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Introduction The clinical course and outcomes are heterogenous among patients (pts) with chronic lymphocytic leukemia (CLL). Somatic hypermutation (SHM) status of the IGHV gene (IGHV-SHM) is an established prognostic marker for pts receiving certain therapeutic regimens. Pts with mutated IGHV (IGHV-M) have a better prognosis vs pts with unmutated IGHV (IGHV-UM). We demonstrate that IGHV-SHM could be accurately assessed utilizing primers located downstream of CDR1. Methods Adaptive's clonoSEQ ® NGS-based assay identifies tumor specific sequence(s) using a set of multiplexed, locus-specific primers that include the Ig heavy chain variable gene (IGHV; starting at the end of CDR1), and the kappa and lambda light chain. Sequences are tagged and used to assess measurable residual disease (MRD). SHM status is calculated as the percentage of somatic mutations deviating from the germline DNA sequence. IGHV-M was defined as ≥2% deviation. For patients with multiple IGHV sequences, the most mutated sequence determined IGHV-SHM status. Pt samples from 5 studies (Table 1) were analyzed by clonoSEQ to determine IGHV-SHM status and compared to IGHV-SHM status determined by institutional standard laboratory (Sanger sequencing or institutional NGS). Four datasets were used from MD Anderson Cancer Center (MDACC). Set1 (n=31) was the positive control as eligibility criteria mandated inclusion of pts with IGHV-M status. Results were correlated with outcome data available from set2 (n=62). Pts with B-cell acute lymphoblastic leukemia served as a negative control (set3; n=246). Validation was performed with CLL samples (set4; n=21 and set5; n=49). IGHV-SHM analysis performed by Sanger sequencing (and NGS in a subset of set1 and set4) were the comparator for Adaptive's IGHV-SHM assessment. Rearranged gDNA sequences from the IMGT database (est. 1989) were ascertained and IgBlast was used to annotate the domains and report the V-gene homology. To approximate a full-length (FL) rearrangement, 9080 sequences were identified where framework region 1 (FR1) had ≥ 75 nucleotides. We compared the mutation status obtained using this FL rearranged sequence to that same sequence beginning at the 5' end of FR2 (consistent with the 5' extent of the primers in the Adaptive Assay). Results In set1, 30/31 pts were IGHV-M by the MDACC sequencing IGHV-SHM analysis. We obtained concordant IGHV-SHM status in 29/30 IGHV-M pts, and the only IGHV-UM pt. From set2, 29 pts with IGHV-M and 24 IGHV-UM were identified; 8 pts had unknown IGHV-SHM status. The IGHV-SHM Assay classified IGHV-M in 24/29 pts and IGHV-UM in 23/24 pts. In the negative control (set3), no IGHV-M pts were identified. In set2, stratifying PFS by Adaptive's IGHV-SHM status a p=0.0067 was non-inferior to the p=0.012 by Sanger sequencing (Figure 1 A-D). Inclusion of 8 pts with unknown IGHV-SHM status by Sanger sequencing yields a p=0.0039 (n=61). In the validation dataset (set4), IGHV-M was concordant for 4/4 IGHV-M pts and 13/14 IGHV-UM pts. In a separate validation dataset (set5), the Adaptive IGHV-SHM Assay was concordant for 16/17 IGHV-M pts and 25/28 IGHV-UM pts. 3 and 4 pts had unknown IGHV-SHM status by MDACC analysis in set4 and set5 respectively. A concordance table compares mutation status with FL rearrangements from IMGT vs excluding FR1 & CDR1 (Table 2). 96.1% of the sequences had concordant SHM status. Of the 3.9% that are discordant, a majority are within ±1% of the 2% threshold, a recognized SHM "gray zone". Only 15 (0.17%) fall outside this borderline category and are categorized as IGHV-M converting to IGHV-UM based on the truncated analysis. Given improvements in sequencing since launching IMGT in 1989, it is likely that some sequencing quality issues may be present within the dataset such that the 0.17% discordance is an upper estimate of discordance. Conclusions 135/147 CLL pts were concordant to independent assessments of IGHV-SHM status; IGHV-SHM status was assessed in additional 15 pts who could not be evaluated by MDACC comparator assay(s). The assessment of IGHV-SHM status correlated with PFS and standard determination of SHM. This analysis highlights the utility of the Adaptive assay to identify trackable sequences and assess IGHV-SHM status from the same baseline sample. The ability of a single platform to determine both SHM and enable subsequent MRD assessment streamlines the clinical management of pts with CLL. Figure 1 Figure 1. Disclosures Lee: Adaptive Biotechnologies: Current Employment. Ching: Adaptive Biotechnologies: Current Employment, Current equity holder in publicly-traded company. Howie: Adaptive Biotechnologies: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Thompson: Janssen: Consultancy, Honoraria; AbbVie: Other: Institution: Advisory/Consultancy, Honoraria, Research Grant/Funding; Gilead: Other: Institution: Advisory/Consultancy, Honoraria; Pharmacyclics: Other: Institution: Advisory/Consultancy, Honoraria, Research Grant/Funding; Adaptive Biotechnologies: Other: Institution: Advisory/Consultancy, Honoraria, Research Grant/Funding, Expert Testimony; Genentech: Other: Institution: Advisory/Consultancy, Honoraria, Research Grant/Funding; Amgen: Other: Institution: Honoraria, Research Grant/Funding. Jain: Beigene: Honoraria; Janssen: Honoraria; Bristol Myers Squibb: Honoraria, Research Funding; TG Therapeutics: Honoraria; Servier: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; ADC Therapeutics: Honoraria, Research Funding; Fate Therapeutics: Research Funding; Cellectis: Honoraria, Research Funding; Adaptive Biotechnologies: Honoraria, Research Funding; Incyte: Research Funding; Genentech: Honoraria, Research Funding; Pfizer: Research Funding; Aprea Therapeutics: Research Funding; Precision Biosciences: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Pharmacyclics: Research Funding. Ferrajoli: BeiGene: Other: Advisory Board, Research Funding; Janssen: Other: Advisory Board ; AstraZeneca: Other: Advisory Board, Research Funding. Burger: TG Therapeutics: Other: Travel/Accommodations/Expenses, Research Funding, Speakers Bureau; AstraZeneca: Consultancy; Beigene: Research Funding, Speakers Bureau; Gilead: Consultancy, Other: Travel/Accommodations/Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC: Consultancy, Other: Travel/Accommodations/Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel/Accommodations/Expenses, Speakers Bureau; Janssen: Consultancy, Other: Travel/Accommodations/Expenses, Speakers Bureau. Wierda: Sunesis: Research Funding; Cyclacel: Research Funding; Miragen: Research Funding; Janssen: Research Funding; KITE Pharma: Research Funding; Karyopharm: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; Gilead Sciences: Research Funding; Oncternal Therapeutics, Inc.: Research Funding; AstraZeneca: Research Funding; GSK/Novartis: Research Funding; Acerta Pharma Inc.: Research Funding; Xencor: Research Funding; Loxo Oncology, Inc.: Research Funding; Juno Therapeutics: Research Funding; Genentech: Research Funding; Genzyme Corporation: Consultancy; AbbVie: Research Funding. Kirsch: Adaptive Biotechnologies: Current Employment, Current equity holder in publicly-traded company.

Chemotherapy-induced cytopenias are a prevalent and significant issue that worsens clinical outcomes and hinders the effective treatment of cancer. While they are classically associated with traditional cytotoxic chemotherapies, they also occur with newer targeted small molecule drugs and the factors that determine the hematotoxicity profiles of chemotherapies are not fully understood. Here, we explore why Aurora kinase inhibitor drugs cause preferential neutropenia when compared to the cytopenic profiles of targeted small molecule cancer drugs that are FDA approved. By studying drug responses of healthy human hematopoietic cells in vitro and analyzing existing published clinical datasets, we provide evidence that the enhanced vulnerability of neutrophil-lineage cells to Aurora kinase inhibitors is acquired at an early stage of differentiation and is caused by developmental changes in the expression pattern of ATP-binding cassette (ABC) transporters. These data show that hematopoietic cell-intrinsic expression of ABC transporters may be an important factor that determines how some chemotherapies affect the bone marrow. Disclosures David: AstraZeneca: Current Employment. Randle: AstraZeneca: Current Employment. Polanska: AstraZeneca: Current Employment. Urosevic: AstraZeneca: Current Employment. Travers: AstraZeneca: Ended employment in the past 24 months. Ingber: Emulate: Membership on an entity's Board of Directors or advisory committees; BOA Biomedical: Membership on an entity's Board of Directors or advisory committees; Freeflow Medical Devices Inc: Membership on an entity's Board of Directors or advisory committees.

Background Measurement of minimal residual disease (MRD) is an established and sensitive prognostic tool to assess the depth of response during and after treatment of chronic lymphocytic leukemia (CLL) and to understand disease dynamics after treatment. Retracing these kinetics is paramount to understand which group of patients is at risk of relapsing despite initial MRD response. To this end, we here provide an analysis of clonal growth patterns in patients treated within the CLL14 trial. Methods In total, 432 patients with previously untreated CLL and co-existing conditions were randomized to receive chlorambucil or venetoclax (216 patients per arm) until completion of cycle 12 (à 28 days), and in combination with obinutuzumab for the first 6 cycles. The primary endpoint was progression-free survival (PFS), MRD was a secondary endpoint. MRD was analyzed by ASO-PCR and, as reported here, by next-generation sequencing (Adaptive clonoSEQ Assay, cut-off: 10-4, 10-5 and 10-6). The limit-of-quantification of the clonoSEQ Assay is less than 10-6. Samples from peripheral blood (PB) are collected every 3 to 6 months until 9 years from last patient enrolled; for this analysis, the longest follow-up MRD sample was from month 24 after treatment completion. A patient-specific clonal growth rate model was formed: MRD level at time t is defined as MRD(t) = λ0 x 10μt, where λ0 is the baseline MRD (i.e. MRD at end of treatment (EoT)), and μ is the growth rate parameter. For the time from MRD status λ0 to new MRD state λ1 , tchange was assumed (1/μ) x log10 (λ1/λ0). An exponential regression model was fitted to each patient to estimate each respective λ0 and μ, by which the patients' MRD clone doubling time was calculated. Data are reported in relation to the intention-to-treat population; p-values are exploratory only. For modelling, only patients with at least two MRD assessments after EoT and who had not experienced PD before EoT were included. Results Two months after treatment completion (follow-up month 3), 40% (7%) of patients in the Ven-Obi arm (Clb-Obi arm) had uMRD levels Most patients (56%) who had undetectable MRD (uMRD) levels at EoT already had uMRD levels at cycle 7, i.e. after completing Ven-Obi therapy. In 25% of the Ven-Obi treated patients, MRD response deepened after continuing with 6 cycles of venetoclax monotherapy (Figure B). In a PFS landmark analysis after EoT, patients in the Ven-Obi arm with MRD levels ≤10-5 had a 2-year PFS after EoT of approximately (approx.) 93%, while patients with detectable MRD >10-2 had a 2-year PFS of approx. 37% (Figure D). Patient-specific clonal growth rates were estimated for 123 patients in the Ven-Obi arm and 143 patients in the Clb-Obi arm (Figure C). Of note, 38 patients in the Ven-Obi arm and 4 patients in the Clb-Obi arm had MRD results below the assays limit of quantification (LOQ), indicating the deepest response beyond the assay's sensitivity. These patients were not included as a growth rate could not be accurately assessed for those very-deep responders. The average clonal growth rate in the Ven-Obi arm (n=123) was µ = 0.0034 (95% CI: 0.0030-0.0038), translating into an MRD doubling time of approx. 89 days (95% CI: 79-102) and an increase of the MRD clone size by 1 with log10 scale within approx. 296 days (95% CI: 263-337) (Figure E). For patients in the Clb-Obi arm (n=143), µ was 0.0042 (95% CI: 0.0038-0.0047), corresponding to a doubling time of approx. 71 days (95% CI: 65-79), and an increase of the MRD clone size by 1 with log10 scale within approx. 237 days (95% CI: 215-263). The average growth rate was lower in the Ven-Obi arm compared to the Clb-Obi arm (p=0.0057). Analyses of growth rate patterns within various biological and clinical risk groups as well as genetic subgroups are currently being conducted and will be presented at the meeting. Conclusions This analysis demonstrates that individual clonal growth rates can be used to estimate the MRD doubling time after a fixed-duration treatment. Clonal growth was lower after Ven-Obi than after Clb-Obi, indicating more effective MRD eradication and clonal growth modulation with Ven-Obi. In a considerable subgroup of Ven-Obi treated patients, no clonal growth was measurable during observation, indicating deepest remissions. Figure Disclosures Al-Sawaf: AstraZeneca: Consultancy, Honoraria, Other: personal fees; BeiGene: Research Funding; AbbVie: Consultancy, Honoraria, Other: personal fees, Research Funding; Janssen: Consultancy, Honoraria, Other: personal fees, Research Funding; Gilead: Consultancy, Honoraria, Other: personal fees; Roche: Consultancy, Honoraria, Other: personal fees, Research Funding. Wilson:Roche Products Limited: Current Employment. Tandon:Roche Products Ltd.: Current equity holder in publicly-traded company. Ching:Adaptive Biotechnologies: Current Employment, Current equity holder in publicly-traded company. Fink:Janssen: Honoraria; Celgene: Research Funding; AbbVie: Other: travel grants. Ritgen:Gilead: Other: travel grants; F. Hoffman-La Roche: Consultancy, Honoraria, Other: travel grants, Research Funding; BMS: Consultancy, Honoraria, Other: travel grants; Pfizer: Consultancy, Honoraria. Tausch:Janssen-Cilag: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding. Kreuzer:AbbVie: Consultancy, Honoraria, Research Funding; Hoffmann-La Roche: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Janssen-Cilag: Consultancy, Honoraria, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding. Schary:AbbVie: Current Employment, Current equity holder in publicly-traded company. Wendtner:AbbVie: Consultancy; MorphoSys: Consultancy; Hoffmann-La Roche: Consultancy; Gilead: Consultancy; Janssen-Cilag: Consultancy. Eichhorst:AstraZeneca: Consultancy, Honoraria, Other: travel support, Research Funding; Celgene: Consultancy, Honoraria, Other: travel support, Research Funding; ArQule: Consultancy, Honoraria, Other: travel support, Research Funding; Novartis: Consultancy, Honoraria, Other: travel support, Research Funding; AbbVie: Consultancy, Honoraria, Other: travel support, Research Funding; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding; Oxford Biomedica: Consultancy, Honoraria, Other: travel support, Research Funding; BeiGene: Consultancy, Honoraria, Other: travel support, Research Funding; Janssen-Cilag: Consultancy, Honoraria, Other: travel support, Research Funding; F. Hoffmann-LaRoche: Consultancy, Honoraria, Other: travel support, Research Funding. Stilgenbauer:Mundipharma: Consultancy, Honoraria, Other, Research Funding; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding; Genzyme: Consultancy, Honoraria, Other: travel support, Research Funding; Celgene: Consultancy, Honoraria, Other: travel support, Research Funding; F. Hoffmann-LaRoche: Consultancy, Honoraria, Other: travel support, Research Funding; Genentech: Consultancy, Honoraria, Other: travel support, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Other: travel support, Research Funding; Amgen: Consultancy, Honoraria, Other: travel support, Research Funding; AbbVie: Consultancy, Honoraria, Other: travel support, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Other: travel support, Research Funding; Novartis: Consultancy, Honoraria, Other, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other, Research Funding; Janssen-Cilag: Consultancy, Honoraria, Other: travel support, Research Funding. Jiang:F. Hoffmann-La Roche: Current equity holder in publicly-traded company; Genentech, Inc.: Current Employment. Hallek:Roche: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Mundipharma: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding. Fischer:F. Hoffmann-La Roche: Honoraria, Other: travel grants; AbbVie: Honoraria.

Acute myeloid leukemia (AML) is an aggressive, heterogeneous malignancy. AML patients whose disease relapses on chemotherapy or are unfit for aggressive induction regimens have limited therapeutic options. Many patients benefit from the combination of venetoclax (BCL2i) and a hypomethylating agent (HMA) but this regimen is rarely curative. The addition of novel agents could provide improved benefit for relapsed/refractory patients. To identify such regimens, we screened a panel of 10 AML cell lines with combinations of venetoclax and novel targeted agents. The agents used spanned multiple mechanisms of action (e.g. DNA damage response, kinase signaling, pro-apoptotic agents) and are all in early clinical development. Cells were treated for 72hrs and viability was assessed by CellTiter-Glo. In several of the cell lines that were insensitive or partially sensitive to venetoclax (OCI-AML3, KG1a, MonoMac6, THP1), combinations with inhibitors of MCL1 (AZD5991), AURKB (AZD2811), and BRD4 (AZD5153) showed synergistic activity (Loewe synergy score >5, growth inhibition > 180%) (Table 1). We next asked if these combinations were active in patient-derived xenograft (PDX) models of AML. We established an ex vivo co-culture assay using the HS-5 bone marrow stromal cell line. AML PDX cells were isolated from mouse spleens and plated in 96-well format in direct co-culture with HS-5 cells or in HS-5-derived conditioned media. Cells were treated with three doses of each monotherapy and three doses of fixed ratio combination. Replicate screens using cells from individual mice on different days confirmed data were reproducible (r2=0.687) across animals engrafted with the same PDX. Drug response was similar between conditioned media and direct co-culture assays (r2=0.81). Venetoclax sensitivity varied across PDX models ex vivo. Notably, 2/5 PDX models screened (DFAM-68555 and DFAM-10360) were insensitive to both venetoclax and the combination of venetoclax + 5-azacytidine (HMA) ex vivo. Both models were established from untreated/1L patients and harbor TP53 mutations. Combination treatments did not add additional benefit over venetoclax monotherapy in the DFAM-10360 model. However, in DFAM-68555, AZD5153, AZD5991, and AZD2811 showed improved activity over venetoclax alone (67%, 54%, and 67% vs. 26% decrease in viability for venetoclax alone, respectively). Since combination strategies will likely be most impactful in patients refractory to or relapsed after venetoclax, we chose this venetoclax insensitive model to prioritize in vivo. To confirm the translatability of these findings, we designed a pilot in vivo study using DFAM-68555. Mice were randomized to receive vehicle, venetoclax + HMA, or venetoclax + AZD5153 when peripheral blood disease reached ~5% (hCD45+hCD33+ cells by flow cytometry). After two weeks of dosing, animals were sacrificed to evaluate disease burden in bone marrow (sternum), spleen, and peripheral blood. The model remained insensitive to venetoclax + HMA in vivo. The combination of AZD5153 with venetoclax decreased disease burden in blood and spleen compared to vehicle (30% and 42% hCD45+CD33+ cells by flow cytometry vs 70% and 95%, respectively) with similar efficacy seen by immunohistochemistry in the bone. Finally, we screened these venetoclax combinations in additional aggressive AML PDX models which were resistant or only partially responsive to venetoclax in vivo. Addition of AZD2811NP and AZD5991 to venetoclax was more effective than venetoclax alone and venetoclax + HMA in the bone marrow. The most active combination varied from model to model. Efficacy screening in additional models is ongoing to further build ex vivo to in vivo translation and prioritize development of specific combinations. Also ongoing is genomic and transcriptomic profiling of these PDXs to identify potential predictive biomarkers of combination activity. In summary, we developed an ex vivo screening platform to test clinically actionable combinations for activity in clinically relevant models. Using this platform and subsequent in vivo efficacy, we identified venetoclax combinations across multiple mechanisms (pro-apoptotic, cell cycle regulation, transcriptional regulation, DNA damage response) with activity in venetoclax-insensitive models. These results suggest potential therapeutic options to explore clinically for AML patients. Disclosures Andersen: AstraZeneca: Employment. Christie:AstraZeneca: Employment. Rosen:Astrazeneca: Employment. Maratea:AstraZeneca: Employment. Hattersley:AstraZeneca: Employment. Travers:AstraZeneca: Employment. Cidado:AstraZeneca: Employment. Pulukuri:AstraZeneca: Employment. Saeh:AstraZeneca: Employment. Clark:AstraZeneca: Employment, Equity Ownership. Reimer:AstraZeneca: Employment. Mettetal:AstraZeneca: Employment.

Background: Aurora kinases (AurK) represent potential targets for anticancer therapy in hematological malignancies and solid tumors. AurK B inhibitor AZD1152 (barasertib) showed benefit (35% CR/CRi) in patients (pts) with untreated AML when given as a 7-day continuous infusion (Lowenberg B et al, Blood 2011, Kantarjian HG et al., Cancer 2013). AZD2811NP, a nanoparticle encapsulated slow-release inhibitor of AurKB, when given as 2-4hr IV on days 1 and 4, offers a prolonged drug exposure in vivo, mimicking the AZD1152 7-day continuous IV infusion. This is an update on the first-in-man dose-escalation study of AZD2811NP in pts with relapsed/refractory AML/MDS or treatment-naïve patients (pts) not eligible for intensive induction therapy (NCT03217838). The primary objectives are to determine the Maximum Tolerated Dose (MTD) and safety profile of AZD2811NP monotherapy and in combination with azacitidine. The secondary objectives are to evaluate the pharmacokinetic (PK) profile, Biologically Effective Dose (BED), and preliminary efficacy (CR, CRi, PR, 6 month OS). Methods: Pts received a 2-hour IV infusion on Day 1 and 4 of each 28-day cycle (Cy) for doses up to 600mg, extending to a 4 h IV infusion for dosages > 600 mg. In the ongoing dose escalation, 3-6 pts have been sequentially enrolled in cohorts ranging from 100 mg to 800 mg per infusion (Day 1 & 4), i.e. from 200 mg to 1,600 mg per cycle in monotherapy setting, according to a modified continuous reassessment method (mCRM) dose escalation design. AZD2811NP was also combined with azacitidine (75 mg day 1 to 7 or the 5-2-2 schedule) starting at an AZD2811NP dose of 400 mg D1 and D4 every 4 weeks. Study treatment was continued until disease progression, intolerability, or when discontinuation criteria were met. Results: Currently, 30 pts have enrolled of which 29 pts (12 females and 17 males) received study treatment in 5 monotherapy cohorts and 2 azacitidine combination cohorts, with ages ranging from 53 to 85 years. Nineteen pts had relapsed/refractory AML, 9 pts had MDS and 1pt had MDS/MPN. Monotherapy cohort 5 (800 mg D1 & D4) and combination cohort 4c (600mg D1 & D4 + Azacitidine) are currently enrolling. Of the 19 pts in monotherapy cohorts 1-5, 18 pts discontinued (due to consent withdrawal [2], early disease related deaths [2], other reason [1], or completed follow up [13; 11 pts after Cy1, 2 pts after Cy2]) and 1 pt is still on therapy. Nine pts were treated in combination with azacitidine, and of these, 3 pts are still on therapy and 6 pts have discontinued AZD2811NP (due to death [1], consent withdrawal [2], or completed follow up [3; 2 pts after Cy2, 1 pt after Cy4]). Adverse events that occurred in ≥ 20% of pts were mainly myelotoxicity, nausea and fatigue. One dose-limiting toxicity (DLT) has been observed in the monotherapy arm (esophageal infection) and one DLT in the combination setting (late neutropenia recovery). Two deaths were due to the underlying disease and 1 due to a serious adverse event of Gr 5 sepsis not related to study drug. AZD2811 total and released blood PK exposure appears broadly dose proportional with a terminal t1/2 of ~ 30-50 hours. Released blood PK exposure is ~ 1% of total PK exposure. Conclusion: AZD2811NP is documented to be well tolerated at doses up to 600 mg on Day 1 & 4 every 28 days in monotherapy setting and up to 400 mg (D1 & 4) in combination with azacitidine. The monotherapy and combination therapy dose escalations are ongoing. Updated results including preliminary efficacy data will be presented. Additional dose finding and expansion cohorts of AZD2811NP in combination with venetoclax are planned. Disclosures Atallah: Pfizer: Consultancy; Helsinn: Consultancy; Jazz: Consultancy; Helsinn: Consultancy; Novartis: Consultancy; Takeda: Consultancy, Research Funding; Jazz: Consultancy. Yang:AstraZeneca: Research Funding; Agios: Consultancy. Eghtedar:Jazz: Consultancy, Honoraria, Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Takeda: Honoraria, Speakers Bureau; Verastem Oncology: Consultancy; Novartis: Consultancy, Honoraria, Speakers Bureau. Borthakur:Merck: Research Funding; Oncoceutics: Research Funding; Cantargia AB: Research Funding; FTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Argenx: Membership on an entity's Board of Directors or advisory committees; Xbiotech USA: Research Funding; Arvinas: Research Funding; Polaris: Research Funding; Strategia Therapeutics: Research Funding; Tetralogic Pharmaceuticals: Research Funding; Agensys: Research Funding; Bayer Healthcare AG: Research Funding; AstraZeneca: Research Funding; BMS: Research Funding; Eli Lilly and Co.: Research Funding; PTC Therapeutics: Consultancy; NKarta: Consultancy; BioLine Rx: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cyclacel: Research Funding; GSK: Research Funding; Janssen: Research Funding; Incyte: Research Funding; AbbVie: Research Funding; Eisai: Research Funding; Novartis: Research Funding; BioTheryX: Membership on an entity's Board of Directors or advisory committees; Oncoceutics, Inc.: Research Funding. Charlton:AstraZeneca: Employment; GSK: Equity Ownership. MacDonald:AstraZeneca: Employment, Equity Ownership. Korzeniowska:AstraZeneca: Employment. Sainsbury:AstraZeneca: Employment, Equity Ownership. Strickland:Sarah Cannon Development Innovations: Employment. Overend:AstraZeneca: Employment, Equity Ownership. Adelman:AstraZeneca: Employment, Equity Ownership. Fabbri:AstraZeneca: Employment. Travers:AstraZeneca: Employment. Smith:AstraZeneca: Employment, Equity Ownership. Pease:AstraZeneca: Employment, Equity Ownership. Cosaert:AstraZeneca: Employment. OffLabel Disclosure: AZD2811NP, a nanoparticle encapsulated slow-release inhibitor of Aurora Kinase B (AurKB), is an investigational agent in clinical trials for human cancers including AML/MDS.

Introduction: Approximately 20% of patients with Hodgkin lymphoma (HL) are aged 60 years or older. There is no standard of care for such patients, who have a markedly inferior prognosis compared with younger patients and are significantly underrepresented in clinical trials. Escalated BEACOPP chemotherapy has been demonstrated to be highly effective in frontline HL management, but when used in older patients (even at non-escalated doses) there is an unacceptable rate of toxicity-related death. We proposed that a modification of the BEACOPP regime by removing bleomycin and etoposide and dose reduction of cyclophosphamide ('ACOPP') would potentially be a well-tolerated and effective regimen for older HL patients. Methods: This is a single centre pilot study investigating the feasibility of ACOPP chemotherapy for frontline management of older patients with HL who were deemed unfit for more intensive management approaches. Patients received intravenous (IV) Doxorubicin (35mg/m2) and Cyclophosphamide (650mg/m2) on day 1 along with oral Procarbazine (100mg/m2) for 7 days and oral Prednisolone (40mg/m2) for 14 days. IV Vincristine (1.4mg/m2, max 2mg) was given on day 8 and subcutaneous granulocyte colony stimulating factor given on days 9-13. Treatment was delivered as an outpatient every 21 days to a maximum of 6 cycles. Interim PET/CT assessment was performed after 2 cycles of treatment. Results: Seven patients with a median age of 81 years (range 58-92) were treated between March 2018 - March 2019. All patients had histologically confirmed classical Hodgkin lymphoma. 6/7 patients had advanced stage disease with median IPS 3. One patient had unfavourable stage 2 disease by EORTC criteria. Median Charlson Comorbidity Index (CCI) score was 6 with further details outlined in Table 1. All 7 patients completed at least 2 cycles of therapy and all had a complete metabolic response on interim PET with ongoing response confirmed on end of treatment CT. Four patients completed 6 cycles of therapy, with the other 3 patients completing 2, 4 and 5 cycles respectively. Reasons for completing less than 6 cycles of therapy were physician concerns over increased frailty in patient 5, concurrent diagnosis of Parkinsons disease with resultant physical deconditioning in patient 3 and postural hypotension (possibly secondary to vincristine) in patient 7. Grade 3/4 neutropenia was seen in 5/7 patients but only 1 patient had an episode of febrile neutropenia. Thrombocytopenia (2 patients, both grade 3, none requiring platelet transfusion) and anaemia (5 patients, all grade 3 and required red cell transfusion) were also seen. Peripheral neuropathy occurred in 2 patients (grade 1, grade 2). 6/7 patients required admission to hospital during therapy - primary reasons for admission were infection (n=2), constipation (n=2), anaemia (n=1) and autonomic neuropathy (n=1). All 7 patients remain alive and in remission at time of analysis (median FU 9 months). Conclusion: We have demonstrated the feasibility of ACOPP as a chemotherapy regimen which can be delivered in an outpatient setting to elderly patients with HL. We would highlight the median age of 81 years and significant co-morbidity burden of the patients in this pilot study. All patients achieved a negative interim PET and remain in remission at the time of analysis. Toxicity was mainly haematological but was manageable with only 1 episode of febrile neutropenia and no platelet transfusion required. The majority had hospital admissions during therapy, with only 4/7 managing to complete six cycles of treatment. Definitive conclusions regarding efficacy and safety cannot be drawn from this small number of patients but the high rates of metabolic response are encouraging and we feel the regimen merits further, prospective assessment in a large clinical trial. Disclosures Travers: Celgene: Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees. McKay:Janssen: Honoraria, Speakers Bureau; Epizyme: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees; MSD: Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Leach:Abbvie: Honoraria; Janssen: Consultancy, Honoraria; Roche: Honoraria.

Background: Recent data suggests that older patients with acute myeloid leukemia (AML) have a higher frequency of poor risk TP53 mutations (TP53mut) than previously suspected. Patients with TP53mut have a very poor prognosis, making this AML sub-group an area of high unmet therapeutic need. Although recent clinical trials suggest promising response rates for the BCL-2 inhibitor venetoclax (Ven) in combination with DNA methyltransferase inhibitors (DNMTi) or low-dose cytarabine, survival outcomes remain poor among patients with TP53mut (Strickland et al, EHA 2018). Methods and Results: To identify novel therapies effective against TP53mut AML, isogenic TP53 knockout cells (TP53 KO) were generated by CRISPR/Cas9 in OCI-AML3, MV4;11 and MOLM-13 human AML cell lines. In a drug screen of 50 compounds, we identified AZD2811, an aurora kinase B inhibitor, which potently killed AML cells independently of TP53 genotype (Figure 1). We hypothesised that AZD2811 would synergise with Ven to overcome venetoclax-resistance in TP53-mutant AML. Consistent with this, AZD2811 and venetoclax showed strong synergy (Loewe score Conclusions: We report for the first time that AZD2811NP can overcome venetoclax resistance in TP53-mutant AML in vitro and in vivo. These findings therefore, support the clinical investigation of combined aurora kinase and BCL-2 targeting in the clinic for patients with TP53-mutant AML, who currently lack effective treatment options. Disclosures Urosevic: AstraZeneca: Employment. Polanska:AstraZeneca: Employment. Cosaert:AstraZeneca: Employment. Pease:AstraZeneca: Employment, Equity Ownership. Travers:AstraZeneca: Employment. Wei:Pfizer: Honoraria; Janssen: Honoraria; Walter and Eliza Hall Institute: Other: former employee, Patents & Royalties: receives a fraction of its royalty stream related to venetoclax; AbbVie: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Macrogenics: Honoraria; Astellas: former employee, Honoraria; Genentech: Honoraria; Servier: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding.

Background: Aurora kinases represent potential targets for anticancer therapy in solid tumors and hematological malignancies. In a phase I/II study, the aurora B kinase inhibitor AZD1152 (barasertib) showed benefit (35% CR/CRi) in patients (pts) with untreated AML when given as a 7-day continuous infusion (Lowenberg B et al, Blood 2011, Kantarjian HG et al., Cancer 2013). AZD2811 nanoparticle is a novel, encapsulated slow-release inhibitor of Aurora kinase B offering several advantages compared with AZD1152, including prolonged drug release in vivo. AZD2811 nanoparticle mimics the AZD1152 7-day continuous infusion when given as a 2-hr infusion on Day 1 and 4, and resulted in increased efficacy and decreased toxicity in vivo. We report the first-in-man dose-escalation of AZD2811 nanoparticle in pts with relapsed AML/MDS or treatment-naïve patients not eligible for intensive induction therapy. The objectives were to determine the safety profile, MTD, PK, dosing schedule and preliminary efficacy of AZD2811 nanoparticle. Methods: Patients received a single 2-hour IV infusion on Day 1 and 4 of each 28-day cycle. Based on the previous experience with AZD1152 in the same patient population, the expected MTD is in a range of 1,200 mg per cycle. In the ongoing dose escalation, cohorts of 3-6 patients have been sequentially enrolled in 4 cohorts ranging from 100 mg to 600 mg per infusion on Day 1 and 4 every 28 days, i.e. from 200 mg to 1,200 mg per cycle. Patients were allowed to continue to receive AZD2811 until disease progression, tolerability, or discontinuation criteria have been met. The study uses a Bayesian adaptive design approach to dose escalation to improve the efficiency and precision of the MTD estimation compared to a traditional 3+3 design. Results: Currently, 10 pts with age ranges from 56 to 86 years have completed DLT assessment period for the first 3 cohorts. 9 patients had relapsed/refractory secondary AML/MDS after failure of hypomethylating agents and 1 patient had a relapsed, therapy-related AML. Cohort 4 (600 mg per infusion D1 and D4) is currently under evaluation. Of the 10 pts in cohorts 1-3, 7 pts discontinued due to disease progression, 1 discontinued due to subject decision/consent withdrawal, 1 discontinued due to physician decision, and 1 pt is active and ongoing. AEs assessed as related to AZD2811 that occurred in one or more patients were Grade 3/4 neutropenia/thrombocytopenia, Grade 3 anemia and Grade 1 fatigue, rash and stomatitis. Thus far, no DLTs and no fatal AEs related to AZD2811 have been observed. 2 deaths have been reported, 1 due to the underlying disease and 1 due to a Serious Adverse Event of Gr 5 Sepsis (not related to study drug). AZD2811 total blood PK appears dose proportional with a t1/2 of 30-50 hours. Conclusion: AZD2811 nanoparticle is safe and well tolerated at a dose up to 400 mg on Day 1 and 4 every 28-days. The monotherapy dose escalation is ongoing and updated results including preliminary efficacy data and supporting preclinical data will be presented. Additional dose finding and expansion cohorts of AZD2811 nanoparticle in combination with azacytidine and venetoclax are planned. Disclosures Atallah: Novartis: Consultancy; Abbvie: Consultancy; BMS: Consultancy; Jazz: Consultancy; Pfizer: Consultancy. Charlton:AstraZeneca: Employment. MacDonald:AstraZeneca: Employment. Young:AstraZeneca: Employment. Sainsbury:AstraZeneca: Employment. Overend:AstraZeneca: Employment. Adelman:AstraZeneca: Employment. Travers:AstraZeneca: Employment. Smith:AstraZeneca: Employment. Pease:AstraZeneca: Employment. Brugger:AstraZeneca: Employment.

The BCR-ABL oncogene is central in the pathogenesis of chronic myeloid leukemia (CML). Here, tandem nanospray mass spectrometry was used to demonstrate cell surface HLA-associated expression of the BCR-ABL peptide KQSSKALQR on class I-negative CML cells transfected with HLA-A*0301, and on primary CML cells from HLA-A3–positive patients. These patients mounted a cytotoxic T-lymphocyte response to KQSSKALQR that also killed autologous CML cells, and tetramer staining demonstrated the presence of circulating KQSSKALQR-specific T cells. The findings are the first demonstration that CML cells express HLA-associated leukemia-specific immunogenic peptides and provide a sound basis for immunization studies against BCR-ABL.

Reactivation of cytomegalovirus (CMV) remains a serious complication after allogeneic stem cell transplantation, but the role of γδ T cells is undefined. We have studied the immune reconstitution of Vδ2negative (Vδ2neg) γδ T cells, including Vδ1 and Vδ3 subsets and Vδ2positive (Vδ2pos) γδ T cells in 40 patients during the first 24 months after stem cell transplantation. Significant long-term expansions of Vδ2neg but not Vδ2pos γδ T cells were observed during CMV reactivation early after transplantation, suggesting direct involvement of γδ T cells in anti-CMV immune responses. Similarly, significantly higher numbers of Vδ2neg γδ T cells were detected in CMV-seropositive healthy persons compared with seronegative donors; the absolute numbers of Vδ2pos cells were not significantly different. The expansion of Vδ2neg γδ T cells appeared to be CMV-related because it was absent in CMV-negative/Epstein-Barr virus-positive patients. T-cell receptor-δ chain determining region 3 spectratyping of Vδ2neg γδ T cells in healthy subjects and patients showed restricted clonality. Polyclonal Vδ2neg cell lines generated from CMV-seropositive healthy donors and from a recipient of a graft from a CMV-positive donor lysed CMV-infected targets in all cases. Our study shows new evidence for role of γδ T cells in the immune response to CMV reactivation in transplantation recipients.

Upon differentiation, hematopoietic stem cells (HSC) give rise to multipotent progenitors (MPP) that retain the ability to produce all blood lineages but have lost their self-renewal capacity. MPP are then orientated towards either the lymphoid or myeloid lineages, developing into common myeloid progenitors (CMP) or lymphoid-primed multipotent progenitors which can still produce certain myeloid cell types. CMP can differentiate into either granulocyte-macrophage progenitors (GMP) or megakaryocyte-erythroid progenitors. GMP finally differentiate into granulocyte or monocyte progenitors (GP/MP). This study aimed to investigate whether acute myeloid leukemia (AML) samples can be sub-classified based on the stage of arrest in differentiation (Stage of Leukemia Arrest, SLA). Understanding which critical differentiation program is specifically altered could be of special interest to design new therapeutic strategies aimed at re-inducing the differentiation process in AML. Based on CD34/CD117/CD13/CD33/MPO expression, we defined phenotypic signatures that identify all normal hematopoietic stem and progenitor cells (HSPC). Applied to leukemic hematopoiesis, these signatures allowed us to sub-classify 932 AML patients from Toulouse University Hospital (test cohort) and 142 AML patients from The Cancer Genome Atlas Network, published in NEJM in 2013 (control cohort). Globally, AML with an HSC phenotype (henceforth termed HSC-L) represented 1% of the cases, while MPP-L, CMP-L, GMP-L and GP/MP-L accounted for 15%, 30%, 22% and 30% of the cases, respectively. To validate our phenotypic signature and SLA, we generated a transcriptomic signature of normal hematopoietic differentiation from three databases including normal HSPC assessment (GSE21973, GSE42414 and E-TABM-978). Unsupervised clustering and gene set enrichment analysis (GSEA) of a test cohort (n=40) and a control cohort (n=142) confirmed that AML shared a common transcriptomic signature with their SLA. To strengthen the relationship between SLA and HSPC, we then compared their energetic metabolism. Using metformin as an enhancer of glycolytic (GLY) metabolism (e.g. Pasteur effect), we constructed transcriptomic signatures for AML cell lines with either a GLY or with an oxidative (OX) metabolism. We showed that, as expected normal HSC and MPP were enriched in GLY genes, whereas CMP and GMP were enriched in OX genes set. Similarly, HSC-L and MPP-L highly expressed genes related to GLY metabolism; CMP-L samples could be subdivided between GLY AML and OX AML; and GMP-L highly expressed genes related to OX metabolism. Each AML subgroup defined accordingly to the SLA had a particular clinical presentation (Table). Interestingly, the least differentiated AML (HSC-L, MPP-L and CMP-L) were enriched in HSC and LSC genes sets. These AML samples blocked at early stage of hematopoiesis gave higher engraftment levels in NSG mice compared to GMP-L and GP/MP-L (21%, vs 5%, panel of 59 AML tested in 376 mice, p We then sought to investigate the distribution of AML mutations according to SLA classification. We took advantage of the fully annotated TCGA database and found that 80% of HSC-L and 25% of MPP-L had RUNX1 mutations, 80% of GMP-L had either CEBPA mutation or RUNXL1-RUNX1/CBFB-MYH11 AML, and 80% of GP/MP-L had NPM1 mutation. The subgroups of CMP-L showed a greater molecular heterogeneity. AML and normal HSPC shared phenotypic, transcriptomic and metabolic signatures. This is of special interested for differentiation therapy as specific critical dysregulated transcriptional factors (e.g. CEBPA) could be related to specific stage of arrest. Identifying each of them could allow to propose targeted differentiating therapy and maximize therapeutic responses. Table. HSC-L MPP-L CMP-L GMP-L GP/MP-L frequency 1% 15% 30% 22% 30% Median WBC at diagnosis (G/L) 4 25 20 35 54 organ infiltration NA spleen varied lymph nodes gengiva or lymph nodes CFU-L (% of blasts) 0.7 8.1 9.6 8.7 6.9 metabolism GLY GLY GLY/OX OX NA oncogenic event RUNX1m RUNX1m/TP53m unknown CEBPAm/CBF NPM1c BAALC/ERG/MN1 expression high high mid mid low stem cell signature high high high low low Median DFS (months) 5 22.9 13.7 41.3 >120 Median OS (months) 6.6 26.6 16.3 >120 83.3 Disclosures Huguet: Novartis: Consultancy, Research Funding; BMS: Consultancy, Speakers Bureau; ARIAD: Consultancy, Speakers Bureau; PFIZER: Consultancy, Speakers Bureau.

The signals that mediate T-cell infiltration during T-cell autoimmune diseases are poorly understood. The chemokine CCL21 (originally isolated by us and others as Exodus-2/6Ckine/SLC/TCA4) is highly potent and highly specific for stimulating T-cell migration. However, it is thought to be expressed only in secondary lymphoid organs, directing naive T cells to areas of antigen presentation. It is not thought to play a role in T-cell effector function during a normal immune response. In this study we tested the expression of T-cell chemokines and their receptors during T-cell autoimmune infiltrative skin diseases. By using immunohistology it was found that the expression of CCL21 but not CCL19 or 20 was highly induced in endothelial cells of T-cell autoimmune diseases. The receptor for CCL21, CCR7, was also found to be highly expressed on the infiltrating T cells, most of which expressed the memory CD45Ro phenotype. These data imply that the usual loss of CCL21 responsiveness in the normal development of memory T-cell effector function does not hold for autoimmune skin diseases.

Abstract 674 Background. Activating mutations in FLT3 occur in ∼30% of adult acute myeloid leukemia (AML) cases, including internal tandem duplication (ITD) mutations (∼25%) and point mutations in the tyrosine kinase domain (KD), primarily at the activation loop (AL) residue D835 (∼5%). FLT3-ITD and FLT3-AL mutations can co-occur in individual patients. AC220 (quizartinib) is a potent selective investigational FLT3/KIT inhibitor with encouraging preliminary clinical activity in FLT3-ITD+ AML, as evidenced by a composite complete remission rate of 45% in 53 chemotherapy refractory/relapsed patients in an interim analysis of a phase II study (Cortes et al, ASH 2011 abstract #614). While some patients without FLT3 mutations in the AC220 phase I dose escalation study responded, no objective response was observed in a small number of FLT3-AL mutant patients (Cortes et al, ASH 2008 abstract #767). We recently reported that secondary mutations at F691, D835 and Y842 in FLT3-ITD confer in vitro resistance to AC220, and that relapse in 8/8 AML patients who initially responded to AC220 was associated with evolution of secondary KD mutations in the FLT3-ITD+ allele (Smith et al., Nature, 2012). In 6 of 8 patients, relapse was associated with D835 substitutions, making this amino acid position the most commonly substituted residue in FLT3-ITD+ patients at the time of disease relapse. Given that D835 mutations in FLT3-ITD appear to confer a high degree of pre-clinical and clinical resistance to AC220, we hypothesized that D835 mutations in the absence of the ITD might also confer resistance to AC220 and could be associated with clinical relapse, especially in patients harboring a mixture of FLT3-WT, FLT3-ITD+, and FLT3-AL mutant blasts. Results. In vitro binding studies revealed that FLT3-D835V and FLT3-D835Y have decreased affinity for AC220 (Kd=4.8 and 7nM, respectively) compared to native FLT3 (Kd=1.8nM). In proliferation assays of BaF/3 cells, FLT3-D835V/Y mutants demonstrated increased relative AC220 resistance (IC50 24 and 6.8nM) compared to FLT3-ITD (IC50 0.13nM). To sensitively and precisely determine the frequency and phase (ITD+ vs ITD-) of mutations in clinical isolates, we utilized single molecule real-time (SMRT; Pacific Biosciences, Menlo Park, CA) sequencing, which can generate reads of sufficient length to enable focused interrogation of the KD of FLT3 ITD+ and ITD- alleles. We analyzed ITD- sequences from pretreatment and relapse samples obtained from the 8 FLT3-ITD-positive AML patients with acquired AC220 resistance reported in our previous analysis. Interestingly, all 6 patients with secondary mutations at D835 in ITD+ alleles at relapse also harbored D835 mutant ITD- alleles. All D835 mutations detected in ITD- alleles were also observed in ITD+ alleles from the same patient sample. In ITD+ alleles, we detected D835Y (n=6), D835V (n=3), and D835F (n=2) (range 2.7–50.6% of ITD+ alleles). In ITD- alleles, we found D835Y (n=6), D835V (n=2), and D835F (n=1) (range 3.8–50% of ITD- alleles). One patient had 2 D835 mutations (D835Y/F) in ITD- alleles at relapse. Although 2 of 8 patients evolved the F691L mutation in ITD+ alleles at relapse, this substitution was not detectable in ITD- sequences in any patient. In 7/8 patients, samples obtained immediately prior to AC220 administration were available for analysis. We were unable to document the presence of AC220-resistant mutations in either ITD+ or ITD- sequences in these pretreatment samples, suggesting that these mutants evolved under the selective pressure of AC220 treatment. We are currently performing single cell RT-PCR to determine if D835 mutations in ITD+ and ITD- alleles occur in distinct leukemic cells, which would suggest a polyclonal blast population at relapse. Conclusions. FLT3-AL mutations at D835 confer resistance to AC220 in vitro. The evolution of D835 substitutions in ITD- alleles in the majority of patients who respond and relapse on AC220 suggests that constitutively activating FLT3-AL mutations at residue D835 can confer acquired clinical resistance to AC220. AML patients with FLT3-D835 mutations may exhibit de novo resistance to AC220 and other FLT3 inhibitors. Our findings predict that the clinical activity of potent FLT3 inhibitors that are tolerant of D835 substitutions will mechanistically involve inhibition of D835 mutants in both the presence and absence of an ITD mutation. Disclosures: Chin: Pacific Biosciences: Employment. Levis:Astellas Pharma: Consultancy; Plexxikon: Consultancy; Symphogen: Consultancy; Ambit: Joined performance of clinical trial, Joined performance of clinical trial Other. Perl:Astellas Pharmaceuticals: Consultancy. Travers:Pacific Biosciences: Employment. Kasarskis:Pacific Biosciences: Equity Ownership. Radich:Ariad: Consultancy; Bristol-Myers Squibb: Consultancy; Pfizer: Consultancy; Novartis: Consultancy, Research Funding. Shah:Ariad: Consultancy, Research Funding.

Abstract 917 Background: Secondary kinase domain (KD) mutations represent the most well-documented mechanism of resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML). In CML, multiple TKIs with different mutation profiles are approved and the ability to detect KD mutations at the time of disease progression can impact therapy choice. To optimize clinical impact, second generation TKI selection must consider the majority TKI-resistant mutant population as well as smaller mutant sub-populations that may be selected with subsequent treatment. Sequential TKI therapy is associated with additional complexity: multiple mutations can coexist separately in an individual patient (“polyclonality”) or can occur in tandem on a single allele (“compound mutations”). Multiple mutations are associated with poor clinical outcome (Parker et al., Blood 2012). Compound mutations can cause in vitro resistance to ponatinib, the only TKI clinically active against the highly resistant T315I mutation (Eide, et. al, ASH 2012 abstract #1416). Currently, no clinically adaptable technology can distinguish polyclonal from compound mutations. Due to the size of the BCR-ABL KD, most next-generation sequencing platforms cannot generate reads of sufficient length to determine if mutations separated by ≥500 nt reside on the same allele. Pacific Biosciences RS Single Molecule Real Time (SMRT) sequencing technology is a third generation deep sequencing technology capable of achieving average read lengths of ∼1000bp and frequently >3000bp, enabling sensitive and accurate sequencing of the entire ABL KD on a single strand of DNA. Though allele-specific detection methods such as MassARRAY offer sensitivity as low as ∼0.5%, these assays are designed to detect a limited number (∼31) of mutations whereas SMRT sequencing offers an unbiased approach capable of detecting novel variants. We sought to (1) develop a potential clinically-applicable SMRT sequencing assay for the detection of BCR-ABLKD mutations capable of distinguishing polyclonal and compound mutations, and (2) compare the accuracy and sensitivity of this method to standard sequencing and MassARRAY. Results: We assessed 54 samples from 36 CML patients who had clinically relapsed on ABL kinase inhibitor therapy and were previously analyzed by standard sequencing, and in a subset, by MassARRAY. We amplified an 863bp area of the BCR-ABLKD from patient-derived cDNA with primers containing 5' barcodes, enabling sequencing of 6 to 8 patient samples on a single SMRT cell on a single run. On average, 2519 reads were obtained for each sample per run (range 330 to 10,240). All of 131 known mutations detected by MassARRAY were identified by SMRT sequencing using a p-value threshold of 1.03e–03. SMRT sequencing also identified all 107 known mutations detected by direct sequencing with a p-value threshold of 6.0e–08. In addition to these known mutations, SMRT sequencing detected an additional 1320 non-silent mutations across all patient samples using a strict p-value threshold cut-off of 6e–08, ranging in abundance from 0.2% to 17% (median 0.75%). Among 47 samples where >1 mutation was detectable by direct sequencing or MassARRAY, SMRT sequencing revealed that 40 (85%) had compound mutations detectable at a frequency of ≥1. In total, we detected 73 different compound mutations at a frequency of ≥1%. In all cases where compound mutations were detected and more than one treatment timepoint was available, at least one compound mutation clearly evolved from a mutation detectable at a prior timepoint. In the most complex case, 4 separate mutations yielded 8 different mutant alleles. Conclusions: Pacific Biosciences RS SMRT sequencing detects KD mutations in patient samples with sensitivity comparable to or better than MassARRAY and can distinguish compound from polyclonal mutant clones. Among patient samples with multiple mutations, compound mutations were detectable in the vast majority of samples by SMRT sequencing, revealing a complex mutational landscape not demonstrable by other clinically viable sequencing methods and previously unappreciated. Given the growing numbers of patients exposed to multiple TKIs in a sequential manner, the ability to accurately and sensitively characterize drug-resistant alleles by SMRT sequencing promises to further facilitate a personalized approach to patient management and inform models of disease evolution. Disclosures: Brown: Pacific Biosciences: Employment. Travers:Pacific Biosciences: Employment. Wang:Pacific Biosciences: Employment. Branford:Novartis : Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad : Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cepheid : Consultancy. Shah:ARIAD, Bristol Myers-Squibb: Consultancy, Research Funding; Novartis: Consultancy.

Abstract 3752 Background: Secondary kinase domain (KD) mutations are the most well-recognized mechanism of resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) and other cancers. In some cases, multiple drug resistant KD mutations can coexist in an individual patient (“polyclonality”). Alternatively, more than one mutation can occur in tandem on a single allele (“compound mutations”) following response and relapse to sequentially administered TKI therapy. Distinguishing between these two scenarios can inform the clinical choice of subsequent TKI treatment. There is currently no clinically adaptable methodology that offers the ability to distinguish polyclonal from compound mutations. Due to the size of the BCR-ABL KD where TKI-resistant mutations are detected, next-generation platforms are unable to generate reads of sufficient length to determine if two mutations separated by 500 nt reside on the same allele. Pacific Biosciences RS Single Molecule Real Time (SMRT) circular consensus sequencing technology is a novel third generation deep sequencing technology capable of rapidly and reliably achieving average read lengths of ∼1000bp (Travers et al, 2010) and frequently beyond 3000bp, allowing sequencing of the entire ABL KD on single strand of DNA. We sought to address the ability of SMRT sequencing technology to distinguish polyclonal from compound mutations using clinical samples obtained from patients who have relapsed on BCR-ABL TKI treatment. Results: We analyzed an 863bp area of the BCR-ABL KD in 6 patients who had clinically relapsed on ABL kinase inhibitor therapy. SMRT sequencing detected mutations at a sensitivity of ∼1–2% of the total sequenced population, and successfully distinguished polyclonal from compound BCR-ABL KD mutations in several patient samples. Results were largely consistent with those obtained by PCR subcloning and sequencing, although SMRT sequencing detected additional mutations and/or mutation combinations. In the most complex case, 7 distinct mutation-bearing alleles were detected in an individual patient after sequential relapse on imatinib and dasatinib. Mutant clones contained single and compound mutations combining distinct mutations (Y253H, T315F, T315A, T315I, T319A, E355G). Three distinct substitutions at residue T315 were detected: T315A, T315I and T315F. Notably, these findings are clinically important as the T315A mutation confers resistance to dasatinib but not imatinib, while the T315F and T315I mutations are resistant to all three clinically approved BCR/ABL inhibitors (imatinib, dasatinib, and nilotinib). Phospho-flow analysis for p-Crkl, a direct substrate of BCR-ABL, was conducted following ex vivo exposure of patient cells from the same time point to all three BCR-ABL inhibitors, and demonstrated the existence of distinct populations of cells with varying sensitivity to each drug (i.e. polyclonal drug sensitivity), underscoring the potential clinical importance of distinguishing polyclonal from compound mutations. Additionally, SMRT sequencing routinely detected alleles harboring compound mutations not detectable by conventional direct sequencing. Data analysis of samples from additional patients is ongoing and will be presented. Conclusions: Pacific Biosciences RS SMRT sequencing sensitively detects KD mutations in patient samples and can distinguish TKI-resistant clones containing compound mutations to reveal a complex mutational landscape in an individual patient not detectable by conventional sequencing. SMRT sequencing of the BCR-ABL KD can feasibly be developed into a rapid and economical clinical test with the additional advantages of increased sensitivity and reliability over current methods. Given the growing numbers of patients exposed to multiple TKIs in a sequential manner, the ability to accurately and sensitively characterize drug-resistant alleles promises to further facilitate a personalized approach to patient management. Disclosures: Brown: Pacific Biosciences: Employment. Chin:Pacific Biosciences: Employment. Travers:Pacific Biosciences: Employment. Wang:Pacific Biosciences: Employment. Kasarskis:Pacific Biosciences: Employment, Equity Ownership. Schadt:Pacific Biosciences: Employment, Equity Ownership.

Abstract 937 Background . Activating in-tandem duplication (ITD) mutations within the FLT3 juxtamembrane region are detected in ∼25% of adult acute myeloid leukemia (AML) cases and portend a poor prognosis. AC220 (quizartinib) is a potent selective investigational FLT3/KIT inhibitor with encouraging preliminary clinical activity, as evidenced by a composite complete remission rate of 43% in 53 chemotherapy refractory/relapsed AML patients evaluable for an interim analysis of the ongoing phase II study in FLT3-ITD+ relapsed/refractory AML (Cortes et al, EHA 2011 abstract 1019). Many patients who initially respond ultimately suffer disease progression. We sought to utilize the clinical efficacy of AC220 to determine if FLT3-ITD is a valid therapeutic target in human AML. A saturation mutagenesis strategy identified amino acid substitutions at three amino acid residues (F691, D835, Y842) in FLT3-ITD that confer a high degree of resistance to AC220 in vitro. Of these, substitutions at F691 and D835 conferred the greatest degree of relative resistance. Results . To assess the validity of FLT3-ITD as a therapeutic target in AML, we analyzed paired pretreatment and relapse samples obtained from a cohort of 9 FLT3-ITD-positive AML patients enrolled in the exploratory part of the ongoing phase II study of AC220 in relapsed/refractory AML (clinicaltrials.gov NCT00989261) who relapsed after initially achieving morphologic clearance of bone marrow blasts to In an expanded analysis of genomic DNA samples from 30 patients enrolled in the exploratory part of the Phase II study who discontinued study drug for any reason, we observed the occurrence of acquired mutations in the kinase domain (D835 and F691) in a total of 10 of 30 (33%) patients at the off study timepoint. One patient had a D835Y mutation prior to going on AC220. Molecular docking studies were undertaken to provide mechanistic insights into the structural basis of resistance conferred by AC220-resistant mutations. These studies revealed that AC220 likely binds strongly to the DFG-out inactive FLT3 kinase domain. AC220 directly interacts with the gatekeeper residue F691, explaining the drug-resistance associated with the F691L mutation. Mutations at D835 and Y842 may potentially de-stabilize the inactive conformation, and result in a more active, AC220 binding-deficient FLT3 kinase. Indeed, substitutions at these residues are known to activate FLT3 in the absence of an ITD mutation. To more precisely assess the frequency and identity of resistance-conferring mutations at relapse, we analyzed a subset of samples using single molecule real-time (SMRT™; Pacific Biosciences, Menlo Park, CA) sequencing, which can generate reads of sufficient length to enable focused interrogation of the kinase domain of FLT3-ITD alleles. With this assay, more than 350 reads of >1000 nucleotides were reliably obtained. Analyses of pretreatment and relapse samples from four patients confirmed the presence of resistance-conferring FLT3-ITD kinase domain mutations at F691 or D835 at relapse in 36–60% of FLT3-ITD sequence reads. Additionally, this method detected polyclonal resistance in two of the four samples assessed. Conclusions . Our studies validate FLT3-ITD as a therapeutic target in a proportion of AML cases, and demonstrate that the clinical activity of AC220 is mediated by FLT3-ITD inhibition. AC220-resistant FLT3-ITD gatekeeper and activation loop mutations identified in clinical samples from relapsing patients represent high-value therapeutic targets for next-generation FLT3 inhibitors. Disclosures: Off Label Use: AC220 is an investigational agent and has no approved drug indication in AML. Chin:Pacific Biosciences: Employment. Hunt:Ambit Biosciences: Employment. Levis:Ambit Biosciences, Inc: Consultancy. Travers:Pacific Biosciences: Employment. Wang:Pacific Biosciences: Employment. Kasarskis:Pacific Biosciences: Employment, Equity Ownership. Schadt:Pacific Biosciences: Employment, Equity Ownership.