Minimal Residual Disease Measurement Research Articles

Analysis of measurable residual disease by IG/TR gene rearrangements: quality assurance and updated EuroMRD guidelines.

Minimal/measurable residual disease (MRD) diagnostics using real-time quantitative PCR analysis of rearranged immunoglobulin and T-cell receptor gene rearrangements are nowadays implemented in most treatment protocols for patients with acute lymphoblastic leukemia (ALL). Within the EuroMRD Consortium, we aim to provide comparable, high-quality MRD diagnostics, allowing appropriate risk-group classification for patients and inter-protocol comparisons. To this end, we set up a quality assessment scheme, that was gradually optimized and updated over the last 20 years, and that now includes participants from around 70 laboratories worldwide. We here describe the design and analysis of our quality assessment scheme. In addition, we here report revised data interpretation guidelines, based on our newly generated data and extensive discussions between experts. The main novelty is the partial re-definition of the "positive below quantitative range" category by two new categories, "MRD low positive, below quantitative range" and "MRD of uncertain significance". The quality assessment program and revised guidelines will ensure reproducible and accurate MRD data for ALL patients. Within the Consortium, similar programs and guidelines have been introduced for other lymphoid diseases (e.g., B-cell lymphoma), for new technological platforms (e.g., digital droplet PCR or Next-Generation Sequencing), and for other patient-specific MRD PCR-based targets (e.g., fusion genes).

Minimal Residual Disease Status in Multiple Myeloma 1 Year After Autologous Hematopoietic Cell Transplantation and Lenalidomide Maintenance Are Associated With Long-Term Overall Survival.

Prognostic Immunophenotyping in Myeloma Response (PRIMeR) is an ancillary study of minimal residual disease (MRD) assessment for multiple myeloma by next-generation multiparameter flow cytometry (MFC). Patients were enrolled on a three-arm randomized control trial (Blood and Marrow Transplants Clinical Trials Network 0702 Stem Cell Transplant for Myeloma in Combination of Novel Agents [STaMINA]; ClinicalTrials.gov identifier: NCT01109004). Four hundred and thirty-five patients consented to the MRD panel, which included 10 monoclonal antibodies measured via six-color MFC. MRD was measured at baseline/preautologous hematopoietic cell transplant (BL/preAutoHCT), premaintenance (PM), and 1 year (Y1) after AutoHCT with a sensitivity of 10-5 to 10-6. The primary objective was to assess MRD-negative (MRDneg) at 1 year after AutoHCT and progression-free survival and overall survival (PFS/OS). Similar to the STaMINA results, at a median follow-up of 70 months, there was no significant difference in PFS/OS by treatment arm in the PRIMeR patients. MRDneg at all three time points was associated with significantly improved PFS, and MRDneg at Y1 had significantly longer OS. Multivariate analysis of PFS, adjusting for disease risk and treatment arm, demonstrated hazard ratios (HRs) in MRD-positive patients compared with MRDneg patients at BL, PM, and Y1 of 1.55 (P = .0074), 1.83 (P = .0007), and 3.61 (P < .0001), respectively. Corresponding HRs for OS were 1.19 (P = .48), 0.88 (P = .68), and 3.36 (P < .001). Patients with sustained MRDneg or who converted to MRDneg by Y1 had similar PFS/OS. To our knowledge, this first, prospective US cooperative group, multicenter study demonstrates that MRDneg at Y1 after AutoHCT with lenalidomide maintenance is prognostic for improved 6-year PFS and OS. Serial MRD measurements may direct trials to test how further therapy may improve long-term PFS and OS.

Abstract PO3-14-10: A single-center prospective cohort study to evaluate circulating tumor cells as a monitoring tool in women with breast cancer treated with neoadjuvant chemotherapy: final results of baseline data

Abstract Background: The presence of liquid biomarkers such as circulating tumor cells (CTCs) among women undergoing neoadjuvant chemotherapy (NAC) for breast cancer may be associated with treatment response and/or an increased risk of recurrence, but limited data is available. Objectives: To detect and enumerate CTCs in blood samples from women with a new diagnosis of non-metastatic breast cancer of any subtype both i) at baseline (prior to commencing NAC), and ii) after completion of NAC and surgery using the Epic Sciences platform. Methods: Women with non-metastatic breast cancer of any subtype who have not yet commenced NAC were included, irrespective of age. Those with a prior history of another invasive cancer (apart from non-melanoma skin cancer identified 5+ years prior to enrollment) were excluded. Blood samples were obtained to measure CTCs prior to commencing NAC, and after completion of NAC and surgery (at least 4 weeks post-operatively). As previously described, CTC identification was based on immunofluorescence analysis using the Epic Sciences platform (Ueno et al 2017). Clinical/pathological data and clinical outcomes were abstracted from patients’ medical records. Associations between CTC detection and clinical/pathologic characteristics were evaluated using Fisher’s exact test for categorical variables and t-test or Wilcoxon rank sum tests for numerical variables. All analyses were performed using the R software package. Results: 50 participants who met eligibility criteria were included; a baseline blood sample was evaluable for CTC detection and enumeration for 47 patients. The median age at breast cancer diagnosis was 52 (29-75). Among them, 18 (38.3%) had HER2+ breast cancer, 17 (36.1%) had hormone receptor (HR)+/HER2- breast cancer and 12 (25.5%) had triple negative breast cancer (TNBC). The majority of patients (91%) received anthracycline and taxane-based NAC. A total of 68 samples were tested for CTC enumeration (5 mL equivalent per sample) including 47 pre-treatment and 21 post-treatment samples. CTCs were detected in 37 (79%) of patients for whom a baseline (pre-NAC) sample was available with a mean of 4.2 (SD 17.0) CTCs/mL and a range of 0 - 115.4 CTCs/mL. Detection of CTCs at baseline was highest among patients with TNBC (n=10/12, 83%), followed by those with HER2+ (n=14/18, 78%) and HR+/HER2- (n=13/17, 76%) breast cancer. CTCs were detected in 43% (n=9/21) of post-treatment samples, with a mean of 1.2 (SD 3.6) CTCs/mL and range of 0 – 16.4 CTCs/mL. Among the 20 patients for whom matched pre- and post-treatment CTC results were available, 16 (80%) had detectable CTCs pre-treatment and 8 (40%) had detectable levels post-NAC and surgery; of the 8 patients with post-treatment CTCs, 4 (50%) had HR+/HER2- breast cancer, 2 had HER2+ (25%) and 2 (25%) had TNBC. Three patients had numerically higher CTC levels after completion of NAC and surgery compared to baseline levels, 2 of whom had HR+/HER2- breast cancer and one of whom had TNBC. To-date, only 8 patients (19% of 43 who have undergone surgery) have achieved a pathological complete response (PCR) to NAC, among whom 3 had matched pre- and post-treatment CTC results available. None of these 3 patients had detectable CTCs post treatment. Conclusions: Approximately 4 in 5 women with non-metastatic breast cancer who undergo NAC have detectable CTCs at baseline (pre-treatment) using the Epic Sciences Platform. Given that CTCs remain detectable in a high proportion (40%) of patients after NAC and surgery, evaluation of CTCs as a potential measure of minimal residual disease warrants further evaluation in this patient population. Citation Format: Rania Chehade, Arushi Jain, Veronika Moravan, Giuseppe Di Caro, Megan Slade, Nadine Hartmann, Rick Wenstrup, Ana Elisa Lohmann, William Tran, Katarzyna Jerzak. A single-center prospective cohort study to evaluate circulating tumor cells as a monitoring tool in women with breast cancer treated with neoadjuvant chemotherapy: final results of baseline data [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 PO3-14-10.

Survival after allogeneic transplantation according to pretransplant minimal residual disease and conditioning intensity in patients with acute myeloid leukemia

BackgroundThe measurement of minimal residual disease (MRD) by multiparametric flow cytometry (MFC) before hematopoietic stem cell transplantation (HSCT) in patients with acute myeloid leukemia (AML) is a powerful prognostic factor. The interaction of pretransplant MRD and the conditioning intensity has not yet been clarified.ObjectiveThe aim of this study is to analyze the transplant outcomes of patients with AML who underwent HSCT in complete remission (CR), comparing patients with positive MRD (MRD+) and negative MRD (MRD−) before HSCT, and the interaction between conditioning intensity and pre-HSCT MRD.Study designWe retrospectively analyzed the transplant outcomes of 118 patients with AML who underwent HSCT in CR in a single institution, comparing patients with MRD+ and MRD− before HSCT using a cutoff of 0.1% on MFC, and the interaction between conditioning intensity and pre-HSCT MRD.ResultsPatients with MRD+ before HSCT had a significantly worse 2-year (2y) event-free survival (EFS) (56.5% vs. 32.0%, p = 0.018) than MRD− patients, due to a higher cumulative incidence of relapse (CIR) at 2 years (49.0% vs. 18.0%, p = 0.002), with no differences in transplant-related mortality (TRM) (2y-TRM, 19.0% and 25.0%, respectively, p = 0.588). In the analysis stratified by conditioning intensity, in patients who received MAC, those with MRD− before HSCT had better EFS (p = 0.009) and overall survival (OS) (p = 0.070) due to lower CIR (p = 0.004) than MRD+ patients. On the other hand, the survival was similar in reduced intensity conditioning (RIC) patients regardless of the MRD status.ConclusionsPatients with MRD+ before HSCT have worse outcomes than MRD− patients. In patients who received MAC, MRD− patients have better EFS and OS due to lower CIR than MRD+ patients, probably because they represent a more chemo-sensitive group. However, among RIC patients, results were similar regardless of the MRD status.

Flow cytometric minimal residual disease measurement accounting for cytogenetics in children with non-high-risk acute lymphoblastic leukemia treated according to the ALL-MB 2008 protocol.

Quantitative measurement of minimal residual disease (MRD) is the "gold standard" for estimating the response to therapy in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Nevertheless, the speed of the MRD response differs for different cytogenetic subgroups. Here we present results of MRD measurement in children with BCP-ALL, in terms of genetic subgroups with relation to clinically defined risk groups. A total of 485 children with non-high-risk BCP-ALL with available cytogenetic data and MRD studied at the end-of-induction (EOI) by multicolor flow cytometry (MFC) were included. All patients were treated with standard-risk (SR) of intermediate-risk (ImR) regimens of "ALL-MB 2008" reduced-intensity protocol. Among all study group patients, 203 were found to have low-risk cytogenetics (ETV6::RUNX1 or high hyperdiploidy), while remaining 282 children were classified in intermediate cytogenetic risk group. For the patients with favorable and intermediate risk cytogenetics, the most significant thresholds for MFC-MRD values were different: 0.03% and 0.04% respectively. Nevertheless, the most meaningful thresholds were different for clinically defined SR and ImR groups. For the SR group, irrespective to presence/absence of favorable genetic lesions, MFC-MRD threshold of 0.1% was the most clinically valuable, although for ImR group the most informative thresholds were different in patients from low-(0.03%) and intermediate (0.01%) cytogenetic risk groups. Our data show that combining clinical risk factors with MFC-MRD measurement is the most useful tool for risk group stratification of children with BCP-ALL in the reduced-intensity protocols. However, this algorithm can be supplemented with cytogenetic data for part of the ImR group.

Refining risk stratification in paediatric B-acute lymphoblastic leukaemia: Combining IKZF1plus and Day 15 MRD positivity.

This study investigates the potential utility of IKZF1 deletion as an additional high-risk marker for paediatric acute lymphoblastic leukaemia (ALL). The prognostic impact of IKZF1 status, in conjunction with minimal/measurable residual disease (MRD), was evaluated within the MRD-guided TPOG-ALL-2013 protocol using 412 newly diagnosed B-ALL patients aged 1-18. IKZF1 status was determined using multiplex ligation-dependent probe amplification. IKZF1 deletions, when co-occurring with CDKN2A, CDKN2B, PAX5 or PAR1 region deletions in the absence of ERG deletions, were termed IKZF1plus . Both IKZF1 deletion (14.6%) and IKZF1plus (7.8%) independently predicted poorer outcomes in B-ALL. IKZF1plus was observed in 4.1% of Philadelphia-negative ALL, with a significantly lower 5-year event-free survival (53.9%) compared to IKZF1 deletion alone (83.8%) and wild-type IKZF1 (91.3%) (p < 0.0001). Among patients with Day 15 MRD ≥0.01%, provisional high-risk patients with IKZF1plus exhibited the worst outcomes in event-free survival (42.0%), relapse-free survival (48.0%) and overall survival (72.7%) compared to other groups (p < 0.0001). Integration of IKZF1plus and positive Day 15 MRD identified a subgroup of Philadelphia-negative B-ALL with a 50% risk of relapse. This study highlights the importance of assessing IKZF1plus alongside Day 15 MRD positivity to identify patients at increased risk of adverse outcomes, potentially minimizing overtreatment.

Influence of Minimal Residual Disease at Day 15 of Induction Therapy on Survival of Children with Acute Lymphoblastic Leukemia.

The aim of the study was to evaluate the impact of minimal residual disease (MRD) on day 15 of induction therapy (d15) on the treatment outcome in children with acute lymphoblastic leukemia (ALL). The study included 74 patients (1-18 years) with ALL, who were treated at the Pediatric Clinic of the University Clinical Center Banja Luka from January 2011 to May 2021. All patients were treated according to ALL IC-BFM 2009 protocol. MRD on bone marrow was assessed d15, using the multiparameter flow cytometry method (FCM). Of all, 59.46% of patients had MRD d15 0.1-10%, MRD<0.1% had 18.92% of patients, and 21.62% had MRD >10%. Patients with the lowest MRD had the highest 5-year overall survival (OS) and event-free survival (89.5% and 91% respectively) and the lowest cumulative risk for relapse or death (9.7% and 8.1%), in contrast to patients with MRD>10% in whom OS was 80.0%, and the risk of recurrence is 20%. Predicted MRD d15 was significantly associated with prednisone response assessed in the peripheral blood on day 8 (P<0.001) and statistically significantly positive correlation (r=0.498; P<0.001) was found. MRD measurement d15 has a great prognostic significance for patients in the standard and high risk groups, but not for patients in the intermediate risk group. The introduction of additional testing is necessary for better identification of patients with an increased risk of disease recurrence.

Applications of Circulating Tumor DNA in Myelodysplastic Syndromes and Acute Myeloid Leukemia: Promises and Challenges.

The term 'liquid biopsy' has become widely used by clinicians with the development of non-invasive diagnostic and monitoring techniques for malignancies. Liquid biopsy can provide genetic information for early diagnosis, risk stratification, treatment selection and postoperative follow-up. In the era of personalized medicine, liquid biopsy is an important research direction. In recent years, research on circulating tumour DNA (ctDNA) in hematological malignancies has also made great progress. This review provides an overview of the current understanding of circulating tumour DNA in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Additionally, recent advancements in the monitoring of minimal/measurable residual disease (MRD) through ctDNA are discussed.

Complex karyotype but not other cytogenetic abnormalities is associated with worse posttransplant survival of patients with nucleophosmin 1-mutated acute myeloid leukemia: A study from the European Society for Blood and Marrow Transplantation Acute Leukemia Working Party.

In the 2022 European LeukemiaNet classification, patients with nucleophosmin 1 (NPM1)-mutated acute myeloid leukemia (AML) were classified in the adverse-risk category in the presence of high-risk cytogenetics (CG). Nonetheless, the impact of various CG aberrations on posttransplant outcomes remains to be unraveled. This registry study analyzed adult patients with NPM1-mutated de novo AML who underwent their first allogeneic hematopoietic cell transplantation in the first complete remission from 2005 to 2021. A total of 3275 patients were identified, 2782 had normal karyotype, 493 had chromosomal aberrations including 160 with adverse-risk CG, 72 patients had complex karyotype (CK), and 66 monosomal karyotype (MK). Overall, 2377 (73%) patients had FLT3-ITD. On univariate analysis, only FLT3-ITD, minimal/measurable residual disease (MRD) positivity and CK, but not abnormal CG, affected posttransplant outcomes. On multivariable analysis, CK was associated with lower overall survival (OS) (hazard ratio [HR] 1.72, p = .009). In the subgroup of 493 patients with aberrant CG, the 2-year leukemia-free survival (LFS) and OS were around 61% and 68%, respectively. On multivariable analysis for this subgroup, CK and MRD positivity were associated with increased risk of relapse (HR 1.7, p = .025; and 1.99, p = .003 respectively) and worse LFS (HR 1.62, p = .018; and 1.64, p = .011 respectively) while FLT3-ITD, MK, or other CG abnormalities had no significant effect. Importantly, CK negatively affected OS (HR 1.91, p = .002). In the first complete remission transplant setting, CK was found as the only cytogenetic risk factor for worse outcomes in NPM1-mutated AML. Nevertheless, even for this subgroup, a significant proportion of patients can achieve long-term posttransplant survival.

Update for the ‘Watch’ Registry, a Real-World Observational Study Using Clonoseq® to Monitor MRD in Lymphoid Malignancies

Background: For patients with the most common B-cell malignancies, assessment of minimal/measurable residual disease (MRD) is routinely incorporated into clinical trials to evaluate patient response and NCCN guidelines include recommendations for incorporation of MRD assessment into clinical practice for ALL, CLL, and MM. Despite that, there is a desire for additional data that reflect the impact of MRD assessment in patient management in the real-world setting. The Watch Registry (NCT04545333) is a prospective, multicenter observational study being conducted in the US to collect data from health care providers who use the clonoSEQ MRD assay as part of routine care for their lymphoid malignancy patients. Data collection is focused on understanding broad practice patterns for clonoSEQ® MRD assessment, including assay timing and subsequent treatment decisions. The ultimate goal of the study is to describe the implementation and impact of the assay in patient management. Here we describe the latest updates from this ongoing study. Methods: Eligible patients are ≥ 18 years old, diagnosed with NHL, MM, ALL, or CLL, not enrolled in an interventional clinical study that dictates the frequency of their MRD testing, and whose treating physician is currently or planning to use clonoSEQ to monitor their MRD as part of their routine management. Patients may be in any phase of treatment at enrollment. Enrollment is now closed and follow-up will continue through December 31, 2024. Results: From October 2020 through data cutoff of June 21, 2023, 465 patients were enrolled across 18 academic and community sites (463 patients with reportable data). Enrollment by disease is 70 ALL, 109 CLL, 140 MM, 34 DLBCL, 27 MCL, 54 FL and 29 other lymphoid malignancies. Of these patients, 91 (20%) have discontinued the study; the most common reasons are death (35; 8%) and failure to detect trackable sequences by clonoSEQ (23; 5%). Other reasons include site closure and patient/physician decision to withdraw. Patients range in age from 18 to 92, 57% are male, and the mean lines of therapy (LOT) prior to enrollment is ~2. The median follow up period is 14 months. To date, 994 clonoSEQ MRD tests have been ordered and 74.5% of patients have received at least 1 MRD test (Table 1). The most frequent testing was reported for ALL patients (4.8 tests/pt) followed by CLL (2.9 tests/pt), MCL (3.3 tests/pt), other lymphoid malignancies (2.9tests/pt), DLBCL (2.6 tests/pt), FL (2.1 tests/pt) and MM (2.2 tests/pt). Across all disease cohorts, most MRD tests were ordered during remission (71.4%). In the ALL cohort, a larger proportion of tests were ordered at other treatment timepoints relative to other disease cohorts: end of induction (6%), pre-HSCT (6%), post-HSCT, including some post-HSCT monitoring (42%) and post-CAR-T (6%). Across all cohorts, 220 patients (48%) had multiple MRD tests ordered with the largest proportion observed in the ALL cohort (67%), followed by MCL (63%), DLBCL (50%), CLL (48%), other lymphoid malignancies (45%), MM (39%) and FL (35%). For patients tested during monitoring, the number of tests/pt was generally larger among patients with ≥1 prior LOT compared to patients with 0 prior LOT. With 979 test results reported (Table 2), there were 583 tests with residual sequences detected (59.5%) and 396 with no residual sequences detected (39.5%). Based on the presence of residual sequences, a change to a patient's treatment plan was reported 66 times (11.3%) and there were 52 separate reports (11.1%) of a change to the frequency of future MRD monitoring. Specific treatment changes included 56 instances of therapy intensification (9.6%) and 10 instances of therapy de-intensification (1.7%). In the case where no change was made based on the presence of residual sequences, reported reasons for not making a change (if reported) included decreasing MRD (24%) and low/stable disease (10.9%). Conclusions: Clinicians are using clonoSEQ to assess MRD across multiple lymphoid malignancies in routine care. In ALL, MRD testing was reported for multiple treatment timepoints whereas in other disease states (CLL, MM, NHL) reported use of the assay was predominantly for monitoring remission. The advent of therapeutic regimens for B-cell malignancies with higher relative response rates denotes the need for sensitive tools that can be used in clinical practice to better assess treatment response and monitor MRD to facilitate future treatment decisions.

Functional Imaging As a Key Diagnostic Tool in the Follow-up of RRMM Patients after CAR-T-Cell Therapy

Introduction: Anti BCMA CAR T-cell therapy (CART) is establishing itself as a new standard of care for relapsed and refractory multiple myeloma (RRMM). However, the optimal follow-up strategy post CART has yet to be determined. Prior studies of our own and others have emphasized the role of functional imaging in monitoring RRMM patients post traditional chemotherapy, revealing residual disease in up to 50 % of otherwise minimal residual disease (MRD) negative patients (Rasche et al. Leukemia 2019). Whether a similar patchy disease pattern is relevant post CART remains unknown. To address this, we implemented a standardized follow-up program post CART, which includes 18F-FDG PET-CT, diffusion-weighted MRI, and bone marrow aspiration with MRD measurements at our center. Methods: We conducted monthly ambulatory visits and serological testing to monitor patients post CART infusion. Additionally, an extended follow-up visit at three months post CART infusion was performed, encompassing whole body PET-CT, whole body diffusion-weighted MRI, and bone marrow aspiration with MRD testing. Response assessments were based on IMWG criteria. MRI classified focal lesions as vital if they exhibited 1) significant diffusion-restriction, 2) a fat fraction less than 20 %, and 3) homogenous hypointensity on T2-weighted images. MRD measurements were performed with flow cytometry with a sensitivity of at least 10 -5 following the Euroflow recommendations. Results: From December 2021 to July 2023 we treated 37 heavily pretreated patients with idecabtagene vicleucel at our center. These patients had a median of 5 prior lines of therapy. High-risk cytogenetics, extra-medullary disease (EMD), and non-secretory disease were seen in 17, 14 and 6 patients, respectively. At data cut off, the ORR, VGPR and CR rates were 78 %, 27 % and 27 %, respectively. We observed primary refractory disease in 14 % of the patients. Among the 22 patients who underwent extended follow-up examinations at three months, the majority achieved MRD negativity, with only 7/22 (32 %) remaining MRD positive after CART. However, functional imaging revealed a higher number of patients with residual disease, with positive results on PET-CT and DW-MRI in 9/22 (41 %) and 10/22 (45 %) of patients, respectively. Notably, 6 of 22 (27 %) patients displayed active lesions on imaging despite MRD negativity at the iliac crest at this time point. Next, we focused on patients who experienced relapse from CART and sought to determine the necessary technology to detect relapse in this setting. Thirteen patients experienced relapse during the study period, and in 8 (61%) of them, functional imaging was the sole method to detect progressive disease. Only 1 patient transitioned from MRD negative to positive during the follow-up period, while the remaining 4 patients showed a significant M protein increase as defined by the IMWG criteria. Conclusion: In our heavily pretreated RRMM population, functional imaging emerged as a crucial diagnostic tool, detecting relapse alone in more than half of the patients. Our data supports inclusion of cross-sectional imaging in a comprehensive follow-plan after CART.

Safety and Efficacy of Bortezomib-Pomalidomide-Dexamethasone (VPD) As Novel Induction Therapy in Newly-Diagnosed Multiple Myeloma: Updated Data from an Ongoing Single Arm Phase-II Investigator- Initiated Clinical Trial (PRIME Study)

Introduction: Pomalidomide is a third-generation immunomodulatory drug approved for relapsed and/or refractory Multiple Myeloma (RRMM). In a phase 3 OPTIMISMM trial, Pomalidomide, Bortezomib, and Dexamethasone demonstrated superior efficacy in patients with RRMM. PRIME study (CTRI/2019/10/021618) is testing Bortezomib, Pomalidomide and Dexamethasone combination in Newly Diagnosed Multiple Myeloma (NDMM). Initial data of 26 patients was presented as abstract in 63rd ASH Annual Meeting and Exposition. Current abstract is reporting the results of 46 patients with a longer follow up of this ongoing study. Objectives: Primary objective is to determine the efficacy of VPD regimen by measuring Minimal Residual Disease (MRD) assessment by flow-cytometry after 9 cycles of VPD. Secondary objectives are to determine response rates, PFS and safety of the regimen Methods: A prospective, single arm, phase II study from a tertiary care center. Both transplant eligible and ineligible patients with NDMM aged between 18-70 years as defined by the International Myeloma Working Group (IMWG) are being recruited in the study. The regimen consists of weekly Bortezomib 1.3mg/sq m, Pomalidomide 2 - 4mg once daily for 21days and Dexamethasone 20mg twice weekly, in a 28 days cycle for 9-12 cycles. Transplant patients planned for 9 cycles and non-transplant patients for 12 cycles of VPD. Eligible patients underwent High dose chemotherapy followed by Autologous HCT after 4-6 cycles of VPD. After Auto HCT 3-5 cycles of VPD given as consolidation therapy. After 9-12 cycles of VPD maintenance treatment given as per institutional practice. A 2-tube 8-colour antibody panel was used for MRD analysis using Flowcytometry (BD FACS CANTO-II) and analysed on Kaluza version. 1.3 software. A valve of &amp;lt; 0.001% was considered as MRD negative. Results: In this ongoing study, out of the proposed 50 patients, 46 patients were enrolled between April 2020 to June 2023. Median follow up is 24.5 months. The median age was 55 years (38-70), and gender ratio 1:1. At disease presentation, bone lesions was the commonest (91.3%, n 42) finding. The most common myeloma subtype was IgG Kappa in 43.5% (n 20). High risk cytogenetics were seen in 43.5% (n 20) and 43.5% (n 20) of patients were in RISS stage III (Table 1). Out of 46 patients 41 (89.1%) have completed 4 cycles and 30 (65.2%) patients have completed 9 cycles of VPD till last follow up (June, 2023). One patient had withdrawn consent, two patients were out of study due to increasing peripheral neuropathy (Grade 3). Five deaths occurred, 2 post cycle 8 and 3 after cycle 9. Four deaths were due to infections (COVID 19 and bacterial) and one due to progressive disease. Five patients underwent auto HCT (between Cycle 4 to 6). No patient failed stem cell mobilization, 2 patients required plerixafor in addition to G-CSF for mobilization. Table-1 shows drug-induced toxicity and response rates. Hematological toxicities were the commonest followed by fever, infections and skin rash. Majority of nonhematological adverse events were grade 1 to 2 and protocol was well tolerated. Protocol adherence was 97.8% (45/46 patients). After 4 cycles (n 43), 13 (30.2%) patients were in stringent complete response (sCR), 2(4.7%) in CR, 24 (55.8%) in very good partial response (VGPR) and 2 (4.7%) in partial response. After 9 cycles (n 35), 16 (45.7%) patients were in sCR, 2(5.7%) in CR and 11 (31.4%) in VGPR. Two (5.7%) patients had progressive disease (PD) and 3 patients were out of study due to consent withdrawn and toxicity. After 4 cycles of VPD 21 (48.8%) patients were MRD negative and MRD negative rate improved after 9 cycles to 74.3% (26 patients). Two years Progression Free survival (PFS) was 77.2% +/- 7.7% (Figure 1), disease free survival was 90.3% +/- 5.3% and overall survival (OS) was 83.1% +/- 7%. Conclusion: Safety data from the PRIME study demonstrates that VPD regimen has a favorable tolerance profile in patients with NDMM and no clinically significant impact on stem cell mobilization or engraftment. Early efficacy signals are encouraging, and recruitment continued. VPD has shown improved depth of response (MRD negativity) and overall response rate. Early follow up results shows promising PFS and OS results. VPD may be a promising induction regimen instead of Dara VRD in resource constrain setting where Daratumumab is not readily accessible. Phase 3 randomized trials are the way forward to its clinical use in frontline setting.

36-Month Follow-up Results of the Gimema ‘Veritas’ Trial of Front-Line Venetoclax and Rituximab (VenR) in Young and Fit Patients with Chronic Lymphocytic Leukemia and an Adverse Biologic Profile

Introduction: Based on the efficacy of the fixed-duration venetoclax and rituximab combination (VenR) in the setting of relapsed/refractory (R/R) patients with chronic lymphocytic leukemia (CLL), we investigated the efficacy and safety of the front-line VenR regimen in young (≤65 years) and fit patients with CLL and an unfavorable biologic profile in the GIMEMA LLC1518 VERITAS trial. Here, we report the 36-month updated results of this phase II, single-arm, multicenter, front-line study for young patients with CLL carrying an unmutated IGHV profile and/or a TP53 disruption. Methods: Seventy-five CLL patients requiring treatment, according to the International Workshop on CLL (iwCLL) criteria, were enrolled between October 2018 and May 2020. Treatment consisted of the five-week Ven ramp-up, six-monthly courses of the VenR combination, followed by six monthly courses of Ven single agent. Patients received tumor lysis syndrome (TLS) prophylaxis with urate-reducing agents and oral or iv hydration. The primary endpoint of this study was the complete remission (CR) rate assessed at the end of treatment (EOT, month 15) according to the iwCLL criteria. Measurable/minimal residual disease (MRD) was assessed on the peripheral blood (PB) and bone marrow (BM) by ASO-PCR at baseline, EOT and during the follow-up. MRD was categorized as undetectable (uMRD) with a cut-off of &amp;lt;1 cell in 10,000 leukocytes. Results: The median age of patients was 54 years (range 38-65); 96% had unmutated IGHV, 9 (12%) had TP53 disruption, and 4% showed mutated IGHV with TP53 disruption. At the EOT, the overall response rate (ORR) was 94.7%, with a CR rate of 76%. On an intention-to-treat basis, 52 (69.3%) patients showed a response with uMRD in the PB and 44 (58.7%) in the BM. At 33 months from the EOT, uMRD was maintained by 50% of patients who had achieved an uMRD in the PB and by 57% of those with a prior uMRD in the BM. The 33-month PB MRD-free survival was 50% (Figure 1). In multivariate analysis, factors associated with a significantly shorter MRD-free survival were the presence of an unmutated IGHV associated with a TP53 disruption (HR: 3.43 [95%CI: 1.13-10.4]; p=0.029), Binet B stage (HR: 0.16 [95%CI: 0.06-0.43]; &amp;lt;0.001) or, Binet C stage (HR: 0.19 [95%CI: 0.07-0.52]; p=0.001), while the clinical response, CR, or PR, did not show a significant impact. Three patients have died due to COVID-19. Despite iv hydratation, the administration of anti-uric acids, fatal TLS occurred in a patient who used self-administered fentanyl patches for analgesis purposes during the ramp-up phase. The 36-month OS was 96%. A further lymphoproliferative disorder was diagnosed in 2 patients (follicular lymphoma, 1; large granular cell leukemia, 1) and a second malignancy in 1 (prostate cancer), while no cases of Richter transformation were recorded. No unexpected adverse events were observed during the follow-up. Conclusions: The updated data from the VERITAS trial show that in young and fit patients with adverse genetic characteristics the front-line VenR combination was an effective regimen with high rates of deep responses with uMRD. Furthermore, at 33 months from the EOT, uMRD was maintained in the PB and BM in about half of the patients.

Multinational Retrospective Analysis of Bridging Therapy Prior to Chimeric Antigen Receptor T Cells for Relapsed/Refractory Acute Lymphoblastic Leukemia in Children and Young Adults

Background: Chimeric antigen receptor (CAR) T cells targeting CD19 are a well-established treatment option for children and young adults suffering from relapsed and/or refractory B-lineage acute lymphoblastic leukemia. Nonetheless, there is still insufficient data about the proper management of bridging therapy between eligibility for therapy and administration of CAR T cells taking into consideration that most of the patients are heavily pretreated. Bridging therapy has been designed to achieve a low leukemia burden prior to CAR T cell infusion. However, systematic data of bridging therapy are still limited and the effect of bridging therapy on outcome, side effects and response to CAR T cell therapy is still poorly understood. With this retrospective, multinational, large-scale study, we strive to understand the impact of low- and high-intensity bridging regimens on a variety of outcome parameters in order to improve the basis for clinical decision making in bridging therapy prior to CAR T cell administration. Methods: Real-world data were collected from 83 patients receiving 88 CAR T cell therapies from twelve different sites in Germany, Austria and Switzerland. Data were collected anonymously via paper case report forms and subsequently analyzed. Performed treatments were classified into the categories 1) no systemic therapy, 2) low-intensity therapy and 3) high-intensity therapy. Bridging therapies were defined as high-intensity if at least one chemotherapeutic agent of the following was given: cyclophosphamide/ifosfamide, etoposide, anthracyclines or other agents with high toxicity potential (intravenous methotrexate, platinum-based antineoplastic drugs, thiotepa, high-dose cytarabine, fludarabine). Low-intensity bridging therapies comprised the administration of steroids, vincristine, low-dose cytarabine, PEG-asparaginase/Erwinia asparaginase and oral maintenance therapy (mercaptopurine, thioguanine, oral methotrexate, hydroxyurea). The administration of specific chemotherapeutic agents as well as immunotherapies and targeted therapies was assessed. CAR therapies comprised CD19 2 nd generation CAR T cell products from commercial and academic providers. We then analyzed the impact of different bridging regimens on several outcome parameters such as overall and disease-free survival, adverse events, tumor burden and performance status at defined time points (eligibility, leukapheresis if performed, lymphodepletion/CAR T cell infusion). Results: 33 of 88 treatments were classified as high-intensity and 34 as low-intensity bridging regimens. Prior to 13 CAR T cell administrations no systemic bridging therapy was given, 8 of 88 bridging regimens could not be stratified due to incomplete data (Figure 1). Between eligibility and apheresis, mostly low-intensity therapy or no systemic therapy was given. Within the period between apheresis and CAR T cell infusion, treatment diversified due to the heterogeneity of the cohort. Patient characteristics are listed in Table 1. Patients receiving a high-intensity bridging therapy had a significantly higher tumor burden at time point of eligibility defined by blasts in bone marrow and by measurement of minimal residual disease (MRD) compared to patients treated with a low-intensity or no systemic bridging therapy. Tumor burden within the two groups converged over the time of bridging therapy. However, at time of lymphodepletion, patients in the high-intensity group showed a significantly lower performance status indicated by Karnofsky/Lansky score than patients in the low-intensity/no systemic therapy group, reflecting the higher toxicity potential. Furthermore, these patients suffered significantly more often from bacterial adverse events and mucositis. Neither overall nor disease-free survival differed significantly between the two bridging regimen groups. Conclusion: In this retrospective cohort data, a high-intensity bridging therapy has not improved the outcome of CAR T cell therapy in terms of overall and disease-free survival. Yet high-intensity bridging therapy has caused more mucositis, bacterial adverse events and worsened the performance status. Our study suggests that a low-intensity bridging regimen may be preferred whenever tumor burden and disease kinetics allow this treatment strategy.

Endpoint Surrogacy in Chronic Lymphocytic Leukemia: A Pooled Analysis of the German CLL Study Group

Introduction Overall survival (OS) is generally considered the most patient-relevant endpoint in oncology trials, however, its implementation can entail several challenges in the context of chronic lymphocytic leukemia (CLL): 1) The competing survival risk due to comorbidities in the mostly elderly CLL patient population; 2) the high efficacy of targeted therapies warrant longer observation periods or extensive patient recruitment, which can be operationally challenging; 3) the interference with increasingly potent relapse therapies, which can neutralize differences of efficacy of first line regimen. Hence, surrogate endpoints, such as progression-free survival (PFS), overall response or complete response rate (ORR, CR) or minimal residual disease (MRD), are commonly used in CLL research. However, data supporting a correlation between surrogate endpoints and OS in the treatment for CLL is scarce. Here, we present a large, systematic analysis of over 4000 patients (pts) treated in studies of the German CLL Study Group (GCLLSG) to determine the adequacy of surrogate endpoints in clinical trials for pts with CLL. Methods We analysed twelve phase II and phase III trials of the GCLLSG spanning from 1999 to 2022. Only pts receiving first-line therapy were included in the analysis. To determine correlation, the Kaplan-Meier survival function was estimated for both OS and PFS for each study separately. From these functions, the probability of survival (OS and PFS) was estimated at different time points. These pairs of time points were then correlated across the studies, with each pair of probabilities weighted by the number of pts in each corresponding study using Spearman's correlation coefficients. Additionally, we compared OS according to MRD and iwCLL response status at end-of-treatment (for fixed-duration regimens) or end-of-induction-treatment (for MRD-guided treatments) (EO[I]T) using Kaplan-Meier functions calculated from the time point of MRD assessment in peripheral blood. EO(I)T ranged from month 9 to month 15 after treatment initiation, depending on the study protocol. Results The full analysis set comprised 4237 pts. The median observation time was 67 months. Median age was 64 years (range 27 - 90). 45.1% of pts were older than 65 years. 3159 pts (74.6%) had received chemo/chemoimmunotherapy (C/CIT), whereas 1078 (25.4%) were treated with targeted therapies. There were 2114 progressive disease (PD) events and 1211 deaths captured within the trial data. The Spearman's Rho of the estimated survival probabilities for the time points 12/24, 24/36, 36/48 and 60/72 months (PFS/OS) was 0.95, 0.96, 0.94 and 0.92, respectively ( Panel A). Focussing on pts with confirmed OS event (n=1211), the Spearman's Rho between PFS and OS in months was 0.66 for pts receiving chemo/chemoimmunotherapy and 0.88 for pts receiving targeted treatment. ORR and CR rate was 89.8% and 31.5%, respectively. Pts with a CR had a significantly longer PFS (HR 0.38, 95% CI 0.34-0.42, p &amp;lt;0.001) and OS (HR 0.39, 95% CI 0.33- 0.46, p &amp;lt;0.001) compared to pts with non-CR. MRD measurements at the EO(I)T were available for 2521 pts (59.5%), of which 39% were treated with targeted agents. Rates of undetectable (&amp;lt;10 -4), intermediate (≥10 -4 and &amp;lt;10 -2) and high MRD (≥10 -2) were 59.1%, 21.7%, 19.3%, respectively, across treatment groups. The median OS for undetectable and intermediate MRD was not reached and 60.7 months for high MRD status ( Panel B). The estimated 60-month OS was 84.6%, 71.3% and 51.1%, respectively. Accordingly, median PFS for uMRD was 61.8 months and 60-month PFS was 52.6%. In pts ≤65 years of age, median OS was not reached with undetectable or intermediate MRD and 70.5 months in pts with high MRD status. In pts &amp;gt;65 years, median OS was not reached for uMRD and was 75.2 and 58.8 months, respectively, for pts with intermediate or high MRD. The corresponding hazard ratios for high versus uMRD status at EO(I)T were 3.33 (95% CI 2.57 - 4.3) in older and 5.89 (4.15 - 8.35) in younger pts. Conclusion In this large analysis of 12 prospective CLL trials, we found a robust correlation between PFS and OS, thereby supporting the use of PFS as a surrogate endpoint in clinical studies. More importantly, the MRD status at EO(I)T was associated with OS across all treatment modalities, demonstrating its utility in identifying pts with potentially dismal outcomes and providing a rationale for MRD-guided treatment strategies.

Post-CAR-T Minimal Residual Disease (MRD) Monitoring in Mantle Cell Lymphoma Enables Early Relapse Detection

INTRODUCTION: Over 40% of patients with mantle cell lymphoma (MCL) will progress within 1 year after treatment with brexucabtagene autoleucel (Brexu-cel)(Michael Wang et al. ZUMA-2 NEJM 2020). Monitoring minimal residual disease (MRD) of circulating tumor cells (CTC) via next-generation sequencing of the immunoglobulin sequences (IgHTS) with a sensitivity of 1 in million (ClonoSeq, Adaptive Biotechnologies) has previously been shown to identify patients at high risk of MCL relapse (Simone Ferrero et al. Blood 2022). However, there is a limited understanding of MRD monitoring after Brexu-cel. A sub-analysis of 27 patients post-Brexu-cel in ZUMA-2 showed predictive performance of MRD measured at months 1, 3, and 6 in estimating relapse potential. METHODS: Clonotypes were identified from paraffin-embedded tissues and underwent PCR amplification of the IgH, IgH-DJ, and Ig Kappa/lambda regions. CTC MRD assessments were monitored at 1, 3, 6, and 12 months (if applicable) following standard-of-care brexu-cel infusion in all patients with MCL at our center who had at least 3 months of follow-up. PFS and overall survival (OS) were determined by Kaplan-Meier analysis and significance by log-rank test. The comparison between the two groups was conducted using a two-tailed Mann-Whitney test. Significances were calculated using a Fisher exact test between two categorical variables or as otherwise specified. Statistical analysis and plots were generated using Prism 8.4.1 (GraphPad) RESULTS: Between January 2020 and December 2022, all treated patients (n=34) were analyzed. 3 patients were excluded as they were unable to reach day 28 (D28) assessments due to death, progressive disease, or refusal of CAR-T infusion. 5 patients were excluded due to a follow-up of &amp;lt;3 months. The median follow-up time for the 26 included patients was 18.9 months. The median age was 68, 73% were male and 27% were female, 4 median lines of therapy prior, including AutoHSCT (34.6%) or BTKi (92%). The best overall response rate at D28 was 100% with a CR rate of 73% which was similar to ZUMA-2. The median OS was 18.9 months and the median PFS was 15.3 months. We chose to analyze the Day 28 MRD assessment and found 17 of 23 patients (74%) had undetectable MRD and 6 patients had detectable MRD (26%). Due to the relatively small sample size, no significant differences were found between Age, Gender, Pre-treatment LDH, MIPI score, Ki-67, TP53 mutational status, and probability of Day 28 undetectable MRD. Notably, however, 3 of the 6 patients with TP53 mutations had detectable Day 28 MRD. Those patients with detectable MRD compared with those with undetectable MRD had a lower median PFS of 10.74 months vs 17.69 months (p-value= 0.21, Figure 1); although the median OS was similar of 17.69 and 17.84 months, (p-value= 0.75) respectively. 88% of patients (15 out of 17) with undetectable MRD on Day 28 not only maintained this status at 6 months but also showed no radiological disease progression in 93% (14 out of 15) of cases at the time of longest follow-up. A total of 7 patients relapsed during follow-up. MRD predicted 6 impending relapses earlier than PET CT with a median of 294 days (range 15-332 days) prior to progression (Figure 2). CONCLUSION: Evidence from the ZUMA-2 study and our study highlights the significance of early post-CAR-T MRD as an independent prognostic marker for predicting disease recurrence and durable remissions in MCL. Combining MRD measurements with PET-CT at months 1, 3, and 6 after CAR-T therapy can effectively identify high-risk patients for disease progression, offering promising potential to improve outcomes in MCL.

The Different Impacts of an MRD-Guided Protocol on Relapse of Childhood Acute Lymphoblastic Leukemia: The Report of Taiwan Pediatric Oncology Group (TPOG)-ALL-2013

Taiwan Pediatric Oncology Group (TPOG)-ALL-2013 protocol (TPOG-2013), modifying from St. Jude Total Therapy XV Study and Total Therapy XVI Study, was launched since January 2013. This is the first nationwide minimal residual disease (MRD)-directed protocol for treatment of childhood acute lymphoblastic leukemia (ALL) in Taiwan. According to TPOG-2013, two MRD measurements were scheduled on days 15-19 of induction (MRD1 time point, TP1) and days 35-42, end of induction (MRD2 time point, TP2) to make the definitive risk stratification to guide subsequent therapy. As of April 30, 2023, 814 newly diagnosed ALLs treated with TPOG-2013 and followed up ≥3 years were enrolled in the study. The outcomes were compared with those of TPOG-ALL-2002 protocol (TPOG-2002) (N=1350, between January 2002 and December 2012), which did not integrate the MRD monitoring. The 5-year event-free survival and overall survival were significantly improved from 76% (95% CI, 74-78) and 81% (80-84) of TPOG-2002 to 86% (83-88) and 89% (87-92) of TPOG-2013, respectively ( P&amp;lt; 0.0001). For further analysis of TPOG-2013, 585 (72%) patients with exact adherence (EA) to both TPs were assigned as MRD EA group; 225 patients who were non-adherence (NA) to either one of TPs as MRD NA group; and four patients were excluded for the comparative outcome analysis. For B-cell precursor (BCP) ALL, the 5-year cumulative incidence of relapse (CIR) significantly decreased from TPOG-2002 (N=1194) to TPOG-2013 MRD EA (N=527) ( P &amp;lt;0.0001). However, there was no significant improvement of CIR between TPOG-2002 and MRD NA (N=202) ( P= 0.21) (Figure 1). In MRD EA of BCP-ALL, compared with TPOG-2002, patients with ETV6::RUNX1 or BCR::ABL1 appeared to have significantly lower CIR. Patients without the common genetic subtypes , denoted as “negative” group, also had significant inferior CIR. And the trend of CIR decrease did not attain significant difference in high hyperdiploidy. In contrast, patients with T CF3::PBX1 or infant with KMT2A-rearrangement did not show CIR improvement (Table 1). In T-ALL, compared with TPOG-2002 (N=156), there was a significant decrease of CIR in TPOG-2013 MRD EA (N=58)( P= 0.01)(Table 1), but not in MRD NA (N=23)( P= 0.86). There was a longer duration to relapse in MRD EA of BCP-ALL. However, a delay of relapse was only demonstrated in patients with BCR::ABL1, which could be addressed by the introduction of MRD-monitoring and TKI inhibitor in TPOG-2013 (Table 1). Further, in term of early relapse before 1.5 year of remission, there was no significant difference in MRD EA of BCP-ALL, compared with TPOG-2002. In conclusion, this study demonstrated diffident impacts on relapse according to genetic subtypes and MRD adherence. In the negative group of BCP-ALL without the discovery of novel genetic alternations, the relapse could be much decreased with the MRD-directed treatment protocol.

Practical and Flexible Genome Profiling Study Using the Halo-Shape Annealing and Defer-Ligation Enrichment (HANDLE) System: HM-Screen-JAPAN02

Background Acute myeloid leukemia (AML) is known to have a relatively small number of genomic mutations compared to solid tumors. With the development and clinical application of drugs targeting genetic mutations involved in the pathogenesis of AML, the Japanese Society of Hematology has defined “fast-track mutations” as those that should be reported promptly due to clinical necessity. A multicenter genomic profiling study (HM-SCREEN-JAPAN01: HM01 study) was conducted to comprehensively sequence 406 DNA mutations and 265 RNA aberrations in 177 AML patients, suggesting its usefulness in clinical practice, but the turnaround time (TAT) until results are reported remains an issue (Hosono, et al. Cancer Sci, 2023). Aims We have launched the HM-SCREEN-JAPAN02 (HM02) study, a practice-oriented genome profiling study that improves on the previous HM01 study and uses a newly developed targeted sequencing method. The HM02 study adopted the novel Amoy Myeloid Panel® method, which uses the HANDLE (Halo-shape ANnealing and Defer-Ligation Enrichment) system to significantly shorten TAT and facilitate specimen submission, and was designed to be completed in 7 days from sequencing to reporting. The HM02 study allows mutation analysis at any treatment timing, such as at initial presentation, during treatment, or at relapse, as well as the capability to track clonal changes through repeat analysis; the HM02 study was designed to determine whether such flexible analysis is useful in determining treatment strategy. Results The study was conducted at 23 centers in Japan, enrolling a total of 154 cases from 2020 to 2022, with 191 samples analyzed to assess the usefulness of the study. Repetitive mutation analysis was performed in 21 cases, and clonal changes could be tracked. Among the mutations detected, we selected those that were determined to be pathogenic or likely pathogenic by ClinVar. Of the 154 cases analyzed, none of the pathogenic mutations were detected in 9 cases, and 6 were of the normal karyotype. The most frequently observed mutations were TET2 (51%), FLT3 (30%), TP53 (18%), and NPM1 (17%) (Figure 1). In multivariate analysis adjusted for age, gender, and stem cell transplantation status, TP53 and NRAS mutations were associated with higher risk for death (HR=6.82 and 14.98, respectively). Of the 19 cases in which pathological mutations were repeatedly investigated, 8 were useful in determining minimal/measurable residual disease (MRD) (2 cases of MRD loss and 6 cases of MRD retained), 6 cases showed the development of new clones and 5 cases showed clonal evolution, indicating that repeated mutation analysis can be used to evaluate detailed disease status. In terms of clinical utility, HM02 mutation analysis was useful in 62% of cases (98 of 158), particularly for risk assessment and determining the indication for stem cell transplantation (Figure 2). Conclusion Flexible analysis using the novel Amoy Myeloid Panel® method suggests that it can be used to assess the risk of relapse, evaluate the need for transplantation, and select optimal therapeutic agents. NGS-based mutation analysis provides useful information at any point in AML treatment and is expected to improve outcomes.

Mutually-Exclusive Pathways between IKZF1 plus and RAS Mutations and TP53 double-Hit Alterations Lead Relapse in B-Other, Ph-like and Hyperdiploidy Genetic Groups in Adult B-Cell Acute Lymphoblastic Leukemia

Introduction: Adult B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a hematologic malignancy characterized by a high genetic heterogeneity and high relapse rate. Despite treatment advances have led to high cure rates in pediatric BCP-ALL (90%), improving survival in adults remains a challenge. Identification of genetic alterations driving relapse and both new prognostic and therapeutic biomarkers are important to improve survival in adults with BCP-ALL. Aim: To identify genetic alterations that lead to relapse in adult BCP-ALL patients (18-72 years) based on the tumor genetic landscape. Methods: Forty-five adult BCP-ALL patients (median age: 43 years; range 18-72 years) were studied at diagnosis and relapse by massive sequencing using a custom panel that allows detection of single nucleotide variants (SNVs) and insertions/deletions (INDELs) in 150 genes involved in BCP-ALL, in addition to copy number variations (CNVs), gene fusions and aneuploidy. Thirty-eight percent of patients belonged to the BCP-other ALL group, 16% were Philadelphia-positive (Ph+), 16% were classified as Philadelphia-like (Ph-like), 11% belonged to the high hyperdiploidy group, 8% had KMT2A rearrangements ( KMT2Ar), 4% TCF3::PBX1 fusion, 3% showed hypodiploidy, 2% low hyperdiploidy and 2% de novo MYC and BCL2 double-hit. Patients were treated according to the Spanish Adult-PETHEMA high-risk ALL protocols. Results: The most frequently deleted gene at diagnosis was IKZF1 (25/45, 56%). Among these cases, 72% (20/25) had IKZF1plus genetic profile, characterized by co-occurrence of IKZF1 deletions with at least one deletion in CDKN2A/B, PAX5 or PAR1, without ERG deletion. The IKZF1plus genetic profile was associated with specific genetic subgroups: Ph+ (5/7, 71%), Ph-like (5/7, 71%) and B-other (10/17, 59%). Moreover, it was significantly related with the presence of minimal/measurable residual disease (MRD) after induction (16/18, 89%) p &amp;lt; 0.001 and persistence at relapse in most patients (18/20, 90%; p &amp;lt; 0.001). Interestingly, among those 5 patients initially diagnosed with IKZF1 deletion but who did not meet the criteria for the IKZF1plus profile, 2 of them acquired the IKZF1plus profile at relapse. This suggests a possible cooperation of these genetic deletions contributing to therapy resistance and disease progression in these genetic subgroups. Eighty-six percent (6/7) of Ph+ patients who received imatinib treatment presented ABL1 mutations that were detected exclusively at relapse. Of these cases, 67% (4/6) were also associated with IKZF1plus at relapse (Figure 1A). Two mutually exclusive genetic patterns were identified at relapse in adult B-other and Ph-like patients. Thus, patients with IKZF1plus profile did not show RAS pathway mutations (59% and 57%, respectively), while patients with RAS pathway mutations lack in IKZF1plus profile (18% and 29%, respectively) (Figure 1B). Moreover age-related differences in the adult B-other ALL patient group were observed. The presence of mutations in RAS pathway was associated with younger age (&amp;lt;40 years) (3/10, 30% vs. 0/7, 0%), whereas older patients (≥40 years) predominated in the IKZF1plus profile (6/7, 86% vs. 3/10, 30%). TP53 alterations were detected at diagnosis and/or relapse in 11 patients (24%), most of which (9/11, 82%) had a heterozygous mutation plus a deletion or a homozygous mutation (double-hit). The high hyperdiploidy subgroup was enriched in TP53 mutations (4/5, 80%), most of which were already present at diagnosis (3/4, 75%). These findings indicate that genetic biomarkers usually linked to a favorable prognosis, such as high hyperdiploidy, can be associated with unfavorable genetic markers, leading to poorer outcomes in this group of patients (Figure 1C). Both the IKZF1plus profile and TP53 alterations were associated with poorer overall survival (OS) rates (P=0.034 and P=0.040, respectively) in adult BCP-ALL (Figure 2). Conclusion: Our results highlight the clinical importance of the TP53 alterations and the IKZF1plus profile as prognostic factors in adult BCP-ALL, suggesting they might help guiding more personalized therapeutic strategies to improve outcomes in this group of BCP-ALL patients. Grants: SACYL(GRS2386/A/21,GRS2385/A/21,GRS2527/A/22,GRS2506/A/22), JCyL SA118P20, FMM21/002 AP176752021, Pfizer 69383919.

Cladribine Combined with Homoharringtonine and Cytarabine Achieves a High Remission Rate in Adult Patients with De Novo Acute Myeloid Leukemia, Especially for Adverse-Risk Group: A Prospective, Single Center, Single-Arm, Phase 2 Study

Background: For adult de novo acute myeloid leukemia (AML), the standard 3+7 induction regimen has approximately 60% complete remission (CR) rate. Recently, BCL-2 inhibitor plus intensive chemotherapies as novel induction regimens have shown an above 90% composite complete remission (CRc) rate, however still have limited response effect in an adverse risk group, especially for patients with specific cytogenetic abnormalities such as KMT2A rearrangements, FLT3, RAS, and TP53 mutations. Multiple studies have shown that homoharringtonine (HHT) can target leukemia diseases that have switched their anti-apoptotic dependence from BCL-2 to MCL-1. We recently demonstrated that cladribine (CdA) could eradicate BCL-2 inhibitor-resistant monocytic leukemia stem cells driven by RAS mutants and KMT2A rearrangements. (Pei S, et al. Cancer Discovery. 2023) Together, these clinical and basic findings suggest a rationale for combining CdA with HHT to treat AML patients. In this study, we report the efficacy and safety of a newly designed “CHA” regimen composed of CdA, HHT, and cytarabine (AraC) in adult patients with de novo AML. Methods: This is a prospective, single-center, single-arm phase 2 trial. Newly diagnosed AML patients aged 18-59 years were enrolled. The diagnosis, risk classification, and response evaluation were determined according to ELN 2022 criteria. All enrolled patients received the CHA regimen: CdA (5mg/m2, days 1-3), HHT (2mg/m2, days 1-5), and AraC (100mg/ m2, days 1-7) administered intravenously. The response was evaluated on day 28 via bone marrow routine and minimal residual disease (MRD) measurement by flow cytometry. The primary endpoint was CRc (CR plus CRi). Secondary endpoints were MRD, overall survival (OS), disease-free survival (DFS), event-free survival (EFS), and adverse events. Bone marrow (BM) samples on day 1 and 7 and peripheral blood (PB) samples on days 1-7 were collected for laboratory flow analysis to monitor the kinetics of leukemia reduction. We also treated primary AML specimens with single, dual, and triple combinations of CdA, HHT, and Ara-C to evaluate their anti-leukemic activity in vitro. Statistical analyses were carried out using SPSS 24.0. Survival curves were prepared using the Kaplan-Meier method. A two-tailed P value &amp;lt;0.05 was considered statistically significant. This trial is registered on ClinicalTrials.gov (NCT05906914). Results: A total of 33 patients were enrolled from August 1, 2021, to April 15, 2023. The median age was 49 years. 33% (11/33) of patients were at favorable risk, 42% (14/33) were at intermediate risk, and 24% (8/24) were at adverse risk. After one course of CHA induction therapy, the CRc rate was 87% (27 of 31 patients), and the CR rate was 84% (26 of 31 patients). The CR rates in the favorable, intermediate, and adverse-risk groups were 82% (9 of 11 patients), 75% (9 of 12 patients), and 100% (8 of 8 patients), respectively. Notably, six out of six patients with KMT2A rearrangement and ten out of ten patients with RAS mutations achieved MRD-negative CR. The overall MRD negativity was 89% (24 of 27 patients with CRc). In 31 evaluable patients, four patients did not reach CR; three of them held FLT3 mutations. 30-days treatment-related mortality (TRM) was 6% (2 of 33 patients), and both died of sepsis. The median neutrophil recovery time (from the end of chemotherapy to the neutrophil counts ≥0.5×109/L) and platelets recovery time (from the end of chemotherapy to platelets counts ≥20×109/L) were both 11 days. With a median follow-up time of 13 months (IQR: 6-17 months), the one-year OS was 86%, 1-year DFS was 89%, and the 1-year EFS was 76%. Flow cytometry analysis of serially collected PB and BM samples from nine randomly selected patients revealed that an average of 91.0% and 94.7% of leukemia burden was cleared in the PB and BM by day 7, respectively, demonstrating a rapid and effective response induced by CHA. Finally, our in vitro assay demonstrated a synergistic effect of CdA, HHT, and Ara-C on primary AML specimens including three patients with adverse-risk. Conclusion: We propose the CHA regimen as a novel, effective, and safe chemotherapy regimen for newly diagnosed adult AML patients with remarkable potency in treating adverse-risk group patients, especially those with KMT2A rearrangements and RAS mutations that are otherwise resistant to BCL-2 inhibitor-based therapies, but a limited effect on FLT3mutated AML patients.