Minimal Residual Disease Assessment Research Articles

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 < 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.

Centralized MRD Assessment at Early Timepoints in Gimema AML1718 Trial, a Phase 2 Open-Label Study of Venetoclax, Fludarabine, Idarubicin and Cytarabine (V-FLAI) in the Induction Therapy of Non Low-Risk AML

Background: New therapeutic strategies are strongly needed to improve the prognosis of high risk Acute Myeloid Leukemia (AML) patients, such as combination of novel agents and conventional chemotherapy. The Italian GIMEMA AML1718 trial (NCT03455504) investigate the safety and efficacy of the BCL-2 inhibitor venetoclax (VEN) in combination with intensive fludarabine-based induction (FLAI) [fludarabine 30 mg/sqm from day 1 to day 5, cytarabine 2000 mg/sqm from day 1 to day 5, and idarubicin 8 mg/sqm on days 1, 3 and 5] as first-line therapy for newly diagnosed non low-risk ELN AML patients. Results from the Planned Interim Analysis of Safety Run-in and Part 1 (early expansion cohorts) were presented at the last 2022 ASH Meeting. Median overall survival (OS) was not reached; probability of 12-month OS was 76%. Median disease-free survival was not reached. With a median follow-up of 10.5 months, 28 patients (49%) received allogeneic stem cell transplantation (HSCT) in first complete remission (CR). Centralized multicolour flow cytometry minimal residual disease (MFC-MRD) assessment was planned during the phase 2, part 2 of the study (confirmatory cohort) where the lower effective dose of VEN (400 mg/day) was administered in association with FLAI. Here we report results from early time-points (TPs) MRD analysis with the aim of identify the most informative TPs for MRD assessment and prognostic correlations. Methods: Erythrocyte-lysed whole bone marrow (BM) samples obtained at diagnosis from patients enrolled in the confirmatory cohort were centralized and analysed with a broad panel of monoclonal antibodies to identify the leukemia-associated phenotype (LAIP) which was used to track residual leukemic cells during follow-up. Eight color flow cytometry analysis was performed at pre-defined TPs (TP1: post-induction I, TP2: post induction II/consolidation I, following TPs: post consolidation/pre-transplantation) (FACSCantoII; BD Facs Diva Software V6.1.3). A positive flow MRD was defined by the presence of no less than 10 clustered leukemic cells/10^4 total events. Enrollment closed on January 2023. Collection and analysis of later TPs is ongoing. Results: Sixty-seven patients from 11 centers were enrolled in the phase 2, part 2. Risk stratification according to ELN 2017 was intermediate in 46% of patients and high risk in 54%. Fifty-eight/67 patients (87%) obtained CR after induction I. In the centralized MRD analysis, 170 samples has been collected and analysed so far (60 baseline samples, 110 MRD samples). Seven patients lacking baseline sample for LAIP identification were excluded from the analysis. TP1 was available in 57/59 patient achieving CR (97%), and TP2 was available in 29 patients, so far. MFC-MRD negativity was obtained in 31/57 (54%) at TP1 (post V-FLAI, Fig. 1). An increase in MFC-MRD negativity rate was observed at TP2 (post Induction II or consolidation I) with 20 MFC-MRD negative patients/29 available samples (69%, Fig. 1). Thirteen/110 follow up samples (8%), albeit resulting MRD negative, were considered inadequate for the analysis due to haemodilution, 9 from TP1 and 4 from TP2 (Fig. 1). TP1 and TP2 MRD assessment were scheduled at day 28 of Induction I and Induction II/consolidation, respectively. However, in most cases, delayed haematological recovery was observed after V-FLAI, thus resulting in suboptimal samples for MRD evaluation. Conclusions: Preliminary results from centralized MRD analysis confirm that the combination therapy is able to induce high-quality remissions with a very high percentage of MFC-MRD negativity in a difficult cohort of patients, with a higher percentage of MFC-MRD-negative after the completion of the second course of therapy. Delayed haematological recovery may impact on reliability of MRD assessment due to hypocellular and regenerative marrow samples, suggesting that in those cases MRD analysis should be postponed. Correlation with survival will be performed as the data collection will be complete.

Fixed-Duration Pirtobrutinib Combined with Venetoclax ± Rituximab in Relapsed/Refractory Chronic Lymphocytic Leukemia: Updated Results, Including MRD Data, from the BRUIN Phase 1b Study

Background: Covalent (c) Bruton tyrosine kinase inhibitors (BTKi) have transformed the management of chronic lymphocytic leukemia (CLL). Recent clinical studies have evaluated the safety and efficacy of fixed-duration regimens with venetoclax and cBTKi. While these combinations are effective, their use may be limited by toxicity. Pirtobrutinib, a highly selective, non-covalent (reversible) BTKi, has shown promising safety and efficacy in heavily pretreated relapsed or refractory (R/R) CLL patients (pts). Here, we report the safety and efficacy of fixed-duration pirtobrutinib combined with venetoclax ± rituximab in pts with R/R CLL. Methods: Pts with R/R CLL were eligible for pirtobrutinib combined with venetoclax ± rituximab in the phase 1b portion of the multicenter phase 1/2 BRUIN study (NCT03740529). Prior cBTKi therapy was allowed, but not prior venetoclax. Pts were enrolled into sequential cohorts, first in the pirtobrutinib + venetoclax (PV) cohort and then the PV + rituximab (PVR) cohort. Pirtobrutinib 200 mg QD was started on cycle 1 day 1, and venetoclax was started on cycle 2 day 1 with the standard 5-week dose ramp to 400 mg QD target dose. PV was given for up to 24 cycles; each cycle was 28 days. For the PVR cohort, rituximab was given at 375 mg/m 2 on cycle 1 day 1 and then 500 mg/m 2 on day 1 of cycles 2-6. The primary endpoint was safety as assessed by treatment-emergent adverse events (TEAEs) graded according to CTCAE v5.0. Other key endpoints included overall response rate (ORR), progression-free survival (PFS), and minimal residual disease (MRD). Undetectable MRD (uMRD) was defined as less than 1 CLL cell/10,000 nucleated cells (<1x10 -4) in peripheral blood by ClonoSEQ® assay (Adaptive Biotechnologies, Seattle, WA). A data cut of May 5, 2023, was utilized. Results: Fifteen pts received PV, and 10 received PVR. Median age was 66 years (range, 49-77) for those who received PV and 69 (range, 39-78) for those who received PVR. Median prior lines of therapy was 1 (range, 1-2) for the PV cohort and 2 (range 1-4) for the PVR cohort. Most pts had received prior chemotherapy (PV=53%; PVR=60%), CD20 monoclonal antibody (73%; 70%), and cBTKi (73%; 60%). The majority of pts had IGHV unmutated CLL (PV=73%; PVR=89%). ORR was 93.3% (95% CI, 68.1-99.8) for the 15 pts receiving PV and 100% (95% CI, 69.2-100.0) for the 10 pts receiving PVR, with 10 complete responses (PV=7; PVR=3). Median duration of follow-up for PFS was 22.1 months (IQR, 20.1-25.7) for PV and 22.1 months (IQR, 20.7-22.1) for PVR; PFS at 18 months was 92.9% (95% CI, 59.1-99.0) for the 15 pts receiving PV and 80.0% (95% CI, 40.9-94.6) for the 10 pts receiving PVR. The overall uMRD rate at cycle 13 was 70.8% (PV=10; PVR=7) for the 24 evaluable pts, with 87.5% (PV=12; PVR=9) of pts achieving uMRD at some time during the trial and all but one pt sustaining uMRD during subsequent MRD assessments ( Figure). Twenty-two pts discontinued treatment (PV=14; PVR=8); of these, 14 completed all 24 cycles of therapy (PV=9; PVR=5) and 8 discontinued early because of progressive disease (PV=2), adverse events (PVR=2), protocol noncompliance (PV=1), death unrelated to treatment (PVR=1), or other reasons (PV=2). Three pts (PV=1; PVR=2) continue to receive treatment. The median relative dose intensity was similar in both cohorts (PV=97.9%; PVR=98.6%). The most common TEAE of any grade included nausea (PV=60.0%; PVR=40.0%), fatigue (53.3%; 50.0%), and diarrhea (46.7%; 60.0%). The most common grade ≥3 TEAE was neutropenia/neutrophil count decreased (PV=46.7%; PVR=60.0%). Grade ≥3 clinical tumor lysis syndrome occurred during venetoclax dose escalation (PV=2), including a grade 3 case that resolved spontaneously after 24 hours and a grade 4 case that resolved after short-term intravenous fluids. Treatment-related adverse events led to dose reductions in three pts (PV=1; PVR=2) and treatment discontinuation in two pts (PVR=2). Conclusions: Fixed-duration pirtobrutinib combined with venetoclax ± rituximab was well tolerated and demonstrated sufficiently promising efficacy to warrant further investigation in pts with R/R CLL. The BRUIN CLL-322 phase 3 trial comparing PVR vs VR in previously treated CLL is currently enrolling pts (NCT04965493).

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 <0.001) and OS (HR 0.39, 95% CI 0.33- 0.46, p <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 (<10 -4), intermediate (≥10 -4 and <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 >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.

Digital Polymerase Chain Reaction (dPCR) Versus Flow Cytometry (FC), a Validation Study for CAR-T Cell Product Optimal Detection

CONCLUSIONS Adoptive immunotherapy with Chimeric Antigen Receptor - T cells (CAR-T), has revolutionized hematological treatment mainly in relapsed/refractory (r/r) B-cell malignancies. CAR-T cells constitute challenging living drugs with heterogeneous in vivo cellular kinetics and adverse side effects that emphasizes the importance of its accurate detection and monitoring. Diagnostic assays are required for precise CAR-T quantification. Digital PCR (dPCR) is characterized by a high specificity, sensitivity and reproducibility, therefore we developed a study to implement this technique for CAR-T detection and compare its efficacy against Flow Cytometry (FC). Duplex dPCR assays were performed to concomitantly amplify CAR and RPP30 (reference gene) sequences using ddPCR Expert Design Assay: CD-19 CAR-T Assay (Bio-Rad, California, USA) with QIAcuity Digital PCR System and data analysis with QIAcuitySoftware Suite (QIAGEN, Germany). To assess sensitivity, a Axicabtagene Ciloleucel (Axi-Cel ®) original product obtained from leftovers's infusion bag patient was employed to perform dPCRs on serial dilutions using genomic DNA(gDNA) from a wild type (WT) patient. Specificity was ensured using WT and no template controls (NTC). Two independent dPCR assays with replicates for each sample were conducted to assess reproducibility. For FC comparative study, CAR-T cells percentage (%CAR-T) was determined by both techniques at variable post-infusion intervals (n=210) in different sample types, peripheral blood (n=205) and cerebrospinal fluid (n=5). Anti-FMC63 scFv Antibody, Mouse IgG1 (Acro biosystems) was employed, using FACSCanto II®, Becton-Dickinson Biosciences with FACSDiva software ® (V8.0; BD) and InfinicytTM software (Cytognos SL, Salamanca, Spain), for data analysis. Correlation coefficients were determined using two-tailed Pearson statistics with a confidence interval of 95%. Statistical analyses were made with SPSS Software (IBM, Germany). The dPCR data collected of 2 independent dPCR assays revealed a good split between the negative and positive partitions for the targets evaluated, showing a progressive decrease of the %CAR-T detected (63,88% - 4,12% - 0,77% - 0,08% - 0,01% - 0,00%). This reduction is correlated with the dilution factor applied to the series with an initial concentration of 50ng/µL both for CAR-T original product and WT gDNA. WT and NTC samples were negative with a SD=0; %CV=0, intra and inter-assays, evidencing thus the specificity of the technique. Positivity of the last (1 pg/µL) samples revealed a maximum sensitivity of 0.009 %CAR-T, threshold below which false positive results would be considered. WT gDNA did not influence the assay's sensitivity. 3 replicates of each sample and 2 independent dPCR assay were performed to study potential variations, exhibiting an excellent intra and inter-assay reproducibility with a minimal SD across dilutions ( Table.1.) Comparative study with FC revealed a light correlation between techniques with a Pearsons's correlation coefficient (R=0,553) ( p<0,001). Differences among both were enhanced for Minimal Residual Disease (MRD) evaluation from 60 days post-infusion monitoring (R=0,58) ( p=0,066) in which CAR-T cells can be detected applying dPCR up to 2 years and 9 months post-infusion, whereas being undetectable by FC. Complex CAR-T in vivo kinetics enhances the need for optimal diagnostic and monitoring techniques. dPCR is an ideal tool for precise CAR-T therapy evaluation with a higher grade of specificity, sensitivity and reproducibility than FC, mainly for MRD assessment. dPCR implementation and the establishment of standardized protocols across centers is required to improve CAR-T biology knowledge with a direct benefit on patient care.

Long-Term Outcomes in Chronic Lymphocytic Leukemia Treated with Ibrutinib: 10-Year Follow-up of a Phase 2 Study

Background: Targeted agents have replaced conventional chemotherapy in treatment of chronic lymphocytic leukemia (CLL) with substantially improved outcomes in CLL with TP53 alterations (del17p or TP53 mutations). Whereas fludarabine, cyclophosphamide, and rituximab chemoimmunotherapy (CIT) had a median progression-free survival (mPFS) of 11.2 months and median overall survival (mOS) of 33.1 months as frontline therapy in del17p CLL (Fisher et al. Blood 2016), we previously reported a 6-year PFS of 61% and OS of 79% in CLL with TP53 alterations receiving frontline continuous ibrutinib (Ahn et al. NEJM 2020). In a similar patient subset, the CLL14 study demonstrated a mPFS of 51.9 months and a 5-year OS of 60.0% using fixed-duration venetoclax and obinutuzumab (Al-Sawaf et al. EHA 2023; Al-Sawaf et al. Nat. Comm 2023). Although more effective than CIT, long-term outcomes in the era of targeted therapy are not yet fully defined. With a median follow-up of nearly 10 years, we analyzed efficacy, safety, and depth of response in CLL patients treated with long-term ibrutinib therapy in a phase 2 study (NCT01500733). Methods: Eligible patients had high-risk CLL, defined by TP53 alterations or age ≥65 years-old (N = 84 evaluable), and received ibrutinib 420 mg orally once daily until disease progression or intolerable side effects. Response assessments were performed annually with CT scans up to five years and subsequently with physical exam. Minimal residual disease (MRD) was performed annually with peripheral blood flow cytometry. Undetectable MRD (uMRD) was defined as <1 CLL cell per 10,000 leukocytes. For patients discontinuing the study, long-term outcomes were obtained when possible through a separate natural history study (NCT00923507). Results: As of December 31, 2022, a median follow-up of 113 months and 117 months (reverse Kaplan-Meier) was available for calculation of PFS and OS, respectively. The mPFS for all patients was 85.9 months (Table 1). mPFS was significantly shorter in those with TP53 alterations (67.3 months, N = 53) vs. those without (not reached, N = 31) and in relapsed disease (49 months, N = 32) vs. frontline treatment (108 months, N = 52). The OS at 117 months was 58.5% for all patients, 73.8% for treatment-naïve patients, and 54.1% in patients with TP53 alterations. Among patients with TP53 alterations receiving frontline ibrutinib (N = 34), the mPFS was 81 months with an OS of 69.7% at 117 months. Most patients (82.1%, 69/84) had discontinued ibrutinib at the time of analysis with 15 patients (17.9%) remaining on therapy. 37 (44.0%) patients discontinued ibrutinib for progressive disease including 6 cases with histologic transformation. 27 (32.1%) patients discontinued therapy related to treatment-emergent adverse events (AEs) including 8 cardiac AEs (four cases were grade ≥2 atrial fibrillation), 7 second cancers, 4 infections, 3 cases of dementia (ages 78, 80, and 85), 2 pleural effusions, and 3 other AEs. Five patients (5.9%) withdrew from the study with 4 switching to commercial supply of ibrutinib and one changing to CAR-T therapy. uMRD was observed in 15.4% (13/84) of all patients. The median time to uMRD was 5 years (range: 2 - 10 years). There was no enrichment in these patients for Rai stage ≥3-4, unmutated IGHV, TP53 alteration, prior treatment, or age. Most patients achieving uMRD also had a complete response (CR) to therapy (10/13) and remained progression-free at last follow-up (12/13). The one patient with progressive disease achieved uMRD and CR at 24 months, had detectable MRD at 36 months, and had nodal progression at 60 months. Among the progression-free patients, 8 had subsequent MRD assessments demonstrating sustained uMRD for 1-6 years including one patient who had been off any CLL-directed therapy for 4 years. Conclusions: These results further define the long-term prognosis of patients treated with BTK inhibitors for CLL, most notably with substantial improvements in the OS of patients with TP53 alterations over CIT. Additionally, these results may inform discussions of continuous BTK inhibitor therapy versus fixed-duration venetoclax combinations in frontline therapy of high-risk CLL. In a subset of ibrutinib-treated patients, depth of response increased over multiple years to the point of uMRD suggesting the possibility that such patients might safely discontinue therapy, following paradigms established in patients with chronic myelogenous leukemia.

Non-invasive Assessment of Pathological Complete Response and Minimal Residual Disease Through Plasma Methylomics in Patients With Triple Positive Breast Cancer

Non-invasive Assessment of Pathological Complete Response and Minimal Residual Disease Through Plasma Methylomics in Patients With Triple Positive Breast Cancer

Ibrutinib maintenance after frontline treatment in patients with mantle cell lymphoma

AbstractMaintenance rituximab in mantle cell lymphoma (MCL) has improved survival and supports exploration of maintenance with novel agents. We evaluated the safety and efficacy of ibrutinib maintenance (I-M) after induction in patients with treatment-naive MCL. Patients with MCL with complete response (CR) or partial response to frontline chemoimmunotherapy ± autologous stem cell transplantation (auto-SCT) received I-M 560 mg daily for up to 4 years. Primary objective was 3-year progression-free survival (PFS) rate from initiation of I-M. Minimal residual disease (MRD) assessments by next-generation sequencing (NGS) on peripheral blood were measured before I-M initiation and at 1, 6, and 18 to 24 months after initiation. Among 36 patients, the median age was 60 years (range, 46-90). For frontline treatment, 18 patients (50%) had consolidation with auto-SCT in CR1 before I-M. At median follow-up of 55.7 months, 17 patients (47%) completed full course I-M (median, 37.5 cycles; range, 2-52). The 3-year PFS and overall survival (OS) rates were 94% and 97%, respectively. With prior auto-SCT, 3-year PFS and OS rates were both 100%. The most common treatment–related adverse event with I-M was infection (n = 31; 86%), typically low grade; the most common grade 3/4 toxicities were hematologic. In 22 patients with MRD assessments, all were MRD negative after induction. Six became MRD positive on I-M, with 2 reverting to MRD-negative status with continued I-M, and all maintain radiographic CR with the exception of 1 with disease progression. I-M is feasible in MCL after frontline chemoimmunotherapy with manageable toxicities although significant. Changes in NGS-MRD were noted in limited patients during maintenance with few progression and survival events. This trial was registered at www.clinicaltrials.gov as #NCT02242097.

Minimal residual disease assessment in B-cell precursor acute lymphoblastic leukemia by semi-automated identification of normal hematopoietic cells: A EuroFlow study.

Presence of minimal residual disease (MRD), detected by flow cytometry, is an important prognostic biomarker in the management of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, data-analysis remains mainly expert-dependent. In this study, we designed and validated an Automated Gating & Identification (AGI) tool for MRD analysis in BCP-ALL patients using the two tubes of the EuroFlow 8-color MRD panel. The accuracy, repeatability, and reproducibility of the AGI tool was validated in a multicenter study using bone marrow follow-up samples from 174 BCP-ALL patients, stained with the EuroFlow BCP-ALL MRD panel. In these patients, MRD was assessed both by manual analysis and by AGI tool supported analysis. Comparison of MRD levels obtained between both approaches showed a concordance rate of 83%, with comparable concordances between MRD tubes (tube 1, 2 or both), treatment received (chemotherapy versus targeted therapy) and flow cytometers (FACSCanto versus FACSLyric). After review of discordant cases by additional experts, the concordance increased to 97%. Furthermore, the AGI tool showed excellent intra-expert concordance (100%) and good inter-expert concordance (90%). In addition to MRD levels, also percentages of normal cell populations showed excellent concordance between manual and AGI tool analysis. We conclude that the AGI tool may facilitate MRD analysis using the EuroFlow BCP-ALL MRD protocol and will contribute to a more standardized and objective MRD assessment. However, appropriate training is required for the correct analysis of MRD data.

POSTER: AML-053 Assessment of Minimal Residual Disease in Patients With Adult Acute Myeloid Leukemia: Single Egyptian Center Experience

POSTER: AML-053 Assessment of Minimal Residual Disease in Patients With Adult Acute Myeloid Leukemia: Single Egyptian Center Experience

AML-053 Assessment of Minimal Residual Disease in Patients With Adult Acute Myeloid Leukemia: Single Egyptian Center Experience

AML-053 Assessment of Minimal Residual Disease in Patients With Adult Acute Myeloid Leukemia: Single Egyptian Center Experience

Prospective use of molecular minimal residual disease for risk stratification in children and adolescents with acute lymphoblastic leukemia : Long-term results of the AIEOP-BFM ALL 2000 trial in Austria.

Since 1979 Austrian children and adolescents with acute lymphoblastic leukemia (ALL) have been treated according to protocols of the Berlin-Frankfurt-Münster (BFM) study group. The Associazione Italiana di Ematologia eOncologia Pediatrica and BFM (AIEOP-BFM) ALL 2000study was designed to prospectively study patient stratification into three risk groups using minimal residual disease (MRD) on two time points during the patient's early disease course. The MRD levels were monitored by detection of clone-specific rearrangements of the immunoglobulin and T‑cell receptor genes applying aquantitative polymerase chain reaction-based technique. The 7‑year event-free survival (EFS) and overall survival rates for all 608 Austrian patients treated between June 1999 and December 2009 within the AIEOP-BFM 2000study were 84 ± 2% and 91 ± 1%, respectively, with amedian observation time of 6.58years. Event-free survival for patients with precursor B‑cell and T‑cell ALL were 84 ± 2% (n = 521) and 84 ± 4% (n = 87; p = 0.460), respectively. The MRD assessment was feasible in 94% of the patients and allowed the definition of precursor B‑cell ALL patients with alow, intermediate or high risk of relapse even on top of clinically relevant subgroups.A similar finding with respect to MRD relevance in T‑ALL patients was not possible due to the small number of patients and events. Since this pivotal international AIEOP-BFM ALL 2000 trial, molecular response to treatment has been continuously used with additional refinements to stratify patients into different risk groups in all successive trials of the AIEOP-BFM ALL study group.

Immune-checkpoint inhibition for resectable non-small-cell lung cancer - opportunities and challenges.

Therapeutic strategies harnessing the immune system to eliminate tumour cells have been successfully used for several cancer types, including in patients with advanced-stage non-small-cell lung cancer (NSCLC). In these patients, immune-checkpoint inhibitors (ICIs) can provide durable responses and improve overall survival either as monotherapy, or combined with chemotherapy or other immunotherapeutic agents. However, the implementation of ICIs in early stage NSCLC has been hampered by the continuous struggle to develop robust end points to assess their efficacy in this setting, especially those enabling a fast and reproducible evaluation of the clinical activity of neoadjuvant strategies. Several trials are testing ICIs, alone or in combination with chemotherapy, in early stage NSCLC as an adjuvant, neoadjuvant or perioperative approach. As a novelty, most trials in the neoadjuvant setting have adopted pathological response as a primary end point. ICIs have been approved for use in the neoadjuvant and adjuvant settings on the basis of event-free survival and disease-free survival benefit, respectively; however, the correlation of these end points with overall survival remains unclear in these settings. Unresolved challenges for the optimal use of ICIs with curative intent include concerns about their applicability in daily clinical practice and about improving patient selection based on predictive biomarkers or assessment of pathological response and minimal residual disease. In this Review, we discuss the rationale, available strategies and current trial landscape for the implementation of ICIs in patients with resectable NSCLC, and we further elaborate on future approaches to optimize their clinical benefit.

Circulating tumor DNA analysis detects micrometastatic disease and predicts recurrence in a patient with colon cancer: A case report.

Colorectal cancer (CRC) is one of the most prevalent and deadly cancers worldwide, and approximately 50% of patients with early-stage disease develop metastases. A critical limitation for successful management of CRC is early disease detection and identification of progression. Next-generation sequencing-based circulating tumor DNA (ctDNA) profiling has emerged as a promising biomarker for the assessment of minimal or molecular residual disease in CRC. The patient was initially diagnosed with resectable CRC with uncertain small lung nodules. The patient was diagnosed with sigmoid colon adenocarcinoma placed at 15 to 20 cm above the anal verge (ypT4N1R0). Lung nodules were found in the apical part of the upper lobe of the right lung and the dorsal segment of the lower lobe of the left lung. The patient received systemic therapy and local treatment and plasma ctDNA-MRD detection was performed for monitoring the molecular disease status after surgery. The patient achieved a complete response after treatment. However, he presented with disease recurrence in liver lesions. The postoperative ctDNA detection suggested the possibility of micrometastatic pulmonary disease, and that was confirmed by follow-up examination. Serial ctDNA detection revealed disease relapse ahead of radiologic imaging by a lead time of 9 months. This case demonstrated the potential of ctDNA analysis to be a sensitive and specific tool for the detection of micrometastatic disease and prediction of recurrence.

Vidjil add-on for MRD quantification of samples processed using the EuroClonality-NGS protocol.

Assessment of minimal residual disease in acute lymphoblastic leukemia by immune repertoire NGS requires spiking CDR3 sequences at known quantities into the patient's sample. Recently, the EuroClonality-NGS group released one of the most comprehensive protocols for this purpose. ARResT/Interrogate is a closed-source software for processing these NGS libraries, developed by this same group. Vidjil, an open-source alternative, currently cannot handle libraries prepared using this protocol. Here, we present a Vidjil add-on to solve this issue. EuroClonality-NGS prepared samples analyzed with Vidjil and ARResT/Interrogate were highly concordant (r=0.998) and presented low error (root-mean-square error, RMSE=0.112).

Response-Adapted Therapy for Newly Diagnosed Multiple Myeloma.

The development of potent novel agents has improved outcomes for patients with multiple myeloma (MM). Heterogeneity of response to therapy, an expanding arsenal of treatment options, and cost are however major challenges for physicians making treatment decisions. Response-adapted therapy is hence an attractive strategy for sequencing of therapy in MM. Despite its successful application in other haematologic malignancies, response-adapted therapy is yet to become a standard of care for MM. We provide our perspective on response-adapted therapeutic strategies evaluated thus far and how they may be implemented and improved on in treatment algorithms of the future. While older studies suggested that early response based on International Myeloma Working Group response criteria could impact long-term outcomes, recent data have contradicted these findings. The advent of minimal residual disease (MRD) as a powerful prognostic factor in MM has raised the promise of MRD-adapted treatment strategies. The development of more sensitive techniques for paraprotein quantification as well as imaging modalities to detect extramedullary disease is likely to change response assessment in MM. These techniques combined with MRD assessment may provide sensitive and holistic response assessments which could be evaluated in clinical trials. Response-adapted treatment algorithms have the potential to allow an individualised treatment strategy, maximising efficacy, while minimising toxicities and cost. Standardisation of MRD methodology, incorporation of imaging into response assessment, and the optimal management of MRD positive patients are key questions to be addressed in future trials.

Circulating Tumor DNA as a Minimal Residual Disease Assessment and Recurrence Risk in Patients Undergoing Curative-Intent Resection with or without Adjuvant Chemotherapy in Colorectal Cancer: A Systematic Review and Meta-Analysis.

Emerging data have suggested that circulating tumor DNA (ctDNA) can be a reliable biomarker for minimal residual disease (MRD) in CRC patients. Recent studies have shown that the ability to detect MRD using ctDNA assay after curative-intent surgery will change how to assess the recurrence risk and patient selection for adjuvant chemotherapy. We performed a meta-analysis of post-operative ctDNA in stage I-IV (oligometastatic) CRC patients after curative-intent resection. We included 23 studies representing 3568 patients with evaluable ctDNA in CRC patient post-curative-intent surgery. Data were extracted from each study to perform a meta-analysis using RevMan 5.4. software. Subsequent subgroup analysis was performed for stages I-III and oligometastatic stage IV CRC patients. Results showed that the pooled hazard ratio (HR) for recurrence-free survival (RFS) in post-surgical ctDNA-positive versus -negative patients in all stages was 7.27 (95% CI 5.49-9.62), p < 0.00001. Subgroup analysis revealed pooled HRs of 8.14 (95% CI 5.60-11.82) and 4.83 (95% CI 3.64-6.39) for stages I-III and IV CRC, respectively. The pooled HR for RFS in post-adjuvant chemotherapy ctDNA-positive versus -negative patients in all stages was 10.59 (95% CI 5.59-20.06), p < 0.00001. Circulating tumor DNA (ctDNA) analysis has revolutionized non-invasive cancer diagnostics and monitoring, with two primary forms of analysis emerging: tumor-informed techniques and tumor-agnostic or tumor-naive techniques. Tumor-informed methods involve the initial identification of somatic mutations in tumor tissue, followed by the targeted sequencing of plasma DNA using a personalized assay. In contrast, the tumor-agnostic approach performs ctDNA analysis without prior knowledge of the patient's tumor tissue molecular profile. This review highlights the distinctive features and implications of each approach. Tumor-informed techniques enable the precise monitoring of known tumor-specific mutations, leveraging the sensitivity and specificity of ctDNA detection. Conversely, the tumor-agnostic approach allows for a broader genetic and epigenetic analysis, potentially revealing novel alterations and enhancing our understanding of tumor heterogeneity. Both approaches have significant implications for personalized medicine and improved patient outcomes in the field of oncology. The subgroup analysis based on the ctDNA method showed pooled HRs of 8.66 (95% CI 6.38-11.75) and 3.76 (95% CI 2.58-5.48) for tumor-informed and tumor-agnostic, respectively. Our analysis emphasizes that post-operative ctDNA is a strong prognostic marker of RFS. Based on our results, ctDNA can be a significant and independent predictor of RFS. This real-time assessment of treatment benefits using ctDNA can be used as a surrogate endpoint for the development of novel drugs in the adjuvant setting.

Association of tumor-informed minimal residual disease (MRD) with clinical outcomes for muscle invasive bladder cancer (MIBC): A multicenter retrospective real-world analysis.

3050 Background: Muscle invasive bladder cancer is associated with a significant elevated risk of mortality and morbidity due to micro metastatic spread at the time of diagnosis. Accurate biomarkers to guide treatment determination and evaluate response are needed. Previous studies using tumor informed ct-DNA have shown that MRD assessment may be informative in these scenarios. Methods: We conducted a retrospective multicenter analysis of real-world data obtained from commercial testing (Signatera; Natera, Inc.). This cohort of MIBC patients included those who underwent radical cystectomy with a minimum follow up of 90 days. Data were collected on patient demographics, clinical and pathological staging, histologic type, and treatments. Correlation of ct-DNA status with clinical outcomes was analyzed using Cox-regression analysis to evaluate utility of the assay for detecting residual disease post-surgery and radiological recurrence. Results: A total of 109 patients with 545 plasma samples were identified that fit the inclusion criteria. The majority of the patients [79, 72.5%] were male, with a median age of 70 (range: 32-88) years. Most patients had pure urothelial carcinoma [73.4%, 80/109]. Median follow-up was 13.63 (range: 3-89.04) months. On analyzing the first time point post-surgery (median 54 days), 91.55% (65/71) of ct-DNA negative patients at the first post-surgical test remained recurrence free while 65.79% (25/38) of positive patients had recurred at data cut-off. Therefore, ct-DNA positivity at the first post-surgical timepoint was associated with shorter disease-free survival (DFS) (HR=9.9, 95%CI: 4.1-24%, p&lt;0.001). ct-DNA detected imaging recurrence with a median lead time of 61 days (range: 12-381). Similarly, on analyzing ct-DNA positivity at any time post-surgery, of those who remained ct-DNA negative (N=63), 96.83% (61/63) showed no evidence of recurrent disease. On the other hand, 63.04% (29/46) of pts with ct-DNA positivity at any time after cystectomy recurred and experienced reduced DFS (HR=25, 95%CI: 5.9-103%, p&lt;0.001). The assay’s sensitivity for predicting radiological recurrence at any time post-surgery was 93.55%. Notably, in the multivariate analysis, anytime ct-DNA positivity after cystectomy was found to be the only significant risk factor associated with shorter DFS (HR=23.07, 95%CI: 5.1-104.4, p&lt;0.001). Conclusions: In this initial analysis of real-world data, MRD detection via tumor-informed ct-DNA testing was highly prognostic of patient outcomes post-surgery and may guide adjuvant treatment decisions. Future studies in this setting will explore the utility of ct-DNA testing as a predictive biomarker in response to systemic therapy.

Three years (yrs) maintenance with bortezomib, lenalidomide, and dexamethasone (RVD) in multiple myeloma (MM): Results of Total Therapy (TT) IIIB.

e20053 Background: Maintenance therapy with lenalidomide in MM is associated with improved overall survival (OS). Addition of proteasome inhibitory (PI) is usually suggested for patients (pts) with high-risk disease to overcome worse outcomes. We report long-term follow up data of TT IIIB in which initial results were reported after a median follow up of 2 years (yrs) ( Nair et.al. Blood 2010). Methods: TT IIIB (NCT00572169) used, for newly diagnosed MM, 2 cycles of VTD-PACE (bortezomib, thalidomide, and dexamethasone and 4-day continuous infusions of cisplatin, doxorubicin, cyclophosphamide, and etoposide) as induction before and consolidation therapy after melphalan-based tandem autologous transplantation (ASCT), which was followed by 3 yrs of RVD with monthly cycles of bortezomib 1.0 mg/m2 (D 1 4 8 11) in yr 1 followed by weekly administration in yrs 2 and 3; lenalidomide was administered at 15 mg on D 1-20 followed by 5 mg on D 21-28 for all 3 yrs; dexamethasone was given at 20 mg on D 1-4 and D8-11 in year 1 and was given weekly with bortezomib in yrs 2 and 3. Data cut-off was October 10th, 2022. Results: 177 pts enrolled between Nov 2006 and Sep 2008. Median age was 57 yrs with 26% of pts &gt; 65yrs. 39% females and 8% black pts. Gene expression profile (GEP) defined high-risk disease in 22% of pts. After median follow up of 14.2 yrs, median progression free survival (PFS) was 6.37 yrs (95%CI:5.36-10 yrs), 10-yr PFS was 42.4% (95%CI: 35.5%-50.5%) and 15-yr PFS was 34.6% (95%CI: 27.8%-43.2%). Median PFS was better in GEP low risk at 8.69 yrs (95%CI: 6.28-NR) and in ISS stage I at 11.04 yrs (95%CI: 6.37-NR). Median OS was 10.01 yrs (95%CI: 7.75-12.41 yrs) with 15-yr OS of 36.7% (95%CI: 29.6 %-45.6 %). Median OS was 11.14 yrs (95%CI:9.19-NR) and 2.79 yrs (95%CI:2.25-6.62 yrs) in GEP low-risk and high-risk, respectively.10-yr OS for GEP high risk was 21.6% (95% CI: 11.7- 39.9 %). Multivariable analysis showed worse OS outcomes with GEP high risk disease (hazard ratio (HR): 3.12 (95% CI: 2.01-4.82, p &lt; 0.001). When compared with TT IIIA that used 1 yr of bortezomib maintenance, no difference in OS was noted. Achieving minimal residual disease (MRD) negativity at any time during treatment and follow up even for pts who subsequently relapsed was associated with improved OS (HR 0.21 (95% CI 0.12-0.38, P &lt; 0.001). Average MRD assessment per pt was 9.36 times (range: 1-29). Secondary hematological malignancies occurred in 11 pts (6%). Conclusions: T IIIB resulted in further improvement of outcomes with more than one third of patients with OS &gt; 15 yrs. Use of 3 yrs of RVD as maintenance therapy in TT IIIB did not result in improvement of outcomes of high-risk MM patients (defined by GEP) when compared to 1 yr use of maintenance bortezomib and 3 yrs use of maintenance immunomodulatory drug (TT IIIA). High-risk MM remains unmet need despite the use of extended triplet-based maintenance therapy. Clinical trial information: NCT00572169 .

Identification of Immunoglobulin Gene Rearrangement Biomarkers in Multiple Myeloma through cfDNA-Based Liquid Biopsy Using tchDNA-Seq.

Multiple myeloma (MM) is a hematological malignancy characterized by the clonal proliferation of pathogenic CD138+ plasma cells (PPCs) in bone marrow (BM). Recent years have seen a significant increase in the treatment options for MM; however, most patients who achieve complete the response ultimately relapse. The earlier detection of tumor-related clonal DNA would thus be very beneficial for patients with MM and would enable timely therapeutic interventions to improve outcomes. Liquid biopsy of "cell-free DNA" (cfDNA) as a minimally invasive approach might be more effective than BM aspiration not only for the diagnosis but also for the detection of early recurrence. Most studies thus far have addressed the comparative quantification of patient-specific biomarkers in cfDNA with PPCs and BM samples, which have shown good correlations. However, there are limitations to this approach, such as the difficulty in obtaining enough circulating free tumor DNA to achieve sufficient sensitivity for the assessment of minimal residual disease. Herein, we summarize current data on methodologies to characterize MM, and we present evidence that targeted capture hybridization DNA sequencing (tchDNA-Seq) can provide robust biomarkers in cfDNA, including immunoglobulin (IG) rearrangements. We also show that detection can be improved by prior purification of the cfDNA. Overall, liquid biopsies of cfDNA to monitor IG rearrangements have the potential to provide important diagnostic, prognostic, and predictive information in patients with MM.