ALBERT

Description: Epithelial growth factor-like 7 (EGFL7) is a protein that is secreted by endothelial cells and plays an important role in angiogenesis. Although EGFL7 is aberrantly overexpressed in solid tumors, its role in leukemia has not been evaluated. Here, we report that levels of bothEGFL7mRNA and EGFL7 protein are increased in blasts of patients with acute myeloid leukemia (AML) compared with normal bone marrow cells. HighEGFL7mRNA expression associates with lower complete remission rates, and shorter event-free and overall survival in older (age ≥60 y) and younger (age

Description: Patients with Chronic Lymphocytic Leukemia (CLL) have a variety of chromosomal abnormalities and mutations. At diagnosis, about 10% of CLL patients have deletions of chromosome 17 (Del17p) leading to the loss of one allele of tumor suppressor protein TP53, which increases to over 30% in relapsed/refractory disease. Additionally, 83% of patients with a Del17p acquire a mutation on their second TP53 allele at one of several sites within the DNA binding domain. While the consequence of some of these "hotspot" mutations (R175H, R179H, G245D, G248Q/W, Y220, R213X, R273H and R282H) has been described in solid tumors and AML, very little is known of their role in CLL. Clinically, patients with Del17p/Mutp53 have worse overall survival, increased disease progression and are more likely to relapse on the current targeted therapies such as ibrutinib. Although relapse to these treatments is largely due to acquired mutations in Bruton's Tyrosine Kinase (BTK) or its downstream target PLCg2, we hypothesize that the biology of mutant 53 bearing CLL is a key driver of resistance and progression. Specifically, we aim to determine the molecular signature and downstream effectors that allow mutant p53 to drive the adverse biology associated with this subtype. Conversely, we hypothesize that targeting the mutant p53 pathway will lead to better outcomes and overall survival for patients bearing this adverse prognosis marker. We performed high-throughput Sequencing of DNA from 270 CLL patients with high coverage in the exonic regions of TP53 prior to ibrutinib therapy as well as during progression. At baseline, 40% of patients had mutations found in the DNA binding region with the most frequent occurring in R248Q, R175H and R273H. We then characterized each p53 mutant (n=106) functionally in terms of their ability to ability to activate p21, PUMA, and Bax which serve as cell cycle checkpoint and apoptotic effectors of wild type p53 in response to DNA damage. Most mutants were incompetent in upregulating p21, PUMA or Bax at the transcript level. A few mutants upregulated p21 protein in a p53 independent fashion. We then evaluated the consequence of mutant p53 in CLL. We performed chromatin immunoprecipitation (ChIP-Seq), open chromatin signatures (ATAC-Seq) and expression analysis (RNA-Seq) on CLL samples with R248Q or R175H as well as in wild-type (WT) p53 samples. Integration of ChIP, ATAC and RNA Seq profiles indicated that mutant p53 activated a unique transcriptomic profile not shared by wt p53 bearing CLL. Several genes that facilitated survival or progression were downstream targets of mutant p53. Of these, we identified PRKCB (PKC-beta), BCL2L1 (Bcl-xL), EZH2, MLL and MALAT as a potential key downstream effectors of mutant p53. To determine whether mutations at R248Q and R175H in p53 were causal in the observed increases in PKC-beta, Bcl-xL, EZH2, MLL, and MALAT we used CRISPR/Cas9 editing to introduce mutations at R175H and R248Q in the p53 wildtype CLL cell lines HG-3 and PGA-1. These were accomplished by electroporating sgRNA-Cas9 ribonucleoprotein complexes (RNPs). Western blotting of mutants revealed an increase in the mRNA and protein expression of PKC-beta, and BCL-xL in mutant p53 compared to WT. Levels of EZH2 and MLL were not increased in these cells indicating that PKC-beta and Bcl-xL may be direct transcriptional targets upregulated by mutations at R248Q and R175H in p53. Ongoing efforts will characterize the transcriptional profile of all p53 mutants in our cohort to determine whether they all have a unifying transcriptomic profile that confers a gain of function phenotype to this subtype of CLL. Disclosures Byrd: Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; BeiGene: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; Novartis: Other: Travel Expenses, Speakers Bureau; Acerta: Research Funding; BeiGene: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Genentech: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau. 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.

Description: Key Points CLL exosomes exhibit a disease-relevant microRNA signature. B-cell receptor signaling enhances exosome secretion in CLL that can be antagonized by ibrutinib.

Description: 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.

Description: Lucas et al explored the clonal dynamics of chronic lymphocytic leukemia (CLL) patients following treatment and subsequent acquired resistance to ibrutinib and then venetoclax. They report different patterns of resistance mutations from previously reported changes following venetoclax treatment in the absence of prior BTK inhibitor therapy.

Description: Acute myeloid leukemia (AML) is the most commonly diagnosed acute leukemia in adults. Despite newly approved treatment, AML still results in poor outcomes especially in older patients (pts). Cytogenetic abnormalities, gene mutations, and their combinations contribute to the pathogenesis and pt outcomes in AML. The PTPN11 gene encodes the phosphatase Shp2, which activates the RAS-MAPK pathway. Despite the relatively high frequency of PTPN11 mutations in AML, little is known about associations of PTPN11 mutations with other genomic features and their influence on outcomes of pts with standard 7+3 chemotherapy. In addition, primary resistance to targeted therapy, such as venetoclax and enasidenib, has been preliminarily noted in PTPN11+ pts. This study sought to determine the type and frequency of PTPN11 mutations as well as associations with clinical, cytogenetic, and genomic features and outcome in adult AML pts treated with 7+3 induction chemotherapy followed by consolidation chemotherapy on Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology trials. 1,725 newly diagnosed AML pts, defined by the European LeukemiaNet 2017 recommendations (excluding acute promyelocytic leukemia), were examined using targeted next generation sequencing analysis and centrally reviewed metaphase cytogenetics. Missense, nonsense, or frameshift variants not reported in the 1000 Genomes database, dbSNP137 or dbSNP142, were considered mutations. Fisher's exact test was used to determine mutation association and complete remission (CR) rates while continuous variables are from Wilcoxon rank sum test. The median follow-up was 9 years. We identified 140 pts (8.1%) with PTPN11 mutations with the majority (61%) located in the N-terminal SH2 domain (Figure 1). 98 younger ( 0.3. NPM1 (61% vs 31%, P 〈 .001) and DNMT3A (R882 or other) mutations (39% vs 22%, P 〈 .001) were more likely to co-occur with PTPN11 mutations than wild-type (WT) PTPN11 (Figure 2). PTPN11 mutations were less common in FLT3-ITD pts than in those without (17% vs 25%, P = .07). PTPN11 mutations were more common in inv(3)(q21q26)/t(3;3)(q21;q26) pts (26%, P = .004) and were rare in pts with core-binding factor AML, inv(16)/t(16;16) (3%, P = .03) and t(8;21) (0%, P =.005). Clinical features of PTPN11+ pts were similar to those of WT pts except for elevated platelet counts (P 〈 .001) and more extramedullary involvement (P = .03). For all pts, there was no difference in CR rate, disease-free (DFS), overall (OS), and event-free (EFS) survival between PTPN11+ and PTPN11- pts. DFS of older PTPN11+ pts was shorter (3-y rates: 5% vs 15%, P = .04). Given that PTPN11 mutations often co-occur with NPM1 mutations, which are typically associated with favorable outcome (in the absence of a high FLT3-ITD ratio), we focused on the contribution of PTPN11 mutations to outcomes in the NPM1+/FLT3-WT subset. Compared with PTPN11-/NPM1+/FLT3-WT, PTPN11+ pts had a lower CR rate (38% vs 64%, P = .001) and shorter EFS (3-y rates: 10% vs 21%, P = .01), whereas there was no significant differences in OS (3-y rates: 23% vs 32%, P = .13) or DFS (3-y rates: 27% vs 33%, P = .75). When considering the role of PTPN11 mutations with WT NPM1, there was a reduction in survival in PTPN11+/NPM1- pts compared with PTPN11-/NPM1- pts. Younger PTPN11+/NPM1- pts had a lower CR rate (45% vs 71%, P = .002) and shorter OS (3-y rates: 30% vs 41%, P = .04), and EFS (3-y rates: 13% vs 27%, P = .008). Compared to older PTPN11-/NPM1-, older PTPN11+/NPM1- pts had a lower CR rate (18% vs 43%, P = .04) and shorter DFS (3-y rates: 0% vs 10%, P = .02) and EFS (3-y rates: 0% vs 4%, P = .02), whereas OS (3-y rates: 12% vs 10%, P = .58) had no significant difference. To our knowledge, this study is the largest cohort of PTPN11+ pts in adult AML and demonstrates specific mutational and cytogenetic associations. When considering PTPN11+ pts based on NPM1 mutation status, we showed that PTPN11 mutations associated with worse outcome in both NPM1+ and NPM1- AML pts when treated with intensive chemotherapy. Developing targeted treatments to this genomic group in AML represents a research priority. Support: U10CA180821, U10CA180882, U24CA196171, R35CA198183; https://acknowledgments.alliancefound.org; Clinicaltrials.gov Identifier: NCT00048958, NCT00899223, NCT00900224 Disclosures Mims: Leukemia and Lymphoma Society: Other: Senior Medical Director for Beat AML Study; Agios: Consultancy; Kura Oncology: Membership on an entity's Board of Directors or advisory committees; Syndax Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau; Jazz Pharmaceuticals: Other: Data Safety Monitoring Board. Blachly:AbbVie, AstraZeneca, KITE Pharma: Consultancy. Stone:Takeda: Consultancy; Trovagene: Consultancy; Pfizer: Consultancy; Gemoab: Consultancy; Janssen: Consultancy; AbbVie: Consultancy, Research Funding; Actinium: Consultancy; Agios: Consultancy, Research Funding; Argenx: Consultancy, Other: Data and safety monitoring board; Arog: Research Funding; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy; Biolinerx: Consultancy; Celgene: Consultancy, Other: Data and safety monitoring board; Jazz: Consultancy; Novartis: Consultancy, Research Funding; Otsuka: Consultancy; Syntrix: Consultancy; Syros: Consultancy; Elevate: Consultancy; Syndax: Consultancy; Daiichi-Sankyo: Consultancy; Stemline: Consultancy; Macrogenics: Consultancy; Hoffman LaRoche: Consultancy. Wang:Bristol Meyers Squibb (Celgene): Consultancy; PTC Therapeutics: Consultancy; Macrogenics: Consultancy; Astellas: Consultancy; Jazz Pharmaceuticals: Consultancy; Stemline: Speakers Bureau; Genentech: Consultancy; Pfizer: Speakers Bureau; Abbvie: Consultancy. Kolitz:Pfizer: Membership on an entity's Board of Directors or advisory committees; Magellan: Membership on an entity's Board of Directors or advisory committees. Powell:Rafael Pharmaceuticals: Consultancy, Other: Advisor, Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Genentech: Research Funding; Jazz Pharmaceuticals: Consultancy, Other: Advisor, Research Funding. Eisfeld:Vigeo Therapeutics: Consultancy; Karyopharm: Current Employment, Current equity holder in publicly-traded company. Byrd:Syndax: Research Funding; Vincera: Research Funding; Novartis: Research Funding; Kartos Therapeutics: Research Funding; Acerta Pharma: Research Funding; Trillium: Research Funding; Leukemia and Lymphoma Society: Other; 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.