Minimal Residual Disease Evaluation Research Articles

Abstract PO5-16-04: Tissue-informed personalized MRD detection assay may outperform tumor-informed fixed panel strategy in Triple Negative Breast Cancer (TNBC)

Abstract Background: Minimal residual disease (MRD) detection by ctDNA analysis enables identifying patients with high recurrence risk and refining risk stratification to guide treatment selection precisely, which may improve patient survival or avoid overtreatment. We previously developed a 1021-gene fixed panel MRD detection assay, MNavigator V1, demonstrating high prognostic evaluation value in various solid tumors, including lung cancer, colorectal cancer, and esophageal carcinoma, and relevant research results have been published. Recently, the assay upgraded to MNavigator V2, a customized patient-specific panel strategy to ultra-deep sequencing, with higher clinical performance and more economical. Here, we head-to-head compared the performance of MNavigator V1 with V2, using the same collection of blood samples from TNBC patients. Methods: The retrospective study recruited 48 patients diagnosed with stage II~III TNBC with adequate samples (NCT04501523/NCT04803539). Five patients in the cohort received neoadjuvant therapy (Neoad-group), and the standard treatment regimen of the remaining 43 patients was adjuvant therapy (ad-group) after surgery. The surgical tumor tissue of all these patients was collected. Peripheral blood samples were collected at various time points, baseline samples before neoadjuvant therapy, 7-day after neoadjuvant therapy, 1-month postoperative, 7-day after adjuvant therapy, and subsequent every 3~6 months follow-up. The personalized, tumor-informed MRD detection approach MNavigator V2 started by identifying somatic mutation of the resected tumor with a 1021-gene panel covering 1.6Mb of the human genome. Then, a customized panel consisting of up to 20 top-ranked patient-specific somatic variants was designed. After that, unique molecular identifiers (UMI) based 100,000X ultra-deep next-generation sequencing on plasma samples was conducted by adopting the mix panel of the patient-specific panel and a 5kb breast cancer universal core panel. MNavigator V1 assay, a fixed panel assay also applied the same off-the-shelf 1021-genes panel with a sequencing depth of 10,000X to detect MRD of blood samples. Results: Until May 2023, the median follow-up time of 660 days, and 8 patients were recurrence confirmed by CT imaging. The median tracked tissue-informed mutations was 7 (2~18). In this study, the screening time point was defined as the combined MRD status of the blood samples collected before and after neoadjuvant therapy, and 1-month post-surgery for the Neoad-group, as for the ad-group, the time point referred to 1-month post-surgery and post-adjuvant therapy. If any one of these time nodes' MRD was positive, the screening period MRD was positive. During the screening period, MNavigator V1 and V2 accurately predicted 6/8 (75.0%) recurrence. MRD-positive status was associated with a worse prognosis, compared to the MRD-negative population, with hazard ratios of 9.0 and 19.9 for V1 and V2 assay, respectively. The longitudinal MRD evaluation yield a higher sensitivity of 7/8 (87.5%) for recurrence prediction with MNavigator V2 at a median lead time of 104 days, whereas the V1 assay predicted 6/8 (75.0%) at 94 days, and the hazard ratio was 40.4 vs 8.5. In one brain-only metastasis patient, MRD was undetectable, which may be due to the limitation of the blood-brain barrier. For recurrence-free patients with 1 year of follow-up, 92.5% (37/40) were MRD-negative with MNavigator V2 assay. These three patients who detected MRD-positive after surgery, ctDNA cleared after adjuvant therapy, and persistently negative during follow-up, may benefit from treatment. Conclusion: Based on the patient-specific tumor mutation profile, the personalized MRD detection assay achieved better clinical performance than the fixed panel strategy. Citation Format: Shunying Li, Wei Gao, Ning Fu, Wuqiang Cao, Xiaoling Zeng, Xinhua Du, Qiang Liu. Tissue-informed personalized MRD detection assay may outperform tumor-informed fixed panel strategy in Triple Negative Breast Cancer (TNBC) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-16-04.

Application and prognostic relevance of CD34 detection in immunophenotyping of pediatric acute B lymphoblastic leukemia.

We aimed to investigate the application of CD34 detection in immunophenotypic discrimination and its prognostic relevance in children with acute B-lymphoblastic leukemia (B-ALL). A retrospective analysis was conducted on clinical follow-up data of 105 children with newly diagnosed B-ALL treated at our hospital from January 2022 to December 2023. Based on the expression of CD34 in the bone marrow, patients were divided into a CD34 positive group (positive cells ≥10%) and a CD34 negative group (positive cells <10%). The study compared the positive rates of common leukemia cell antigens, clinical characteristics, initial treatment responses, and long-term follow-up outcomes between the two groups. Among all 105 B-ALL cases, 87 children (82.9%) had bone marrow CD34 positive cells ≥10%, classified into the CD34 positive group, while the remaining 18 children (17.1%) had bone marrow CD34 positive cells <10%, classified into the CD34 negative group. The CD34 positive group exhibited significantly higher positive rates of CD13 expression, standard-risk B-ALL, and risk stratification than the CD34 negative group. In contrast, the proportions of early pre-B-ALL, E2A-PBX1 fusion gene, and MLL-AF4 fusion gene were significantly lower in the CD34 negative group, with statistically significant differences (p<0.05). No significant differences were found in the positive rates of leukemia cell antigens such as CD10, CD19, CD20, CD22, CD79a, CD13, CD33, and CD38 between the two groups (p>0.05). The occurrence rates of minimal residual disease (MRD) and relapse after induction chemotherapy in the CD34 positive group were significantly lower than those in the CD34 negative group (p<0.05). However, the sensitivity to the first prednisone treatment and bone marrow treatment efficacy on the 19th and 33rd days after chemotherapy showed no significant differences between the groups (p>0.05). A higher positive rate of bone marrow CD34 expression in children with B-ALL is associated with a favorable prognosis. Children with negative CD34 expression are relatively more prone to MRD and tumor relapse after chemotherapy.

Droplet digital PCR: An effective method for monitoring and prognostic evaluation of minimal residual disease in JMML.

Juvenile myelomonocytic leukaemia (JMML) is a rare myeloproliferative neoplasm requiring haematopoietic stem cell transplantation (HSCT) for potential cure. Relapse poses a significant obstacle to JMML HSCT treatment, as the lack of effective minimal residual disease (MRD)-monitoring methods leads to delayed interventions. This retrospective study utilized the droplet digital PCR (ddPCR) technique, a highly sensitive nucleic acid detection and quantification technique, to monitor MRD in 32 JMML patients. The results demonstrated that ddPCR detected relapse manifestations earlier than traditional methods and uncovered molecular insights into JMML MRD dynamics. The findings emphasized a critical 1- to 3-month window post-HSCT for detecting molecular relapse, with 66.7% (8/12) of relapses occurring within this period. Slow MRD clearance post-HSCT was observed, as 65% (13/20) of non-relapse patients took over 6 months to achieve ddPCR-MRD negativity. Furthermore, bone marrow ddPCR-MRD levels at 1-month post-HSCT proved to be prognostically significant. Relapsed patients exhibited significantly elevated ddPCR-MRD levels at this time point (p = 0.026), with a cut-off of 0.465% effectively stratifying overall survival (p = 0.007), event-free survival (p = 0.035) and cumulative incidence of relapse (p = 0.035). In conclusion, this study underscored ddPCR's superiority in JMML MRD monitoring post-HSCT. It provided valuable insights into JMML MRD dynamics, offering guidance for the effective management of JMML.

The Role of Autologous Stem Cell Transplantation in the Treatment of Newly Diagnosed Multiple Myeloma: Is It Time to Rethink the Paradigm in the Era of Targeted Therapy?

High-dose melphalan (HDM) plus autologous stem cell transplant (ASCT) remains a standard-of-care treatment approach for eligible patients with newly diagnosed multiple myeloma (NDMM) based on demonstrated superiority in terms of progression-free survival (PFS) versus nontransplant approaches. Very high rates of minimal residual disease (MRD)-negative responses are also being seen with novel triplet and quadruplet induction regimens plus HDM-ASCT. However, recent clinical trials have shown no overall survival benefit with transplant versus nontransplant approaches. Furthermore, HDM is associated with several important downsides, including acute and long-term toxicities, transient decreases in quality of life, the need for hospitalization, an increased mutational burden at relapse, and an elevated risk of second primary malignancies. In this context, given the highly heterogeneous nature of MM in the NDMM patient population, as well as the continued emergence of novel agents and treatment approaches, there is an increasing rationale for considering deferred HDM-ASCT approaches in selected patients. Approaches under investigation include MRD-adapted therapy and the use of novel immune-based therapies as alternatives to HDM-ASCT. Ongoing developments in understanding the pathobiology and prognostic factors in NDMM, plus immune profiling and routine MRD evaluation, will result in novel, HDM-sparing treatment paradigms, enabling further improvement in patient outcomes.

Clinical Analysis of Mitoxantrone Liposome in the Treatment of Children with High-Risk Acute Myeloid Leukemia

To investigate the safety and efficacy of mitoxantrone liposome in the treatment of children with high-risk acute myeloid leukemia (AML). The children with high-risk AML who received the mitoxantrone liposome regimen at Wuhan Children's Hospital from January 2022 to February 2023 were collected as the observation group, and the children with high-risk AML who received idarubicin regimen were enrolled as controls, and their clinical data were analyzed. Time to bone marrow recovery, the complete remission rate of bone marrow cytology, the clearance rate of minimal residual disease, and treatment-related adverse reactions were compared between the two groups. The patients treated with mitoxantrone liposome showed shorter time to recovery of leukocytes(17 vs 21 day), granulocytes(18 vs 24 day), platelets(17 vs 24 day), and hemoglobin(20 vs 26 day) compared with those treated with idarubicin, there were statistical differences (P <0.05). The effective rate and MRD turning negative rate in the observation group were 90.9% and 72.7%, respectively, while those in the control group were 94.1% and 76.4%, with no statistical difference (P >0.05). The overall response rate of the two groups of patients was similar. The efficacy of mitoxantrone liposome is not inferior to that of idarubicin in children with high-risk AML, but mitoxantrone liposome allows a significantly shorter duration of bone marrow suppression and the safety is better.

Abstract 3659: Development of customized circulating tumor DNA panel for minimal residual disease detection in biliary tract cancer

Abstract Biliary tract cancer (BTC) is a rare type of abdominal cancer and is more frequent in Asian countries than in Western. The 5-year survival rate for BTC is very low 7 to 20% and only 20% of BTC patients could undergo curative-intent surgery. BTC diagnosis based on biopsy and cytology are low in sensitivity that prompted the necessity to identify alternative biopsy. Liquid biopsy, specifically circulating tumor DNA (ctDNA) is known for the accessibility and low invasiveness received great interest for their potential uses in various clinical applications for patients with insufficient tumor tissue, including mutation profiling, treatment monitoring, and cancer detection. However, the panel available in the market up-to-date focused mainly targeted, actionable alterations and thus not suitable for minimal residual disease (MRD) monitoring. In this study, we aimed to develop a customized ctDNA panel for MRD monitoring in BTC. In this study, 124 BTC patients undergone curative resections were enrolled at Hokkaido University and Sapporo Medical University from May 2019 to April 22. All enrolled patients have BTC diagnosis confirmed by pathological diagnosis after surgery, and without other malignant tumors. Genomic profiles of FFPE tissue were conducted using Ampliseq CCP. 13 patients were excluded for high fragmentation. Referring to the genomic profiles of 111 patients in our study and 1924 patients in TCGA established a customized panel using Ion Ampliseq HD technology. The Ampliseq HD panel claims to detect variants at 0.1% LOD for ctDNA detection. It also integrates molecular barcodes to reduce NGS-related errors. The designed panel was 258 Amplicons and 20 genes including eleven therapeutically targetable genes. Clinical characteristics of ctDNA detection were also assessed. Results:The panel validation: concordance of the mutation allele frequency (MAF) between the NGS standard control and Ampliseq HD panel showed a coefficient of determination (R2) of 0.96. Also, concordance of MAF Ampliseq CCP and Ampliseq HD panel on FFPE tumor profiling showed R2 of 0.78. When assessing 70% of BTC patients were covered to have at least one tumor-derived Ampliseq HD panel. When assessing using plasma cell-free DNA, 55% of BTC patients were detected to have at least one tumor-derived ctDNA. Distribution of detected altered top4 genes using Ampliseq HD panels shows TP53 35%, KRAS 10%, SMAD4 5%, ARID1A 2.5%. Clinical characteristics may affected the concordance of plasma and FFPE tumor tissue; stage (P=0.035), size of tumors (P=0.0088), subtype (P=0.013), LN metastasis (P=0.11). Conclusions: A highly sensitive ctDNA customized panel for BTC was established. We also found that clinical characteristics of the BTC affects the detection of ctDNA from blood liquid biopsy. Future study: MRD evaluation using this customized panel. Citation Format: Yoshihito Shinohara, Siew-Kee Low, Toru Nakamura, Yasutoshi Kimura, Takeshi Murakami, Kazuma Kiyotani, Satoshi Hirano. Development of customized circulating tumor DNA panel for minimal residual disease detection in biliary tract cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3659.

Mutations of epigenetic modifier genes predict poor outcome in adult acute lymphoblastic leukemia.

Epigenetic modifier (EM) genes play important roles in the occurrence and progression of acute lymphoblastic leukemia (ALL). However, the prognostic significance of EM mutations in ALL has not yet been thoroughly investigated. This retrospective study included 205 adult patients with ALL engaged in a pediatric-type regimen. Based on targeted next-generation sequencing, they were divided into EM mutation group (EM-mut, n = 75) and EM wild-type group (EM-wt, n = 130). The EM-mut group showed a higher positive rate of minimal residual disease (MRD) on treatment day24 and before consolidation therapy (P = 0.026, 0.020). Multivariate Cox regression analysis showed that EM-mut was an independent adverse factor for overall survival (OS) and event-free survival (EFS) (HR = 2.123, 1.742; P = 0.009, 0.007). Survival analysis revealed that the OS and EFS rates were significantly lower in the EM-mut group than in the EM-wt group (3-year OS rate, 45.8% vs. 65.0%, P = 0.0041; 3-year EFS rate, 36.7% vs. 53.2%, P = 0.011). In conclusion, EM was frequently mutated in adult ALL and was characterized by poor response to induction therapy and inferior clinical outcomes.

Abstract A077: The utility of surgical minimal residual disease after frontline treatment for prognostic and investigational opportunities in advanced ovarian cancer

Abstract Cure rates for patients with advanced stage ovarian cancer have not changed significantly over the last four decades likely due to inability to eradicate residual cancer cells after frontline therapy. Identifying clinically undetectable minimal residual disease (MRD) after frontline therapy by second look laparoscopy (SLL) has potential to a) uncover mechanisms of chemoresistance and tumor dormancy, b) identify MRD-specific therapeutic targets, and c) allow for earlier intervention with investigational therapies. The objective of this study is to describe the initial safety and feasibility of SLL and illustrate clinical and pathological correlates. Methods: This IRB-approved observational, single-center study includes patients with epithelial high-grade ovarian cancer with complete CA-125 and radiologic response after frontline treatment (combination of surgery and platinum-taxane chemotherapy) who underwent SLL from 4/2017-6/2023. Patients were subsequently offered standard of care or investigational maintenance therapy options based on homologous recombination deficiency (HRD) and MRD status. Summary statistics described the study population, chi-square tests compared categorical variables, and progression free survival (PFS) based on MRD status was estimated using Kaplan-Meier method and compared using log rank test. Results: 70 patients underwent SLL with a median of 8.5 biopsies (range 3-16). The majority of patients had stage III-IV disease (90%), and high-grade serous histology (90%). Approximately half (51.4%) of patients underwent neoadjuvant chemotherapy (NACT) with interval tumor reductive surgery (TRS). MRD was present in 32 patients (45.7%), and the presence of MRD was associated with a worse PFS (HR 3.0, 95% CI 1.5-5.8; 12.5 vs. 24.8 months; p=0.001). MRD was more frequent among NACT recipients compared to those who underwent primary TRS (63% vs 26%, P=0.002), those with HRD negative tumors (58% vs 24%, P=0.025), and those with R1 versus R0 resections (71% vs 39%, P=0.024). Of patients with MRD, 22 patients (71%) received bevacizumab until progression as part of a clinical trial (NCT02884648), 3 (10%) received bevacizumab off trial, 5 (16%) received a PARP inhibitor (all HRD positive), and 1 received other therapy. There were two (2.9%) intraoperative complications (both enterotomies) requiring conversion to laparotomy. Conclusions: SLL is feasible, overall safe, and detected MRD in 46% of ovarian cancer patients who were in radiologic and CA-125 remission after frontline therapy. PFS was significantly shorter for patients with residual disease, despite those patients all receiving additional therapy. Knowledge of MRD status in this patient population enables interventional, observational, and translational opportunities, as well as personalized prognostic counseling. MRD rate may also have utility as an early surrogate for efficacy in trials evaluating novel frontline investigational therapies. Future studies will compare the performance of circulating tumor DNA (ctDNA) versus surgical MRD. Citation Format: Anne Knisely, Roni Nitecki, J. Alejandro Rauh-Hain, R. Tyler Hillman, Jolyn S. Taylor, Lois M. Ramondetta, Michaela O. Grinsfelder, Lauren P. Cobb, David M. Boruta, Gwyn Richardson, Pamela T. Soliman, Aaron Shafer, Shannon N. Westin, Nicole D. Fleming, Travis T. Sims, Anil K. Sood, Pedro T. Ramirez, Karen H. Lu, Amir A. Jazaeri. The utility of surgical minimal residual disease after frontline treatment for prognostic and investigational opportunities in advanced ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A077.

The therapeutic effect and prognostic value of oligoclonal bands after autologous stem cell transplant in patients with multiple myeloma

Objective: To investigate the therapeutic effect and prognostic value of oligoclonal bands (OB) in multiple myeloma (MM) patients after autologous stem cell transplant (ASCT). Methods: The data of 156 patients with MM who underwent ASCT after inductive treatment in the Department of Hematology, Jiangsu Provincial People's Hospital from December 2013 to February 2022 were retrospectively analyzed, including 91 males and 65 females. The median age was 56 (26, 71) years. Patients were divided into two groups according to OB formation after ASCT treatment, including OB group (n=60) and non-OB group (n=96). The last follow-up date was August 31, 2023, and the follow-up period was 42 (18, 117) months. The clinical baseline characteristics and efficacy of the two groups were compared. Progression-free survival (PFS) and overall survival (OS) were compared between the two groups by Kaplan-Meier method. Cox risk regression modal was used to analyze the risk factors associated with prognosis. Results: There were no significant differences in age, type, stage, risk stratification, extramedullary disease (EMD), proportion of circulating plasma cells and induction therapy regimen between OB and non-OB groups (all P>0.05). The proportion of patients in OB group who achieved complete response (CR) or above after ASCT treatment was 93.3% (56/60), which was higher than that in non-OB group (80.2%, 77/96) (P=0.024). The negative rate of minimal residual disease (MRD) in OB group was 66.7% (40/60), which was higher than that in non-OB group (34.4%, 33/96) (P=0.001). The median PFS and OS in the OB group were not reached, and the median PFS and OS in the non-OB group were 28 (2, 80) months and 86 (2, 100) months, respectively. The PFS (P<0.001) and OS (P=0.017) of patients with OB were considerably longer. In the Cox multivariate analysis, OB was an independent prognostic factor for PFS in MM patients (HR=0.314, 95%CI: 0.153-0.644, P=0.002). Subgroup analysis showed that among high-risk patients with mSMART, the OS of patients in OB group was not reached, which was significantly better than that of non-OB group [71 (2, 90) months, P=0.046]. However, no significant difference was observed in the OS of patients with OB and those with non-OB in standard risk group (not reached vs not reached, P=0.103). In those with EMD at diagnosis, patients with OB had significantly better OS than those with non-OB [not reached vs 47 (6, 74) months, P=0.037]. However, no significant difference was observed in the OS of patients with OB and those with non-OB in those without EMD at diagnosis [not reached vs 86 (2, 100) months, P=0.130]. Conclusions: OB formation after ASCT treatment in MM patients is related to the efficacy and prognosis. OB formation can increase the negative MRD rate, prolong the OS and improve the prognosis, especially for newly diagnosed patients with extramedullary disease or patients with high-risk genetic characteristics.

Minimal residual disease assessment and progress in multiple myeloma

With the rapid iteration of multiple myeloma therapeutics over the last two decades, as well as increasing remission rates and depth of remission in patients, traditional methods for monitoring disease response are insufficient to meet the clinical needs of new drugs. Minimal residual disease (MRD) is a more sensitive test for determining the depth of response, and data from multiple clinical trials and meta-analyses show that a negative MRD correlates with a better prognosis than a traditional complete response. MM is at the forefront of MRD evaluation and treatment. MRD detection methods have been continuously updated. The current MRD assessment has three dimensions: bone marrow-based MRD testing, MRD testing based on images of residual metabolic of focal lesions, and peripheral blood-based MRD testing. The various MRD assessment methods complement one another. The goal of this article is to discuss the currently used MRD assays, the progress, and challenges of MRD in MM, and to provide a reference for clinicians to better use the techniques.

Role of minimal residual disease assessment in multiple myeloma.

Multiple myeloma (MM) is a hematologic malignancy characterized by clonal proliferation of plasma cells. MM is a heterogeneous disease, featured by various molecular subtypes with different outcomes. With the advent of very efficient therapies including monoclonal antibodies, bispecific T cell engagers and chimeric antigen receptor T cells (CAR T cells), most of MM patients have now a prolonged survival. However, the disease remains incurable, and a subgroup of high-risk patients continue to have early relapse and short survival. Novel and highly sensitive methods have been developed allowing to detect minimal residual disease (MRD) during or after treatment. Achievement of MRD negativity is a strong and independent prognosis factor in both prospective randomized clinical trials and in real-world setting. While MRD assessment is now a validated endpoint in clinical trials, its incorporation in clinical practice is not yet established and its potential impact on guiding therapy remains under deep evaluation. We discuss in this chapter, the different available methods allowing MRD assessment and the role of MRD evaluation in multiple myeloma management.

Comparison of Multiparameter Flow Cytometry and ClonoSEQ Studies for Precursor B-Lymphoblastic Leukemia Minimal Residual Disease Detection on Bone Marrow Samples

Minimal residual disease (MRD) detection for precursor B-lymphoblastic leukemia (B-ALL) has become standard of care. We retrospectively compared MRD evaluation using COG Multiparameteric flow cytometry (MFC) and ClonoSEQ technique to assess the test characteristics.

Upfront or Deferred Autologous Stem Cell Transplantation for Newly Diagnosed Multiple Myeloma in the Era of Triplet and Quadruplet Induction and Minimal Residual Disease/Risk-Adapted Therapy.

The standards of care for the initial treatment of patients with newly diagnosed multiple myeloma (NDMM) who are eligible for high-dose melphalan and autologous stem cell transplantation (HDM-ASCT) include highly active triplet and quadruplet regimens based on proteasome inhibitors, immunomodulatory drugs, and monoclonal antibodies. These regimens are resulting in improved outcomes and increasingly high rates of minimal residual disease (MRD)-negative responses without HDM-ASCT as part of the upfront therapy. Furthermore, recent randomized studies have shown that, while transplant-based approaches as a frontline therapy result in significantly longer progression-free survival compared to non-transplant approaches, this has not translated into an overall survival benefit. Given these developments, and in the context of the treatment burden of undergoing HDM-ASCT, in addition to the acute toxicities and long-term sequelae of HDM, which are associated with the genotoxicity of melphalan, there is an increasing rationale for considering deferring upfront HDM-ASCT in select transplant-eligible patients and saving it as a treatment option for later salvage therapy. Here, we review the latest clinical trial data on upfront or deferred HDM-ASCT and on the activity of quadruplet induction regimens, including rates of MRD-negative responses, and summarize emerging treatment approaches in the upfront setting such as the use of MRD-directed therapy and alternatives to HDM-ASCT.

Relationship Between Venetoclax Exposure and Undetectable Minimal Residual Disease Rates in Relapsed/Refractory Patients With Chronic Lymphocytic Leukemia: A Pooled Analysis of Six Clinical Studies.

Relationship Between Venetoclax Exposure and Undetectable Minimal Residual Disease Rates in Relapsed/Refractory Patients With Chronic Lymphocytic Leukemia: A Pooled Analysis of Six Clinical Studies.

Final Analysis of a Phase 2 Trial of Daratumumab, Carfilzomib, Lenalidomide, and Dexamethasone in Newly Diagnosed Multiple Myeloma (NDMM) without Autologous Stem Cell Transplantation (ASCT)

Introduction: A previous trial showed that Dara-KRd for 8 cycles without ASCT led to a minimal residual disease (MRD) negativity rate of 71% at 10 -5 in NDMM (Landgren et al. JAMA Oncology 2021). MRD-adapted Dara-KRd with ASCT led to an MRD(-) rate of 71% at 10 -6 with a 3-year progression-free survival (PFS) of 88%, 79%, and 50% in pts with 0, 1 or 2+ high-risk cytogenetic abnormalities (HRCA), respectively (Costa et al. EHA 2022). In this phase 2 study, we evaluated the safety and efficacy of 24 cycles of Dara-KRd without ASCT in pts with NDMM. Methods: Pts with NDMM with up to 1 cycle of prior therapy were enrolled regardless of ASCT eligibility. Pts received Dara-KRd for planned 24 cycles: daratumumab 16 mg/kg IV weekly for cycles (C) 1 &amp; 2, q2weeks for C3-8, then q4weeks for C9-24; carfilzomib 20/36 mg/m 2 on days 1, 2, 8, 9, 15, 16 for C1-8, then 36 mg/m 2 on days 1, 2, 15, 16 for C9-24; lenalidomide 25 mg PO days 1-21 of a 28 day cycle for 24 cycles; dexamethasone 40 mg PO weekly (20 mg if age &amp;gt; 75) for C1-9, and 20 mg PO weekly for C9-24. Pts had the option to harvest stem cells (SC) to permit ASCT in the future. MRD testing was performed by NGS (clonoSEQ, Adaptive Biotechnologies) with sensitivity &amp;lt;10 -6 at the end of cycles 8 (C8), C12, and C24. Mass spectrometry (MS) of peripheral blood samples were also performed using MALDI and liquid-chromatography (LCMS) (The Binding Site Group). The primary endpoint was the rate of stringent CR and/or MRD(-) &amp;lt;10 -5 at the end of C8; the hypothesis was that &amp;gt;50% would meet the primary endpoint with Dara-KRd compared to historical 30% with KRd (85% power, one-sided alpha=0.10). Results: 42 pts enrolled from March 2019-January 2022. Median age was 58 years (range 39-79). 13 (31%) were Black and 5 (12%) were Hispanic. HRCA (t(4;14), t(14;16), t(14;20), +1q, del17p) were present in 24/42 (57%); 10 (24%) had 2+ HRCA. 34 pts (81%) underwent SC collection, all with plerixafor (median yield 8.91x10 6 CD34+/kg). 40 pts (95%) were evaluable for the primary endpoint at C8. Two pts withdrew before C8 for reasons unrelated to treatment. There were 32 (80%) MRD-evaluable pts at C8. The sCR and/or MRD(-) rate (&amp;lt;10 -5) was 30/40 (75%, 95% CI 61%-89%). The C8 10 -5 MRD(-) rate was 20/32 (63%) and at 10 -6 it was 12/32 (38%). Responses deepened over time; 1/12 (8%) and 6/20 (30%) pts converted from MRD(+) at C8 to MRD(-) at later timepoints at 10 -5 and 10 -6 thresholds, respectively, for a best MRD(-) rate of 21/32 (66%) at 10 -5 and 18/32 (56%) at 10 -6 . Sustained 10 -5 MRD(-) &amp;gt;12 months was achieved in 11 (26%) pts; 6 pts were &amp;lt;1 year from first MRD(-) result. Overall response rate at C8 was 38/40 (95%) with 27 (68%) sCR, 38 (95%) VGPR+, and 2 PD. Of the 39 pts with trackable mass spectra in the peripheral blood, the MALDI(-) rate was 22/39 (56%) after C8 and 24/39 (62%) as best response. The LCMS(-) rate was 7/39 (18%) after C8 and 12/39 (31%) as best response. Median follow-up was 27 months. 21 pts (50%) completed all 24 cycles, and 11 (26%) remain on protocol. 7 pts (17%) had disease progression (6 on protocol), including 4 pts who reached the primary endpoint at C8. 4 pts (10%) discontinued protocol therapy early (additional 2 pts due to nonadherence). 1 pt received ASCT after discontinuing while in response. 2 deaths occurred, both early in treatment and due to primary refractory disease. The 3-year PFS was 85%: 100% for standard-risk disease, 92% for 1 HRCA, and 60% for 2+ HRCA. Of the 7 pts with progression, 6 had at least one of: extramedullary disease (4), 2+ HRCA (4), or circulating plasma cells (1). Neither NGS nor mass spectrometry status at C8 were associated with PFS differences using the landmark method. The 3-year OS was 95%. All-grade/grade 3+ (G3) neutropenia occurred in 26%/21%, anemia in 59%/2%, and thrombocytopenia in 64%/26%. Notable nonhematologic adverse events were hyperglycemia (all 76%/ G3+ 7%), diarrhea (71%/5%), hypertension (57%/17%), and neuropathy (40%/0%). COVID infections occurred in 38% (2% G3) and upper respiratory infections in 45% (0% G3+). There was one case of thrombotic microangiopathy leading to discontinuation of carfilzomib. G3 atrial fibrillation and heart failure each occurred in 1 pt. Conclusions: Extended frontline Dara-KRd for NDMM without ASCT induced high rates of sCR and/or MRD(-) within 8 cycles, meeting the primary endpoint. The rate and depth of MRD(-) improved beyond C8. This ASCT-free approach led to lower PFS for pts with 2+ HRCA, as also seen with ASCT, but excellent PFS in those with standard-risk disease and 1 HRCA.

Early-Stage Follicular Lymphoma: Learnings from the Final Analysis of the Multicenter Phase II FIL (Fondazione Italiana Linfomi) “Miro” Trial, Combining Local Radiotherapy and MRD-Driven Immunotherapy

Introduction. Early-stage follicular lymphoma (FL) is usually managed with involved field radiotherapy (IFRT), allowing a complete and long-lasting eradication of the disease in 40-50% of patients (pts). The aim of this multicenter phase II prospective study was to evaluate if a minimal residual disease (MRD)-driven immunotherapy consolidation after IFRT can improve the outcome of pts unlikely to be cured by IFRT only, and to assess the therapeutic effect of IFRT and anti-CD20 consolidation by MRD evaluation. Methods. One hundred and ten pts with stage I/II FL were enrolled; 106 were evaluable and treated with 24 Gy IFRT. Peripheral blood (PB) and bone marrow (BM) samples were centralized to the FIL (Fondazione Italiani Linfomi) MRD Network of EuroMRD-certified laboratories. In pts BCL2::IGH+ at baseline by both nested PCR (NEST) and RQ-PCR (RQ) in the BM and/or (a/o) PB, MRD was analyzed after IFRT and every 6 months over a 3-year period. Pts who were MRD+ by both NEST and RQ in the BM a/o PB after IFRT or who became MRD+ during the follow-up were treated with 8 weekly doses of the anti-CD20 monoclonal antibody ofatumumab (OFA). The primary objective of the study was to define the efficacy of immunotherapy in obtaining a negative MRD. Results. Of the 106 evaluable pts, 50 were males (47%). Median age was 55.5 years (29-83). The FLIPI score was 0 in 60% of pts, 1 in 34%, 2 in 6%; 69% of pts had inguinal site involvement, significantly more frequent in young patients. At baseline, 30.5% of pts had a BCL2::IGH rearrangement (30 MBR, 1 MBR and mcr, 1 mcr) in the BM a/o PB; the concordance between compartments was 87%. BCL2::IGH+ and BCL2::IGH- pts were not significantly different, except for a younger age (p 0.009) and a more frequent FLIPI 0 (p 0.052) among marker negative cases. All but 1 pt achieved a clinical response after IFRT; 1 pt died soon after IFRT of unrelated causes. MRD evaluation after IFRT revealed the persistence of BCL2::IGH+ cells in the PB a/o BM in 60% of pts. MRD+ pts, either after IFRT (n=18) or in case of conversion to MRD+ during the follow-up (n=8), received OFA, obtaining a conversion to MRD- in 23/25 evaluable pts (92%, CI 74-99%), significantly superior to the expected 50%. After a median follow-up of 46.3 months (m) (range 11-66), of the 23 pts who achieved a MRD- with OFA, 5 returned MRD+ (4 underwent OFA re-treatment, achieving a second MRD-, 1 relapsed as a transformation), 4 relapsed being MRD-, 14 remain in complete remission and MRD-, with a duration of molecular response at the last molecular assessment of 30 months (range 6-36). Overall, a clinical relapse was observed in 29 pts submitted to IFRT and 1 patient progressed soon after IFRT. A confirmed histologic transformation to DLBCL was observed in 5 of these patients. Among the 73 “marker negative” patients, 20 relapsed (27.4%) over time, whilst among the 32 BCL2::IGH+ at baseline 9 (28.1%) were resistant or relapsed (p=0.939). The administration of OFA was associated to a significantly reduced risk of POD24 (p= 0.035). Overall, the 24-m and 36-m overall survival (OS) were 100% and 98.9%, respectively; the 24-m and 36-m PFS were 83.7% and 77.4%, respectively. PFS was not different according to the presence or absence of BCL2::IGH at enrollment (p=0.820). PFS was significantly different according to the FLIPI score (p=0.041), but not different according to gender (p=0.696), stage I-II (p=0.122), histological grade (p=0.212), inguinal nodal site vs others (p=0.492). The Kaplan Meier landmark analysis, stratified by MRD, at m+6 (p=0.228), m+12 (p=0.003), m+18 (p=0.0002), m+24 (p=0.004) showed that PFS significantly improved from m+12 onwards when a negative MRD was present (Figure 1). Conclusions. IFRT alone is often insufficient to eradicate FL, inducing a MRD- status only in 40% of pts, long-lasting only in a third of them. An immunotherapy consolidation with OFA after IFRT in MRD+ pts was capable of increasing the molecular responses to 92%, allowing to achieve the primary objective of the study, though this effect was not always persistent. The achievement of a molecular remission correlated significantly with a reduction in the incidence of relapse over time, although this strategy was applicable only to 30% BCL2::IGH+ PET/CT staged FL pts. A consolidation strategy with more effective agent should be advisable. With these limits, a clinical advantage of the MRD-driven treatment strategy is suggested, although not largely applicable.

Mutational Burden in High Risk Genes Do Not Affect Remission Rates and MRD Clearance in Elderly AML Patients Receiving CPX-351 Induction

Background: CPX-351 has recently been approved for the treatment of patients diagnosed with Acute Myeloid Leukemia (AML) arising from a previous myelodisplastic syndrome (s-AML) or secondary to chemotherapy (t-AML) as per former WHO 2016 classification. Minimal residual disease (MRD) assessment is a strong prognostic factor for relapse and shorter survival in AML patients undergoing intensive induction chemotherapy. Recent studies explored the role of genomic profile of AML influencing clinical outcome. In particular, Papaemmanuil et al showed that mutations in critical genes such as TP53, ASXL1, SRSF2 and RUNX1 are independently associated with a worse prognosis; moreover, co-occurrence of different high-risk mutations are predictors of dismal outcome when conventional chemotherapy is administered. Adverse risk mutations are particularly frequent in s-AML and t-AML. As clinical trials evaluating the prognostic relevance of MRD in the context of different molecular subsets mainly involve younger patients affected by de novo AML and receiving conventional 3+7 chemotherapy, there is a lack of data to define the impact of specific mutations on MRD clearance and prognosis in elderly s- AML and t-AML patients receiving CPX-351 induction. Aims: The aim of this study was to evaluate the prognostic impact of recurrent genic mutations and mutational burden at diagnosis on CR probability, MRD clearance and prognosis in a cohort of elderly s-AML or t-AML patients treated with CPX-351 induction chemotherapy. Methods: The study included 67 elderly patients (&amp;gt;60 year, median age 69, range 60-77) with a diagnosis of s-AML or t-AML according to WHO2016 classification, treated in our Center with CPX-351. Next generation sequencing (NGS) was performed using the Myeloid Solution panel by SOPHiA Genetics, encompassing 34 critical genes mutations. Sample have been processed on a Illumina MiSeq platform and the analysis performed with SOPHiA DDM® Software. MRD was analysed in all patients achieving complete remission (CR) with multicolour flow-cytometry, with a threshold of 0.1%. Results: AML patients were re-classified according to ELN 2022 risk score which was adverse, intermediate and favourable in 35, 29 and 3 patients, respectively. Median number of mutations for single patient (mutational burden) by NGS was 4 (range 2-9). Most frequent mutations involved: RUNX1 (44%), ASXL1 (37%), TP53 (33%), IDH1 (18%), IDH2 (14%), DNMT3A (14%), TET2 (8%). Four patients died before response assessment, mainly due to infections. Fifty-four patients (81%) achieved complete remission (CR) after first induction cycle; MRD evaluation was negative in 28/54 responding patients (52%). Univariate and multivariate analysis showed that factors traditionally associated with poor outcome such as ELN risk group, leukocytosis at diagnosis or previous treatment with hypomethylating agents did not affect the probability of CR rate and MRD negativity. Interestingly, having any high risk mutation, the combination of ≥2 high risk mutations or high mutational burden (having ≥ 4) did not affect CR probability and MRD negativity rate both in univariate and multivariate analysis. After cycle 2, the cumulative CR rate was 56/67 (84%), with 33/56 (59%) responding patients obtaining MFC MRD negativity. After a median follow up of 36 months (CI 95%; 22.4 -48.5 months), median OS was 19 months (CI 95% 13-24), whereas 2 year OS was 24%. Multivariate OS analysis revealed that negative MRD was the strongest independent predictor of longer survival (p&amp;lt;0.05). Notably, OS was not affected by mutational burden (29.2% and 27.6% for patients with less or more than 4 mutations, respectively, p=n.s, Fig.1). In landmark analysis, patients achieving CR and proceeding to allogeneic stem cell transplantation consolidation (HSCT) within 3 months from CR (N=13) had a significantly better outcome if compared to CR patients who did not receive HSCT or proceeded to transplant later (N= 41, 2-year OS: 84.6% and 31.7, respectively, p&amp;lt;0.03, Fig. 2). Conclusion : CPX-351 is able to induce good quality remission with high CR rate and MRD negativity, regardless of mutational burden, allowing a high number of elderly AML patients to undergo to HSCT. MRD evaluation has proven to be a strong prognostic tool also in the setting of elderly s-AML and t-AML patients receiving CPX-351. The prognostic value of high risk mutation seems to be less relevant in CPX-351 treated patients.

Prospective Evaluation of Minimal Residual Disease in Waldenström Macroglobulinemia across Different Tissues and Treatments: Results of the “BIO-WM” Trial of the Fondazione Italiana Linfomi (FIL)

Introduction. MYD88 L265P is the hallmark mutation in Waldenström Macroglobulinemia (WM) and is becoming increasingly important in the management of IgM-gammopathies, due to its role as prognostic and predictive biomarker. Recently, MYD88 L265P detection by allele-specific quantitative PCR was proposed as reliable minimal residual disease (MRD) marker in bone marrow (BM) samples (Varettoni, Hematol Oncol 2022). Novel, more sensitive, techniques as droplet digital PCR (ddPCR) might extend the feasibility of MRD detection in WM also in non-invasive tissues, as peripheral blood (PB) or plasmatic cell-free (cf) DNA. This was a secondary endpoint of the multicenter, observational “BIOWM” (NCT03521596) trial, sponsored by the Fondazione Italiana Linfomi (FIL) and the International WM Foundation/Leukemia and Lymphoma Society. From 2018 to 2020 this trial enrolled 300 consecutive patients with primary diagnosis of WM or IgM-MGUS and a systematic biobanking was performed. Here are presented the first results of the MRD study on the patient subset who received frontline treatment, with the aim of driving correlations with clinical response, type of therapy received and outcome prediction. Methods. Paired BM, PB and plasma samples were collected for each patient at baseline (T0) and follow-up and MYD88 L265P detection was performed by ddPCR in all specimens (Drandi, Haematologica 2018). As of today, 59/300 patients (58 WM and 1 IgM-MGUS) received frontline treatment and MRD levels after therapy (T2) were detected by ddPCR: actually, T0-T2 paired samples were available in 49 (BM), 56 (PB) and 48 (cfDNA) cases, respectively. Moreover, MRD was evaluated also by 8-color multiparametric flow cytometry (MFC) in 23 BM and 24 PB samples with a minimum of 10 million cells acquired. Results. Median age of the 59 treated patients was 68 years (24-85), 32% were female, median IgM value was 2420 mg/dL (101-8840), median Hb 10.5 g/dL (8 -17) and IPSS-WM was high in 35% and intermediate in 46%. MYD88 L265P mutation rate was 94% (46/49) in BM, 80% (45/56) in PB and 90% (43/48) in cfDNA samples, respectively. Treatment was started because of anemia in 32% cases, lymphadenopathy or splenomegaly in 34%, hyperviscosity in 20%, other reasons in 13%, and the only MGUS patient required treatment for anti-myelin-associated glycoprotein polyneuropathy. Thirty-one out of 59 patients received bendamustine-rituximab (BR), 23/59 dexamethasone, rituximab and cyclophosphamide (DRC) or DRC-like regimen and 5/59 a single agent therapy (namely, 3 rituximab, 1 cyclophosphamide, 1 ibrutinib). Overall response rate was 90% (87% for BR and 96% for DRC), with 19% CR, 29% VGPR and 35% PR in BR and 4% CR, 0% VGPR and 91% PR in DRC, respectively. Overall, MRD negativity after treatment was 30% (14/46) in BM, 89% (40/45) in PB and 54% (23/43) in cfDNA, respectively. Moreover, among patients still MRD positive after treatment, median MYD88 L265Ptumor burden shrinkage was about 2 Logs in BM (5.5E-03 vs 2.1E-01) and around 1 Log in cfDNA (5.9E-03 vs 3.7E-02) and in PB samples (3.6E-03 vs 2.5E-02), respectively (Figure 1). Interestingly, the MRD shrinkage in BM was deeper among patients receiving BR (48% MRD negative with a median MYD88 L265P decrease of more than 2 Logs among patients still MRD positive) vs patients receiving DRC (10% MRD negative with a median decrease of 1 Log), Figure 2. Similar trends in favor of BR in inducing MRD negativity were reported in PB (96% vs 88%) and cfDNA (70% vs 35%), respectively. MRD results by MFC were overall in line with ddPCR results: namely 27% of patients reached MRD negativity in BM and 69% in PB. The median follow-up for the whole series was 41 months, resulting in a 3-years PFS of 71% and 3-years OS of 89%, respectively. Interestingly, despite the current limited follow-up for such an indolent disease, MRD positivity by ddPCR in PB predicted a dismal clinical outcome if compared with MRD negative patients (3-years PFS 40% vs 73%, p=0.038). Conclusions. To the best of our knowledge, this is the first report of prospective MRD evaluation in a WM clinical trial. Our preliminary results suggest that MYD88 L265P monitoringby ddPCR is a suitable target for MRD analysis, in the contest of common immunochemotherapeutic schedules. Moreover, different levels of disease persistence were described across BM, PB and cfDNA, with MRD monitoring in non-invasive tissues showing promising predictive value for outcome discrimination.

High Efficacy and Feasibility of a Full Pediatric Induction in Adults Aged up to 55 Years with Philadelphia-Negative Acute Lymphoblastic Leukemia

Background: Treatment of acute lymphoblastic leukemia (ALL) in adults has historically shown unsatisfactory outcomes when compared to the outstanding results obtained in pediatric patients. Pediatric protocols rely on increased drug intensity and on a tighter evaluation of minimal residual disease (MRD) for early intensification. Nowadays, pediatric-inspired protocols have been established as the standard of care for adolescents and young adult patients (AYA) leading to improved results. In the setting of older patients, probably due to the limited data available, the feasibility of such intensive therapies, and in particular the administration of high doses PEG-asparaginase (PEGASP), is still a matter of debate. In this context, the NOPHO group reported that a full-pediatric protocol was feasible and highly effective in adult patients, up to 45 years of age. Aim: The aim of our study was to evaluate the feasibility and efficacy of the entire full pediatric AIEOP-BFM LAL 2009 protocol in an older cohort of adult patients up to 55 years. Methods: Since May 2013, 28 consecutive adults diagnosed with Ph-neg ALL received first line therapy according to AIEOP- BFM-ALL 2009 protocol, which includes high PEG-asparaginase (PEGASP) doses, with an accurate MRD-driven therapeutic strategy. The protocol consists of 2 induction blocks: IA (Vincristine, Daunorubicin, PEGASP, IT methotrexate) and IB (Cytarabine, 6-mercaptopurine, cyclophosphamide, IT methotrexate), I consolidation block (high-dose methotrexate, 6-mercaptopurine, IT methotrexate), maintenance therapy (6-mercaptopurine, weekly MTX, IT Methotrexate). Median age in our study was 45 years (range 17-55), equal to the maximum age allowed in the NOPHO study. Fifteen patients had B-ALL, 13 T-ALL/T including 5 Early-T phenotype. Dose intensification and stem cell transplantation (SCT) were scheduled according to baseline and MRD-driven risk assessment. MRD was evaluated by multicolor-flow cytometry (MFC), with a threshold for positivity of 0.025%, and molecular PCR for JH rearrangement (MOL), with a sensitivity greater or equal to 10-4 in all patients. Patients with MOL MRD &amp;gt;10-4 but &amp;lt;10-3 were defined as low-level positive. MRD time points were day 33 and day 78 (MFC and MOL). Results: All patients achieved complete hematological remission following the first Induction course (100%). One patient died during induction due to infection by multidrug resistant P. aeruginosa. In general, therapy was well tolerated. Remarkably, patients in this study received high dose of PEGASP (median 10000 UI/sqm, equal to 4 administrations at 2500 UI/sqm each) without experimenting life threatening toxicities, which mostly consisted in asymptomatic elevation of transaminase (7 patients G3, 1 G4) and/or bilirubin (9 patients G3, 1 G4) only requiring supportive therapy. No major bleeding or thrombotic events o other &amp;gt;G2 toxicities were observed. All except one patient achieved CR at day 33 and all patients achieved CR at day 78. MFC MRD evaluation at day 33 resulted negative in 24/25 evaluable patients (95%). Day 33 MOL-MRD was evaluated in 22 patients, and resulted negative, low level positive or positive in 14, 5 and 3 patients, respectively. MRD assessment at day 78 showed both MFC and MOL negativity achieved in all 25 and 22 evaluated patients, respectively (Fig. 1). Four very high-risk patients underwent SCT in negative MRD status. After a median follow-up of 59 months (CI 95% 38.45-78.63), median survival was not reached, 5-years OS was 88.7% (Fig. 2). One patient died due to progressive transverse myelitis after isolated CNS relapse and one patient died due to sudden cardiac arrest while in complete MRD negative remission, 6 months after completion of therapy. All other 25 patients are alive and in complete MRD negative remission. Conclusion: The application of a full pediatric induction regimen in a cohort of adult ALL patients with a median age of 45 years proved to be feasible, with tolerable toxicities. In particular, we didn't observe major toxicities during the intensified PEGASP administration and all patients were able to receive the planned PEGASP doses. The possibility to maintain the correct timing translated in a very high MRD negativity rate and promising OS survival.

Phase 1 Study of CART-Ddbcma for the Treatment of Patients with Relapsed and/or Refractory Multiple Myeloma: Results from at Least 1-Year Follow-up in All Patients

Introduction: CART-ddBCMA, an autologous anti-B-cell maturation antigen (BCMA) chimeric antigen receptor (CAR) T-cell therapy with a unique, synthetic binding domain, is being studied in a first-in-human clinical trial in patients (pts.) with relapsed &amp;/or refractory multiple myeloma (RRMM). One-year or more follow-up clinical data from all patients are presented in this report. Methods: Details of study design have been previously reported (Frigault et al Blood Adv 2023). Briefly, pts with RRMM who have received ≥3 prior lines of therapy were enrolled &amp; received a single infusion of CART-ddBCMA following lymphodepletion chemotherapy (fludarabine: 30 mg/m 2/d &amp; cyclophosphamide: 300 mg/m 2/d daily for 3 days). Two dose levels (DL1 &amp; DL2 respectively) of 100 &amp; 300 (±20%) ×10 6 CAR+ cells were evaluated. The primary endpoints included incidence of adverse events (AEs) &amp; dose-limiting toxicities (DLTs). Additional endpoints included quality &amp; duration of clinical response assessed according to the IMWG Uniform Response Criteria for MM, evaluation of minimal residual disease (MRD), progression-free (PFS) &amp; overall survival (OS). Correlative analyses related to CART-ddBCMA expansion, characterization, patient &amp; disease-related features were also evaluated. Results: As of June 2, 2023, 40 pts; median age 66 years (range: 44-76) were enrolled; 38 received CART-ddBCMA (32, DL1; 6, DL2) &amp; 38 were evaluable for initial safety &amp; clinical response. Two pts who were not dosed had cell product manufactured but were not eligible for cell infusion due to medical complications. Pts had a median of 4 (range: 3-16) prior lines of therapy. All infused pts (100%) were triple-refractory, &amp; 26 (68%) were penta-refractory; 34 pts (89%) were refractory to last-line of treatment; 9 pts (24%) had high tumor burden with ≥60% bone marrow plasma cells; 13 (34%) pts had extramedullary disease; &amp; 11 (29%) pts had high-risk cytogenetics (Del 17p, t(14;16), t(4;14)) at baseline. Median follow-up after CART-ddBCMA infusion was 22 months (range: 9-40 months). CAR+ cells comprised a median 70% (range: 48-87%) of total CD3+ T cells; median vector copy number was 2.2 copies/cell (range: 1.1-3.5); median cell viability was 98% (range: 90-100%), &amp; median cell manufacturing yield was 1174 ×10 6 CAR + cells (range: 470-1626 ×10 6). CART-ddBCMA product characteristics were consistent with the specifications in all the lots, &amp; there were no manufacturing failures. CRS occurred in 36/38 (95%) pts but only 1 pt in DL2 had grade (Gr) 3 CRS &amp; all other cases were Gr≤2. ICANS occurred in 7 pts (5, Gr≤2; 2, Gr3), with 1 Gr3 case in each DL. All cases of CRS &amp; ICANS resolved without further sequalae with management. No cases of off-tumor cell mediated toxicity, delayed neurotoxicity events (i.e., occurring after day 28), or Parkinsonian-like symptoms were observed. All 38 evaluable pts demonstrated investigator-assessed clinical response per 2016 IMWG criteria (ORR, 100%) with 22 sCR, 7 CR (≥CR rate, 76%), 6 VGPR (≥VGPR rate, 92%), &amp; 3 PR. Responses deepened over time &amp; conversion to CR/sCR was observed with longer follow-up (as late as month 12). Of those evaluable for MRD testing to date (n=29), 25 (86%) were MRD-neg at 10 -5. Median duration of response, PFS, &amp; OS were not reached at the time of data-cut because 25 of 38 evaluable pts (66%) had ongoing response. The Kaplan-Meier estimated PFS rates for 6, 12 &amp; 18 months were 92%, 74%, &amp; 67% respectively. Durable responses were also observed in patients with high-risk features (EMD, BMPC ≥ 60%, or B2M ≥ 5.5 at baseline) &amp; high-risk cytogenetics. PFS rates at 6-, 12-, &amp; 18-months are shown in Table 1. Based on the results from the study, a dose of 115 ± 10 ×10 6 cells, consistent with DL1, was recommended for the phase 2 study. Conclusions: Adverse events with CART-ddBCMA, including CRS &amp; ICANS, were manageable &amp; no off-tumor tissue-targeted toxicity, delayed neurotoxicity, or Parkinsonian-like events were observed in the entire cohort at the time of data-cut. Ongoing efficacy results are encouraging, with 100% ORR, including 35 (92%) response of VGPR or better &amp; 29 (76%) with CR/sCR. More importantly, clinical responses were durable with an overall estimated 18-mo PFS rate of 67% with comparable clinical responses seen in ‘high-risk’ patients known to have poor prognosis. Updated data with additional follow-up based on later data-cut will be presented.