Sustained Molecular Remission Research Articles

VEXAS syndrome: complete molecular remission after hypomethylating therapy

The VEXAS syndrome, a genetically defined autoimmune disease, associated with various hematological neoplasms has been attracting growing attention since its initial description in 2020. While various therapeutic strategies have been explored in case studies, the optimal treatment strategy is still under investigation and allogeneic cell transplantation is considered the only curative treatment. Here, we describe 2 patients who achieved complete molecular remission of the underlying UBA1 mutant clone outside the context of allogeneic HCT. Both patients received treatment with the hypomethylating agent azacitidine, and deep molecular remission triggered treatment de-escalation and even cessation with sustained molecular remission in one of them. Prospective studies are necessary to clarify which VEXAS patients will benefit most from hypomethylating therapy and to understand the variability in the response to different treatment strategies.

Phase 2 Study of Dasatinib with and without Venetoclax in Patients with Early Chronic Phase Chronic Myeloid Leukemia (ECP-CML)

Background Treatment-free remission (TFR) is a therapeutic goal for patients with ECP-CML once patients maintain a deep molecular remission (DMR) on tyrosine kinase inhibitors (TKIs). Only a fraction of patients (pts) achieved sustained (3+ years) molecular remission with 4.0- to 4.5-log reduction (MR4.5). Preclinical studies showed that BCL-2 is a key survival factor for CML stem/progenitor cells. Therefore, the combination of TKIs and venetoclax, targeting BCR::ABL1 and BCL-2, has the potential to significantly improve DMR and subsequently the rates of TFR. The aim of this study is to evaluate the safety and efficacy of the combination. Methods Patients with ECP-CML or accelerated phase (AP; defined by clonal evolution only) were eligible; pts with < 1 month of prior TKI therapy were eligible. Pts received single-agent dasatinib 50 mg/d for the first 3 months. From Month 4 and onwards, venetoclax was administered from Days 1 to 14 each month concomitantly with dasatinib for a total of 3 years. The first 16 patients received venetoclax 200 mg/d, with a rapid ramp-up starting 50 mg on Day 1, then 100 mg on Day 2, then 200 mg daily on Day 3 and onwards. Due to neutropenia observed with continuous dose of venetoclax 200 mg/d, the protocol was ameded: from patient #17 and onwards, venetoclax was administered at 200 mg/d for 14 days per month and upon achievement of MR4.5, venetoclax was reduced to 100 mg/d for 7 days per month. After 3 years of combination, patients received single-agent dasatinib. TFR was offered after ≥ 3 years of DMR. The primary endpoint was the rate of major molecular response (MMR) by 12 months of the combination. The secondary endpoints were the 12-month rate of MR4.5 and the cumulative overall rate of MR4.5; the rates of MR4.5 at 6-, 12-, 18-, 24, and 36-months of the combination; the proportion of patients with sustained MR4.5 of 3 years and more; the TFR rate; overall survival; and the safety of the combination. Cumulative response rates were calculated with treatment failure as a competing event. Failure-free survival (FFS) was calculated from the initiation of therapy to loss of major cytogenetic response (MCyR), transformation to AP or blast phase, discontinuation of therapy for any reason except of TFR, or death. Results Sixty-five pts were enrolled and treated between April 2018 and August 2021. The median age at treatment initation was 46 years (range, 23-73) and 57% of the pts were females. Twenty-one (32%) pts had splenomegaly, 46 (71%) had low Sokal risk disease and 3 (5%) had high Sokal risk disease (Table 1). By 12 months of the combination, 85% of the pts had achieved MMR or deeper response. The median time to MMR, MR4, and MR4.5 were 6.2 months, 13 months, and 23.9 months from the time of TKI start, respectively. After a median follow-up of 25.7 months (range, 8.7-48.1), two pts discontinued therapy due to adverse events (GI intolerance, and pleural effusion); the 2 -year FFS was 96%. One pt switched TKI therapy due to the lack of 18-month complete cytogenetic response (CCyR); no loss of CCyR, transformation, or death was observed. We then compared the outcomes with the combination to the outcomes of 85 pts treated with single-agent dasatinib. From the start of TKI therapy, the 12-month cumulative MMR rates were 78% and 79% in the dasatinib single-agent and dasatinib + venetoclax groups, respectively. The cumulative 24-month rates of CCyR, MMR, MR4, and MR4.5 are summarized in Table 2. In the dasatinib single-agent group, 3 pts switched TKI therapy due to the lack of 12-month MCyR, 18-month CCyR, and the acquisition of ABL1 mutation (1 each); 3 pts switched TKI therapy due to loss of CCyR; 2 pts died during therapy (deaths unrelated to CML). Within one year of therapy, the incidence of grade 1/2 neutropenia (absolute neutrophil count, below lower range of normal to 1,000 x10 9/L) was 21% and 52% in the dasatinib single agent and dasatinib + venetoclax groups, respectively ( P<0.001); the incidence of grade 3/4 neutropenia (absolute neutrophil count, below 1,000 x10 9/L) was 8% and 17%, respectively ( P<0.001). There was no incidence of neutropenic fever in the dasatinib + venetoclax. Conclusion The combination of dasatinib 50 mg/d and venetoclax is safe and effective in pts with ECP-CML. With a median follow-up of 2 years, the cumulative resposne of dasatinib + venetoclax is similar to that of dasatinib single-agent without adding significant toxicities. Further follow-up is needed for the evaluation of TFR.

Involvement of the JAK-STAT Pathway in the Molecular Landscape of Fusion-Free Myeloid Neoplasms with Eosinophilia

Introduction. Gene fusions leading to the constitutive activation of tyrosine kinases (TK) such as PDGFRA, PDGFRB or FGFR1 have been the first recurrent genetic defects involved in myeloid hypereosinophilic syndromes (HES). Although activation of the Janus kinase (JAK)/Signal Transducer and Activator of Transcription(STAT) pathway is critical for eosinophil production and survival, genes involved in the JAK/STAT pathway are not included in most next-generation sequencing (NGS) panels used for the etiological workup of hypereosinophilia (HE). Methods. A custom 149-gene NGS panel including subunits of the IL3/IL5/GM-CSF receptors, TK ( PDGFRA/B, FGFR1, ABL1, FLT3, KIT), intracellular proteins of the JAK-STAT and RAS-MAPK pathways was performed in 64 consecutive adult patients (experimental group) referred between March 2012 to June 2023 to the French Reference Center for Hypereosinophilic Syndromes (CEREO) for HE/HES displaying at least one clinic-biological feature suggestive of myeloid neoplasm ( i.e. splenomegaly, other unexplained CBC abnormality besides HE, increased serum tryptase and/or vitamin B12 levels, corticosteroid-refractory HE and/or sensitivity to either TK inhibitors or JAK inhibitors). All of them were negative by PCR and/or FISH analyses for gene rearrangements involving PDGFRA, PDGFRB or FGFR1. Patients with lymphocytic HES (n=7), idiopathic HES (n=26) and HE of undetermined significance (n=11) were used as controls (total, n=44). Results. Concordant with the latest recommendations of the international cooperative working group on eosinophil-associated disorders, at least one mutation was reported in 50/64 (78%) patients of the experimental group versus 8/44 (18%) patients in the control group (p<0.001) when applying a threshold of at least 3% of variant allele frequency (VAF). Mutations in the control group implied genes involved in age-related clonal hematopoiesis ( e.g.DNMT3A, TET2), with low VAF in almost all cases. All 35 patients with at least one mutation involving the JAK/STAT pathway belonged to the experimental group, among whom all 22 patients treated with steroids were refractory to therapy. Eighteen patients had STAT5B mutations, including 13 (72%) with the somatic N642H mutational hotspot. Two patients harbored JAK2Ex13InDel mutations, including one with eosinophilia and erythrocytosis. Previously unreported molecular alterations were also evidenced, including seven patients with JAK1 mutations and three STAT5A-mutated patients who shared common features i.e. the co-occurrence of BCOR mutations, high hemoglobin levels and eosinophil hyperplasia. Of note, the latter mutations were not reported both in a public database (GnomAD) as well as in a second cohort of 613 samples referred for suspicion of myeloid malignancies (yet without HE) studied with the same NGS workflow, thereby strongly supporting their association with myeloid HES. Deciphering the data from bulk sequencing also suggests that JAK-STAT mutations were frequently preceded by (or associated with) myelodysplasia-related gene mutations, with SF3B1 (12/36) and ASXL1 (10/36) mutations being the most common. Overall, both KIT D816V (n=2) and RAS/MAPK pathway activation mutations (n=3) were rare. No mutation of either PDGFRA/B, FGFR1 or IL3/IL5/GM-CSF-receptor genes was evidenced.In the experimental group, 17/18 (94%) patients (including 12 with JAK-STAT mutations) treated with ruxolitinib and with > 3 months of follow-up responded to treatment (12 complete and 5 partial hematological responses). Discussion. These dataemphasize the usefulness of NGS in daily practice for the workup of fusion-free HES patients harboring features suggestive of myeloid neoplasms. In such patients, druggable mutations involving the JAK-STAT pathway (including yet unidentified STAT5A mutations) are frequent. Most JAK/STAT mutations occur in the setting of a preexisting myelodysplastic or myelodysplastic/myeloproliferative disease harboring mutations in RNA-splicing genes or chromatin modifiers. These findings provide a rationale for refining treatment algorithms in fusion-free myeloid HES patients, supporting the use of JAK inhibitors as frontline therapy. More data are warranted to assess whether JAK inhibition enables sustained molecular remission in all disease subtypes.

The Importance of Bone Marrow Lymphocyte Subtypes As Predicting Factors for Molecular Recurrence in Patients with Chronic Myeloid Leukemia after Discontinuation of Imatinib

Introduction: in recent years the feasibility of the discontinuation of tyrosine kinase treatment in chronic myeloid leukemia (CML) has been proven, and several clinical factors influencing the duration of treatment-free remission (TFR) after discontinuation have been studied.Aim: we analyzed the influence of bone marrow (BM) lymphocyte subsets on the molecular recurrence after discontinuation of Imatinib (IM) in CML.Methods: in a recent discontinuation study performed at our Institution (EDI-PIO trial) we assessed BM lymphocyte subsets before and after introduction of pioglitazone which was given 3 months before discontinuation of IM. Criterias for discontinuation were: patient in chronic phase at diagnosis, a minimum of 3 years on TKI and sustained molecular remission MR4.5 for at least 2 years. Lymphocyte populations studied: B, TCD8, TCD4, T CD4+CD8+ and T CD4- CD8- besides T naïve and memory, TCRαβ, TCRγδ, NK-t and NK cells. The influence of Sokal score at diagnosis, the duration of imatinib treatment together with the BM lymphoid subsets on the time of treatment-free remission (TFR) were examined by uni- and multivariate Cox regressions.Results: we studied 30 out of 32 patients diagnosed between 1998 and 2013 that reached criteria for discontinuation that were included in EDI-PIO trial: 13 male and 17 female. Median age at diagnosis: 41 years (22-65). Median time of IM treatment: 116.3 months (38.2-209.3); 11 patients (36%) had a molecular recurrence in a median of 5,17 months (2.4-29.4). For patients remaining in TFR the median time of follow-up was 46 months (26.3-55.9). Median overall time of IM treatment (IM-Tr) was 65 months for patients recurring and 124 months for those remaining in TFR. In the univariate Cox regression a significant value was found for IM-Tr, higher percentages of T CD4+CD8+ and lower ones of TCRγδ lymphocytes at discontinuation. In the multivariate model only the T CD4+CD8+ remained.Conclusion: discontinuation of IM is feasible in patients remaining in continuous MR4.5 for more than 2 years. A longer time of IM treatment and higher values of BM T CD4+CD8+ predict a lower risk of relapse.Key words: chronic myeloid leukemia, imatinib, discontinuation, lymphocytes, pioglitazone DisclosuresPagnano: Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pintpharma: Other: Lecture; EMS: Other: Lecture; Jansenn: Other: Lecture.

CML-158: Analysis of TKI Discontinuation in Tunisian Chronic Myeloid Leukemia Patients

Context Discontinuation of tyrosine kinase inhibitor (TKI) therapy to achieve a treatment-free remission (TFR) has become an important goal in routine clinical practice for patients with chronic myeloid leukemia (CML) in sustained deep molecular remission (DMR). Objective The aim of this study was to evaluate discontinuations of tyrosine kinase inhibitor (TKI) treatment in Tunisian patients with chronic myeloid leukemia (CML). Design This retrospective study collected data of CML patients who had discontinued ITK treatment as part of daily clinical practice in Sfax, Tunisia. Setting University Hospital. Patients or other participants 14 Tunisian CML patients who discontinued TKIs were followed. Interventions Quantitative assessment of the BCR-ABL transcript was performed using the Cepheid Xpert BCR-ABL ultra assay. Main outcome measures Duration of TKI therapy, depth of molecular response, and the reasons for TKI discontinuation were collected. Results In our series, 13 patients and one discontinued imatinib and second-generation TKIs, respectively. Discontinuation was programmed in 11 patients (78.5%) and 3 discontinued for other reasons (1 for adverse effects and 2 for pregnancy). 5 patients (35.7%) experienced a molecular relapse defined as the loss of a major molecular response (MMR). Relapses occurred after a mean time of 7 months (range: 1-21). For the 9 patients (64.2%) who maintain a TFR, mean duration of TKI therapy before discontinuation was 120 months, with a mean follow-up after discontinuation of 30.69 months. Conclusions Despite the limit of our study, these results highlight the importance of sustained deep molecular responses to safely suspend therapy and attempt treatment-free remission (TFR).

Successful Treatment of Chronic Myeloid Leukemia With Dasatinib After Kidney Transplantation: A Case Report

ObjectiveChronic myeloid leukemia (CML) is a rare malignancy in kidney transplant (KT) recipients. Although dasatinib is the first-line treatment for CML, it has inhibitory activity against CYP3A4; this might increase the blood concentration of tacrolimus (administered to KT patients for immune suppression). Furthermore, tacrolimus can also increase blood concentrations of dasatinib through P-glycoprotein inhibition. MethodsHere, we report a case of sustained molecular remission of CML with prolonged first-line dasatinib therapy in a KT recipient being treated with tacrolimus. A 61-year-old woman developed CML-chronic phase (CML-CP) 38 months post KT. Her maintenance immunosuppressive therapy consisted of tacrolimus, mycophenolate mofetil, and methylprednisolone. Considering the potential drug interaction with tacrolimus, dasatinib was administered at a low dose of 50 mg/day. Her immune status was evaluated regularly by assessing the mixed lymphocyte reaction (MLR) using an intracellular carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeling technique; immunosuppressive therapy was adjusted accordingly. ResultsThe patient achieved complete hematologic remission (CHR) after 1 month of dasatinib treatment. Six months after dasatinib treatment, she achieved a major molecular response. During the observation period, neither antibody-mediated nor acute cellular rejection were encountered in the patient. She remained in CHR with a major molecular response 12 months after the diagnosis of CML-CP. ConclusionData obtained from immune monitoring assays using CFSE-MLR helped us to successfully manage a KT recipient with CML-CP being treated with dasatinib. Drug-drug interactions are a key consideration while designing treatment regimens; such strategies would ensure that drug-drug interactions do not negatively affect the treatment outcomes.

Discontinuation of Tyrosine Kinase Inhibitors (TKIs) in Philadelphia Chromosome-Positive (Ph+) Acute Lymphoblastic Leukemia (ALL)

Background: Treatment of Ph+ ALL has significantly improved since the addition of TKIs to chemotherapy, with improvement in complete molecular remission (CMR) and overall survival (OS) rates. However, the optimal duration of TKI is not yet established and the common practice is to continue indefinitely unless allogeneic stem cell transplant (ASCT) is performed. In pediatric setting, when TKIs were discontinued after 2 years of maintenance, high rates of relapse were seen, fortunately salvaged with ASCT and other approaches (Slayton WB et al; JCO. 2018 and Schultz KR; Leukemia 2014). In chronic myeloid leukemia, patients (pts) who achieve deep and sustained molecular remissions on TKI may be able to stop therapy successfully. Herein, we reviewed outcome of pts with Ph+ ALL treated with chemotherapy + TKI without ASCT who later discontinued TKI mainly due to adverse events. Methods: We reviewed 240 pts treated at our institution on sequential protocols with Hyper-CVAD chemotherapy + TKI (dasatinib [n=100], ponatinib [n=84], or imatinib [n=56]) between 2001 and 2019. We identified 9 pts (4%) in whom TKI was discontinued, 4 (44.5%) post dasatinib therapy, 4 (44.5%) post imatinib therapy, and 1 (11%) post ponatinib. We analyzed their characteristics and outcomes including molecular relapse rates and treatment-free remission (TFR). Pts were closely monitored with monthly PCR for the first 3 months, then every other month for 3 months, then every 3 months thereafter. Molecular relapse was defined as the loss of MMR (PCR>0.1%) or positivity of PCR at two assessments within a 2-week period. TKI was resumed upon molecular relapse. TFR was defined from the date of TKI discontinuation to molecular relapse or last-follow-up. Kaplan-Meier method was used for survival analysis. Results: Baseline characteristics are summarized in table 1. The median follow up from the time of diagnosis was 138 months (range: 40-190). The median age at diagnosis was 60 years (range: 20-80). Transcript type was p190 BCR-ABL1 in 7 pts (78%). Median time to CMR was 3 months (range: 0.4-120). Median duration of TKI therapy prior to discontinuation was 70 months (range: 23-143). Median duration of CMR before TKI stop was 52 months (range: 22-141). Reasons for stopping TKI were side effects in 8/9 pts, and physician's choice in 1 pt (after completing 2+ years of maintenance). At the time of TKI stop, 8 pts were in CMR, and 1 pt with low positive transcript level (0.01%). Median follow-up post TKI discontinuation was 37 months (range: 9-75). None of the pts had morphological relapse. Three pts (33%) had molecular relapse within a median of 6 months (range: 0.8-13.2 months). All 3 resumed TKI therapy: 2 of them regained MMR after a median of 4 months (range: 4.0-4.6 months); third pt continues to respond; the BCR-ABL1 transcripts down from 17.68% to 0.36% after 7 months (Table 2). Six pts remain alive and 3 pts died of disease-unrelated causes. The median TFR was not reached; 3-y TFR was 65% (Figure 1). Though the number of pts was only 9, the duration of CMR had a tendency of successful TFR (P=0.062; HR, 0.09; [95% CI, 0.009-1.119] with duration of CMR for 2 years, and P=0.137; HR, 0.15; [95% CI, 0.01-1.80] with duration of CMR for 3 years, as a binomial variable). The median duration of CMR in pts who relapsed and who did not relapse was 22 months (range, 0-39.9) and 58 months (range, 30.9-140.6), respectively (P= 0.096). Conclusions: Our anecdotal experience reflects the feasibility of stopping TKI in a subset of pts with Ph+ ALL and sustained molecular remissions. Longer follow up and validation of these findings on a larger cohort are highly needed before attempting to discontinue TKI. Disclosures Kantarjian: Astex: Research Funding; Ariad: Research Funding; Jazz Pharma: Research Funding; AbbVie: Honoraria, Research Funding; Cyclacel: Research Funding; Immunogen: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Takeda: Honoraria; Agios: Honoraria, Research Funding; Novartis: Research Funding; Amgen: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Daiichi-Sankyo: Research Funding. Sasaki:Otsuka: Honoraria; Pfizer: Consultancy. Konopleva:Ascentage: Research Funding; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Agios: Research Funding; Ablynx: Research Funding; Cellectis: Research Funding; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria; Eli Lilly: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Kisoji: Consultancy, Honoraria; Astra Zeneca: Research Funding; Genentech: Honoraria, Research Funding. O'Brien:Acerta: Research Funding; AbbVie: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Research Funding; Sunesis: Consultancy, Research Funding; Janssen: Consultancy, Honoraria; Kite: Research Funding; GlaxoSmithKline: Consultancy; Gilead: Consultancy, Research Funding; Eisai: Consultancy; Celgene: Consultancy; Astellas: Consultancy; Aptose Biosciences, Inc: Consultancy; Amgen: Consultancy; Alexion: Consultancy; TG Therapeutics: Consultancy, Research Funding; Vaniam Group LLC: Consultancy; Verastem: Consultancy; Regeneron: Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding. Kadia:Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Bioline RX: Research Funding; BMS: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Research Funding. Jabbour:Takeda: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Adaptive: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Cyclacel LTD: Research Funding.

Long-Term Clinical and Molecular Remissions in Patients with Mantle Cell Lymphoma (MCL) Following High-Dose Therapy (HDT) and Autologous Stem Cell Transplantation (ASCT)

Background:Long-term clinical and molecular remissions in patients with MCL following HDT and ASCT have been evaluated in only a few studies. Results are especially limited for remission duration over 5 years. So the curability of this disease remains an open question. Patients and methods:Altogether 61 patients with MCL were treated in our institution with ASCT from 1998 to April 2019 (50 1st-line ASCT, ten 2nd-line ASCT, one 3rd-line ASCT). The data were collected retrospectively in 29 and prospectively in 32 patients who participated in two clinical trials: the 1st-line therapy trials of the German Low Grade Lymphoma Study Group (GLSG, principal investigator W. Hiddemann [Dreyling M, Blood 2005] and the European Mantle Cell Lymphoma Network [Hermine O, Lancet 2016], respectively. The diagnosis was regularly approved by the reference pathology of the GLSG (W.K.). The induction therapy before 1st-line ASCT consisted of 6 courses CHOP (n=11), mostly combined with rituximab and followed by Dexa-BEAM, and 6 alternating courses of R-CHOP and R-DHAP (n=39), respectively. For the salvage treatment patients usually received three to four courses of the DHAP protocol or the ESHAP protocol, since 2001 also combined with rituximab.Stem cell apheresis was carried out in the Blood Transfusion Service following these protocols in remission. High-dose protocols: 1) total body irradiation with cyclophosphamide or melphalan and cytarabin (n=32) or 2) BEAM (n=29). Patients with partial remission after ASCT received a radiotherapy (RT) with 30-36 Gy in the field of persisting lymphoma, if possible (n=4). Since 09/2016 patients received a maintenance therapy with rituximab after ASCT (every 2 months, planned for 3 years, n=14). Further details are described in an earlier publication of our first 36 patients (Metzner B, Ann Hematol 2013). Response assessment was performed by careful clinical examination and by ultrasound, chest x-ray and partly CT at regular 3 - 12 monthly time points. In the case of long-term remission (≥ 5 years; n = 18), peripheral blood was regularly tested twice a year for minimal residual disease (MRD) by quantitative t(11;14) or allel specific IGH RQ-PCR and/or IGH-consensus PCR.Calculations were done using IBM SPSS Statistics Version 25. Data were analysed as of 01 July 2019. Results:With a median follow-up of 5 years (range 0.1-20) 10-year overall survival, progression-free survival (PFS) and freedom from progression (FFP) after 1st-line ASCT were 54%, 46% and 52%, respectively, after 2nd-line ASCT 42%, 20%and 20%, with a significant difference for PFS (p=0.045) and FFP (p=0.014) between 1st-line and 2nd-line cohort.Further prognostic factors (like sex, age, MIPI, bone marrow involvement, remission grade at ASCT: CR vs. PR, type of HDT: TBI vs. BEAM…) seemed to be without relevance (considering the small subgroups). Only one clinical relapse occurred after 5 years following ASCT in 1stor 2ndremission, respectively (one patient 6 years after 1st-line ASCT and another patient 7 years after 2nd-line ASCT and subsequent radiotherapy). So far, 18 patients experienced long-term remissions of at least 5 years (median 9 years, from 5 to 16 years). Fifteen of 17 tested patients were MRD negative at last follow-up, the two MRD positive patients (positive at a low level below the quantitative measuring range) had no clinical signs of relapse at last follow-up. None of these 18 patients had received rituximab maintenance therapy. None showed clinical criteria of "smoldering mantle cell lymphoma" at induction therapy. Treatment-related mortality at 100 days after ASCT was 1.6% (pneumonitis following TBI). One patient developed a secondary invasive malignancy in remission after ASCT (acute myeloid leukemia 4 years following TBI). Conclusion: Sustained long-term clinical and molecular remissions up to 16 years can be achieved following ASCT (without rituximab maintenance therapy), indicating the potential curative impact of 1st-line ASCT in MCL. The 2nd-line ASCT was obviously less effective. Disclosures Dreyling: Celgene: Consultancy, Other: scientific advisory board, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: scientific advisory board, Research Funding, Speakers Bureau; Mundipharma: Consultancy, Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: scientific advisory board, Research Funding, Speakers Bureau; Bayer: Consultancy, Other: scientific advisory board, Speakers Bureau; Gilead: Consultancy, Other: scientific advisory board, Speakers Bureau; Acerta: Other: scientific advisory board; Novartis: Other: scientific advisory board; Sandoz: Other: scientific advisory board. Klapper:Roche, Takeda, Amgen, Regeneron: Honoraria, Research Funding.

U.S. Food and Drug Administration Benefit-Risk Assessment of Nilotinib Treatment Discontinuation in Patients with Chronic Phase Chronic Myeloid Leukemia in a Sustained Molecular Remission.

On December 22, 2017, the U.S. Food and Drug Administration (FDA) updated the product label for nilotinib to include information for providers on how to discontinue this drug in certain patients. With the updated dosing recommendations, select patients with chronic phase myeloid leukemia (CML) taking nilotinib for 3 years or more and whose leukemia has responded with sustained molecular remission (MR4.5, BCR-ABL transcripts of ≤0.0032%) as determined by a FDA-approved test may be eligible to discontinue nilotinib. The updated dosing regimen was based on the efficacy results from two trials that measured how long patients could stop taking nilotinib without the leukemia returning (treatment-free remission). Trial results demonstrated that, among selected patients who received nilotinib as first-line therapy or after transition from imatinib, approximately 50% continued to be in remission at 96 weeks after stopping therapy. Relapses continued to occur throughout the study, indicating that long-term monitoring is needed for safety and disease monitoring. Discontinuation of treatment was associated with an increased risk of low grade musculoskeletal adverse events, some of which were prolonged. Overall, the results support the approval of updates to the dosing recommendations with regard to treatment discontinuation in selected patients who have received nilotinib for at least 3 years, are in a sustained molecular remission, and who can undergo appropriate monitoring. IMPLICATIONS FOR PRACTICE: The updated dosing information provides eligibility criteria for treatment discontinuation, strict monitoring criteria after nilotinib discontinuation, and guidance for treatment reinitiation in eligible patients with chronic phase myeloid leukemia. About half of appropriately selected patients remained in remission 96 weeks after treatment discontinuation. Patients may experience musculoskeletal pain on withdrawal of treatment, incidence of which appears to decrease over time; however, some patients may have long lasting events. The decision to withdraw or continue treatment with nilotinib should be based on clinical condition and patient preferences.

MP-3 - Recent advances in treatment for hematological malignancies

Treatment for hematological malignancies had progressed to obtain 30-40% long-term remission in some lymphomas and acute leukemias up to a year 2000 by cytotoxic anti-cancer agents. However, there had been little further improvement only with cytotoxic drugs. At turning the century, molecularly-targeting agents became available and treatment strategy was dramatically changed to enter a new era. I want to congratulate the JSMO founders who had a great keen insight to establish the society in this era (2002). Since then, we have experienced tremendous progress in this field. In diffuse large B-cell lymphoma which had no change in a cure rate with the standard chemotherapy (CHOP) for 30 years, 10% improvement was reported by adding anti-CD20 monoclonal antibody, rituximab (2001). It was surprised by not only hematologists but also oncologists that chronic myelogenous leukemia had an excellent response to a single oral agent, imatinib (2005), targeting the bcr-abl chimeric protein. Furthermore, over 1/3 patients who stopped imatinib after acquisition of undetectable residual disease for at least 2 years sustained molecular remission for longer than 5 years. Multiple myeloma is a chronic uncurable disease in the elderly which had no improvement in survival with melphalan or other intensive cytotoxic drugs for years. A proteasome inhibitor, bortezomib (2006) and immunomodulatory drugs, thalidomide (2008) or lenalidomide (2010), had improved a median survival of 3-5 years to longer than 5 years. Introduction of arsenic trioxide (2004) and all trans-retinoic acid (2007) made it possible to control DIC more easily and raised a cure rate in acute promyelocytic leukemia, and hypomethylating agents gave rise to prolong the survival in MDS. Supportive care has also advanced to control grave complications and prolong the survival. Development of new drugs along with new discovery of tumor biology in hematology will continue to improve treatment outcomes in the future.

Tyrosine Kinase Inhibitor Discontinuation in Chronic Myeloid Leukemia: Single US Center Experience

Imatinib (IM) can be safely discontinued in patients with chronic myeloid leukemia (CML) with sustained complete molecular remission. Previous publications from France and Australia have shown that overall 40% maintain MMR or MR5 for up to 5 years after IM is stopped. We report single US center experience with tyrosine kinase inhibitor (TKI) discontinuation. Between 06/2010 and 7/2015, 22 patients with CML in chronic (CP, n=19), accelerated (AP, n=2) and lymphoid blast phase (LBP, n=1) discontinued IM (n=17), dasatinib (DAS, n=3) or bosutinib (BOS, n=2), and were monitored by qPCR for BCR-ABL1 monthly for the first 3 months, quarterly for the following 2 years and then bi-annually. TKI was restarted in case of confirmed loss of MMR on a repeat qPCR. Reason for TKI discontinuation was predominantly driven by patients' request and TKI intolerance. Median age was 66 (range, 21-84). The 3 who discontinued DAS had IM-resistant (loss of CCyR; n=1), IM-intolerant CP (n=1), or received DAS as first-line agent (n=1). BOS was discontinued for IM-intolerant CP (n=1), or while in CR2 in a patient with LBP that transformed from IM resistant CP and relapsed following chemotherapy (HCVAD). Median duration of TKI therapy pre-discontinuation for the entire cohort was 89 months (range, 26-106). Three patients are not evaluable due to short follow-up (TKI stopped between 5/2015 and 7/2015). With a median follow-up of 40 months (range, 8-60), 7 (41%, 6 CP and 1 AP), all previously on IM lost MMR a median of 3 months (range, 3-24) after TKI was stopped and restarted IM. Loss of CHR occurred 13 months after loss of MMR in 1 patient who elected not to restart IM at the time MMR was lost, due to complications from cardiac transplant rejection. All 7 achieved MMR following restart of IM. Median duration of TKI therapy pre-discontinuation for these 7 patients was 60 months (range, 48-98). 12 patients (59%, 10 CP, 1 AP, 1 LBP) remain off TKI and have not lost MMR, 8 with continuously undetectable BCR-ABL1; and 4 had 1-2 transient detectable BCR-ABL1 at MR4 levels. Median duration of TKI therapy pre-discontinuation for these 12 patients was 87 months (range, 26-106). Loss of MMR-free survival is depicted in the Figure. We conclude that, similar to previous reports, TKI can be safely discontinued in patients with CML without reappearance of BCR-ABL1 in 50-60%. Figure 1. Figure 1. Disclosures Jillella: Seattle Genetics, Inc.: Research Funding. Kota:Leukemia Lymphoma Society: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees.

Molecular Detection of Minimal Residual Disease Provides the Most Powerful Independent Prognostic Factor Irrespective of Clonal Architecture in Nucleophosmin (NPM1) Mutant Acute Myeloid Leukemia

▪NPM1 mutation (NPM1c) defines the commonest molecular subtype of AML, which is molecularly heterogeneous, with outcome influenced by the pattern of cooperating mutations. This has generated interest in molecular profiling to guide treatment approach, particularly concerning allogeneic transplantation (SCT) in first complete remission (CR1). Patients with NPM1c AML with FLT3-ITD and/or DNMT3A mutations have been associated with poorer outcome and are widely considered candidates for SCT. Conversely, those with the NPM1c/FLT3-ITDneg genotype are no longer routinely transplanted in CR1, due to their generally favorable outcome. Given the molecular heterogeneity, use of real time quantitative PCR (RT-qPCR) to detect residual leukemia (MRD) could improve outcome prediction. However, its value has been questioned by recent studies providing evidence that relapse can arise from preleukemic clones.To address these issues we analysed sequential samples from 346 patients (median age 50y, 6-81y) with NPM1c AML treated in the UK NCRI AML17 trial (median follow-up 35mo). An established RT-qPCR assay with mutation-specific primers was used, allowing MRD detection in all patients, covering 27 NPM1 mutations. Assays were confirmed to be mutant specific, with a median sensitivity of 1 in 105 (1 in 103.7-7.1). Overall 2,569 follow-up samples (902 BM, 1667 PB) were analysed (median 6 samples/pt). To determine if MRD assessment provided independent prognostic information, targeted sequencing (Haloplex, Agilent Technologies) of 51 genes was undertaken in 223 cases with available diagnostic DNA. The panel included established recurrent mutation targets in NPM1c AML and 14 genes found to be mutated in whole exome sequencing of 27 cases with differing kinetics of relapse or sustained molecular remission (CRm).For patients in documented morphological CR, early MRD assessment distinguished those at markedly differing risk of relapse and overall survival (OS). For patients with NPM1 mutant transcripts still detectable in peripheral blood (PB) following chemotherapy course 2 (30 of 194, 15%), cumulative incidence of relapse was 77% at 3 years, compared to 28% in those testing PCR negative (p<0.0001), associated with poorer OS (25% vs 77%, p<0.0001). While PCR positive patients had a higher rate of FLT3-ITD (66% vs 34%, p=0.002) and were more likely to have poor risk features (47% vs 17% p=0.002) considering age, cytogenetics, WBC and 2° AML, multivariable analysis found MRD status was the most significant prognostic factor (HR 4.51 (2.43-8.35) p<0.0001), identifying patients with poorer outcome within the NPM1c/FLT3-ITDneg group. Conversely, achievement of PCR negativity in PB by post-course 2 distinguished a major cohort (93 of 117, 79%) with relatively favorable outcome (OS 76%) despite an adverse genotype (FLT3-ITD &/or DNMT3A mutant). After adjustment for MRD status neither FLT3-ITD (p=0.8), DNMT3A (p=0.7) nor risk group (n=0.8) provided additional discrimination.Serial monitoring of paired BM and PB samples showed that analysis of marrow increases MRD detection rate, affording a median 1-log increment in sensitivity, associated with a longer time from molecular conversion to relapse (BM median 144d vs 88d for PB, p=0.3). Detection of MRD beyond consolidation predicted progression in 53 of 64 patients, preceded by a median 0.7-log rise in transcripts (range 0.2-3.6) per month; 8 patients remain in CR following pre-emptive therapy including SCT or epigenetic therapy and only 3 have not yet relapsed at 2-10 months follow-up. Targeted sequencing analysis at molecular or clinical relapse, showed mutant allele frequencies of genes including DNMT3A and TET2 exceeding the NPM1 mutation in 21 of 51 (41%) cases, consistent with presence of preleukemic clones. Moreover, amongst patients achieving CRm with long-term follow-up BM samples (NPM1c neg, sensitivity >10-4), DNMT3A-R882 remained readily detectable by digital PCR at 27-40 months (allele frequency 7-49%) in all 8 patients treated with chemotherapy alone; whereas the DNMT3A mutant clone was eliminated in the 3 patients after SCT. Irrespective of the clonal architecture, the NPM1mutation was found to be a stable marker of AML status, detected at relapse in 69 of 70 cases analysed.Therefore the NPM1c RT-qPCR assays track the leukemic clone with high fidelity and merit adoption into routine practice to assess response and inform patient management. DisclosuresNo relevant conflicts of interest to declare.

Therapy-Free Remission after Interferon Alfa Maintenance with Imatinib Discontinuation in Patients with Chronic Myeloid Leukemia

Introduction: Imatinib mesylate (IM) induces sustained molecular remissions in patients with chronic myeloid leukemia (CML) but a life-long therapy is required for most of these patients. We previously reported that CD8+ memory T cells showed significant predominance over naive T cells in the patients with sustained therapy-free major molecular response (MMR), all of that had previously received IFN [1]. We have been performing a phase 2 study of treatment discontinuation after the drug change from IM to IFN (Japanese Imatinib Stop And Interferon Study; JISAS). This trial is comprised of 2 parts; the first part is to change therapy drug from imatinib to IFN and the second part is to stop IFN for therapy-free. We present here a final result of this trial.Patients: Patients with a confirmed diagnosis of CML in 1st chronic phase (CP1) as well as in CMR (undetectable BCR-ABL transcript) following over 2 years of MMR on IM were enrolled in this pilot study after obtaining the written informed consent. All the eligible patients were recruited from September 2009 to December 2011 whether or not any therapeutic drug had been given before IM.Evaluation and response criteria:Since a conversion factor for International scale was not available in Japan until recently, for entry to the study, BCR-ABL transcripts at a level equal to or below 100 copy/mg RNA in a real-time quantitative-polymerase chain reaction (RQ-PCR) assay or equal to or below 50 copy/assay in a highly sensitive transcription-mediated amplification (TMA) method were defined as MMR. CMR was defined as detection of no BCR-ABL transcript in RQ-PCR assay, nested reverse transcriptase-polymerase chain reaction (RT-PCR) assay, or TMA.During the study, molecular response was assessed at the baseline and every month thereafter, by determining the BCR-ABL to ABL mRNA transcript ratio isolated from the peripheral blood using RQ-PCR and RT-PCR. The BCR-ABL to ABL transcript ratio at a level below 0.001 was defined as MMR and no detectable BCR-ABL transcript in RT-PCR was defined as CMR. Molecular relapse defined as a loss of MMR was taken into account if confirmed in 2 successive assessments.Study design and treatment: Administration of IFN is started at a dose of 3 million units 2-5 times per week within 4 weeks after IM discontinuation. IFN administration was stopped after 2 years of IFN maintenance. In case of molecular relapse, IM was resumed at 400 mg daily.Statistical analysis: Non-parametric values or numbers were compared between the two groups using the Mann-Whitney test. Relapse-free survival was estimated using the Kaplan-Meier method.Results: Fifteen patients were enrolled from September 2009 to December 2011. Median age was 50 years (range, 28-67 years) and male to female ratio was 1.5. Sokal score at the diagnosis was low in 13 patients and high in 2 patients. Four patients had been treated before IM. Previous therapies comprised IFN in all these patients, including 1 patient who relapsed after allogeneic stem cell transplantation (SCT). The clinical stage of this patient was amended to be accelerated phase at the time of diagnosis. Two other patients withdrew the consent. Excluding these 3 patients, the remaining 12 patients with low Sokal score were analyzed.The average follow-up period is 1220 days (range: 797-1519 days). Four patients lost MMR (1, 3, and 6 months in IFN maintenance phase and 10 months after IFN discontinuation, respectively) and other 8 maintained CMR. Estimated molecular relapse-free survival at 1 year after discontinuation of IFN is 60%. The patients in therapy-free remission revealed significantly longer CMR period on IM (median 31 months, range 26-79 months) compared with relapsed patients (0, 9, and 14 months, respectively; p=0.0142). Molecular relapse free survival was significantly better in patients with over 2 years of CMR on IM than otherwise (80% vs 25%; log-rank test, p=0.0013). All the relapsed patients achieved MMR after 2, 2, 4, and 5 months of IM resumption, respectively.Conclusion: IFN maintenance can provide high probability of therapy-free remission in patients with sustained molecular response of IM treatment for CML, especially with over 2 years of sustained CMR of IM treatment.

Use of High-Throughput Sequencing (HTS) of TCRß to Determine the Kinetics of Graft-Versus-Lymphoma (GVL) Effect and T-Cell Repertoire Profiles after Allogeneic Transplant

▪Introduction:While allogeneic transplant using non-myeloablative preparative regimen provides a viable alternative with low TRM, the kinetics of GVL effect and the T-cell reconstitution may differ from the myeloablative transplant. In general, the donor cell engraftment is slower using a non-myeloablative regimen and a delayed GVL effect is expected. In this study, we applied the TCRß HTS to determine (1) the kinetics of the GVL effect by quantifying the tumor cell burden prior to and after transplant and (2) the pace of the T-cell reconstitution after transplant.Method: This report included a cohort of 24 patients with advanced stage mycosis fungoides or Sézary Syndrome who underwent allogeneic transplant using a non-myeloablative regimen with total skin electron beam therapy (TSEBT, 24-36 Gy), total lymphoid irradiation (TLI, 8 Gy) and anti-thymocyte globulin (ATG). All patients received G-CSF-mobilized peripheral blood hematopoietic cells with a median donor CD34+ cell dose of 6.9 x 106/kg (range 2.0-12.4) and a median donor CD3+ cell dose of 278.1 x 106/kg (range 134.4-631.0). The unique malignant T-cell clonotype of each individual patient was identified from diagnostic blood/skin samples as the single dominant sequence by TCRß HTS. Blood samples were collected prior to and at different time points after transplant. DNA extracted from PBMC corresponding to approximately 200,000 genomes was used for HTS (Sci. Transl. Med.5:214ra171, 2013) (ImmunoSEQ, Adaptive Biotech). The tumor cell burden (including minimal residual disease, MRD) was expressed as percentage of the malignant clonotype found in the entire T-cell repertoire.Results: Prior to transplant, 21 patients (88%) had detectable disease by TCRß HTS in the blood (<1%: 10 patients, 1-5%: 3 patients, >5%: 8 patients), and 3 patients had no detectable disease in the blood. The percentage of malignant clone decreased in 19 of these 21 patients at day+30 post-transplant, and 2 patients showed stable minimal disease (0.03 and 0.30%, respectively). The reduction of tumor burden was most pronounced in patients with >5% involvement prior to transplant. In these 8 patients, the pre- and day+30 post-transplant disease burden decreased from 77.9 to 0.9%, 23.6 to 9.0%, 21.1 to 8.8%, 11.8 to 1.9%, 11.3 to 0.9%, 10.0 to 0.1%, 6.0 to 1.5% and 5.5 to 0.2%, respectively. This immediate post-transplant GVL effect was not associated with full donor T-cell engraftment (donor CD3+ >95%). The donor T-cell chimerism at day+30 was 94%, 79%, 1%, 90%, 93%, 93%, 23% and 91% for these 8 patients, respectively. Subsequently, 11 of the 24 patients achieved sustained molecular remission in the blood with a median time to achieve molecular remission of 60 days (range 30-540). Patients with full donor T-cell chimerism (n=16) had a higher chance of achieving molecular remission in the blood than those with mixed donor T-cell chimerism (69% vs 0%, p = 0.002). Of these 11 patients who achieved molecular remission in the blood, 8 also achieved molecular remission in the skin at the same time. Of the remaining 3 patients, 2 achieved molecular remission in the skin more than 4 months after achieving remission in the blood, while one patient has yet to achieve molecular remission in the skin.We then analyzed the “T-cell repertoire” at different time points post-transplant by assessing the number of unique T-cell clonotypes in each blood sample from 14 patients who had at least 1-year follow-up. While the size of the T-cell repertoire varied significantly between patients, we observed an overall upward trend within individual patients after transplant (Table). The size of T-cell repertoire did not correlate with the donor CD34+ or CD3+ cell dose in the allograft.Conclusion:By using an extremely sensitive and specific TCRß HTS, we have shown an immediate post-transplant GVL effect in which a full donor engraftment was not required, followed by a subsequent sustained GVL effect that may depend on full donor T-cell engraftment. Our results suggest a different kinetics of GVL effect in different compartments (blood vs skin). We also demonstrate continued expansion of T-cell repertoire profile after allogeneic transplant. Abstract 2473Table. The number of unique T-cell clonotypes at different time points post-transplantDay+30Day+60Day+90Day+180Day+270Day+360Median7,55010,4177,9428,40013,62820,057Range629 - 60,6441,916 - 63,6911,297 - 82,0591,572 - 66,5916,510 - 42,5651,914 - 57,358 DisclosuresNo relevant conflicts of interest to declare.

Sustained complete molecular remission after imatinib discontinuation in children with chronic myeloid leukemia.

Approximately 40% of adults with chronic myeloid leukemia (CML) in prolonged complete molecular response (CMR) remain in CMR after imatinib discontinuation. Corresponding information in children is lacking. Two children with CML in CMR for 48 and 19 months after imatinib discontinuation showed low-level fluctuating disease at RNA transcript and genomic DNA levels. Both patients were low risk according to adult criteria. Since adults with molecular relapse responded to re-introduction of imatinib, we postulated that treatment discontinuation in low risk children might be justified within clinical trials with close monitoring. This may help to minimize exposure to imatinib and its potential side effects.

Long-term clinical and molecular remissions in patients with mantle cell lymphoma following high-dose therapy and autologous stem cell transplantation

Long-term clinical and molecular remissions in patients with mantle cell lymphoma (MCL) following high-dose therapy (HDT) and autologous stem cell transplantation (ASCT) have been evaluated in only a few studies. Thirty-six patients with MCL received ASCT in our institution (27 patients undergoing first-line therapy, 8 patients undergoing second-line therapy, and 1 patient undergoing third-line therapy). In the case of long-term remission (≥5years; n = 8), peripheral blood was tested for minimal residual disease (MRD) by t(11; 14) polymerase chain reaction (PCR) and immunoglobulin heavy-chain (IGH) PCR at the last follow-up. Ten-year overall survival (OS), progression-free survival (PFS), and freedom from progression (FFP) after first-line ASCT were 42%, 43%, and 54%; after second-line ASCT, these were all 0%. Four-year OS, PFS, and FFP for the first-line cohort were 75%, 48%, and 61%, respectively. Four-year OS, PFS, and FFP after second-line ASCT were 55%, 30%, and 30%, respectively. Treatment-related mortality (3months after ASCT) was 0%. The only prognostic factor for OS, PFS, and FFP was treatment line (p = 0.011, p = 0.046, and p = 0.023, respectively). No relapses occurred after 5years following ASCT. So far, eight patients developed sustained long-term clinical and molecular complete remissions of up to 14.6years following ASCT in the first treatment line. Sustained long-term clinical and molecular remissions can be achieved following ASCT in the first treatment line and apparently less frequent in the second treatment line.

A Case of Therapy-Related Acute Myeloid Leukemia With a Normal Karyotype After Sustained Molecular Complete Remission of Acute Promyelocytic Leukemia

vealed a scattered distribution of blasts (60%) and abnormal promyelocytes (16%) with occasional Auer rods. The patient’s karyotype was 46,XY,t(15;17;19)(q22;q21q13.3)[20], consistent with APL with a t(15;17) variant clone. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect a breakpoint cluster region 1 (bcr1) isoform of the PML-RARα fusion transcript. The PML-RARα fusion transcript was quantified in the BM sample, using the Real-Q PML-RARα Quantification kit (BioSewoom, Seoul, Korea), showing a PML-RARα/ABL ratio of 0.91. On the basis of these results, the patient was diagnosed as having APL, and induction chemotherapy with idarubicin (10 mg/m 2 for 4 days) and ATRA (200 mg/m 2 for 7 days) was administered. After 4 weeks of treatment, the patient acquired complete remission (CR) and his PML-RARα/ABL quantification ratio reduced to 0.006. He was finally assessed as negative in April 2010. Maintenance chemotherapy with methotrexate (15 mg/m 2 per week) and 6-mercaptopurine (90 mg/m 2 per day) was administered for 20 months, and the PML-RARα fusion transcript was not detected for 32 months. In March 2013, the patient was re-admitted because of the presence of blasts in the blood. The morphology of blasts in the peripheral blood (37%) and BM (23.2%) (large amount of cytoplasm without azurophilic granules) was different from that

Long-Term Functional Persistence, B Cell Aplasia and Anti-Leukemia Efficacy In Refractory B Cell Malignancies Following T Cell Immunotherapy Using CAR-Redirected T Cells Targeting CD19

▪ BackgroundRecent advances in T cell engineering have enabled clinical trials to evaluate the potential for adoptive transfer of T cells to target malignancy. A single treatment with engineered gene-modified T cells has the potential to generate potent and long-lasting anti-tumor immunity. We have reported initial clinical data on the use of T cells engineered to express a Chimeric Antigen Receptor (CAR) that targets CD19 (CTL019) in patients with advanced treatment-refractory CLL and pediatric ALL (Porter, et al NEJM 2011; Kalos et al. Sci Trans Med 2011, Grupp et al. NEJM 2013). The initial cohort of patients is now disease free between 1 and 3 years post-infusion. In this report we present data on the functional persistence, trafficking, and bioactivity of CTL019 cells from the initial cohort of CLL patients, a larger and more recently treated cohort of CLL patients, a cohort of pediatric ALL patients, as well as an initial cohort of adult ALL patients. MethodsCAR engineering of patient T cells was accomplished by lentivirus transduction followed by in-vitro expansion using anti-CD3 and anti-CD28 antibody-coated beads. Persistence of gene-modified T cells was assessed by quantitative PCR. CAR19 surface expression was detected by flow. B cell aplasia was evaluated by multiparametric flow cytometry. Multiplex cytokine analyses were performed using LuminexTMtechnology. ResultsDetailed clinical outcomes for each patient cohort will be reported separately at this meeting (Porter, D.L.et al, Grupp S. et al). In summary, as of July 15, 2013: For CLL: A total of 24 adult patients with advanced relapsed and/or treatment-refractory CLL have been treated under two separate protocols. To date 5 patients have achieved ongoing CR, 7 patients PR and 12 patients were NR at the primary endpoint (within 3 months post infusion). For ALL (pediatric): A total of 14 pediatric patients with treatment refractory ALL have been treated and were evaluable under a single protocol. To date 8 patients have ongoing CR, 4 patients have relapsed including 2 with CD19-negative disease and 2 patients were NR. For ALL (Adult): A total of 3 adult patients with advanced relapsed and/or treatment-refractory ALL have been treated under a single protocol. All 3 patients have ongoing CR; one patient went into an allogeneic transplant while in CTL019-induced CR.In all patients with CR, robust in vivo expansion of CTL019 cells was observed, as assessed by both molecular and flow cytometric analysis, followed by contraction and in all but one patient ongoing stable persistence of engineered cells, elimination of tumor B cells and ongoing B cell aplasia in blood and marrow at all evaluated time points (minimum 3 months, maximum ongoing at 35 months). In CR patients, peak marking exceeded 5% of total CD3+. Patients with PR demonstrated less robust in-vivo expansion accompanied by transient B cell elimination, while NR patients demonstrated minimal in-vivo expansion.Clinical MRD analysis in patients was supplemented with Illumina-HiSeq/MiSeq-deep sequencing based molecular MRD analysis. These analyses demonstrate that CRs were accompanied with deep and sustained molecular remissions as assessed by the absence of the tumor specific clonotype defined in the enrollment samples.All patients in CR experienced on-target cytokine release syndrome (CRS) and macrophage activation syndrome (MAS). Multiplex-cytokine analysis of serum samples from patients in CR demonstrated a broad pro-inflammatory signature with significant elevation in a subset of soluble immune modulators including IFN-g, IL-6, and IL2ra. In contrast, patients with NR did not have elevated serum cytokines. Elevation of cytokines coincided with expansion of CTL019 cells, elimination of B cells, and toxicity suggesting the potential for a cytokine-based diagnostic signature to monitor CTL019 treatment efficacy. ConclusionsAdoptive transfer of CTL019 cells engineered to express CD137 and TCR-zeta signaling domains can result in in-vivo expansion, homing to marrow, and long-term functional persistence of engineered cells, accompanied by ongoing complete clinical responses and long-term B cell aplasia in a substantial fraction of patients with advanced, refractory and high risk CLL and relapsed refractory pre- B cell ALL. These results highlight the potential of CTL019 therapy to effectively target CD19-positive malignancy. Disclosures:Kalos:Novartis corporation: CART19 technology, CART19 technology Patents & Royalties; Adaptive biotechnologies: Member scientific advisory board , Member scientific advisory board Other. Zheng:Novartis: Patents & Royalties. Levine:Novartis: cell and gene therapy IP, cell and gene therapy IP Patents & Royalties. Grupp:Novartis: Research Funding. June:Novartis: Patents & Royalties, Research Funding.

High-Throughput Sequencing Of T Cell Receptors Detects Signatures Of T Cell Repertoire That Predict MRD Status In Patients With Mantle Cell Lymphoma Undergoing Immunotransplantation

Background A clinical trial is ongoing at Stanford for MCL patients in first remission that interdigitates an autologous CpG-stimulated tumor cell vaccine with autologous peripheral blood stem cell transplant (PSCT) (NCT00490529). In this trial, blood samples collected before and after vaccination and serially post-transplant are assayed for minimal residual disease (MRD) and for T cell repertoire using the LymphoSIGHT™ sequencing method (Faham et al., Blood 2012). We identified a set of T cell clones that appear to be responding to the vaccine, and therefore we investigated whether the number of these clonotypes was correlated with MRD status. Methods Using universal primer sets, we amplified rearranged IgH variable (V), diversity, and joining (J) gene segments from genomic DNA. Amplified products were sequenced to obtain &gt;1 million reads. The B cell tumor-specific sequence was identified for each patient based on its high frequency in the original tumor biopsy. The presence of the tumor cells was then monitored in serial blood samples with a sensitivity of 1 cell per million leukocytes. The same blood samples were used for amplification, sequencing and analysis of the entire TCRβ repertoire. To facilitate identification of tumor vaccine-induced TCRβ clonotypes, we sequenced the TCRβ repertoire immediately before and after the administration of both the priming vaccination and a booster vaccination. We developed a metric called the vaccine response score (V score). This metric is calculated for each clonotype and reflects the increase in frequency after the initial vaccination AND after the boost. The formula for calculating V score is: V = F1 x F2 x square root [1/ (|F1 – F2| + 1)], where F1 and F2 represent the fold-change of the priming and boost vaccinations, respectively. Clonotypes with a V score &gt;10 were deemed to be vaccination-induced by virtue of these frequency changes. Results In a series of 12 vaccinated patients, the number of clonotypes with V score ≥ 10 ranged between 0 and 262, with a median of 57. We utilized an antigen-specific analysis to validate that clones with high V scores (≥ 10) were in fact tumor-specific. For this analysis, we incubated peripheral blood mononuclear cells (PBMCs) with the tumor and then sequenced the TCRβ repertoire from cells obtained after culture. Clones that were enriched after culture compared to pre-stimulation PBMCs were deemed to be antigen-specific. These clones that are antigen-specific are highly likely to have a high V score compared to a random frequency-matched set of clones (P two tailed = 1.8 x 10-10), providing further evidence that clones with a high V score are tumor-specific. We then analyzed the relationship between V score and clinical outcome. Patients could be stratified into two groups with “high” (&gt; 25) or “low” (&lt;25) numbers of vaccine-responsive clonotypes. Patients in the high V score group, who had larger numbers of putative tumor-specific T cells, were more likely to have sustained molecular remission during the first-year post-transplant compared with patients in the low V score group (P = 0.018) (Figure 1). Conclusions T cell repertoire analysis identified clonotypes responding to the vaccination, and the presence of these vaccine-specific clonotypes correlates with MRD positivity at the important landmark of one year post-PSCT. Further analysis of additional patients enrolled on the MCL trial is ongoing. This data underscores the prognostic relevance of the sequencing-based V score metric and provides a novel approach for assessment of cancer immunotherapy responses. Disclosures: Faham: Sequenta: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

Safety and Efficacy Of BEZ235, a Dual PI3-Kinase /mTOR Inhibitor, In Adult Patients With Relapsed Or Refractory Acute Leukemia: Results Of a Phase I Study

Activation of phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling plays a role in cell proliferation, survival, and drug resistance in solid tumors and hematologic malignancies including chronic myeloid leukemia (CML), B-cell precursor acute lymphoblastic leukemia (BCP-ALL), T-ALL and acute myeloid leukemia (AML). The investigational compound BEZ235 is a potent dual pan-class I PI3K and mTOR complex C1 and C2 inhibitor and an attractive agent for relapsed or refractory leukemias.Primary objectives of this phase I study were determination of the dose-limiting toxicity (DLT), maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) in pts. with advanced acute leukemia. Secondary objectives included assessment of pharmacokinetics (PK), pharmakodynamic (PD) parameters and preliminary evidence of anti-leukemic activity.Inclusion criteria included age > 18years, relapsed or refractory AML, ALL or CML-BP considered ineligible for intensive or established treatment. Pts. with a fasting blood glucose >160mg/dl or an HbA1c >8% were excluded. The starting dose of BEZ235 was 400 mg twice daily (BID), administered orally during 28d cycles. Dose escalation was based on a “rolling-six”design, followed by an expansion phase at the RP2D. PK analyses were performed on days 1 and 15 by HPLC and fluorescence detection, PD analysis included assessment of phosphorylation of AKT, S6 and 4EBP1 by Western blotting (WB) and flow cytometry. The presence of PI3KCA, AKT or PTEN mutations was evaluated by direct sequencing of exons with known mutation hotspots. All pts. gave informed written consent, the study was approved by the Ethics Committee of the University of Frankfurt.22 pts. (13m, 9f), median age 62.5 years (range 29-82), were enrolled. Types of leukemia were AML (n=11), BCP-ALL (n=9), T-ALL (n=1) and CML in myeloid blast phase (CML-BP, n=1). 6 pts. were in first and 9 pts. in second or later relapse, 7 refractory or in refractory relapse, 7 pts. had extramedullary leukemia, 14 pts. previously received an allogeneic stem cell transplant (SCT). Six pts. were evaluated at the starting dose of BEZ235 (400 mg BID). No DLTs were observed, but BEZ235-related AEs (stomatitis and GI toxicity grades 1-3) necessitated treatment interruptions in 3 of 6 pts. 400 mg BID was considered not tolerable for prolonged administration and 16 pts. were subsequently treated at dose level -1 (300 mg BID). The most frequent non-hematologic AEs were gastrointestinal primarily of grades 1 and 2 with diarrhea (n=20), nausea/vomiting (n=18/6), stomatitis/mucositis (n=20), decreased appetite (n=14), fatigue (n=10) and hyperglycemia (n=21). Grade 3/4 AEs included sepsis (n=6), pneumonia (n=4), diarrhea (n=3), hyperglycemia (n=2), mucositis and fatigue (2 each). No patient started at dose level -1 was dose-reduced and none discontinued BEZ235 because of toxicity, 300 mg BID was selected as the RP2D. Clinical responses were observed in 4 of 22 pts. (3/10 ALL): one pat. with pro-B ALL achieved a complete hematologic and molecular remission with full donor chimerism, ongoing after 11 cycles of BEZ235. Hematologic improvement was observed in two pts. with BCP-ALL (1 Ph+, 1 Ph neg) and stable disease of 4 mos. duration in an AML patient. Nineteen of 22 pts. discontinued because of disease progression, median time to progression was 28 days (5d-112d). PK analysis revealed substantial interpatient variability of peak and trough levels at steady state, with no clear dose-dependency. All three responders in whom PK data are already available had low steady state trough levels below 100 ng/ml. No activating mutations of PIK3CA, AKT or PTEN were identified in any of the 22 pts. Phospho-flow and WB analysis provided no evidence of PI3K pathway activation, even in responding pts.In conclusion, the RP2D for BEZ235 was determined to be 300 mg BID, without formal definition of DLTs and an MTD. Single-agent anti-leukemic efficacy was most pronounced in ALL, with an overall response rate of 30% and a sustained molecular remission in one patient. Results of PK analysis and assessment of PD markers associated with PI3K signaling did not correlate with response. The PI3K pathway appears to be a “driver pathway” in only a small minority of pts. with ALL or AML, but more comprehensive genomic analysis may identify a subset of patients likely to benefit from treatment with dual PI3K-mTOR inhibitors. Disclosures:Ottmann:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.