Abstract: Background: The Canadian tyrosine kinase inhibitor (TKI) discontinuation trial, named "Treatment Free Remission Accomplished By Dasatinib " (NCT#02268370), has reported 56.8% molecular relapse-free survival (mRFS) rate at 12 months after imatinib (IM) discontinuation. MMR loss occurred quickly after IM discontinuation, typically within 2-4 months, while those who lost MR4 on two consecutive measurements tended to lose their molecular response more gradually. BCR-ABL transcript doubling time (DT) after TKI discontinuation is a reciprocal concept to transcript halving time following TKI therapy. Due to inter-individual differences in DT after TKI discontinuation, DT can be used as a potential biomarker to identify those patients at high-risk for TFR failure when measured before they experience a clinically significant event of molecular relapse. The present study has not only evaluated the kinetics of BCR-ABL transcript rise after IM discontinuation, but also explored the predictive/prognostic role of DT of BCR-ABL transcript level as an early predictor of TFR failure. Patients and methods: Changes of BCR-ABL1 transcript level in each patient were assessed monthly by estimating the number of days required for BCR-ABL1 to double from the previous expression level/measurement, termed the DT. Based on the BCR-ABL1 qPCR value taken monthly in the first 6 months after IM discontinuation, DT was calculated monthly, as x = ln(2)/K, where x is the DT and K is the fold BCR-ABL1 change from the previous value divided by the number of days between each measurement. K was determined as follows: K = [ln(b) - ln(a)]/t, where a is the BCR-ABL1 value of the previous measurement, b is the BCR-ABL1 at the relevant time point, and t is the number of days between measurements. The baseline qPCR level from the prior month to TKI discontinuation was referenced. The distribution of DT was assessed at each time point of DT measurement within the first 6 months. In order to define cut-off levels for BCR-ABL1 qPCR and DT for the first 6 months, multiple statistical parameters were taken into account including positive (PPV) and negative predictive value (NPV), accuracy and F1 score of DT value, resulting in the DT value of 12.75 days at 2 months as the optimal cut-off value of DT value. Patients were stratified into the 3 groups based on the DT value of 12.75 days at 2 months after IM discontinuation. The high-risk group was defined as the patients showing DT & lt; 12.75 days but above 0, i.e. rapidly proliferating CML cells, implying a high risk for TFR failure with a shorter DT. The intermediate-risk group was defined as those patients with DT ≥ 12.75 days, i.e. more slowly proliferating CML cells implying intermediate risk for TFR failure. The low-risk group was defined as patients showing DT of zero or below, i.e. no increase in the size of the pool of cells expressing BCR-ABL, implying a low risk for TFR failure. The mRFS was analyzed for each of these risk groups at 6 monthly intervals after TKI discontinuation. Results: We compared the DT values of the patients that failed TFR with those from the patients who maintained their molecular response at last follow-up. The DT values at 2 months were much shorter in patients who failed TFR after IM cessation (median 8.32 days) compared to those who maintained molecular response (median 20.7 days; p & lt;0.001 by Mann-Whitney U-test). The DT value of 12.75 days was defined as the optimal value for DT at 2 months with the NPV, PPV, accuracy and F1 score of 80.90%, 96.43%, 84.62% and 0.75, respectively, as the -log10(p-value), accuracy and F1 score reached a plateau at a DT of 12.75 days as presented in the Figure A. At a DT value of 12.75 days at 2 months after IM discontinuation, patients were stratified into 3 groups: high- (n=26), intermediate- (n=16) and low-risk groups (n=71; Figure B). With respect to mRFS rate, the high-risk group showed 7.7% mRFS rate at 12 months compared to 53.6% in the intermediate-risk group or 90.0% in the low-risk group (p & lt;0.001; Figure C). Thus, this risk stratification system based on DT value at 2 months can stratify patients according to their risk of TFR failure after IM cessation. Conclusion: Monthly assessment of DT based on the monthly BCR-ABL qPCR is useful to identify the patients with an imminent risk of molecular recurrence after IM cessation for TFR. Figure Disclosures Bence-Bruckler: Merck: Membership on an entity's Board of Directors or advisory committees. Keating:Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Hoffman La Roche: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy; Merck: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Shire: Membership on an entity's Board of Directors or advisory committees; Taiho: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Busque:Novartis: Honoraria; BMS: Honoraria; Pfizer: Honoraria. Delage:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Lipton:BMS: Consultancy, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Ariad: Consultancy, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Research Funding. Leber:Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS/Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Treadwell: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda/Palladin: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Lundbeck: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Otsuka Pharmaceutical: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Abstract: Background: Several clinical factors have been proposed to predict successful tyrosine kinase inhibitor (TKI) discontinuation for treatment-free remission (TFR), among which a longer duration of total TKI or deep molecular response (DMR) duration correlate with increased success of TFR. Although many guidelines have been proposed to safely select patients who would be candidates for TFR attempt, there is some discrepancy regarding which duration of DMR and/or Imatinib (IM) treatment optimally stratifies patients according to their probability of TFR success. These suggested DMR or IM treatment durations are often the result of a panel consensus, and the method of calculation of these thresholds as categorical values is not provided. Thus, there is a practical need for an evidence-based determination of duration of IM therapy while awaiting TFR attempt to establish if the likelihood of successful TKI discontinuation reaches an optimal maximum after a certain duration of treatment and/or DMR. The present study attempted to define the optimal (i.e. shortest) duration of IM treatment or DMR that predicts TFR success at a specified level of confidence. Patients and methods: The Canadian TKI discontinuation study has enrolled 131 patients with the longest Imatinib treatment duration, at a median of 9 years. A Cox's proportional hazard ratio model was applied using molecular relapse-free survival (mRFS) as the endpoint. Continuous variables were initially tested using Cox's proportional hazard model and were converted into categorical variables according to the optimal cut-off values derived from the current analysis. We have evaluated six statistical parameters to determine the optimal cut-off of IM treatment duration and MR4 response duration for TFR prediction: 1) mRFS rate at 12 months between the groups, stratified according to the cut-off value, 2) proportion of patients divided by the cut-off value, 3) negative predictive value (NPV), 4) positive predictive value (PPV), 5) accuracy, and 6) the p-values as a measure of risk stratification power. The optimal cut-off was sought that met the joint criteria of a p-value ≤ 0.05, PPV≥60% and NPV≥60%. Results: Out of 131 patients enrolled, 123 patients completed a planned follow-up of 2.5 years. The mRFS rate was calculated as 56.8% (47.8-64.8%) at 12 months. One additional year of IM therapy increased the chance of TFR success by 5.5%, while one additional year of MR4 duration increased its likelihood by 5.1% by assessing the mRFS rates after 5 versus 9 years. The formula generated from this linear regression analysis is as follows: The probability of mRFS (%) = 0.05146 x (IM duration in year) + 0.08379; or 0.0555 x (MR4 duration in year) + 0.1844 For example, as shown in Figure A, a patient with a total IM treatment duration of less than 6 years has a mRFS rate of 36.0% (n=25), implying a high risk of TFR failure, while those patients with IM duration of above 6 years showed a mRFS rate of 61.8% (n=106). PPV is defined as the probability of TFR failure in a subject at high risk for TFR failure at the proposed cut-off, while NPV is defined as the probability of TFR success in a subject at low risk for TFR failure (i.e. at low chance of TFR success) at the proposed cut-off. An ideal cut-off value should have both a high PPV (68%) and NPV (61.3%), thus defining 6 years as the optimal cut-off. The next parameter evaluated is the p-value of each cut-off value as a measure for risk stratification power; an acceptable p-value is ≤ 0.05. IM duration above 5.6 years and MR4 duration in the range of 4.2-11 years meet these criteria, respectively. Accordingly, the optimal cut-off value for IM treatment duration is ~ 6 years (Figure A), while that for MR4 duration is ~ 4.5 years (Figure B). According to these cut-off values evaluated, patients treated with IM duration of 6 years or longer showed a superior mRFS rate at 12 months (61.8%) than those with less treatment (36.0%; p=0.01). Patients with MR4 duration of 4.5 years or longer showed a higher mRFS rate at 12 months (64.2%) than those with a shorter duration of deep molecular response (41.9%; p=0.003). Conclusion: In summary, we propose 6 years as the cut-off for IM duration with p-value=0.01, 68% PPV and 62% of NPV, while 4.5 years' cut-off value for MR4 duration is proposed with p-value=0.003, 63% of PPV and 61% of NPV. These results can be incorporated into clinical guidelines as optimal IM duration or MR4 duration for IM discontinuation to achieve successful TFR. Disclosures Bence-Bruckler: Merck: Membership on an entity's Board of Directors or advisory committees. Busque:Novartis: Honoraria; Pfizer: Honoraria; BMS: Honoraria. Delage:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Keating:Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffman La Roche: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy; Servier: Membership on an entity's Board of Directors or advisory committees; Shire: Membership on an entity's Board of Directors or advisory committees; Taiho: Membership on an entity's Board of Directors or advisory committees. Lipton:Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Honoraria; Ariad: Consultancy, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding. Leber:Otsuka Pharmaceutical: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS/Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Treadwell: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda/Palladin: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Lundbeck: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Abstract: Abstract 4437 This multi-center, open-label, single-arm study evaluated the safety and efficacy of nilotinib in patients with imatinib resistant/intolerant chronic myeloid leukemia (CML) in blast crisis, accelerated phase and chronic phase. This study provided expanded access to nilotinib prior to commercial availability. Methods: Patients received nilotinib orally at a dose of 400 mg twice daily. Adverse events were monitored throughout the study and assessments of cardiac, hematology and blood chemistry were performed every 3 months at a minimum. The efficacy assessments were performed every 6 months at a minimum and comprised of cytogenetic analysis of bone marrow, evaluation of extramedullary disease, and cancer related symptoms. Results: Sixty-five patients were enrolled in the study. The median age was 56 (range 21–85) years; 50.8% were male, and 89.2% Caucasian. The majority of patients (92.3%) were in chronic phase; 3.1% and 4.6% of the patients were in blast crisis and accelerated phase respectively. Twenty-five (38.5%) patients were imatinib-resistant, thirty-seven (56.9%) imatinib-intolerant and three (4.6%) both imatinib-resistant and -intolerant. The overall median duration of treatment with nilotinib was 27.2 (range 0.3 – 48.8) months. Thirty-six patients (55.4%) were still on treatment at study end. The main reasons for discontinuation were adverse events (16.9%) and unsatisfactory therapeutic effect (12.3%). Treatment related adverse events reported in at least 10% of patients, irrespective of severity, included rash (23.1%), pruritus (20%), fatigue (18.5%), muscle spasms (15.4%), headache (16.9%), alopecia (13.8%), abdominal pain (10.8%) and lipase elevation (10.8%). Grade 3 and 4 adverse events reported in at least 5% of patients, irrespective of study drug relationship, included thrombocytopenia (12.3 %), anemia (6.2%), neutropenia (6.2%) and lipase elevation (6.2%). Three patients (4.6%) discontinued due to cardiac disorders including tachycardia (1), myocardial infarction (1), and palpitations (1). Clinically significant abnormal ECG results were uncommon and mostly transient. There was no evidence of toxicity to a major organ system. One patient died within 28 days after study discontinuation due to progression of recently diagnosed multiple myeloma and chronic obstructive pulmonary disease deterioration not suspected to be related to study drug. Thirty patients received treatment for at least 30 months with nilotinib on study. At month 30, 14 (82.4%) of 17 patients who underwent a bone marrow aspirate achieved a complete cytogenetic response. Seven of these fourteen patients (50%) had never previously achieved complete cytogenetic response with imatinib. Conclusions: This phase IIIB trial provided nilotinib to 65 patients with imatinib-resistant or -intolerant chronic CML in all phases on expanded access for 44 months, allowing evaluation of its safety profile. Nilotinib treatment was reasonably well-tolerated with an overall safety profile similar to that reported in previous studies, without the occurrence of unexpected adverse events. Real-time quantitative RT-PCR of BCR-ABL mRNA levels was not a study endpoint but is used routinely in Canada to monitor patients in complete cytogenetic response instead of cytogenetic assessments which likely explains the low compliance with cytogenetic assessments in this study. Disclosures: Turner: Novartis Pharmaceuticals Canada: Research Funding, Speakers Bureau. Leber:Novartis Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau. Hasegawa:Novartis Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees. Leitch:Novartis Pharmaceuticals: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Bence-Bruckler:Novartis Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees. Sandeep:Novartis Pharmaceuticals: Honoraria. Laneuville:Novartis Pharmaceuticals: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau. Woo:Novartis Pharmaceuticals Canada Inc.: Employment. Beauparlant:Novartis Pharmaceuticals Canada Inc: Employment. Lipton:Novartis Pharmaceuticals Canada Inc: Honoraria, Membership on an entity’s Board of Directors or advisory committees.

Abstract: CD32b (FcγR2b), the sole inhibitory Fcγ receptor, negatively regulates immune function and is expressed throughout B cell development and on their malignant counterparts with the highest expression found on multiple myeloma. Additionally, CD32b expression on tumor cells is known to sequester IgG Fc thereby providing a mechanism of resistance to therapeutic monoclonal antibodies (mAb) with Fc dependent activity. Taken together, CD32b represents an attractive tumor antigen for targeting with a mAb. To this end, two anti-CD32b mAbs, NVS32b1 and NVS32b2, were developed. The complementarity-determining regions (CDRs) of these antibodies bind the CD32b Fc binding domain with high specificity and affinity while the Fc region is afucosylated enabling enhanced activation of FcγR on immune effector cells. This specificity and optimized potency is highlighted in whole blood assays where NVS32b2 depletes CD32b positive B cells but spares immune subsets with low CD32b expression or expression of the homologous CD32a. The antibodies mediate potent killing of opsonized cells via antibody dependent cell-mediated cytotoxicity, antibody dependent cellular phagocytosis, and complement dependent cytotoxicity. Additionally, NVS32b mAbs’ CDR block the CD32b Fc binding domain, thereby minimizing CD32b mediated resistance to therapeutic mAbs with Fc dependent activity including rituximab, obinutuzumab, and daratumumab. In vivo, NVS32b mAbs demonstrate robust antitumor activity against CD32b positive xenografts and immunomodulatory activity including recruitment of intratumoral macrophages. The NVS32b mAbs’ activity against malignant B and plasma cells featuring a range of CD32b expression demonstrates their therapeutic potential, as a single agent or in combination with therapeutics including mAbs with Fc dependent activity. Citation Format: Haihui Lu, Dongshu Chen, Sunyoung Jang, Babette Wolf, Stefan Ewert, Meghan Flaherty, Fangmin Xu, Sinan Isim, Yeonjiu Shim, Christina Dornelas, Nicole Balke, Xavier Charles Leber, Meike Scharenberg, Johanna Koelln, Eugene Choi, Rebecca Ward, Jennifer Johnson, Thomas Calzascia, Isabelle Isnardi, Juliet Williams, Heather Huet, Emma Lees, Matthew J Meyer. Discovery and characterization of next generation monoclonal antibodies targeting the inhibitory Fc gamma receptor CD32b for the treatment of B and plasma cell malignancies [abstract] . In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C028. doi:10.1158/1535-7163.TARG-19-C028

Abstract: Introduction: Tyrosine kinase inhibitor (TKI) therapy can result in the adverse events of prolonged anemia, thrombocytopenia and/or leukopenia via c-kit blockade in hematopoietic stem cells. Previous studies have reported that even low-grade adverse events could impair a patient's health-related quality of life. One of the benefits of TKI discontinuation is to allow patients to live drug-free, thereby preventing drug-related adverse events including cytopenias. The Canadian tyrosine kinase inhibitor (TKI) discontinuation trial has reported 59.1% and 21.5% molecular relapse-free survival (mRFS) rates after first and second attempts at treatment free remission (TFR) with imatinib (IM) or Dasatinib (DA) discontinuation, respectively. In the present study, we attempted to analyze the impact of TKI discontinuation on changes in hematological parameters, and its impact on TFR success after TKI discontinuation. Methods and materials: Throughout the trial, we have collected the hematoloigc parameters at 22 timepoints in 131 patients. These included Hb level, WBC count with differentials, and platelet count during IM discontinuation (7 times), DA rechallenge (10 times), and DA discontinuation (5 times). Results: With IM discontinuation, most of the hematological parameters showed a significant improvement within 3 months: Hb level rise by +10.47g/L (+8.86%; p=1.67x10-22), WBC count rise by +1.43x109/L (+30.08%; p=2.03x10-16), neutrophil rise by +0.99x109/L (36.76%; p=4.48x10-11), lymphocyte rise by +0.24x109/L (+20.64%; p=6.72x10-9), monocyte rise by +0.13x109/L (+35.9 0%; p=3.33x10-14), platelet count rise by +22.65 x109/L (+12.76%; p=1.03x10-7). Eosinophil counts were not significantly changed (p=0.475). With DA rechallenge, mixed changes were observed in hematologic parameters within 1 month: Hb level significant dropped by 11.57g/L (-8.26%; p=6.38x10-14) and platelet counts also showed a decreasing trend (-9.39x109/L or -4.57%; p=0.07), while significant increases were noted in lymphocyte (+0.41x109/L or +22.22%; p=0.00027), and monocyte counts (+0.14x109/L or +14.29%; p=0.001). No significant changes were noted in WBC counts (+0.32x109/L or +1.64%; p=0.234), neutrophil counts (-0.18x109/L or -10.42%; p=0.285), or eosinophil counts (+0.03 x109/L or 0%; p=0.185). With DA discontinuation, the Hb level rebounded by +7.08g/L within 3 months (+9.45%; p=0.0003). However, there was no significant change in the other parameters 3 months after DA discontinuation, including WBC (p=0.841), neutrophil (p=0.309), lymphocyte (p=0.995), monocyte (p=0.451), eosinophil (p=0.826) and platelet counts (p=0.533). When the changes in hematologic parameters were analyzed in correlation with mRFS, there was no association of those parameter changes with RFS after DA discontinuation. However, associations of mRFS following IM discontinuation were noted as follows: higher mRFS after IM discontinuation was observed in the group with a smaller change in Hb level (≤+1.17%, p=0.004), lymphocyte count (≤+1.06%; p=0.006), and monocyte count (≤+1.43%; 0.005) compared to those with a larger change. In other words, the group showing a rebounded Hb level after IM discontinuation showed a lower mRFS rate compared to those in whom the Hb did not rebound. A lower mRFS was noted in the group with a smaller change in neutrophil count (≤+1.07%) compared to those with a larger change (p=0.008), implying that the group with rebounded neutrophil count showed a higher mRFS compared to those not. Multivariate analysis confirmed: 1) IM treatment duration longer than 8.75 years is associated with a decrease in loss of molecular response by 13% per year (p=0.001, HR 0.871), 2) Hb level rebound above 22gm/L showed 2.8 times higher risk of molecular relapse (p=0.021, HR 2.801), 3) rebound rise of neutrophil count by 1.075% or above reduced the risk of molecular relapse by 52% (p=0.06, HR 0.485). Conclusion: Further research is warranted to explore the functional role of the hematopoietic stem cell fraction following prolonged TKI therapy in CML patients. Hematopoiesis in Ph-negative cell population could contribute to TFR after TKI discontinuation. Figure Disclosures Busque: ExCellThera: Patents & Royalties; BMS: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Paladin: Consultancy. Savoie:BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy. Keating:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Seattle Genetics: Consultancy; Janssen: Membership on an entity's Board of Directors or advisory committees; Shire: Membership on an entity's Board of Directors or advisory committees; Hoffman La Roche: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees. Delage:Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Liew:Novartis: Consultancy, Honoraria. Leber:Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.