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Background: Clonal hematopoiesis of indeterminate potential (CHIP) is a clinical entity of aging humans that is characterized by cancer-associated mutations in white blood cells, without evidence of overt neoplasia. CHIP has been associated with an increased risk of hematologic cancers, cardiovascular disease, and all-cause mortality. We hypothesized that somatic mutations in specific genes associated with human CHIP would be detectable in the blood of aged dogs not known to have hematologic disorders. Methods: DNA from paired germline and whole blood samples from 93 geriatric canine patients affected by solid cancer were subjected to targeted next generation sequencing. Impact of the variants was predicted using Polymorphism Phenotyping version 2 software (PolyPhen-2, Harvard). Clinical and demographic data were extracted from medical records. Results: Somatic variants were detected in peripheral blood of four (4.3%) female dogs aged 12-15 years. Affected genes were ASXL1, KIT, SF3B1, TET2, RUNX1, and PPM1D. The variant in PPM1D was a nonsense mutation, while the other five variants were single nucleotide non-synonymous variants in protein coding regions of the genes. Following analysis by PolyPhen-2, the single nucleotide variants in KIT and SF3B1 were predicted to be benign, while the variants in ASXL1, TET2, and RUNX1 were predicted to be damaging. Conclusion: These results support the presence of variants in CHIP-associated genes in geriatric canids at a frequency similar to that observed in people, and the dog represents the first species in which the genetic lesion of CHIP has been documented. Further investigations are needed to confirm the association of this genetic lesion with clinical outcomes. Citation Format: Kimberley N. Sebastian, Hatice Gülçin Özer, Cory Howard, Laura Chadsey, Arletta Lozanski, Tzyy-Jye Doong, Gerard Lozanski, Wenjuan Ma, William C. Kisseberth, John C. Byrd, Bonnie K. Harrington. Clonal hematopoiesis of indeterminate potential in the companion dog [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1620.

7540 Background: Targeted agent combinations for chronic lymphocytic leukemia (CLL) have resulted in safe regimens with improved progression-free survival (PFS) compared to chemoimmunotherapy. The optimal regimen and long-term outcomes remain unknown. We conducted a phase 2 study of obinutuzumab (OBIN), ibrutinib (IBR), and venetoclax (VEN) in relapsed/refractory (RR) and treatment-naïve (TN) CLL patients (pts) and report extended follow up on two previously reported cohorts (RR, TN 1) and a new cohort of identically treated TN pts (TN 2). Methods: Pts with TN or RR (≥1 prior treatment) CLL requiring treatment were eligible. Treatment was given for 14 28-day cycles (C). OBIN was started C1, IBR C2, and VEN C3 with standard dose escalation. Response was assessed 2 months (mos) after C14 (EOT) by iwCLL 2008 criteria. Minimal residual disease (MRD) was measured by 10-color flow cytometry with a cutoff of < 1x10-4. The primary endpoint was complete remission (CR) with undetectable MRD (uMRD) in blood and bone marrow at EOT. A sample size of 25 pts per cohort achieves 90% power to test the null hypothesis of 10% against 30% with 1-sided α of 10%. The Kaplan-Meier method was used to estimate PFS and overall survival (OS). Results: 75 pts were treated in 3 cohorts (Table). Data are as of 1/3/2022. In the TN 2 cohort, the overall response rate (ORR) at EOT was 96% (95% CI: 80-100) and CR with uMRD was 20% (95% CI: 7-41). The ORR for TN 1 and TN 2 combined was 90% (95% CI: 78-97) and CR with uMRD was 24% (95% CI: 13-38). Median follow up was 56 (0-65), 57 (7-63), and 30 (24-35) mos, respectively, for RR, TN 1, and TN 2. There were 3 deaths (RR = 1, TN 1 = 1, TN 2 = 1) and 6 disease progressions (RR = 4, TN 1 = 2, TN 2 = 0). Estimated 48-month PFS for TN 1 and RR cohorts were both 96% (95% CI: 72-99) and OS was 85% (95% CI: 60-95) and 100%, respectively. The estimated 24-month PFS and OS for TN 2 were both 96% (95% CI: 75-99). The most frequent adverse events were neutropenia (95%, 73% grade ≥3), leukopenia (95%, 45% grade ≥3), lymphopenia (93%, 40% grade ≥3), and thrombocytopenia (91%, 28% grade ≥3). The only grade ≥3 non-hematologic toxicity occurring at ≥20% was hypertension (85%, 39% grade ≥3). Atrial fibrillation occurred in 11% of pts. Conclusions: At extended follow-up, remissions remain durable after fixed duration OBIN, IBR, and VEN. The efficacy and acceptable safety justify further study and are being compared to IBR and OBIN in 2 phase 3 US cooperative group trials. Clinical trial information: NCT02427451. [Table: see text]

The existence of "leukemia-initiating cells" (LICs) in chronic lymphocytic leukemia (CLL) remains controversial due to the difficulty in isolating and identifying the tumor-initiating cells. Here, we demonstrate a microchannel electroporation (MEP) microarray that injects RNA-detecting probes into single live cells, allowing the imaging and characterization of heterogeneous LICs by intracellular RNA expression. Using limited-cell FACS sequencing (LC-FACSeq), we can detect and monitor rare live LICs during leukemogenesis and characterize their differential drug sensitivity. Disease-associated mutation accumulation in developing B lymphoid but not myeloid lineage in CLL patient hematopoietic stem cells (CLL-HSCs), and development of independent clonal CLL-like cells in murine patient-derived xenograft models, suggests the existence of CLL LICs. Furthermore, we identify differential protein ubiquitination and unfolding response signatures in GATA2

Hematopoiesis is hierarchical, and it has been postulated that acute myeloid leukemia (AML) is organized similarly with leukemia stem cells (LSCs) residing at the apex. Limited cells acquired by fluorescence activated cell sorting in tandem with targeted amplicon-based sequencing (LC-FACSeq) enables identification of mutations in small subpopulations of cells, such as LSCs. Leveraging this, we studied clonal compositions of immunophenotypically-defined compartments in AML through genomic and functional analyses at diagnosis, remission and relapse in 88 AML patients. Mutations involving DNA methylation pathways, transcription factors and spliceosomal machinery did not differ across compartments, while signaling pathway mutations were less frequent in putative LSCs. We also provide insights into TP53-mutated AML by demonstrating stepwise acquisition of mutations beginning from the preleukemic hematopoietic stem cell stage. In 10 analyzed cases, acquisition of additional mutations and del(17p) led to genetic and functional heterogeneity within the LSC pool with subclones harboring varying degrees of clonogenic potential. Finally, we use LC-FACSeq to track clonal evolution in serial samples, which can also be a powerful tool to direct targeted therapy against measurable residual disease. Therefore, studying clinically significant small subpopulations of cells can improve our understanding of AML biology and offers advantages over bulk sequencing to monitor the evolution of disease.

Detecting, characterizing, and monitoring rare populations of cells can increase testing sensitivity, give insight into disease mechanism, and inform clinical decision making. One area that can benefit from increased resolution is management of cancers in clinical remission but with measurable residual disease (MRD) by multicolor FACS. Detecting and monitoring genomic clonal resistance to treatment in the setting of MRD is technically difficult and resource intensive due to the limited amounts of disease cells. Here, we describe limited-cell FACS sequencing (LC-FACSeq), a reproducible, highly sensitive method of characterizing clonal evolution in rare cells relevant to different types of acute and chronic leukemias. We demonstrate the utility of LC-FACSeq for broad multigene gene panels and its application for monitoring sequential acquisition of mutations conferring therapy resistance and clonal evolution in long-term ibrutinib treatment of patients with chronic lymphocytic leukemia. This technique is generalizable for monitoring of other blood and marrow infiltrating cancers.

Background Venous thromboembolism (VTE) is the third most common cardiovascular disease with an incidence over 1% in the elderly. About 50% of VTE is unprovoked, which is associated with higher rates of recurrent VTE and complications such as post-thrombotic syndrome. Clonal hematopoiesis (CH) refers to recurrent somatic mutations associated with hematologic malignancies that are present in otherwise healthy individuals. CH is more common with increased age, occurring in more than 10% of people over the age of 70 and has been associated with an increased risk of coronary artery disease. The rates of CH in patients with VTE or complications arising from this is not known. Methods Adult patients (age ≥ 18) with VTE were enrolled at the Ohio State University Thrombosis Clinic between 2017 and 2020. Clinical data was obtained at the time of routine clinic follow-up or ad hoc, and a research blood sample was collected with routine blood work. DNA extracted from peripheral blood mononuclear cells were analyzed for the presence of CH via error corrected next generation sequencing (NGS) using the Ion Gene Studio S5 System. A custom AmpliSeq panel IAH150087 was designed to detect CH, which included whole gene sequencing and hotspots in 25 common genes associated with myeloid malignancies, including DNMT3A, TET2, ASXL1, among others, with a sequencing depth of up to 50,000X. Data was collected and analyzed using Ion Torrent S5/GS-S5 Instrument with Torrent Suite 5.12.0 version. Final analysis of sequence data was performed using combination of software: Variant Caller v.5.12.10-1, IGV5.01 (0) and Ion Reporter v.5.12.3.0. The hg19 reference sequence was used to assess for deviation. CH was defined as a variable allele frequency ≥ 2%. Two sample t-test and chi-square test were used to compare patients with and without CH for continuous and categorical variables, respectively. Results A total of 167 patients are included the study. Median age is 60.3 years and 55% of patients are female. 80% of patients are white. The majority of patients have unprovoked VTE (81%); 23 patients (13.8%) have VTE associated with cancer. 84% of patients were treated with a direct oral anticoagulant. Over the median follow-up duration of 400 days, 7 patients had recurrent VTE and 19 patients had a total of 21 bleeding events (7 major bleeding and 14 clinically relevant non-major bleeding [CRNMB] by ISTH criteria). Overall, 22 patients (13.2%) have detectable CH. Patients with CH are significantly older than those without CH (mean age 70 vs 59, p 30 distinct CH mutations are noted in these 22 patients. Most patients have a single gene mutation (77%). Gene mutations in DNMT3A are the most common (46%), followed by TET2 (13%), and PPM1D (10%) (see figure). Most mutations are missense (57%), followed by nonsense (30%) and insertion/deletion mutations (13%). One patient has a JAK2 V617F mutation without known history of myeloproliferative disease. 8 CH patients with serial samples collected over a median duration of 136 days (range 91 - 490 days) showed no significant change in CH clone size over time. Patients with CH have no differences in rates of recurrent VTE or arterial thrombosis compared to those without CH. Patients with CH have a trend towards increased rate of combined major bleeding and CRNMB events (23% vs 10%, p=0.14). Patients with CH also had a trend towards increased mean corpuscular volume (MCV) (mean 93.5 vs 90.7, p=0.13), although no other cytopenias or differences in complete blood count parameters were noted. Conclusion The CH rate in patients with VTE was not found to be much higher than what might be expected by age alone. We found no change in the CH rate after excluding cancer patients. A larger VTE population with age-matched controls will better determine the CH rate in patients with VTE. CH can be a consideration in designing future clinical VTE studies that assess bleeding risk. Figure 1 Figure 1. Disclosures Byrd: Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Newave: Membership on an entity's Board of Directors or advisory committees; Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria. Wang: Servier: Membership on an entity's Board of Directors or advisory committees; Leo Pharma: Research Funding.

Purpose Therapeutic targeting of Bruton tyrosine kinase (BTK) with ibrutinib in chronic lymphocytic leukemia has led to a paradigm shift in therapy, and relapse has been uncommon with current follow-up. Acquired mutations in BTK and PLCG2 can cause relapse, but data regarding the prevalence and natural history of these mutations are limited. Patients and Methods Patients accrued to four sequential studies of ibrutinib were included in these analyses. Deep sequencing for BTK and PLCG2 was performed retrospectively on patients who experienced relapse and prospectively on a screening population. Results With a median follow-up time of 3.4 years, the estimated cumulative incidence of progression at 4 years is 19% (95% CI, 14% to 24%). Baseline karyotypic complexity, presence of del(17)(p13.1), and age less than 65 years were risk factors for progression. Among patients who experienced relapse, acquired mutations of BTK or PLCG2 were found in 85% (95% CI, 71% to 94%), and these mutations were detected an estimated median of 9.3 months (95% CI, 7.6 to 11.7 months) before relapse. Of a group of 112 patients examined prospectively, eight patients have experienced relapse, and all of these patients had acquired resistance mutations before relapse. A resistance mutation was detected in an additional eight patients who have not yet met criteria for clinical relapse. Conclusion Relapse of chronic lymphocytic leukemia after ibrutinib is an issue of increasing clinical significance. We show that mutations in BTK and PLCG2 appear early and have the potential to be used as a biomarker for future relapse, suggesting an opportunity for intervention.

Normal hematopoiesis is organized in a hierarchical manner and it has been hypothesized that acute myeloid leukemia (AML) is organized in a similar way with leukemia-initiating cells (LIC) at the top of the hierarchy, giving rise to more differentiated blasts to sustain AML. Therefore, elimination of LIC population is critical for cure. This may be accomplished via novel molecular targeted therapies. The mutational composition of LIC and non-LIC compartments in AML has not been fully elucidated and could provide new insights into biology and treatment. We investigated the distribution and variant allelic frequencies (VAFs) of recurrent gene mutations within these compartments in newly diagnosed CD34+ AML patients (pts). We studied a total of 88 pts. CD34- AML cases, defined as Clinical characteristics are summarized in Table 1. The median frequency of the LIC population was 0.5% (range, 0.01% - 69%). The prevalence of high LIC frequency (≥0.5%) was significantly higher in pts with adverse risk (AR) AML, as compared to intermediate (IR) and favorable risk groups (94% vs 34% vs 16%, respectively, p We re-validated the commonly used LIC markers with ELDA of primary AML cells. In one IR sample, stem cell frequencies in sorted CD34+CD38-, CD34+CD38+ and CD34- compartments were 1:3, 1:15 and 1:16, respectively (p Relapse samples obtained from 6 pts were analyzed and compared with diagnosis. In all cases, we could identify LIC clones that persisted after chemotherapy and led to relapse (see example in Figure). Similarly, 3 pts who were primary refractory showed persistence of LIC clones that were resistant to treatment. On the contrary, 6 pts in whom LIC clones could be eradicated with treatment did not experience disease recurrence. LICs exist at a very low frequency in pre-treatment AML samples. The mutational composition of LIC-enriched compartment shows differences from blasts constituting the bulk of leukemia, which is consistent with the sequence of mutations observed during the evolution of AML. LC-FACS-seq is an ultrasensitive method to detect mutations in a tiny population of residual LICs in pts at remission. Therapies targeting mutations that are concentrated in LICs may re-shape the clonal hierarchy and impact on disease course. Disclosures Behbehani: Fluidigm corporation: Other: Travel funding. Byrd:Ohio State University: Patents & Royalties: OSU-2S; Genentech: Research Funding; Genentech: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; BeiGene: Research Funding; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Ohio State University: Patents & Royalties: OSU-2S; Acerta: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau. Lozanski:Boehringer Ingelheim: Research Funding; Beckman Coulter: Research Funding; Stemline Therapeutics Inc.: Research Funding; Genentec: Research Funding.

Introduction Acalabrutinib (A) is a selective, second-generation irreversible Bruton's Tyrosine Kinase inhibitor (BTKi) that has shown outstanding clinical activity in chronic lymphocytic leukemia (CLL) and other hematologic malignancies. Acquired resistance to the first-generation BTKi, ibrutinib (I), is mediated most commonly by C481S mutations in BTK, which decrease binding affinity and change binding from irreversible to reversible. The mechanism of resistance to A has not been investigated. Patients and Methods All patients (pts) treated at The Ohio State University and enrolled on an IRB approved phase 1b/2 study in CLL were included in this analysis. Beginning 12 months (mos) after the initiation of A, pts underwent deep sequencing every 3-6 cycles using a digital droplet PCR assay for BTK C481S or Ion Torrent Sequencing for any BTK or PLCG2 mutations. At relapse, samples from each pt underwent full BTK and PLCG2 sequencing. Correlation between baseline characteristics and relapse was assessed using proportional sub-distribution hazards model, pts who were still on treatment were censored at last follow-up. Results 105 pts were included in this analysis. 38 (36%) were treatment-naïve (TN), 50 (48%) were relapsed/refractory (RR), and 17 (16%) were previously intolerant of I (intol). The median age was 62 (range 33-84) and median number of prior therapies was 1 (range 0-11). The pts were generally high-risk, with 66% having unmutated IGHV, 24% with del(11)(q22.3), 15% with del(17)(p13.1), and 28% with complex karyotype (≥ 3 abnormalities). With a median follow-up of 47.5 mos (range 37.7-58.5) 30% of pts (n=31) have discontinued therapy, 17 for progression of disease (CLL in 16 pts, Richter's transformation in 1), and 14 for other reasons. Cumulative incidence rate (CIR) of progression varied significantly by cohort. At 12 mos, CIR of progression ranged from 0% in both the TN and RR cohorts to 11.8% (95% CI 1.8-32%) in those who were intol. By 36 mos, CIR of progression was 5.3% (95% CI 0.9-15.7%) in TN cohort, 6% (95% CI 1.5-15%) in the RR cohort, and 35.3% (95% CI 13.7-58%) in the intol cohort (Figure 1). In univariable analysis, higher risk of progression was associated with del(17)(p13.1) on baseline FISH (p All 16 pts with CLL relapse had a sample evaluated for BTK C481S at relapse, and 14 had samples evaluated for full BTK and PLCG2 mutations using Ion Torrent deep sequencing. BTK C481 mutations were found in 11/16 (69%; C481S in 10, C481R and C481Y in 1). One pt with a BTK C481S mutation also had a BTK T474I gatekeeper mutation, and one had co-existing C481R mutation. Two pts with BTK C481S mutations had co-existing PLCG2 mutations previously associated with I resistance at 103/105 pts were screened every 3-6 cycles for BTK mutations. 22 pts have had mutations detected at a median of 31.6 mos from A initiation. TN and RR pts had median time to mutation of 38.8 and 32.9 mo, respectively. For intol pts median time to mutation detection was 24.8 mos from start of A, 33.8 mos from start of any BTKi. Median time from BTK mutation detection to clinical relapse is 12 mos (95% CI: 4.8-not reached), with no significant difference among cohorts in time from mutation to disease progression. Conclusions Here we show for the first time that CLL relapse on A is mediated predominantly by mutations in BTK similar to I. While not unexpected, this is significant as resistant patterns could be different given the more selective nature of A as well as potentially higher BTK occupancy over time due to twice daily dosing. Monitoring for BTK resistance offers the opportunity to intervene clinically before symptomatic disease. Disclosures Woyach: Janssen: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; AbbVie: Research Funding; Karyopharm: Research Funding; Loxo: Research Funding; Morphosys: Research Funding; Verastem: Research Funding. Rogers:AbbVie: Research Funding; Acerta Pharma: Consultancy; Genentech: Research Funding; Janssen: Research Funding. Bhat:Janssen: Consultancy; Pharmacyclics: Consultancy. Grever:Acerta Pharma, LLC: Membership on an entity's Board of Directors or advisory committees. Lozanski:Boehringer Ingelheim: Research Funding; Beckman Coulter: Research Funding; Stemline Therapeutics Inc.: Research Funding; Genentec: Research Funding. Byrd:Ohio State University: Patents & Royalties: OSU-2S; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Genentech: Research Funding; Genentech: Research Funding; Acerta: Research Funding; Acerta: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; BeiGene: Research Funding.

Introduction: Targeted irreversible Bruton's Tyrosine Kinase (BTK) inhibitors ibrutinib and acalabrutinib, have revolutionized treatment for chronic lymphocytic leukemia (CLL). While BTK inhibition (BTKi) achieves durable responses in 90% of patients, only 10% achieve minimal residual disease (MRD) negative status. MRD positive patients have persistent residual CD5+CD19+ tumor B cells at approximately 1-5 /mm3 in peripheral blood. These cells may represent a subpopulation of B-cell lymphocytosis pre-malignant cells or may carry a BTK C481, PLCG2, or other CLL mutation that is ultimately responsible for disease relapse. Alternatively, MRD could be derived from the original clones present at initial disease presentation that are not dependent on BTK signaling. Readily available clinical DNA sequencing and MRD monitoring techniques lack the ability to characterize these cells adequately due to their rarity in peripheral blood. To address this problem, we developed a novel method for limited-cells using fluorescence activated cell sorting in tandem with next generation sequencing (LC-FACSeq) to characterize rare tumor subpopulations in the blood and bone marrow. LC-FACSeq may be useful not only for CLL but also other leukemias. Methods: LC-FACSeq uses fluorescent activated cell sorting (FACS) to isolate pure populations of rare tumor cells after which targeted deep sequencing is performed to monitor CLL-related mutations in NOTCH1, SF3B1, and TP53, as well as genes associated with BTKi relapse and resistance: BTK and PLCG2. For validation of this method, we generated libraries from DNA isolated from FACS isolated bulk (n >15000) versus n= 50, 100, 300, or 500 CD5+/CD19+ cells from CLL patients (n=5). Results: All samples analyzed had an average read depth of 1212 (SEM=56) per gene and an average coverage uniformity of 88.24% (SEM=.01). We show that showed that 300-cell LC-FACSeq libraries demonstrated comparable variant calling and minimal noise to standard libraries generated from purified DNA from bulk cells. Using samples from patients with previously identified BTK C481S mutations, we found that both sensitivity and specificity of LC-FACSeq for BTK C481S was 100%. Furthermore, LC-FACSeq reliably amplified BTK C481S signals from subclones as small as 6 in 300 total cells (2%) when mutated tumor cells were serially diluted into BTK wild type tumor cells. In using LC-FACSeq to retrospectively analyze four independent patients who developed Ibrutinib resistance, we found that we could see the emergence of small BTKi resistant subclones as early as 10 months before clinical detection. We next extended LC-FACSeq to examine the clonal architecture of long-term (> 12 months) ibrutinib-treated MRD positive patients. Median treatment time was 5 years. BTK C481S mutations were observed in the latest available on-treatment samples of only one patient. Using LC-FACSeq we observed canonical CLL-associated clonal mutations similar to those observed in previous studies. Of the 14 MRD positive patients, 7 showed subclonal changes in TP53, NOTCH1, POT1, SF3B1, and MYD88 over the course of ibrutinib treatment although we found no correlation or consensus in these clonal shifts. Conclusion: LC-FACSeq is a highly sensitive method of characterizing clonal evolution in rare cells. Our data shows that LC-FACSeq is useful for monitoring sequential acquisition of mutations conferring therapy resistance and clonal evolution in long-term ibrutinib treated chronic lymphocytic leukemia (CLL) patients. We also observe that in most cases, MRD clones after long-term ibrutinib treatment are genetically similar to disease clones from pretreatment baseline. Compared to current MRD monitoring strategies, the main advantages of LC-FACSeq are that 1) variants can be confidently called from rare sorted tumor populations and subpopulations, 2) library generation can be completed in less than a day in a diagnostic laboratory compared to the labor-intensive protocols of traditional NGS approaches, and 3) amplicon panels can be easily customized for application to other types of leukemia and lymphoma. (EH is supported by the Graduate Pelotonia Fellowship and the NIH F30) Disclosures Bhat: Janssen: Consultancy; Pharmacyclics: Consultancy. Rogers:Janssen: Research Funding; AbbVie: Research Funding; Genentech: Research Funding; Acerta Pharma: Consultancy. Woyach:AbbVie: Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; Karyopharm: Research Funding; Loxo: Research Funding; Morphosys: Research Funding; Verastem: Research Funding. Lozanski:Beckman Coulter: Research Funding; Stemline Therapeutics Inc.: Research Funding; Genentec: Research Funding; Boehringer Ingelheim: Research Funding. Muthusamy:Ohio State University: Patents & Royalties: OSU-2S. Byrd:Novartis: Other: Travel Expenses, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; BeiGene: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Genentech: Research Funding; Acerta: Research Funding.

Ibrutinib has significantly improved the outcome of patients with relapsed chronic lymphocytic leukemia (CLL). Recent reports attribute ibrutinib resistance to acquired mutations in Bruton agammaglobulinemia tyrosine kinase (BTK), the target of ibrutinib, as well as the immediate downstream effector phospholipase C, γ2 (PLCG2). Although the C481S mutation found in BTK has been shown to disable ibrutinib's capacity to irreversibly bind this primary target, the detailed mechanisms of mutations in PLCG2 have yet to be established. Herein, we characterize the enhanced signaling competence, BTK independence, and surface immunoglobulin dependence of the PLCG2 mutation at R665W, which has been documented in ibrutinib-resistant CLL. Our data demonstrate that this missense alteration elicits BTK-independent activation after B-cell receptor engagement, implying the formation of a novel BTK-bypass pathway. Consistent with previous results, PLCG2(R665W) confers hypermorphic induction of downstream signaling events. Our studies reveal that proximal kinases SYK and LYN are critical for the activation of mutant PLCG2 and that therapeutics targeting SYK and LYN can combat molecular resistance in cell line models and primary CLL cells from ibrutinib-resistant patients. Altogether, our results engender a molecular understanding of the identified aberration at PLCG2 and explore its functional dependency on BTK, SYK, and LYN, suggesting alternative strategies to combat acquired ibrutinib resistance.

Fludarabine (F) and cyclophosphamide (C) remain backbones of up-front chemotherapy regimens for chronic lymphocytic leukemia (CLL). We report long-term follow-up of a randomized F versus FC trial in untreated, symptomatic CLL (NCT00003764, clinicaltrials.gov). With median follow-up of 88 months, estimated median progression-free survival (PFS) was 19.3 versus 48.1 months for F (N=109) and FC (N=118) respectively (p

Significance B-cell chronic lymphocytic leukemia (CLL) is the most common adult leukemia. We previously found that dysregulation of the T-cell leukemia/lymphoma 1 ( TCL1 ) oncogene is a critical contributing event in the pathogenesis of this disease. In this study we investigated molecular causes of TCL1 overexpression in CLL. We identified miR-3676 as a powerful regulator of TCL1 expression. We found that miR-3676 is down-regulated on all groups of CLLs and mutated in 1% of CLLs. Interestingly, miR-3676 is located at 17p13, only 500-kb centromeric of tumor protein p53 ( Tp53 ), and is codeleted with Tp53 in 17p-deleted CLL. Loss of miR-3676 causes high levels of TCL1 expression contributing to CLL progression. Thus, this study uncovers a major mechanism in the pathogenesis of CLL.

De novo CD5+ diffuse large B-cell lymphomas (DLBCL) are a distinct subgroup of DLBCL with poor prognosis. However the role of rituximab-containing therapy and salvage stem cell transplantation in this patients' population remain to be defined. We retrospectively reviewed clinical features and outcomes of 102 patients with de novo CD5+ DLBCL treated with rituximab-containing therapy at nine different institutions. By Hans' criteria, 64 patients had activated B-cell (ABC) subtype, 24 germinal center B-cell (GCB) subtype, and 14 were not evaluated. No patients had a myc translocation. Eighty-three patients were treated with rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone (R-CHOP), 7 with rituximab, etoposide, cyclophosphamide, doxorubicin, vincristine, prednisone (R-EPOCH), and 6 with R-CHOP with methotrexate, 3 g/m(2) . The overall response rate to front-line therapy was 85%. The 3-year progression free survival (PFS) and overall survival (OS) for all patients were 40 and 65%, respectively. The 3-year PFS for ABC- and GCB-subtypes was 34 and 45%, respectively. The 3-year OS for ABC- and GCB-subtypes was 62 and 67%, respectively. The median time to second treatment failure was 3 months and 1 month for ABC- and GCB-subtypes, respectively. Twenty of 28 (71%) transplanted patients with autologous, allogeneic, or both, relapsed. This study confirms the poor prognosis of de novo CD5+ DLBCL in a large multi-center cohort despite initial rituximab-containing chemotherapy and suggests that stem cell transplantation fails to salvage the majority of these patients. Approaches to prevent recurrence and/or novel therapies for relapsed disease are needed for this subgroup of DLBCL patients.

The impact of mutation of the ATM (ataxia telangiectasia mutated) gene in chronic lymphocytic leukemia (CLL) treatment outcome has not been examined. We studied ATM mutations in 73 patients treated with fludarabine and rituximab. ATM gene mutation analysis was performed using temperature gradient capillary electrophoresis. The impact of detected variants on overall survival (OS) and progression-free survival (PFS) was tested with proportional hazards models. None of the 73 patients demonstrated truncating ATM mutations; 17 (23%, 95% confidence interval 14 – 35%) had non-silent variants (ATM-NSVs), including 13 known ATM polymorphisms and four missense variants. ATM-NSVs were not significantly associated with any baseline characteristics including immunoglobulin heavy chain variable gene (IGVH) status. In multivariable models, no significant differences in complete response (p = 0.70), PFS (p = 0.59) or OS (p = 0.13) were observed. Our data indicate that truncating ATM mutations are rare in patients with CLL. Furthermore, in this dataset, these non-silent variants had limited impact on PFS and OS.

Outcome of patients with relapsed or refractory chronic lymphocytic leukemia treated with flavopiridol: impact of genetic features

Ibrutinib is an irreversible inhibitor of Bruton's tyrosine kinase (BTK) and is effective in chronic lymphocytic leukemia (CLL). Resistance to irreversible kinase inhibitors and resistance associated with BTK inhibition have not been characterized. Although only a small proportion of patients have had a relapse during ibrutinib therapy, an understanding of resistance mechanisms is important. We evaluated patients with relapsed disease to identify mutations that may mediate ibrutinib resistance.We performed whole-exome sequencing at baseline and the time of relapse on samples from six patients with acquired resistance to ibrutinib therapy. We then performed functional analysis of identified mutations. In addition, we performed Ion Torrent sequencing for identified resistance mutations on samples from nine patients with prolonged lymphocytosis.We identified a cysteine-to-serine mutation in BTK at the binding site of ibrutinib in five patients and identified three distinct mutations in PLCγ2 in two patients. Functional analysis showed that the C481S mutation of BTK results in a protein that is only reversibly inhibited by ibrutinib. The R665W and L845F mutations in PLCγ2 are both potentially gain-of-function mutations that lead to autonomous B-cell-receptor activity. These mutations were not found in any of the patients with prolonged lymphocytosis who were taking ibrutinib.Resistance to the irreversible BTK inhibitor ibrutinib often involves mutation of a cysteine residue where ibrutinib binding occurs. This finding, combined with two additional mutations in PLCγ2 that are immediately downstream of BTK, underscores the importance of the B-cell-receptor pathway in the mechanism of action of ibrutinib in CLL. (Funded by the National Cancer Institute and others.).

The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib has outstanding activity in patients with chronic lymphocytic leukemia. Most patients experience lymphocytosis, representing lymphocyte egress from nodal compartments. This resolves within 8 months in the majority of patients, but a subgroup has lymphocytosis lasting >12 months. Here we report a detailed characterization of patients with persistent lymphocytosis during ibrutinib therapy. Signaling evaluation showed that while BTK is inhibited, downstream mediators of B-cell receptor (BCR) signaling are activated in persistent lymphocytes. These cells cannot be stimulated through the BCR and do not show evidence of target gene activation. Flow cytometry for κ and λ expression, IGHV sequencing, Zap-70 methylation, and targeted gene sequencing in these patients are identical at baseline and later time points, suggesting that persistent lymphocytes do not represent clonal evolution. In vitro treatment with targeted kinase inhibitors shows that they are not addicted to a single survival pathway. Finally, progression-free survival is not inferior for patients with prolonged lymphocytosis vs those with traditional responses. Thus, prolonged lymphocytosis is common following ibrutinib treatment, likely represents the persistence of a quiescent clone, and does not predict a subgroup of patients likely to relapse early.

The Bruton's Tyrosine Kinase (BTK) inhibitor ibrutinib is very effective in chronic lymphocytic leukemia (CLL). Resistance in CLL is at least in part mediated by acquired mutations in BTK or down-stream PLCG2. Here we describe the largest institutional cohort of CLL patients treated with ibrutinib, focusing on risk factors for relapse, prevalence of known resistance mutations, and development of a monitoring strategy to identify patients at risk for relapse. All 308 patients from The Ohio State University Comprehensive Cancer Center participating in 4 clinical trials of ibrutinib were included. Deep sequencing for BTK and PLCG2 was performed using Ion Torrent Personal Genome Machine and covered coding regions of both genes. Preclinical experiments used XLA cell lines (Coriell Institute) infected with lentiviral constructs containing empty vector, wild type BTK, or C481S BTK. With a median follow-up of 40.5 months (range 4-71 months), 136 patients remain on study, 14 have received transplant or therapy elsewhere, and 158 have discontinued. 83 patients experienced disease progression, classified as Richter's or PLL transformation (n=28) and progressive CLL (n=55). Using multivariable models, baseline risk factors for ibrutinib discontinuation due to transformation include complex karyotype (p 46 patients with progressive CLL had samples at relapse available for deep sequencing. Of these, 39 had mutations in BTK and/or PLCG2 (84.8%; 95% CI 71.1-93.7). Distribution of mutation included patients with BTK C481 mutation only (n=30), mutation in PLCG2 only (n=3) and both BTK/PLCG2 genes (n=6). Given the poor prognosis at relapse and presence of acquired mutations in the majority of progressive CLL patients, we were interested to evaluate whether the emergence of small clones of mutated cells could be used as a biomarker to predict relapse. For 15 patients with BTK or PLCG2 mutations, serial samples were available prior to relapse, and a clone of resistant cells could first be detected a median of 9.3 months prior to clinical relapse (95% CI: 7.6-11.7). Based on the finding that patients who relapsed on ibrutinib often had rapid progression and poor outcomes, we initiated a clinical-grade monitoring strategy in our institutional CLIA-certified molecular lab starting in November 2014. Mutational analysis of the entire coding regions of BTK and PLCG2 was performed on a cohort of 112 patients every 3 months. To date, 8 patients have clinically relapsed and all 8 had BTK C481S mutations with expansion of the clone prior to relapse. BTK C481S mutations of over 1% allelic frequency were detected in an additional 8 patients. All patients have had expansion of the resistant clones and all but one, who discontinued therapy without clinical relapse, have had increasing circulating CLL cells in the peripheral blood. Four of these 7 patients have also had increasing lymph node size on CT scan. In 11 patients who relapsed with BTK C481S, samples were available following discontinuation. In 5 patients treated subsequently with venetoclax, allelic fractions of C481S decreased dramatically or were eliminated, but in 6 patients treated with other agents, the mutant clone persisted. To investigate whether this was due to differences in biology between wild type and C481S BTK, we created XLA cell lines without BTK protein expression stably expressing BTK, or C481S BTK. C481S BTK showed enhanced BCR signaling as evidenced by pERK and cMYC expression, and enhanced migration compared to wild type BTK (p=0.04). This demonstrates for the first time that this acquired resistance mutation fundamentally alters the tumor cell biology. These data confirm our initial findings that relapse on ibrutinib is primarily mediated through mutations in BTK and PLCG2 and suggest that the presence of these mutations may have utility as an early biomarker to predict clinical relapse. Early identification of relapsing patients could improve outcomes by facilitating initiation of adjunct therapies such as venetoclax to eliminate the small resistant clone. Disclosures Woyach: Karyopharm: Research Funding; Morphosys: Research Funding; Acerta: Research Funding. Jones:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding. Andritsos:Hairy Cell Leukemia Foundation: Research Funding. Awan:Innate Pharma: Research Funding; Pharmacyclics: Consultancy; Novartis Oncology: Consultancy. Lozanski:Beckman Coulter: Research Funding; Stemline Therapeutics Inc.: Research Funding; Boehringer Ingelheim: Research Funding; Genentech: Research Funding.

SummaryTetraspanins are commonly believed to act only as “molecular facilitators,” with no direct role in signal transduction. We herein demonstrate that upon ligation, CD37, a tetraspanin molecule expressed on mature normal and transformed B cells, becomes tyrosine phosphorylated, associates with proximal signaling molecules, and initiates a cascade of events leading to apoptosis. Moreover, we have identified two tyrosine residues with opposing regulatory functions: one lies in the N-terminal domain of CD37 in a predicted “ITIM-like” motif and mediates SHP1-dependent death, whereas the second lies in a predicted “ITAM motif” in the C-terminal domain of CD37 and counteracts death signals by mediating phosphatidylinositol 3-kinase-dependent survival.

Importance The Bruton tyrosine kinase (BTK) inhibitor ibrutinib is effective in patients with chronic lymphocytic leukemia (CLL). Reasons for discontinuing therapy with this drug and outcomes following discontinuation have not been evaluated outside of clinical trials with relatively short follow-up. Objective To determine features associated with discontinuation of ibrutinib therapy and outcomes. Design, Setting, and Participants A total of 308 patients participating in 4 sequential trials of ibrutinib at The Ohio State University Comprehensive Cancer Center were included. These clinical trials accrued patients included in this analysis from May 2010 until April 2014, and data were locked in June 2014. Main Outcomes and Measures Patients were evaluated for time to therapy discontinuation, reasons for discontinuation, and survival following discontinuation. For patients who discontinued therapy because of disease progression, targeted deep sequencing was performed in samples at baseline and time of relapse. Results With a median follow-up of 20 months, 232 patients remained on therapy, 31 had discontinued because of disease progression, and 45 had discontinued for other reasons. Disease progression includes Richter’s transformation (RT) or progressive CLL. Richter’s transformation appeared to occur early and CLL progressions later (cumulative incidence at 12 months, 4.5% [95% CI, 2.0%-7.0%] and 0.3% [95% CI, 0%-1.0%], respectively). Median survival following RT was 3.5 months (95% CI, 0.3-6.0 months) and 17.6 months (95% CI, 4.7 months–“not reached”) following CLL progression. Sequencing on peripheral blood from 8 patients with RT revealed 2 with mutations in BTK , and a lymph node sample showed no mutations in BTK or PLCG2 . Deep sequencing on 11 patients with CLL progression revealed BTK or PLCG2 mutations in all. These mutations were not identified before treatment in any patient. Conclusions and Relevance This single-institution experience with ibrutinib confirms it to be an effective therapy and identifies, for the first time, baseline factors associated with ibrutinib therapy discontinuation. Outcomes data show poor prognosis after discontinuation, especially for those patients with RT. Finally, sequencing data confirm initial reports associating mutations in BTK and PLCG2 with progression and clearly show that CLL progressions are associated with these mutations, while RT is likely not. Trial Registrations clinicaltrials.gov Identifiers:NCT01105247,NCT01217749,NCT01589302, andNCT01578707

7010 Background: The Bruton’s Tyrosine Kinase (BTK) inhibitor ibrutinib (I) is very effective in chronic lymphocytic leukemia (CLL), with progression free survival of 76% at 26 months (mo) for patients (pts) with relapsed disease (Byrd J et al, NEJM 2013). We evaluated pts treated with I to explore features associated with progressive disease (PD) and subsequent outcomes. Methods: 267 pts from The Ohio State University Comprehensive Cancer Center participating in 3 previously reported Institutional Review Board approved trials of I were included; 196 pts received single agent I and 71 received I plus ofatumumab. A subset of pts with PD had Ion Torrent deep sequencing (DS) performed on peripheral blood at baseline and relapse. Results: With a median follow-up of 16 mo (

Studies of chronic lymphocytic leukemia (CLL) have yielded substantial progress, however a lack of immortalized cell lines representative of the primary disease has hampered a full understanding of disease pathogenesis and development of new treatments. Here we describe a novel CLL cell line (OSU-CLL) generated by EBV transformation, which displays a similar cytogenetic and immunophenotype observed in the patient's CLL (CD5 positive with trisomy 12 and 19). A companion cell line was also generated from the same patient (OSU-NB). This cell line lacked typical CLL characteristics, and is likely derived from the patient's normal B cells. In vitro migration assays demonstrated that OSU-CLL exhibits migratory properties similar to primary CLL cells whereas OSU-NB has significantly reduced ability to migrate spontaneously or towards chemokine. Microarray analysis demonstrated distinct gene expression patterns in the two cell lines, including genes on chromosomes 12 and 19, which is consistent with the cytogenetic profile in this cell line. Finally, OSU-CLL was readily transplantable into NOG mice, producing uniform engraftment by three weeks with leukemic cells detectable in the peripheral blood spleen and bone marrow. These studies describe a new CLL cell line that extends currently available models to study gene function in this disease.