.18, CI:1.17-4.05, p<0.01) with a HR lower than TP53ab (CLL4 HR:3.38, CI:2.13-5.37 & ARC/ADM HR:4.94, CI:2.58-9.48, p<0.001). DME emerged as significant for PFS in the CLL4 cohort (n-CLL HR:2.35, CI:1.37-4.05, p<0.01), along with SF3B1 mutation and treatment arm (Figure 2). In ARC/ADM, U-CLL, TP53ab, TL-S, del11q and biATM emerged as significant predictors of PFS (Figure 2). For OS, in both cohorts, TP53ab (CLL4 HR:2.77, CI:1.74-4.4, p<0.001 & ARC/ADM HR:3.28, CI:1.64-6.55, p<0.001) and DME (CLL4 HR:2.07, CI:1.15-3.73, p<0.05 & ARC/ADM, HR:3.4, CI:1.14-10.12, p<0.05) were found to predict shorter survival (Figure 2). Currently, we are integrating additional IGHV/IGLV data into our analysis, including the presence of IGLV3-21R110. In conclusion, by assessing the individual contribution of DME and TL to disease survival, we found that both variables offer valuable independent prognostic information when included in statistical models with poor-risk genomic lesions. TL and DME could help identify IGHV-mutated patients destined to respond poorly to (immuno-)chemotherapy, that might be more favourably treated with targeted agents.

Clinical Outcomes with Venetoclax-Based Treatment Regimens in Patients with Chronic Lymphocytic Leukemia (CLL)

INTRODUCTION We aim to identify factors that impact outcomes of venetoclax for patients with CLL treated in routine practice at a tertiary center. We report on the use of venetoclax in the most frequently encountered disease scenarios: first-line, relapsed/Bruton tyrosine kinase inhibitor (BTKi)-naïve, and relapsed/BTKi-exposed. METHODS We identified patients who received venetoclax therapy for CLL (between 4/2012-4/2023) from the Mayo Clinic CLL Database. Undetectable measurable residual disease (uMRD) was defined as <1 CLL cell per 10,000 leukocytes using 8-color flow cytometry on peripheral blood (PB) or bone marrow (BM). Overall survival (OS) was defined as the time from venetoclax start until date of death or last known to be alive. Treatment-free survival (TFS) after venetoclax was defined as the time from venetoclax start until the earliest of date of next treatment, or death. Kaplan-Meier was used to display OS and TFS. Multivariable Cox proportional hazards regression models were used to estimate associations of factors with time-to-event outcomes. RESULTS A total of 155 patients received venetoclax: firstline therapy (in combination with obinutuzumab, n=55) and relapsed CLL (n=100; 17 had relapsed/BTKi-naïve CLL, and 83 had previously received BTKi [55 with progression after BTKi], relapsed/BTKi-exposed). The median follow-up for the cohorts of first-line therapy, relapsed/BTKi-naïve, and relapsed/BTKi-exposed was 12.9 months, 37.0 months, and 27.6 months, respectively. Baseline characteristics at the time of venetoclax initiation for all patients are shown in Table 1. The median TFS for the overall cohort was 39.0 months. The median OS was 54.6 months. Among patients treated with venetoclax as first-line therapy (n=55), the 2-year TFS ( Figure 1) and 2-year OS rates were both 91%. MRD testing was performed in 28 patients and was uMRD in 23 (82%) patients (only PB assessed, n=7; only BM assessed, n=2; PB and BM assessed, n=14). Detectable MRD was identified in 3 patients, and 2 patients had discordant results (PB uMRD and BM detectable disease). Among patients treated with venetoclax in the relapsed/BTKi-naïve setting (n=17), the 2-year TFS rate was 73% ( Figure 1) and the 2-year OS rate was 100%. MRD testing was performed in 7 patients and was uMRD in all 7 (100%) (only PB assessed, n=3; only BM assessed, n=2; PB and BM assessed, n=2). The median time to first uMRD result was 14.1 months. Among relapsed/BTKi-exposed venetoclax-treated patients (n=83), the median TFS was 26.9 months ( Figure 1), and the median OS was 39.4 months.In this subgroup, the median TFS for patients with (n=55) and without (n=28) prior disease progression on prior BTKi were 22.3 and 42.3 months, respectively. Median TFS with venetoclax monotherapy (n=30) was 24.0 months, venetoclax in combination with rituximab (n=37) was 26.9 months, and venetoclax in combination with obinutuzumab (n=16) was 39.0 months. BTKi-exposed patients that were chemotherapy-naïve (n=27) and chemotherapy-exposed (n=56) had median TFS of 29.1 and 24.0 months, respectively. MRD testing was performed in 28 patients and was uMRD in 16 (57%) patients (only PB assessed, n=9; only BM assessed, n=2; PB and BM assessed, n=5). Detectable MRD was identified in 11 patients, and 1 had discordant results (PB uMRD and BM detectable disease). The median time to first uMRD result was 11.5 months. TP53 disruption, unmutated IGHV genes, older age, complex karyotype (CK; defined as more than 3 chromosomal aberrations on CpG stimulated karyotype), and disease progression on prior BTKi were associated with shorter TFS in the overall cohort on univariate analysis. TP53 disruption, older age, CK, and disease progression on prior BTKi were associated with shorter OS in the overall cohort on univariate analysis. Multivariable analysis was performed by including only those patients where all variables significant in univariable analysis were available (OS model n=65, TFS model n=53). In these models, only CK was significantly associated with shorter TFS (HR 8.5; 95%CI 2.5-29.1; P<0.001) and shorter OS (HR 4.1; 95%CI 1.2-14; P=0.03). CONCLUSIONS Patients with BTKi-exposed CLL, particularly those with prior disease progression on BTKi, had worse outcomes. Our study identified CK as one of the most important baseline predictors of adverse TFS and OS in the overall cohort of patients, supporting karyotype assessment for prognostication prior to venetoclax treatment.

Evidence of Defective Cell-Autonomous Signalling By B Cell Receptors in CLL Stereotyped Subset #1

B cell receptor (BCR)-mediated signalling is central in the pathogenesis of chronic lymphocytic leukaemia (CLL), and represents a target for pharmacological intervention. Notably, so far characterized CLL-derived BCRs are capable of triggering cell-autonomous intracellular signalling through homotypic interactions without requirement for exogenous antigens. Approximately 41% of all CLL cases can be classified in subsets based on homologous, otherwise known as stereotyped, BCRs expressed by the malignant clones, sharing remarkably similar sequences in the VH CDR3 region and highly restricted immunoglobulin heavy and/or light variable genes. Since cases within a subset display clinicobiological consistency in contrast with the pronounced heterogeneity of CLL at large, analysis of the mechanism of BCR-mediated signalling can unveil correlations between molecular details of BCR activation and the course of the disease. Our present study focused on BCRs of stereotyped subset #1, the second largest stereotyped subset, known for a particularly aggressive clinical course. Cases belonging to subset #1 express unmutated IGHV genes of phylogenetic clan I (namely, IGHV1, IGHV5 and IGHV7 subgroup genes) partnered by light chains encoded by the IGKV1-39/1D-39 gene. First, we used X-ray crystallography to determine the high-resolution crystal structures of the BCR Fab fragments from three subset #1 cases utilizing the IGHV1-2, IGHV1-3, and IGHV5-10 genes partnered with IGKV1-39/1D-39 to obtain information on the combining sites' structures and possible modes of intermolecular interactions that may mediate cell-autonomous signalling. Analysis of the experimental structures revealed that, despite the high sequence homology of the three proteins, the heavy and light chain CDR loops in the three BCR Fabs are arranged differently to shape binding cavities with distinct dimensions and polarity, thus suggesting that distinct antigens may be the physiological target of each subgroup. Unlike BCRs from other subsets previously characterised, the intermolecular contacts between the subset #1 Fab molecules in the three crystals involve diverse residues that are not indicative of a shared homotypic interaction. In addition, the surfaces involved in the contacts are small and the computed energies for the protein-protein contacts observed are suggestive at best of very weak interactions. In accordance to this, when we used analytical ultracentrifugation along with chromatographic and calorimetric methods to detect BCR Fab oligomerization in solution, no evidence of self-association was obtained, thus indicating that the receptors in solution either do not interact, or are characterized by self-dissociation constants that exceed the sensitivity limit of the techniques employed. To assess whether the cellular environment could promote self-association and signalling activity of the full-length BCR, we expressed subset #1 BCRs in the model triple knockout (TKO) pre-B-cell line, designed to measure Ca 2+ ion influx using cytofluorimetry. All subset #1 BCRs tested were unable to mobilize autonomously the Ca 2+ ions, despite being fully expressed on the cell surface and capable of signalling upon stimulation with anti-BCR antibody as a proxy for exogenous ligand. This result is in sharp contrast with the behaviour of all other CLL-derived BCRs so far characterised in this system that could activate a cell-autonomous signal as evidenced from a persistent Ca 2+ ion influx in the transduced cells without requiring external stimuli. Hence, subset #1 BCRs differ from the so far characterized BCRs from CLL cases belonging to other stereotyped subsets, including clinically aggressive subset #2 and its satellite subset #169 as well as the clinically indolent stereotyped subset #4. In conclusion, our combined biochemical and cellular analysis demonstrates that a constitutively active cell-autonomous signalling may not be a unifying feature of all CLL clones as previously proposed, and that other mechanisms may be at the roots of the BCR-mediated signal required for leukemic cell proliferation and development of CLL.

Expression and Activity of the NF-κB Subunits in Chronic Lymphocytic Leukaemia: A Role for RelB and Non-Canonical Signalling.

Canonical NF-κB signalling by p65 (RelA) confers chemo-resistance and poor survival in chronic lymphocytic leukaemia (CLL). The role of non-canonical NF-κB signalling (leading to RelB and p52 subunit activation) in CLL is less understood, but given its importance in other B-cell tumour types, we theorised that RelB and p52 may also contribute to the pathology of CLL. DNA binding activity of all five NF-kB subunits, p65, p50, RelB, p52, and c-Rel, was quantified using ELISA and correlated to ex vivo chemoresistance, CD40L-stimulated signalling (to mimic the lymph node microenvironment), and clinical data. Importantly, we show for the first time that high basal levels of RelB DNA binding correlate with nuclear RelB protein expression and are associated with del(11q), ATM dysfunction, unmutated IGHV genes, and shorter survival. High levels of nuclear p65 are prevalent in del(17p) cases (including treatment-naïve patients) and also correlate with the outcome. CD40L-stimulation resulted in rapid RelB activation, phosphorylation and processing of p100, and subsequent CLL cell proliferation. These data highlight a role for RelB in driving CLL cell tumour growth in a subset of patients and therefore strategies designed to inhibit non-canonical NF-κB signalling represent a novel approach that will have therapeutic benefit in CLL.

Clonal composition and differentiation stage of human CD30+ B cells in reactive lymph nodes.

Normal CD30+ B cells represent a distinct B-cell differentiation stage with features of strong activation. We lack an in depth understanding of these cells, because they are not present in peripheral blood and are typically very rare in reactive lymphoid organs. CD30+ B cells have been discussed as a potential precursor population for the malignant CD30+ Hodgkin and Reed-Sternberg cells in classical Hodgkin lymphoma. As CD30+ B cells can be more numerous in some cases of reactive lymphadenitis, we aimed to characterize these CD30+ B cells in terms of their differentiation stage and clonal composition, also as a means to clarify whether such CD30+ B-cell populations may represent potential precursor lesions of Hodgkin lymphoma. We microdissected single CD30+ B cells from tissue sections of eight reactive lymph nodes with substantial numbers of such cells and sequenced their rearranged immunoglobulin (Ig) heavy chain V region (IGHV) genes. The CD30+ B cells were polyclonal B cells in all instances, and they not only encompass post-germinal center (GC) B cells with mutated IGHV genes, but also include a substantial fraction of pre-germinal center B cells with unmutated IGHV genes. In five of the lymph nodes, mostly small clonal expansions were detected among the CD30+ B cells. Most of the expanded clones carried somatically mutated IGHV genes and about half of the mutated clones showed intraclonal diversity. We conclude that in human reactive lymph nodes with relatively many CD30+ B cells, these cells are a heterogenous population of polyclonal B cells encompassing activated pre-GC B cells as well as GC and post-GC B cells, with some clonal expansions. Because of their polyclonality and frequent pre-GC differentiation stage, there is no indication that such cell-rich CD30+ B-cell populations represent precursor lesions of Hodgkin lymphoma.

Immunoglobulin genes and stereotyped antigenic receptors in chronic lymphocytic leukemia and other lymphoproliferative diseases

Introduction. The mutational status of immunoglobulin heavy chain variable region genes (IGHV) is the most important prognostic factor in chronic lymphocytic leukemia (CLL). Furthermore, a significant narrowing of the IGHV gene repertoire is found in CLL and other lymphoproliferative diseases. Aim — to review the publication data on the IGHV genes repertoire and mutational status in CLL and other lymphoproliferative diseases regarding their clinical significance. General information. Nucleotide sequence of rearranged IGHV genes is a unique marker of a tumor clone. CLL patients with unmutated IGHV genes have an extremely unfavorable disease outcome in contrast to the patients with mutated IGHV genes. Patients with mutated IGHV genes benefit from conventional immunochemotherapy, while non-mutated IGHV patients require therapy escalation with new targeted drugs. The study of IGHV genes and stereotyped antigen receptors repertoire makes possible to identify additional groups of CLL patients with specific genetic and clinical features. Stereotype receptors are also detected in other lymphoproliferative diseases, but their clinical significance has not yet been defined. However, stereotyped receptors are found to be disease-specific.

EVALUATION OF CD38 ANTIGEN EXPRESSION IN CHRONIC LYMPHOCYTIC LEUKEMIA: THE EXPERIENCE OF CLINICAL IMMUNOLOGY DEPARTMENT, NATIONAL RESEARCH CENTER FOR RADIATION MEDICINE, NATIONAL ACADEMY OF MEDICAL SCIENCES OF UKRAINE

Summary. CD38 antigen is a negative prognostic marker of chronic lymphocytic leukemia (CLL). The question of what cut-off level of CD38 antigen expression should be considered as prognostically significant, remains debatable. Aim: to evaluate the optimal cut-off level for CD38 antigen in primary untreated CLL patients for the prognosis of disease and its association with the mutational status of immunoglobulin heavy chain genes (IGHV genes). Objects and methods: flow cytometric and molecular-genetic analysis (mutational status of IGHV genes using polymerase chain reaction followed by direct sequencing) were performed in the groups of 106 primary previously untreated CLL patients. Time-to-treatment, progression-free survival and overall survival were evaluated using the Kaplan-Meier method and Cox regression. Results: The prognostic value of cut-off level CD38 antigen expression ≥ 20% or ≥ 30% does not differ significantly. Lowering of cut-off level to 7% or 5% was unsignificant. The specificity of the evaluation of the mutational status of IGHV genes based on CD38 antigen expression was low, since CD38 expression in almost half of CLL cases with unmutated IGHV genes not differ from CLL cases with mutated IGHV genes. Conclusion: the obtained data confirmed the prognostic value of CD38 antigen expression in patients at the initial stages of disease (A0-AI), primarily for assessing the duration of overall survival.

Obinutuzumab in combination with chlorambucil in first line treatment of elderly patients with chronic lymphocytic leukemia

Introduction. Optimal therapy for elderly patients with chronic lymphocytic leukemia (CLL) is the subject of intensive research.Aim – to study the safety of obinutuzumab, as well as the selection of the optimal scheme of its use in patients with CLL, complicated by diabetes mellitus, renal insuffi ciency, cardiac comorbidity.Materials and methods. The study included primary patients with CLL having indications requiring therapy. The inclusion criteria were: Cumulative Illness Rating Scale, CIRS) (CIRS) > 6 and or glomerular fi ltration rate (GFR) < 70 mL/min, the lower limit of GFR was not restricted. The study focused on patients with diabetes mellitus, renal failure and signifi cant cardiac pathology. Patients with Richter’s syndrome, CNS involvement, HBs-antigen, and 17p deletion were not included. In the fi rst cycle obinutuzumab was administered at a dose of 100 or 25 mg on the fi rst day and 900 or 975 mg on the second day, then at a dose of 1000 mg on days 8 and 15. For all subsequent cycles, obinutuzumab was given at a dose of 1000 mg on day 1. The dosage of chlorambucil was 10 mg/m2 from days 1 to 7. Treatment cycles in totals of 6 were repeated every 28 days.Results. The study included 90 patients. Median age was 73.5 years, range – 60–89 years, there were 49 men (54 %) and 41 women (46 %). Twenty-four patients (27 %) had stage C, IGHV unmutated status was detected in 76 % of patients. The median creatinine clearance was 48.6 mL/min (25–110). The median CIRS score was 3 (range – 1–14). Thirty-one patients (34 %) had signifi cant cardiovascular comorbidity (previous myocardial infarction, coronary artery stenting or bypass, HF ≥ II NYHA, peripheral artery disease) as well as hemodynamically signifi cant valvular disease. Fifteen patients (17 %) had diabetes mellitus and 71 patients (79 %) had creatinine clearance < 70 ml/min. Infusion reactions to obinutuzumab grade ≥ II were reported in 29 patients (32 %). Hospitalization on the day of administration or the next day after the fi rst administration was required in 5 cases (5.5 %). Twenty-seven (30 %) patients could not complete 6 cycles. The largest number of patients (14 people, 15.5 %) stopped treatment after 1 course. The causes were the development of persistent cytopenia (n = 4), grade IV reaction to obinutuzumab (n = 3), patient’s refusal (n = 2), infectious complications (n = 2), severe tumor lysis syndrome (n = 1), acute pancreatitis (n = 1) and toxicodermia (n = 1). The leading cause of premature discontinuation on subsequent cycles was persistent neutropenia. Progression during treatment occurred in 3 patients only. Overall survival was signifi cantly predicted by CIRS (maximum discriminatory value of 3, p = 0.013) as well as GFR < 50 mL/min (p = 0.03). No other associations were identifi ed. At least 1 episode of grade III–IV neutropenia occurred in 41 % of patients. Grade IV neutropenia was associated with creatinine clearance < 60 mL/min (p = 0.05), baseline neutrophil level < 2 × 109/L (p = 0.0001), baseline monocyte level < 0.3 × 109/L (p = 0.007) and age > 70 years (p = 0.01). The effectiveness of treatment was evaluated in patients who completed at least 3 cycles of therapy. Complete remission was achieved in 26 patients (35 %), partial remission – in 41 (54 %), stabilization – in 4 (5 %), progression was noted in 3 (4 %). Sixteen patients (18 %) were not available to respond to the assessment. Minimal residual disease < 0.01 % in the bone marrow after completion of treatment was found in 17 patients (19 %), within 0.01–0.9 % – in 25 (28 %) patients. The median follow-up from the date of therapy initiation was 39.7 months (range – 0.6–72 months). The median relapse-free survival was not reached, and 2- and 3-year survival rates were 81 and 62 %, respectively. Poor relapse-free survival signifi cantly correlated with unmutated IGHV genes (HR = 2.4, 95 % CI: 1.12–5.0, p = 0.02) and partial response as opposed to complete response (HR = 3.35; 95 % CI: 1.45–7.7, p = 0.03).Conclusion. The results of our study have practical implications as obinutuzumab is actively integrated into modern treatment regimens. Infusion reactions pose a high risk of complications in elderly patients. The dose of obinutuzumab on day 1 of administration in elderly patients, should not exceed 25 mg. The G-Clb regimen may not be optimal in patients over 75 years of age due to the unpredictable risk of complications. In patients at high risk of neutropenia, it may be appropriate to consider primary prophylaxis. ClbG is an effective regimen that resulted in high rate of MRD-negative responses and prolonged relapse-free survival.

High Cell Death Rates at Start of Ibrutinib Therapy Predict for Deeper Remissions in Patients with Chronic Lymphocytic Leukemia (CLL)

Introduction: The BTK inhibitor ibrutinib blocks B-cell receptor and other signaling pathways in CLL cells and has become a standard CLL therapy. Isotopic labeling with deuterated water (2H2O) to measure the effects of ibrutinib on CLL cell proliferation and death directly in patients revealed that ibrutinib profoundly inhibits proliferation and induces high rates of leukemia cell death (registered at clinicaltrials.gov as NCT01752426; Burger et al., JCI Insight, 2017). Here, we report a long-term follow-up of this trial in which we analyzed long-term outcomes and correlated CLL cell birth and death rates with depth of response over time. Methods: Thirty patients with previously untreated CLL/SLL enrolled in the study to receive continuous long-term ibrutinib monotherapy, after one month of labeling with deuterated water. For our analyses, we stratified patients in groups based on high or low CLL cell death rates in the peripheral blood (PB) or lymphatic tissues (lymph nodes, spleen), i.e. death rates above or below the median, assessed over the first months on therapy, as detailed in the original publication. Responses were assessed according to 2008 IWCLL guidelines, with the exception that lymphocytosis was not used as a sole criterion for progression. PB MRD was quantified by flow cytometry with a sensitivity 10-4. Results: The median age of the participants was 64 years (range, 48 - 78); 63.3% were male. Thirteen patients (43%) had advanced stage disease, 57% had unmutated IGHV genes, and 10% carried 17p deletion (del17p). Twenty-two patients (73%) completed 5 years of protocol treatment and subsequently continued treatment with commercial supply ibrutinib. The patients were followed for a median time of 107 months (range, 6 - 111). The reasons for eventual treatment discontinuation (n=21) were adverse events (n=8, 38%), progressive disease (n=4, 19%), change to another treatment (n=5, 24%), death (n=2, 7%) and other reasons (n=2, 7%). To date, nine patients are still receiving ibrutinib. Five patients (17%) achieved a complete remission, 24 (80%) achieved partial remission, and one patient (3%) had stable disease, resulting in an overall response rate of 97%. Median progression-free (PFS) and overall survival (OS) were not reached at a median follow-up of 107 months. Five-year PFS was 80.6%, and OS was 90%. PB MRD was measured in 18 patients after 5 years of ibrutinib (54 to 60 cycles); all patients had detectable MRD (median 11.8%, range, 0.06 - 47.30%). We found that CLL cell death rates in PB and tissues after treatment initiation inversely correlated with the level of PB MRD after 5 years (r=-0.5872, P=0.0104 and r=-0.4407, P=0.0672). MRD levels were significantly lower in patients with high CLL cell death rates in PB (7.03% ± 3.68% vs. 29.46% ± 5.53%, mean ± SEM, P=0.0056) and in tissues (9.73% ± 5.49% vs. 28.89% ± 4.42%, P=0.0155) as compared with patients with lower CLL cell death rates (Figure 1). Tissue CLL cell death rates were significantly higher in patients with IGHV-unmutated CLL compared to mutated (28.88% ± 2.75% vs. 13.16% ± 2.82%, P=0.0005), and MRD levels trended lower (IGHV-unmutated: 10.71% ± 5.39% vs. IGHV-mutated: 28.36% ± 6.02%, P=0.0878). No statistically significant correlation was found between CLL cell birth rate at baseline or after start of ibrutinib and MRD levels after 5 years of treatment. Conclusion: CLL cell death rates during early treatment correlated with achieving lower MRD levels. We found that CLL cell death rates are higher in IGHV-unmutated patients suggesting a higher dependency of IGHV-unmutated clones on BCR signaling and, therefore, resulting in deeper responses. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Identification of Three Unique Clusters of Serum Proteins with Distinctive Functionalities in IGHV-Mutated MBL

Background. CLL-like monoclonal B-cell lymphocytosis (MBL) is considered a requisite precursor of CLL. Since MBL is also associated with inflammatory disorders, other cancers, and increased incidence of infections (Landgren et al, 2007; Moreira et al, 2013; Solomon et al, 2016), an underlying immune alteration might be at the root of the condition's onset and progression to CLL. Patients with CLL whose clone expresses mutated (M-CLL) and unmutated (U-CLL) IGHV genes differ in inflammatory profiles, with the latter exhibiting a more activated immune system, in line with the distinct capabilities of the two CLL subtypes to interact with the microenvironment with M-CLL being more anergic. Here, we studied the serum protein profiles in MBL, CLL patients, and age-matched healthy donors (HD). Since a sufficient number of MBL subjects with unmutated IGHV was not available, only MBL with mutated IGHV (M-MBL) and M-CLL patients were analyzed. Objectives. 1. Compare serum proteomic profiles of the three subsets of individuals. 2. Identify protein clusters more restricted to M-MBL. Methods. In all, 34 subjects were studied: 10 M-MBL, 12 M-CLL, and 12 HD. All individuals were treatment naive at sample collection, and all MBL subjects remained stable over a median follow-up of 53 months. Serum protein levels were quantified using a library of modified single-stranded DNA aptamers (SOMAmers) that measure 1,310 protein targets (SomaLogic). P-values <0.05 were required to consider protein levels as differentially expressed (DE) between groups. The protein-specific STRING database v11.5 was used for analyses. Functional enrichments were selected according to i) confidence > 0.4; ii) lowest significant (< 0.05) FDR P-values; iii) network description, focusing only on informative disease-related pathways. The Markov Cluster (MCL) algorithm was used to identify clusters of non-redundant, upregulated proteins related to functionally-associated networks. Serum levels of selected molecules were validated using the U-PLEX Platform (MSD) in 24 HD, 29 M-MBL and 25 M-CLL. Results: Levels of 34 proteins were DE in MBL vs. HD [19 upregulated (u), 56%; and 15 downregulated (d)]; 288 in CLL vs. HD [16u, 6%; and 272d]; and 419 in MBL vs. CLL [411u, 98%; and 8d]. U-PLEX assays validated the significantly increased levels of IL8 (P = 0.020) and CXCL11 (P = 0.038) in MBL vs. HD and IL4 (P = 0.029) in MBL vs. CLL. STRING analysis of the DE proteins between groups is shown in Table 1. Pathways related to viral infections were only highlighted in MBL vs. HD. Cytokine activity, receptor signaling, and the PI3K-Akt pathway that promotes proliferation, were detected in both MBL vs. HD (mainly upregulated) and CLL vs. HD (mostly downregulated). The last three pathways were also observed for MBL vs. CLL (upregulated in MBL). To unravel the previous comparison, the MCL algorithm was used, and this disclosed 3 main clusters of non-redundant, upregulated proteins in MBL (Table 2). Cluster #1 involved pathways related to receptor binding (including growth factor receptors such as the ErbB family) and signal transduction, with an emphasis on the PI3K-Akt pathway. The activation of these pathways promotes cell survival and migration. Cluster #2 included cell division pathways and upregulated HMGB1 and MYC, both allowing gene transcription and proliferation. Cluster #3 concerned immune system activation, in particular inflammatory responses involving innate immune cells, viral interactions, IL17, and the JAK-STAT pathway, the latter playing a major role in cytokine receptor signaling. Conclusions: The serum proteins in M-MBL suggest immune stimulation, which underwent considerable change after transition to M-CLL. This highlights an evolving serum proteome functionality with a slight activation of the immune system in MBL that declines upon transition to CLL. Clustering analysis identified 3 non-redundant, upregulated protein clusters in M-MBL compared to M-CLL. These findings suggest immune stimulation as a characteristic feature in MBL that is not operative in CLL and point out 3 groups of distinct pathological networks that may have a role in disease onset and tumor control. Future studies, focusing on specific cell subsets, will be performed to disclose the main cell orchestrators and their exact role within each cluster, to ultimately develop preemptive strategies. Acknowledgments. GB was supported by Fundación Alfonso Martín Escudero. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Enhanced IFN Sensing by Aggressive Chronic Lymphocytic Leukemia Cells.

Type I IFN is made by cells in response to stress. Cancer cells exist in a state of stress, but their IFN response is complex and not completely understood. This study investigated the role of autocrine IFN in human chronic lymphocytic leukemia (CLL) cells. CLL cells were found to make low amounts of IFN via TANK-binding kinase 1 pathways, but p-STAT1 and -STAT2 proteins along with IFN-stimulated genes that reflect IFN activation were variably downregulated in cultured CLL cells by the neutralizing IFNAR1 Ab anifrolumab. Patients with CLL were segregated into two groups based on the response of their leukemia cells to anifrolumab. Samples associated with more aggressive clinical behavior indicated by unmutated IGHV genes along with high CD38 and p-Bruton's tyrosine kinase expression exhibited responses to low amounts of IFN that were blocked by anifrolumab. Samples with more indolent behavior were unaffected by anifrolumab. Hypersensitivity to IFN was associated with higher expression of IFNAR1, MX1, STAT1, and STAT2 proteins and lower activity of negative regulatory tyrosine phosphatases. Autocrine IFN protected responsive CLL cells from stressful tissue culture environments and therapeutic drugs such as ibrutinib and venetoclax in vitro, in part by upregulating Mcl-1 expression. These findings suggest hypersensitivity to IFN may promote aggressive clinical behavior. Specific blockade of IFN signaling may improve outcomes for patients with CLL with higher-risk disease.

STEREOTYPED CASES IN UKRAINIAN COHORT OF CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS DEPENDING ON THE IONIZING RADIATION EXPOSURE.

to analyze the stereotyped subsets in cohort of Ukrainian chronic lymphocytic leukemia (CLL) patients in general and depending on the ionizing radiation (IR) exposure. Analysis was performed in the groups of 118 CLL patients irradiated due to the Chornobyl NPP accident (95 clean-up workers, 17 inhabitants of radionuclide contaminated areas, and 6 evacuees) and 294 IR non-exposed patients. The IGHV (immunoglobulin heavy chain variable region) gene mutational status, mutations of NOTCH1, TP53 and SF3B1 genes were studied by polymerase chain reaction followed by direct sequencing. Associations between clinical and molecular data of patients were analyzed with the SPSS software package, version 20.0. The incidence of stereotyped CLL cases in Ukrainian cohort was high (50.5 %) and comparable in IR-exposed and non-exposed patients. The ratio of major and minor clusters as well as the frequency of individual clusters was comparable with reported data with some exceptions: a low incidence of subset #2; absence of subset #8; high frequency of minor subset #V4|J4.5.6|18|5. The distinctive features of IR-exposed CLL patients found were:1) comparable frequency of stereotyped cases among mutated and unmutated (UM) IGHV genes cases (p = 0.557);2) lack of differences IGHV gene repertoires among stereotyped and heterogeneous cases (p = 0.508); 3) «heterogeneity» of stereotyped cases: all identified stereotyped clusters, with the exception of cluster #1, consisted of one case. Stereotyped cases with expression of UM IGHV clan I genes (except IGHV1-69 gene) were more susceptible to the appearance of NOTCH1 mutations. Patients of cluster #4 were younger, tended to have a longer time-to-treatment period and overall survival (OS) compared to subset #2. Patients of cluster #2 are more likely to have autoimmune hemolytic anemia (AIHA) and SF3F1 mutations. IGHV3-21 expression was associated with worse OS in univariate and multivariate analysis. AIHA was more common in patients with UM IGHV4-59 and IGHV3-11 genes. The revealed differences in distribution of stereotyped CLL cases in Ukrainian cohort are most likely to reflect variations in the genetic background, environmental factors (including IR exposure), and their interactions in different geographic areas.

The Outcome of CLL Patients According to IGHV Mutational Status: An Israeli Perspective

Introduction: The prognostic significance of immunoglobulin heavy-chain variable region gene (IGHV) mutational status in chronic lymphocytic leukemia (CLL) is well established. Previous studies have shown that CLL patients with mutated IGHV (M-IGHV) have a better prognosis, manifested in a longer time-to first treatment (TTFT) and overall survival (OS). Here we present an analysis on the impact of IGHV mutational status in an Israeli cohort of patients with CLL.Methods: A total of 254 patients with CLL (diagnosed from 1991 to 2020), followed at the Tel-Aviv Sourasky Medical Center were included. The IGHV mutational status has been determined by next-generation sequencing (NGS) or cDNA Sanger. The sequences with a germline homology 98% or higher were considered unmutated, and those with a homology less than 98% as mutated CLL. IGHV subsets were analyzed using the ARResT/AssignSubsets website. All data were statistically analyzed by IBM SPSS Statistics 27 (IBM Corporation, Armonk, NY, USA), P-values were two-sided, and the significance level was determined at a<0.05 were considered significant.Results: Out of 254 patients, 132 (52.0%) had an unmutated IGHV gene (UM-IGHV), and 122 (48.0%) were mutated (M-IGHV). At diagnosis, most patients (Table 1) were ≤65 years of age (n=157, 61.8%), males (n=160, 63.0%) and had an absolute lymphocytic count equal to or less than (≤)15.0 x10 9/L (n=115, 52.5%). Advanced Binet stage was more commonly associated with UM-IGHV (n=44, 57.9%) (P=0.033). Among 240 patients (94.1%), the most frequently used VH gene segments (Figure 1) included; VH1-69 (n=35, 14.6%), VH4-34 (n=24, 10.0%), VH1-2 (n=17, 7.1%), and VH3-23 (n=16, 6.7%). Six major B-cell receptors (BCR) subsets (Figure 2) were identified in 21/180 of patients (11.7%), which most commonly included: CLL#1 (n=6, 28.6%), CLL#4 (n=5, 23.8%), and CLL#2(n=3, 14.3%). Patients with M-IGHV had a longer time to first treatment (TTFT) (P<0.001, hazard ratio (HR)=2.5, 95% confidence interval [1.8-3.5], Figure 3A) and a better overall survival (OS) (P=0.002, HR=2.7, [1.4-5.1]) compared to those with UM-IGHV gene (Figure 3B).In multivariate analyses of the entire cohort; for TTFT (Table 2A), males (P=0.002, HR=1.9, [1.3-2.9]), >65 years of age at diagnosis (P=0.008, HR=1.8, [1.2-2.7]), advanced Binet stage (P<0.001, HR=2.4, [1.5-3.8]) and UM-IGHV mutational status (P=<0.001, HR=2.0, [1.3-2.9]) were found to be significant predictors for shorter TTFT, while in a multivariate analysis for OS (Table 2B), only >65 years of age at diagnosis (P=0.010, HR=2.6, [1.3-5.5]) and UM-IGHV mutational status (P=0.004, HR=2.9, [1.4-5.9]) were found to be significant factors for shorter OS.In multivariate analyses performed separately for each IGHV mutational status group; males (P=0.021, HR=2.1, [1.1-4.0]), age >65 years (P=0.002, HR=3.2, [1.5-6.4]) and advanced Binet stage (P<0.001, HR=4.1, [1.9-8.7]) were found as independent predictors for a shorter TTFT in M-IGHV patients, while only males retained a statistically significance in UM-IGHV patients (P=0.017 HR=2.0, [1.1-3.5]). Furthermore, in a multivariate analysis for OS; age>65 years at diagnosis (P=0.039, HR=4.3, [1.1-17.0]) was the only independent predictor in M-IGHV patients, while no variable maintained its statistical significance in UM-IGHV cases.Conclusion: As previously studied, our cohort demonstrates that M-IGHV CLL patients have better TTFT and OS. However, the rate of BCR subsets in Israel appears to be lower than expected. A separated multivariate analysis for M-IGHV and UM-IGHV patients revealed different independent predictors for TTFT and OS to each of the IGHV mutational status groups. Further studies with larger cohorts are required to deeply study these differences and provide further clinical insight into the pathophysiology of CLL. [Display omitted] DisclosuresJoffe: AstraZeneca: Consultancy; Epizyme: Consultancy. Herishanu: AbbVie: Honoraria, Research Funding; Janssen: Honoraria; Roche: Honoraria; Medison: Honoraria.

Real-World Evidence on Therapeutic Strategies and Treatment-Sequencing in Patients with Chronic Lymphocytic Leukemia: An International Study of Eric, the European Research Initiative on CLL

Real-World Evidence on Therapeutic Strategies and Treatment-Sequencing in Patients with Chronic Lymphocytic Leukemia: An International Study of Eric, the European Research Initiative on CLL

The Clonotypic BCR IG of CLL Patients Contain Predicted T-Cell Class I Epitopes with Shared Structural Properties

Targeted therapies have revolutionized the treatment of chronic lymphocytic leukemia (CLL) with remarkable overall response rates. Against that, however, CLL remains incurable, indicating a need for novel strategies towards disease control and eventual eradication, including reinvigoration of anti-tumor immune responses. T cells in CLL display an oligoclonal profile and appear selected by restricted antigens, with recent evidence suggesting that the selecting epitopes may lie within the clonotypic B-cell receptor immunoglobulins (BcR IG). Should this prove to be the case, such neoepitopes could be exploited as idiotypic targets for cellular therapy or for peptide vaccine design, aiming to augment response to current treatments.Here we performed ad hoc prediction of putative T-cell class I neoepitopes contained within the clonotypic BcR IGs of CLL patients, with an intended bias towards major stereotyped CLL subsets. We selected 27 patients to represent the full spectrum of CLL: (i) with mutated IGHV genes (M-CLL, n=5), (ii) with unmutated IGHV genes (U-CLL, n=5), (iii) assigned to major stereotyped subsets (subset #1, n=7; subset #2, n=5; subset #4, n=5). RT-PCR was performed for the heavy (H) and the light (K/L) IG chains using subgroup-specific Leader primers for the IGHV/IGK/LV gene and universal primers annealing to the constant domain (IGHG, IGHM, IGKC, IGLC), in order to produce the full-length V-(D)-J gene rearrangement sequence, plus the start of the constant domain. PCR products were subjected to direct double-strand Sanger sequencing with a quality-optimized protocol. The amino acid sequences were subsequently parsed in peptides and subjected to NetMHCpan. The rank score was calculated, considering the 4-digit HLA-A and -B typing for each individual patient. High- and medium-binding peptides (rank score <2%) were selected. Exact matches to germline and/or proteome databases were excluded.Overall, 1,007 predicted neoepitopes were identified. All patients had predicted CD8 + T-cell epitopes within the clonotypic BcR IG, either in the heavy chain (26/27 pts, n=632 epitopes) or the light chain (26/27 pts, n=375 epitopes). The majority of the peptides resulted from somatic hypermutations (SHMs) across the IGHV gene outside the complementarity-determining region 3 (CDR3; n=538, 53.4%). With the exception of few peptides located within the FR4 region (n=11, 0.1%), the remaining (n=458, 45.5%) involved (at least part of) the CDR3, which is particularly relevant given its small length (9-27 aa) within the full sequence (331-660 aa). There was no statistically significant difference in the rank score of peptides involving the CDR3 vs. all others. Peptide clustering assigned most of the predicted neoepitopes (970/1007, 96.3%) in 54 clusters of similar length and amino acid composition. Also, it revealed similar or identical predicted neoepitopes among different patients (30 clusters of two, 10 clusters of three, 8 clusters of four, 4 clusters of five, 1 cluster of six and 1 cluster of eight). Importantly, these clusters involved: (i) shared CDR3 patterns in patients assigned to the same stereotyped subset, but also (ii) subset-specific recurrent SHMs across the rearranged IGHV gene, e.g. G-to-E SHM at position 28 in the VH CDR1 of subset #4, a recurrent SHM in this subset. Also of note, the two most highly populated clusters involved peptides within the VL CDR3, and were biased towards specific subsets; the cluster of eight patients contained 4 patients assigned to subset #1 and the cluster of six patients included 4 patients assigned to subset #2.In conclusion, in silico prediction identified a significant number of putative T-cell class I neoepitopes contained within the clonotypic BcR IG of CLL patients. The majority of these neoepitopes can be assigned to clusters based on amino acid similarity and are shared among different patients. Many of them culminate from subset-specific (‘stereotyped‘) CDR3 patterns or recurrent SHMs, suggesting that the targeted SHM which shapes the CLL BcR IG repertoire may produce immunogenic CD8 + T-cell epitopes. Their actual immunogenicity has to be tested in ex vivo studies, currently underway by our group. DisclosuresAnagnostopoulos: Abbvie: Other: clinical trials; Sanofi: Other: clinical trials ; Ocopeptides: Other: clinical trials ; GSK: Other: clinical trials; Incyte: Other: clinical trials ; Takeda: Other: clinical trials ; Amgen: Other: clinical trials ; Janssen: Other: clinical trials; novartis: Other: clinical trials; Celgene: Other: clinical trials; Roche: Other: clinical trials; Astellas: Other: clinical trials . Chatzidimitriou: Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.

Venetoclax, Obinutuzumab and Atezolizumab (PD-L1 Checkpoint Inhibitor) for First-Line Treatment for Patients with Chronic Lymphocytic Leukemia (CLL)

Venetoclax, Obinutuzumab and Atezolizumab (PD-L1 Checkpoint Inhibitor) for First-Line Treatment for Patients with Chronic Lymphocytic Leukemia (CLL)

Optimal Management of Chronic Lymphocytic Leukemia and Economic Constraints.

In this article, we carry out an overview on the management options available for chronic lymphocytic leukemia (CLL) patients and discuss possible treatment decisions, taking into account the issue of sustainability and availability. Targeted agents have shown to be superior compared with chemoimmunotherapy (CIT) in terms of progression-free survival in high-risk CLL. In the majority of studies, however, continuous treatment was compared with fixed-duration CIT and no overall survival or progression-free survival-2 (time from randomization to second progression or death) advantage could be documented. Meanwhile, a substantial financial burden on both patients and payers has raised issues about affordability and adherence to treatment. Therefore, value-based pricing of new drugs has been used to set up price negotiation policies in several countries, and fixed-duration therapy has shown to be less costly than continuous treatment. Thus, CIT continues to have a role in the treatment of CLL patients with a favorable genetic profile, that is, with a mutated IGHV gene profile and a wild-type TP53. Targeted treatment represents the preferred choice in patients with an unmutated IGHV gene configuration and/or a TP53 disruption, provided that adherence to treatment is guaranteed and bearing in mind that should costly drugs not be available for frontline treatment, new agents can be very effective as first salvage treatment.