Post-treatment Minimal Residual Disease Research Articles

The Charactex Protocol for Blood-Derived Cytological Preparation of Nonhematological Cancer

Introduction: Liquid biopsy, especially when performed by the isolation, expansion, and examination of circulating tumor cells (CTCs) from peripheral blood, has become an innovative and transforming diagnostic tool in Clinical Oncology. The CTCs have already entered the clinical practice as an alternative method to invasive tumor biopsy for detecting postsurgical and/or posttreatment minimal residual disease, to predict cancer recurrence and real-time treatment response. In this context, the retrospective observational project, known as CHARACTEX, has permitted to state that it is possible to exploit blood-based cytologic samples through short-term culture and in vitro CTC expansion. Methods: This method is based initially on a gradient-sedimentation technique, which impoverishes without completely depriving the obtained sample from the hematological cells, followed by short-term (14 days) in vitro culture and expansion and cytomorphological and flow cytometric analysis to investigate whether the expanded cell population possesses proliferative advantage and fits with criteria, which are consistent to the known primary tumor. Results: The originality of this method is that, apart from the above exposed criteria, there is no selection bias for the isolation of the cells from peripheral blood (like immunomagnetic bead treatment or preliminary immunocytochemistry), which can potentially introduce some limitation to the cell population under evaluation. Conclusion: The examination of the expanded cell population obtained by this method is very rewarding for both the pathologist – who can assess multiple tumor-related variables (like immunocytochemistry, flow cytometry of several parameters, and molecular pathology on cell suspensions and cell blocks obtained from them) – and the clinician.

MODL-05. DISCOVERY OF DYNAMIC MINIMAL RESIDUAL DISEASE STATES IN ADULT GLIOBLASTOMA USING SINGLE CELL TECHNOLOGY

Abstract Persister states are proposed oncogenic substrates of disease recurrence in cancer. Recent concepts suggest persisters may occur in dynamic states of the cell cycle (such as cycling and non-cycling states). We therefore investigated biological programs at the post-treatment minimal residual disease (MRD) state following standard chemoradiotherapy in patient-derived xenograft models of GBM. Our analysis of single cell RNA sequencing (scRNA-seq) data from 2704 tumor cells (929 cells post treatment, 1775 matched controls) yielded a cellular profile for non-cycling and cycling persister states. We validated these programs in 3 independent scRNA-seq datasets of human glioblastoma specimens consisting of over 16,000 cells from various genetic backgrounds, including an internal two patient-matched primary and recurrent GBM pairs with over 13,000 cells. Finally, we determined that clones identified based on large scale chromosomal rearrangements converge on previously identified persister states including the dynamic states discovered in our study. Our results provide new evidence towards dynamic persister states in glioblastoma, further analysis of these dynamic states is critical to targeting this incurable disease.

Time-Limited Venetoclax and Ibrutinib for Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia (R/R CLL) Who Have Undetectable MRD - Primary Analysis from the Randomized Phase II Vision HO141 Trial

Time-Limited Venetoclax and Ibrutinib for Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia (R/R CLL) Who Have Undetectable MRD - Primary Analysis from the Randomized Phase II Vision HO141 Trial

Liquid Biopsy-Based Biosensors for MRD Detection and Treatment Monitoring in Non-Small Cell Lung Cancer (NSCLC).

Globally, non-small cell lung cancer (NSCLC) is the leading cause of cancer deaths. Despite advancements in chemotherapy and targeted therapies, the 5-year survival rate has remained at 16% for the past forty years. Minimal residual disease (MRD) is described as the existence of either isolated tumour cells or circulating tumour cells in biological liquid of patients after removal of the primary tumour without any clinical signs of cancer. Recently, liquid biopsy has been promising as a non-invasive method of disease monitoring and treatment guidelines as an MRD marker. Liquid biopsy could be used to detect and assess earlier stages of NSCLC, post-treatment MRD, resistance to targeted therapies, immune checkpoint inhibitors (ICIs) and tumour mutational burden. MRD surveillance has been proposed as a potential marker for lung cancer relapse. Principally, biosensors provide the quantitative analysis of various materials by converting biological functions into quantifiable signals. Biosensors are usually operated to detect antibodies, enzymes, DNA, RNA, extracellular vesicles (EVs) and whole cells. Here, we present a category of biosensors based on the signal transduction method for identifying biosensor-based biomarkers in liquid biopsy specimens to monitor lung cancer treatment.

SO-34 Clinical experience of a personalized and tumor-informed circulating tumor DNA assay for minimal residual disease detection in oligometastatic colorectal cancer patients

For patients with oligometastatic colorectal cancer (CRC) treated with definitive management with curative intent, upwards of 50% still recur post-resection.1,2 Circulating tumor DNA (ctDNA) testing can be used to assess minimal residual disease (MRD) in patients with oligometastatic CRC and predict the risk of recurrence. Prospective evaluation of this methodology in clinical practice has been limited to date and the use of chemotherapy after metastasectomy is largely based on data extrapolated from earlier stage CRC. A personalized and tumor-informed multiplex PCR assay (Signatera™ bespoke mPCR NGS assay) was used for the detection and quantification of ctDNA for MRD assessment. We analyzed and present results from an ongoing early adopter program of ctDNA testing in patients with oligometastatic CRC (largely isolated to the liver) treated with surgery, radiation, or other definitive metastasis therapy. Adjuvant therapy was given at the discretion of the treating physician. ctDNA levels were analyzed on an average of 55 days post oligometastatic disease resection/ablation (range 3 – 150 days, median 52 days) in a total of 31 oligometastatic CRC patients. Post-treatment MRD was detected in 15 patients (48.4%), with an average of 74 mean tumor molecules per mL (range 0.11 - 673 MTM/mL; median 4.83 MTM/mL). Using the same assay, MRD rates after surgery were lower (up to 24%) in historical stage I-III CRC patients. Longitudinal case studies describing the ctDNA clearance effects of systemic therapy on MRD rates in the post-metastasectomy setting will be presented. This is the largest set of clinical experience data analyzing ctDNA in oligometastatic CRC patients and shows definitive evidence that a highly sensitive MRD assay can detect residual disease after curative intent procedures. An average MRD rate of 48.4% is consistent with published relapse rates of ∼50% post-oligometastatic resection, suggesting this assay accurately measures residual disease and may be prognostic for relapse, as well as potentially predictive for who might benefit from post-metastasectomy adjuvant chemotherapy.

Clinical Utility of Next-Generation Sequencing in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a genetically heterogeneous disease that, even with current advancements in therapy, continues to have a poor prognosis. Recurrent somatic mutations have been identified in a core set of pathogenic genes including FLT3 (25-30% prevalence), NPM1 (25-30%), DNMT3A (25-30%), IDH1/2 (5-15%), and TET2 (5-15%), with direct diagnostic, prognostic, and targeted therapeutic implications. Advances in the understanding of the complex mechanisms of AML leukemogenesis have led to the development and recent US Food and Drug Administration (FDA) approval of several targeted therapies: midostaurin and gilteritinib targeting activated FLT3, and ivosidenib and enasidenib targeting mutated IDH1/2. Several additional drug candidates targeting other recurrently mutated gene pathways in AML are also being actively developed. Furthermore, outside of the realm of predicting responses to targeted therapies, many other mutated genes, which comprise the so-called long tail of oncogenic drivers in AML, have been shown to provide clinically useful diagnostic and prognostic information for AML patients. Many of these recurrently mutated genes have also been shown to be excellent biomarkers for post-treatment minimal residual disease (MRD) monitoring for assessing treatment response and predicting future relapse. In addition, the identification of germline mutations in a set of genes predisposing to myeloid malignancies may directly inform treatment decisions (particularly stem cell transplantation) and impact other family members. Recent advances in sequencing technology have made it practically and economically feasible to evaluate many genes simultaneously using next-generation sequencing (NGS). Mutation screening with NGS panels has been recommended by national and international professional guidelines as the standard of care for AML patients. NGS-based detection of the heterogeneous genes commonly mutated in AML has practical clinical utility for disease diagnosis, prognosis, prediction of targeted therapy response, and MRD monitoring.

Identification of Gene Expression Signatures in Leukemia Stem Cells and Minimal Residual Disease Following Treatment of Adverse Risk Acute Myeloid Leukemia

Identification of Gene Expression Signatures in Leukemia Stem Cells and Minimal Residual Disease Following Treatment of Adverse Risk Acute Myeloid Leukemia

Venetoclax and Ibrutinib for Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia (R/R CLL) - 15-Month Safety, Response and MRD Evaluation: Third Interim Analysis from the Phase II Vision HO141 Trial

Venetoclax and Ibrutinib for Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia (R/R CLL) - 15-Month Safety, Response and MRD Evaluation: Third Interim Analysis from the Phase II Vision HO141 Trial

Next-generation sequencing-defined minimal residual disease before stem cell transplantation predicts acute myeloid leukemia relapse.

In acute myeloid leukemia (AML), the assessment of post-treatment minimal residual disease (MRD) may inform a more effective management approach. We investigated the prognostic utility of next-generation sequencing (NGS)-based MRD detection undertaken before hematopoietic stem cell transplantation (HSCT). Forty-two AML subjects underwent serial disease monitoring both by standard methods, and a targeted 42-gene NGS assay, able to detect leukemia-specific mutant alleles (with >0.5% VAF) (mean 5.1 samples per subject). The prognostic relevance of any persisting diagnostic mutation before transplant (≤27 days) was assessed during 22.1 months (median) of post-transplant follow-up. The sensitivity of the NGS assay (27 MRD-positive subjects) exceeded that of the non-molecular methods (morphology, FISH, and flow cytometry) (11 positive subjects). Only one of the 13 subjects who relapsed after HSCT was NGS MRD-negative (92% assay sensitivity). The cumulative incidence of post-transplant leukemic relapse was significantly higher in the pre-transplant NGS MRD-positive (vs MRD-negative) subjects (P = .014). After adjusting for TP53 mutation and transplant conditioning regimen, NGS MRD-positivity retained independent prognostic significance for leukemic relapse (subdistribution hazard ratio = 7.3; P = .05). The pre-transplant NGS MRD-positive subjects also had significantly shortened progression-free survival (P = .038), and marginally shortened overall survival (P = .068). In patients with AML undergoing HSCT, the pre-transplant persistence of NGS-defined MRD imparts a significant, sensitive, strong, and independent increased risk for subsequent leukemic relapse and death. Given that NGS can simultaneously detect multiple leukemia-associated mutations, it can be used in the majority of AML patients to monitor disease burdens and inform treatment decisions.

Distinct Gene Expression Patterns of Minimal Residual Disease (MRD) Cells in High-Risk AML Patients Identified By RNA-Sequencing

INTRODUCTIONEvolving techniques have made possible the direct detection, physical isolation, and study of AML minimal residual disease (MRD) after treatment. This could allow for better identification of therapeutic vulnerabilities in AML. Prior studies have focused on cells that initiate leukemia in mouse models, known as leukemia-initiating cells (LIC), generally with a foundational CD34+CD38- immunophenotype. LIC are typically derived from diagnostic samples of untreated patients. Such stem-like cells do not necessarily represent the residual fraction of AML after treatment. Relapse may originate from non-LIC, and the presence of phenotypically and molecularly defined MRD is now firmly established as a critical prognostic factor for patients. High-risk AML is characterized by relapse, despite morphologic complete remission with initial therapy in most cases. RNA-sequencing was performed on pre- and post-treatment AML subpopulations, including MRD, from high-risk patients, to determine differences in gene expression.METHODSMatched primary AML samples were collected from marrow and peripheral blood of patients with high-risk AML (including patients with unfavorable karyotype and/or TP53 mutation) at diagnosis and after treatment. Mononuclear cells were flow-sorted for bulk (CD45dim) and LIC (Lin-CD34+CD38-CD123+) from diagnostic samples. Post-treatment samples were sorted for bulk mononuclear cells (MNC) and MRD, based on difference-from-normal/MRD immunophenotype specific for each patient as determined from established 20-marker clinical flow cytometry analysis. RNA was isolated using low-input methodology, and RNA-sequencing was performed using Illumina HiSeq 2000. Gene expression was assessed using GO-Elite, and differences between patients and subpopulations were assessed using rank product method.RESULTSGene expression in MRD was analyzed by RNA-sequencing in comparison to diagnostic samples in eight patients with high-risk AML. Four patients had unfavorable karyotype, including two with TP53 mutations. Patients had additional high-risk features, such as FLT3-ITD or RUNX1 mutations, or secondary/therapy-related AML. Treatment consisted of chemotherapy (6/8) or hypomethylating agents (2/8), with or without other targeted drugs. Residual leukemia was detected in post-treatment samples in all study patients. Significant differences in gene expression were detected between MRD and other sorted populations, including diagnostic bulk AML and LIC. Relevant MRD pathways included those with strong interactions with the microenvironment. Anti-apoptotic mechanisms, cytoskeletal, and cell adhesion related genes, WNT/beta-catenin signaling, and TGFbeta signaling ranked among the most relevant processes in AML MRD subpopulations (Figure 1A, GO-Elite interactome of highly expressed genes in AML MRD). To identify potentially critical and unique MRD-specific genes, rank product method was applied using 1) the most highly expressed genes in AML MRD, 2) the most differential expressed genes between MRD and bulk AML at diagnosis, and 3) the most differentially expressed genes between MRD and bulk MNC after treatment. Among the top 50 scoring genes using this approach (Figure 1B), 16 genes were among the top 5% of genes expressed in MRD among all patients and 20 genes have cell surface gene-products (shown in yellow). Several potential leukemia- and cancer-related genes of interest were identified (shown in bold).CONCLUSIONSKey differences exist between the gene expression profiles of post-treatment MRD from high-risk AML patients, in comparison to other populations and subpopulations of sorted cells before and after treatment. The highlighted differences suggest that MRD relies on specific intrinsic gene expression changes and microenvironmental interactions, and therefore may be targetable after elimination of bulk AML with initial therapy. Accessible surfacesome targets are among top hits. [Display omitted] DisclosuresKonopleva:cellectis: Research Funding; Immunogen: Research Funding; abbvie: Research Funding; Stemline Therapeutics: Research Funding. Andreeff:Astra Zeneca: Research Funding; Amgen: Consultancy, Research Funding; Jazz Pharma: Consultancy; Celgene: Consultancy; Reata: Equity Ownership; SentiBio: Equity Ownership; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Equity Ownership; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Early Achievement of MRD-Negativity in IGHV-Mutated (IGHV-M) Patients Portends Highly Favorable Outcomes after First-Line Treatment of CLL with Fludarabine, Cyclophosphamide and Rituximab (FCR). Serial Monitoring for Minimal Residual Disease (MRD) in Blood after Achieving MRD-Negativity Predicts Subsequent Clinical Relapse

IntroductionAchieving MRD-negative remission after FCR therapy is an important predictor of longer progression-free (PFS) and overall survival (OS); however, with long-term follow-up, many MRD-negative patients subsequently relapse. The time course and predictive factors for relapse in initially MRD-negative patients have not been extensively characterized.MethodsWe prospectively analyzed 289 patients treated first-line with FCR for CLL at M.D. Anderson Cancer Center from 2008-15, to determine pre-treatment characteristics associated with post-treatment MRD negativity and the time course of relapse in MRD-negative patients. MRD analysis was performed in BM after course 3 (n=239) and at end of therapy (EOT) (n=231) using standardized 4-color flow cytometry (FLC). Ninety-five MRD-negative patients had 12-monthly serial MRD monitoring on blood.ResultsPretreatment characteristics are shown below (N=289).For the total cohort, the only pre-treatment characteristic significantly associated with PFS was IGHV-unmutated (IGHV-UM) (HR 3.0 [1.6-5.5], p<0.001). Analyzed separately, among IGHV-mutated (IGHV-M) patients, only ZAP70 positivity (38/92 [41%]) by immunohistochemistry was significantly associated with shorter PFS (HR 3.2 [1.3-8.2], p=0.01); among IGHV-UM patients only B2M ≥4.0mg/L (64/146 [44%]) was significantly associated with shorter PFS (HR 1.9 [1.2-3.0], p=0.005).Overall response rate was 96%. 46/239 patients (19%) after 3 courses and 120/231 (51%) at EOT achieved MRD negativity in BM. The following pre-treatment characteristics were significantly associated in univariableanalysis with achieving MRD-negativity at EOT in all patients: negative FISH (OR 3.1 [1.3-7.0], p=0.01), trisomy 12 (OR 2.7 [1.1-6.3], p=0.03, IGHV-M [OR 3.2 (1.8-5.9), p<0.001], β2-microglobulin <4.0mg/L [OR 1.85 (1.1-3.1), p=0.02] and ZAP70 negativity [OR 1.8 (1.03-3.2), p=0.04]. On multivariable analysis, only IGHV-M was significantly associated with MRD-negativity [OR 3.7 (1.7-8.2), p=0.001]. There were trends toward association between negative FISH [OR 5.4 (0.9-31.8), p=0.07] and trisomy 12 [OR 4.9 (0.8-29.8), p=0.08] with MRD-negativity. MRD negativity at EOT correlated with PFS, independent of IWCLL response category, and correlated with PFS in both IGHV-M and IGHV-UM patients (Figure A). In IGHV-M patients who were MRD-negative at EOT, there was a trend toward superior PFS if MRD-negativity was attained after 3 courses of FCR, even though 11/22 patients who were MRD-negative after 3 courses received no further therapy and only 3/22 received 6 courses (Figure B).Re-emergence of MRD in blood was detected in 45/95 MRD negative patients, at a median of 49 months post- treatment; this appeared less frequent in IGHV-M patients (Figure C). Re-emergence of MRD preceded clinical relapse by a median of 25 months. There were no significant differences in time-to-subsequent clinical relapse after re-emergence of MRD according to mutation status (Figure D). Among IGHV-M patients who were MRD-negative after 3 cycles, 4/22 had subsequent re-emergence of MRD.ConclusionsAmong patients with CLL receiving first-line FCR, the best pre-treatment predictor of achieving MRD-negativity and subsequent longer PFS was IGHV-M. Post-treatment MRD-negativity correlated with subsequent PFS; patients with IGHV-M who achieved MRD-negativity after 3 courses had a particularly favorable outcome, even after abbreviation of therapy. However, despite achieving MRD-negativity, many patients subsequently relapse; serial MRD monitoring in peripheral blood can herald relapse with a lead-time of approximately 2 years and potentially direct monitoring and/or early intervention strategies. [Display omitted] [Display omitted] DisclosuresThompson:Pharmacyclics: Consultancy, Honoraria. O'Brien:Janssen: Consultancy, Honoraria; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding. Jain:Incyte: Research Funding; Celgene: Research Funding; Seattle Genetics: Research Funding; Servier: Consultancy, Honoraria; Abbvie: Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Infinity: Research Funding; Novimmune: Consultancy, Honoraria; BMS: Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Genentech: Research Funding; ADC Therapeutics: Consultancy, Honoraria, Research Funding. Kadia:Novartis: Honoraria; BMS: Research Funding. DiNardo:Novartis: Research Funding; Daiichi Sankyo: Research Funding; Agios: Research Funding; Abbvie: Research Funding; Celgene: Research Funding. Wierda:GSK/Novartis: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Genentech/Roche: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Emergent: Consultancy, Research Funding; Sanofi: Consultancy; Celgene: Consultancy; Genzyme: Consultancy; Merck: Consultancy; Karyopharm: Research Funding; Acerta: Research Funding; Janssen: Research Funding; Juno Therapeutics: Research Funding; KITE Pharma: Research Funding.

Risk-Adapted Induction and Maintenance with Ofatumumab in Previously Untreated Patients with Chronic Lymphocytic Leukemia (CLL) / Small Lymphocytic Lymphoma (SLL)

Introduction: Pre-treatment cytogenetics and post-treatment minimal residual disease (MRD) status are predictive biomarkers in CLL. We hypothesized the two markers can guide a risk-adapted treatment strategy to achieve deep and durable responses in previously untreated CLL/SLL.Methods and Patients: 31 subjects were enrolled. Risk assessment was performed before treatment and after induction. Based on pre-treatment FISH, patients were categorized into either high-risk (with deletion 17p or 11q) or standard-risk (no deletion 17p or 11q) and treated with up to 6 cycles of OFC (Ofatumumab 300mg i.v. on cycle 1 day 1, 1000mg i.v. on cycle 1 day 8 and on day 1 of subsequent cycles; Fludarabine 25mg/m2 i.v. on day 1-3; Cyclophosphamide 250mg/m2 i.v. on day 1-3;) or OF (Ofatumumab given as described for the high-risk group; Fludarabine 25mg/m2 /d i.v. on day 1-5), respectively. Three months after completion of chemoimmunotherapy, a second risk stratification was performed based on presence or absence of MRD. MRD negative (MRDneg) state was defined as <1 monoclonal B cell among 104 leukocytes based on 8-color peripheral blood flow cytometry (PBFC). MRDneg patients were observed and MRD positive (MRDpos) patients received ofatumumab maintenance (1000mg i.v. every two months for 4 cycles). Serial PBFCs were performed pre- and post-induction, pre- and post-maintenance, and at 24 months. The primary endpoint was progression free survival at 24 months.Results: 25 patients who completed ≥3 cycles of chemoimmunotherapy were considered evaluable; median age 60 years (range 26-79), 56% male, 48% in high Rai stage, 68.6% having unmutated IGHV. Nine (36%) patients received OFC, 16 (64%) received OF. 72% of all patients required dose reductions and 64% completed 6 cycles. After induction, 15 (60%) patients were MRDpos and received maintenance, while 10 MRDneg patients were observed. More patients on the OFC arm were MRDneg than on the OF arm (66.7 vs. 25%, P =0.087). At a median follow-up of 32 months, 7 patients had progressed. The projected PFS was 95% at 24 months (85-100%), 82.5% at 32 months (66-100%), and 75% at 36 months (56-100%), with no significant difference between subgroups divided by treatment regimen or MRD status after induction. The estimated median PFS was 36 months for MRDpos vs. 49 months for MRDneg patients (P =0.13). At 24 months, 3 out of 7 MRDneg patients maintained MRD negativity without further treatment. For MRDpos patients, the number of residual CLL cells appeared to decrease during maintenance ofatumumab and two patients became transiently MRDneg. However, the change in MRD burden was not statistically significant (median CLL cell count per 104 leukocytes: 50 vs. 36 at pre- vs. post-maintenance, P =0.13) and none of the patients receiving maintenance was MRDneg at 24 months. The median CLL count per 104 leukocytes increased from 36 at post-maintenance to 260 at 24 months (P =0.047). Taken together, MRD after chemoimmunotherapy was not eradicated with additional doses of ofatumumab. We hypothesized that loss of CD20 expression through trogocytosis (Fc-receptor mediated "stripping" of CD20 from CLL cells) or internalization may limit the efficacy of ofatumumab. In fact, among MRDpos patients, CD20 expression was negative by PBFC in 90% of patients at post-induction and in all patients at post-maintenance. On bone marrow biopsies, immunohistochemical staining for CD20 using an antibody against an intracellular epitope confirmed CD20 loss or demonstrated substantially decreased CD20 expression compared to matching baseline samples.Conclusion: Pre-treatment risk stratification with FISH cytogenetics can guide the choice of induction therapy. Maintenance therapy with four additional doses of ofatumumab in MRDpos patients did not improve the depth of response but appeared to reduce the risk of disease progression. Loss of CD20 expression on residual tumor cells may limit the efficacy of prolonged therapy with an anti-CD20 monoclonal antibody. DisclosuresWiestner:Pharmacyclics: Research Funding.

Implications of Minimal Residual Disease Negative Complete Remission (MRD-CR) and Allogeneic Stem Cell Transplant on Safety and Clinical Outcome of CD19-Targeted 19-28z CAR Modified T Cells in Adult Patients with Relapsed, Refractory B-Cell ALL

Background: We have previously reported high anti-tumor activity of autologous T cells genetically modified to express 19-28z chimeric antigen receptor (CAR) targeting CD19 in adult patients with relapsed or refractory (R/R) ALL (Park et al. ASH 2014). Herein, we further report the long-term outcome of a larger cohort from our phase 1 clinical trial in adults with R/R ALL (NCT01044069) with a focused analysis on the role of post-treatment minimal residual disease (MRD) negativity as a predictive marker of survival as well as the effect of allogeneic hematopoietic stem cell transplant (allo-HSCT) prior to or after CAR T cell infusion on safety and clinical outcome. Patients and Methods: Adult patients with R/R B-cell ALL (B-ALL) were enrolled. Eligible patients underwent leukapheresis, and T cells were transduced with a retroviral vector encoding a CAR comprising a CD19-specific scFv and CD28 and CD3ζ signaling domains (19-28z). All patients received lymphodepleting chemotherapy followed 2 days later by 1x106 - 3x106 19-28z CAR T cells/kg. The primary objective of the study was to evaluate the safety and anti-tumor activity of 19-28z CAR T cells. Post-treatment MRD was assessed at day 14-28 by multiparameter flow cytometry in bone marrow (BM) samples. Results: 44 patients have been treated to date. The median age was 45 years (range, 22-74). 14 patients (32%) had Philadelphia chromosome positive (Ph+) ALL (T315I mutation in 5 patients), 17 patients (39%) had prior allo-HSCT, and 24 patients (55%) had ≥ 3 prior lines of ALL therapy. Of the 44 patients, 43 patients were evaluable for response. At the time of 19-28z CAR T cell infusion, 22 of the 43 patients (51%) had morphologic disease (≥5% blasts in BM or measurable extramedullary disease) and the remaining 21 patients had minimal disease (&lt;5% blasts in BM). 36 patients (84%) were in complete remission (CR) after 19-28z CAR T-cell infusion. MRD analysis was performed in 35 of 36 CR patients, and 29 of these 35 patients (83%) achieved an MRD-negative CR (MRD-CR). As of July 13, 2015, the median follow-up was 4.2 months (range 1-45), with 16 patients having at least 6 months of follow-up. Responses appear durable with 7 patients remaining disease-free beyond 1 year up to 45 months. A median overall survival (OS) of all patients and patients who achieved MRD-CR is 8.5 months and 10.8 months, respectively. Post-treatment MRD status emerged as a strong predictive marker of OS: OS at 6 months was 76% (95% CI: 51-89) in the MRD-CR cohort vs. 14% (95% CI: 8-45) in the MRD+CR cohort. In contrast, allo-HSCT after achieving CR with CAR T cell infusion did not affect the survival rate. Of the 36 patients in CR following the T cell infusion, 12 patients underwent allo-HSCT. OS at 6 months was 70% (95% CI: 33-89) in patients who underwent post-CAR allo-HSCT vs. 64% (95% CI: 36-82) in patients who did not get allo-HSCT after CAR T cells. Comparing baseline disease characteristics of patients who had prior allo-HSCT before the CAR T cell treatment vs. no prior allo-HSCT, patients who had prior allo-HSCT (n=17) were similar in age (median age 45 vs. 46), but had higher disease burden (65% with morphologic disease vs. 44%), were more heavily pretreated (59% of patients with ≥4 lines of therapy vs. 15%), and included more high-risk disease (41% with Ph+ ALL vs. 26%). However, there was no statistically significant difference in CR rates (75%, CI: 48-93 vs. 89%, CI: 71-98), incidences of severe cytokine release syndrome (24% vs. 22%), and OS at 6 months (57% vs. 60%) between these two cohorts. Fewer patients who had prior allo-HSCT underwent another allo-HSCT following CAR T cell infusion: 2 patients vs. 10 patients with no prior all-HSCT. Although no obvious case of graft-versus-host disease (GvHD) was noted, one patient experienced a grade 3 gastrointestinal toxicity that may have been related to GvHD. Conclusions: These data confirm the potent anti-tumor efficacy of 19-28z CAR T cells (JCAR015) in adult patients with R/R ALL. MRD negativity following the 19-28z CAR T cell treatment is highly predictive of survival, and allo-HSCT post-CAR T cell infusion had no significant impact on survival. Furthermore, 19-28z CAR T cells appear to be safe in patients who had prior allo-HSCT, and may represent an attractive alternative option to second allo-HSCT. These findings are being confirmed in an ongoing multi-center, pivotal phase 2 trial evaluating JCAR015 in adult patients with R/R ALL. Disclosures Park: Amgen: Consultancy; Genentech: Research Funding; Juno Therapeutics: Other: Advisory Board, Research Funding. Riviere:Juno Therapeutics: Other: Co-founder, stockholder and consultant. Curran:Juno Therapeutics: Consultancy. Sadelain:Juno Therapeutics: Consultancy, Equity Ownership, Other: Co-Founder, stockholder, Patents &amp; Royalties: Licensed patents on CARs. Brentjens:Juno Therapeutics: Other: Co-founder, stockholder and consultant.

CD19-Targeted 19-28z CAR Modified Autologous T Cells Induce High Rates of Complete Remission and Durable Responses in Adult Patients with Relapsed, Refractory B-Cell ALL

Background: Relapsed adult acute lymphoblastic leukemia (ALL) is associated with high reinduction mortality, chemotherapy resistance, and dismal prognosis with a median overall survival (OS) < 6 months and 5-year OS ≤10%. We have previously reported a high anti-tumor activity of autologous T cells genetically modified to express 19-28z chimeric antigen receptor (19-28z CAR) targeting CD19 in adult patients with CLL and ALL (Brentjens R et al. Blood 2011; Davila M et al. Sci Transl Med2014). Herein, for the first time, we further report the long-term outcome of our phase I clinical trial in adults with relapsed/refractory (R/R) ALL (NCT01044069) with analysis on potential predictive markers of response and neurological toxicities.Patients and Methods: Adult patients with R/R B-ALL were enrolled. Eligible patients underwent leukapheresis, and T cells were transduced with a retrovirus encoding a CAR construct composed of anti-CD19 scFV linked to CD28 and CD3ζ signaling domains (19-28z). All patients received lymphodepleting chemotherapy followed 2 days later by 1x106 – 3x10619-28z CAR T cells/kg. The primary objective of the study was to evaluate the safety and anti-tumor activity of 19-28z CAR T cells in ALL. Post-treatment minimal residual disease (MRD) was assessed at day 14-28 by multiparameter flow cytometry and deep sequencing in the bone marrow (BM) samples (Adaptive Biotech Corp.)Results: 24 patients have been treated. The median age was 56 years (range, 23-74). 6 patients (25%) had Ph+ B-ALL (T315I mutation in 2 patients), 6 patients (25%) had prior allogeneic hematopoietic stem cell transplant (allo-HSCT), and 11 patients (46%) had 3 or more prior lines of ALL therapy before receiving the 19-28z CAR T cell therapy.Of the 24 patients, 22 patients were evaluable for response. At the time of 19-28z CAR T cell infusion, 12 of 22 patients had morphologic disease (6 to 97% blasts in the BM) and the remaining 10 patients had MRD. Twenty out of 22 patients (91%) were in complete remission (CR) after 19-28z CAR T-cell infusion, and 18 of these 20 patients (90%) achieved an MRD-negative CR. Ten of the 13 transplant eligible patients (77%) successfully underwent allo-HSCT following the 19-28z CAR T cell therapy. As of July 1, 2014, the median follow-up was 7.4 months (range 1-34), with 13 patients having at least 6 months of follow-up. Responses appear durable with 6 patients remaining disease-free beyond 1 year (range 12.6 – 34 months). Median overall survial (OS) is 9 months. 5 patients relapsed during the follow-up, including 1 patient with CD19 negative relapse. Three of the relapsed patients were treated again with the 19-28z CAR T cells, and two patients achieved a second CR.Comparing responders to non-responders, no association was observed between response and age (<60 vs. ≥60), prior allo-HSCT, number of prior therapies, or pre-treatment blast percentage.While none of the 10 patients with MRD at the time of T cell infusion developed cytokine release syndrome (CRS), 9 of 13 patients with morphologic disease at the time of the T cell infusion developed CRS with or without neurological symptoms that required intervention with an IL-6R antagonist or corticosteroid. A detailed analysis of serum cytokines demonstrated a consistent peak of IL-6 (22.2 to 553-fold increase) immediately prior to the development of neurological toxicities. Based on these data, we have developed a multi-disciplinary CRS management algorithm for patients at high risk in order to reduce the severity of CRS and improve safety of the 19-28z CAR T cell therapy.Conclusions: While longer follow-up is needed to confirm the durability of the observed responses, the potent induction of MRD-negative responses and successful long-term outcomes, including subsequent allo-HSCT without apparent additional post-transplant toxicities, strongly support the use of 19-28z CAR T cells in adult patients with B-ALL. A temporal relationship between serum IL-6 levels and neurological toxicities indicates that early intervention with IL-6 directed therapy may be more effective in ameliorating neurological toxicities in patients with morphologic disease at the time of T-cell infusion. These findings will need to be evaluated systematically and confirmed in a larger phase 2 trial. DisclosuresPark:Juno Therapeutics: Research Funding. Riviere:Juno Therapeutics: Consultancy, scientific co-founders Other. Sadelain:Juno Therapeutics: Consultancy, Scientific co-founder and Stock holder Other. Brentjens:Juno Therapeutics: Consultancy, Scientific co-founder and Stock holder Other.

On-Going Evolution Of IGH In B-Cell Precursor Acute Lymphoblastic Leukemia Does Not Substantially Affect Day 29, Post-Treatment MRD Quantification By High-Throughput Sequencing

High-throughput sequencing (HTS) of immunoglobulin heavy chain genes (IGH) may be useful for detecting minimal residual disease (MRD) in B-cell precursor acute lymphoblastic leukemia (BPC-ALL), particularly in the context of massive clonal evolution at the IGH locus, as previously identified by others (Gawad et al., Blood 120(22):4407-17, 2012; and Faham et al., Blood 120(26):5173-80, 2012). This on-going rearrangement of IGH may limit detection of MRD in post-treatment samples by traditional molecular-based methods, typically real-time PCR using patient-specific primers or probes. Here, we examine the extent to which evolution of IGH in unselected pre-treatment samples from patients with BPC-ALL affects detection of MRD in day 29 post-treatment samples by high-throughput sequencing of IGH.Of 99 samples from an unselected series from the Children's Oncology Group trial AALL0932, we find that 92 of 98 samples have a clonal IGH gene rearrangement in pre-treatment samples. One sample failed at the outset during the DNA extraction step. Of the remaining 92 cases with pre-treatment VDJ or D-J rearrangements, 82 had evidence of on-going recombination in which VH replacement was identified in clones, each having conserved D-J rearrangements. The average number of clones was 192, but ranged from 1 to over 2000 unique sequences. In cases with VH replacement, an average of 4.12% of IGH sequences was made up of VH-replaced sequences. In post-treatment samples that were MRD positive, the predominant clone in pre-treatment samples was typically the most frequent clone. Clones consistent with VH replacement were found in 19 patients; in one patient, the only MRD detected was a single clone consistent with VH replacement at a level of ∼1 in 1,000,000. In the other 18 post-treatment MRD positive cases, the dominant clone identified pre-treatment was also dominant post-treatment: on average, 3.2% of total IGH rearrangements matched the dominant clone post-treatment, while only 0.027% of IGH rearrangements were consistent with VH replacement of the major clone. Among pre-treatment samples in which VH replaced clones were detected, all VH replaced clones together were 12% as large as the dominant clone on average. Among post-treatment samples, VH replaced clones were on average 14% as large as the dominant clone, indicating little change in the relative proportions of the dominant clone and VH replaced sub-clones.These findings together suggest that on-going rearrangement of the IGH locus is not likely to be important for clonal tumor evolution within the time frame of initial chemotherapy, as no substantial change in clonal diversity as assessed by IGH sequencing is evident. In other words, on-going rearrangement of IGH appears to be neutral with respect to therapy-induced selection of tumor clones that may represent early (day 29) relapse. Disclosures:Emerson:Adaptive Biotechnologies: Employment, Equity Ownership. Sherwood:Adaptive Biotechnologies: Employment, Equity Ownership. Kirsch:Adaptive Biotechnologies: Employment, Equity Ownership. Carlson:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties. Williamson:Adaptive Biotechnologies: Employment, Equity Ownership. Wood:Becton Dickinson and Company, NJ, USA: Research Funding. Robins:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties.

Molecular and Cytogenetic Risk Stratification For Core-Binding Factor-Positive Adult AML With Analysis Of Post-Remission Treatment Outcomes Including Transplantation

Core-binding factor (CBF)-positive acute myeloid leukemia (AML) is regarded as a favorable group with good complete remission (CR) rate after induction chemotherapy and is generally treated by repeated high-dose cytarabine consolidation or autologous hematopoietic stem cell transplantation (auto-HSCT). However, emerging molecular studies have recently identified the unfavorable CBF-AML subgroup which should be treated by further intensified treatments.This single center retrospective study enrolled 264 adult CBF-AML patients from 2002 to 2011. Except 15 patients, 217 patients were treated by intensive induction chemotherapy and 32 were treated by reduced intensity treatment. After CR achievement, patients with available donor were treated by allogeneic (allo)-HSCT and the rest were treated by auto-HSCT or chemotherapy alone. We evaluated 206 patients who achieved CR after intensive chemotherapy regarding survival outcomes according to post-remission therapies and prognostic factors which affected the outcomes. The factors included cytogenetic study and subgroup analysis with additional chromosomes and normal karyotype (NK) mosaicism, c-kit mutation, minimal residual disease (MRD) qPCR level, BAALC and WT1 expression.We achieved CR in 94.9% with intensive chemotherapy and 115 patients went on allo-HSCT and 72 were treated by auto-HSCT. There were no significant OS differences between CBF¥â/MYH11 and RUNX1/RUNX1T1 (p=0.173), and auto-HSCT showed favorable EFS (p=0.038) compared to allo-HSCT and chemotherapy alone. For cytogenetic analysis, inv(16) or t(16;16) with NK mosaicism showed the most favorable OS compared to t(8;21) with additional chromosome (¡Ã2) which showed the worst OS. c-kit mutation was a poor prognostic factor with lower reduction rate of post-induction MRD qPCR, however the effect was not definite after HSCT. For HSCT patients, we analyzed post-HSCT MRD qPCR and WT1 expression level. We found that undetected level of post-HSCT MRD qPCR and lower level of WT1 expression (<0.015) showed the most favorable OS with no relapse cases.For CBF-AML, the role of auto-HSCT and allo-HSCT for selected patients should be re-evaluated by large prospective studies and the values like post-treatment MRD qPCR and WT1 expression level can be used for prediction of patients who might relapse with higher probability.▪▪ Disclosures:No relevant conflicts of interest to declare.

Minimal Residual Disease Stratification in Fludarabine Ccyclophosphamide Rituximab (FCR) Therapy in Chronic Lymphocytic Leukaemia (CLL

AbstractAbstract 1791Six courses of FCR is considered standard therapy for fit, non-(17p) deleted patients with Chronic Lymphocytic Leukaemia (CLL), however the optimal dose of Rituximab in combined chemotherapy for CLL or the number of FCR courses required has never been formally assessed. Minimal residual disease (MRD) eradication (assessed by 6 colour flow-cytometry(FC) is associated with an increased disease free interval and is a logical endpoint in determining length/efficacy of treatment. Serial MRD testing following therapy provides an objective test of disease re-emergence.A multi-centre prospective phase II study in treatment-naïve CLL patients with modified FCR (Rituximab 375mg/m2) using MRD to determine treatment length/efficacy and identify disease emergence recruited from 2009–2012. Standard pre-treatment assessment plus C-T scan, FISH and Ig somatic hypermutation (SHM) analyses were performed. Patient in radiological and MRD-ve remission after 4 courses of FCR stopped therapy and the remainder received 6 courses. All were followed by 6 monthly MRD assessment.Fifty-two patients were included (35M/17F), mean age 52 years (range 37–72), mean WCC 51 × 109/L (range 8–386), elevated LDH 23 of 45 (51%), lymphadenopathy 43 (83%) and hepatosplenomegaly in 37 (71%). Abnormal FISH results were, del(11q) in 15, +12 in 5, del(13q) in 18; SHM 15(29%) mutated and 37(71%) unmutated or with V3–21 gene usage. Post-treatment MRD is available in 43 patients; 36 (70%) were MRD –ve including 18 (42%) after 4 courses. Nine patients did not complete treatment (toxicity −7, progression-1, non-compliance-1). With a mean follow-up of 20.7 months (range 4.5–38), 6 patients have reverted to MRD +ve at a mean of 13 months (range 10–19) from therapy; only the patient with primary treatment failure has required further chemotherapy. Myelotoxoicty resulted in 23 NCI grade 3 episodes and 7 treatment delays of a mean duration of 25 days (range15–32). One further grade 3 toxicity of pneumonia was identified.Modified FCR was effective in this patient cohort with high risk features (38% unfavourable FISH, 71% unfavourable SHM), with 70% patients achieving MRD-ve status on completion of therapy. 42% of patients became MRD-ve after 4 courses of FCR, suggesting that some patients may not require 6 courses of therapy. Myelotoxicity remains an issue with 7 patients not completing therapy. Longer follow-up will clarify whether shortened therapy will have an impact on MRD+ve reversion, time to re-treatment and survival. Disclosures:No relevant conflicts of interest to declare.

Immunophenotypical Sequential MRD Assessment in AML for Prediction of Relapse and Definition of Putative Future Intervention Time Points.

Immunophenotypical assessment of minimal residual disease (MRD) has been shown by us (Feller et al, Leukemia 18:1380, 2004) and others (San Miguel et al., Blood 98: 1746, 2001, Venditti et al., Blood 96: 3948, 2000) to be predictive for clinical outcome in AML both in patients treated with standard chemotherapy (SD) and high dose chemotherapy followed by autologous (AutoTQ) or allogeneic (AlloTQ) transplantation. Directly after consolidation therapy using a cut-off of 0.11% in the high MRD group the risk of relapse was 7.2 fold than in the low MRD group (Feller et al., 2004). Using that cut-off level, in the present study another important issue, i.e. sequential MRD assessments after end of treatment were done to i) prove consistancy of low MRD% in patients that remain in CR, ii) prove the ability to predict relapse using increase of MRD% and iii) assess the minimally required inter-sampling period to make MRD assessment clinically useful. To enable such 124 samples of 33 patients (<60 years) were included: 16 after SD, 8 after AutoTQ and 9 after AlloTQ. All time points refer to post-treatment periods, time point zero reflecting end of therapy.Of 16 patients studied after SD, 15 had MRD <0.11%. Of these, 12 are in CR with a median follow-up of 30.5 (range 9–46) months. In these 12 patients MRD remained consistently <0.11 %, (mean of 3 sequential MRD assessments per patient). Of these 15 patients 3 relapsed after 9, 9 and 26 months, but in these cases the last time point of MRD assessment (still <0.11%) unfortunately was at least 6 months before relapse. One patient who started with MRD>0.11 relapsed within 2 months after that MRD assessment.Of 8 patients studied after AutoTQ, 6 had MRD <0.11%. Of these, 4 are in CR with a follow-up of 6, 15, 40 and 72 months. Again, MRD remained consistently <0.11% (mean of 3 assessments per patient). Of these 6 patients 2 relapsed after 13 and 43 months, with relapse predictable using 3 months assessment intervals. Two patients had >0.11% MRD and relapsed after 5 and 11 months and within 3 months after the last MRD assessment, with no previous sign of forthcoming increase. Of 9 patients studied after AlloTQ, 2 had MRD <0.11%; thet are still in remission after 47 and 53 months. Again, MRD remained consistently <0.11 % (6 and 7 MRD assessments in these 2 patients).Of these 9 patients 2 relapsed too fast to have the opportunity to assess MRD. In addition, 5/9 had MRD >0.11%, of whom 2/5 relapsed after 6 and 7 months which was 4 and 2 months resp., after their last MRD assessment, with no previous sign of forthcoming increase. A remarkable phenomenon was observed in the other 3/5 patients: a large drop in MRD% down to detection level (0.01%) occurred 3–10 months after end of therapy. 2 of these 3 patients are still in CR (at 11 and 33 months), 1/3 relapsed after 26 months, 5 months after last MRD assessment with no previous sign of increase. The unique decrease of MRD% may be speculated to result from graft versus leukemia effects.These results show: 1) consistently low MRD% in sequential MRD assessments during persisting CR; 2) increases of MRD% which consistently predict relapse; 3) a minimally required inter-sampling period of 3 months for patients with MRD <0.11% and probably <3 months for patients with MRD>0.11%; 4) a strong decrease of MRD in part of the allogeneically transplanted patients.Results show the feasibility of sequential post-treatment MRD assessment that ultimately will contribute to design of post-treatment strategies in AML.