Pembrolizumab in Microsatellite-Instability-High Advanced Colorectal Cancer.

BACKGROUND: Programmed death 1 (PD-1) blockade has clinical benefit in microsatellite-instability-high (MSI-H) or mismatch-repair-deficient (dMMR) tumors after previous therapy. The efficacy of PD-1 blockade as compared with chemotherapy as first-line therapy for MSI-H-dMMR advanced or metastatic colorectal cancer is unknown. METHODS: In this phase 3, open-label trial, 307 patients with metastatic MSI-H-dMMR colorectal cancer who had not previously received treatment were randomly assigned, in a 1:1 ratio, to receive pembrolizumab at a dose of 200 mg every 3 weeks or chemotherapy (5-fluorouracil-based therapy with or without bevacizumab or cetuximab) every 2 weeks. Patients receiving chemotherapy could cross over to pembrolizumab therapy after disease progression. The two primary end points were progression-free survival and overall survival. RESULTS: At the second interim analysis, after a median follow-up (from randomization to data cutoff) of 32.4 months (range, 24.0 to 48.3), pembrolizumab was superior to chemotherapy with respect to progression-free survival (median, 16.5 vs. 8.2 months; hazard ratio, 0.60; 95% confidence interval [CI], 0.45 to 0.80; P = 0.0002). The estimated restricted mean survival after 24 months of follow-up was 13.7 months (range, 12.0 to 15.4) as compared with 10.8 months (range, 9.4 to 12.2). As of the data cutoff date, 56 patients in the pembrolizumab group and 69 in the chemotherapy group had died. Data on overall survival were still evolving (66% of required events had occurred) and remain blinded until the final analysis. An overall response (complete or partial response), as evaluated with Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1, was observed in 43.8% of the patients in the pembrolizumab group and 33.1% in the chemotherapy group. Among patients with an overall response, 83% in the pembrolizumab group, as compared with 35% of patients in the chemotherapy group, had ongoing responses at 24 months. Treatment-related adverse events of grade 3 or higher occurred in 22% of the patients in the pembrolizumab group, as compared with 66% (including one patient who died) in the chemotherapy group. CONCLUSIONS: Pembrolizumab led to significantly longer progression-free survival than chemotherapy when received as first-line therapy for MSI-H-dMMR metastatic colorectal cancer, with fewer treatment-related adverse events. (Funded by Merck Sharp and Dohme and by Stand Up to Cancer; KEYNOTE-177 ClinicalTrials.gov number, NCT02563002.).

Clinical and biological implications of target occupancy in CLL treated with the BTK inhibitor acalabrutinib.

Inhibition of the B-cell receptor pathway, and specifically of Bruton tyrosine kinase (BTK), is a leading therapeutic strategy in B-cell malignancies, including chronic lymphocytic leukemia (CLL). Target occupancy is a measure of covalent binding to BTK and has been applied as a pharmacodynamic parameter in clinical studies of BTK inhibitors. However, the kinetics of de novo BTK synthesis, which determines occupancy, and the relationship between occupancy, pathway inhibition and clinical outcomes remain undefined. This randomized phase 2 study investigated the safety, efficacy, and pharmacodynamics of a selective BTK inhibitor acalabrutinib at 100 mg twice daily (BID) or 200 mg once daily (QD) in 48 patients with relapsed/refractory or high-risk treatment-naïve CLL. Acalabrutinib was well tolerated and yielded an overall response rate (ORR) of partial response or better of 95.8% (95% confidence interval [CI], 78.9-99.9) and an estimated progression-free survival (PFS) rate at 24 months of 91.5% (95% CI, 70.0-97.8) with BID dosing and an ORR of 79.2% (95% CI, 57.9-92.9) and an estimated PFS rate at 24 months of 87.2% (95% CI, 57.2-96.7) with QD dosing. BTK resynthesis was faster in patients with CLL than in healthy volunteers. BID dosing maintained higher BTK occupancy and achieved more potent pathway inhibition compared with QD dosing. Small increments in occupancy attained by BID dosing relative to QD dosing compounded over time to augment downstream biological effects. The impact of BTK occupancy on long-term clinical outcomes remains to be determined. This trial was registered at www.clinicaltrials.gov as #NCT02337829.

Depth and durability of response to ibrutinib in CLL: 5-year follow-up of a phase 2 study.

The safety and efficacy of ibrutinib (420 mg) in chronic lymphocytic leukemia (CLL) were evaluated in a phase 2 study; 51 patients had TP53 aberration (TP53 cohort) and 35 were enrolled because of age 65 years or older (elderly cohort). Both cohorts included patients with treatment-naive (TN) and relapsed/refractory (RR) CLL. With the median follow-up of 4.8 years, 49 (57.0%) of 86 patients remain on study. Treatment was discontinued for progressive disease in 20 (23.3%) patients and for adverse events in 5 (5.8%). Atrial fibrillation occurred in 18 (20.9%) patients for a rate of 6.4 per 100 patient-years. No serious bleeding occurred. The overall response rate at 6 months, the primary study endpoint, was 95.8% for the TP53 cohort (95% confidence interval, 85.7%-99.5%) and 93.9% for the elderly cohort (95% confidence interval, 79.8%-99.3%). Depth of response improved with time: at best response, 14 (29.2%) of 48 patients in the TP53 cohort and 9 (27.3%) of 33 in the elderly cohort achieved a complete response. Median minimal residual disease (MRD) in peripheral blood was 3.8 × 10-2 at 4 years, with MRD-negative (<10-4) remissions in 5 (10.2%) patients. In the TP53 cohort, the estimated 5-year progression-free survival (PFS) was 74.4% in TN-CLL compared with 19.4% in RR-CLL (P = .0002), and overall survival (OS) was 85.3% vs 53.7%, respectively (P = .023). In the elderly cohort, the estimated 5-year PFS and OS in RR-CLL were 64.8% and 71.6%, respectively, and no event occurred in TN-CLL. Long-term administration of ibrutinib was well tolerated and provided durable disease control for most patients. This trial was registered at www.clinicaltrials.gov as #NCT01500733.

Ibrutinib for previously untreated and relapsed or refractory chronic lymphocytic leukaemia with TP53 aberrations: a phase 2, single-arm trial.

BACKGROUND: Patients with chronic lymphocytic leukaemia (CLL) with TP53 aberrations respond poorly to first-line chemoimmunotherapy, resulting in early relapse and short survival. We investigated the safety and activity of ibrutinib in previously untreated and relapsed or refractory CLL with TP53 aberrations. METHODS: In this investigator-initiated, single-arm phase 2 study, we enrolled eligible adult patients with active CLL with TP53 aberrations at the National Institutes of Health Clinical Center (Bethesda, MD, USA). Patients received 28-day cycles of ibrutinib 420 mg orally once daily until disease progression or the occurrence of limiting toxicities. The primary endpoint was overall response to treatment at 24 weeks in all evaluable patients. This study is registered with ClinicalTrials.gov, number NCT01500733, and is fully enrolled. FINDINGS: Between Dec 22, 2011, and Jan 2, 2014, we enrolled 51 patients; 47 had CLL with deletion 17p13.1 and four carried a TP53 mutation in the absence of deletion 17p13.1. All patients had active disease requiring therapy. 35 enrolled patients had previously untreated CLL and 16 had relapsed or refractory disease. Median follow-up was 24 months (IQR 12.9-27.0). 33 previously untreated patients and 15 patients with relapsed or refractory CLL were evaluable for response at 24 weeks. 32 (97%; 95% CI 86-100) of 33 previously untreated patients achieved an objective response, including partial response in 18 patients (55%) and partial response with lymphocytosis in 14 (42%). One patient had progressive disease at 0.4 months. 12 (80%; 95% CI 52-96) of the 15 patients with relapsed or refractory CLL had an objective response: six (40%) achieved a partial response and six (40%) a partial response with lymphocytosis; the remaining three (20%) patients had stable disease. Grade 3 or worse treatment-related adverse events were neutropenia in 12 (24%) patients (grade 4 in one [2%] patient), anaemia in seven (14%) patients, and thrombocytopenia in five (10%) patients (grade 4 in one [2%] patient). Grade 3 pneumonia occurred in three (6%) patients, and grade 3 rash in one (2%) patient. INTERPRETATION: The activity and safety profile of single-agent ibrutinib in CLL with TP53 aberrations is encouraging and supports its consideration as a novel treatment option for patients with this high-risk disease in both first-line and second-line settings. FUNDING: Intramural Research Program of the National Heart, Lung, and Blood Institute and the National Cancer Institute, Danish Cancer Society, Novo Nordisk Foundation, National Institutes of Health Medical Research Scholars Program, and Pharmacyclics Inc.

Incidence and risk factors of bleeding-related adverse events in patients with chronic lymphocytic leukemia treated with ibrutinib.

Ibrutinib is associated with bleeding-related adverse events of grade ≤ 2 in severity, and infrequently with grade ≥ 3 events. To investigate the mechanisms of bleeding and identify patients at risk, we prospectively assessed platelet function and coagulation factors in our investigator-initiated trial of single-agent ibrutinib for chronic lymphocytic leukemia. At a median follow-up of 24 months we recorded grade ≤ 2 bleeding-related adverse events in 55% of 85 patients. No grade ≥ 3 events occurred. Median time to event was 49 days. The cumulative incidence of an event plateaued by 6 months, suggesting that the risk of bleeding decreases with continued therapy. At baseline, von Willebrand factor and factor VIII levels were often high and normalized on treatment. Platelet function measured via the platelet function analyzer (PFA-100™) was impaired in 22 patients at baseline and in an additional 19 patients on ibrutinib (often transiently). Collagen and adenosine diphosphate induced platelet aggregation was tested using whole blood aggregometry. Compared to normal controls, response to both agonists was decreased in all patients with chronic lymphocytic leukemia, whether on ibrutinib or not. Compared to untreated chronic lymphocytic leukemia patients, response to collagen showed a mild further decrement on ibrutinib, while response to adenosine diphosphate improved. All parameters associated with a significantly increased risk of bleeding-related events were present at baseline, including prolonged epinephrine closure time (HR 2.74, P=0.012), lower levels of von Willebrand factor activity (HR 2.73, P=0.009) and factor VIII (HR 3.73, P=0.0004). In conclusion, both disease and treatment-related factors influence the risk of bleeding. Patients at greater risk for bleeding of grade ≤ 2 can be identified by clinical laboratory tests and counseled to avoid aspirin, non-steroidal anti-inflammatory drugs and fish oils. ClinicalTrials.gov identifier NCT01500733.

Real-world patterns and sequences of targeted therapy use in chronic lymphocytic leukemia and small lymphocytic lymphoma in the United States: a longitudinal study.

With increasing focus on novel targeted therapies for chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), this longitudinal claims-based study evaluated real-world CLL/SLL treatment sequences, particularly sequential targeted therapy. Among patients with first-line (1 L) treatment in 2014-2017 (N = 2,612; median follow-up = 3 years), the most common 1 L treatment was chemoimmunotherapy (CIT; 44.6%), followed by CD20 (25.2%) and Bruton's tyrosine kinase inhibitors (BTKi; 21.7%). Among those with 1 L in 2018-2021 (N = 4,534; median follow-up = 1 year), these were BTKi (45.5%), CD20 (20.4%), CIT (17.5%), and B-cell lymphoma 2 inhibitor (8.3%). In 2014-2017, the proportion of patients receiving sequential targeted therapy in the first 2 LOTs was 11.2% (80.2% was BTKi→BTKi); in 2018-2021, this proportion was 34.3% (66.4% was BTKi→BTKi). Over time, there was a substantial increase in targeted therapy use in 1 L and sequential targeted therapy, particularly with BTKi→BTKi. Future studies should assess clinical outcomes to determine optimal sequences for CLL/SLL and reasons for restarting BTKi.

First-in-Human Study of the Reversible BTK Inhibitor Nemtabrutinib in Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia and B-Cell Non-Hodgkin Lymphoma.

Nemtabrutinib is an orally bioavailable, reversible inhibitor of Bruton tyrosine kinase (BTK) and C481S mutant BTK. We evaluated the safety, pharmacology, and antitumor activity of nemtabrutinib in relapsed/refractory hematologic malignancies. Forty-eight patients with chronic lymphocytic leukemia (CLL), B-cell non-Hodgkin lymphoma (NHL), or Waldenström macroglobulinemia (WM), relapsed/refractory after ≥2 prior therapies were enrolled in the open-label, single-arm, phase I MK-1026-001 study (NCT03162536) to receive nemtabrutinib 5 to 75 mg once daily in 28-day cycles. Dose finding progressed using a 3 + 3 dose escalation design. Primary endpoints were safety and the recommended phase II dose (RP2D). Among 47 treated patients, 29 had CLL, 17 had NHL, and 1 had WM. Grade ≥3 treatment-emergent adverse events occurred in 37 (89%), most commonly neutropenia (11; 23.4%), febrile neutropenia (7; 14.9%), and pneumonia (7; 14.9%). The RP2D was 65 mg daily. An overall response rate of 75% was observed in patients with CLL at 65 mg daily. SIGNIFICANCE: This first-in-human phase I study demonstrates the safety and preliminary efficacy of nemtabrutinib in patients with relapsed/refractory B-cell malignancies. These data support further exploration of nemtabrutinib in larger clinical studies. This article is featured in Selected Articles from This Issue, p. 5.

Using artificial intelligence tools in answering important clinical questions: The KEYNOTE-183 multiple myeloma experience.

The phase III, randomized, active-controlled, multicenter, open-label KEYNOTE-183 study (NCT02576977) evaluating pomalidomide and low dose dexamethasone (standard-of-care [SOC]) with or without pembrolizumab in patients with refractory or relapsed and refractory multiple myeloma (rrMM) was placed on full clinical hold by the US FDA on July 03, 2017 due to an imbalance in the number of deaths between arms. Clinically-led subgroup analyses are typically used to shed light on clinical findings. However, this approach is not always successful. We propose a systematic approach using the artificial intelligence tools to identifying risk factors and subgroups contributing to the overall death (prognostic) or to the excess death observed in the pembrolizumab plus SOC arm (predictive) of the KEYNOTE-183 study. In KEYNOTE-183, with a data cutoff date of June 02, 2017, we identified plasmacytoma as a prognostic factor, and ECOG performance status as a predictive factor of death. In addition, a qualitative interaction was observed between ECOG performance status and the treatment arm. The subsequent subgroup analysis based on ECOG performance status confirmed that more deaths were associated with pembrolizumab plus SOC versus SOC alone in patients with ECOG performance status 1.

Pembrolizumab plus lenalidomide and dexamethasone for patients with treatment-naive multiple myeloma (KEYNOTE-185): a randomised, open-label, phase 3 trial.

BACKGROUND: Lenalidomide and dexamethasone has been a standard of care in transplant-ineligible patients with newly diagnosed multiple myeloma. The addition of a third drug to the combination is likely to improve treatment efficacy. KEYNOTE-185 assessed the efficacy and safety of lenalidomide and dexamethasone with and without pembrolizumab in patients with previously untreated multiple myeloma. Here, we present the results of an unplanned interim analysis done to assess the benefit-risk of the combination at the request of the US Food and Drug Administration (FDA). METHODS: KEYNOTE-185 was a randomised, open-label, phase 3 trial done at 95 medical centres across 15 countries (Australia, Canada, France, Germany, Ireland, Israel, Italy, Japan, New Zealand, Norway, Russia, South Africa, Spain, UK, and USA). Transplantation-ineligible patients aged 18 years and older with newly diagnosed multiple myeloma, Eastern Cooperative Oncology Group performance status of 0 or 1, and who were treatment naive were enrolled, and randomly assigned 1:1 to receive either pembrolizumab plus lenalidomide and dexamethasone or lenalidomide and dexamethasone alone using an interactive voice or integrated web response system. Patients received oral lenalidomide 25 mg on days 1-21 and oral dexamethasone 40 mg on days 1, 8, 15, and 22 of repeated 28-day cycles, with or without intravenous pembrolizumab 200 mg every 3 weeks. The primary endpoint was progression-free survival, which was investigator-assessed because of early trial termination. Efficacy was analysed in all randomly assigned patients and safety was analysed in all patients who received at least one dose of study drug. This trial is registered at ClinicalTrials.gov, number NCT02579863, and it is closed for accrual. FINDINGS: Between Jan 7, 2016, and June 9, 2017, 301 patients were randomly assigned to the pembrolizumab plus lenalidomide and dexamethasone group (n=151) or the lenalidomide and dexamethasone group (n=150). On July 3, 2017, the FDA decided to halt the study because of the imbalance in the proportion of death between groups. At database cutoff (June 2, 2017), with a median follow-up of 6·6 months (IQR 3·4-9·6), 149 patients in the pembrolizumab plus lenalidomide and dexamethasone group and 145 in the lenalidomide and dexamethasone group had received their assigned study drug. Median progression-free survival was not reached in either group; progression-free survival estimates at 6-months were 82·0% (95% CI 73·2-88·1) versus 85·0% (76·8-90·5; hazard ratio [HR] 1·22; 95% CI 0·67-2·22; p=0·75). Serious adverse events were reported in 81 (54%) patients in the pembrolizumab plus lenalidomide and dexamethasone group versus 57 (39%) patients in the lenalidomide and dexamethasone group; the most common serious adverse events were pneumonia (nine [6%]) and pyrexia (seven [5%]) in the pembrolizumab plus lenalidomide and dexamethasone group and pneumonia (eight [6%]) and sepsis (two [1%]) in the lenalidomide and dexamethasone group. Six (4%) treatment-related deaths occurred in the pembrolizumab plus lenalidomide and dexamethasone group (cardiac arrest, cardiac failure, myocarditis, large intestine perforation, pneumonia, and pulmonary embolism) and two (1%) in the lenalidomide and dexamethasone group (upper gastrointestinal haemorrhage and respiratory failure). INTERPRETATION: The results from this unplanned, FDA-requested, interim analysis showed that the benefit-risk profile of pembrolizumab plus lenalidomide and dexamethasone is unfavourable for patients with newly diagnosed, previously untreated multiple myeloma. Long-term safety and survival follow-up is ongoing. FUNDING: Merck Sharp & Dohme, a subsidiary of Merck & Co, Inc (Kenilworth, NJ, USA).

The evolutionary landscape of chronic lymphocytic leukemia treated with ibrutinib targeted therapy.

Treatment of chronic lymphocytic leukemia (CLL) has shifted from chemo-immunotherapy to targeted agents. To define the evolutionary dynamics induced by targeted therapy in CLL, we perform serial exome and transcriptome sequencing for 61 ibrutinib-treated CLLs. Here, we report clonal shifts (change >0.1 in clonal cancer cell fraction, Q < 0.1) in 31% of patients during the first year of therapy, associated with adverse outcome. We also observe transcriptional downregulation of pathways mediating energy metabolism, cell cycle, and B cell receptor signaling. Known and previously undescribed mutations in BTK and PLCG2, or uncommonly, other candidate alterations are present in seventeen subjects at the time of progression. Thus, the frequently observed clonal shifts during the early treatment period and its potential association with adverse outcome may reflect greater evolutionary capacity, heralding the emergence of drug-resistant clones.

Direct in vivo evidence for increased proliferation of CLL cells in lymph nodes compared to bone marrow and peripheral blood.

Chronic lymphocytic leukemia (CLL) is a progressive malignancy of mature B-cells that involves the peripheral blood (PB), lymph nodes (LNs) and bone marrow (BM). Although the majority of CLL cells are in a resting state, small populations of proliferating cells exist; however, the anatomical site of active cell proliferation remains to be definitively determined. Based on findings that CLL cells in LNs have increased expression of B-cell activation genes, we tested the hypothesis that the fraction of 'newly born' cells would be highest in the LNs. Using a deuterium oxide (2H) in vivo labeling method in which patients consumed deuterated (heavy) water (2H2O), we determined CLL cell kinetics in concurrently obtained samples from LN, PB and BM. The LN was identified as the anatomical site harboring the largest fraction of newly born cells, compared to PB and BM. In fact, the calculated birth rate in the LN reached as high a 3.3% of the clone per day. Subdivision of the bulk CLL population by flow cytometry identified the subpopulation with the CXCR4dimCD5bright phenotype as containing the highest proportion of newly born cells within each compartment, including the LN, identifying this subclonal population as an important target for novel treatment approaches.

Hair and Nail Changes During Long-term Therapy With Ibrutinib for Chronic Lymphocytic Leukemia.

IMPORTANCE: Ibrutinib, a Bruton tyrosine kinase inhibitor, is a new targeted agent approved by the US Food and Drug Administration for the treatment of chronic lymphocytic leukemia (CLL), mantle cell lymphoma, and Waldenström macroglobulinemia. Ibrutinib is overall well tolerated but long-term treatment is required until disease progression or intolerable toxic effects occur. Little is known regarding its cutaneous adverse effects. OBJECTIVE: To describe the hair and nail manifestations associated with the long-term use of ibrutinib for the treatment of CLL. DESIGN, SETTING, AND PARTICIPANTS: Prospective study of 66 patients with CLL enrolled in a single-arm phase 2 clinical trial of ibrutinib for CLL between March 2014 and October 2015 at the National Institutes of Health. MAIN OUTCOMES AND MEASURES: The primary outcome, nail and hair changes associated with ibrutinib therapy, was assessed by an 11-question survey. In addition, the severity of nail changes was determined from a 0 to 3 rating scale for both onychoschizia and onychorrhexis. RESULTS: Among 66 patients (43 men and 23 women with ages ranging from 55 to 85 years), 44 (67%) reported brittle fingernails at a median of 6.5 (95% CI, 6-12) months after starting ibrutinib therapy. Fifteen patients (23%) developed brittle toenails after a median of 9 (95% CI, 6-15) months of ibrutinib therapy. Textural hair changes were reported in 17 patients (26%), at a median of 9 (95% CI, 6-12) months of ibrutinib treatment. CONCLUSIONS AND RELEVANCE: Hair and nail abnormalities are commonly associated with ibrutinib and appear several months after initiating therapy. Ibrutinib inhibits Bruton tyrosine kinase by covalently binding to cysteine 481. Whether ibrutinib affects the hair and nails by binding and altering cysteine-rich proteins of hair and nails or by means of another mechanism remains unknown. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01500733.

Interactions between Ibrutinib and Anti-CD20 Antibodies: Competing Effects on the Outcome of Combination Therapy.

PURPOSE: Clinical trials of ibrutinib combined with anti-CD20 monoclonal antibodies (mAb) for chronic lymphocytic leukemia (CLL) report encouraging results. Paradoxically, in preclinical studies, in vitro ibrutinib was reported to decrease CD20 expression and inhibit cellular effector mechanisms. We therefore set out to investigate effects of in vivo ibrutinib treatment that could explain this paradox. EXPERIMENTAL DESIGN: Patients received single-agent ibrutinib (420 mg daily) on an investigator-initiated phase II trial. Serial blood samples were collected pretreatment and during treatment for ex vivo functional assays to examine the effects on CLL cell susceptibility to anti-CD20 mAbs. RESULTS: We demonstrate that CD20 expression on ibrutinib was rapidly and persistently downregulated (median reduction 74%, day 28, P < 0.001) compared with baseline. Concomitantly, CD20 mRNA was decreased concurrent with reduced NF-κB signaling. An NF-κB binding site in the promoter of MS4A1 (encoding CD20) and downregulation of CD20 by NF-κB inhibitors support a direct transcriptional effect. Ex vivo, tumor cells from patients on ibrutinib were less susceptible to anti-CD20 mAb-mediated complement-dependent cytotoxicity than pretreatment cells (median reduction 75%, P < 0.001); however, opsonization by the complement protein C3d, which targets cells for phagocytosis, was relatively maintained. Expression of decay-accelerating factor (CD55) decreased on ibrutinib, providing a likely mechanism for the preserved C3d opsonization. In addition, ibrutinib significantly inhibited trogocytosis, a major contributor to antigen loss and tumor escape during mAb therapy. CONCLUSIONS: Our data indicate that ibrutinib promotes both positive and negative interactions with anti-CD20 mAbs, suggesting that successfully harnessing maximal antitumor effects of such combinations requires further investigation.

Disruption of in vivo Chronic Lymphocytic Leukemia Tumor-Microenvironment Interactions by Ibrutinib--Findings from an Investigator-Initiated Phase II Study.

PURPOSE: Chronic lymphocytic leukemia (CLL) cells depend on microenvironmental interactions for proliferation and survival that are at least partially mediated through B-cell receptor (BCR) signaling. Ibrutinib, a Bruton tyrosine kinase inhibitor, disrupts BCR signaling and leads to the egress of tumor cells from the microenvironment. Although the on-target effects on CLL cells are well defined, the impact on the microenvironment is less well studied. We therefore sought to characterize the in vivo effects of ibrutinib on the tumor microenvironment. EXPERIMENTAL DESIGN: Patients received single-agent ibrutinib on an investigator-initiated phase II trial. Serial blood and tissue samples were collected pretreatment and during treatment. Changes in cytokine levels, cellular subsets, and microenvironmental interactions were assessed. RESULTS: Serum levels of key chemokines and inflammatory cytokines decreased significantly in patients on ibrutinib. Furthermore, ibrutinib treatment decreased circulating tumor cells and overall T-cell numbers. Most notably, a reduced frequency of the Th17 subset of CD4(+)T cells was observed concurrent with reduced expression of activation markers and PD-1 on T cells. Consistent with direct inhibition of T cells, ibrutinib inhibited Th17 differentiation of murine CD4(+)T cells in vitro Finally, in the bone marrow microenvironment, we found that ibrutinib disaggregated the interactions of macrophages and CLL cells, inhibited secretion of CXCL13, and decreased the chemoattraction of CLL cells. CONCLUSIONS: In conjunction with inhibition of BCR signaling, these changes in the tumor microenvironment likely contribute to the antitumor activity of ibrutinib and may impact the efficacy of immunotherapeutic strategies in patients with CLL. See related commentary by Bachireddy and Wu, p. 1547.

Bcl-2 level as a biomarker for 13q14 deletion in CLL.

BACKGROUND: Deletion 13q14.3 is the most common cytogenetic abnormality in chronic lymphocytic leukemia (CLL). Previously it was reported that miR-15/16 is the target of 13q14 deletions and plays a tumor suppressor role by suppressing Bcl-2. Therefore, Bcl-2 expression was examined more closely to determine whether it would predict 13q14 deletion status. METHODS: A multi-color flow panel consisting of anti-Bcl-2/anti-lambda/anti-kappa/CD19/CD5/CD3/CD20 was performed. The ability of Bcl-2 to predict 13q14 deletion was tested using the conventional Bcl-2 index (c-index): mean fluorescence intensity (MFI) of CLL clone/MFI of residual T cells. Fifty-four untreated CLL/MBL patients were studied. Bimodal Bcl-2 expression was evaluated to test the ability of Bcl-2 to detect intraclonal heterogeneity. Other CLL prognostic markers including CD38, CD49d, CD26, and CD69 were evaluated. FISH was performed on selected sorted populations. RESULTS: The Bcl-2 c-index strongly predicts del13q14 P < 0.0001. A statistically significant association was observed between the percentage of cells carrying the deletion and the level of Bcl-2 expression P < 0.05. Cells sorted based on Bcl-2 expression showed enrichment of both hemizygous and homozygous del 13q14 cells. Also, we observed that an alteration in Bcl-2 level over time predicts changes in 13q14 deletion status. And a statistically significant correlation between the bimodal pattern of CD69 expression and the presence of 13q14 deletion was found P < 0.0001. CONCLUSION: Bcl-2 expression using the c-index strongly predicts 13q14 deletion and can be used to distinguish homozygous, heterozygous, and diploid CLL clonal cells. Further systematic studies of this biomarker are needed for confirmation and expansion of these findings.

Combined normal donor and CLL: Single tube ZAP-70 analysis.

INTRODUCTION: Zeta-chain-associated protein kinase 70 (ZAP-70) has been identified as an independent prognostic marker in chronic lymphocytic leukemia (CLL). Based on our previous studies, we have developed a combined one-tube technology with multiple internal controls to optimize ZAP-70 assessment. METHODS: Forty-eight untreated CLL cases were examined for ZAP-70 expression using a modified 7-color one-tube assay. Normal donor (ND) whole blood is stained with CD3 APC-Cy7 and CD19 APC. In a second tube, patient whole blood is stained with CD5 PE-Cy7, CD19 PerCP-Cy5.5, and CD20 eFluor450. After surface staining and fixation, these two tubes are combined. After saponin permeabilization, the cells were stained with two anti-ZAP-70 clones (1E7.2/AF488 and SBZAP/PE). The results obtained from this modified tube were compared with those obtained concurrently using the non-mixed single sample tubes. Five different methods of ZAP-70 expression analysis were evaluated: percentage positive cells using ND T-cells as a reference; the internal patient T-cell/clone ratio; ND T-cell/clone ratio; clone/ND B-cell ratio; and modified Z-index. RESULT: Overall, the combined patient and ND mix tube performed better than the non-mixed single sample tube. The strongest correlations between ZAP-70 expression and immunoglobulin heavy chain variable (IGHV) mutational status were seen with percentage positive ND T-cell, ND T-cell/clone ratio, and clone/ND B-cell ratio for both 1E7.2 and SBZAP clone (P < 0.0001). CONCLUSION: The modified one tube method combining the ND and patient sample provides highly reliable results that correlate with the IGHV mutational status. This method should be considered as part of the next step in standardization of the ZAP-70 assay in CLL.

Methodological comparison of two anti-ZAP-70 antibodies.

BACKGROUND: ZAP-70 expression is a stage independent prognostic marker in CLL. However, interlaboratory variation is large, and there is neither a consensus nor a regulatory approved methodology. METHODS: Two anti-ZAP70 clones (1E7.2 and SBZAP) were compared in 45 untreated CLL patients. Nine different methods for ZAP-70 expression analysis were evaluated: M1, isotype control to determine negative; M2, internal residual T-cell to determine positive; M3, normal donor (ND) T-cell to determine positive; M4, internal T-cell/clone ratio; M5, ND residual T-cell/clone ratio; M6, clone/normal remaining B-cell ratio; M7, clone/ND B- cell ratio; M8, CLL-Z score; M9, modified CLL-Z score. A scoring system was designed integrating both 1E7.2 and SBZAP clones to assign ZAP-70 expression. RESULTS: The correlation coefficients for the four selected highest statistically significant methods were as follows (M1 = 0.71, M3 = 0.72, M7 = 0.67, and M9 = 0.64). These four methods were used to generate a combined score. The two reagents showed agreement using the designed scoring system for 37/45 samples (82%), and 8/45 (18%) showed equivocal result with one of the two clones. Seven of the eight equivocal samples were resolved using the scoring system. CONCLUSIONS: Four of the nine methods of analysis were compared for each reagent. The use of two independent ZAP-70 reagents increases analytical certitude and the scoring method aids in the resolution of equivocal results. The combined use of two reagents, four methods of analysis, and a scoring method allowed for assignment of ZAP-70 expression in 44/45 samples (98%) tested and improved performance of this important prognostic assay.

Improved ZAP-70 assay using two clones, multiple methods of analysis and clinical correlation.

INTRODUCTION: In a companion methodological study, we compared two anti-ZAP-70 clones (1E7.2 AF 488 and SBZAP PE) and four selected methods of analysis. Clinical correlations are required for validation. METHODS: Multicolor flow-cytometric evaluation of ZAP-70, CD38, CD69, CD26, CD49d, and CD27 was tested in 45 untreated-CLL patients. Four methods of ZAP-70 expression analysis and a scoring system were designed. A correlation analysis between ZAP-70 score, immunoglobulin heavy chain variable (IGHV) mutational status, fluorescence in situ hybridization, and these biomarkers was undertaken. RESULTS: There is a strong correlation between ZAP-70 expression and IGHV mutational status. The scoring system for a single reagent (P = 0.0006 or 0.0002) favors the use of multiple methods of analysis. The combined score was substantially equivalent (P = 0.0003). There was also a correlation with del 13q14 (P = 0.017) and trisomy12 (P = 0.011). A correlation for CD38 and ZAP-70 score was seen using both 1E7.2 AF488 and SBZAP PE when ≥20% or ≥7% cutoff was used. A positive correlation was seen for CD49d expression using both reagents. CD26 showed a correlation with ZAP-70 expression, but it was dependent upon the method of analysis. CD69 and CD27 showed no statistically significant correlation. CONCLUSION: In our study population, ZAP-70 expression is the better predictor of the IGHV mutational status. The correlation analysis confirms that the use of four methods of analysis with a single reagent or both reagents is superior to the use of a single method of analysis. The routine use of CD38, CD49d, and CD26 will require standardization.