ALBERT

Description: Abstract 915 Background: Many patients (pts) with advanced Ph+ leukemias experience failure of all currently available tyrosine kinase inhibitors (TKIs) targeting BCR-ABL and have limited treatment options. Ponatinib is a potent pan-BCR-ABL inhibitor that is active against native and mutated forms of BCR-ABL, including the TKI resistant T315I mutant. The efficacy and safety of ponatinib (45 mg orally once daily) in pts with AP-CML, BP-CML, or Ph+ ALL were evaluated in a phase 2, international, open-label clinical trial. Methods: The PACE trial enrolled 449 pts, including 85 AP-CML, 62 BP-CML, and 32 Ph+ ALL. Pts were resistant or intolerant (R/I) to dasatinib or nilotinib, or had the T315I mutation at baseline. AP-CML, BP-CML, and Ph+ ALL pts were assigned to 1 of 4 cohorts: AP-CML R/I, AP-CML T315I, BP-CML/Ph+ ALL R/I, BP-CML/Ph+ ALL T315I. Two AP-CML pts were not assigned to a cohort (post-imatinib, did not have T315I at baseline) and were excluded from efficacy analyses and included in safety analyses. The primary endpoint was major hematologic response (MaHR) at any time within 6 mos after treatment initiation. Data as of 23 July 2012 are reported, with a minimum follow-up of 9 mos (median 13 [4 to 21], 6 [0.1 to 18], and 6 [0.1 to 16] mos for AP-CML, BP-CML, and Ph+ ALL, respectively). Results: The median age for AP-CML, BP-CML, and Ph+ ALL pts was 60, 53, and 62 yrs, respectively. Median time from initial disease diagnosis to start of ponatinib was 7, 4, and 1.5 yrs, respectively. Pts were heavily pretreated: 94% received prior imatinib, 88% dasatinib, 61% nilotinib; 8% received 1 prior approved TKI, 39% received 2, and 53% received 3. Sixteen percent had undergone prior stem cell transplant. In pts previously treated with dasatinib or nilotinib (N=171), 94% had a history of resistance to dasatinib or nilotinib, 6% were purely intolerant. Reported MaHR rates with the most recent dasatinib or nilotinib therapy were 35% AP-CML, 16% BP-CML, 43% Ph+ ALL. At the time of analysis, 59% of AP-CML, 8% of BP-CML, and 9% of Ph+ ALL pts remained on study. Overall, the most common reasons for discontinuation were progressive disease (19%, 50%, and 53%, respectively) and adverse events (AEs; 11%, 16%, and 6%, respectively). Hematologic and cytogenetic response rates are shown in the table; MaHR and MCyR were observed across cohorts. MMR was achieved by 14% of AP-CML pts (14% R/I, 17% T315I). There was a trend for higher response rates among pts who received fewer prior approved TKIs. In AP-CML pts, the differences in MaHR rates by number of prior approved TKIs (1: 3/4 [75%]; 2: 20/33 [61%]; 3: 24/46 [52%]) were not significant (Fisher's Exact); differences in MCyR rates (1: 4/4 [100%]); 2: 13/33 [39%]; 3: 15/46 [33%]) were significant for pts treated with 1 vs 2 (p=0.0360) and 1 vs 3 prior approved TKIs (p=0.0168). Of pts achieving MaHR, 42% of AP-CML and 35% of BP-CML/Ph+ ALL pts were projected (Kaplan-Meier) to remain in MaHR at 1 yr. In AP-CML, the median progression-free survival (PFS) was estimated (Kaplan-Meier) as 80 (range 6 to 88) wks; the probability of maintaining PFS at 6 mos and 1 yr was estimated as 80% and 57%, respectively. Median overall survival (OS) had not yet been reached; the probability of OS at 6 mos and 1 yr was estimated (Kaplan-Meier) as 96% and 85%, respectively. In BP-CML/Ph+ ALL, median PFS was estimated as 18 (range 0.1 to 74) wks; the probability of maintaining PFS at 6 mos and 1 yr was estimated as 34% and 20%, respectively. Median OS was estimated as 30 (range 0.4 to 77) wks; the probability of OS at 6 mos and 1 yr was estimated as 54% and 34%, respectively. Ponatinib was generally well-tolerated; the most common treatment-related AEs were thrombocytopenia (29%), rash (25%), and neutropenia (22%). The most common serious treatment-related AEs were thrombocytopenia (3%) and pancreatitis (3%). Rash was generally grade 1 or 2 in severity. Thrombocytopenia, neutropenia, and pancreatitis were typically reported early in treatment and were manageable with dose modification. Conclusions: Ponatinib was generally well-tolerated and had substantial activity in pts with AP-CML, BP-CML, or Ph+ ALL, regardless of mutation status or prior therapy. Data with a minimum follow-up of 12 mos will be presented Disclosures: Kantarjian: Novartis: Consultancy; Pfizer: Research Funding; BMS: Research Funding; Novartis: Research Funding; ARIAD: Research Funding. Off Label Use: ponatinib. Kim:Novartis, Bristol Myers-Squibb, Pfizer, ARIAD, and Il-Yang: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Pinilla-Ibarz:Novartis, BMS: Research Funding, Speakers Bureau. le Coutre:Novartis and BMS: Honoraria. Paquette:ARIAD: Consultancy. Chuah:Novartis and Bristol Myers-Squibb: Honoraria. Nicolini:Novartis, Bristol Myers Squibb, Pfizer, ARIAD: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Apperley:Novartis, Bristol Myers-Squibb, and ARIAD: Honoraria, Research Funding. Talpaz:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millenium: Research Funding; Celgene: Research Funding; ARIAD: Research Funding; Deciphera: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees. Abruzzese:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees. Rea:Bristol Myers-Squibb, Novartis, and Teva: Honoraria. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Muller:ARIAD: Consultancy. Wong:MolecularMD Corp: Employment, Equity Ownership. Lustgarten:ARIAD Pharmaceuticals, Inc.: Employment, Equity Ownership. Rivera:ARIAD Pharmaceuticals, Inc.: Employment, Equity Ownership. Clackson:ARIAD: Employment, Equity Ownership. Turner:ARIAD: Employment, Equity Ownership. Haluska:ARIAD: Employment, Equity Ownership. Guilhot:ARIAD: Honoraria. Hochhaus:ARIAD, Novartis, Bristol Myers-Squibb, Pfizer, and MSD: Membership on an entity's Board of Directors or advisory committees, Research Funding. Hughes:Novartis, BMS, ARIAD: Honoraria, Research Funding. Goldman:Novartis, Bristol Myers Squibb, Amgen: Honoraria. Shah:ARIAD Pharmaceuticals: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Novartis: Consultancy. Cortes:Novartis, BMS, ARIAD, Pfizer, and Chemgenex: Consultancy, Research Funding.

Description: Abstract 163 Background: Despite progress in Ph+ leukemia therapy, patients who experience failure of tyrosine kinase inhibitors (TKIs) and those with the T315I BCR-ABL mutation have limited treatment options. Ponatinib is an oral TKI developed using computational and structure-based design with optimal binding to the BCR-ABL active site. At clinically achievable concentrations, ponatinib demonstrated potent in vitro activity against native BCR-ABL and all BCR-ABL mutants tested, including T315I. The efficacy and safety of ponatinib (45 mg orally once daily) in patients with Ph+ leukemia were evaluated in a phase 2, international, open-label clinical trial. Methods: 449 patients resistant or intolerant (R/I) to dasatinib or nilotinib or with the T315I mutation confirmed at entry were enrolled and assigned to 1 of 6 cohorts: chronic phase (CP)-CML R/I (N=203), CP-CML T315I (N=64), accelerated phase (AP)-CML R/I (N=65), AP-CML T315I (N=18), blast phase (BP)-CML/Ph+ALL R/I (N=48), BP-CML/Ph+ALL T315I (N=46). Five patients (3 CP-CML, 2 AP-CML) without confirmed T315I and not R/I to dasatinib or nilotinib were treated, but not assigned to a cohort; they were included in safety analyses. The primary endpoint was major cytogenetic response (MCyR) at any time within 12 months for CP-CML and major hematologic response (MaHR) at any time within 6 months for advanced Ph+ leukemia. The trial is ongoing. Data as of 23 July 2012 are reported: median follow-up 11 (0.1 to 21) months; minimum follow-up 9 months. Results: Median age was 59 (18–94) yrs; 53% were male. Median time from diagnosis to ponatinib was 6 (0.3–28) yrs. Patients were heavily pretreated: 96% received prior imatinib, 84% dasatinib, 65% nilotinib; median number of prior TKIs was 3, with 53% exposed to all 3 approved TKIs. In patients previously treated with dasatinib or nilotinib (N=427), 88% had a history of resistance and 12% were purely intolerant to dasatinib or nilotinib. Best prior response to most recent dasatinib or nilotinib was 26% MCyR or better in CP-CML, and 23% MaHR or better in advanced Ph+ leukemia. Frequent BCR-ABL mutations confirmed at entry were: 29% T315I, 8% F317L, 4% E255K, 4% F359V, 3% G250E. No mutations were detected in 44%. The primary endpoint response rates (see Table) in each cohort exceeded the prespecified statistical criteria for success. In CP-CML and AP-CML R/I (the 3 largest cohorts), 95% CIs exceeded the prespecified response rate. Median time to response (for responders) was 84 days in CP-CML, 112 days in AP-CML, 55 days in BP-CML/Ph+ALL. Responses were durable; the estimated (Kaplan-Meier) probability of responders maintaining the primary endpoint at 1 yr was 91% in CP-CML, 42% in AP-CML, 35% in BP-CML/Ph+ALL. In CP-CML, 46% had complete cytogenetic response and molecular response rates were 32% MMR, 20% MR4, and 12% MR4.5. Response rates were higher in patients exposed to fewer prior TKIs and those with shorter disease duration. Similar response rates were observed in patients with and without BCR-ABL mutations. In CP-CML, response rates were higher in those with T315I; however, a post hoc analysis found that presence of T315I was not a predictor of response. Instead, the difference in response rate was explained by T315I patients' younger age, shorter duration of leukemia, and exposure to less prior therapy. At the time of analysis, 52% of patients remained on therapy (66% CP-CML). The most frequent reasons for discontinuation were progression (18%) and AEs (12%). The most common drug-related AEs were thrombocytopenia (36%), rash (33%), and dry skin (31%). Pancreatitis was the most common drug-related SAE (5%); however, it occurred early and was managed with dose modification (1 patient discontinued due to pancreatitis). Conclusions: Ponatinib has substantial activity and is generally well tolerated in these heavily pretreated Ph+ leukemia patients who have limited available treatment options. Data with a minimum follow-up of 12 months will be presented. Disclosures: Cortes: Novartis, BMS, ARIAD, Pfizer, and Chemgenex: Consultancy, Research Funding. Kim:Novartis, BMS, Pfizer, ARIAD, Il-Yang: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Pinilla-Ibarz:Novartis, BMS: Research Funding, Speakers Bureau. le Coutre:Novartis and BMS: Honoraria. Paquette:ARIAD: Consultancy. Chuah:Novartis, Bristol-Myers Squibb: Honoraria. Nicolini:Novartis, Bristol Myers Squibb, Pfizer, ARIAD: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Apperley:Novartis, Bristol Myers-Squibb, and ARIAD: Honoraria, Research Funding. Talpaz:Deciphera: Research Funding; ARIAD: Research Funding; Celgene: Research Funding; Millenium: Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees. Abruzzese:BMS, Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Rea:Bristol Myers-Squibb, Novartis, and Teva: Honoraria. Baccarani:ARIAD, Novartis, Bristol Myers Squibb, Pfizer: Consultancy, Honoraria, Speakers Bureau. Muller:ARIAD: Consultancy. Wong:MolecularMD Corp: Employment, Equity Ownership. Lustgarten:ARIAD Pharmaceuticals, Inc.: Employment, Equity Ownership. Rivera:ARIAD: Employment, Equity Ownership. Clackson:ARIAD: Employment, Equity Ownership. Turner:ARIAD: Employment, Equity Ownership. Haluska:ARIAD: Employment, Equity Ownership. Guilhot:ARIAD: Honoraria. Hochhaus:ARIAD, Novartis, BMS, Pfizer, MSD: Membership on an entity's Board of Directors or advisory committees, Research Funding. Hughes:Novartis, BMS, ARIAD: Honoraria, Research Funding. Goldman:Novartis, Bristol Myers-Squibb, and Amgen: Honoraria. Shah:ARIAD: Consultancy, Research Funding; Briston-Myers Squibb: Consultancy, Research Funding; Novartis: Consultancy. Kantarjian:Novartis: Consultancy, Research Funding; BMS: Research Funding; ARIAD: Research Funding; Pfizer: Research Funding.

Description: Chronic myeloid leukemia is effectively treated with imatinib, but reactivation of BCR-ABL frequently occurs through acquisition of kinase domain mutations. The additional approved ABL tyrosine kinase inhibitors (TKIs) nilotinib and dasatinib, along with investigational TKIs such as ponatinib (AP24534) and DCC-2036, support the possibility that mutation-mediated resistance in chronic myeloid leukemia can be fully controlled; however, the molecular events underlying resistance in patients lacking BCR-ABL point mutations are largely unknown. We previously reported on an insertion/truncation mutant, BCR-ABL35INS, in which structural integrity of the kinase domain is compromised and all ABL sequence beyond the kinase domain is eliminated. Although we speculated that BCR-ABL35INS is kinase-inactive, recent reports propose this mutant contributes to ABL TKI resistance. We present cell-based and biochemical evidence establishing that BCR-ABL35INS is kinase-inactive and does not contribute to TKI resistance, and we find that detection of BCR-ABL35INS does not consistently track with or explain resistance in clinical samples from chronic myeloid leukemia patients.

Description: BCR-ABL and v-ABL are oncogenic forms of the Abl tyrosine kinase that can cause leukemias in mice and humans. ABL oncogenes trigger multiple signaling pathways whose contribution to transformation varies among cell types. Activation of phosphoinositide 3-kinase (PI3K) is essential for ABL-dependent proliferation and survival in some cell types, and global PI3K inhibitors can enhance the antileukemia effects of the Abl kinase inhibitor imatinib. Although a significant fraction of BCR-ABL-induced human leukemias are of B-cell origin, little is known about PI3K signaling mechanisms in B-lineage cells transformed by ABL oncogenes. Here we show that activation of class IA PI3K and downstream inactivation of FOXO transcription factors are essential for survival of murine pro/pre-B cells transformed by v-ABL or BCR-ABL. In addition, analysis of mice lacking individual PI3K genes indicates that products of the Pik3r1 gene contribute to transformation efficiency by BCR-ABL. These findings establish a role for PI3K signaling in B-lineage transformation by ABL oncogenes. (Blood. 2004;103:4268-4275)

Description: Abstract 2549 Background: BCR-ABL1 mutation testing is recommended for CML and Ph+ ALL patients who fail first line tyrosine kinase inhibitor (TKI) therapy or who have a suboptimal response to therapy. BCR-ABL1 mutations in the kinase domain (KD) of ABL1 account for at least 40–50% of all TKI resistant cases. Rare mutations such as E123Q and T212R in the regulatory domain of ABL upstream of the kinase domain have also been reported to lead to resistance to imatinib. The current gold standard for BCR-ABL1 mutation detection is Sanger sequencing, which has an analytical sensitivity of ∼10–30%. Based on recent findings that mass spectrometry can identify low level BCR-ABL1 mutations that confer clinical resistance in patients sooner than Sanger sequencing, it is likely useful to have a significantly more sensitive BCR-ABL1 test than Sanger sequencing. Although commercial NGS cancer panels have included ABL1 in the region of interest, ABL1 resistance mutations should be sequenced from BCR-ABL1 fusion transcripts instead of being sequenced from genomic DNA as in the commercial panels. Here we developed a fusion transcript based BCR-ABL1 mutation assay on the scalable and cost-effective Ion Torrent platform that has 1–5% sensitivity and comprehensive coverage of the kinase domain, regulatory domain, and the SH2/SH3 domains. The assay was designed to detect both the major and minor BCR-ABL1 fusion gene products and can also detect the micro BCR-ABL1 fusion product accounting for over 99% of all CML and Ph+ ALL patients. Methods: RT and long range PCR was performed to amplify BCR-ABL1 e1, e13, and e14 fusion transcripts and the PCR products were enzymatically fragmented and ligated with Ion Torrent sequencing adaptors. Size-selected libraries were quantified, pooled, amplified with OneTouch system and sequenced with Ion Torrent PGM. Sequencing data was analyzed with Torrent Suite 2.2 and the associated variant caller with variant frequency cutoff adjusted to 1%. Results: Initial work with cell lines harboring the T315I mutation in both e1 and e14 BCR-ABL1 transcript types diluted into wild type cell line demonstrated that Ion Torrent NGS can detect T315I at least down to 1%. In a set of 17 blinded clinical samples, Ion Torrent NGS not only identified all the mutations found by Sanger sequencing but additionally identified rare imatinib resistant mutations such as K357E (MMD-3) present at 7.6%; this mutation was previously reported in patient CD34+CD38-stem cells (Table 1). This patient also expressed the E255K resistance mutation; in patients, the presence of multiple resistance mutations has been shown to be an important predictor of poor response. Understanding whether compound mutations are present in cis or in trans may be important in understanding therapy resistance. For patient MMD-9, although both the predominant mutation G250E (79%) and subclone E255V(12.6%) were identified by both Sanger sequencing and NGS, only Ion Torrent was able to show that the two mutations were on different reads, indicating that the mutations are on different alleles. The BCR-ABL1 Ion Torrent based assay reads DNA fragments between 150–200bps in size and can identify cis and trans mutations from individual fragments with this read length, which is not possible by Sanger sequencing. In MMD-9, another low frequency subclone F359C (3%) was detected by only NGS and may have important implications due to its reported sensitivity to dasatinib and not imatinib or nilotinib. Similarly, in MMD-10 the E255V predominant mutation was identified by both NGS and Sanger sequencing, while the F317L mutation with a low frequency of 1.70% was solely detected by NGS. In this patient, a combination of dasatinib and nilotinib treatment may be required to eliminate the dasatinib sensitive E255V dominant clone and F317L nilotinib sensitive subclone. Ion Torrent NGS was also capable of identifying and calling the 35 base pair 475 insertion from 4 samples (MMD-2 and 8–10), only two of which were detected by Sanger sequencing. In our ongoing study, the low-level mutations not detected in Sanger sequencing will be confirmed with analyses on the MiSeq platform and mutation enrichment methods. Additional patient samples representing 30 CML patients will be analyzed and presented. Disclosures: Wong: MolecularMD: Equity Ownership.

Description: Background: The evolution of BCR-ABL1 tyrosine kinase inhibitors (TKIs), from imatinib to newer agents such as nilotinib, has led to progressively lower levels of disease burden in patients with CML receiving TKI therapy. Currently, real-time quantitative PCR (RQ-PCR) is the most commonly used test for assessing MR in patients with CML and is able to detect BCR-ABL1 transcript levels down to MR4.0 to MR4.5 on the IS. It has been reported that dPCR can detect BCR-ABL1 with greater sensitivity and precision than RQ-PCR. In addition, unlike RQ-PCR, dPCR is able to perform absolute copy number quantification without the use of calibrators; thus, it should be free from calibrator biases. To date, validation of dPCR BCR-ABL1 assays with multiple control genes and standardization to the World Health Organization (WHO) IS reference material have not been reported. Standardization to the IS is critically important to enable appropriate assessment of patient response to therapy and to establish a standard of care. In this study, we describe the development, validation, and IS standardization of a sensitive BCR-ABL1 dPCR assay using 3 different control genes. We also tested the comparability of 3 different dPCR platforms. Methods: Four experiments were included in this study: (1) Three BCR-ABL1 dPCR assays were developed using ABL1, BCR, and GUS as control genes on the Bio-Rad dPCR platform. The ABL1 assay was standardized to the IS using the WHO Reference Panel. The BCR and GUS IS alignments are currently in process. All 3 assays were validated for linearity, precision, accuracy, limit of detection, and limit of blank, following in vitro diagnostics industry best practices and Clinical and Laboratory Standards Institute (CLSI) guidelines. (2) Multiple BCR-ABL1 and ABL1 primer/probe sets were tested on both RQ-PCR and dPCR for the selection of the best primer/probe design. (3) BCR-ABL1 and ABL1 quantifications were compared between 3 dPCR platforms: Bio-Rad QX200, RainDance RainDrop System, and Applied Biosystems QuantStudio3D. (4) Sensitivity of the BCR-ABL1 dPCR assay will be compared in CML PAXgene vs K2EDTA blood tube samples to assess the potential impact of blood-collection tube type on deep MR measurement in patients with CML. Results: (1) The BCR-ABL1/ABL1 dPCR assay WHO IS alignment resulted in a conversion factor of 1. Using the WHO IS-calibrated BCR-ABL1/ABL dPCR assay, MR in blood samples from patients with CML were quantified with sensitivity beyond MR4.5 to levels between MR5.0 and MR5.5 (Figure 1). Combining multiple dPCR wells, which enables analysis of thousands to millions of reactions together, improved the sensitivity and precision of the assay (Figure 2). (2) Evaluation of 10 BCR-ABL1 and 9 ABL1 primer/probe sets using the Bio-Rad dPCR platform resulted in 〈 2-fold differences in copy number detection compared with the 16- to 32-fold difference using RQ-PCR. This is likely due to dPCR measurement being an endpoint PCR reaction. (3) Less than 2-fold differences in both detected copy number and BCR-ABL1/ABL1 ratios were observed using 1 selected primer/probe set when a contrived sample panel representing key BCR-ABL1 levels from 10% to 0.0032% IS was tested across 3 different dPCR platforms, suggesting good cross-platform robustness of dPCR (Table). (4) The PAXgene vs K2EDTA blood tube comparison study is ongoing; data will be presented upon completion of the study. Conclusion: Three dPCR BCR-ABL1 assays were developed. The BCR-ABL1 dPCR assay demonstrated detection capability at levels below MR4.5, down to MR5.0 to MR5.5 in contrived samples from patients with CML. This increased sensitivity relative to RQ-PCR may aid future comparisons of deep MR rates across different CML therapies. dPCR assay performance is also more robust against primer/probe design changes than RQ-PCR, thus requiring less assay optimization. Our results demonstrate that dPCR offers robust BCR-ABL1 quantification on the WHO IS across commonly used control genes without requiring calibrators or extensive assay optimization. Disclosures Huang: Novartis: Employment. Salcius:Novartis: Employment, Equity Ownership. Wong:Novartis: Employment, Equity Ownership. Wong:Novartis: Employment. Wang:Novartis: Employment, Equity Ownership.

Description: Background Quantitative polymerase chain reaction (qPCR) is an analytical method that has been used to investigate the in vivo kinetics of chimeric receptor antigen (CAR) transgene following the infusion of tisagenlecleucel. B cell aplasia, likely an "on-target toxicity" of tisagenlecleucel, has been considered a measure of functional persistence. (Maude SL et al. Blood 2015;125(26):4017-4023) Although the CAR transgene can be detected in peripheral blood of tisagenlecleucel treated patients, it is unclear whether CAR transgene detection by qPCR could be reliably used to inform treatment decision in an individual patient. Methods Transgene levels in blood measured by qPCR from pivotal phase II studies in relapsed/refractory (r/r) pediatric and young adult acute lymphoblastic leukemia (B-ALL) patients (pts) (ELIANA [NCT02435849, N=75]; ENSIGN [NCT02228096, N=29]) and adult diffuse large B cell lymphoma (DLBCL) pts (JULIET [NCT02445248, N=93]) were used to investigate the relationship between transgene persistence and clinical response. Results To determine whether CAR qPCR measurements are associated with or predictive of response, CAR transgene levels and timing of peak levels were examined. In both ALL and DLBCL pts, there were detectable CAR transgene levels by qPCR in both responders and non-responders. The geometric mean maximal expansion (geo mean Cmax) was similar between responding and non-responding adult DLBCL pts, while 1.7 fold differences were observed in pediatric ALL pts (geo mean Cmax in copies/µg: responders, 32700, n=79; non-responders, 19500, n=10; Table 1). For both DLBCL and ALL pts, high inter-individual variability in transgene levels was noted. Similarly, higher CAR-T cell expansion from flow cytometry data pooled from responding pediatric ALL and chronic lymphocytic leukemia (CLL) pts were observed relative to non-responding pts (Mueller KT et al. Blood 2017;130(21):2317-2325), while the levels in DLBCL pts were comparatively lower in blood, likely due to partitioning of functional CAR-T cells to the target sites including lymph nodes. The median time to maximal transgene level ranged from 9-10 days in DLBCL responders and non-responders and pediatric ALL responders, while non-responding pediatric ALL pts showed delayed expansion with median Tmax of 20 days. The median time corresponding to last quantifiable transgene level (Tlast), an indicator of persistence, was higher in responding pts compared to non-responding pts, indicating a trend for longer persistence in both DLBCL and ALL pts with continued response (Table 1). Similarly, the half-life estimated from the terminal slope of the cellular kinetic profile, an additional indicator of persistence, was higher in responding pts relative to non-responding pts for both DLBCL and ALL (Table 1). Despite this general trend, in some cases, transgene levels were not detectable at later time points in pts with continued response. The swimmer plot for representative responder ALL (Figure 1a) and DLBCL pts (Figure 1b) with responses and transgene levels demonstrate that although the majority of responding pts show persistent transgene levels, some pts maintained a favorable clinical response despite a decline in transgene levels to below the level of quantification of 50 copies/µg. Conclusion In both ALL and DLBCL, CAR transgene is initially detected at high levels with high variability in both responders and non-responders. While the majority of responding pts tend to have persistent transgene levels, some pts maintain favorable clinical responses despite a lack of quantifiable transgene. These results indicate that qPCR testing for CAR transgene in blood of tisagenlecleucel treated pts should not be used for making treatment decisions for individual pts. In addition, the qPCR measurements in peripheral blood do not reflect on the trafficking of CAR positive cells to sites outside peripheral blood. The assessment by flow cytometry remains an important assay to distinguish high expression in responding vs non-responding pts in ALL and CLL, and further evaluation of target tissue is needed in DLBCL to understand the utility of CAR expression as a means to distinguish responder and non-responders. Also, further data are needed to improve our understanding of how CAR transgene levels relate to disease burden and duration of response and whether this information is clinically useful. Disclosures Awasthi: Exelixis: Equity Ownership; Celgene: Equity Ownership; Novartis Institutes for Biomedical Research: Employment. Mueller:Novartis Institutes for Biomedical Research: Employment; Novartis Pharmaceuticals Corporation: Equity Ownership, Other: Patent pending. Tam:Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BeiGene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Research Funding. Rives:Amgen: Consultancy, Other: advisory board ; Novartis Pharmaceuticals Corporation: Consultancy, Other: Symposia, advisory boards ; Jazz Pharma: Consultancy, Other: Symposia, advisory boards ; Shire: Consultancy, Other: Symposia, advisory boards . McGuirk:Bellicum Pharmaceuticals: Research Funding; Fresenius Biotech: Research Funding; Novartis Pharmaceuticals Corporation: Honoraria, Other: speaker, Research Funding; Astellas Pharma: Research Funding; Gamida Cell: Research Funding; Kite Pharma: Honoraria, Other: travel accommodations, expenses, speaker ; Pluristem Ltd: Research Funding. Pulsipher:Adaptive Biotech: Consultancy, Research Funding; Amgen: Honoraria; CSL Behring: Consultancy; Novartis: Consultancy, Honoraria, Speakers Bureau. Jaeger:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria; Takeda-Millenium: Membership on an entity's Board of Directors or advisory committees; Takeda-Millenium: Membership on an entity's Board of Directors or advisory committees; AOP Orphan: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Infinity: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; MSD: Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Baruchel:Novartis: Membership on an entity's Board of Directors or advisory committees; Shire: Research Funding; Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Travel, accommodations or expenses; Amgen: Consultancy; Roche: Consultancy; Servier: Consultancy; Celgene: Consultancy. Myers:Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding, Speakers Bureau. Balke-Want:Novartis Pharmaceuticals Corporation: Honoraria. Schuster:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Consultancy, Honoraria, Research Funding; Dava Oncology: Consultancy, Honoraria; Nordic Nanovector: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Research Funding. Stefanski:Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Speakers Bureau. Bishop:Novartis Pharmaceuticals Corporation: Speakers Bureau; Juneau Therapeutics: Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees; United Healthcare: Employment. Waldron:Novartis Pharmaceuticals Corporation: Employment, Equity Ownership. Anak:Novartis Pharma AG: Employment. Chakraborty:Novartis Institutes for Biomedical Research: Employment. Bleickardt:Novartis Pharmaceuticals Corporation: Employment. Wong:Novartis Pharmaceuticals Corporation: Employment, Equity Ownership. Bubuteishvili Pacaud:Novartis Pharmaceuticals Corporation: Employment, Equity Ownership. Waller:Kalytera: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celldex: Research Funding; Pharmacyclics: Other: Travel Expenses, EHA, Research Funding; Cambium Medical Technologies: Consultancy, Equity Ownership. Maude:Novartis Pharmaceuticals Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Description: Introduction Detection of minimal residual disease (MRD) is an important predictor of patient outcome following treatment of B-cell acute lymphoblastic leukemia (B-ALL). We assessed concordance between two MRD assays, with different assay sensitivities, to determine which MRD detection method could support early relapse detection. Immunoglobulin next generation sequencing (Ig NGS) and flow cytometry (FC) were tested in samples from two clinical trials ELIANA (NCT02435849) and ENSIGN (NCT02228096) for pediatric relapsed and refractory B-ALL patients treated with tisagenlecleucel. We also assessed whether using blood with Ig NGS would be comparable to BM testing with FC. Finally we analyzed whether clonal evolution, as detected by Ig NGS, occurred during of the course of therapy for both CD19+ and CD19- relapse patients. Methods In this analysis, bone marrow and peripheral blood specimens at screening (pre-tisagenlecleucel infusion), post-infusion and relapse were tested. Ig NGS was performed in 300 samples from 88 patients. 237 samples from 83 patients also had FC MRD results available. MRD was measured on fresh blood and bone marrow using a 3-tube FC assay (CD10, CD19, CD13, CD20, CD22, CD33, CD34, CD38, CD45, CD58, CD123). The FC MRD assay has a lower limit of sensitivity of 0.01% of white blood cells. Ig NGS detection of MRD was performed using the Adaptive Biotechnology's NGS MRD assay. MRD quantitative values, along with the qualitative MRD calls at each assay sensitivity level (10-4, 10-5 and 10-6) were reported. At baseline, 85 out of 88 samples had informative clones. Results and Conclusions To examine the comparability of flow cytometry and Ig NGS methods in assessing MRD, baseline and post-treatment samples were tested. Baseline samples, which had a high disease burden, showed 100% MRD concordance between both assays. However, post-treatment, where the leukemic burden was dramatically reduced, Ig NGS detected a greater number of MRD positive samples compared to FC, at each sensitivity level tested (10-4, 10-5 and 10-6). At the highest sensitivity level of 10-6, Ig NGS was able to detect 18% more MRD positive post-treatment samples. Importantly, Ig NGS was able to detect MRD positivity 1-4 months ahead of clinical relapse in a small number of relapsed patients, whether relapse was CD19+ or CD19-. This may provide an important window of opportunity for pre-emptive treatment while a patients' tumor burden is still low. In B-ALL, it has previously been described that MRD levels can be one to three logs lower in blood compared to bone marrow (VanDongen JJ et al. Blood 2015). Our results support these findings whereby MRD burden in bone marrow was higher than in blood using both FC and Ig NGS. We next set out to determine if the increased sensitivity afforded by the Ig NGS assay could provide a level of MRD detection in the blood comparable to FC in the bone marrow. In patients with matching data available, Ig NGS was able to detect more MRD positive blood samples than FC MRD positive bone marrow samples. This suggests that monitoring of MRD using Ig NGS in the blood holds the potential to be used as a surrogate for FC MRD in bone marrow. The relationship between MRD and prognosis was examined. Patients who were MRD negative by both Ig NGS and FC at the end of first month post-infusion had better progression-free survival and overall survival compared to those with detectable MRD. Tumor clonality will be further analyzed to understand sub-clone composition at baseline and clonal evolution following tisagenlecleucel treatment. Taken together, these results highlight the importance of using a highly sensitive assay, such as Ig NGS, when monitoring for MRD. MRD detection by Ig NGS holds the potential to identify early response/relapse in patients, which could provide a window of opportunity for additional intervention before morphological relapse. Ongoing studies with larger patient groups will provide further information on the applicability of Ig NGS MRD detection and its association with long-term outcome in tisagenlecleucel-treated pediatric r/r B-ALL patients. Disclosures Pulsipher: Novartis: Consultancy, Honoraria, Speakers Bureau; CSL Behring: Consultancy; Amgen: Honoraria; Adaptive Biotech: Consultancy, Research Funding. Han:Novartis Pharmaceuticals Corporation: Employment, Equity Ownership. Quigley:Novartis Pharmaceuticals Corporation: Employment. Kari:Adaptimmune LLC: Other: previous employment within 2 years; Novartis Pharmaceuticals Corporation: Employment. Rives:Shire: Consultancy, Other: Symposia, advisory boards ; Amgen: Consultancy, Other: advisory board ; Novartis Pharmaceuticals Corporation: Consultancy, Other: Symposia, advisory boards ; Jazz Pharma: Consultancy, Other: Symposia, advisory boards . Laetsch:Bayer: Consultancy; Eli Lilly: Consultancy; Pfizer: Equity Ownership; Novartis Pharmaceuticals Corporation: Consultancy; Loxo Oncology: Consultancy. Myers:Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding, Speakers Bureau. Qayed:Novartis: Consultancy. Stefanski:Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Speakers Bureau. Baruchel:Shire: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Servier: Consultancy; Roche: Consultancy; Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Travel, accommodations or expenses; Celgene: Consultancy. Bader:Cellgene: Consultancy; Riemser: Research Funding; Medac: Patents & Royalties, Research Funding; Neovii: Research Funding; Novartis: Consultancy, Speakers Bureau. Yi:Novartis Pharmaceuticals Corporation: Employment. Kalfoglou:Novartis Pharmaceuticals Corporation: Employment. Robins:Adaptive Biotechnologies: Consultancy, Employment, Equity Ownership, Patents & Royalties. Yusko:Adaptive Biotechnologies: Employment, Equity Ownership. Görgün:Novartis Pharmaceuticals Corporation: Employment. Bleickardt:Novartis Pharmaceuticals Corporation: Employment. Wong:Novartis Pharmaceuticals Corporation: Employment, Equity Ownership. Grupp:Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy; Adaptimmune: Consultancy; University of Pennsylvania: Patents & Royalties.

Description: Abstract 3747 Introduction: Ponatinib is a potent, oral, pan-BCR-ABL inhibitor with activity against native and mutant forms of BCR-ABL, including the tyrosine kinase inhibitor (TKI)-resistant T315I mutant. The efficacy and safety of ponatinib (45 mg orally QD) were evaluated in a phase 2, international, open-label clinical trial (PACE). These multivariate analyses explored the impact of dose intensity and several prognostic and predictive factors on clinical responses, adverse events (AEs), and laboratory changes. Methods: Enrolled patients were resistant or intolerant (R/I) to dasatinib or nilotinib, or had the T315I BCR-ABL mutation at baseline. A total of 267 chronic phase (CP), 83 accelerated phase (AP), and 94 blast phase (BP) CML/Ph+ ALL patients were assigned to 1 of 6 cohorts according to disease phase (CP-, AP-, or BP-CML/Ph+ ALL), R/I to dasatinib or nilotinib, and presence of T315I. Three CP-CML and 2 AP-CML patients were treated, but not assigned to a cohort (post-imatinib, did not have T315I at baseline); these patients were excluded from efficacy analyses and included in safety analyses. For the purposes of the efficacy multivariate analyses, AP-CML, BP-CML, and Ph+ ALL patients were combined. The baseline covariates analyzed were age, time since diagnosis, number of prior TKIs, presence or absence of the T315I mutation, neutrophil and platelet counts, and weight. The primary efficacy outcome analyzed was major cytogenetic response (MCyR) in CP-CML and major hematologic response (MaHR) for all other patients. The safety outcomes analyzed were the following AEs: pancreatitis, elevated lipase, alanine aminotransferase (ALT) increase, aspartate aminotransferase (AST) increase, rash, neutropenia, thrombocytopenia, arthralgia, and hypertriglyceridemia. The impact on neutrophils, platelets, bilirubin, ALT, AST, creatinine, lipase, and triglycerides was also examined. Binary event outcomes were analyzed using logistic regression models. Data values over time were analyzed using linear mixed effects models. Laboratory values were log-transformed. Data as of 27 April 2012 were used in these analyses. Results: Median baseline characteristics of the CP-CML R/I and T315I cohorts, respectively, were: 61 vs 51 yrs of age, 8 vs 5 yrs since initial diagnosis, 3 vs 2 prior TKIs. The median dose intensity for the CP-CML R/I and T315I cohorts was 30 and 39 mg/day, respectively. In general, other baseline characteristics were balanced between these 2 cohorts. Multivariate analysis found statistically significant associations between MCyR and increasing dose intensity (mg/day) (p0.2). This was likely because patients with T315I received a greater dose intensity, were younger, and were previously treated with fewer TKIs. The probability of achieving MaHR in patients with AP-CML, BP-CML, and Ph+ ALL increased with increasing dose intensity (p