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Patients receiving ibrutinib for CLL rarely achieve undetectable measurable residual disease (U-MRD), necessitating indefinite therapy, with cumulative risks of treatment discontinuation due to progression or adverse events. This study added venetoclax to ibrutinib for up to 2 years, in patients who had received ibrutinib for ≥12 months (mo) and had ≥1 high risk feature (TP53mutation and/or deletion, ATMdeletion, complex karyotype or persistently elevated β2-microglobulin). The primary endpoint was U-MRD with 10–4sensitivity (U-MRD4) in bone marrow (BM) at 12mo. Forty-five patients were treated. On intention-to-treat analysis, 23/42 (55%) patients improved their response to CR (2 pts were in MRD + CR at venetoclax initiation). U-MRD4 at 12mo was 57%. 32/45 (71%) had U-MRD at the completion of venetoclax: 22/32 stopped ibrutinib; 10 continued ibrutinib. At a median of 41 months from venetoclax initiation, 5/45 patients have progressed; none have died from CLL or Richter Transformation. In 32 patients with BM U-MRD4, peripheral blood (PB) MRD4 was analyzed every 6 months; 10/32 have had PB MRD re-emergence at a median of 13 months post-venetoclax. In summary, the addition of venetoclax in patients treated with ≥12mo of ibrutinib achieved high rate of BM U-MRD4 and may achieve durable treatment-free remission.

The ionic liquid (IL) 1-butyl-1-methyl-pyrrolidinium bis(trifluoromethanesulfonyl)imide ([Pyr14][TFSI]) with organic solvent methyl propionate (MP) was investigated to gain a fundamental understanding of the thermal, physical and electrochemical properties. Estimation of the coulombic efficiencies on a copper electrode was additionally carried out using cyclic voltammetry as an initial screening to test the promise for lithium metal batteries. The addition of MP to [Pyr14][TFSI] suppressed the melting point, dramatically increased conductivity (from 0.56 mS/cm at 25 °C in 0.8 m LiTFSI in [Pyr14][TFSI] to 11 mS/cm at 25 °C in 0.8 m LiTFSI in a mixture of 1:7 mol ratio of [Pyr14][TFSI] to MP) and still maintained the electrochemical window (>5 V). While the addition of MP reduced the melting point and increased conductivity, the presence of cyclic carbonates in the cosolvent was necessary to enable deposition and stripping of lithium with high coulombic efficiencies. The fluorinated ethylene carbonate (FEC) appeared to enhance the SEI formation and suppress the Claisen reaction that MP enabled.

Fadraciclib (CYC065) is a second-generation aminopurine CDK2/9 inhibitor with increased potency and selectivity toward CDK2 and CDK9 compared to seliciclib (R-roscovitine). In chronic lymphocytic leukemia (CLL), a disease that depends on the over-expression of anti-apoptotic proteins for its survival, inhibition of CDK9 by fadraciclib reduced phosphorylation of the C-terminal domain of RNA polymerase II and blocked transcription in vitro; these actions depleted the intrinsically short-lived anti-apoptotic protein Mcl-1 and induced apoptosis. While the simulated bone marrow and lymph node microenvironments induced Mcl-1 expression and protected CLL cells from apoptosis, these conditions did not prolong the turnover rate of Mcl-1, and fadraciclib efficiently abrogated the protective effect. Further, fadraciclib was synergistic with the Bcl-2 antagonist venetoclax, inducing more profound CLL cell death, especially in samples with 17p deletion. While fadraciclib, venetoclax, and the combination each had distinct kinetics of cell death induction, their activities were reversible, as no additional cell death was induced upon removal of the drugs. The best combination effects were achieved when both drugs were maintained together. Altogether, this study provides a rationale for the clinical development of fadraciclib in CLL, either alone or in combination with a Bcl-2 antagonist.

Richter’s Syndrome (RS) is an aggressive transformation of CLL, usually clonally-related diffuse large B-cell lymphoma (DLBCL), characterized by frequent TP53 mutations, intrinsic chemoresistance and poor survival. TP53-independent treatments are needed. We conducted a single center, phase 2, investigator-initiated study of high dose blinatumomab (maximum 112 mcg/d after initial, weekly dose escalation), NCT03121534, given for an 8-week induction and 4-week consolidation cycle. Responses were assessed by Lugano 2014 criteria. Serial multi-parameter flow cytometry from blood was performed to identify patient-specific biomarkers for response. Nine patients were treated. Patients had received a median of 4 and 2 prior therapies for CLL and RS, respectively. Five of 9 had del(17p) and 100% had complex karyotype. Four patients had reduction in nodal disease, including one durable complete response lasting >1 y. Treatment was well tolerated, with no grade >3 cytokine release syndrome and 1 case of grade 3, reversible neurotoxicity. Immunophenotyping demonstrated the majority of patients expressed multiple immune checkpoints, especially PD1, TIM3 and TIGIT. The patient who achieved CR had the lowest levels of immune checkpoint expression. Simultaneous targeting with immune checkpoint blockade, especially PD1 inhibition, which has already demonstrated single-agent efficacy in RS, could achieve synergistic killing and enhance outcomes.

The chemoattractant CXCL13 organizes the cellular architecture of B-cell follicles and germinal centers. During adaptive immune responses, CXCL13 plasma concentrations transiently increase and function as a biomarker for normal germinal center activity. Chronic lymphocytic leukemia (CLL) cells express high levels of CXCR5, the receptor for CXCL13, and proliferate in pseudofollicles within secondary lymphoid organs (SLO). Given the morphologic and functional similarities between normal and CLL B-cell expansion in SLO, we hypothesized that CXCL13 plasma concentrations would correlate with CLL disease activity and progression. We analyzed CXCL13 plasma concentrations in 400 CLL patients and correlated the findings with other prognostic markers, time to treatment (TTT), CCL3 and CCL4 plasma concentrations, and in vivo CLL cell proliferation. We found that CXCL13 plasma concentrations were higher in CLL patients with active and advanced stage disease, resulting in a significantly shorter TTT. Accordingly, high CXCL13 levels correlated with other markers of disease activity and CCL3 levels. Higher CLL cell birth rates in vivo also associated with higher CXCL13 plasma concentrations. Interestingly, elevated CXCL13 plasma levels normalized during ibrutinib therapy, and increased in ibrutinib resistance patients. Collectively, these studies emphasize the importance of CXCL13 in crosstalk between CLL cells and the SLO microenvironment.

Despite major improvements in treatment outcome with novel targeted therapies, such as the Bruton tyrosine kinase (BTK) inhibitor ibrutinib, chronic lymphocytic leukemia (CLL) remains incurable in the majority of patients. Activation of PI3K, NF-κB, and/or MYC has been linked to residual disease and/or resistance in ibrutinib-treated patients. These pathways can be targeted by inhibitors of bromodomain and extra-terminal (BET) proteins. Here we report about the preclinical activity of GS-5829, a novel BET inhibitor, in CLL. GS-5829 inhibited CLL cell proliferation and induced leukemia cell apoptosis through deregulation of key signaling pathways, such as BLK, AKT, ERK1/2, and MYC. IκBα modulation indicates that GS-5829 also inhibited NF-κB signaling. GS-5829-induced apoptosis resulted from an imbalance between positive (BIM) and negative regulators (BCL-XL) of the intrinsic apoptosis pathway. The antileukemia activity of GS-5829 increased synergistically in combinations with B-cell receptor signaling inhibitors, the BTK inhibitor ibrutinib, the PI3Kδ inhibitor idelalisib, and the SYK inhibitor entospletinib. In cocultures that mimic the lymph node microenvironment, GS-5829 inhibited signaling pathways within nurselike cells and their growth, indicating that BET inhibitors also can target the supportive CLL microenvironment. Collectively, these data provide a rationale for the clinical evaluation of BET inhibitors in CLL.

Patients with chronic lymphocytic leukemia (CLL) who achieve blood or bone marrow (BM) undetectable minimal residual disease (U-MRD) status after first-line fludarabine, cyclophosphamide, and rituximab (FCR) have prolonged progression-free survival (PFS), when assessed by an assay with sensitivity 10-4 (MRD4). Despite reaching U-MRD4, many patients, especially those with unmutated IGHV, subsequently relapse, suggesting residual disease <10-4 threshold and the need for more sensitive MRD evaluation. MRD evaluation by next-generation sequencing (NGS) has a sensitivity of 10-6 (MRD6). To better assess the depth of remission following first-line FCR treatment, we used NGS (Adaptive Biotechnologies Corporation) to assess MRD in 62 patients, all of whom had BM U-MRD by multicolor flow cytometry (sensitivity 10-4) at end-of-FCR treatment. Samples from these patients included 57 BM samples, 29 peripheral blood mononuclear cell (PBMC) samples, and 32 plasma samples. Only 27.4% of the 62 patients had U-MRD by NGS. Rate of U-MRD by NGS was lowest in BM (25%), compared with PBMC (55%) or plasma (75%). No patient with U-MRD by NGS in BM or PBMC was MRD+ in plasma. Patients with mutated IGHV were more likely to have U-MRD by NGS at the end of treatment (EOT; 41% vs 13%, P = .02) than those with unmutated IGHV. Median follow-up was 81.6 months. Patients with U-MRD at EOT had superior PFS vs MRD+ patients, regardless of sample type assessed (BM, P = .02, median not reached [NR] vs 67 months; PBMC, P = .02, median NR vs 74 months). More sensitive MRD6 testing increases prognostic discrimination over MRD4 testing.

Patients with chronic lymphocytic leukemia (CLL) who achieve blood or bone marrow (BM) undetectable minimal residual disease (U-MRD) status after first-line fludarabine, cyclophosphamide, and rituximab (FCR) have prolonged progression-free survival (PFS), when assessed by an assay with sensitivity 10−4(MRD4). Despite reaching U-MRD4, many patients, especially those with unmutated IGHV, subsequently relapse, suggesting residual disease <10−4threshold and the need for more sensitive MRD evaluation. MRD evaluation by next-generation sequencing (NGS) has a sensitivity of 10−6(MRD6). To better assess the depth of remission following first-line FCR treatment, we used NGS (Adaptive Biotechnologies Corporation) to assess MRD in 62 patients, all of whom had BM U-MRD by multicolor flow cytometry (sensitivity 10−4) at end-of-FCR treatment. Samples from these patients included 57 BM samples, 29 peripheral blood mononuclear cell (PBMC) samples, and 32 plasma samples. Only 27.4% of the 62 patients had U-MRD by NGS. Rate of U-MRD by NGS was lowest in BM (25%), compared with PBMC (55%) or plasma (75%). No patient with U-MRD by NGS in BM or PBMC was MRD+in plasma. Patients with mutated IGHVwere more likely to have U-MRD by NGS at the end of treatment (EOT; 41% vs 13%, P= .02) than those with unmutated IGHV. Median follow-up was 81.6 months. Patients with U-MRD at EOT had superior PFS vs MRD+patients, regardless of sample type assessed (BM, P= .02, median not reached [NR] vs 67 months; PBMC, P= .02, median NR vs 74 months). More sensitive MRD6 testing increases prognostic discrimination over MRD4 testing.

Background:Ibrutinib (IBR) and venetoclax (VEN) combination is an effective therapy for patients (pts) with CLL. We previously reported results of the first-line cohort of a phase II trial of combined IBR and VEN for high-risk pts with CLL (Jain, NEJM 2019, JAMA Oncology 2021). Here we report updated data for 120 pts (80 pts in the original published cohort and 40 pts in an expansion cohort) with a median follow-up of 61.5 months.

CLL is characterized by immune and hematopoietic abnormalities such as T-cell dysfunction, hypogammaglobulinemia, increased levels of inflammatory cytokines, aberrant monocyte polarity, anemia, neutropenia, and low-grade bone marrow fibrosis. As a result, CLL patients become susceptible to increased rates of infection and/or second neoplasms.

Background

This phase 2 study was conducted to prospectively evaluate how clinical and biological factors correlate with outcome in patients with treatment-naive (TN) and relapsed (R) chronic lymphocytic leukemia (CLL) treated with lenalidomide and rituximab. Oral lenalidomide 10 mg was administered daily starting on day 9 of cycle 1. IV rituximab 375 mg/m2 was administered weekly during cycle 1 and every 4 weeks for cycles 3 to 12. Sequencing of a custom panel of 295 genes was performed in pretreatment bone marrow samples. The study included 61 patients with TN CLL and 59 with R CLL; the overall response rate (ORR) was 73% and 64%, respectively. A baseline β2-microglobulin level <4 mg/L was associated with higher ORR in both groups (both, P = .03), and absence of mutations in the NOTCH signaling pathway showed a trend for association with higher ORR in R CLL (P = .10). Median PFS was 50 months in TN patients and 28 months in R patients. On multivariate analysis, age ≥65 years (P = .02) was associated with shorter PFS in TN patients, whereas according to univariate analysis, >2 previous therapies (P = .02) was the only factor associated with shorter PFS in R patients. A trend for association between mutations in the NOTCH pathway and shorter PFS was observed in TN CLL (P = .15). Further exploration of the NOTCH pathway may help optimize the efficacy of this combination in patients with CLL. This study protocol was approved by the University of Texas MD Anderson Cancer Center institutional review board and registered at clinicaltrials.gov (#NCT01446133).

This phase 2 study was conducted to prospectively evaluate how clinical and biological factors correlate with outcome in patients with treatment-naive (TN) and relapsed (R) chronic lymphocytic leukemia (CLL) treated with lenalidomide and rituximab. Oral lenalidomide 10 mg was administered daily starting on day 9 of cycle 1. IV rituximab 375 mg/m2was administered weekly during cycle 1 and every 4 weeks for cycles 3 to 12. Sequencing of a custom panel of 295 genes was performed in pretreatment bone marrow samples. The study included 61 patients with TN CLL and 59 with R CLL; the overall response rate (ORR) was 73% and 64%, respectively. A baseline β2-microglobulin level <4 mg/L was associated with higher ORR in both groups (both, P= .03), and absence of mutations in the NOTCH signaling pathway showed a trend for association with higher ORR in R CLL (P= .10). Median PFS was 50 months in TN patients and 28 months in R patients. On multivariate analysis, age ≥65 years (P= .02) was associated with shorter PFS in TN patients, whereas according to univariate analysis, >2 previous therapies (P= .02) was the only factor associated with shorter PFS in R patients. A trend for association between mutations in the NOTCH pathway and shorter PFS was observed in TN CLL (P= .15). Further exploration of the NOTCH pathway may help optimize the efficacy of this combination in patients with CLL. This study protocol was approved by the University of Texas MD Anderson Cancer Center institutional review board and registered at clinicaltrials.gov(#NCT01446133).

Ibrutinib, an oral covalent inhibitor of Bruton's tyrosine kinase, is an effective therapy for patients with chronic lymphocytic leukemia (CLL). To determine whether rituximab provides added benefit to ibrutinib, we conducted a randomized single-center trial of ibrutinib vs ibrutinib plus rituximab. Patients with CLL requiring therapy were randomized to receive 28-day cycles of once-daily ibrutinib 420 mg, either as a single agent (n = 104), or together with rituximab (375 mg/m2; n = 104), given weekly during cycle 1, then once per cycle until cycle 6. The primary end point was progression-free survival (PFS) in the intention-to-treat population. We enrolled 208 patients with CLL, 181 with relapsed CLL and 27 treatment-naive patients with high-risk disease (17p deletion or TP53mutation). After a median follow-up of 36 months, the Kaplan-Meier estimates of PFS were 86% (95% confidence interval [CI], 76.6-91.9) for patients receiving ibrutinib, and 86.9% (95% CI, 77.3-92.6) for patients receiving ibrutinib plus rituximab. Similarly, response rates were the same in both arms (overall response rate, 92%). However, time to normalization of peripheral blood lymphocyte counts and time to complete remission were shorter, and residual disease levels in the bone marrow were lower, in patients receiving ibrutinib plus rituximab. We conclude that the addition of rituximab to ibrutinib in relapsed and treatment-naive high-risk patients with CLL failed to show improvement in PFS. However, patients treated with ibrutinib plus rituximab reached their remissions faster and achieved significantly lower residual disease levels. Given these results, ibrutinib as single-agent therapy remains current standard-of-care treatment in CLL. This trial was registered at www.clinicaltrials.govas #NCT02007044.

Ibrutinib, an oral covalent inhibitor of Bruton’s tyrosine kinase, is an effective therapy for patients with chronic lymphocytic leukemia (CLL). To determine whether rituximab provides added benefit to ibrutinib, we conducted a randomized single-center trial of ibrutinib vs ibrutinib plus rituximab. Patients with CLL requiring therapy were randomized to receive 28-day cycles of once-daily ibrutinib 420 mg, either as a single agent (n = 104), or together with rituximab (375 mg/m2; n = 104), given weekly during cycle 1, then once per cycle until cycle 6. The primary end point was progression-free survival (PFS) in the intention-to-treat population. We enrolled 208 patients with CLL, 181 with relapsed CLL and 27 treatment-naive patients with high-risk disease (17p deletion or TP53 mutation). After a median follow-up of 36 months, the Kaplan-Meier estimates of PFS were 86% (95% confidence interval [CI], 76.6-91.9) for patients receiving ibrutinib, and 86.9% (95% CI, 77.3-92.6) for patients receiving ibrutinib plus rituximab. Similarly, response rates were the same in both arms (overall response rate, 92%). However, time to normalization of peripheral blood lymphocyte counts and time to complete remission were shorter, and residual disease levels in the bone marrow were lower, in patients receiving ibrutinib plus rituximab. We conclude that the addition of rituximab to ibrutinib in relapsed and treatment-naive high-risk patients with CLL failed to show improvement in PFS. However, patients treated with ibrutinib plus rituximab reached their remissions faster and achieved significantly lower residual disease levels. Given these results, ibrutinib as single-agent therapy remains current standard-of-care treatment in CLL. This trial was registered at www.clinicaltrials.gov as #NCT02007044.

Ibrutinib is highly efficacious and used at 420 mg/d for treatment of chronic lymphocytic leukemia (CLL). We previously demonstrated a decline in Bruton’s tyrosine kinase (BTK) protein levels in CLL cells after 1 cycle of ibrutinib, suggesting ibrutinib dose could be lowered after the first cycle without loss of biological effect. To test this postulate, a pilot study (NCT02801578) was designed to systematically reduce ibrutinib dosing within the same patient with CLL over the course of three 28-day cycles. After an initial cycle of 420 mg/d, the dose was reduced to 280 mg/d in cycle 2, and then to 140 mg/d in cycle 3. Eleven patients began study treatment, and 9 completed the 3 cycles. Plasma and intracellular pharmacokinetics (PK), BTK occupancy, and pharmacodynamic (PD) response at different doses of ibrutinib were compared. Plasma and intracellular levels of ibrutinib were dose-dependent, and even the lowest dose was sufficient to occupy, on average, more than 95% of BTK protein. In concert, BTK downstream signaling inhibition was maintained with 140 mg/d ibrutinib in cycle 3, and there were comparable reductions in total and phospho-BTK (Tyr223) protein levels across 3 cycles. Reductions of plasma chemokine CCL3 and CCL4 levels, considered to be biomarkers of ibrutinib response, were similar during the 3 cycles. These PK/PD data demonstrate that after 1 cycle of ibrutinib at the standard 420 mg/d dose, the dose can be reduced without losing biological activity. Clinical efficacy of lower doses needs to be systematically evaluated. Such dose reductions would lower drug cost, lessen untoward toxicity, and facilitate rationale-based combinations. This trial was registered at www.clinicaltrials.gov as #NCT02801578.

Ibrutinib is highly efficacious and used at 420 mg/d for treatment of chronic lymphocytic leukemia (CLL). We previously demonstrated a decline in Bruton's tyrosine kinase (BTK) protein levels in CLL cells after 1 cycle of ibrutinib, suggesting ibrutinib dose could be lowered after the first cycle without loss of biological effect. To test this postulate, a pilot study (NCT02801578) was designed to systematically reduce ibrutinib dosing within the same patient with CLL over the course of three 28-day cycles. After an initial cycle of 420 mg/d, the dose was reduced to 280 mg/d in cycle 2, and then to 140 mg/d in cycle 3. Eleven patients began study treatment, and 9 completed the 3 cycles. Plasma and intracellular pharmacokinetics (PK), BTK occupancy, and pharmacodynamic (PD) response at different doses of ibrutinib were compared. Plasma and intracellular levels of ibrutinib were dose-dependent, and even the lowest dose was sufficient to occupy, on average, more than 95% of BTK protein. In concert, BTK downstream signaling inhibition was maintained with 140 mg/d ibrutinib in cycle 3, and there were comparable reductions in total and phospho-BTK (Tyr223) protein levels across 3 cycles. Reductions of plasma chemokine CCL3 and CCL4 levels, considered to be biomarkers of ibrutinib response, were similar during the 3 cycles. These PK/PD data demonstrate that after 1 cycle of ibrutinib at the standard 420 mg/d dose, the dose can be reduced without losing biological activity. Clinical efficacy of lower doses needs to be systematically evaluated. Such dose reductions would lower drug cost, lessen untoward toxicity, and facilitate rationale-based combinations. This trial was registered at www.clinicaltrials.govas #NCT02801578.

ABSTRACTIntroduction: The treatment of relapsed/refractory (RR) CLL has been revolutionized by the advent of the new oral inhibitors of B-cell receptor (BCR) signaling and the pro-survival protein, B-cell lymphoma 2 (BCL2). Additionally, new and more potent monoclonal antibodies against CD20 have replaced/may replace rituximab in many settings.Areas covered: Herein, we review the entire therapeutic landscape of RR CLL, with particular attention to the new small-molecule kinase inhibitors and BH3-mimetics. We discuss preclinical data with these agents in CLL, cover available efficacy and safety information, and examine potential resistance mechanisms and possible rational combinations to circumvent them.Expert opinion: The availability of potent and selective inhibitors of BCR signaling and of the anti-apoptotic functions of BCL2 has enormously enhanced our therapeutic armamentarium, with unprecedented efficacy now observed in patients who historically had poor outcomes with chemoimmunotherapy (CIT), e.g., those with deletion 17p/11q and/or IGHV-unmutated disease. The next challenge is to optimally sequence these agents and develop rational combinations that will hopefully lead to deeper and more durable remissions than ever seen before. Indeed, long term relapse free survival, already achievable with CIT in patients with genetically favorable-risk disease, now appears to be a realistic possibility for most patients with CLL.

Cancer cells experience higher oxidative stress from reactive oxygen species (ROS) than do non-malignant cells because of genetic alterations and abnormal growth; as a result, maintenance of the antioxidant glutathione (GSH) is essential for their survival and proliferation. Under conditions of elevated ROS, endogenous L-cysteine (L-Cys) production is insufficient for GSH synthesis. This necessitates uptake of L-Cys that is predominantly in its disulfide form, L-cystine (CSSC), via the xCT(−) transporter. We show that administration of an engineered and pharmacologically optimized human cyst(e)inase enzyme mediates sustained depletion of the extracellular L-Cys and CSSC pool in mice and non-human primates. Treatment with this enzyme selectively causes cell cycle arrest and death in cancer cells due to depletion of intracellular GSH and ensuing elevated ROS; yet this treatment results in no apparent toxicities in mice even after months of continuous treatment. Cyst(e)inase suppressed the growth of prostate carcinoma allografts, reduced tumor growth in both prostate and breast cancer xenografts and doubled the median survival time of TCL1-Tg:p53−/−mice, which develop disease resembling human chronic lymphocytic leukemia. It was observed that enzyme-mediated depletion of the serum L-Cys and CSSC pool suppresses the growth of multiple tumors, yet is very well tolerated for prolonged periods, suggesting that cyst(e)inase represents a safe and effective therapeutic modality for inactivating antioxidant cellular responses in a wide range of malignancies.

•Patients with IGHV-M have favorable very-long–term PFS after FCR, although later relapses (>10 years) can occur, albeit rarely.•Cumulative risk of tMNs in all patients was 6.3%; patients with IGHV-M are more likely to die from causes other than CLL.

Ibrutinib, a potent and irreversible small-molecule inhibitor of both Bruton’s tyrosine kinase and interleukin-2 inducible kinase (ITK), has been used to treat relapsed/refractory chronic lymphocytic leukemia (CLL) with prolongation of progression-free and overall survival. Here, we present 27 patients with relapsed CLL following allogeneic hematopoietic cell transplant (HCT) who subsequently received ibrutinib salvage therapy. Sixteen of these patients were part of multi-institutional clinical trials and achieved an overall response rate of 87.5%. An additional 11 patients were treated at Stanford University following US Food and Drug Administration approval of ibrutinib; 7 (64%) achieved a complete response, and 3 (27%) achieved a partial response. Of the 9 patients treated at Stanford who had mixed chimerism–associated CLL relapse, 4 (44%) converted to full donor chimerism following ibrutinib initiation, in association with disease response. Four of 11 (36%) patients evaluated by ClonoSeq achieved minimal residual disease negativity with CLL <1/10 000 white blood cells, which persisted even after ibrutinib was discontinued, in 1 case even after 26 months. None of the 27 patients developed graft-versus-host-disease (GVHD) following ibrutinib initiation. We postulate that ibrutinib augments the graft-versus-leukemia (GVL) benefit through a T-cell–mediated effect, most likely due to ITK inhibition. To investigate the immune modulatory effects of ibrutinib, we completed comprehensive immune phenotype characterization of peripheral B and T cells from treated patients. Our results show that ibrutinib selectively targets pre–germinal B cells and depletes Th2 helper cells. Furthermore, these effects persisted after drug discontinuation. In total, our results provide evidence that ibrutinib effectively augments GVL without causing GVHD.

Ibrutinib, a potent and irreversible small-molecule inhibitor of both Bruton's tyrosine kinase and interleukin-2 inducible kinase (ITK), has been used to treat relapsed/refractory chronic lymphocytic leukemia (CLL) with prolongation of progression-free and overall survival. Here, we present 27 patients with relapsed CLL following allogeneic hematopoietic cell transplant (HCT) who subsequently received ibrutinib salvage therapy. Sixteen of these patients were part of multi-institutional clinical trials and achieved an overall response rate of 87.5%. An additional 11 patients were treated at Stanford University following US Food and Drug Administration approval of ibrutinib; 7 (64%) achieved a complete response, and 3 (27%) achieved a partial response. Of the 9 patients treated at Stanford who had mixed chimerism–associated CLL relapse, 4 (44%) converted to full donor chimerism following ibrutinib initiation, in association with disease response. Four of 11 (36%) patients evaluated by ClonoSeq achieved minimal residual disease negativity with CLL <1/10 000 white blood cells, which persisted even after ibrutinib was discontinued, in 1 case even after 26 months. None of the 27 patients developed graft-versus-host-disease (GVHD) following ibrutinib initiation. We postulate that ibrutinib augments the graft-versus-leukemia (GVL) benefit through a T-cell–mediated effect, most likely due to ITK inhibition. To investigate the immune modulatory effects of ibrutinib, we completed comprehensive immune phenotype characterization of peripheral B and T cells from treated patients. Our results show that ibrutinib selectively targets pre–germinal B cells and depletes Th2 helper cells. Furthermore, these effects persisted after drug discontinuation. In total, our results provide evidence that ibrutinib effectively augments GVL without causing GVHD.

ABSTRACTIntroduction:Ibrutinib, a first-in-class covalent inhibitor of Bruton’s tyrosine kinase (BTK), is approved in many countries for the treatment of relapsed/refractory chronic lymphocytic leukemia (CLL) and for previously untreated disease with a 17p deletion and, most recently, as a frontline therapy for CLL. In controlled trials in CLL, ibrutinib produced high response rates and improved survival in both the frontline and relapsed settings. While ibrutinib controls CLL with impressive efficacy, it only infrequently induces complete remissions, particularly of relapsed CLL, and does not eradicate minimal residual disease. Finally, ibrutinib is extremely expensive, has off-target toxicities, and requires indefinite therapy.Areas covered:In this article, we provide an overview of the CLL therapeutic landscape and discuss the pharmacokinetic and pharmacodynamic aspects of ibrutinib. Major clinical trials of ibrutinib in CLL are summarized, and its safety profile explored.Expert opinion:Ibrutinib represents a transformative advance in CLL management and has validated BTK as a therapeutic target in this disease, but has some limitations, leading to the emergence of other BTK inhibitors and mechanism-based combination strategies. Given complete BTK occupancy at lower doses of ibrutinib and declining levels of BTK on ibrutinib therapy, lower doses of ibrutinib in CLL are being explored.

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

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

Deletion 20q (Del(20q)), a common cytogenetic abnormality in myeloid neoplasms, is rare in chronic lymphocytic leukemia. We report 64 patients with chronic lymphocytic leukemia and del(20q), as the sole abnormality in 40, a stemline abnormality in 21, and a secondary abnormality in 3 cases. Fluorescence in situ hybridization (FISH) analysis revealed an additional high-risk abnormality, del(11q) or del(17p), in 25/64 (39%) cases. In most cases, the leukemic cells showed atypical cytologic features, unmutated IGHV (immunoglobulin heavy-chain variable region) genes, and ZAP70 positivity. The del(20q) was detected only after chemotherapy in all 27 cases with initial karyotypes available. With a median follow-up of 90 months, 30 patients (47%) died, most as a direct consequence of chronic lymphocytic leukemia. Eight patients developed a therapy-related myeloid neoplasm, seven with a complex karyotype. Combined morphologic and FISH analysis for del(20q) performed in 12 cases without morphologic evidence of a myeloid neoplasm localized the del(20q) to the chronic lymphocytic leukemia cells in 5 (42%) cases, and to myeloid/erythroid cells in 7 (58)% cases. The del(20q) was detected in myeloid cells in all 4 cases of myelodysplastic syndrome. In aggregate, these data indicate that chronic lymphocytic leukemia with del(20q) acquired after therapy is heterogeneous. In cases with morphologic evidence of dysplasia, the del(20q) likely resides in the myeloid lineage. However, in cases without morphologic evidence of dysplasia, the del(20q) may represent clonal evolution and disease progression. Combining morphologic analysis with FISH for del(20q) or performing FISH on immunomagnetically selected sub-populations to localize the cell population with this abnormality may help guide patient management.

Deletion 20q (Del(20q)), a common cytogenetic abnormality in myeloid neoplasms, is rare in chronic lymphocytic leukemia. We report 64 patients with chronic lymphocytic leukemia and del(20q), as the sole abnormality in 40, a stemline abnormality in 21, and a secondary abnormality in 3 cases. Fluorescence in situhybridization (FISH) analysis revealed an additional high-risk abnormality, del(11q) or del(17p), in 25/64 (39%) cases. In most cases, the leukemic cells showed atypical cytologic features, unmutated IGHV(immunoglobulin heavy-chain variable region) genes, and ZAP70 positivity. The del(20q) was detected only after chemotherapy in all 27 cases with initial karyotypes available. With a median follow-up of 90 months, 30 patients (47%) died, most as a direct consequence of chronic lymphocytic leukemia. Eight patients developed a therapy-related myeloid neoplasm, seven with a complex karyotype. Combined morphologic and FISH analysis for del(20q) performed in 12 cases without morphologic evidence of a myeloid neoplasm localized the del(20q) to the chronic lymphocytic leukemia cells in 5 (42%) cases, and to myeloid/erythroid cells in 7 (58)% cases. The del(20q) was detected in myeloid cells in all 4 cases of myelodysplastic syndrome. In aggregate, these data indicate that chronic lymphocytic leukemia with del(20q) acquired after therapy is heterogeneous. In cases with morphologic evidence of dysplasia, the del(20q) likely resides in the myeloid lineage. However, in cases without morphologic evidence of dysplasia, the del(20q) may represent clonal evolution and disease progression. Combining morphologic analysis with FISH for del(20q) or performing FISH on immunomagnetically selected sub-populations to localize the cell population with this abnormality may help guide patient management.

Intrinsic and extrinsic apoptotic pathways converge to activate common downstream executioner caspases (caspase-3, -6, and -7), resulting in cell death. In chronic lymphocytic leukemia (CLL), neoplastic B cells evade apoptosis owing to the overexpression of survival proteins. We hypothesized that direct activation of procaspases could bypass the apoptosis resistance induced by the upstream prosurvival proteins. The procaspase-activating compounds (PAC-1), including B-PAC-1 (L14R8), convert inactive executioner procaspases to their active cleaved forms by chelation of labile zinc ions. Both at transcript and protein levels, primary CLL cells express high levels of latent procaspases (3, -7, and -9). B-PAC-1 treatment induced CLL lymphocyte death which was higher than that in normal peripheral blood mononuclear cells or B cells, and was independent of prognostic markers and microenvironmental factors. Mechanistically, B-PAC-1 treatment activated executioner procaspases and not other Zn-dependent enzymes. Exogenous zinc completely, and pancaspase inhibitors partially, reversed B-PAC-1–induced apoptosis, elucidating the zinc-mediated mechanism of action. The cell demise relied on the presence of caspase-3/7 but not caspase-8 or Bax/Bak proteins. B-PAC-1 in combination with an inhibitor of apoptosis protein antagonist (Smac066) synergistically induced apoptosis in CLL samples. Our investigations demonstrated that direct activation of executioner procaspases via B-PAC-1 treatment bypasses apoptosis resistance and is a novel approach for CLL therapeutics.

Although fludarabine, cyclophosphamide, and rituximab (FCR) together are established as a standard first-line treatment of younger patients with chronic lymphocytic leukemia (CLL), there is little information to guide the management of patients with CLL refractory to, or who have relapsed after, receiving frontline FCR treatment. To define optimal salvage strategy and identify patients unsuitable for retreatment with FCR, we examined the survival and treatment outcome of 300 patients enrolled in a phase 2 study of FCR. After a median 142 months of follow-up, 156 patients developed progressive CLL, with a median survival of 51 months after disease progression. The duration of first remission (REM1) was a key determinant of survival after disease progression and first salvage. Patients with a short REM1 (<3 years) had a short survival period, irrespective of salvage therapy received; these patients have high unmet medical needs and are good candidates for investigation of novel therapies. In patients with a long REM1 (≥3 years), salvage treatment with either repeat FCR or lenalidomide-based therapy results in subsequent median survival exceeding 5 years; for these patients, FCR rechallenge represents a reasonable standard of care.

Although fludarabine, cyclophosphamide, and rituximab (FCR) together are established as a standard first-line treatment of younger patients with chronic lymphocytic leukemia (CLL), there is little information to guide the management of patients with CLL refractory to, or who have relapsed after, receiving frontline FCR treatment. To define optimal salvage strategy and identify patients unsuitable for retreatment with FCR, we examined the survival and treatment outcome of 300 patients enrolled in a phase 2 study of FCR. After a median 142 months of follow-up, 156 patients developed progressive CLL, with a median survival of 51 months after disease progression. The duration of first remission (REM1) was a key determinant of survival after disease progression and first salvage. Patients with a short REM1 (<3 years) had a short survival period, irrespective of salvage therapy received; these patients have high unmet medical needs and are good candidates for investigation of novel therapies. In patients with a long REM1 (≥3 years), salvage treatment with either repeat FCR or lenalidomide-based therapy results in subsequent median survival exceeding 5 years; for these patients, FCR rechallenge represents a reasonable standard of care.