Measurable Residual Disease Assessment Research Articles

SWIGH-SCORE: A translational light-weight approach in computational detection of rearranged immunoglobulin heavy chain to be used in monoclonal lymphoproliferative disorders

We present a lightweight tool for clonotyping and measurable residual disease (MRD) assessment in monoclonal lymphoproliferative disorders. It is a translational method that enables computational detection of rearranged immunoglobulin heavy chain gene sequences.•The swigh-score clonotyping tool emphasizes parallelization and applicability across sequencing platforms.•The algorithm is based on an adaptation of the Smith-Waterman algorithm for local alignment of reads generated by 2nd and 3rd generation of sequencers.For method validation, we demonstrate the targeted sequences of immunoglobulin heavy chain genes from diagnostic bone marrow using serial dilutions of CD138+ plasma cells from a patient with multiple myeloma. Sequencing libraries from diagnostic samples were prepared for the three sequencing platforms, Ion S5 (Thermo Fisher Scientific), MiSeq (Illumina), and MinION (Oxford Nanopore), using the LymphoTrack assay. Basic quality filtering was performed, and a Smith-Waterman-based swigh-score algorithm was developed in shell and C for clonotyping and MRD assessment using FASTQ data files. Performance is demonstrated across the three different sequencing platforms.

Clonal relapse dynamics in acute myeloid leukemia following allogeneic hematopoietic cell transplantation

AbstractPatients with acute myeloid leukemia (AML) who experience relapse following allogeneic hematopoietic cell transplantation (alloHCT) face unfavorable outcomes regardless of the chosen relapse treatment. Early detection of relapse at the molecular level by measurable residual disease (MRD) assessment enables timely intervention, which may prevent hematological recurrence of the disease. It remains unclear whether molecular MRD assessment can detect MRD before impending relapse and, if so, how long in advance. This study elucidates the molecular architecture and kinetics preceding AML relapse by using error-corrected next-generation sequencing (NGS) in 74 patients with AML relapsing after alloHCT, evaluating 140 samples from peripheral blood collected 0.6 to 14 months before relapse. At least 1 MRD marker became detectable in 10%, 38%, and 64% of patients at 6, 3, and 1 month before relapse, respectively. By translating these proportions into monitoring intervals, 38% of relapses would have been detected through MRD monitoring every 3 months, whereas 64% of relapses would have been detected with monthly intervals. The relapse kinetics after alloHCT are influenced by the functional class of mutations and their stability during molecular progression. Notably, mutations in epigenetic modifier genes exhibited a higher prevalence of MRD positivity and greater stability before relapse, whereas mutations in signaling genes demonstrated a shorter lead time to relapse. Both DTA (DNMT3A, TET2, and ASXL1) and non-DTA mutations displayed similar relapse kinetics during the follow-up period after alloHCT. Our study sets a framework for MRD monitoring after alloHCT by NGS, supporting monthly monitoring from peripheral blood using all variants that are known from diagnosis.

Acalabrutinib, venetoclax, and obinutuzumab in relapsed/refractory CLL: final efficacy and ctDNA analysis of the CLL2-BAAG trial

AbstractThe phase 2 CLL2-BAAG trial tested the measurable residual disease (MRD)–guided triple combination of acalabrutinib, venetoclax, and obinutuzumab after optional bendamustine debulking in 45 patients with relapsed/refractory chronic lymphocytic leukemia (CLL; 1 patient was excluded from the analysis because of a violation of exclusion criteria). MRD was measured by flow cytometry (FCM; undetectable MRD <10–4) in peripheral blood (PB) and circulating tumor DNA (ctDNA) using digital droplet polymerase chain reaction of variable-diversity-joining (VDJ) rearrangements and CLL-related mutations in plasma. MRD recurrence was defined as detectable ctDNA and/or MRD ≥10–4 after achieving both undetectable MRD (uMRD) and undetectable ctDNA. The median number of previous treatments was 1 (range, 1-4); 18 patients (40%) had received a Bruton tyrosine kinase inhibitor (BTKi) and/or venetoclax before inclusion, 14 of 44 (31.8%) had TP53 aberrations, and 34 (75.6%) had unmutated IGHV. With a median observation time of 36.3 months and all patients off-treatment for a median of 21.9 months, uMRD <10–4 in PB was achieved in 42 of the 45 patients (93.3%) at any time point, including 17 of 18 (94.4%) previously exposed to venetoclax/BTKi and 13 of 14 (92.9%) with TP53 aberrations. The estimated 3-year progression-free and overall survival rates were 85.0% and 93.8%, respectively. Overall, 585 paired FCM/ctDNA samples were analyzed and 18 MRD recurrences (5 with and 13 without clinical progression) occurred after the end of treatment. Twelve samples were first detected by ctDNA, 3 by FCM, and 3 synchronously. Patients with earlier detection of ctDNA appeared to have a genetically higher risk of disease. In conclusion, time-limited MRD-guided acalabrutinib, venetoclax, and obinutuzumab achieved deep remissions in almost all patients with relapsed/refractory CLL. The addition of ctDNA-based analyses to FCM MRD assessment seems to improve early detection of relapses. This trial was registered at www.ClinicalTrials.gov #NCT03787264.

Circulating tumor plasma cells and peripheral blood measurable residual disease assessment in multiple myeloma patients not planned for upfront transplant.

Circulating tumor plasma cells (CTPCs) provide a noninvasive alternative for measuring tumor burden in newly diagnosed multiple myeloma (NDMM). Moreover, measurable residual disease (MRD) assessment in peripheral blood (PBMRD) can provide an ideal alternative to bone marrow MRD, which is limited by its painful nature and technical challenges. However, the clinical significance of PBMRD in NDMM still remains uncertain. Additionally, data on CTPC in NDMM patients not treated with transplant arescarce. We prospectively studied CTPC and PBMRD in 141 NDMM patients using highly sensitive multicolor flow cytometry (HS-MFC). PBMRD was monitored at the end of three cycles (PBMRD1) and six cycles (PBMRD2) of chemotherapy in patients with detectable baseline CTPC. Patients received bortezomib-based triplet therapy and were not planned for an upfront transplant. Among baseline risk factors, CTPC ≥ 0.01% was independently associated with poor progression-free survival (PFS) (hazard ratio [HR] = 2.77; p = 0.0047) and overall survival (OS) (HR = 2.9; p = 0.023) on multivariate analysis. In patients with detectable baseline CTPC, undetectable PBMRD at both subsequent time points was associated with longer PFS (HR = 0.46; p = 0.0037), whereas detectable PBMRD at any time point was associated with short OS (HR = 3.25; p = 0.004). Undetectable combined PBMRD (PBMRD1 and PBMRD2) outperformed the serum-immunofixation-based response. On multivariate analysis, detectable PBMRD at any time point was independently associated with poor PFS (HR = 2.0; p = 0.025) and OS (HR = 3.97; p = 0.013). Thus, our findings showed that CTPC and PBMRD assessment using HS-MFC provides a robust, noninvasive biomarker for NDMM patients not planned for an upfront transplant. Sequential PBMRD monitoring has great potential to improve the impact of the existing risk stratification and response assessment models.

Performance of a novel eight-color flow cytometry panel for measurable residual disease assessment of chronic lymphocytic leukemia.

Measurable residual disease (MRD) is an important prognostic indicator of chronic lymphocytic leukemia (CLL). Different flow cytometric panels have been developed for the MRD assessment of CLL in Western countries; however, the application of these panels in China remains largely unexplored. Owing to the requirements for high accuracy, reproducibility, and comparability of MRD assessment in China, we investigated the performance of a flow cytometric approach (CD45-ROR1 panel) to assess MRD in patients with CLL. The European Research Initiative on CLL (ERIC) eight-color panel was used as the "gold standard." The sensitivity, specificity, and concordance rate of the CD45-ROR1 panel in the MRD assessment of CLL were 100% (87/87), 88.5% (23/26), and 97.3% (110/113), respectively. Two of the three inconsistent samples were further verified using next-generation sequencing. In addition, the MRD results obtained from the CD45-ROR1 panel were positively associated with the ERIC eight-color panel results for MRD assessment (R = 0.98, p < 0.0001). MRD detection at low levels (≤1.0%) demonstrated a smaller difference between the two methods (bias, -0.11; 95% CI, -0.90 to 0.68) than that at high levels (>1%). In the reproducibility assessment, the bias was smaller at three data points (within 24, 48, and 72 h) in the CD45-ROR1 panel than in the ERIC eight-color panel. Moreover, MRD levels detected using the CD45-ROR1 panel for the same samples from different laboratories showed a strong statistical correlation (R = 0.99, p < 0.0001) with trivial interlaboratory variation (bias, 0.135; 95% CI, -0.439 to 0.709). In addition, the positivity rate of MRD in the bone marrow samples was higher than that in the peripheral blood samples. Collectively, this study demonstrated that the CD45-ROR1 panel is a reliable method for MRD assessment of CLL with high sensitivity, reproducibility, and reliability.

Assessment of Measurable Residual Disease on Day 28 of First Induction Chemotherapy in Acute Myeloid Leukaemia Patients

Background: Measurable residual disease (MRD) is an independent, strong, prognostic marker in acute myeloid leukaemia (AML) that is important for risk stratification and treatment planning. This study assessed the MRD on day-28 after first induction chemotherapy in AML patients. Objective: To assess measurable residual disease status on day 28 after the first induction chemotherapy in newly diagnosed acute myeloid leukaemia patients. Methodology: This cross-sectional study was conducted at the Department of Haematology, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka from April 2021 to March 2022 among a total of 25 newly diagnosed AML patients who were admitted and received induction chemotherapy with DA (7+3). On Day-28, bone marrow aspiration was done with standard procedure and first pull of 2ml bone marrow sample was sent to a laboratory for MRD evaluation by multiparameter flow cytometry. MRD was considered positive when value was ?0.1%. Results: On day 28 of post induction chemotherapy most of the patients (92%) achieved morphological remission and among them more than half (52.2%) were MRD positive. The presence of CD14 myeloid marker and baseline higher serum uric acid level was significant in MRD positive patients and cytarabine dose 150 mg/m2 was found significant in MRD negative patients. Conclusion: More than half of the Acute Myeloid Leukaemia patients, treated with standard induction chemotherapy and achieved morphological remission on day 28 of chemotherapy, had positive measurable residual disease assessed by multiparameter flow cytometry. Positive MRD indicates relatively poorer prognosis and may necessitate more cycles of induction followed by more intensive consolidation which may include haematopoietic stem cell transplantation.

Impact of hemodilution on flow cytometry based measurable residual disease assessment in acute myeloid leukemia

Measurable residual disease (MRD) measured in the bone marrow (BM) of acute myeloid leukemia (AML) patients after induction chemotherapy is an established prognostic factor. Hemodilution, stemming from peripheral blood (PB) mixing within BM during aspiration, can yield false-negative MRD results. We prospectively examined hemodilution by measuring MRD in BM aspirates obtained from three consecutive 2 mL pulls, along with PB samples. Our results demonstrated a significant decrease in MRD percentages between the first and second pulls (P = 0.025) and between the second and third pulls (P = 0.025), highlighting the impact of hemodilution. Initially, 39% of MRD levels (18/46 leukemia-associated immunophenotypes) exceeded the 0.1% cut-off, decreasing to 30% (14/46) in the third pull. Additionally, we assessed the performance of six published methods and parameters for distinguishing BM from PB samples, addressing or compensating for hemodilution. The most promising results relied on the percentages of CD16dim granulocytic population (scarce in BM) and CD117high mast cells (exclusive to BM). Our findings highlight the importance of estimating hemodilution in MRD assessment to qualify MRD results, particularly near the common 0.1% cut-off. To avoid false-negative results by hemodilution, it is essential to collect high-quality BM aspirations and preferably utilizing the initial pull for MRD testing.

Comparison of Measurable Residual Disease in Pediatric B-Lymphoblastic Leukemia Using Multiparametric Flow Cytometry and Next-Generation Sequencing.

Measurable residual disease (MRD) testing, a standard procedure in B-lymphoblastic leukemia (B-ALL) diagnostics, is assessed using multiparametric flow cytometry (MFC) and next-generation sequencing (NGS) analysis of immunoglobulin gene rearrangements. We evaluated the concordance between eight-color, two-tube MFC-MRD the LymphoTrack NGS-MRD assays using 139 follow-up samples from 54 pediatric patients with B-ALL. We also assessed the effect of hemodilution in MFC-MRD assays. The MRD-concordance rate was 79.9% (N=111), with 25 (18.0%) and 3 (2.2%) samples testing positive only by NGS-MRD (MFC-NGS+MRD) and MFC-MRD (MFC+NGS-MRD), respectively. We found a significant correlation in MRD values from total nucleated cells between the two methods (r=0.736 [0.647-0.806], P<0.001). The median MRD value of MFC-NGS+MRD samples was estimated to be 0.0012% (0.0001%-0.0263%) using the NGS-MRD assays. Notably, 14.3% of MFC-NGS+MRD samples showed NGS-MRD values below the limit of detection in the MFC-MRD assays. The percentages of hematogones detected in MFC-MRD assays significantly differed between the discordant and concordant cases (P<0.001). MFC and NGS-MRD assays showed relatively high concordance and correlation in MRD assessment, whereas the NGS-MRD assay detected MRD more frequently than the MFC-MRD assay in pediatric B-ALL. Evaluating the hematogone percentages can aid in assessing the impact of sample hemodilution.

Quantification of Measurable Residual Disease Detection by Next-Generation Sequencing–Based Clonality Testing in B-Cell and Plasma Cell Neoplasms

Next generation sequencing (NGS)-based measurable residual disease (MRD) monitoring in post-treatment settings can be crucial for relapse risk stratification in patients with B-cell and plasma cell neoplasms. Prior studies have focused on validation of various technical aspects of the MRD assays, but more studies are warranted to establish the performance characteristics and enable standardization and broad utilization in routine clinical practice. Here, we describe our approach and clinical experience with an NGS-based IGH MRD quantification assay, incorporating a spike-in calibrator for monitoring B-cell and plasma cell neoplasms based on their unique IGH rearrangement status. Comparison of MRD status (positive or undetectable) by NGS and flow cytometry (FC) assays showed high concordance (91%, 471/519 cases) and overall good linear correlation in MRD quantitation, particularly for chronic lymphocytic leukemia and B-lymphoblastic leukemia/lymphoma (R=0.85). Quantitative correlation was lower for plasma cell neoplasms, where underestimation by FC is a known limitation. No significant effects on sequencing efficiency by the spike-in calibrator were observed, with excellent inter- and intra-assay reproducibility within our laboratory, and in comparison to an external laboratory, using the same assay and protocols. Assays performed both at internal and external laboratories showed highly concordant MRD detection (100%) and quantitation (R=0.97). Overall, this NGS-based MRD assay showed highly reproducible results with quantitation that correlated well with FC MRD assessment, particularly for B-cell neoplasms.

Minimally Invasive Assessment of Measurable Residual Disease (MRD) in Multiple Myeloma (MM)

BACKGROUND MRD is an established test in MM clinical trials but not in routine practice. A hindrance to its wider adoption is invasive bone marrow (BM) aspirates. Thus, the possibility of assessing MRD using minimally invasive methods could be paramount to facilitate its transition from clinical trials to routine practice, but also to improve patients' (pts) quality of life and to generate more meaningful MRD results based on periodic assessments instead of limited time points. However, the methods developed for MRD evaluation in BM are less sensitive when used in peripheral blood (PB). AIM Investigate the complementarity and prognostic value of new multimodal minimally invasive MRD assessment in MM. METHODS This study included 243 transplant eligible and ineligible pts who were on maintenance or observation at the time of MRD assessment in PETHEMA/GEM clinical trials. In all cases, MRD was evaluated in PB using BloodFlow, which is a highly sensitive method (10 -7) that combines immunomagnetic enrichment with next-generation flow (NGF) cytometry. Periodic assessment of MRD in peripheral blood (PB) using BloodFlow was performed every six months. In 27 patients, MRD was further investigated in cfDNA using CloneSight, a highly sensitive (10 -4) next-generation sequencing method based on pt-specific mini-panels of multiplexed amplicons covering somatic mutations identified at diagnosis. In 169 of the 243 patients, MRD was analyzed in the serum using Quantitative Immunoprecipitation Mass Spectrometry (QIP-MS) with anti-IgG/A/M, total κ and λ beads. RESULTS BloodFlow was performed in a total of 867 PB samples, out of which 77 (9%) were MRD positive. The median number of CTCs/µL was 0.016 (range, 0.0003 - 0.29). In 506 of the 867 assessments, MRD was simultaneously analyzed in the BM using NGF. The concordance between BloodFlow in PB and NGF in BM was 78.9% (70.4% double negative and 8.5% double positive MRD results). The frequency of BloodFlow-/NGF+ and BloodFlow+/NGF- discordant assessments was 20.3% and 0.8%, respectively. Serial assessment of MRD in PB using BloodFlow showed that 221 of the 243 (91%) pts had sustained MRD negativity and 13 (5%) were persistently positive. MRD conversions from negative to positive were noted in 9 (4%) pts. The landmark median PFS from the latest MRD assessment was not reached (NR) vs 3 months in negative vs positive patients (HR: 0.09, P &amp;lt; .001). Of note, only 11 of the 221 (5%) MRD negative pts relapsed thus far. Of the 27 pts in whom MRD was simultaneously analyzed in cfDNA using CloneSight, five were MRD positive. The landmark median PFS from the latest MRD assessment was NR vs 5 months in negative vs positive pts (HR: 0.23, P = .057). The number of patients with BloodFlow-/CloneSight-, BloodFlow+/CloneSight-, BloodFlow-/CloneSight+ and BloodFlow+/CloneSight+ results were 17, 5, 2 and 3. The landmark median PFS from the latest MRD assessment in patients with double negative MRD vs positive by either method was NR vs 8 months (HR: 0.20, P = .056). Of note, only 2 of the 17 double negative MRD patients relapsed thus far. Of the 169 pts in whom MRD was simultaneously analyzed in serum using QIP-MS, 37 (22%) were MRD positive. The landmark median PFS from the latest MRD assessment was NR in negative and positive patients (HR: 0.13, P &amp;lt; .001). The number of patients with BloodFlow-/QIPMS-, BloodFlow+/QIPMS-, BloodFlow-/QIPMS+ and BloodFlow+/QIPMS+ results were 130, 2, 26 and 11. The landmark median PFS from the latest MRD assessment in patients with double negative MRD vs positive by either method was NR and 8 months, respectively (HR: 0.07, P &amp;lt; .001). Of note, only 3 of the 130 (2%) double negative MRD patients relapsed thus far. The negative predictive value (NPV) of a double negative MRD detection in PB/serum using BloodFlow/QIP-MS and MRD negativity in BM using NGF was 86%. CONCLUSIONS This is the first study of multimodal minimally invasive MRD assessment in MM. Our results show that BloodFlow, CloneSight and QIP-MS are empowered to detect MRD with high sensitivity in PB, cfDNA and serum. The presence of CTCs, mutations and M-component was systematically associated with dismal PFS. The complementarity between these methods achieved a high NPV of MRD negativity in BM, and enabled the identification of multimodal MRD negative pts with very low risk of relapse. Thus, this study paves the way towards minimally invasive MRD assessment in MM pts on maintenance or observation.

Prospective Evaluation of Measurable Residual Disease (MRD) and Sustainability of the Response in Patients with Systemic AL Amyloidosis Treated with Daratumumab

Background: Daratumumab can provide deep hematologic responses for patients with light chain (AL) amyloidosis, both through its direct anti-clonal activity and immunomodulatory effects on other cell populations in the bone marrow (BM) microenvironment. Promising efficacy in the frontline setting when combined with CyBorD (per the ANDROMEDA trial), as well as in the relapsed/refractory setting as monotherapy, has led to daratumumab becoming the first FDA-approved therapy for AL amyloidosis. Though it is administered for up to 24 cycles, the optimal duration of treatment is unclear. Dynamic monitoring of measurable residual disease (MRD) may provide insights into changes in the aberrant plasma cell (PC) clone and sustainability of the hematologic response in relation to daratumumab therapy. Aims: We sought to evaluate the rates of MRD clearance and resurgence both during and after daratumumab therapy for AL amyloidosis. Methods: MRD status was assessed for patients followed at the Boston University Amyloidosis Center, who were treated with a daratumumab-based regimen and achieved a hematologic very good partial response (hemVGPR) or complete response (hemCR) according to consensus criteria (Palladini, 2012). MRD assessments were performed by multiparametric 10-color flow cytometry of BM aspirates (sensitivity level of ≤10 -5) and repeated at 12-month intervals during follow-up. Clonal progression was defined as growth in the aberrant PC clone size of &amp;gt;1-log. Results: Between 2019 and 2023, a total of 66 patients achieved ≥hemVGPR and were tested for MRD; 43 (65%) received daratumumab monotherapy in the relapsed/refractory setting and 23 (35%) in combination with CyBorD as frontline therapy. Collectively, 48 (73%) achieved a hemCR and 18 (27%) a hemVGPR. The median age at diagnosis was 60 years (range 35-78); 19 (29%) patients were female; and 11 (17%) identified as non-white race. MRD assessment was performed once, twice or ≥3 times for 33, 18 and 15 patients, respectively. The first MRD evaluation occurred while still receiving daratumumab for 33 patients (at median 12 cycles, range 6-23); the remaining patients had their first MRD evaluation after therapy completion (median one month after, range 0-25). Of the entire cohort, 28 (42%) patients achieved MRD negativity and 38 (58%) MRD positivity (median estimated aberrant PC clone size 1.5 x 10 -4, range 1.4 x 10 -5 to 1.1 x 10 -2, representing 46.9% ± 32.8% of total PCs) at first evaluation. All but one patient with a hemVGPR had detectable MRD. Of the 23 patients who received frontline daratumumab-CyBorD (18 hemCR and 5 hemVGPR), 8 (35%) achieved MRD negativity at first evaluation; whereas 20 (47%) of the 43 patients who received daratumumab as monotherapy (30 hemCR and 13 hemVGPR) achieved MRD negativity. We surveyed MRD profiles based on the number of daratumumab treatment cycles received (Table 1) and found no apparent patterns with increasing cycles. Serial MRD monitoring was performed for 33 patients (Figure 1). Of 10 patients who continued to receive daratumumab between MRD assessments (group 1), 6 had sustained MRD negativity; 2 had persistent MRD positivity (of whom one had clonal progression); and 2 had MRD clearance (conversion from positive to negative). Of 23 patients who underwent serial MRD assessments after completing daratumumab (group 2), 9 (39%) had sustained MRD negativity; 10 (43%) had persistent MRD positivity; 2 (9%) had MRD clearance; and 2 (9%) had MRD resurgence (conversion from negative to positive), which occurred 17 and 26 months after daratumumab cessation. Clonal progression was observed in 5 of the 10 patients with persistent positivity, of whom 3 experienced a hematologic relapse. Conclusions: Our findings show that patients with AL amyloidosis can attain deep responses with daratumumab-based treatments, even to the depth of undetectable MRD. Clearance of MRD was frequent not only during treatment with daratumumab but also after completion of therapy, suggesting continuing effects on the plasma cell clone. Clonal progression after daratumumab completion was often associated with hematologic relapse. Prospective studies are needed to investigate whether daratumumab may be stopped early for those with undetectable MRD, or whether those with persistent detectable disease may benefit from extending daratumumab treatment beyond 24 cycles.

Reshaping MRD Detection Strategies: Next-Generation Sequencing and High DNA Input Identify Previously Missed Measurable Residual Disease in Peripheral Blood of B-Cell Precursor Acute Lmyphoblastic Leukemia

Background: In acute lymphoblastic leukemia (ALL), measurable residual disease (MRD) is the most important prognostic factor. MRD is traditionally quantified in bone marrow aspirates (BM) using real-time quantitative polymerase chain reaction (RQ-PCR) according to EuroMRD standards. Since the emergence of modern molecular methods, it has been a continuous effort to enable MRD assessment from peripheral blood (PB). The less invasive material collection would allow closer MRD monitoring and solve technical challenges of BM aspiration, such as skewed or non-representative MRD values due to hemodilution and unequal distribution of leukemic cells. While several studies demonstrated comparable MRD values of BM and PB in T-ALL, in B-cell precursor ALL (BCP-ALL) MRD determination from PB is still a challenge. Mean MRD levels in PB are considerably lower than in BM and frequently escape detection. Additionally, paired samples show a highly variable MRD level ratio in both pediatric and adult cases (Kotrova et al, Leukemia 2020). Objective: BCP-ALL cases with known MRD positivity in the BM likely also harbor MRD positive cells in the PB, even when missed by traditional methods. With more sensitive assays, either by increased DNA input or by application of amplicon-based next generation sequencing (NGS), MRD detection from PB may improve in these cases. Methods: We retrospectively selected PB - BM sample pairs of 74 BCP-ALL patients with BM MRD positivity (43 with quantifiable MRD positivity and 31 with positivity below quantitative range) and sufficient leftover DNA from a cohort of patients treated according to GMALL protocols, where conventional RQ-PCR of clonal immunoglobuline heavy chain (IGH) VJ/DJ gene rearrangements failed to detect PB MRD. MRD had been determined by standard RQ-PCR with 1.5 µg DNA in BM and PB, respectively, according to the EuroMRD guidelines (van der Velden et al, Leukemia 2007). PB samples were re-tested for MRD with high DNA input RQ-PCR using 6.6 µg DNA and amplicon-based NGS, each using 6.6 µg DNA, as well as standard input NGS. NGS analyses of corresponding PB and BM samples were performed with the EuroClonality amplicon-based NGS assay (Brüggemann et al, Leukemia 2019). Identification and quantification of leukemia-derived clonotypes was done using ARResT/Interrogate. Results: In total, 74 BCP-ALL patients with pre/c-B-ALL (n=60) and pro-B-ALL (n=14) were included. Patient age ranged from 19 - 71 years with a median of 44.5. In all included samples (56 in first-line therapy and 18 salvage treatment), BM was MRD positive and PB was MRD negative in standard RQ-PCR at a sensitivity of at least 10 -4. To assess the effect of high DNA input alone, RQ-PCR was repeated in PB with high DNA input for 59 patients and 63 IGH VJ/DJ markers. At least one positive signal was detected in 19/59 patients (32%) and 20/63 markers (32%) (FIGURE 1A). NGS was performed for 73 PB samples and 84 markers using standard and high DNA input. With standard DNA input, MRD could be detected for 19/73 patients (26%) and 21/84 markers (25%) (FIGURE 1A). In the high input approach, MRD was detected in 36/73 patients (49%) and 40/84 markers (48%) (FIGURE 1A). A quantifiable MRD value was identified in 17 patients and 19 markers. All markers with quantifiable NGS MRD positivity in PB showed higher MRD values in the corresponding BM sample and MRD values below 10 -4 (FIGURE 1B). No correlation of MRD in paired samples could be demonstrated (Pearson correlation coefficient: 0.09) (FIGURE 1B). Conclusion: In BCP-ALL, MRD detection from PB is challenging with conventional methods. Increased DNA input and amplicon-based NGS allowed detection of MRD in a subset of PB samples that were negative in standard RQ-PCR. With identical DNA input, NGS showed a higher sensitivity than RQ-PCR and best results were achieved when high DNA input and NGS were combined, identifying MRD in nearly half of the PB samples. These results demonstrate that PB can be a valuable tool for early MRD detection in BCP-ALL when adjusting traditional MRD assessment strategies. However, in half of the samples, MRD was still missed by high input NGS. Even with assays improved for sensitivity, PB therefore cannot fully replace BM for MRD assessment in BCP-ALL yet and should rather be considered to complement BM analyses or in cases where BM is not accessible.

Impact of Pre-Transplant Molecular and Cytogenetic Remission on Outcomes of Allogeneic Stem Cell Transplant in Patients with Myelodysplastic Syndrome

Background: Allogeneic stem cell transplant (allo-SCT) carries a high relapse rate (~35% within 2 years) in patients (pts) with myelodysplastic syndrome (MDS). Pre-transplant measurable residual disease (MRD) increases the risk of relapse and death in acute myeloid leukemia (Thol et al, Blood 2018). However, the impact of pre-transplant MRD on post-transplant outcomes is less clear in MDS. Prior studies have compared outcomes between myeloablative (MAC) and reduced intensity conditioning (RIC) in pts with pre-transplant MRD persistence based on either cytogenetic remission alone or ultra-deep genomic sequencing with a limited 10-gene panel, suggesting reduced risk of relapse in pts receiving MAC (Festuccia et al, Biol Blood Marrow Transplant 2016; Dillon et al, JCO PO 2021). However, pre-transplant MRD assessment using a commercial next-generation sequencing (NGS) panel in addition to cytogenetic testing to assess post-transplant outcomes has not been evaluated in MDS. Methods: We conducted a multicenter retrospective review of MDS pts who underwent allo-SCT from 2015-2022. Pts with FISH/cytogenetic (FC) and/or molecular abnormalities at diagnosis and MRD assessment by commercial myeloid-panel NGS (with a variant-allele frequency detection limit up to 5%) and FC performed on peripheral blood (PB) or bone marrow (BM) aspirate within 3 months prior to transplant were included for analysis. The Kaplan-Meier method and log-rank tests were used to estimate overall survival (OS). Gray's test/cumulative incidence functions were used to estimate relapse-free survival (RFS) with non-relapse mortality (NRM) as competing risk. Results: Table 1 includes baseline pt characteristics. Of 81 pts, 69 pts (85.2%) had residual FC and/or molecular abnormalities (MRD+) at pre-transplant PB/BM assessment. Twelve (14.8%) pts were in both FC and molecular remission (MRD-) prior to transplant. With a median follow-up of 13.9 months [IQR 6.8 - 38.9], 43 (53%) died and 24 (30%) pts experienced a relapse. Twenty-three of 69 (33%) MRD+ pts had relapsed disease with a 38% cumulative incidence of relapse (CIR) at 2 years. In contrast, only 1/12 (8.3%) MRD- pts experienced a relapse after 7 years, with chromosomal loss of TP53 not present at initial diagnosis. CIR at 2 years was 33% and 41% (p=0.207) for MRD+ pts who received MAC vs RIC, respectively with NRM of 34.5% associated with MAC vs 28.2% for RIC (p=0.61). TP53 mutations were most frequently associated with relapse in 8/24 (33%) pts with a median OS of 9.8 months [8.2 - NR]. Besides the previously defined 10-gene panel (Dillon et al, JCO PO 2021), relapses were also driven by SRSF2, U2AF1, KRAS, SETBP1 and NF1 mutations present in the original clone. Six pts had DNMT3A, TET2 and/or ASXL1 (DTA) mutations only on pre-transplant NGS, with 2/6 pts relapsing without the re-emergence of DTA mutations. Median OS was not reached for MRD- pts and was 14.1 months [11.35-NR] for MRD+ pts, p = 0.061 [Fig.1]. OS probability at 2 years was 35% for MRD+ pts vs 75% for MRD- pts. Type of conditioning regimen, RIC vs MAC, did not impact OS (p= 0.6). An equal number of deaths, 35% each, were attributable to GvHD and relapse, respectively. Thirteen (30%) pts died from other causes, primarily infections and bleeding complications. NRM was 25% and 36% for MRD- and MRD+ cohorts, respectively. Pts received a median of 6 cycles [range, 2-36] of hypomethylating agent (HMA) +/- venetoclax prior to transplant with no association between the number of cycles and MRD status (p=0.80). The incidence of grade 3-4 acute GvHD and chronic GvHD requiring systemic therapy did not significantly differ, at 25% and 18.8% (p= 0.696) and 33.3% and 31.8% (p= 1.00) in MRD+ and MRD- pts, respectively. Conclusions: MRD assessment using commercially available NGS panels and cytogenetic testing that are routinely performed in MDS pts in real-world practice can predict risk of relapse. Pre-transplant MRD is associated with worse OS irrespective of the intensity of conditioning regimen. Although limited in statistical power due to a small sample size of MRD- pts in our cohort, further studies with a larger cohort are underway to better clarify the role of MRD on transplant outcomes in MDS. Our data also highlights that only a small minority of MDS pts (&amp;lt;15%) achieve complete molecular and cytogenetic remission at the time of transplant; strategies to eliminate the pre-transplant MDS clone are urgently needed.

Real-World Experience with Selinexor -Containing Regimens in Post-Transplant AML/MDS Patients Who MRD Still Positive after First-Line Preemptive Treatment

Background: Hematopoietic stem cell transplantation (HSCT) is the only cure for acute myeloid leukemia (AML), but relapse is the most important cause of transplant failure. Patients with remission but measurable residual disease (MRD) positive have a higher probability of hematologic relapse. The MRD-directed preemptive treatments can spare some patients in remission from further therapy. The conventional first-line preemptive treatment has been ineffective in some patients, so it's necessary to find a new preemptive treatment to prevent relapse and improve the prognosis. Our study aimed to evaluate the efficacy and safety of selinexor-containing regimens in the preemptive treatment of MRD positive in post-transplant patients with AML or myelodysplastic syndrome (MDS). Methods: The study was conducted in Tongji Hospital from March 2022 to May 2023. The inclusion criteria of post-transplant AML/MDS patients included the followings: (a) Persistent MRD positive. (b) MRD turned negative after first-line preemptive treatment but was positive again. (c) Patients at high risk for hematologic relapse after transplantation; (d) AML-M4/5. The end of the follow-up was in June 2023. Bone marrow aspiration was assessed for MRD using multiparameter flow cytometry (MFC) combined with real-time quantitative polymerase chain reaction (RT-qPCR) as follows: 1) MRD assessed by MFC: MRD negativity was defined as &amp;lt;0.1%. 2) MRD assessed by RT-qPCR: Abnormal genes associated with prognosis were used for molecular MRD detection. 3) WT1 was used for molecular MRD assessment if no molecular marker was available at diagnosis. MRD negativity was defined as &amp;lt;0.6%. Results: A total of 11 patients were included in the study. There were 10 patients with AML and one patient with MDS, with a median age of 43 years old. Two patients had received autologous HSCT, nine patients had received allogeneic HSCT, of which two matched sibling donor, one matched unrelated donor, and six were haploidentical donor. All patients were WT1 positive and two of the cases were both WT1 and MFC positive. Seven patients with post-transplant MRD positive were treated with selinexor-containing regimens after first-line preemptive therapy (azacytidine, azacytidine combined with venetoclax, decitabine combined with venetoclax, etc.) failed or MRD positive again. The remaining four patients were treated directly with selinexor-containing regimens for personal reasons or with a diagnosis of AML-M4/5 may be resistant to venetoclax. The selinexor-containing regimens used in this study were either selinexor (35mg/m 2 biw) alone or in combination with other drugs (decitabine, azacytidine, venetoclax, low-dose cytarabine, and CAG (cytarabine, aclarubicin, G-CSF) regimen). MRD was evaluated after two weeks of the selinexor-containing regimens. And selinexor 20 to 40 mg per week was used as maintenance therapy after MRD converted to negative. Eight patients (72.7%) successfully cleared the MRD. Of the three patients who didn't clear the MRD, two patients successfully converted to negative after continuing the selinexor-containing regimens. It is worth mentioning that 7 of the 11 patients were treated with the selinexor-containing regimens after first-linepreemptive treatment failure, and six of them had successfully cleared the MRD (71.4%). At the end of follow-up, 10 (90.9%) patients survived and one (9.1%) died due to disease progression. In terms of hematological adverse events (AEs), grade 3-4 neutropenia occurred in nine (81.8%) patients, the median duration was 2 days (IQR 1,4). Grade 3-4 thrombocytopenia occurred in three (27.3%) patients, and the median duration was 5 days (range 4-7). Although the number of people with grade 3-4 neutropenia was large, the duration was short after using granulocyte colony-stimulating factor, and no patient developed a severe infection. Most of the non-hematological AEs were mild, including nausea (100.0%), fatigue (63.6%), hyponatremia (58.3%), and vomiting (45.5%). The AEs can be alleviated after symptomatic treatment and no patient discontinued treatment. Conclusion: Selinexor-containing regimens have been shown to be effective and well tolerated in post-transplant AML/MDS patients with MRD-positive, including those who have failed first-line preemptive treatment.

Myelodysplasia-Related Gene Mutations for NGS-Based MRD Assessment in Acute Myeloid Leukemia

Introduction: Measurable residual disease (MRD) plays an increasing role for treatment management of AML patients. While next-generation-sequencing (NGS) based MRD assessment can be well standardized, there is uncertainty about the type of gene mutations that associate with prognosis and are useful for MRD assessment. The 2022 ELN recommendations assign patients with at least one mutation in the genes ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1 or ZRSR2 to the subgroup of myelodysplasia-related gene mutations (MRGM), which is associated with poor prognosis. We aimed to define the prognostic effect of MRD after induction chemotherapy in AML patients with MRGM using MRG mutations for MRD assessment by NGS. Methods: We retrospectively collected bone marrow and peripheral blood samples from AML patients in complete remission (CR) or CR with incomplete hematologic recovery (CRi) after one to two cycles of 7+3-based standard induction chemotherapy (IC) with at least one MRGM detected at diagnosis by myeloid panel sequencing covering 48 AML associated genes. Patients treated with one course of IC were included if they proceeded to allogeneic hematopoietic cell transplantation (alloHCT) already after one cycle. Molecular MRD status was assessed using targeted NGS and bioinformatics error-correction with 0.01% sensitivity as described before using only MRGM as MRD markers. Results: MRD was assessed on 104 adult newly-diagnosed AML patients (median age 58.6) with a total of 166 MRGM, resulting in a median of one MRGM per patient (range 1-4). 43 (41.3%) patients had secondary or therapy-related AML and 20 (19.2%) had myelodysplasia-related cytogenetic abnormalities. 68 (65.4%) patients were MRD positive for at least one MRGM after one (18 patients (17.3%)) or two (86 patients (82.7%)) cycles of intensive chemotherapy, while 36 (34.6%) became MRD negative. Measured variant allele frequencies (VAF) of positive samples ranged from 0.02-43.1% (median VAF 1.34%).MRD positive patients were older and more likely to be male compared to MRD negative patients, while cytogenetic risk, WBC count, Hgb, platelet count and ECOG status at diagnosis were similar to MRD negative patients. 53 (77.9%) MRD positive and 23 (63.9%) MRD negative patients underwent alloHCT in first CR/CRi.Median follow-up was 3.35 years. OS (P = 0.92), RFS (P = 0.99) and CIR (P = 0.98) were similar in MRD positive (at least one MRGM detectable) and negative (no MRGM detectable) patients (Figure 1). OS, RFS and CIR were also similar between MRD positive and negative patients, when MRD negativity was defined by conversion of at least one MRGM to negativity (55 (52.9%) MRD positive, 49 (47.1%) MRD negative), and when ASXL1 was excluded from the analysis. As variants with VAF &amp;gt;5% in CR/CRi samples may indicate clonal hematopoiesis, we excluded all variants with VAF &amp;gt;5% and repeated the analysis with the 88 (84.6%) remaining patients. Again, OS, RFS and CIR were similar for MRD positive and negative patients. We next reasoned that reduction of VAF correlates with the sensitivity of leukemic cells and possibly with depth of remission and long-term outcome. The fold-reduction of VAFs from diagnosis to the time of MRD analysis was calculated for all variants. For patients with several MRGMs, the variant with the largest fold-reduction was considered including undetectable variants. Patients with more or less than a 2-log, 3-log or 4-log reduction (log 10) had similar OS, RFS and CIR, respectively. Evaluating each MRGM individually, only RUNX1-MRD associated with RFS and by trend CIR (comparing MRD positive to negative patients for OS, HR 3.41, 95% CI 0.43-26.82, P = 0.24; RFS, HR 4.37, 95% CI 1.07-17.79, P = 0.04; CIR, HR 3.30, 95% CI 0.79-13.82, P = 0.1). Conclusion: 65.4% of AML-MRGM patients in CR/CRi remained MRD positive when MRGMs were used for MRD assessment by NGS. MRD positivity was associated with age above 60 years and male sex.Using all MRG mutations for MRD assessment at a threshold of 0.01% or by various degrees of log-reduction was not predictive of outcome after intensive induction chemotherapy. This limits the number of potential MRD markers for NGS analysis before alloHCT, although individual markers like RUNX1 may be of prognostic value.

Outcome of Risk- and Response-Adapted Post-Remission Therapy in Patients with NPM1 AML Treated Intensively

Introduction: Molecular assessment of measurable residual disease (MRD) is a powerful prognostic tool to identify high risk of relapse. Specifically, large studies have shown that MRD negativity of NPM1 after two cycles of intensive chemotherapy (IC) predicts long term remission. The translation of MRD results to treatment decisions such as treatment augmentation (MRD erasing) and referral to an allogeneic stem cell transplant (allo-SCT) is still lacking. The purpose of this study is to report the outcome of AML patients with NPM1 mutation who received IC and achieved remission, in whom NPM1 MRD post cycle 2 of IC, combined with FLT3-ITD status, were used systematically to decide whether patients were to complete intensive consolidations or refer to alloSCT. Methods: This is a retrospective analysis from 3 large hospitals in Israel. Patients were treated with 7+3 for induction and a repeat induction based on day 14 marrow. Midostaurin was added for FLT3mut leukemia since 2018. Consolidation consisted of 3-4 HiDAC cycles at doses of 1-3 g/msqrX6. Gemtuzumab ozogamicin and maintenance with FLT3 inhibitors or CC486 could be added. NPM1 transcript levels (normalized to ABL copies) were collected at diagnosis, after 2 cycles of IC, at the end of IC cycles and during follow-up. Patients were transplanted if a donor was available and any of the following: NPM1 transcript level was not reduced by 3 logs by the end of second cycle chemotherapy, absolute level was &amp;gt;1%, poor risk AML (ELN 2017 guidelines) or high AR FLT3-ITD mutation was present. Patients were followed for relapse (type and timing) and whether a second remission was achieved, as well as for EFS and overall survival. Results: A database search yielded 90 patients with NPM1 AML who were treated according the abovementioned protocol between 5/2015 and 11/2022 and achieved remission, with a median follow-up of 872 days (range 103-2751). Forty-seven patients were to receive consolidation cycles without transplantation in CR1, based on the aforementioned criteria. All but one patient (death from sepsis) completed treatment cycles. During follow-up, 21 (45%) patients relapsed at a median of 343 days. Two patients died while in CR1 during follow-up (extensive burns; sudden death at home). In a subgroup analysis of patients &amp;lt;60y (N=28), there were 11 events (9 relapses and 2 deaths), whereas in patients &amp;gt;60y (N=19) there were 13 events (12 relapses and 1 death during follow-up). Of note, 4 patients &amp;gt;60y were treated with CC486, of whom only 1 relapsed. Of the 18 patients who relapsed and received further treatment (3 had palliative care only), only 1 patient did not enter CR2 (died during salvage). The other 17 patients entered CR2 after salvage regimen (N=7, 4 were qPCR MRDneg, 5 were subsequently transplanted in CR2) or after upfront alloSCT without any salvage regimen (N=10, 4 were qPCR MRDneg). Forty-three patients were planned for alloSCT at CR1 (27 - inadequate molecular response, 16 - ELN risk group). Only one of these patients failed to undergo alloSCT in CR1 as she relapsed. The other 42 patients underwent alloSCT at CR1. In this group of patients, the median progression-free survival was 5 years, with no difference in patients above or below the age of 60. One half of the events (9/18) were relapses and the other half were deaths in remission, almost all were GVHD associated (figure1). Discussion: NPM1 mut AML without FLT3 is still considered by the ELN to have favorable outcome and most patients are not being transplanted at CR1. Yet, many relapse. This study evaluated the outcome of AML patients according to molecular risk stratification and alloSCT consideration by the end of cycle 2 of IC. In line with previous publications, younger patients with standard-risk AML and with adequate molecular response had a favorable outcome, with &amp;gt;60% of them having a disease-free long-term survival without transplantation. Moreover, even in a case of relapse, patients can achieve CR2 with MRDneg and attain long-term remission (90% OS in long-term follow-up). Older patients have a less favorable outcome even when they achieve adequate molecular response with two thirds of them experiencing relapse. Hence, the potential long-term outcome without transplantation needs to be balanced by the high risk of relapse. Nevertheless, even these patients can be successfully treated with either a salvage regimen or immediate transplantation, and &amp;gt;40% of them can be long-term survivors.

Measurable Residual Disease Assessment in Patients with Acute Myeloid Leukemia Aged ≥60 Years Treated with a 10-Day Decitabine Schedule Versus Intensive Chemotherapy in the AML21 Study (NCT02172872)

Introduction Several studies have demonstrated that patients with acute myeloid leukemia (AML) who test measurable residual disease (MRD) positive during or after treatment have a higher probability of relapse and a shorter duration of survival, as compared to those who are persistently MRD negative. Indeed, a status of MRD positivity reflects a condition of resistant disease, existing below the threshold of the morphologic evaluation of complete remission (CR). The bulk of our knowledge in terms of MRD assessment has been generated in the setting of intensive chemotherapy; much less is known for older patients receiving less intensive treatments, such as decitabine (DEC) using a 10-day schedule. Methods The AML21 study was a randomized open-label phase 3 clinical trial conducted by the EORTC Leukemia Group, GIMEMA, and GMDS-SG. Patients with a newly diagnosed AML, aged ≥60 years and eligible for intensive chemotherapy were randomized 1:1 to receive DEC (20mg/m2 a day) using a 10-day schedule or intensive chemotherapy regimen consisting of daunorubicin 60 mg/m2 x 3 days and cytarabine 200 mg/m2 x 7 days (3+7), followed by 1-4 additional chemotherapy cycles. Patients who had an HLA-matched donor and at least stable disease were encouraged to undergo HSCT after &amp;gt;1 treatment cycle. Patients from the DEC arm not receiving HSCT could continue DEC treatment. A subset of patients treated in GIMEMA sites underwent a prospectively planned MRD evaluation post cycle 3 (DEC arm)/post consolidation (3+7 arm), and at 6, 12 and 18 months after the randomization, in case they were in a first CR and had not been allografted. MRD was studied by multiparameter flow cytometry. Based on several retrospective validations in the context of former EORTC/GIMEMA protocols, the threshold for discriminating MRD negative from MRD positive cases was set at 3.5x10-4 (0.035%) residual leukemic cells, upon full blood count recovery. Results In total, 130 out of 154 patients treated in GIMEMA centers provided consent for participation in translational research, had leukemia associated immunophenotype suitable for MRD monitoring, and initiated the protocol treatment, DEC (N=66) or 3+7 (N=64). Median age was 68 years (range 60-78), 33% of patients were ≥70 years old, 54% were male, and 31% and 30% had good and adverse European LeukemiaNet 2017 risk profile, respectively. At the MRD evaluation post cycle 3/consolidation, 9 out of 66 (14%) patients from the DEC and 12 out of 64 (19%) patients from the 3+7 arm had documented MRD negativity. The number of patients who either had documented MRD negativity or had undergone allografting and were alive and progression-free was 12 (18%) and 17 (27%) at 6 months, 14 (21%) and 13 (20%) at 12 months, and 14 (21%) and 7 (11%) at 18 months in the DEC and 3+7 arms, respectively. The inspection of individual profiles of patients who underwent the first MRD evaluation (and were at remission at that time) indicated that all MRD-positive patients from the DEC arm who were not allografted eventually relapsed, which often occurred just after the discontinuation of DEC maintenance. Conclusions 10-day DEC treatment can induce MRD-negativity. However, early MRD-negativity appeared more difficult to achieve with 10-day DEC than with 3+7. Among patients in whom 10-day DEC did not induce MRD-negativity, allografting or DEC maintenance appeared necessary to avoid a disease relapse.

Association of Measurable Residual Disease with Progression-Free Survival in Patients with Chronic Lymphocytic Leukemia: A Systematic Review and Meta-Analysis in the Era of Novel Agents

Introduction: Achieving undetectable measurable residual disease (uMRD) following treatment has shown significant prognostic value in several hematological malignancies. In chronic lymphocytic leukemia (CLL), uMRD is associated with improved outcomes in the era of chemoimmunotherapy (CIT). However, the role of measurable residual disease (MRD) in the context of novel agents such as Bruton tyrosine kinase inhibitors (BTKi) and/or BCL2 inhibitors (e.g. venetoclax) is uncertain. To address this question, we conducted a systematic review of the literature and performed a comprehensive meta-analysis of trials that included novel agents to evaluate the association between MRD status after treatment and PFS in patients with CLL. Methods: We conducted a comprehensive search of PubMed, EMBASE, Scopus, and Web of Science focusing on CLL trials reporting MRD and its association with PFS. Results were independently screened by two reviewers. Inclusion criteria included prospective phase 3 trials involving a novel agent in one of the treatment arms, while studies with incomplete information on MRD status related to PFS, reviews, abstracts, and retrospective analyses were excluded. The initial search identified 185 studies in PubMed and 402 studies in EMBASE. After applying prespecified criteria, a total of 10 phase 3 trials were included in our analysis. We extracted sample size, patient age, follow-up time, time point and tissue of MRD assessment, MRD cut-off and rate, performance status and molecular status when available. We only considered treatment arms with complete MRD information. The comparison involved patients who achieved uMRD with those exhibiting detectable MRD. When available, hazard ratios (HR) and confidence intervals (CI) were used to evaluate the relationship between MRD status and PFS. If not, we used Kaplan-Meier (KM) curves from both uMRD and MRD subgroups and extracted the coordinates points of the curves using DigitizeIt software (version 2.5.10, I. Bormann) and using the number at risk provided we estimated the HRs and CIs. A meta-analysis was performed using a random effects model from the R package metafor (Viechtbauer, W. (2010). Conducting meta-analyses in R with the metafor package. Journal of Statistical Software, 36(3), 1-48). The pooled hazard ratio estimates, and the corresponding 95% CI and prediction intervals (PI) were reported. Funnel and Baujat plots were generated to assess study heterogeneity and bias. All analysis was performed using R version 4.1.2 (R Foundation for Statistical Computing, Vienna, Austria). Results: A total of 10 phase 3 trials, encompassing 14 treatment arms and involving 3152 patients, met the stringent inclusion criteria. All but one study used a uMRD cut-off of &amp;lt; 0.01%. Detailed information on the treatment arms and key trial characteristics is presented in Table 1. Notably, ten treatment arms were derived from first-line treatment trials, while the remaining arms pertained to the relapsed/refractory population. Only two trials (4 treatment arms) assessed MRD using next generation sequencing (NGS), while the rest used multicolor flow cytometry (MFC) (9 treatment arms) and allele-specific oligonucleotides polymerase chain reaction (ASO-PCR) (2 treatment arms). All but 3 trials assessed MRD in both bone marrow (BM) and peripheral blood (PB). One trial (2 treatment arms) was excluded from the met-analysis due to higher MRD cut-off. All studies included in the meta-analysis measured uMRD (&amp;lt;0.01) versus MRD. The PFS pooled HR estimate for achieving uMRD at 0.01 cut off from the meta-analysis was 0.32 (95% CI [0.22, 0.45]), with minimal bias observed on the Funnel Plot. Conclusions: In this comprehensive meta-analysis, comprising 9 phase 3 trials, achieving uMRD was strongly associated with PFS. These findings further establish the prognostic role of MRD in CLL management and its potential use as a surrogate marker in clinical trials. As the body of evidence on MRD in CLL continues to grow, it becomes imperative to standardize the optimal time point, methodology, and tissue for MRD assessment. Substantial heterogeneity in these aspects underscores the necessity for unified guidelines to ensure the accurate and consistent evaluation of MRD status in clinical practice.

Venetoclax and Blinatumomab for Adult Patients with Relapsed/Refractory or MRD Positive Ph-Negative B-Precursor ALL: First Results of the GMALL-Bliven Trial

Introduction Prognosis of adult patients with relapsed/refractory (R/R) B-precursor acute lymphoblastic leukemia (BCP-ALL) remains poor. Monotherapy with Blinatumomab in ALL induced a complete remission (CR) in 44% of patients and measurable residual disease (MRD) negative molecular remission (MOL-CR) in 33%. Median overall survival of 7.7 months remains unsatisfactory. In the MRD setting, median survival was 36 months in the context of subsequent allogeneic stem cell transplantation (SCT). The combination of Blinatumomab with Venetoclax may be an effective option in ALL. Objectives In this multicenter phase 1 clinical trial the GMALL study group evaluates the safety of Blinatumomab in combination with Venetoclax in R/R or MRD positive BCP-ALL (NCT05182385). Using RNA sequencing, functional intracellular BH3 profiling, T cell immunomonitoring and effector cell cytotoxicity assays we aim to further elucidate synergy of this combination. Methods The BLIVEN trial includes patients with CD19-positive Ph-negative BCP-ALL aged 18 years and older, with no upper age limit. Eligible patients had either R/R ALL (bone marrow blasts &amp;gt;5%) or molecular failure/relapse (MRD &amp;gt;1xE-04). To assess safety, we determined maximum tolerated dose (MTD) as primary endpoint of the phase I part of the trial as designated recommended phase 2 dose (RP2D). Key secondary endpoint was the MOL-CR rate. Treatment consisted of oral Venetoclax in continuous dosing on day -7 to day 42 of target doses ranging between 200-800 mg once daily, and Blinatumomab as continuous intravenous infusion of 28 µg/d 4 weeks on and 2 weeks off, starting on day 1. For patients with R/R ALL, a cautionary dose step of 9 µg/d Blinatumomab was implemented for one week. MRD was analyzed in the reference laboratory using real-time quantitative PCR (RQ-PCR) of immunoglobulin and T-cell receptor (IG/TR) rearrangements. Results Between February 2022 and May 2023 a total of 7 patients were enrolled in the phase I part at four German centers (Figure 1). Venetoclax target doses were 400 mg (n=3), 600 mg (n=3), and 800 mg (n=1). Median age was 48.5 years (range 22 - 72 years). MRD assessment was available in 7/7 patients. Molecular BCP-ALL subtypes were identified by RNA sequencing in 5/5 analyzed patients, including ZNF384-rearranged ALL (n=2), BCR::ABL1-like (n=2; IGH::CRLF2 rearranged), and KMT2A-rearranged ALL (n=1). Three of seven patients were treated for hematological relapse, while 4/7 patients had molecular failure/relapse at time of enrolment. Among patients with R/R ALL, 2/3 were refractory to front-line therapy with intensive GMALL induction/consolidation therapy, while 1/3 suffered relapse shortly after failure of second allogeneic SCT. CTCAE grade IV neutropenia was identified as adverse reaction related to Venetoclax in one patient. Neutropenia resolved before dose limiting toxicity (DLT) evaluation time (i.e. 42 days after Venetoclax initiation). Overall, no DLT was identified throughout the trial and MTD was not reached. No treatment interruption was noted during cycle 1. Two of seven patients received one treatment cycle of BLIVEN only due to treatment failure, 5/7 patients received two treatment cycles. Response assessment was available for 7/7 patients. Treatment with Blinatumomab/Venetoclax of R/R ALL resulted in CR in 1/3 patients, with the responding patient showing a MOL-CR. The MOL-CR rate in molecular failure/relapse patients was 3/4. Conclusion Salvage therapy with Blinatumomab and Venetoclax exhibited good tolerability without treatment-related mortality. No treatment withdrawals were attributed to safety concerns. Two patients did not respond to BLIVEN combination therapy. Interestingly, both patients exhibited IGH::CRLF2 fusion transcripts.The remaining 5 patients obtained MRD responses, including complete MRD responses, of which 2/2 occurred in ZNF384 rearranged BCP-ALL. Further follow-up and larger patient numbers are needed to establish the potential benefit of this regimen in molecular driver subtypes of BCP-ALL. Enrollment to complete phase I of the BLIVEN trial is currently ongoing. Updated results including RP2D will be presented at the meeting. Acknowledgments This is an independent academic early phase I clinical trial conducted under the sponsor role of the Goethe University, Frankfurt, Germany. It received financial support and supply of Venetoclax by AbbVie Inc..

Detection of Isolated Extramedullary AML Using Next-Generation Sequencing of Cell-Free DNA

Introduction Isolated extramedullary (IEM) manifestation of AML is a recurrent event especially after allogenic hematopoietic cell transplantation (alloHCT). To date, measurable residual disease (MRD) assessment for early detection and monitoring of this difficult-to-treat patient cohort has not been established. Using cell-free DNA (cfDNA) from plasma or serum for MRD measurement shows comparable sensitivity to bone marrow mononuclear cells (BMMCs) and improved sensitivity over peripheral blood mononuclear cells (PBMCs) in medullary AML (Nakamura et al., Blood, 2019). Here, we evaluate next-generation sequencing (NGS) for MRD assessment of cfDNA in a cohort of patients with IEM AML. Methods We retrospectively collected tumor tissue and matched BMMC/PBMCs and cfDNA serum or plasma samples from IEM AML patients at our institution. BMMC/PBMCs and IEM tumor tissue were sequenced with 35- to 48-gene panels at diagnosis and relapse. Amplicon-based NGS-MRD (sensitivity 0.01%) of matched cfDNA and BMMC/PBMCs was quantified at time of IEM manifestation and in all available samples collected up to 12 months before IEM manifestation using the known mutations from IEM AML tissue. Amplicon-based NGS and bioinformatic error-correction was conducted and analyzed as previously described (Thol et al., Blood, 2018). Results We identified a cohort of 15 IEM AML patients at our institution. One patient was diagnosed with IEM AML at first diagnosis, 14 patients suffered from IEM relapse. For three patients, matched BMMC/PBMCs and cfDNA samples were collected from a second IEM AML relapse during the disease course. 15 of 18 IEM AML episodes (83%) occurred as relapse after alloHCT. 42 AML-related mutations were identified in the IEM AML tissue with a median of three mutations per patient (range 1-5). 33 of 39 mutations (85%) found in IEM relapse tissue had been already present at first diagnosis. All mutations were then evaluated in cfDNA and BMMCs at the time of the IEM manifestation by sensitive NGS-MRD analysis for all 18 IEM AML episodes. For two IEM episodes, BMMCs were not available and PBMCs were used instead. 17 of 18 (94%) cfDNA samples were NGS-MRD positive at the time of IEM manifestation, while only 8 of 18 (44%) BMMC/PBMC samples were positive. 20 of 53 mutations (38%) at first and second IEM episode could only be detected using cfDNA, 15 mutations (28%) were detected in both cfDNA and BMMC/PBMCs and only one mutation was solely found in BMMCs. 17 mutations (32%) could not be detected by either method (Figure 1). The variant allele frequency (VAF) of mutations detected by both methods was significantly higher using cfDNA with a median VAF of 2.45% (range 0.12 - 24%) compared to BMMC/PBMCs with a median VAF of 0.048% (range 0.13 - 10%; p &amp;lt; 0.01). Median VAF of mutations that were only found in cfDNA was 0.63% (range 0.017 - 39.6) and did not significantly differ from the cfDNA VAF of mutations that were found by both methods (p=0.43), indicating that these mutations represented the major clone. However, no correlation was found between IEM tumor VAF and cfDNA VAF (r=0.01; p=0.95). For ten IEM episodes from nine patients at least one cfDNA sample was available within 12 months before IEM relapse. In six IEM episodes (60%) cfDNA samples were MRD positive, one at 6 months, one at 5 months, two at 3 months and two at 2 months before IEM relapse. In four IEM episodes (40%) cfDNA samples were MRD negative, one at 10 months, one at 5 months and two at 2 months before IEM relapse. Conclusion In summary, cfDNA analysis from serum or plasma of AML patients can identify the molecular characteristics of a large proportion of patients with IEM AML. More than one third of clinically relevant mutations could only be detected using cfDNA and were missed by NGS-MRD of BMMC/PBMCs. Our data suggest that MRD monitoring from cfDNA detects IEM relapse earlier than routine monitoring from PB or BM in a significant proportion of patients.