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Reversing or preventing immunodeficiency in patients with chronic lymphocytic leukemia (CLL) is of the highest priority. The past decade of research has met the challenge of treating CLL for most patients. Patients continue to struggle, however, with infections and second primary malignancies related to immunodeficiency. Strategies addressing this need currently are limited to vaccinations, with suboptimal efficacy, and immunoglobulin replacement. Correlative studies have provided insights into immunologic alterations on treatment. Understanding vulnerabilities in the immune system may help identify potential interventions to boost immunity. An emphasis on systematically testing such interventions is required to restore immunocompetence in patients with CLL.

Purpose: In chronic lymphocytic leukemia (CLL), the T-cell receptor (TCR) repertoire is skewed and tumor-derived antigens are hypothesized as drivers of oligoclonal expansion. Ibrutinib, a standard treatment for CLL, inhibits not only Bruton tyrosine kinase of the B-cell receptor signaling pathway, but also IL2-inducible kinase of the TCR signaling pathway. T-cell polarization and activation are affected by ibrutinib, but it is unknown whether T cells contribute to clinical response. Experimental Design: High-throughput TCRβ sequencing was performed in 77 longitudinal samples from 26 patients with CLL treated with ibrutinib. TCRβ usage in CD4+ and CD8+ T cells and granzyme B expression were assessed by flow cytometric analysis. Antitumor cytotoxicity of T cells expanded with autologous CLL cells or with antigen-independent anti-CD3/CD28/CD137 beads was tested. Results: The clonality of the TCR repertoire increased at the time of response. With extended treatment, TCR clonality remained stable in patients with sustained remission and decreased in patients with disease progression. Expanded clonotypes were rarely shared between patients, indicating specificity for private antigens. Flow cytometry demonstrated a predominance of CD8+ cells among expanded clonotypes. Importantly, bulk T cells from responding patients were cytotoxic against autologous CLL cells in vitro and selective depletion of major expanded clonotypes reduced CLL cell killing. Conclusions: In patients with CLL, established T-cell responses directed against tumor are suppressed by disease and reactivated by ibrutinib. See related commentary by Zent, p. 4465

We demonstrate the prognostic utility of antigen quantitation in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and monoclonal B-cell lymphocytosis (MBL). Median antibody-bound-per-cell (ABC) of CD20, CD22, CD25, CD19, and %CD38(+) was determined in CLL (185/208), SLL (8/208) and MBL (15/208) cases by flow cytometry, then compared to Dohner-classification, immunoglobulin status (mutated, IGHV-M; unmutated, IGHV-U), CLL-IPI risk and time to first treatment (TTFT). Trisomy 12 cases showed increased %CD38-expression (p=0.0379). Higher %CD38 was observed in IGHV-U versus IGHV-M (p=0.0003). CD20ABC was increased in IGHV-U versus IGHV-M (p=0.006). Del13q cases demonstrated lower CD22ABC (p=0.0198). Cases without cytogenetic abnormality exhibited higher CD19ABC (p=0.0295) and CD22ABC (p=0.0078). Del17p cases demonstrated lower CD25ABC (p=0.0097). High and very-high CLL-IPI risk groups were associated with high CD38-expression (p=0.02) and low CD25ABC (p=0.0004). Shortened TTFT was associated with high CD38-expression (p

Publisher's Note: There is a Blood Commentary on this article in this issue., Key Points De novo immune response to hepatitis B vaccine was nearly absent in CLL patients on BTKi and impaired in treatment-naïve patients. Recall immune response to zoster vaccine was not significantly different between CLL patients on BTKi and treatment-naïve patients., Vaccinations are effective in preventing infections; however, it is unknown if patients with chronic lymphocytic leukemia (CLL) who are treatment naïve (TN) or receiving Bruton tyrosine kinase inhibitors (BTKi's) respond to novel adjuvanted vaccines. Understanding the effect of BTKi's on humoral immunity is timely because BTKi's are widely used and vaccination against coronavirus disease 2019 is urgently needed. In 2 open-label, single-arm clinical trials, we measured the effect of BTKi's on de novo immune response against recombinant hepatitis B vaccine (HepB-CpG) and recall response against recombinant zoster vaccine (RZV) in CLL patients who were TN or on BTKi. The primary end point was serologic response to HepB-CpG (anti-hepatitis B surface antibodies ≥10 mIU/mL) and RZV (≥fourfold increase in anti-glycoprotein E). The response rate to HepB-CpG was lower in patients on BTKi (3.8%; 95% confidence interval [CI], 0.7-18.9) than patients who were TN (28.1%; 95% CI, 15.6-45.4; P = .017). In contrast, the response rate to RZV did not differ significantly between the BTKi (41.5%; 95% CI, 27.8-56.6) and TN cohorts (59.1%; 95% CI, 38.7-76.7; P = .2). BTKi's were associated with a decreased de novo immune response following HepB-CpG, whereas recall immune response following RZV was not significantly affected by BTKi therapy. These trials were registered at www.clinicaltrials.gov as #NCT03685708 (Hep-CpG) and #NCT03702231 (RZV)., Visual Abstract

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.

Cardiovascular (CV) toxicities of the Bruton tyrosine kinase (BTK) inhibitor ibrutinib may limit use of this effective therapy in patients with chronic lymphocytic leukemia (CLL). Acalabrutinib is a second-generation BTK inhibitor with greater BTK selectivity. This analysis characterizes pooled CV adverse events (AE) data in patients with CLL who received acalabrutinib monotherapy in clinical trials (clinicaltrials gov. Identifier: NCT02029443, NCT02475681, NCT02970318 and NCT02337829). Acalabrutinib was given orally at total daily doses of 100–400 mg, later switched to 100 mg twice daily, and continued until disease progression or toxicity. Data from 762 patients (median age: 67 years [range, 32–89]; median follow-up: 25.9 months [range, 0–58.5]) were analyzed. Cardiac AE of any grade were reported in 129 patients (17%; grade ≥3, n=37 [5%]) and led to treatment discontinuation in seven patients (1%). The most common any-grade cardiac AE were atrial fibrillation/flutter (5%), palpitations (3%), and tachycardia (2%). Overall, 91% of patients with cardiac AE had CV risk factors before acalabrutinib treatment. Among 38 patients with atrial fibrillation/flutter events, seven (18%) had prior history of arrhythmia or atrial fibrillation/flutter. Hypertension AE were reported in 67 patients (9%), 43 (64%) of whom had a preexisting history of hypertension; no patients discontinued treatment due to hypertension. No sudden cardiac deaths were reported. Overall, these data demonstrate a low incidence of new-onset cardiac AE with acalabrutinib in patients with CLL. Findings from the head-to-head, randomized trial of ibrutinib and acalabrutinib in patients with highrisk CLL (clinicaltrials gov. Identifier: NCT02477696) prospectively assess differences in CV toxicity between the two agents.

Bruton tyrosine kinase inhibitors (BTKis) are a preferred treatment of patients with chronic lymphocytic leukemia (CLL). Indefinite therapy with BTKis, although effective, presents clinical challenges. Combination therapy can deepen responses, shorten treatment duration, and possibly prevent or overcome drug resistance. We previously reported on a CD19/CD3-bispecific antibody (bsAb) that recruits autologous T-cell cytotoxicity against CLL cells in vitro. Compared with observations with samples from treatment-naïve patients, T cells from patients being treated with ibrutinib expanded more rapidly and exerted superior cytotoxic activity in response to the bsAb. In addition to BTK, ibrutinib also inhibits interleukin-2 inducible T-cell kinase (ITK). In contrast, acalabrutinib, does not inhibit ITK. Whether ITK inhibition contributes to the observed immune effects is unknown. To better understand how BTKis modulate T-cell function and cytotoxic activity, we cultured peripheral blood mononuclear cells (PBMCs) from BTKi-naive and ibrutinib- or acalabrutinib-treated CLL patients with CD19/CD3 bsAb in vitro. T-cell expansion, activation, differentiation, and cytotoxicity were increased in PBMCs from patients on treatment with either BTKi compared with that observed for BKTi-naïve patients. BTKi therapy transcriptionally downregulated immunosuppressive effectors expressed by CLL cells, including cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and CD200. CTLA-4 blockade with ipilimumab in vitro increased the cytotoxic activity of the bsAb in BTKi-naïve but not BTKi-treated PBMCS. Taken together, BTKis enhance bsAb-induced cytotoxicity by relieving T cells of immunosuppressive restraints imposed by CLL cells. The benefit of combining bsAb immunotherapy with BTKis needs to be confirmed in clinical trials.

Chronic lymphocytic leukemia (CLL) is characterized by disordered DNA methylation, suggesting these epigenetic changes might play a critical role in disease onset and progression. The methyltransferase DNMT3A is a key regulator of DNA methylation. Although DNMT3A somatic mutations in CLL are rare, we found that low DNMT3A expression is associated with more aggressive disease. A conditional knockout mouse model showed that homozygous depletion of Dnmt3a from B cells results in the development of CLL with 100% penetrance at a median age of onset of 5.3 months, and heterozygous Dnmt3a depletion yields a disease penetrance of 89% with a median onset at 18.5 months, confirming its role as a haploinsufficient tumor suppressor. B1a cells were confirmed as the cell of origin of disease in this model, and Dnmt3a depletion resulted in focal hypomethylation and activation of Notch and Myc signaling. Amplification of chromosome 15 containing the Myc gene was detected in all CLL mice tested, and infiltration of high-Myc–expressing CLL cells in the spleen was observed. Notably, hyperactivation of Notch and Myc signaling was exclusively observed in the Dnmt3a CLL mice, but not in three other CLL mouse models tested (Sf3b1-Atm, Ikzf3, and MDR), and Dnmt3a-depleted CLL were sensitive to pharmacologic inhibition of Notch signaling in vitro and in vivo. Consistent with these findings, human CLL samples with lower DNMT3A expression were more sensitive to Notch inhibition than those with higher DNMT3A expression. Altogether, these results suggest that Dnmt3a depletion induces CLL that is highly dependent on activation of Notch and Myc signaling. Significance: Loss of DNMT3A expression is a driving event in CLL and is associated with aggressive disease, activation of Notch and Myc signaling, and enhanced sensitivity to Notch inhibition.

The use of Bruton tyrosine kinase (BTK) inhibitors to block B-cell receptor (BCR)–dependent NF-κB activation in lymphoid malignancies has been a major clinical advance, yet acquired therapeutic resistance is a recurring problem. We modeled the development of resistance to the BTK inhibitor ibrutinib in the activated B-cell (ABC) subtype of diffuse large B-cell lymphoma, which relies on chronic active BCR signaling for survival. The primary mode of resistance was epigenetic, driven in part by the transcription factor TCF4. The resultant phenotypic shift altered BCR signaling such that the GTPase RAC2 substituted for BTK in the activation of phospholipase Cγ2, thereby sustaining NF-κB activity. The interaction of RAC2 with phospholipase Cγ2 was also increased in chronic lymphocytic leukemia cells from patients with persistent or progressive disease on BTK inhibitor treatment. We identified clinically available drugs that can treat epigenetic ibrutinib resistance, suggesting combination therapeutic strategies. Significance: In diffuse large B-cell lymphoma, we show that primary resistance to BTK inhibitors is due to epigenetic rather than genetic changes that circumvent the BTK blockade. We also observed this resistance mechanism in chronic lymphocytic leukemia, suggesting that epigenetic alterations may contribute more to BTK inhibitor resistance than currently thought. See related commentary by Pasqualucci, p. 555. This article is highlighted in the In This Issue feature, p. 549

Background: Bruton tyrosine kinase (BTK) inhibitors are effective treatments for B-cell malignancies, but an increased incidence of cardiovascular (CV) toxicities has been observed with ibrutinib. Acalabrutinib (acala) is a next-generation, potent, highly selective, covalent BTK inhibitor approved for chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma and mantle cell lymphoma. The objective of this analysis was to characterize CV adverse events (AEs) in patients (pts) with CLL who received acala monotherapy. Methods: Data from pts with CLL in 4 studies (ACE-CL-001 [NCT02029443]; ACE-CL-007 [ELEVATE-TN, NCT02475681]; ACE-CL-309 [ASCEND, NCT02970318]; 15-H-0016 [NCT02337829]) were pooled. Cutoff dates ranged from December 2018 to February 2019. Pts who received ≥1 dose of acala monotherapy were included. For pts who crossed over from control arms to acala, only AEs recorded after crossover were included. Acala was given orally at total daily doses of 100 mg to 400 mg, later switched to 100 mg twice daily, and continued until disease progression (PD) or toxicity. Cardiac AEs and hypertension (htn) were examined. Results: 762 pts were included (treatment-naïve: n=352 [46%]; relapsed/refractory: n=410 [54%]; median age: 67 years [range: 32-89]; Eastern Cooperative Oncology Group performance status ≤1: 93%; median acala exposure: 24.9 mo [range: 0-58.5]; median follow-up: 25.9 mo [range: 0-58.5]). A total of 199 cardiac AEs of any grade (irrespective of treatment relationship) were reported in 129 pts (17%). Cardiac AEs led to treatment discontinuation in 7 pts (0.9%). The most frequent cardiac AEs reported in ≥2% of pts were atrial fibrillation (afib: n=34; 4%; afib/flutter: n=38; 5%), palpitations (n=23; 3%), and tachycardia (n=17; 2%). The median time to afib/flutter onset was 521 days (range: 8-1280). Overall, 91% (117/129) of pts with vs 79% (503/633) without cardiac AEs had ≥1 CV risk factor before acala initiation. The most prevalent CV risk factors (≥20%) among the 129 pts with cardiac AEs were htn (n=86; 67%), hyperlipidemia (n=38; 29%), and arrhythmias (n=29; 22% [afib: n=16; 12%]). Htn AEs were reported in 9% (67/762) of pts, among whom 46 (69%) had pre-existing htn and 18 (27%) had htn risk factors. The median time to htn onset was 197 days (range: 2-1345). Thirty-seven pts (4%) had 51 grade ≥3 cardiac AEs (grade 3: n=37; grade 4: n=12; grade 5: n=2). Grade ≥3 cardiac AEs of interest included afib (n=10; 1.3%), complete atrioventricular (AV) block (n=2; 0.3%), acute coronary syndrome (n=1; 0.1%), atrial flutter (n=1; 0.1%), second degree AV block (n=1; 0.1%), and ventricular fibrillation (n=1; 0.1%). Two patients experienced grade 5 AEs (cardiac failure congestive [n=1], acute myocardial infarction [n=1]). Among the 37 pts with grade ≥3 AEs, 18 (49%) were continuing acala at data cutoff; 6 (16%) had discontinued due to the grade ≥3 cardiac AEs, 4 (11%) to other AEs, 5 (14%) to PD, 3 (8%) to death, and 1 (3%) to other reasons. Among the 51 grade ≥3 cardiac AEs, 16 (31%) led to dose delay and 36 (71%) were managed with concomitant medications. Most events (43/51 [84%]) resolved (dose delay: n=15; drug withdrawal: n=4; no dose change: n=24). Cardiac AEs occurring in the first 6 mo on acala were assessed based on a predominance of AEs (afib) during this time period with ibrutinib (Brown JR, et al, Haematologica. 2017;102:1796). Overall, 48% of pts with any-grade cardiac AEs experienced them in the first 6 mo on acala. Thirteen grade ≥3 cardiac AEs (25% of total) were observed in 9 pts in the first 6 mo (Table); all but 1 AE (grade 4 cardiac tamponade resulting in hospitalization) were managed with concomitant medications. Two of the 13 AEs resulted in treatment discontinuation (Table). Conclusions: At a median exposure of 24.9 mo, cardiac AEs occurred infrequently in pts with CLL treated with acala monotherapy; only 0.9% discontinued treatment due to cardiac AEs. Among grade ≥3 cardiac AEs, 25% were reported during the first 6 mo on treatment. Most pts with cardiac AEs had pre-existing risk factors that may have contributed to their development. The incidence of afib with acala (4%) was comparable to that of the general CLL population (6.1%; Shanafelt TD, et al. Leuk Lymphoma. 2017;58:1630). These data suggest a low risk of cardiac AEs with acala treatment in pts with CLL. The safety of acala vs ibrutinib in pts with high-risk CLL will be investigated in the phase 3, randomized ACE-CL-006 trial (NCT02477696). Disclosures Brown: Janssen, Teva: Speakers Bureau; Abbvie, Acerta, AstraZeneca, Beigene, Invectys, Juno/Celgene, Kite, Morphosys, Novartis, Octapharma, Pharmacyclics, Sunesis, TG Therapeutics, Verastem: Consultancy; Gilead, Loxo, Sun, Verastem: Research Funding. Byrd:Acerta Pharma: Research Funding; Syndax: Research Funding; Leukemia and Lymphoma Society: Other; Trillium: Research Funding; Kartos Therapeutics: Research Funding; Vincera: Research Funding; Novartis: Research Funding; Janssen: Consultancy; Pharmacyclics LLC, an AbbVie Company, Gilead, TG Therapeutics, BeiGene: Research Funding; Pharmacyclics LLC, an AbbVie Company, Gilead, TG Therapeutics, Novartis, Janssen: Speakers Bureau; Pharmacyclics LLC, an AbbVie Company, Janssen, Novartis, Gilead, TG Therapeutics: Other. Ghia:Adaptive, Dynamo: Consultancy, Honoraria; Novartis: Research Funding; Acerta/AstraZeneca: Consultancy, Honoraria; ArQule: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; BeiGene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Research Funding; Celgene/Juno: Consultancy, Honoraria; Lilly: Consultancy, Honoraria; MEI: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Research Funding. Sharman:TG Therapeutics: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; AstraZeneca: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Acerta: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Bristol Meyers Squibb: Consultancy, Research Funding; BeiGene: Research Funding. Hillmen:F. Hoffmann-La Roche: Honoraria, Research Funding; Astra Zeneca: Honoraria; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Research Funding, Speakers Bureau; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Research Funding, Speakers Bureau; Pharmacyclics: Research Funding; Gilead: Research Funding. Stephens:MingSight: Research Funding; Acerta: Research Funding; Karyopharm: Consultancy, Research Funding; Gilead: Research Funding; Arqule: Research Funding; Pharmacyclics: Consultancy; Verastem: Research Funding; Beigene: Consultancy; Juno: Research Funding; Innate: Consultancy; Janssen: Consultancy. Sun:VERASTEM, GENMAB: Research Funding. Jurczak:Janssen, MeiPharma, Merck, Pharmacyclics, Roche, Tekeda, TG Therapeutics: Research Funding; Maria Sklodowska-Curie National Research Institute of Oncology: Consultancy, Current Employment; Jagiellonian University: Ended employment in the past 24 months, Research Funding. Patel:AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Baek:Acerta Pharma: Current Employment. Lezhava:Astra Zeneca: Current Employment; Melinta Therapeutics Inc: Ended employment in the past 24 months. Kuptsova-Clarkson:AstraZeneca: Current Employment. Moslehi:AstraZeneca, Janssen, BMS, Boston Biomedical, Immunocure, Myovant, Boston Biomedical, Deciphera: Consultancy. Furman:Acerta: Consultancy; Abbvie: Consultancy; Verastem: Consultancy; Sunesis: Consultancy; Pharmacyclics: Consultancy; Oncotarget: Consultancy; Loxo Oncology: Consultancy; Janssen: Consultancy, Speakers Bureau; Incyte: Consultancy; TG Therapeutics: Consultancy, Research Funding; AstraZeneca: Consultancy, Research Funding; Beigene: Consultancy; Genentech: Consultancy.

High-risk cytogenetics and minimal residual disease (MRD) after chemoimmunotherapy (CIT) predict unfavorable outcome in chronic lymphocytic leukemia (CLL). This phase 2 study investigated risk-adapted CIT in treatment-naïve CLL (NCT01145209). Patients with high-risk cytogenetics received induction with fludarabine, cyclophosphamide, and ofatumumab. Those without high-risk cytogenetics received fludarabine and ofatumumab. After induction, MRD positive (MRD+) patients received 4 doses of ofatumumab consolidation. MRD negative (MRD−) patients had no intervention. Of 28 evaluable for response, all responded to induction and 10 (36%) achieved MRD−. Two-year progression-free survival (PFS) was 71.4% (CI(95), 56.5–90.3%). There was no significant difference in median PFS between high-risk and standard-risk group. Ofatumumab consolidation didn’t convert MRD+ to MRD−. In MRD+ group, we saw selective loss of CD20 antigens during therapy. In conclusion, risk-adapted CIT is feasible in treatment-naïve CLL. Ofatumumab consolidation didn’t improve depth of response in MRD+ patients. Loss of targetable CD20 likely reduces efficacy of consolidation therapy.

Bruton tyrosine kinase (BTK) inhibition is an effective therapy for many B-cell malignancies. Acalabrutinib is a next-generation, potent, highly selective, covalent BTK inhibitor. To characterize acalabrutinib tolerability, we pooled safety data from 1040 patients with mature B-cell malignancies treated with acalabrutinib monotherapy in nine clinical studies (treatment-naive: n = 366 [35%], relapsed/refractory: n = 674 [65%]; median [range] age: 67 [32–90] years; median [range] prior treatments: 1 [0–13]; median [range] duration of exposure: 24.6 [0.0–58.5] months). The most common adverse events (AEs) were headache (38%), diarrhea (37%), upper respiratory tract infection (22%), contusion (22%), nausea (22%), fatigue (21%), and cough (21%). Serious AEs (SAEs) occurred in 39% of patients; pneumonia (6%) was the only SAE that occurred in ≥2%. Deaths due to AEs occurred in 52 patients (5%); pneumonia (n = 8) was the only fatal AE to occur in ≥3 patients. AEs led to treatment discontinuation in 9%. Rates for the AEs of interest (all grades) included infections (67%), hemorrhages (46%), neutropenia (16%), anemia (14%), second primary malignancies (12%), thrombocytopenia (9%), hypertension (8%), and atrial fibrillation (4%). This pooled analysis confirmed acalabrutinib’s tolerability and identified no newly emerging late toxicities, supporting acalabrutinib as a long-term treatment for patients with mature B-cell malignancies.

Immune dysregulation in chronic lymphocytic leukemia (CLL) contributes to a high rate of infections and morbidity. The Bruton tyrosine kinase (BTK) inhibitors ibrutinib and acalabrutinib mark major breakthroughs in the treatment of CLL, however many patients require long-term therapy with these agents. Despite receiving effective therapy for CLL, patients on BTK inhibitors remain immunocompromised and at risk of infectious complications. We previously reported that treatment of CLL with ibrutinib leads to partial reconstitution of humoral immunity and fewer infections during the first two years of therapy. It is currently unclear whether the positive effects of ibrutinib on the immune system are sustained during long-term therapy. Acalabrutinib is a newer, more selective BTK inhibitor than ibrutinib; however a detailed evaluation of the immunologic impact of acalabrutinib therapy is lacking. Herein, utilizing two independent trials, we assessed the immunological effects and infectious risk of ibrutinib and acalabrutinib treatment in patients with CLL.

B-cell receptor (BCR) signaling and tumor–microenvironment crosstalk both drive chronic lymphocytic leukemia (CLL) pathogenesis. Within the microenvironment, tumor cells shape the T-cell compartment, which in turn supports tumor growth and survival. Targeting BCR signaling using Bruton tyrosine kinase inhibitors (BTKi) has become a highly successful treatment modality for CLL. Ibrutinib, the first-in-class BTKi, also inhibits Tec family kinases such as interleukin-2–inducible kinase (ITK), a proximal member of the T-cell receptor signaling cascade. It is increasingly recognized that ibrutinib modulates the T-cell compartment of patients with CLL. Understanding these T-cell changes is important for immunotherapy-based approaches aiming to increase the depth of response and to prevent or treat the emergence of resistant disease. Ibrutinib has been shown to improve T-cell function in CLL, resulting in the expansion of memory T cells, Th1 polarization, reduced expression of inhibitory receptors and improved immune synapse formation between T cells and CLL cells. Investigating the modulation of BTKi on the T-cell antitumoral function, and having a more complete understanding of changes in T cell behavior and function during treatment with BTKi therapy will inform the design of immunotherapy-based combination approaches and increase the efficacy of CLL therapy.

Ibrutinib and idelalisib are kinase inhibitors that have revolutionized the treatment of chronic lymphocytic leukemia (CLL). Capable of inducing durable remissions, these agents also modulate the immune system. Both ibrutinib and idelalisib abrogate the tumor-supporting microenvironment by disrupting cell-cell interactions, modulating the T-cell compartment, and altering the cytokine milieu. Ibrutinib also partially restores T-cell and myeloid defects associated with CLL. In contrast, immune-related adverse effects, including pneumonitis, colitis, hepatotoxicity, and infections are of particular concern with idelalisib. While opportunistic infections and viral reactivations occur with both ibrutinib and idelalisib, these complications are less common and less severe with ibrutinib, especially when used as monotherapy without additional immunosuppressive agents. This review discusses the impact of ibrutinib and idelalisib on the immune system, including infectious and auto-immune complications as well as their specific effects on the B-cell, T-cell, and myeloid compartment.

A 52- year-old male with chronic lymphocytic leukemia was hospitalized with disseminated adenovirus disease. More than a month following recovery, hepatic necrotizing granulomas secondary to adenovirus were found. This case illustrates the protracted course that adenovirus disease may take and emphasizes an unusual presentation with hepatic necrotizing granulomas.

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, In a subset of patients with chronic lymphocytic leukemia (CLL) treated with targeted agents, such as ibrutinib, drug resistant subclones emerge. Here, the authors report on transcriptional changes in CLL patients treated with ibrutinib and identify early clonal shifts associated with evolution of resistant clones.

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.

A novel therapeutic approach in cancer, attempting to stimulate host anti-tumor immunity, involves blocking of immune checkpoints. Lymphocyte activation gene 3 (LAG3) is an immune checkpoint receptor expressed on activated/exhausted T cells. When engaged by the major histocompatibility complex (MHC) class II molecules, LAG3 negatively regulates T-cell function, thereby contributing to tumor escape. Intriguingly, a soluble LAG3 variant activates both immune and malignant MHC class II-presenting cells. In the study herein, we examined the role of LAG3 in the pathogenesis of chronic lymphocytic leukemia, an MHC class II-presenting malignancy, and show that chronic lymphocytic leukemia cells express and secrete LAG3. High levels of surface and soluble LAG3 were associated with the unmutated immunoglobulin variable heavy chain leukemic subtype and a shorter median time from diagnosis to first treatment. Utilizing a mechanism mediated through MHC class II engagement, recombinant soluble LAG3-Ig fusion protein, LAG3-Fc, activated chronic lymphocytic leukemia cells, induced anti-apoptotic pathways and protected the cells from spontaneous apoptosis, effects mediated by SYK, BTK and MAPK signaling. Moreover, LAG3 blocking antibody enhanced in vitro T-cell activation. Our data suggest that soluble LAG3 promotes leukemic cell activation and anti-apoptotic effects through its engagement with MHC class II. Furthermore, MHC class II-presenting chronic lymphocytic leukemia cells may affect LAG3-presenting T cells and impose immune exhaustion on their microenvironment; hence, blocking LAG3-MHC class II interactions is a potential therapeutic target in chronic lymphocytic leukemia.

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. Clin Cancer Res; 22(7); 1572–82. ©2015 AACR. See related commentary by Bachireddy and Wu, p. 1547

8064 Background: Acala is a next-generation, highly selective, covalent Bruton tyrosine kinase inhibitor approved in the US for patients (pts) with chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) and previously treated mantle cell lymphoma (MCL). We evaluated the safety profile of acala monotherapy (monotx) in multiple B cell malignancies. Methods: Data from pts with activated B-cell diffuse large B-cell lymphoma, CLL, follicular lymphoma, MCL, multiple myeloma, prolymphocytic leukemia, Richter syndrome, SLL, or Waldenström macroglobulinemia treated with ≥1 dose of acala monotx in 9 studies were pooled. Acala was administered at 100 mg BID in most pts (100–400 mg total dose daily). Adverse events (AE) were assessed. Results: A total of 1040 pts were included (median age: 67 y [range: 32–90]; ECOG status ≤1: 93%; median exposure duration: 24.6 mo [range: 0–58.5]). A total of 360 (34%) pts discontinued acala, most commonly due to progressive disease (PD; 17%). AEs led to acala discontinuation in 97 (9%) pts; those in > 2 pts were pneumonia (n = 5) and thrombocytopenia (n = 4). Incidence of AEs, including the most common (any grade and grade ≥3), are shown in the Table. Events of clinical interest (ECIs) included atrial fibrillation (afib) of any grade in 46 (4%) pts and grade ≥3 in 13 (1%) pts; major hemorrhage (any grade) in 37 (4%) pts; grade ≥3 infection in 183 (18%) pts; hypertension (any grade) in 79 (8%) pts and grade ≥3 in 36 (4%) pts; and second primary malignancies (SPM) excluding non-melanoma skin cancer (NMSC; any grade) in 68 (7%) pts. Median (range) time to first onset in days for each ECI (any grade) was: afib, 522 (8–1280); major hemorrhage, 293 (4–1327); infections, 92 (1–1317); hypertension, 157 (2–1345); SPM excluding NMSC, 339 (7–1499). Death was reported in 139 (13%) pts, most commonly due to PD (6%) and AEs (5%). Conclusions: Acala monotx has a favorable tolerability profile with increased exposure across multiple mature B cell malignancies. Additional analyses will further explore the longitudinal characteristics of AEs. [Table: see text]

The Bruton tyrosine kinase inhibitor ibrutinib induces high rates of clinical response in chronic lymphocytic leukemia (CLL). However, there remains a need for adjunct treatments to deepen response and to overcome drug resistance. Blinatumomab, a CD19/CD3 bispecific antibody (bsAb) designed in the BiTE (bispecific T-cell engager) format, is approved by the US Food and Drug Administration for the treatment of relapsed or refractory B-cell precursor acute lymphoblastic leukemia. Because of its short half-life of 2.1 hours, blinatumomab requires continuous intravenous dosing for efficacy. We developed a novel CD19/CD3 bsAb in the single-chain Fv-Fc format (CD19/CD3-scFv-Fc) with a half-life of ∼5 days. In in vitro experiments, both CD19/CD3-scFv-Fc and blinatumomab induced >90% killing of CLL cells from treatment-naive patients. Antileukemic activity was associated with increased autologous CD8 and CD4 T-cell proliferation, activation, and granzyme B expression. In the NOD/SCID/IL2Rγnull patient-derived xenograft mouse model, once-weekly treatment with CD19/CD3-scFv-Fc eliminated >98% of treatment-naive CLL cells in blood and spleen. By contrast, blinatumomab failed to induce a response, even when administered daily. We next explored the activity of CD19/CD3-scFv-Fc in the context of ibrutinib treatment and ibrutinib resistance. CD19/CD3-scFv-Fc induced more rapid killing of CLL cells from ibrutinib-treated patients than those from treatment-naive patients. CD19/CD3-scFv-Fc also demonstrated potent activity against CLL cells from patients with acquired ibrutinib-resistance harboring BTK and/or PLCG2 mutations in vitro and in vivo using patient-derived xenograft models. Taken together, these data support investigation of CD19/CD3 bsAb’s and other T cell-recruiting bsAb’s as immunotherapies for CLL, especially in combination with ibrutinib or as rescue therapy in ibrutinib-resistant disease.

Richter transformation (RT) of chronic lymphocytic leukemia (CLL) into high-grade lymphoma is a complication in 2–10% of patients. While development of diffuse large B-cell lymphoma (DLBCL) is most...

Immune stimulation contributes to lenalidomide’s antitumor activity. Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of mature, autoreactive B cells in secondary lymphoid tissues, blood, and bone marrow and progressive immune dysfunction. Previous studies in CLL indicated that lenalidomide can repair defective T cell function in vitro. Whether T cell activation is required for clinical response to lenalidomide remains unclear. In this study, we report changes in the immune microenvironment in patients with CLL treated with single-agent lenalidomide and associate the immunologic effects of lenalidomide with antitumor response. Within days of starting lenalidomide, T cells increased in the tumor microenvironment and showed Th1-type polarization. Gene expression profiling of pretreatment and on-treatment lymph node biopsy specimens revealed upregulation of IFN-γ and many of its target genes in response to lenalidomide. The IFN-γ–mediated Th1 response was limited to patients achieving a clinical response defined by a reduction in lymphadenopathy. Deep sequencing of TCR genes revealed decreasing diversity of the T cell repertoire and an expansion of select clonotypes in responders. To validate our observations, we stimulated T cells and CLL cells with lenalidomide in culture and detected lenalidomide-dependent increases in T cell proliferation. Taken together, our data demonstrate that lenalidomide induced Th1 immunity in the lymph node that is associated with clinical response.

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 (

Introduction Immune dysregulation is a hallmark of CLL, making these patients particularly vulnerable to infectious complications. Patients with CLL are at increased risk of developing varicella zoster virus (VZV) reactivation (shingles) due to their advanced age and immunocompromised status. A new recombinant (non-live) adjuvanted shingles vaccine (SHINGRIX; RZV) has the potential to reduce VZV reactivation, without the risk of giving a live vaccine to immunocompromised individuals. RZV is proven to reduce the risks of herpes zoster and postherpetic neuralgia in healthy adults ≥ 50 years of age, however its efficacy in immunocompromised individuals, including CLL, remains unknown. We report preliminary safety and efficacy of RZV in treated and untreated CLL patients. Methods In this phase II open-label study (NCT03702231), patients with CLL who were either treatment naïve or receiving treatment with a Bruton's tyrosine kinase inhibitor (BTK-I) (ibrutinib or acalabrutinib) received 2 doses of RZV via intramuscular injection at 0- and 3-months. Subjects were followed for 6 months and received assessment of serologic response at 3- and 6-months. Based on results of prior published studies, serologic response was defined as a ≥ four-fold rise in VZV anti-glycoprotein E (anti-gE) blood IgG serum titer after completing the RZV vaccine series. Additionally, serologic response was analyzed 3 months following the first vaccine administration to study the kinetics of the humoral vaccine response. All subjects completed an adverse event (AE) diary documenting any local (injection site) or systemic AE that started within 7 days after receiving the first and second vaccine dose. The data reported herein are as of July 12th 2019; updated results will be presented at the meeting. Results Safety data are available on 57 subjects who received at least one vaccine dose. The most frequent local and systemic AEs were injection site pain (67%), injection site reaction (32%) and generalized myalgias (25%) (Table 1). All adverse reactions were grade 1-2, except for 2 (4%) grade 3 reactions. No serious AEs were reported. All AEs resolved or returned to baseline within 7 days of vaccine administration. Seven subjects have completed the primary endpoint and have had serologic response assessment at 6-months. Serologic responses were observed in 3 (43%) patients. All patients (n = 7) achieved ≥ 2.2-fold rise in VZV anti-gE titers at 6-months. Preliminary analysis of 3-month samples (n = 43) shows early evidence of increasing titers after the first dose of RZV. Conclusions RZV administration appears to be safe in CLL patients that are treatment naïve or receiving treatment with a BTK-I. Compared to previously reported toxicities in the healthy population, no increase in frequency or severity of AEs were observed. Preliminary data suggests that RZV induces humoral immune responses in patients with CLL. Disclosures Wiestner: Pharmayclics: Research Funding; Acerta: Research Funding; Merck: Research Funding; Nurix: Research Funding.

Targeting B-cell receptor signaling with ibrutinib, the first-in-class irreversible inhibitor of Bruton tyrosine kinase, has become a highly successful treatment modality for Chronic Lymphocytic Leukemia (CLL) patients. However, there remains a need for adjunct treatments to deepen responses and prevent or treat resistant disease. Ibrutinib also inhibits inducible T-cell kinase (ITK) which is hypothesized to improve antitumor T-cell immunity. We developed a CD19/CD3 bispecific antibody (bsAb) in a 100 kDa single chain-Fv-Fc format (19/3-scFv-Fc). We previously reported increased T-cell activation and cytotoxic activity by the bsAb in blood samples from ibrutinib-treated patients compared to treatment-naïve patients. To better understand how ibrutinib affects T-cell function and increases cytotoxic activity we assessed T-cell responses to the bsAb in samples from ibrutinib-treated patients (12 ± 2 months on ibrutinib) and treatment-naïve (TN) patients. We cultured peripheral blood mononuclear cells (PBMCs) from both patient groups with bsAb and measured CLL cell death, T-cell expansion, activation, and differentiation by flow cytometry. After 5 days of exposure, the CD19/CD3 bsAb induced superior killing of ibrutinib-treated CLL cells, with a median specific-killing of 97% for ibrutinib-treated compared to 77 % for treatment-naïve patient samples (n = 15; p= 0.008). In vitro treatment with CD19/CD3 bsAb induced ≥ 5-fold expansion of autologous CD8 and CD4 T cells and an increase of CD45RO+CCR7- effector memory and CD45RO+CCR7+ central memory CD4 T-cells in response to bsAb (Table 1). We also observed a shift towards Th1 (CCR6-CXCR3+) polarization, and higher T-cell granzyme B expression in response to CD19/CD3 bsAb. While T cells from both patient groups responded to the bsAb, all these effects were more pronounced in ibrutinib-treated samples compared to treatment-naïve samples (Table 1). There was a 13-fold increase of the Th1/Th2 ratio in PBMCs from ibrutinib-treated patients compared to a 7-fold increase in the treatment-naïve group. Moreover, the bsAb induced a significant increase of activation markers HLADR (p=0.0013; p=0.0006) and CD27 (p< 0.001; p=0.0114) on CD4 and CD8 T cells in the ibrutinib-treated patient samples, while there was no significant change in T-cell activation in the treatment-naïve group. Granzyme B expression in CD8 T cells was high even in untreated samples and its upregulation by the bsAb was comparable in both patient groups. However, we observed a 25-fold increase in granzyme B positive CD4 T cells in ibrutinib-treated samples, 5-fold higher than in the treatment-naïve samples. In summary, the enhanced cytotoxic activity of T cells in response to the bsAb in samples from ibrutinib-treated patients correlated with increased memory cell differentiation, higher T-cell activation, and a strong shift towards a Th1 phenotype. As ibrutinib enhances anti-CLL activity of autologous T cells, bsAb immunotherapy may work well with concurrent ibrutinib treatment. These data support the investigation of bsAbs in combination with ibrutinib for immunotherapy of CLL. The mechanisms underlying the enhanced T-cell responses remain to be fully elucidated. In particular, it will be important to define whether inhibition of ITK by ibrutinib plays a role in enhancing T-cell dependent cytotoxicity. Supported by the intramural program of the NHLBI/NIH. Disclosures Rader: NIH: Patents & Royalties: ROR1 mAb 2A2. Wiestner:Acerta: Research Funding; Merck: Research Funding; Nurix: Research Funding; Pharmayclics: Research Funding.

Background: Of the several drugs and drug combinations approved for treatment of relapsed and refractory chronic lymphocytic leukemia (CLL), the reported complete response rates are no greater than 30%. Obinutuzumab is a glycoengineered, humanized type 2 anti-CD20 monoclonal antibody thought to engage the immune system by directly activating antibody- dependent, cell-mediated cytotoxicity (ADCC); it is approved for treatment of chronic lymphocytic leukemia in combination with chlorambucil. The key mediators of ADCC are polymorphonuclear neutrophils, monocytes, and natural killer (NK) cells. Recombinant human Interleukin-15 (rhIL-15) is a stimulatory cytokine that promotes the differentiation and activation of NK cells, monocytes, and long-term CD8+ memory T-cells. In a Phase I trial, administration of rhIL-15 as a 5-day continuous intravenous infusion (civ) was associated with up to 45-fold increase in the number of NK cells at well- tolerated dose levels. Preclinical murine lymphoid malignancy models have shown increased efficacy of monoclonal antibodies when administered together with rhIL-15; BL/6 mice inoculated with EL4-CD20 cells (a syngeneic lymphoma line); including significant prolongation of survival with the IL-15/Rituximab combination compared to either drug given as single agent (90% v. 30% alive at 75 days). We hypothesized that rhIL-15-associated increase in NK cell number and activity would improve efficacy of obinutuzumab in treatment of relapsed and refractory CLL, and would increase the duration and depth of response; this phase I trial is testing the safety of the combination. Primary objective: determine the safety, toxicity profile, dose-limiting toxicity (DLT) and the maximum tolerated dose (MTD) of civ rhIL-15 administration in combination with obinutuzumab treatment Secondary objectives: 1) evaluate the potential antitumor activity of the combination of rhIL-15 and obinutuzumab by assessing the clinical response rate, minimal residual disease (MRD) status, progression-free survival, and overall survival in patients with relapsed and refractory CLL; 2) define the effects of rhIL-15 on the ADCC mediated by obinutuzumab using ex vivo peripheral blood mononuclear cells (PBMCs); 3) characterize the biological effects of rhIL-15 administered with obinutuzumab on the percentages and absolute numbers of circulating lymphocytes (T and NK cells) and the T- cell subsets (including naïve, central, and effector memory subsets) by flow cytometry Exploratory objectives: identify biomarkers predictive of response to rhIL-15 and obinutuzumab treatment, such as circulating tumor DNA (ctDNA) and baseline cytokine levels Eligibility criteria: 1) age ≥ 18 years; 2) ECOG ≤ 1; 3) Diagnosis of CLL or small lymphocytic lymphoma (SLL) with ≥ 50% of B cells expressing CD20; 4) measurable or evaluable disease that is refractory or relapsed following therapy with a BTK inhibitor OR have relapsed/refractory CLL and are intolerant to BTK inhibitor therapy; patients with del(17p) must also be refractory or relapsed after, or intolerant to, therapy with venetoclax; 5) adequate organ function parameters as defined within the protocol; 6) active disease requiring treatment, as defined within the protocol. Study design: a single institution non-randomized phase I dose escalation study evaluating increasing doses of civ rhIL-15 in combination with obinutuzumab using a 3 + 3 dose escalation design. On days 1-5 of each 4-week cycle, rhIL-15 will be administered by civ at dose levels 0.5, 1, and 2 mcg/kg/day. During the first cycle, obinutuzumab will be administered at a dose of 100 mg by IV on day 4, 900 mg on day 5, 1,000 mg on day 11, and 1,000 mg on day 18; then 1,000 mg on day 4 of each subsequent cycle. Infusion reaction, antimicrobial, and tumor lysis syndrome prophylaxis will be administered per manufacturer's recommendations. Treatment will continue up to 6 cycles, or until unacceptable toxicity or progressive disease. Up to 24 patients will be enrolled in the study. Correlative studies: 1) optional lymph node biopsy at baseline and after the first week of treatment (C1D8) for tissue immune cell subsets and quantifying antibody penetration; 2) lymphocyte subset testing, ctDNA, and cell and plasma banking on days 1, 4, 8, and 11 of cycle 1, and days 1 and 8 of cycles 2-6; 3) ADCC capacity of ex vivo NK cells on C1D1, 4, and 8. One patient has started treatment to date. Enrollment is ongoing. Figure Disclosures Wiestner: Acerta: Research Funding; Pharmayclics: Research Funding; Merck: Research Funding; Nurix: Research Funding. Waldmann:Bioniz: Membership on an entity's Board of Directors or advisory committees.

Activation and proliferation of chronic lymphocytic leukemia (CLL) cells depend on signals from the lymph node (LN) tumor microenvironment (TME). Separately, the genetic makeup of CLL has been closely linked to disease aggressiveness and its capacity to evolve under the selective pressures of treatment. Here, we investigated the intersection between the TME and molecular events in CLL pathogenesis. Whole exome and RNA sequencing (RNA-seq) were performed on CD19+ cells of paired peripheral blood (PB) and LN samples and matched germline DNA from 14 patients with treatment-naïve CLL. RNA-seq was also done on unsorted LN samples containing tumor and non-tumor cells from the same patients. A median of 27 (range 11-69) somatic single nucleotide variants (sSNVs) and 3 (0-10) insertions and deletions (sIndels) were detected per exome. All but one patient had copy number alterations (CNAs), most commonly del 11q and del 13q. Cancer cell fractions (CCFs) of sSNVs, sIndels, and CNAs were inferred from variant allele frequencies then clustered over the two anatomic compartments for each patient. Genetic compartmentalization (ΔCCF > 0.25, false discovery rate [FDR] < 0.1) was observed in 7 patients (50%), of whom 6 demonstrated subclonal expansion in LN. To understand factors contributing to spatial heterogeneity, we compared the tumor transcriptome based on the presence (shifted group) or absence (stable group) of an expanded subclone in LN. Most differentially expressed genes between PB and LN were shared by all patients and reflected the activation of CLL cells in the LN TME as previously shown. However, cell cycle genes (e.g. E2F2, CDC25A) were more upregulated (log2FC > 0.5, FDR < 0.05) in LN of the shifted group, while lymphocyte activation markers (e.g. CD83, CD69) were more upregulated in LN of the stable group. We hypothesized the latter finding could indicate immune-mediated control of clonal outgrowth. We therefore evaluated the expression of an 18-gene T-cell associated inflammatory signature in unsorted LN samples. This signature was originally developed as a predictive biomarker for response to immune checkpoint blockade in multiple cancer types. Unsupervised hierarchical clustering of signature genes revealed an inflamed TME in the stable group relative to the shifted group. In summary, genetic compartmentalization is a common phenomenon in CLL. Clonal equilibrium is maintained by a T-cell inflamed TME. When immune surveillance is inactivated, subclones with a competitive advantage may expand in response to support signals provided by the TME. An immunotherapy-based clinical study using checkpoint blockade to restrict clonal evolution is currently in progress (NCT03204188). This research was supported by the Intramural Research Program of the NIH, NHLBI. Disclosures Getz: Pharmacyclics: Research Funding; IBM: Research Funding; MuTect, ABSOLTUE, MutSig and POLYSOLVER: Patents & Royalties: MuTect, ABSOLTUE, MutSig and POLYSOLVER. Wu:Neon Therapeutics: Other: Member, Advisory Board; Pharmacyclics: Research Funding. Wiestner:Merck: Research Funding; Pharmayclics: Research Funding; Acerta: Research Funding; Nurix: Research Funding.

The lymph node (LN) is the site of chronic lymphocytic leukemia (CLL) cell activation and proliferation. Aberrant microRNA (miRNA) expression has been shown to have a role in CLL pathogenesis; however, a comparison of miRNA expression between CLL cells in the LN and the peripheral blood (PB) has previously not been reported. On the basis of the analysis of 17 paired LN and PB samples from CLL patients, we identify a panel of miRNAs that are increased in LN CLL cells correlating with an activation phenotype. When evaluated in CLL cells from 38 patients pre and post treatment with ibrutinib, a subset of these miRNAs (miR-22, miR-34a, miR-146b and miR-181b) was significantly decreased in response to ibrutinib. A concomitant increase in putative miRNA target transcripts (ARID1B, ARID2, ATM, CYLD, FOXP1, HDAC1, IBTK, PTEN and SMAD4) was also observed. Functional studies confirmed targets of ibrutinib-responsive miRNAs to include messenger RNA transcripts of multiple tumor suppressors. Knockdown of endogenous miR-34a and miR146b resulted in increased transcription of tumor suppressors and inhibition of cell proliferation. These findings demonstrate that ibrutinib downregulates the expression of a subset of miRNAs related to B-cell activation leading to increased expression of miRNA targets including tumor suppressors and a reduction in cell proliferation.

Background: Bruton tyrosine kinase (BTK) is a critical component of B-cell receptor signaling and a validated target for CLL. Acalabrutinib is a highly selective, potent, covalent BTK inhibitor, which has shown promising efficacy and safety in patients with CLL, including high-risk patients. We present preliminary efficacy, safety, and pharmacodynamic results from an ongoing single-center, open-label, phase 2 study of acalabrutinib monotherapy in patients with R/R and high-risk, TN CLL. Methods: Patients with R/R or high-risk (chromosome 17p deletion [del17p] or mutation in TP53 or NOTCH1) TN CLL/small lymphocytic lymphoma (SLL) who met International Workshop on Chronic Lymphocytic Leukemia (IWCLL) 2008 criteria for treatment and had an Eastern Cooperative Oncology Group performance status ≤2 were eligible. Patients who had prior BTK inhibitor therapy were excluded. Patients were randomized to receive oral acalabrutinib 100 mg twice daily (BID) or 200 mg daily (QD) until progressive disease or unacceptable toxicity. The primary endpoint was investigator-assessed overall response rate (ORR) by IWCLL 2008 criteria with modification for lymphocytosis. Secondary endpoints included safety and BTK occupancy. BTK occupancy was measured with a biotin-tagged analogue probe in peripheral blood cells at drug trough time points after 3 days of dosing and after 1, 6, and 12 mo of treatment. BTK occupancy in lymph node samples was measured at drug trough time points after 3 days of dosing. Results: Forty-six patients were enrolled and treated (100 mg BID, n=22; 200 mg QD, n=24). The median age was 64 years (range, 45-83), and 35% (16/46) were TN. Approximately 39% of patients (25% of TN) had bulky lymph nodes ≥5 cm, 37% (50% of TN) had Rai stage III-IV disease at baseline, 76% (88% of TN) had unmutatedIGHV, 21% (40% of TN) had del(17p), 21% (23% of TN) had TP53 mutation, and 47% (54% of TN) had NOTCH1 mutation. As of April 13, 2018, the median time on study for all treated patients was 20 mo (range 1-39), with 89% (41/46) remaining on acalabrutinib. Two patients (9%) in the BID group and 3 patients (13%) in the QD group discontinued treatment due to an adverse event (AE; n=1), progressive disease (n=1), and other reasons (n=3). The patient who discontinued due to progressive disease (BID group) achieved partial response at 2 mo and developed Richter transformation at 6 mo. The ORR was 90% (95% CI: 76, 97) for efficacy evaluable patients (N=39), defined per protocol as patients who had ≥ 6 mo of acalabrutinib (Table). ORR was 95% (75, 100) and 84% (60, 97) for the BID and QD group, respectively. For the intent-to-treat population (N=46), ORR was 80% (66, 91). Most AEs were grade 1/2 and did not require dose delays or modifications. The most common AEs (all grades; >25%) were headache (63%), contusion (50%), diarrhea (43%), upper respiratory tract infection (43%), arthralgia (33%), influenza-like illness (28%), maculo-papular rash (28%), myalgia (26%), and nausea (26%). Grade 3/4 AEs occurred in 33% (15/46) of patients (BID, 27% [6/22]; QD, 38% [9/24]), most commonly (>10%) infections (13%; urinary tract infection, lung infection, hepatitis B reactivation, which led to treatment discontinuation and fatal hepatic failure after 10 mo of treatment, and an invasive pulmonary aspergillosis at 2 mo in the setting of prolonged neutropenia and recent systemic corticosteroid use that led to treatment discontinuation) and neutropenia (11%). Approximately 33% (15/46) of patients (BID, 23% [5/22]; QD, 42% [10/24]) reported serious AEs (all grades), most commonly (>5%) lung infection (7%). No atrial fibrillation was reported, and one grade 1 atrial flutter occurred (BID). On day 4 of cycle 1, median trough BTK occupancy was significantly higher for the BID group versus the QD group in the peripheral blood (95% vs 87%; P Conclusion: Acalabrutinib monotherapy produced high ORR in R/R and high-risk TN CLL, with an acceptable safety profile. The study was not designed to detect a statistically significant difference in clinical outcomes between the dosing groups. Near complete target coverage (>95%) was more rapidly achieved with 100 mg BID than 200 mg QD dosing in the lymph node and peripheral blood. Disclosures Nierman: National Institutes of Health: Employment. Covey:Acerta Pharma: Employment; AstraZeneca: Equity Ownership. Hamdy:Acerta Pharma: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: various patents for ACP-196. Izumi:Acerta Pharma: Employment, Equity Ownership, Patents & Royalties: Acerta Pharma, various patents for ACP-196. Liu:Acerta Pharma: Employment. Patel:Acerta Pharma: Employment, Equity Ownership. Wiestner:Pharmacyclics LLC, an AbbVie Company: Research Funding.

Chronic lymphocytic leukemia (CLL) cells actively participate in the formation of the tumor microenvironment (TME). The interplay of CLL cells and leukemia-supporting cells such as Th2 cells and regulatory T cells (Tregs) promotes a leukemia-supportive, immune-tolerant TME. In these supportive/tolerogenic niches of lymph nodes (LN) and bone marrow (BM), CLL cells slowly proliferate, and the rate of proliferation correlates with disease progression. However, how these dividing/recently-divided cells and other intraclonal CLL fractions interact with non-neoplastic cells to shape the TME to support growth and accelerate disease is unclear. To address these questions, matched LN and PB samples collected at day 13 after 2H2O ingestion from treatment-naïve patients (7 with stable disease and 7 with active disease) were sorted to determine in vivo growth rates of CLL cells within the proliferative fraction (PF, CXCR4DimCD5Bright), resting fraction (RF, CXCR4BrightCD5Dim) and intermediate fraction (IF, CXCR4IntCD5Int). In LN, PF cells had the highest 2H-DNA levels, and only the growth rate of PF but not IF or RF cells correlated with disease aggressiveness. In the PB, PF cells also had the highest growth rate; however, all 3 fractions (PF, IF, RF) had 2H-DNA levels that correlated with disease aggressiveness. Thus, PF cells from patients with aggressive clinical course undergo quicker transitions to the IF and then to the RF which might promote faster tissue homing and disease progression. Indeed, more PF cells from active than stable disease patients were found in the spleen (SP) and BM and less remained in PB, 18 hours post-injection into NSG mice. Gene expression profiling (GEP) was then performed on RF, IF, and PF from 7 paired PB and LN samples. GEP signatures of the PF from PB and LN were similar, consistent with 2H2O data that the PF are recent emigrants from TME. These GEP also inferred enhanced cell proliferation (CCND2, CDK2AP1), adhesion and motility (FERMT3, CD49d, CD11a, CD21), antigen presentation (CD1C), and promotion of T-cell trafficking (CCL3, CCL4) in LN CLL cells within the PF but not the IF or RF. Thus, CLL cells within the three intraclonal fractions might promote distinct biologic functions. To test this, we studied T cell responses stimulated by the CXCR4/CD5 fractions in vitro and in vivo. In an antigen-driven allo-MLR, the whole clone of CLL cells triggered the division of normal T cells, but PF induced the highest level of T-cell division that is ≥ 3 times more than any other fraction. Similarly, in an autologous polyclonal CD4 T cell response stimulated by anti-CD3/28 Dynabeads and IL-2, T-cell division was suppressed by unseparated CLL cells and each fraction. However, the least suppression was seen in T cells co-cultured with PF cells compared to those cultured alone or with IF or RF. In both settings, PF cells induced significantly more IL-4+ T cells, and RF cells triggered more Tregs. Similar numbers of Th1 cells were seen in all cultures. The RF and IF, but not the PF, produced the immunosuppressive cytokine IL-10. Finally, when dividing cases based on disease aggressiveness, significantly more T-cell division was triggered by PF and RF from active patients than stable patients; the percentages of Th1 and Th2 cells however were similar. These results were confirmed in vivo; in NSG mice injected with autologous T cells together with the PF, IF or RF sorted from 2 sets of active versus stable disease patients, PF from all 4 cases induced the highest levels of CLL B and T cell expansion in SP and BM, and RF from active but not stable disease patients triggered the growth of CLL T and B cells. In summary, CLL disease progression correlates with the rate of CLL cell division, and the rapidity that CLL cells home to the TME. The intraclonal fractions of CLL clones exhibit distinct biologic properties that can be further differentiated based on disease aggressiveness. This appears especially relevant for the development of a leukemia-supportive, immune-tolerant TME contributed by all 3 CXCR4/CD5 fractions, albeit by different mechanisms. The PF creates this by superior antigen presentation capacity and skewing T cell function to an immunosuppressive Th2 phenotype. For the IF and RF, this is done by inducing IL-10 secretion and amplifying Tregs. Together, these findings suggest the possibility of targeting specific subpopulations in CLL clones with distinct immunoregulatory modalities as a novel form of therapy. Disclosures Chen: Beigene: Research Funding; Pharmacyclics: Research Funding; Verastem: Research Funding. Wiestner:Pharmacyclics LLC, an AbbVie Company: Research Funding. Rai:Cellectis: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees. Chiorazzi:Janssen, Inc: Consultancy; AR Pharma: Equity Ownership.

Introduction Immune dysregulation in chronic lymphocytic leukemia (CLL) contributes to a high rate of infections and morbidity. We previously reported that treatment of CLL with ibrutinib, a Bruton tyrosine kinase (BTK) inhibitor, leads to partial reconstitution of humoral immunity and fewer infections, especially in patients who achieved a ≥50% increase in serum IgA levels. Acalabrutinib is also an irreversible BTK inhibitor that is more selective than ibrutinib and has demonstrated safety and efficacy in the treatment of relapsed or refractory CLL. It is currently unclear how the increased specificity of acalabrutinib affects immune reconstitution and infection rates. We assessed the immunological impact of acalabrutinib in patients with CLL treated with single-agent acalabrutinib. Methods Samples originated from a phase 2, single-center trial studying acalabrutinib 100 mg twice daily (BID) or acalabrutinib 200 mg once daily (QD) in patients with relapsed/refractory (RR) CLL or high-risk, treatment naïve (TN) CLL (chromosome 17p deletion or mutation in TP53 or NOTCH1) (NCT02337829). Patients who received at least 6 months of acalabrutinib and had paired longitudinal data available were included in the analyses. Patients receiving IV immunoglobulin replacement were excluded from analysis of IgG levels. Additionally, patients with detectable monoclonal IgG, IgA and/or IgM proteins on serum immunofixation were excluded from analysis of the corresponding immunoglobulin isotype. The analysis of free light chains was stratified based on k or λ restriction of CLL cells determined by flow cytometry. Immunohistochemical staining of bone marrow biopsies was performed: T cell numbers were estimated by CD3 staining and the degree of CD68-positive macrophage extensions in contact with CLL cells were semi-quantitatively assessed on a scale from 0 (no extensions) to 4 (maximum number of extensions). The Wilcoxon signed-rank test and the Mann-Whitney U test were used to compare paired and unpaired data, respectively. Differences in the rate of infection between groups were examined using the Cox regression model for recurrent events. Results Serum IgA levels increased as early as 3 months after the initiation of acalabrutinib (median increase 35.7%, P = .0001), with levels sustained up to 24 months (Figure 1), whereas serum IgG and IgM levels were not affected by acalabrutinib. There was no difference (P > .05) in IgA, IgG, IgM trend between TN or RR CLL. Furthermore, there was no difference (P > .05) in IgA, IgG and IgM trends between patients treated with QD compared to BID dosing of acalabrutinib. Among 20 k-restricted and 18 λ-restricted CLL cases, the involved (tumor-derived) free light chain was elevated at baseline and trended toward the normal range after 3 months of acalabrutinib therapy consistent with an anti-tumor effect (k: median decrease 55.4%; P < .0001 and λ: median decrease 49.1%; P = .0003). The uninvolved free light chain did not change (P > .05). Peripheral blood CD3+, CD4+ and CD8+ T cell counts were elevated above the laboratory reference range at baseline and normalized after 6 months (CD4+ median decrease 49.2%; P = .0074 and CD8+ median decrease 54.8%; P = .003). T cell numbers in the bone marrow did not appreciably change. However, treatment-induced changes of the immune microenvironment were apparent in tumor-macrophage interactions. At baseline, macrophages tightly interacted with CLL cells, often with multiple podocytes making contact with CLL cells. On acalabrutinib, we observed a decrease in these macrophage podocyte interactions (P = .0007). At a median follow-up of 20 months, 31 (68.9%) patients developed a total of 68 infections, including 7 (10.3%) grade 3 and 1 (1.5%) grade 4 infections. Patients with superior immune reconstitution, as defined by an increase in serum IgA of ≥ a median of 36% from baseline to 3 months, had a significantly lower rate of infections (risk ratio = 0.52, P = 0.029). Conclusions These data indicate that acalabrutinib allows for partial reconstitution of humoral and cell-mediated immunity and disrupts macrophage-CLL cell interaction in the bone marrow microenvironment in patients with CLL. Furthermore, acalabrutinib did not interfere with uninvolved free light chains, suggesting that acalabrutinib selectively inhibits CLL B-cells and does not impair normal B-cell function. Disclosures Izumi: Acerta Pharma: Employment, Equity Ownership, Patents & Royalties: Acerta Pharma, various patents for ACP-196. Hamdy:Acerta Pharma: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Acerta Pharma, various patents for ACP-196. Wiestner:Pharmacyclics LLC, an AbbVie Company: Research Funding.

Inhibitors of B-cell receptor (BCR) signaling, in particular of Bruton tyrosine kinase (BTK), are effective in patients with chronic lymphocytic leukemia (CLL). Acalabrutinib is a highly selective, covalent BTK inhibitor with rapid absorption and fast elimination (Barf et al., J Pharmacol Exp Ther 2017; 363:240-252). In a previous clinical trial of patients with relapsed/refractory CLL, acalabrutinib therapy was well tolerated and efficacious and had high target occupancy (Byrd et al., N Engl J Med 2016; 374:323-21). Here, we report on BTK occupancy, BTK resynthesis rates, and the relationship between BTK occupancy and BCR signaling in patients with CLL enrolled in a single center, phase 2 study of acalabrutinib monotherapy (NCT02337829). Patients were randomized to receive either 200 mg of acalabrutinib once daily (QD) or 100 mg twice daily (BID). The percentage of BTK bound by acalabrutinib (target occupancy) was measured with the aid of a biotin-tagged analogue probe. After 3 days of dosing, the median BTK occupancy in peripheral blood at peak (4 hours postdose) was 99% in both dosing groups (n=21 per group). However, at the drug trough time points (12 hours for BID; 24 hours for QD), median occupancy was greater in the BID than QD group (95% vs 87%, respectively; P As acalabrutinib binds covalently to BTK, reactivation of the pathway requires de novo synthesis of BTK. We estimated the rate of BTK synthesis in vivo by measuring the accumulation of free BTK (% unbound by acalabrutinib) in serial blood samples taken 4, 12, 24, 36, and 48 hours postdose during a preplanned dose interruption on days 4 and 5. BTK was synthesized at a median rate of 13.4% per day, which was highly variable (fast vs slow) across patients (Figure). Interestingly, we found the median BTK de novo synthesis rate to be roughly twice as fast as observed in B cells from healthy volunteers (P We next correlated BTK occupancy levels and activity of BCR signaling in 10 representative patients. On Day 3, four hours after receiving the drug, significant reductions were observed in phospho-BTK (P=0.05), pNF-κB (P=0.03), and CD69 expression (P=0.001) compared with pretreatment. Next, we evaluated the change in BCR signaling from trough (for each dose group) to 36 (BID) or 48 (QD) hours after dosing (i.e., during the short window of drug withholding). A trend for increased signaling was driven by individual patients with low occupancy (≲70%). To mimic microenvironmental activation, we stimulated overnight with anti-IgM, all the peripheral blood samples collected during drug withholding (4 to 48 hours postdose). We found that upon BCR activation, B-CLL cells showed the ability to reactivate BCR signaling as measured by an increase in CD69 expression. Moreover, the extent of reactivation in BCR signaling positively correlated with the percentage of free BTK (R=0.73; P=0.0001). In conclusion, higher target coverage was achieved when acalabrutinib was administered 100 mg BID compared with 200 mg QD. Given that BTK de novo synthesis rates do vary across patients with CLL, and that reactivation of BCR signaling correlates with lower occupancy (higher free BTK), BID dosing provides the highest target coverage for a greater number of patients. This work was supported by the Intramural Research Program of National Heart, Lung, and Blood Institute, the National Institutes of Health, and Acerta Pharma. We thank our patients for donating blood and tissue samples to make this research possible. Figure. Figure. Disclosures Cheung: Acerta Pharma: Employment, Equity Ownership; AstraZeneca: Equity Ownership. Gulrajani:Acerta Pharma: Employment, Equity Ownership. Davies-Hill:NIH/NCI: Employment. Izumi:Acerta Pharma: Employment, Equity Ownership, Patents & Royalties: Acerta Pharma, various patents for ACP-196. Covey:AstraZeneca: Equity Ownership; Acerta Pharma: Employment. Wiestner:Pharmacyclics LLC, an AbbVie Company: Research Funding.

The T-cell receptor (TCR) repertoire in patients with chronic lymphocytic leukemia (CLL) is more clonal than in healthy individuals. Shared TCR clonotypes among patients may reflect the expansion of T-cell clones that recognize common antigens. It is unknown whether these antigens are tumor-derived to which T cells react, or microbial or auto-antigens that promote expansion of both T cells and the leukemic clone. Ibrutinib inhibits Bruton tyrosine kinase and interleukin-2-inducible kinase and has been shown to modulate the T-cell compartment of patients with CLL. We performed deep sequencing of the TCRβ repertoire (Adaptive Biotechnologies) in 26 patients with CLL prior to treatment with ibrutinib, at the time of response, and either at disease progression or during sustained remission. TCR clonality was calculated as the inverse of Shannon entropy normalized to the number of productive clones in a sample and it accounts for both the number of unique clonotypes and the extent to which one or a few clonotypes dominate the sample repertoire. Clonality values may range between 0 and 1 with values closer to 1 indicating a more oligoclonal repertoire. Nonparametric tests were applied for statistical analyses, and paired analyses were performed to compare metrics across different time points. The median productive clonality in CLL patients before treatment was .1795, more than double the clonality reported for healthy donors (.0750, provided by Adaptive Biotechnologies). Differences in clonality were studied between subgroups of patients defined by clinical and biologic features. Overall, the TCR repertoire in treatment naïve patients (clonality = .1454, n = 15) was less clonal than in patients with relapsed/refractory CLL (clonality = .2468, n = 11, P = .002). β2-microglobulin ≥ 3.5 mg/L and CD8+ T-cell counts above the normal reference range were also associated with higher clonality (P = .030 and .003, respectively). Further, CD8+ T-cell counts (median 1134 /uL, IQR 670 - 2474) when assessed as a continuous variable, were correlated with clonality (ρ = .620, P = .001). Next, we evaluated the TCR repertoire at partial (n = 23) or complete (n = 2) response after a median duration of 12 months (range 6-36) on ibrutinib. Clonality increased at the time of response compared to baseline (median increase 33.2%, P = .048) despite a concomitant decrease in CD4+ and CD8+ T-cell counts. In 12 patients who subsequently developed progressive disease, analysis of the TCR repertoire revealed a decrease in clonality (median decrease 5.7%, P = .026) from the time of response to relapse (median 13 months, range 4-32) while CD4+ and CD8+ T-cell counts increased. In contrast, clonality did not significantly change among patients with sustained response to ibrutinib measured 12 months after the first response time point. Overrepresented clonotypes comprising more than .05% of the TCR repertoire at baseline or at the time of response were selected for further analyses (median of 95 clonotypes per patient). Eighteen out of 25 patients shared at least one clonotype. In total, 20 shared clonotypes were identified and interestingly, their frequencies decreased with ibrutinib treatment (P = .003). Interrogation of overrepresented clonotypes in public databases identified 46 clonotypes previously reported in viral infections (77%) followed by those associated with autoimmune conditions (13%) and cancer (10%). The most commonly implicated viruses were Epstein Barr virus, cytomegalovirus and influenza. We observed the convergence of multiple clonotypes recognizing the same viral epitopes and 8 clonotypes were shared between at least 2 patients. However, they behaved differently on treatment with an increase in frequency in some patients and decrease in others. In summary, we show that ibrutinib induces a more clonal TCR repertoire and that it is lost at relapse. Overrepresented clonotypes were generally patient-specific and correlated with CD8+ T cells. These findings suggest that CD8+ T-cell clonotypes recognizing private antigens expand and mediate anti-tumor activity during treatment with ibrutinib. This research was in part supported by the Intramural Research Program of the NIH, NHLBI. Disclosures Wiestner: Pharmacyclics LLC, an AbbVie Company: Research Funding.

Chronic lymphocytic leukemia (CLL) is characterized by immune dysregulation, often including hypogammaglobulinemia, which contributes to a high rate of infections and morbidity. Ibrutinib, a covalent inhibitor of Bruton tyrosine kinase (BTK), inhibits B-cell receptor signaling and is an effective, US Food and Drug Administration (FDA)-approved treatment of CLL. Inactivating germline mutations in BTK cause a severe B-cell defect and agammaglobulinemia. Therefore, we assessed the impact of ibrutinib on immunoglobulin levels, normal B cells, and infection rate in patients with CLL treated with single-agent ibrutinib on a phase 2 investigator-initiated trial. Consistent with previous reports, immunoglobulin G (IgG) levels remained stable during the first 6 months on treatment, but decreased thereafter. In contrast, there were a transient increase in IgM and a sustained increase in IgA (median increase 45% at 12 months, P < .0001). To distinguish the effects on clonal B cells from normal B cells, we measured serum free light chains (FLCs). In κ-clonal CLL cases, clonal (κ) FLCs were elevated at baseline and normalized by 6 months. Nonclonal (λ) FLCs, which were often depressed at baseline, increased, suggesting the recovery of normal B cells. Consistently, we observed normal B-cell precursors in the bone marrow and an increase in normal B-cell numbers in the peripheral blood. Patients with superior immune reconstitution, as defined by an increase in serum IgA of ≥50% from baseline to 12 months, had a significantly lower rate of infections (P = .03). These data indicate that ibrutinib allows for a clinically meaningful recovery of humoral immune function in patients with CLL. This trial was registered at www.clinicaltrials.gov as #NCT015007330.

Chronic lymphocytic leukemia (CLL) is characterized by a highly variable clinical course that has guided treatment principles in as much as anti-leukemic therapy is reserved for patients with active disease. This heterogeneity is somewhat dissected by prognostic markers, many of which represent pathogenic mechanisms. Recently, the introduction of highly active targeted agents and maturing data on predictive markers may lead to more individualized therapeutic approaches. In this chapter, we review key prognostic markers, current and emerging therapy, and will attempt to outline a future “where the two may connect”.

Ibrutinib covalently binds Bruton tyrosine kinase (BTK) leading to sustained target inhibition. On-target effects include reductions in BCR and NF-κB signaling and inhibition of tumor proliferation and survival (Herman, Blood 2014). In randomized trials, ibrutinib induced superior responses and extended the survival of patients with chronic lymphocytic leukemia (CLL) in both the relapsed/refractory and frontline setting (Byrd, NEJM 2014; Burger, NEJM 2015). However, deep remissions are uncommon and most patients have readily detectable residual disease in the blood. To better understand the biology of persistent disease on ibrutinib, we performed RNA-sequencing on CD19+ selected tumor cells from 14 of 86 patients treated with single-agent ibrutinib (ClinicalTrials.gov NCT01500733); 8/14 (57%) had an unmutated IGVHsequence, 8/14 (57%) had del(17p), 6/14 (43%) were previously untreated and 4/14 (29%) had relapsed on ibrutinib at the time of analysis (range: 8-47 months). At best response, 3/14 (21%) achieved a CR, 10/14 (71%) a PR or PRL and 1/14 (7%) had SD. Three samples per patient were analyzed: T1 (baseline), T2 (day 28) and T3 (6 months), for a total of 42 samples. Principal component analysis (PCA) grouped samples by patient, as observed in a similar study (Herishanu, Blood 2011). After adjusting for patient-specific effects, PCA clearly revealed the effect of treatment, separating on-treatment samples (T2, T3) from baseline (T1). To identify differentially expressed (DE) genes, we selected transcripts that showed at least a 2-fold change between baseline and treatment samples (FDR To identify the biologic impact of treatment, we dissected the gene expression changes using pathway-specific gene signatures represented in the KEGG pathways with the molecular signatures database tool (MSigDB; Broad Institute, Boston, MA). Strikingly, no pathways were significantly enriched on treatment. In contrast, multiple gene signatures (n=46) were significantly turned down on ibrutinib (FDR50% reduction in signature scores, ranging from a 71% reduction for BCR to 54% for p53. The degree of inhibition of the signaling pathways was independent of biologic and clinical features, including IGVH mutation status, presence or absence of del(17p), and prior treatment status. In summary, ibrutinib induces widespread changes in gene expression in CLL cells in vivo reflecting strong inhibitory effects across multiple pathways that suggest a broader impact on tumor biology than expected from pure loss of BTK function. The surprising extent of changes across multiple diverse pathways could reflect inhibition of tumor-microenvironment crosstalk and consequent changes in the tumor milieu. This notion is consistent with the more profound inhibition observed with longer treatment duration. Equally notable, we found no distinct biologic signature(s) upregulated in response to ibrutinib which may indicate the absence of a cellular stress response and is consistent with the apparent resting state of the residual tumor and the observed slow attrition of cells over time. This research was supported by the Intramural Research Program of the NHLBI. Disclosures Farooqui: Merck: Employment. Wu:Neon Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Co-founder. Wiestner:Pharmacyclics: Research Funding; Acerta Pharma: Research Funding.

Cancer evolution poses a significant challenge to the ability of therapy to eliminate malignant cells. In chronic lymphocytic leukemia (CLL), clonal evolution was reported as a key feature of disease relapse after standard chemoimmunotherapy and after targeted therapy with the BTK inhibitor ibrutinib. To study potential selection of pre-existing sub-clones, we analyzed changes in clonal architecture at early time points in patients receiving ibrutinib therapy. We therefore performed in-depth longitudinal analysis of CLL samples collected from 59 patients treated with ibrutinib alone (n=44) or in combination with rituximab (n=15). Across this cohort, we performed whole-exome sequencing (WES) on a median of 3 samples per patient with median coverage of 105x (range 50-225x). Pretreatment samples were available for all patients, with post-treatment samples at 1 and 6 months for more than 85% of patients. Additional samples were available at 2, 3, or 12 months for 22 patients. The median age of the cohort was 65 (range 33-85) years. Thirty-one patients (53%) had del(17p) by FISH, 37 patients (63%) had IGHV unmutated CLL and 32 patients (54%) had relapsed or refractory disease. We observed a median of 56 (range 17-201) somatic silent and non-silent single nucleotide variants (SNVs) and insertions and deletions (Indels) per patient. The number of SNVs and indels did not differ between patients receiving ibrutinib alone or with rituximab (Wilcoxon, P=0.2). Thirteen patients had somatic mutations of BCR pathway genes prior to treatment, including one with CARD11-L251P, previously reported to confer resistance to ibrutinib in diffuse large B-cell lymphoma. Comparing pre-treatment and the latest samples within the first year (range 56-357 days, median 168), 26 of 59 (44%) CLLs showed significant changes in cancer cell fraction (CCF) over time, defined as a shift > 5% CCF of the clone with the greatest change (BH-FDR The likelihood for clonal shifts was not impacted by whether patients were treated with ibrutinib alone or with rituximab (45% vs. 40%, P=0.77), nor by exposure to prior therapy (47% vs. 41% with and without prior therapy, P=0.79). We observed trends towards higher likelihood of early clonal evolution in CLLs with unmutated IGHV status, with TP53 somatic aberrations, and those with a pre-treatment subclonal driver (subclones containing CHD2, BIRC3, SF3B1, MAP2K1, CARD11, XPO1, TP53, PTPN11, KRAS, HIST1H1E, ATM). Finally, patients with a decrease in ALC at 6 months compared to pre-treatment were more likely to exhibit significant CCF shifts (P=0.01). This last observation suggests that effective treatment activity and resulting therapeutic bottlenecks indeed contribute to the shift in clonal proportion within the first year of ibrutinib therapy. Relapse samples were available for 10 CLL cases, 6 of whom showed early clonal shifts during the first year of therapy. These included 2 cases of transformation to prolymphocytic leukemia (PLL), 2 with Richter transformation (RT) and 6 cases of progressive CLL disease. All relapse cases were clonally related to the pre-treatment samples. Mutations in BTK or PLCg2 were found in 5 cases by either WES or deep targeted sequencing of known resistance hotspots (both PLL, one RT, two progressive CLL). The remaining 5 cases showed marked clonal shifts suggestive of selection of other resistant genotypes. Altogether, targeted therapy with ibrutinib is associated with overall clonal stability in more than half of CLLs over the first year of treatment. Nevertheless, in many patients, clonal selection occurs even during the first months of therapy and may provide clues regarding the future evolutionary and clinical outcome of the disease. Disclosures Farooqui: Merck: Employment. Burger:Pharmacyclics, LLC, an AbbVie Company: Research Funding; Gilead: Research Funding; Janssen: Consultancy, Other: Travel, Accommodations, Expenses; Roche: Other: Travel, Accommodations, Expenses; Portola: Consultancy. Wiestner:Acerta Pharma: Research Funding; Pharmacyclics: Research Funding. Wu:Neon Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Co-founder.

A novel therapeutic approach in cancer attempting to stimulate host anti-tumor immunity involves blocking of immune checkpoints. Lymphocyte-activation gene 3 (LAG3) is an immune checkpoint receptor expressed on activated/exhausted T-cells. When engaged by MHC Class II (MHCII) molecules, LAG3 negatively regulates T-cell function thereby contributing to tumor escape. Intriguingly, a soluble LAG3 variant was reported to activate both immune and malignant MHCII-presenting cells. In this study, we examined the role of LAG3 in the pathogenesis of CLL and show that CLL cells express and secrete LAG3. High levels of surface and soluble LAG3 were associated with the unmutated immunoglobulin variable heavy chain CLL and a more aggressive disease. It was evident that recombinant soluble LAG3-Ig fusion protein activated CLL cells, induced increased levels of Bcl-2 and Mcl-1 and protected the cells from spontaneous apoptosis, utilizing a mechanism mediated through MHCII engagement. Our data suggest that autocrine secretion of soluble LAG3 from CLL cells and its subsequent interaction with MHCII on their surface, promotes CLL cell activation and anti-apoptotic effects. Moreover, CLL cells may impose immune exhaustion on their microenvironment by expressing MHCII, thus affecting the surrounding, LAG3-presenting immune cells. Hence, blocking LAG3-MHCII interactions is a potential therapeutic target in CLL. Disclosures Avivi: Tel Aviv Sourasky Medical center: Consultancy, Other: consultancy to :BMS Roche. Wiestner:Pharmacyclics: Research Funding; Acerta Pharma: Research Funding.

MLL (mixed-lineage leukemia)-fusion genes induce the development of leukemia through deregulation of normal MLL target genes, such as HOXA9 and MEIS1. Both HOXA9 and MEIS1 are required for MLL-fusion gene-induced leukemogenesis. Co-expression of HOXA9 and MEIS1 induces acute myeloid leukemia (AML) similar to that seen in mice in which MLL-fusion genes are over-expressed. p27(kip1) (p27 hereafter), a negative regulator of the cell cycle, has also been defined as an MLL target, the expression of which is up-regulated in MLL leukemic cells (LCs). To investigate whether p27 plays a role in the pathogenesis of MLL-leukemia, we examined the effects of p27 deletion (p27(-/-)) on MLL-AF9 (MA9)-induced murine AML development. HOXA9/MEIS1 (H/M)-induced, p27 wild-type (p27(+/+)) and p27(-/-) AML were studied in parallel as controls. We found that LCs from both MA9-AML and H/M-AML can be separated into three fractions, a CD117(-)CD11b(hi) differentiated fraction as well as CD117(+)CD11b(hi) and CD117(+)CD11b(lo), two less differentiated fractions. The CD117(+)CD11b(lo) fraction, comprising only 1-3% of total LCs, expresses higher levels of early hematopoietic progenitor markers but lower levels of mature myeloid cell markers compared to other populations of LCs. p27 is expressed and is required for maintaining the quiescent and drug-resistant states of the CD117(+)CD11b(lo) fraction of MA9-LCs but not of H/M-LCs. p27 deletion significantly compromises the leukemogenic capacity of CD117(+)CD11b(lo) MA9-LCs by reducing the frequency of leukemic stem cells (LSCs) but does not do so in H/M-LCs. In addition, we found that p27 is highly expressed and required for cell cycle arrest in the CD117(-)CD11b(hi) fraction in both types of LCs. Furthermore, we found that c-Myc expression is required for maintaining LCs in an undifferentiated state independently of proliferation. We concluded that p27 represses the proliferation of LCs, which is specifically required for maintaining the quiescent and drug-resistant states of a small subset of MA9-LSCs in collaboration with the differentiation blockage function of c-Myc.

Chronic lymphocytic leukemia (CLL) cells rely on signals from the microenvironment to for cell activation, proliferation and survival. Microarray analysis of CLL cells harvested from patient-matched lymph node and peripheral blood demonstrated an up-regulation of B-cell receptor (BCR) signaling in the tissue microenvironment (Herishanu, Blood 2011). Concurrent with increased BCR signaling a dynamic increase in NF-κB signaling was also noted in the activated CLL cells in the tissue microenvironment (Herishanu, Blood 2011). As NF-κB can be activated downstream of multiple signaling pathways, in addition to the BCR, we sought to determine the role of alternative pathways on NF-κB activation. In gene expression analysis of CD19+ selected CLL cells, we found that in addition to an increase in BCR signaling the Toll-like receptor (TLR) signaling pathway was also significantly up-regulated in CLL cells in the lymph node microenvironment compared to circulating CLL cells. Activation of TLR signaling can cooperate with BCR signaling to overcome anergy and promote the expansion of auto-reactive B-cells (Leadbetter, Nature 2002). In addition, TLR signaling can induce proliferation of CLL cells and upregulates co-stimulatory molecules that may make CLL cells more effective antigen presenting cells. The latter effect has been explored as a therapeutic strategy to enhance the efficacy of immunotherapy of CLL by rendering CLL cells more susceptible to attack by cytotoxic T-cells (Spaner and Masellis, Leukemia 2007). We hypothesize that inhibition of TLR signaling could contribute directly to anti-leukemic effects by inhibiting survival and proliferation pathways. To test the effect of TLR signaling in CLL, CLL PBMCs were stimulated in vitro with a CpG oligonucleotide, which activates TLR9, for 6 hours. Increased pIκBα expression along with significant increases in pSTAT3 (P=0.001), IL-10 production (P=0.008) and up-regulation of the cell surface activation markers CD54, CD69 and CD86 (P Recently, the BTK inhibitor ibrutinib was shown to be clinically active in Waldenstrom's macrogloublinemia (WM), a chronic B-cell malignancy not dissimilar to CLL that is characterized by mutations in the TLR adaptor MyD88 (Treon, NEJM 2012; Treon, NEJM 2015). We therefore investigated whether ibrutinib could inhibit TLR signaling in CLL cells. Indeed, even at doses as low as 100nM, ibrutinib significantly inhibited CpG dependent activation of NF-κB signaling (P In summary, TLR signaling is engaged in CLL cells in the lymph node microenvironment and upregulates proliferation and survival pathways. Ibrutinib appears to be as effective as a direct IRAK inhibitor in blocking TLR signaling in CLL cells, suggesting that treatment with ibrutinib is sufficient to inhibit both BCR and TLR signaling. This work was supported by the Intramural Research Program of National Heart Lung and Blood Institute of the National Institutes of Health. Disclosures Wiestner: Pharmacyclics: Research Funding.

Abstract 2980 Acute myeloid leukemia (AML) is developmentally similar to normal hematopoiesis and driven by a subpopulation of leukemia stem cells (LSCs). LSCs are defined by their ability to self-renew and differentiate into more mature leukemia cells. These properties are influenced by the stem cell niche in which they reside. A number of key cytokines within this microenvironment have been identified, including CXCL12 and stem cell factor (SCF), while others have altered the lineage fate of leukemia by in vitro manipulation of growth factor conditions. Activating mutations in fms-like tyrosine kinase receptor-3 (FLT3) have suggested a role for FLT3 signaling in LSC development and function, but little is known about the role of wild type FLT3 and its ligand (FLT3-L) in the LSC niche. In our study, we examined the impact of different hematopoietic growth factors, specifically FLT3-L, on leukemia stem cell properties in a mouse model of MLL-rearranged AML. Murine CD117+ hematopoietic progenitor cells retrovirally transduced with the MLL-AF9 oncogene were selected and cultured in the presence of either FLT3-L or a combination of interleukin-3 (IL-3), interleukin-6 (IL-6), and SCF. We found that FLT3-L selectively expanded a population of CD117+/CD11blo/CD16/32lo cells with a common myeloid progenitor (CMP)-like surface phenotype. This population was present at a frequency of only 1.3% in cells cultured in IL-3/IL-6/ SCF, with remaining cells being mostly CD117+/CD11bhi/CD16/32hi of a granulocyte-macrophage progenitor (GMP)-like surface phenotype. Wright-Giemsa staining showed that cells expanded in FLT3-L also appeared small and morphologically primitive compared to IL-3/IL-6/ SCF cells. When myeloid growth factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-3 were added, FLT3-L expanded cells differentiated into a heterogeneous population of CD117+/CD11bhi/CD16/32hi and CD117−/CD11bhi/CD16/32hi cells, and regained the typical myelomonocytic blast morphology seen in MLL-AF9 AML. Flow cytometry with Hoechst 33342 and Pyronin Y staining showed that the cells expanded in FLT3-L were more quiescent, with 47.1% in G0 phase compared to only 1.5% in IL-3/IL-6/SCF expanded cells. Consistent with this, FLT3-L expanded cells showed increased resistance to daunorubicin in colony forming assays. Upon transplantation into lethally irradiated mice, cells expanded in FLT3-L produced leukemia at a remarkably higher frequency (8.7 log-fold using limiting dilution analysis) than cells expanded in IL-3/IL-6/SCF. To explain the differences observed, immunoblotting revealed selective upregulation of the canonical Wnt/β-catenin pathway in FLT3-L expanded cells. In AML, Wnt signaling has been shown to mediate leukemogenesis and LSC self-renewal and homing, and thus is a plausible mechanism for regulating the leukemia stem cell properties enhanced by FLT3-L. Our findings suggest a key role for FLT3 signaling in the development and function of LSCs in MLL-AF9 AML through upregulation of the Wnt/β-catenin pathway. Disclosures: No relevant conflicts of interest to declare.