Immunoglobulin Heavy Chain Variable Region Research Articles

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

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

A Review of Resistance Mechanisms to Bruton's Kinase Inhibitors in Chronic Lymphocytic Leukemia.

Bruton's Tyrosine Kinase (BTK) inhibitors have become one of the most vital drugs in the therapy of chronic lymphocytic leukemia (CLL). Inactivation of BTK disrupts the B-cell antigen receptor (BCR) signaling pathway, which leads to the inhibition of the proliferation and survival of CLL cells. BTK inhibitors (BTKi) are established as leading drugs in the treatment of both treatment-naïve (TN) and relapsed or refractory (R/R) CLL. Furthermore, BTKi demonstrate outstanding efficacy in high-risk CLL, including patients with chromosome 17p deletion, TP53 mutations, and unmutated status of the immunoglobulin heavy-chain variable region (IGHV) gene. Ibrutinib is the first-in-class BTKi which has changed the treatment landscape of CLL. Over the last few years, novel, covalent (acalabrutinib, zanubrutinib), and non-covalent (pirtobrutinib) BTKi have been approved for the treatment of CLL. Unfortunately, continuous therapy with BTKi contributes to the acquisition of secondary resistance leading to clinical relapse. In recent years, it has been demonstrated that the predominant mechanisms of resistance to BTKi are mutations in BTK or phospholipase Cγ2 (PLCG2). Some differences in the mechanisms of resistance to covalent BTKi have been identified despite their similar mechanism of action. Moreover, novel mutations resulting in resistance to non-covalent BTKi have been recently suggested. This article summarizes the clinical efficacy and the latest data regarding resistance to all of the registered BTKi.

Acalabrutinib-based regimens in frontline or relapsed/refractory higher-risk CLL: pooled analysis of 5 clinical trials

AbstractBefore targeted therapies, patients with higher-risk chronic lymphocytic leukemia (CLL), defined as del(17p) and/or TP53 mutation (TP53m), unmutated immunoglobulin heavy chain variable region genes (uIGHV), or complex karyotype (CK), had poorer prognosis with chemoimmunotherapy. Bruton tyrosine kinase inhibitors (BTKis) have demonstrated benefit in higher-risk patient populations with CLL in individual trials. To better understand the impact of the second-generation BTKi acalabrutinib, we pooled data from 5 prospective clinical studies of acalabrutinib as monotherapy or in combination with obinutuzumab (ACE-CL-001, ACE-CL-003, ELEVATE-TN, ELEVATE-RR, and ASCEND) in patients with higher-risk CLL in treatment-naive (TN) or relapsed/refractory (R/R) cohorts. A total of 808 patients were included (TN cohort, n = 320; R/R cohort, n = 488). Median follow-up was 59.1 months (TN cohort) and 44.3 months (R/R cohort); 51.3% and 26.8% of patients in the TN and R/R cohorts, respectively, remained on treatment at last follow-up. In the del(17p)/TP53m, uIGHV, and CK subgroups in the TN cohort, median progression-free survival (PFS) and median overall survival (OS) were not reached (NR). In the del(17p)/TP53m, uIGHV, and CK subgroups in the R/R cohort, median PFS was 38.6 months, 46.9 months, and 38.6 months, respectively, and median OS was 60.6 months, NR, and NR, respectively. The safety profile of acalabrutinib-based therapy in this population was consistent with the known safety profile of acalabrutinib in a broad CLL population. Our analysis demonstrates long-term benefit of acalabrutinib-based regimens in patients with higher-risk CLL, regardless of line of therapy.

Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma, Version 2.2024, NCCN Clinical Practice Guidelines in Oncology

Chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL) are essentially different manifestations of the same disease that are similarly managed. A number of molecular and cytogenetic variables with prognostic implications have been identified. Undetectable minimal residual disease at the end of treatment with chemoimmunotherapy or venetoclax-based combination regimens is an independent predictor of improved survival among patients with previously untreated or relapsed/refractory CLL/SLL. The selection of treatment is based on the disease stage, presence or absence of del(17p) or TP53 mutation, immunoglobulin heavy chain variable region mutation status, patient age, performance status, comorbid conditions, and the agent's toxicity profile. This manuscript discusses the recommendations outlined in the NCCN Guidelines for the diagnosis and management of patients with CLL/SLL.

Hairy cell leukemia 2024: Update on diagnosis, risk-stratification, and treatment-Annual updates in hematological malignancies.

Hairy cell leukemia (HCL) and HCL-like disorders, including HCL variant (HCL-V) and splenic diffuse red pulp lymphoma (SDRPL), are a very heterogenous group of mature lymphoid B-cell disorders characterized by the identification of hairy cells, a specific genetic profile, a different clinical course and the need for appropriate treatment. Diagnosis of HCL is based on morphological evidence of hairy cells, an HCL immunologic score of 3 or 4 based on the CD11c, CD103, CD123, and CD25 expression, the trephine biopsy which makes it possible to specify the degree of tumoral bone marrow infiltration and the presence of BRAFV600E somatic mutation. Progression of patients with HCL is based on a large splenomegaly, leukocytosis, a high number of hairy cells in the peripheral blood, and the immunoglobulin heavy chain variable region gene mutational status. VH4-34 positive HCL cases are associated with a poor prognosis, as well as HCL with TP53 mutations and HCL-V. Patients should be treated only if HCL is symptomatic. Chemotherapy with risk-adapted therapy purine analogs (PNAs) are indicated in first-line HCL patients. The use of chemo-immunotherapy combining cladribine (CDA) and rituximab (R) represents an increasingly used therapeutic approach. Management of relapsed/refractory disease is based on the use of BRAF inhibitors (BRAFi) plus R, MEK inhibitors (MEKi), recombinant immunoconjugates targeting CD22, Bruton tyrosine kinase inhibitors (BTKi), and Bcl-2 inhibitors (Bcl-2i). However, the optimal sequence of the different treatments remains to be determined.

Low incidence of tumor lysis syndrome in elderly patients with chronic lymphocytic leukemia treated with venetoclax under real-world conditions: results from the prospective observational VeRVe study.

Venetoclax is active in both frontline and relapsed/refractory settings for the treatment of chronic lymphocytic leukemia (CLL). Although the prevalence and severity of tumor lysis syndrome (TLS) are well characterized in clinical trials, laboratory and clinical TLS remain relatively unexplored in real-world clinical practice.In this prospective, real-world observational study, we aimed to determine the incidence and outcomes of TLS in patients with CLL receiving venetoclax outside a clinical trial. The study (VeRVe) was conducted in centers in Austria, Germany, and Switzerland.Two hundred and thirty-nine patients were treated according to local label with at least one dose of venetoclax. Patient demographics, baseline characteristics, and blood chemistry at baseline were documented, and descriptive statistical analyses were conducted.Seventy eight patients (33%) were treated with venetoclax monotherapy, 101 (42%) with venetoclax in combination with rituximab and 60 (25%) with venetoclax in combination with obinutuzumab. In all cases, the TLS risk mitigation strategy adhered to the ramp-up protocol. Median age was 73 years and 66% of patients were male. The majority of patients (75%) had relapsed/refractory CLL, 63/192 (32.8%) patients tested had a del(17p) and 93/134 (69.4%) patients tested had unmutated immunoglobulin heavy chain variable region gene (IGHV). Clinical TLS occurred in 5 patients (2.1%) and laboratory TLS occurred in 15 patients (6.3%). Ten patients received specific treatment, of which 6 were hospitalized. There were no deaths due to a TLS event and venetoclax was well-tolerated. Of the 5 clinical TLS events reported, none were fatal or resulted in renal failure (NCT03342144, registered on Nov 10, 2017).

Risk Factors for Fragility Fractures in Chronic Lymphocytic Leukemia.

Abnormal bone health and fragility fractures (FF) are more common in patients with chronic lymphocytic leukemia (CLL). We hypothesize that there may be risk factors in CLL patients with osteoporosis that increasethe risk of FFs. We conducted a cohort study encompassing all patients diagnosed with CLL from January 1, 2000, to July 31, 2020, utilizing International Classification of Diseases (ICD) codes related to abnormal bone health (osteopenia, osteoporosis, and/or presence of FF) within a single tertiary care institution.Of the 89 patients included, 55 (62%) were female with a mean age of 68 ± 11 years at cohort entry. Fifty-nine (66%) had at least one FF present (pFF) and 30 (34%) did not have an FF (nFF). There were no differences in IGHV (Immunoglobulin heavy chain variable region gene) mutation status, chromosomal abnormalities, or the presence of a complex karyotype. The spine accounted for 81% of identified FF. T-score <-2.5 was more common in those without FF (pFF 38% vs. nFF 71%, P= 0.02). DXA evaluation was not conducted for 36 (40%)individuals within the cohort. Risk factors for fragility fractures included male sex (relative risk [RR] 8.1, 95% confidence interval [CI] 2.1-31.7), diabetes mellitus (RR 1.4, 95% CI 1.04-1.8), smoking (RR 1.3, 95% CI 1.02-1.8), Rai stage >0 (RR 1.4, 95% CI 1.04-1.9), and T-score >-2.5 (RR 1.8, 95% CI 1.1-3.1).There is a high frequency of vertebral FFs in people with CLL despite T-scores not being in the osteoporotic range. Increased awareness to screen and treat vertebral FFs in people with CLL is needed.

Эффективность ибрутиниба в первой линии терапии у пациентов из группы высокого риска и во второй, третьей линиях при резистентном течении хронического лимфоцитарного лейкоза

Risk stratification appears to be the most valid criterion in decision-making regarding optimal specific therapy in chronic lymphocytic leukemia (CLL). The CLL International Prognostic Index takes account of unfavorable cytogenetic abnormalities (del(17p)/del(11q) and/or TP53 gene mutations) as well as the mutation status of immunoglobulin heavy chain variable (IGHV) region genes. Unmutated V(H)-status is commonly associated with such prognostically unfavorable genetic markers as del(17p)/del(11q), trisomy 12, and TP53 mutation. The combination of unmutated V(H)-status with unfavorable karyotype abnormalities (del(17p)/del(11q)) negatively affects the prognosis and overall survival rate. Besides, in high-risk CLL, the efficacy of therapy is rather low, and the development of refractoriness is possible. Targeted therapy (Bruton tyrosine kinase inhibitors) both in first line and in resistant CLL considerably increases the probability of achieving long-term remission. The present paper provides the comparative analysis of clinical and hematological efficacy and tolerability of ibrutinib in first-line CLL therapy of high-risk patients as well as second- and third-line therapies of resistant CLL. Ibrutinib shows high efficacy and low toxicity. First-line ibrutinib treatment results in a faster response and effectively reduces the probability of CLL progression. Second- and third-line ibrutinib treatment allows to overcome CLL resistance without impairing patients’ quality of life.

Five-year follow-up of a phase 2 study of ibrutinib plus fludarabine, cyclophosphamide, and rituximab as initial therapy in CLL

AbstractWe previously reported high rates of undetectable minimal residual disease <10−4 (uMRD4) with ibrutinib plus fludarabine, cyclophosphamide, and rituximab (iFCR) followed by 2-year ibrutinib maintenance (I-M) in treatment-naïve chronic lymphocytic leukemia (CLL). Here, we report updated data from this phase 2 study with a median follow-up of 63 months. Of 85 patients enrolled, including 5 (6%) with deletion 17p or TP53 mutation, 91% completed iFCR and 2-year I-M. Five-year progression-free survival (PFS) and overall survival were 94% (95% confidence interval [CI], 89%-100%) and 99% (95% CI, 96%-100%), respectively. No additional deaths have occurred with this extended follow-up. No difference in PFS was observed by immunoglobulin heavy-chain variable region gene status or duration of I-M. High rates of peripheral blood (PB) uMRD4 were maintained (72% at the end of iFCR, 66% at the end of 2-year I-M, and 44% at 4.5 years from treatment initiation). Thirteen patients developed MRD conversion without clinical progression, mostly (77%) after stopping ibrutinib. None had Bruton tyrosine kinase (BTK) mutations. One patient had PLCG2 mutation. Six of these patients underwent ibrutinib retreatment per protocol. Median time on ibrutinib retreatment was 34 months. The cumulative incidence of atrial fibrillation was 8%. Second malignancy or nonmalignant hematologic disease occurred in 13%, mostly nonmelanoma skin cancer. Overall, iFCR with 2-year I-M achieved durably deep responses in patients with diverse CLL genetic markers. Re-emergent clones lacked BTK mutation and retained sensitivity to ibrutinib upon retreatment. This trial is registered at www.clinicaltrials.gov as #NCT02251548.

Association of Cytogenetics Aberrations and IGHV Mutations with Outcome in Chronic Lymphocytic Leukemia Patients in a Real-World Clinical Setting.

Immunoglobulin heavy chain variable ( IGHV ) region mutations, TP53 mutation, fluorescence in situ hybridization (FISH), and cytogenetic analysis are the most important prognostic biomarkers used in chronic lymphocytic leukemia (CLL) patients in our daily practice. In real-life environment, there are scarce studies that analyze the correlation of these factors with outcome, mainly referred to time to first treatment (TTFT) and overall survival (OS). This study aimed to typify IGHV mutation status, family usage, FISH aberrations, and complex karyotype (CK) and to analyze the prognostic impact in TTFT and OS in retrospective study of 375 CLL patients from a Spanish cohort. We found unmutated CLL (U-CLL) was associated with more aggressive disease, shorter TTFT (48 vs. 133 months, p < 0.0001), and shorter OS (112 vs. 246 months, p < 0.0001) than the mutated CLL. IGHV3 was the most frequently used IGHV family (46%), followed by IGHV1 (30%) and IGHV4 (16%). IGHV5-51 and IGHV1-69 subfamilies were associated with poor prognosis, while IGHV4 and IGHV2 showed the best outcomes. The prevalence of CK was 15% and was significantly associated with U-CLL. In the multivariable analysis, IGHV2 gene usage and del13q were associated with longer TTFT, while VH1-02, +12, del11q, del17p, and U-CLL with shorter TTFT. Moreover, VH1-69 usage, del11q, del17p, and U-CLL were significantly associated with shorter OS. A comprehensive analysis of genetic prognostic factors provides a more precise information on the outcome of CLL patients. In addition to FISH cytogenetic aberrations, IGHV and TP53 mutations, IGHV gene families, and CK information could help clinicians in the decision-making process.

Prediction of Time-to-First Treatment (TTFT) in Favorable-Risk Chronic Lymphocytic Leukemia (CLL) Patients Using a Novel Stratification through Transcriptome Risk Scoring

Background: CLL is the most prevalent adult leukemia with a highly heterogenous genetic landscape leading to extremely varied clinical outcomes. Previously, CLL patients with immunoglobulin heavy chain variable region ( IGHV) mutated status and presence of deletion 13q14 [del(13q)] were identified as favorable-risk with most patients remaining on lifetime observation. However, other intrinsic factors contributing to CLL disease progression and leading to treatment initiation remain unknown. Our goal was to identify patients at high-risk of progression with added predictive value (APV) beyond IGHV-status and presence of del(13q). Methods: Flow cytometry was used to sort CD19+CD5+ malignant cells in peripheral blood from CLL patients at Huntsman Cancer Institute at University of Utah and triple sequencing generated (tumor whole transcriptome and paired tumor-normal whole exome data). We used SPECTRA, a novel dimension reduction transcriptome approach, to derive a set of independent, intrinsic, quantitative transcriptome tumor variables from 249 CLL patients with quality-controlled transcriptome sequence data. From this set, 182 treatment-naïve (TN) patients with quality-controlled clinical variables were used as a discovery cohort set to derive a SPECTRA-based risk score for TTFT using multivariable Cox proportional hazards modeling. Event time was date of patient specimen acquisition to initiation of frontline therapy with patients censored at last follow-up or death. Gaussian mixture modeling (GMM) was performed to identify evidence for distinct risk groups within the quantitative risk score. GMM-risk groups, and further stratifications by IGHV mutational and del(13q) status, were used in hazard ratios (HR) and to illustrate differences in Kaplan-Meier time-to-event curves. An independent publicly available dataset of 548 TN CLL patients with clinical and transcriptome data for CD19+ enriched cells was used for replication. Results: SPECTRA produced 19 quantitative spectra variables to describe most of the expression variation across the 249 CLL patients. In multivariable Cox modeling for TTFT, 7 spectra were significant and retained in a final model, which GMM separated into a bi-modal distribution and defined high (N=83) and low-risk (N=99) groups (HR=5.7, 95% confidence interval [CI] 3.4-9.7). In a post-hoc model including IGHV mutational and del(13q) status, the spectra-risk score showed significant APV beyond both factors (p&amp;lt;0.001 for both). The discovery study timeframe was 6.3 years (y) and included 109 IGHV-mutated and 75 del(13q) patients. In the IGHV-mutated patients, median TTFT (mTTFT) was significantly shorter for spectra-high risk patients (3.1y) vs. spectra-low risk patients (median not reached [NR]; p&amp;lt;0.001). Similarly, in the del(13q) patients, mTTFT was significantly shorter in high-risk patients (3.3y) compared to median NR for spectra-low (p&amp;lt;0.001) ( Figure). Importantly, stratification of patients into low- and high-risk SPECTRA scores provided significant additional information for prediction of TTFT than IGHV mutational and del(13q) status ( Figure). For validation, we applied the same spectra-risk score to the independent data set containing 301 IGHV-mutated and 278 del(13q) CLL patients. In the replication set, the mTTFT in the IGHV-mutated patients who were spectra-high was 3.3y vs. 11.2y in spectra-low (p&amp;lt;0.001). Similarly, in the replication set of del(13q) patients, spectra-high-risk patients had a mTTFT of 2.7y vs. 10.7y for spectra-low (p&amp;lt;0.001). Conclusion: A transcriptome risk score utilizing novel SPECTRA variables identified a substantial subset of CLL patients with IGHV-mutated status and presence of del(13q) who have uncharacteristically short TTFT, but who would be classified as favorable risk under current best practice stratification. Future directions include validating our results with external patient cohorts from Mayo Clinic and investigating the genetic and transcriptomic profile of SPECTRA low- and high-risk cohorts to determine targetable pathways for treatment.

Overall Survival Outcomes in Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma Patients with High-Risk Molecular-Cytogenetic Features Treated with 1L Ibrutinib: A Comparative Effectiveness Study Using the Flatiron Health Data

Introduction: In individuals diagnosed with chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), high-risk features are associated with an unfavorable outcome. Evidence is lacking in comparing the overall survival (OS) of a high-risk group (HRG) vs. a non-high-risk group (NHRG). This large retrospective cohort study, using data from the Flatiron Health CLL Core Registry electronic medical records (EMR), included 1,242 CLL/SLL patients treated with first-line (1L) single-agent ibrutinib. This study compared the OS of patients from the HRG (presence of deletion 17p [del(17p)], or deletion 11q [del(11q)], or unmutated immunoglobulin heavy-chain variable region gene [IGHV]) with that of patients from the NHRG (del[17p] absence, del[11q] absence, and mutated IGHV). This study also included a sensitivity analysis with an alternative high-risk definition and a subgroup analysis for Medicare patients. Methods: The Flatiron Health EMR data includes patients with confirmed CLL/SLL diagnosis using both diagnosis code and chart abstraction. Patients aged ≥18 years were included if they initiated single-agent ibrutinib 28 days before index date (1L initiation+28 days) between 04 March 2016 and 31 January 2023. Patients were excluded if there was missing or incomplete information on their first systemic therapy or on the genetic variables of interest, or if they had other malignancies (≥2 diagnosis codes ≥30 days apart) within the prior 12 months before 1L initiation. OS (the time interval between the index date and all-cause mortality date) was assessed using Kaplan-Meier survival estimators, censoring patients without mortality information at the last activity date. COX proportional hazard model was used for pairwise comparison analysis, treating NHRG as the reference group. Potential confounders (including baseline covariates such as gender, age, race, stage, ECOG PS, comorbidities, practice type, disease type, and other biomarkers) were adjusted using inverse probability of treatment weighting (IPTW). The Medicare subgroup included patients with Medicare as the single payer or one of the multiple payers at ibrutinib initiation, which serves as a proxy for the elderly group (aged ≥65 years). A sensitivity analysis was conducted by defining HRG as those with del(17p) presence and/or unmutated IGHV and NHRG as those with del(17p) absence and mutated IGHV. Flatiron Health mortality data were obtained from multiple sources including EMR, the U.S. Social Security Death Index, obituaries, funeral homes, which had been benchmarked against the U.S. National Death Index, demonstrating 87-92% sensitivity, 94%-99% specificity, and 96%-98% positive predictive value. Results: Of the 1,242 patients included, 969 were from HRG and 273 from NHRG, with mean age of 70.0 vs 70.8 years. For HRG and NHRG, 36.2% vs 43.6% were female, 72.0% vs 74.7% were White, 37.5% vs 39.6% were Medicare patients (due to data limitation, 18.5% and 19.8% patients had missing data for payer type, respectively), 91.6% vs 93.8% were from the community setting, 48.8% vs 53.1% were between Rai stage 0 and II, 74% vs 70.1% had ECOG ≤ 2, 47% vs 63% had deletion 13q presence, 23.5% vs 24.2% had trisomy 12 presence, respectively. Mean (Standard Deviation) time from diagnosis to 1L initiation was 35.0 (48.0) vs 64.4 (73.2) months for HRG and NHRG. Within the HRG, 32.9%, 36.7%, and 58.7% of the patients had the presence of del(17p), del(11q), and unmutated IGHV, respectively. After applying IPTW with trimming, OS hazard ratio (HR; 95% confidence interval [CI]) was 1.09 (0.79, 1.51) and median OS was not reached for either group (Figure 1). Median follow up (interquartile range [IQR]) was 32 (17, 48) months and 31 (19, 44) months, for HRG and NHRG, respectively. The HRG sensitivity analysis showed similar results to the main analysis, with OS HR (95% CI) being 1.19 (0.86, 1.64) and median OS not reached for either group. Within the Medicare subgroup, OS HR (95% CI) was 0.98 (0.63, 1.53), and median OS was not reached for either group (Figure 2). Conclusions: In this real-world study using a large community healthcare dataset, CLL/SLL patients with high-risk molecular-cytogenetic features (del[17p], or del[11q], or unmutated IGHV) treated with 1L ibrutinib had similar OS compared to patients without high-risk features. Importantly, similar findings were observed in the high-risk sensitivity analysis and the Medicare subgroup.

MHC Tetramers Specifically Identify High- and Low-avidity Donor-specific B Cells in Transplantation Tolerance and Rejection.

Although donor-specific antibody pre- and posttransplantation is routinely assessed, accurate quantification of memory alloreactive B cells that mediate recall antibody response remains challenging. Major histocompatibility complex (MHC) tetramers have been used to identify alloreactive B cells in mice and humans, but the specificity of this approach has not been rigorously assessed. B-cell receptors from MHC tetramer-binding single B cells were expressed as mouse recombinant immunoglobulin G1 (rIgG1) monoclonal antibodies, and the specificity was assessed with a multiplex bead assay. Relative binding avidity of rIgG1 was measured by modified dilution series technique and surface plasmon resonance. Additionally, immunoglobulin heavy chain variable regions of 50 individual B-cell receptors were sequenced to analyze the rate of somatic hypermutation. The multiplex bead assay confirmed that expressed rIgG1 monoclonal antibodies were preferentially bound to bait MHC class II I-E d over control I-A d and I-A b tetramers. Furthermore, the dissociation constant 50 binding avidities of the rIgG1 ranged from 10 mM to 7 nM. The majority of tetramer-binding B cells were low avidity, and ~12.8% to 15.2% from naive and tolerant mice and 30.9% from acute rejecting mice were higher avidity (dissociation constant 50 <1 mM). Collectively, these studies demonstrate that donor MHC tetramers, under stringent binding conditions with decoy self-MHC tetramers, can specifically identify a broad repertoire of donor-specific B cells under conditions of rejection and tolerance.

Acalabrutinib ± Obinutuzumab Vs Obinutuzumab + Chlorambucil in Treatment-Naive Chronic Lymphocytic Leukemia: 6-Year Follow-up of Elevate-TN

Background: Previous reports of ELEVATE-TN (NCT02475681) at median follow-up of up to 58.2 months (mo) demonstrated superior efficacy of acalabrutinib (A) ± obinutuzumab (O) compared with O + chlorambucil (Clb) in patients (pts) with treatment-naive (TN) chronic lymphocytic leukemia (CLL). Herein, updated results at 74.5 mo of follow-up are reported. Methods: Pts received A monotherapy, A+O, or O+Clb. Pts who progressed on O+Clb could cross over to A monotherapy. Investigator (INV)-assessed progression-free survival (PFS), INV-assessed overall response rate (ORR), overall survival (OS), and safety were evaluated. All analyses are ad-hoc and P-values are descriptive. Results: A total of 535 pts (A, n=179; A+O, n=179; O+Clb, n=177) were randomized: median age was 70 years, 63% had unmutated immunoglobulin heavy chain variable region genes (uIGHV), and 14% had del(17p) and/or TP53 mutation ( TP53m). At a median follow-up of 74.5 mo (range, 0.0-89.0; data cutoff March 3, 2023), median PFS was not reached (NR) for A+O and A vs 27.8 mo for O+Clb (hazard ratio [HR] vs O+Clb: 0.14 and 0.23, respectively; P&amp;lt;0.0001 for both; HR A+O vs A: 0.58; P=0.0229); estimated 72-mo PFS rates were 78%, 62%, and 17%, respectively (Figure 1). For the 79 pts who crossed over from O+Clb to A, median PFS2 (time to second disease progression or death) was NR; estimated 72-mo PFS2 rate was 54%. Median OS was NR in any treatment arm and was significantly longer with A+O vs O+Clb (HR: 0.62; P=0.0349); estimated 72-mo OS rates were 84% for A+O, 76% for A, and 75% for O+Clb ( Figure 2). In 337 pts with uIGHV, median PFS was NR in both the A+O and A arms vs 22.2 mo for O+Clb (HR: 0.08 and 0.12, respectively; P&amp;lt;0.0001 for both), and estimated 72-mo PFS rates were 75%, 60%, and 5%, respectively, with median OS NR in all treatment arms and estimated 72-mo OS rates of 84%, 76%, and 74%, respectively. In 73 pts with del(17p) and/or TP53m, median PFS was 73.1 mo for A+O and NR for A vs 17.5 mo for O+Clb (HR: 0.28 and 0.23, respectively; P≤0.0009 for both), and estimated 72-mo PFS rates were 56%, 56%, and 18%, respectively. Median OS was NR in both the A+O and A arms vs 74.9 mo for O+Clb (HR: 0.53 and 0.46, respectively, neither statistically significant) and estimated 72-mo OS rates were 68%, 72%, and 53%, respectively. ORR was significantly higher with A+O (96%; 95% confidence interval [CI]: 92-98; P&amp;lt;0.0001) and A (90%; 95% CI: 85-94; P=0.0499) vs O+Clb (83%; 95% CI: 77-88). Combined complete response (CR) plus CR with incomplete hematologic recovery rates were higher with A+O (37%) and A (19%) vs O+Clb (14%; P≤0.0499 for both). Median treatment exposure was 74.4 mo and 72.0 mo for A in the A+O and A arms, respectively; 5.5 mo and 5.6 mo for O in the A+O and O+Clb arms, respectively; and 5.5 mo for Clb in O+Clb arm. Adverse events (AEs) for the O+Clb arm have been previously reported. The most common (≥5% of pts) grade ≥3 AEs for A+O and A, respectively, were neutropenia (31% and 12%), thrombocytopenia (8% and 3%), diarrhea (6% and 1%), COVID-19 (9% and 7%), pneumonia (7% and 6%), syncope (5% and 2%), and hypertension (4% and 5%). For events of clinical interest, grade ≥3 atrial fibrillation, hypertension, and secondary primary malignances were reported in 2%, 4%, and 10% of pts treated with A+O and 2%, 5%, and 5% of pts treated with A, respectively. Treatment is ongoing in 54% (A+O; n=96) and 47% (A; n=84) of pts; the most common reasons for treatment discontinuation were AEs, observed in 21% (n=38) of pts treated with A+O and 18% (n=32) of pts treated with A, and progressive disease, observed in 6% (n=10) of pts treated with A+O and 14% (n=25) of pts treated with A. For pts who crossed over from O+Clb to A, 41% (n=32) discontinued A due to AEs in 13% (n=10) of pts and progressive disease in 16% (n=13) of pts. Conclusions: With a median follow-up of 74.5 mo, the efficacy and safety of A+O and A monotherapy were maintained in pts with TN CLL, including in pts with high-risk genetic features. At 6 years of follow-up, PFS was significantly longer in pts treated with A+O vs A. Median OS was NR in any treatment arm and was significantly longer in pts treated with A+O vs O+Clb.

Characterization of B-cell receptor clonality and immunoglobulin gene usage at multiple time points during active SARS-CoV-2 infection.

Although monoclonal antibodies to the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are known, B-cell receptor repertoire and its change in patients during coronavirus disease-2019 (COVID-19) progression is underreported. We aimed to study this molecularly. We used immunoglobulin heavy chain (IGH) variable region (IGHV) spectratyping and next-generation sequencing of peripheral blood B-cell genomic DNA collected at multiple time points during disease evolution to study B-cell response to SARS-CoV-2 infection in 14 individuals with acute COVID-19. We found a broad distribution of responding B-cell clones. The IGH gene usage was not significantly skewed but frequencies of individual IGH genes changed repeatedly. We found predominant usage of unmutated and low mutation-loaded IGHV rearrangements characterizing naïve and extrafollicular B cells among the majority of expanded peripheral B-cell clonal lineages at most tested time points in most patients. IGH rearrangement usage showed no apparent relation to anti-SARS-CoV-2 antibody titers. Some patients demonstrated mono/oligoclonal populations carrying highly mutated IGHV rearrangements indicating antigen experience at some of the time points tested, including even before anti-SARS-CoV-2 antibodies were detected. We present evidence demonstrating that the B-cell response to SARS-CoV-2 is individual and includes different lineages of B cells at various time points during COVID-19 progression.

Metformin targets intestinal immune system signaling pathways in a high-fat diet-induced mouse model of obesity and insulin resistance.

Research findings of the past decade have highlighted the gut as the main site of action of the oral antihyperglycemic agent metformin despite its pharmacological role in the liver. Extensive evidence supports metformin's modulatory effect on the composition and function of gut microbiota, nevertheless, the underlying mechanisms of the host responses remain elusive. Our study aimed to evaluate metformin-induced alterations in the intestinal transcriptome profiles at different metabolic states. The high-fat diet-induced mouse model of obesity and insulin resistance of both sexes was developed in a randomized block experiment and bulk RNA-Seq of the ileum tissue was the method of choice for comparative transcriptional profiling after metformin intervention for ten weeks. We found a prominent transcriptional effect of the diet itself with comparatively fewer genes responding to metformin intervention. The overrepresentation of immune-related genes was observed, including pronounced metformin-induced upregulation of immunoglobulin heavy-chain variable region coding Ighv1-7 gene in both high-fat diet and control diet-fed animals. Moreover, we provide evidence of the downregulation NF-kappa B signaling pathway in the small intestine of both obese and insulin-resistant animals as well as control animals after metformin treatment. Finally, our data pinpoint the gut microbiota as a crucial component in the metformin-mediated downregulation of NF-kappa B signaling evidenced by a positive correlation between the Rel and Rela gene expression levels and abundances of Parabacteroides distasonis, Bacteroides spp., and Lactobacillus spp. in the gut microbiota of the same animals. Our study supports the immunomodulatory effect of metformin in the ileum of obese and insulin-resistant C57BL/6N mice contributed by intestinal immunoglobulin responses, with a prominent emphasis on the downregulation of NF-kappa B signaling pathway, associated with alterations in the composition of the gut microbiome.

Targeting conditioned media dependencies and FLT-3 in chronic lymphocytic leukemia

AbstractThe importance of the stromal microenvironment in chronic lymphocytic leukemia (CLL) pathogenesis and drug resistance is well established. Despite recent advances in CLL therapy, identifying novel ways to disrupt interactions between CLL and its microenvironment may identify new combination partners for the drugs currently in use. To understand the role of microenvironmental factors on primary CLL cells, we took advantage of an observation that conditioned media (CM) collected from stroma was protective of CLL cells from spontaneous cell death ex vivo. The cytokine in the CM-dependent cells that most supports CLL survival in short-term ex vivo culture was CCL2. Pretreatment of CLL cells with anti-CCL2 antibody enhanced venetoclax-mediated killing. Surprisingly, we found a group of CLL samples (9/23 cases) that are less likely to undergo cell death in the absence of CM support. Functional studies revealed that CM-independent (CMI) CLL cells are less sensitive to apoptosis than conventional stroma-dependent CLL. In addition, a majority of the CMI CLL samples (80%) harbored unmutated immunoglobulin heavy-chain variable (IGHV) region. Bulk-RNA sequence analysis revealed upregulation of the focal adhesion and RAS signaling pathways in this group, along with expression of fms-like tyrosine kinase 3 (FLT3) and CD135. Treatment with FLT3 inhibitors caused a significant reduction in cell viability among CMI samples. In summary, we were able to discriminate and target 2 biologically distinct subgroups of CLL based on CM dependence with distinct microenvironmental vulnerabilities.

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

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

Contribution of the IGCR1 regulatory element and the 3′Igh CTCF-binding elements to regulation of Igh V(D)J recombination

Immunoglobulin heavy chain variable region exons are assembled in progenitor-B cells, from VH, D, and JH gene segments located in separate clusters across the Igh locus. RAG endonuclease initiates V(D)J recombination from a JH-based recombination center (RC). Cohesin-mediated extrusion of upstream chromatin past RC-bound RAG presents Ds for joining to JHs to form a DJH-RC. Igh has a provocative number and organization of CTCF-binding elements (CBEs) that can impede loop extrusion. Thus, Igh has two divergently oriented CBEs (CBE1 and CBE2) in the IGCR1 element between the VH and D/JH domains, over 100 CBEs across the VH domain convergent to CBE1, and 10 clustered 3'Igh-CBEs convergent to CBE2 and VH CBEs. IGCR1 CBEs segregate D/JH and VH domains by impeding loop extrusion-mediated RAG-scanning. Downregulation of WAPL, a cohesin unloader, in progenitor-B cells neutralizes CBEs, allowing DJH-RC-bound RAG to scan the VH domain and perform VH-to-DJH rearrangements. To elucidate potential roles of IGCR1-based CBEs and 3'Igh-CBEs in regulating RAG-scanning and elucidate the mechanism of the ordered transition from D-to-JH to VH-to-DJH recombination, we tested effects of inverting and/or deleting IGCR1 or 3'Igh-CBEs in mice and/or progenitor-B cell lines. These studies revealed that normal IGCR1 CBE orientation augments RAG-scanning impediment activity and suggest that 3'Igh-CBEs reinforce ability of the RC to function as a dynamic loop extrusion impediment to promote optimal RAG scanning activity. Finally, our findings indicate that ordered V(D)J recombination can be explained by a gradual WAPL downregulation mechanism in progenitor-B cells as opposed to a strict developmental switch.

Impact of Minimal Residual Disease on Progression-Free Survival Outcomes After Fixed-Duration Ibrutinib-Venetoclax Versus Chlorambucil-Obinutuzumab in the GLOW Study.

In GLOW, fixed-duration ibrutinib + venetoclax showed superior progression-free survival (PFS) versus chlorambucil + obinutuzumab in older/comorbid patients with previously untreated chronic lymphocytic leukemia (CLL). The current analysis describes minimal residual disease (MRD) kinetics and any potential predictive value for PFS, as it has not yet been evaluated for ibrutinib + venetoclax treatment. Undetectable MRD (uMRD) was assessed by next-generation sequencing at <1 CLL cell per 10,000 (<10-4) and <1 CLL cell per 100,000 (<10-5) leukocytes. PFS was analyzed by MRD status at 3 months after treatment (EOT+3). Ibrutinib + venetoclax achieved deeper uMRD (<10-5) rates in bone marrow (BM) and peripheral blood (PB), respectively, in 40.6% and 43.4% of patients at EOT+3 versus 7.6% and 18.1% of patients receiving chlorambucil + obinutuzumab. Of these patients, uMRD (<10-5) in PB was sustained during the first year post-treatment (EOT+12) in 80.4% of patients receiving ibrutinib + venetoclax and 26.3% receiving chlorambucil + obinutuzumab. Patients with detectable MRD (dMRD; ≥10-4) in PB at EOT+3 were more likely to sustain MRD levels through EOT+12 with ibrutinib + venetoclax versus chlorambucil + obinutuzumab. PFS rates at EOT+12 were high among patients treated with ibrutinib + venetoclax regardless of MRD status at EOT+3: 96.3% and 93.3% in patients with uMRD (<10-4) and dMRD (≥10-4) in BM, respectively, versus 83.3% and 58.7% for patients receiving chlorambucil + obinutuzumab. PFS rates at EOT+12 also remained high in patients with unmutated immunoglobulin heavy-chain variable region (IGHV) receiving ibrutinib + venetoclax, independent of MRD status in BM. Molecular and clinical relapses were less frequent during the first year post-treatment with ibrutinib + venetoclax versus chlorambucil + obinutuzumab regardless of MRD status at EOT+3 and IGHV status. Even for patients not achieving uMRD (<10-4), PFS rates remained high with ibrutinib + venetoclax; this is a novel finding and requires additional follow-up to confirm its persistence over time.