Positive Minimal Residual Disease Research Articles

The prognosis of haploidentical hematopoietic stem cell transplantation in infants and patients under 3 years old with acute leukemia.

The role of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in patients<3 years of age remains controversial. Data on haploidentical donor (HID) transplants in this age group is limited. We retrospectively analyzed the prognosis of 97 patients with acute leukemia aged<3 years who underwent HID transplantation at our institute. With a median follow-up of 45 months, the 3-year disease-free survival (DFS), overall survival (OS), and 3-year cumulative incidence rate of treatment-related mortality were 69.3% (95% confidence interval (CI): 59.9%-78.7%), 74.2% (95% CI: 65.2%-83.2%), and 3.6% (95% CI: 0.9%-9.7%) in all 97 patients, respectively. The 3-year DFS and OS rate in patients diagnosed<1 year and patients diagnosed ≥1 year were comparable: 77.8% (95% CI: 62.2%-93.4%) versus 66.3% (95% CI: 55.0%-77.6%, p=.253) and 82.5% (95% CI: 66.3-98.7%) versus 72.8% (95% CI: 61.9%-83.7%, p=.153), respectively. At the last follow-up, 23 patients had died, and 20 had died of relapse. Multivariate analysis revealed that positive pre-HSCT flow cytometric minimal residual disease (hazard ratio 5.605, p=.000) and AML-M7 expression (hazard ratio 2.906, p=.014) were independent adverse prognostic variables for relapse. HID transplantation is potent and safe for infants and young patients with acute leukemia. Relapse is the primary cause of treatment failure.

A randomized controlled trial of CAPEOX vs observation in patients with early-stage colorectal cancer with positive MRD after curative surgery (CAREME).

TPS225 Background: Most patients (pts) with early-stage colorectal cancer (CRC) have a good prognosis, but some still relapse shortly after surgery. Effective methods to assess the risk of recurrence in these pts and guide adjuvant chemotherapy (ACT) decision-making are lacking. Increasing studies have shown that circulating tumor DNA (ctDNA) can detect minimal residual disease (MRD) and identify pts with a high risk of recurrence. ACT could improve survival of MRD-positive pts with stage Ⅱ CRC suggested by recent studies, whether it is appropriate for those with stage Ⅰ or clinically low-risk stage Ⅱ CRC remains unknown. Methods: CAREME is a multicenter randomized controlled clinical trial aimed at investigating the benefit of chemotherapy for MRD-positive pts with early-stage CRC. Pts with resected stage Ⅰ or Ⅱ CRC will be evaluated by oncologists. Those who did not receive neoadjuvant therapy and are deemed suitable for active surveillance (i.e., ACT is not needed) will undergo a postoperative ctDNA test using MinerVa MRD assay (a tumor-informed assay covering 769 cancer-related genes). Pts with mismatch repair-deficient tumors will be excluded and stage Ⅱ pts should have no traditional high-risk features according to clinical practice guidelines. Pts with positive ctDNA (N = 38) will be equally randomized into two groups: treatment group (Arm A) or surveillance group (Arm B). Arm A will receive 8 cycles of CAPEOX therapy while pts in Arm B will not receive any ACT; both groups will undergo ctDNA tests at 6 months after randomization and be followed up every 3 months. The primary endpoint is 18-month recurrence-free survival, and a key secondary endpoint is ctDNA clearance at 6 months after randomization. Tumor samples from the pts will be collected for MRD assay and exploratory research. Accrual started in February 2023. Support: Genecast Biotechnology Company. Clinical trial information: NCT05699746 .

Prognostic value of minimal residual disease profiling in resectable hepatocellular carcinoma.

530 Background: Detecting post-surgical residual disease is a critical clinical requirement in resectable hepatocellular carcinoma (HCC). Previous studies focused on specific genomic regions have indicated that ctDNA holds promise as a tool for prognostic assessment. Nevertheless, these methods exhibited limited sensitivity and failed to meet the minimal residual disease (MRD) threshold. Here we report the prognostic value of MRD profiling in HCC patients who have undergone hepatectomy. Methods: This retrospective study involved 88 HCC patients who underwent surgical resections in China, from January to May in 2016. During surgery, tumor and normal tissue specimens were collected. Plasma samples were obtained 7 days post-surgery. PredicineBEACON, a baseline tissue- or blood-informed MRD assay, was used for MRD profiling. This entailed identifying tumor-specific mutations through whole exon sequencing of tissue samples. Subsequently, a personalized MRD panel, selecting up to 50 mutations via bioinformatics pipelines merged with a fixed panel featuring 500 tumor actionable hotspots, was created for each patient. To detect MRD in post-surgery plasma samples, we employed ultra-deep sequencing at a coverage of 100,000X, utilizing the designed panel. Results: In the cohort of 88 patients, the distribution based on Barcelona Clinic Liver Cancer (BCLC) staging was as follows: 79.5% stage A (N=70), 12.5% stage B (N=11), and 8.0% stage C (N=7,). The MRD assay identified 36 patients as MRD+ and 52 patients as MRD-. We observed significant correlations between MRD status and both relapse-free survival (RFS) and overall survival (OS). For MRD+ patients, the median RFS was 17.1 months, while it was not reached for MRD- patients (p=0.0013). Likewise, the median OS for MRD+ patients was 40.1 months, in contrast to it not being reached for MRD- patients (p=0.0012). The MRD positivity rate stood at 100% in stage C patients, a significantly higher percentage compared to the rates observed in stage B (36.4%, p=0.0256) and stage A (36.2%, p=0.0042). Significantly, MRD status emerged as a pivotal prognostic differentiator among clinically low-risk patients in stage A: among MRD+ patients, the median RFS was 23.3 months, whereas it was not reached for MRD- patients (p=0.05). Additionally, the median OS of both MRD+ and MRD- was not reached but their survival curves showed significant differences (p=0.038). Conclusions: This study demonstrated the clinical utility of ctDNA MRD assay in patients with resectable HCC, as notably evident in the robust detection of MRD using plasma samples collected 7 days after surgery. Furthermore, the assessment of post-surgical MRD status provided valuable prognostic insights into both patient survival and the risk of disease relapse. ctDNA MRD status played a pivotal role as a prognostic differentiator, particularly among clinically low-risk patients.

Abstract C092: T cell responses and clinical outcomes in pancreatic and colorectal cancer patients with minimal residual disease in AMPLIFY-201, a phase 1 trial of a first-in-class amphiphile lymph node targeted mutant KRAS vaccine

Abstract Background: Mutations in RAS occur in &amp;gt; 25% of solid tumors and &amp;gt; 90% of patients (pts) with PDAC and &amp;gt; 40% of pts with CRC. ELI-002 2P is a vaccine comprised of lymph-node targeted Amphiphile (Amph)-modified G12D and G12R mutant KRAS peptides together with an Amph-modified CpG oligonucleotide adjuvant designed to expand polyfunctional mutant KRAS-specific T cells. Interim phase 1 AMPLIFY-201 data (O’Reilly et al; ASCO, 2023, #2528) showed that adjuvant administration of ELI-002 was well-tolerated, with T cell responses in 87%, and tumor biomarker responses in 77% (a subset of 32% had ctDNA clearance). Here we report relapse free survival data and correlations between ELI-002 2P- induced T cell levels and outcomes in pts with PDAC and colorectal cancer (CRC) at high risk for relapse following locoregional treatment. Methods: Resected PDAC (n=20) and CRC (n=5) pts with tumors harboring KRAS G12D or G12R who had minimal residual disease positivity (MRD+) defined as elevated circulating tumor DNA (ctDNA) and/or serum tumor biomarker (CA19-9/CEA), received up to 6 priming doses and 4 booster doses separated by a 3-month rest period of subcutaneous ELI-002 2P vaccine monotherapy comprised of Amph-peptides (700 mcg each G12D/G12R), admixed with Amph-CpG-7909 at 0.1, 0.5, 2.5, 5.0 and 10.0 mg per cohort dose level. Primary endpoints included safety and recommended phase 2 dose (RP2D) of Amph-CPG-7909; secondary endpoints: biomarker reduction/clearance; exploratory endpoints: included relapse free survival (RFS) using immune Response Evaluation Criteria in Solid Tumors (iRECIST) and immunogenicity assessed by direct ex vivo Fluorospot and intracellular cytokine staining of peripheral blood mononuclear cells. Results: Direct ex vivo polyfunctional mKRAS-specific T cell responses to ELI-002 2P were observed in 20/23 (87%; 50% induced both CD4+ and CD8+ T cells, median 13-fold and mean 56-fold increase from baseline), with response in 9/9 (100%) pts treated at highest two dose levels including the 10 mg RP2D. Clinical efficacy correlated with T cell response in evaluable pts (n=22): median tumor biomarker reduction/clearance was -86.9% vs -4.5% in above vs below median T cell responders, respectively (p &amp;lt; 0.0124). At 25 weeks median follow-up, the median RFS was not reached compared to 17.1 weeks in above versus below median T cell responders (HR 0.11; 95% CI 0.027-0.446; p = 0.0108). The association of RFS with T cell response was not confounded by other baseline prognostic variables (including tumor stage, recovery from prior cytotoxic therapy as assessed by absolute neutrophil count, or immune system subsets such as %CD4+ or %CD8+of CD3+ lymphocytes). Conclusions: The association of ELI-002 2P vaccine T cell responses with clinical outcomes in a novel MRD+ trial design underscores the potential to reduce recurrence in the setting of microsatellite stable PDAC and CRC. A new phase 1/2 trial, AMPLIFY-7P (NCT05726864), is evaluating a new seven peptide (G12D, G12R, G12V, G12C, G12A, G12S, G13D) ELI-002 7P formulation. Citation Format: Zev A. Wainberg, Shubham Pant, Colin D. Weekes, Muhammad Furqan, Pashtoon M. Kasi, Craig E. Devoe, Alexis D. Leal, Vincent Chung, James R. Perry, Lochana Seenappa, Lisa K. McNeil, Esther Welkowsky, Peter C. DeMuth, Christopher M. Haqq, Eileen M. O'Reilly. T cell responses and clinical outcomes in pancreatic and colorectal cancer patients with minimal residual disease in AMPLIFY-201, a phase 1 trial of a first-in-class amphiphile lymph node targeted mutant KRAS vaccine [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C092.

Intensified conditioning regimens with total marrow irradiation/etoposide/cyclophosphamide and busulfan/etoposide/cyclophosphamide overcome the impact of pre-transplant minimal residual disease on outcomes in high-risk acute lymphoblastic leukemia patients in complete remission.

Among high-risk acute lymphoblastic leukemia (ALL) patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT), those with positive minimal residual disease (MRD) are susceptible to poor outcomes. Therefore, it is necessary to determine the most suitable preparatory regimen for these patients. Data were analyzed from 141 patients who received allo-HSCT and were diagnosed with high-risk ALL. These patients underwent intensified conditioning regimens, including either total marrow and lymphoid irradiation (TMLI)-etoposide (VP16)-cyclophosphamide (CY) or busulfan (BU)-VP16-CY between October 2016 and November 2022. A total of 141 individuals were in complete remission (CR) before transplantation and, among all patients, 90 individuals exhibited a negative MRD status and 51 patients had a positive MRD status. In patients who tested negative for MRD, the incidence of relapse within a 2-year timeframe was 25.0% (24.8%-25.5%), compared with 32.2% (31.2%-33.2%) in MRD-positive patients; however, this difference was not statistically significant. There were no significant differences in the 2-year disease-free survival (DFS) and 2-year overall survival (OS) rates between the MRD-negative and MRD-positive groups (DFS: 67.2% (57.9%-78.1%) vs. 55.5% (42.6%-72.3%); OS: 69.0% (61.9%-88.2%) vs. 66.7% (53.9%-82.5%)). Furthermore, no notable variations were observed in the occurrence of transplant-related mortality (TRM) and graft-versus-host disease (GVHD) across the two groups. This study reveals the benefits of TMLI-VP16-CY and BU-VP16-CY conditioning regimens in high-risk ALL patients with CR and MRD-positive status. A large-scale prospective clinical trial is warranted in the future.

Combination of minimal residual disease on day 15 and copy number alterations results in BCR-ABL1-negative pediatric B-ALL: A powerful tool for prediction of induction failure

The current genomic abnormalities provide prognostic value in pediatric Acute Lymphoblastic Leukemia (ALL). Furthermore, Copy Number Alteration (CNA) has recently been used to improve the genetic risk stratification of patients. This study aimed to evaluate CNA profiles in BCR-ABL1-negative pediatric B-ALL patients and correlate the data with Minimal Residual Disease (MRD) results after induction therapy. We examined 82 bone marrow samples from pediatric BCR-ABL1-negative B-ALL using the MLPA method for the most common CNAs, including IKZF1, CDKN2A/B, PAX5, RB1, BTG1, ETV6, EBF1, JAK2, and PAR1 region. Subsequently, patients were followed-up by multiparameter Flow Cytometry for MRD (MFC-MRD) assessment on days 15 and 33 after induction. Data showed that 58.5 % of patients carried at least one gene deletion, whereas 41.7 % of them carried more than one gene deletion simultaneously. The most frequent gene deletions were CDKN2A/B, ETV6, and IKZF1 (30.5 %, 14.6 %, and 14.6 %, respectively), while the PAR1 region showed predominantly duplication (30.5 %). CDKN2A/B and IKZF1 were related to positive MRD results on day 15 (p = 0.003 and p = 0.007, respectively). The simultaneous presence of more than one deletion was significantly associated with high induction failure (p = 0.001). Also, according to the CNA profile criteria, the CNA with poor risk (CNA-PR) profile was statistically associated with older age and positive MRD results on day 15 (p = 0.014 and p = 0.013, respectively). According to our results, the combined use of CNAs with MRD results on day 15 can predict induction failure and be helpful in ameliorating B-ALL risk stratification and treatment approaches.

Sequential Imaging with Diffusion-Weighted Whole-Body MRI (DW-MRI) and PET-CT Identifies Patients at High Risk of Relapse in Multiple Myeloma

Introduction: Although the vast majority of patients with newly diagnosed multiple myeloma (NDMM) achieve deep responses during first-line therapy, most of them face relapse over time. Focal lesions, which are seen in up to 80% of NDMM, play a crucial role in MM evolution and can act as reservoirs for relapse disease. As a consequence, the combination of imaging- and minimal residual disease (MRD) negativity has been defined as a new response category. However, the dynamics and location of focal lesions during the treatment course remains poorly characterized and the answer to what imaging modality should be used to visualize focal residual disease remains elusive. In this study, we employed two techniques, PET-CT and DW-MRI, at multiple pre-defined time points to longitudinally track resistant lesions and investigate the combined prognostic value of these imaging modalities with other prognostic markers. Methods: We included 170 NDMM patients who were diagnosed between the years of 2010-2015 to ensure sufficient median follow-up time, which was 10 years. Median age of the study cohort was 60 years (range: 34-76), 104 were male (61%), and 31 patients (18%) had high-risk according to gene-expression profiles. All patients underwent PET-CT and DW-MRI evaluation at three pre-defined timepoints: baseline, post autologous stem-cell transplantation (ASCT) and at relapse. Minimal residual disease testing (MRD) by 8 color flow cytometry at a sensitivity level of 10 -5 post ASCT was available for 70 patients. Results: At baseline, 144/170 patients (85%) presented with focal lesions. Both imaging modalities, PET-CT and DW-MRI, showed positive results in 116 patients (68%). However, 21 patients (12%) had focal lesions detectable only by DW-MRI, while 7 patients (4%) showed focal lesions solely in PET-CT. After ASCT, only 12 patients had PET-CT positive lesions, whereas DW-MRI detected focal lesions in 53 patients, indicating a much higher detection rate with DW-MRI. Overall, 55 out of 170 patients (32%) showed residual lesions at this time point. The 26 patients w/o focal lesions in both PET-CT and DW-MRI at baseline remained negative post ASCT. Except for one patient, focal lesions after ASCT were persistent from initial diagnosis. Of note, two-thirds of the patients had residual focal lesions in the humeri and/or femora, which is surprising given that myeloma is considered a disease of the axial skeleton. Next, we correlated the presence of residual lesions with baseline patient characteristics, including age, gender, stage, and cytogenetic risk factors. Compared to patients w/o residual lesions, PET-positive patients showed an enrichment of rISS stage III (7 vs 50%, p&amp;lt;0.001) and deletion 17p (10 vs 33%, p=0.04). In contrast, patients who were DW-MRI positive only showed no significant association with any of the investigated variables. Both PET- and DW-MRI positive lesions post ASCT were associated with poor PFS (median 1.5 and 2.4 years, respectively vs. 9.4 years in imaging-negative patients, p&amp;lt;0.001) and OS (median 3.7 and 5.7 years, respectively vs. 11.3 years, p&amp;lt;0.001). Importantly, in a multivariate model the adverse prognostic effect of residual lesions was independent of baseline risk factors. To increase the power of the following combined imaging/MRD analysis, we defined an “Imaging+” group, which included the patients with PET+ and/or DW-MRI+ focal lesions. Among the 20 patients who achieved MRD-negative and imaging-negative status, an excellent PFS was observed, with only three events occurring in an 8-year follow-up. Residual lesions despite MRD negativity were only seen in two patients, and one of them progressed after 1.5 years. Dismal PFS was seen for double-positive patients (median 1.4 years), significantly worse compared to patients with MRD positivity only (8.9 years). Conclusion: Taken together, DW-MRI captures more patients with prognostically relevant residual focal lesions compared to PET-CT and should therefore be considered as a preferable imaging modality post ASCT. The high number of residual lesions in the extremities suggests a role of humeri and femora as reservoirs for treatment-resistant cells, deserving further investigation. For patients with MRD and imaging double positivity post ASCT, prognosis is dismal and alternate therapeutic approaches should be considered.

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

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

GWAS Identifies Variants Associated with Minimal Residual Disease after Induction I in Pediatric Patients with Newly Diagnosed Acute Myeloid Leukemia

Acute myeloid leukemia (AML) patients' clinical outcomes are associated with genetic abnormalities present in leukemic blasts. However, the significance of common genetic variants with regards to variation in AML treatment outcome remains elusive. To that end, we conducted a genome wide association study (GWAS) on 400 pediatric patients diagnosed with de novo AML to identify single nucleotide polymorphisms (SNPs) that may be associated with minimal residual disease (MRD) after the initial induction chemotherapy. Integrating our top SNPs from GWAS results with matched AML-derived transcriptomic data, we performed an expression quantitative trait loci (eQTL) analysis to determine which variants may affect the expression of one or more genes. Array-based whole-genome genotyping with imputation was used to investigate SNPs for association with MRD outcome in 2 pediatric AML cohorts: the multi-site AML02 [NCT00136084, n=167] and AML08 [NCT00703820, n=233] trials. Genotype imputation was performed via TOPMED Imputation Server and yielded 6,741,200 SNPs. MRD positivity was defined as 1 or more leukemic cells per 1000 mononuclear bone-marrow cells. Association analysis with adjustment for ancestry was conducted using logistic regression to model MRD positivity with SNP as a predictor. Genome-wide significance level was set at Bonferroni-corrected P&amp;lt;5x10 -8 and a suggestive significance level of P&amp;lt;1x10 -4. For gene expression profiling, the mRNA expression levels for 137 patients were obtained at diagnosis using GeneChip Human Genome U133A array from patients in the AML02 cohort. For eQTL analysis, we evaluated the association of gene expression using a 500kb window surrounding the top SNPs identified in the GWAS using a linear regression model. A total of 92 SNPs reached the suggestive significance level but none passed the genome-wide threshold. Of the 92 SNPs, 10 SNPs were mapped to the Major Histocompatibility Complex, Class II, DP Alpha 1 gene (HLA-DPA1), with 8 of the 10 SNPs associated with an increased risk of MRD positivity. The top SNP in HLA-DPA1 is located in intron 1 with presence of the variant A allele conferring increased MRD positivity (OR=2.17, 95% CI (1.55-3.04), P=6.64x10 -6, Figure 1A). Of note, HLA-DPA1 SNP was not significantly associated with event free survival (EFS; HR: 1.21, 95% CI (0.95-1.56), P=0.13) or overall survival (OS; HR: 1.28, 95% CI (0.96-1.71), P=0.09), however the direction of association was consistent with the GWAS results. HLA-DPA1 is a protein coding gene and plays an integral role in the immune response by presenting peptides derived from extracellular proteins. HLA-DPA1 and several other MHC class II genes have shown to be downregulated in patients diagnosed with AML who experienced a relapse (PMID: 30380364). The eQTL analysis revealed a significant association with presence of the variant A associated with reduced mRNA expression of HLA-DPA1 (P=0.01, Figure 1B). In addition to the cis-eQTL association of our top SNP with HLA-DPA1 expression, significant associations were also observed with neighboring genes: HLA-DOA, HLA-DQA1, HLA-DQB1, and DAXX. Furthermore, when examining transcriptomic data for association with survival outcomes, we observed high HLA-DPA1 gene expression was significantly associated with favorable OS (HR: 0.46, 95%CI (0.23-0.91), P=0.025), though this association had similar direction it was not significant for association with EFS (HR: 0.68, 95% CI (0.39-1.18), P=0.169). Although limited by numbers, this study is one of the largest GWAS conducted in pediatric AML to identify common SNPs associated with MRD status after initial chemotherapy induction using an integrated genomic and transcriptomic validation approach. None of the SNPs reached genome-wide significant levels, however our top results show that genetic variation in several genes of biological relevance to have an impact on an early outcome endpoint. Moreover, we also have shown that our top SNP located in HLA-DPA1 gene is an eQTL and its mRNA expression is significantly associated with OS. Future studies are aimed at integrating other clinical endpoints such as EFS and OS with hope of creating a polygenic risk score to personalize treatment approaches. In addition, validation of these findings in larger cohorts of AML patients are warranted with future analyses to include known prognostic factors such cytogenetically defined risk.

Zanubrutinib and Venetoclax As Initial Therapy for CLL/SLL with Obinutuzumab Triplet Consolidation in Patients with Minimal Residual Disease Positivity (BruVenG)

Background: Bruton's tyrosine kinase inhibitors (BTKi), anti-CD20 antibodies, and B cell lymphoma 2 inhibitors (BCL-2i) are essential therapeutic drug classes in the treatment of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). Clinical synergy of BTKi and BCL-2i combinations have been observed, which allow for an oral regimen, that can be safely administered in a clinic setting for most patients (Tam et al., 2022; Wierda et al., 2021). Still, it remains uncertain if anti-CD20 antibodies meaningfully add to the effectiveness of oral combination therapy. Recent reports of triplet regimens demonstrate modest improvement in minimal residual disease (MRD) negativity with similar response rates when compared to oral doublets (Davids et al., 2021; Soumerai et al., 2021; Wierda et al., 2021). Additionally, anti-CD20 antibody use causes significant B cell depletion resulting in prolonged immune suppression. Recent reports suggest BTKi use may in fact impair clinical activity of anti-CD20 antibodies, which can be mitigated by using selective BTKi and incorporating anti-CD20 later in the treatment course when lower disease burden is present (Da Roit et al., 2015; Flinsenberg et al., 2020; Pavlasova et al., 2016; Rawstron et al., 2018). Therefore, anti-CD20 therapy may not be required for all patients and could be optimized within a response-adapted strategy. This trial has been developed to assess efficacy and safety of a response adapted consolidative treatment approach utilizing obinutuzumab triplet consolidation in CLL/SLL patients who remain MRD positive after an oral doublet fixed duration treatment with zanubrutinib and venetoclax. This approach looks to maximize MRD negative remissions, spare toxicity by limiting anti-CD20 exposure, and provide a finite therapy. This study will utilize once daily zanubrutinib for which there is limited data in regard to safety and efficacy particularly in combination with venetoclax. Study Design and Endpoints: This is a single center, open label, phase II, investigator initiated clinical trial enrolling 50 subjects evaluating the efficacy and safety of zanubrutinib and venetoclax with obinutuzumab triplet consolidation in subjects who remain MRD positive (BruVenG). Eligible patients must have treatment naïve CLL/SLL and treatment indications per the International Workshop on CLL 2018 guidelines. Subjects with Richter's transformation are excluded. Treatment will consist of lead-in, combination, and consolidation phases. All subjects will be treated with a 3-cycle lead-in of 320mg oral, once daily zanubrutinib monotherapy. Venetoclax will be incorporated at cycle 4, per the standard prescriber insert ramp-up schedule. A final dose of 400mg oral, once daily venetoclax, in combination with 320mg oral, once daily zanubrutinib will be used in all subsequent cycles after ramp-up. At cycle 16, MRD will be assessed in the peripheral blood (PB) and bone marrow (BM) via clonoSEQ (Adaptive Biotechnologies). Subjects who are MRD negative (&amp;lt;1x10 -4 ) in both compartments will stop therapy. Subjects who are MRD positive in PB or BM will continue zanubrutinib and venetoclax for an additional 6 cycles. Obinutuzumab will be added to the oral doublet starting at cycle 17. Standard intravenous obinutuzumab lead-in will occur during cycle 17, followed by standard monthly intravenous dosing of 1000mg through cycle 22. Depending on MRD status, subjects will be treated for 16 or 22 cycles. All subjects who receive consolidation will stop treatment at cycle 23 regardless of end of treatment MRD status. The primary endpoint is cycle 16 PB and BM MRD negativity rates. A co-primary endpoint will be cycle 23 PB and BM MRD negativity rates in subjects who were MRD positive at C16 and who went on to receive at least 1 dose of obinutuzumab. Key secondary endpoints will include cycle 16 overall response rate, 36-month progression free survival, 36-month overall survival, 36-month time to next treatment, 24- and 36-month PB MRD negativity rates, tumor lysis risk reduction rates, and safety. Exploratory endpoints will assess the positive predicative value of the ∆400 biomarker (Soumerai et al., 2021) on MRD negativity at cycle 16. This biomarker will be assessed between cycle 4 and 6. The MRD negative rates at cycle 16 and cycle 23 will be estimated with a 90% confidence interval using the Clopper-Pearson method based on exact binomial distribution.

Prognostic Features of CD9 in Childhood Acute Lymphoblastic Leukemia - a Retrospective Analysis of a Nation-Wide Multicenter Study in China

Background and objectives: The outcomes of children with acute lymphoblastic leukemia (ALL) have been incrementally improved with risk-directed chemotherapy but therapy responses remain heterogeneous in clinically defined risk groups. Parameters with added prognostic values are therefore warranted to refine the current risk stratification system and inform appropriate management. CD9, a cell surface protein presented on malignant lymphoblasts, may have such predictive characteristics as shown by our previous single-center study conducted in Hong Kong (Leung et al, Leukemia, 2020). This study aims to determine the precise prognostic features of CD9 in a nation-wide, multicenter childhood ALL cohort in China, and deliver algorithms for identification of patients who may benefit from early interventions. Study design and outcome measures: This multicenter cohort study included childhood ALL patients (aged &amp;lt;18 years) enrolled into the Chinese Children Cancer Group (CCCG)-ALL-2015 study from January 2015 to December 2019, with 7,640 patients from 20 tertiary hospitals in China treated with a uniform protocol. A total of 3,781 subjects (49.5%) from 16 sites had flow cytometry data on lymphoblast CD9 at diagnosis. The final study cohort comprised 3,395 B-ALL and 386 T-ALL patients with a median follow-up of 53.9 months. The primary outcomes were 5-year overall survival (OS), event-free survival (EFS), and cumulative incidence of relapse (CIR) for patients with CD9 + or CD9 - phenotypes (positivity defined by the presence of ≥20% CD9 + blasts evaluated through a univariate analysis demonstrating the highest odds for adverse events or relapse at this cut-off). The secondary outcomes were 5-year OS, EFS, and CIR rates of CD9 + and CD9 - patients after inclusion of known prognostic factors, including risk assignment, cytogenetic anomalies, and minimal residual disease (MRD) status. Survival benefits of CD9 + subjects from treatment intensification or hematopoietic stem cell transplantation (HSCT) were analyzed. Results: CD9 was expressed in 88.5% of B-ALL and 27.9% of T-ALL cases. It conferred a dismal EFS (82.1% vs. 89.3%, P=0.001) and a higher CIR (15.5% vs. 7.8%, P&amp;lt;0.001) in B-ALL but not T-ALL patients. The prognostic impact of CD9 is much more prominent in subjects with adverse presenting features or poor early treatment responses, as best exemplified in those assigned to the intermediate/high-risk arms (EFS: 72.8% vs. 83.2%, P=0.028; CIR: 23.1% vs. 10.2%, P=0.003) or those with positive MRD at day 19 (EFS: 65.2% vs. 82.0%, P=0.053; CIR: 30.3% vs. 9.7%, P=0.007). The adverse impact of CD9 is confined to specific cytogenetics, including BCR-ABL1 (CIR: 39.5% vs. 0%, P=0.019) and normal karyotype (CIR: 15.0% vs. 6.8%, P=0.034). In multivariate analyses, CD9 remains an independent factor predicting event-free survival (HR=1.917, P=0.001) and relapse (HR=2.208, P&amp;lt;0.001). The time to and site of relapse did not differ between CD9 + and CD9 - patients. For CD9 + patients with MRD at the end of induction, treatment upstaging only benefited initial low-risk subjects. HSCT at first remission did not improve the outcomes of high-risk subjects with a CD9 + phenotype. Conclusions: This is the largest study to evaluate the significance of CD9 in childhood ALL. We confirmed the results of our previous single-center study with this multicenter national study. CD9 positivity is definitively associated with a higher relapse probability particularly for patients with intermediate/high risk diseases, MRD positivity or specific cytogenetic background. BCR-ABL1 + patients with a CD9 - phenotype had excellent outcomes and HSCT may not be required. Whereas MRD + patients who were CD9 + had poor outcomes, and therefore should have early interventions with innovative treatments to reduce the risk of relapse. With these, we propose to incorporate CD9 into the diagnostic immunophenotyping panel to inform risk stratification and management of childhood ALL.

Analysis of Peripheral Blood and Bone Marrow Residual Disease Dynamics during Intensive Treatment and Maintenance in Patients with Multiple Myeloma Included in the GEM12MENOS65 and GEM14MAIN Clinical Trials

Introduction: In patients (pts) with multiple myeloma (MM), analysis of minimal residual disease (MRD) dynamics in bone marrow by Next Generation Flow Cytometry (NGF) has shown that conversion of minimal residual disease (MRD) status modulates the risk of progression. Mass spectrometry measuring low-level monoclonal immunoglobulins has shown potential for Peripheral-blood based Residual Disease (PRD) assessment. In this study, we have analyzed the clinical impact of PRD and MRD dynamics in MM pts receiving intensive treatment as per the GEM2012MENOS65 trial and during maintenance as per the GEM2014MAIN trial. Patients and Methods: In GEM2012MENOS65 trial, pts received six cycles of VRD-GEM induction, autologous stem cell transplantation conditioned with melphalan or busulfan plus melphalan and consolidation with two cycles of VRD-GEM. Patients achieving at least minimal response were offered to be enrolled in the GEM2014MAIN and randomized to maintenance with lenalidomide and low-dose dexamethasone (Rd) or Rd plus ixazomib for two years; if not reaching MRD negativity at this point, pts received three more years of Rd. PRD dynamics were analyzed separately in each trial by Quantitative Immunoprecipitation Mass Spectrometry with anti IgG/A/M, total k and total l beads using the EXENT ® Solution (The Binding Site, part of Thermo Fisher Scientific). MRD was analyzed following the recommendations of the IMWG and according to the Euroflow guidelines. Only those pts with available samples at all the time points analyzed (GEM2012: post-induction, post-ASCT and after-consolidation; GEM014: post-consolidation and after 1 and 2 years of maintenance) were included. Results: 134 out of the 458 pts enrolled in the GEM2012MENOS65 trial and receiving intensive treatment as per protocol were analyzed. At treatment completion (post-consolidation), PRD and MRD status were associated with almost identical prognostic value: median progression-free survival (mPFS) in PRD - not reached (n.r.) vs 3.98 years in PRD + cases (p=0.0006) and in MRD - n.r. vs 3.99 years in MRD + cases (p=0.0001). When dynamics were analyzed, sustained PRD and MRD negativitywas observed in 58 (43.3%) and 44 (33.8%) pts and sustained positivity in 42 (31.2%) and 57 (42.5%) pts, respectively. In 35 (26.1%) and 33 (24.6%) pts, PRD and MRD converted from positive to negative, respectively. Sustained PRD or MRD positivity at the 3 time points analyzed was associated with a significantly shorter PFS (mPFS 4.04 years, p=0.0042 and 3.9 years, p=0.0008, respectively), compared to pts remaining negative or converting from positive to negative, in whom mPFS was not reached (Fig.1A). 109 pts out of the 332 pts enrolled in the GEM2014MAIN trial and receiving maintenance treatment as per protocol were analyzed. At treatment completion (after two years of maintenance), PRD or MRD status were associated with comparable prognostic value: mPFS in PRD - and PRD + cases n.r. (p=0.0039) and in MRD - cases n.r. vs MRD + 2.87 years (p&amp;lt;0.0001). When dynamics were analyzed, sustained PRDand MRD negativity was observed in 68 pts (62.4%) and 61 (55.9%) pts and sustained positivity in 19 (17%) and 19 (17%) cases, respectively. In 17 (15.6%) pts PRD and MRD converted from positive to negative and in 5 (5.6%) and 10 (0.91%) from negative to positive, respectively. Sustained PRD positivity at the 3 time points analyzed was associated with a shorter PFS (p=0.0185), compared to pts who remained PRD - or converted from PRD + to PRD -, in whom mPFS was not reached. The mPFS in pts remaining MRD - or converting from MRD + to MRD - was similar and has not been reached yet; the mPFS in pts with sustained MRD positivity at the 3 time points analyzed or converting from MRD - to MRD + was also similar (2.99 years and 2.88 years, respectively). Interestingly, pts converting from PRD - to PRD + had a poorer PFS (mPFS of 0.86 years) as compared to patients converting from MRD - to MRD + (mPFS of 2.28 years) (Fig.1B). Conclusions: In conclusion, assessment of PRD and MRD dynamics during intensive treatment and maintenance allows to define more precisely the patients' clinical outcome as compared to the analysis of isolated time points. Reaching MRD or PRD negativity post-consolidation or after 2 years of maintenance is associated with a similar prognostic value, thus highlighting the clinical utility of mass spectrometry as an alternative, non-invasive technique for disease evaluation.

Outcome of Infants Treated on Total Therapy for Infants with Acute Lymphoblastic Leukemia I: Results from a Non-Randomized Multi-Center Study

Infants with KMT2A rearranged (KMT2Ar) acute lymphoblastic leukemia (ALL) have a poor prognosis with an event-free-survival (EFS) of 33.6-36.9% on recent Children's Oncology Group and Interfant trials. In preclinical studies, we identified bortezomib and vorinostat as novel active agents in primary KMT2Ar infant ALL specimens that have not been included in infant regimens to date. Total therapy for infants with acute lymphoblastic leukemia I (TINI) evaluated this novel combination in a multi-institutional pilot trial for infants with de novo ALL. In this study, bortezomib and vorinostat were incorporated into an ALL chemotherapy backbone containing dexamethasone, mitoxantrone, and pegasparaginase during induction and reinduction chemotherapy cycles. A run-in dose escalation evaluated 3 dose levels of vorinostat which was then followed by a dose expansion phase. The primary endpoint of the study was tolerability; secondary endpoints included minimal residual disease (MRD), 3-year EFS and overall survival (OS). Fifty patients enrolled between 2017-2021. Median follow up was 3.4 years (range 1.6-6.8 years). The six patients in the dose escalation phase had no dose-limiting toxicities. Forty-four patients received vorinostat at the third dose level (DL3) of 180 mg/m 2/dose. 68% of patients treated at DL3 had CNS involvement at diagnosis. 68% of DL3 patients carried a KMT2Ar. The most frequent KMT2A partner genes were AFF1 (40%), MLLT1 (20%), and MLLT3 (13%). The most frequent grade 3-4 non-hematologic adverse events during induction include neutropenic fever (45%), hypertension (43%), infection (40%), anorexia (32%), diarrhea (18%) and perineal skin ulceration (16%). There were 3 episodes of sepsis and 1 induction death due to parainfluenza pneumonia. Fewer events and no deaths or proven sepsis occurred during reinduction, with only grade 3 hypertriglyceridemia exceeding 10% of patients affected (18%). 79% of all patients and 69% of KMT2Ar patients became MRD negative by flow cytometry on day 22 of induction following our investigational block. Following induction intensification with cyclophosphamide, cytarabine, and mercaptopurine, 77% and 66% of all patients and KMT2Ar patients respectively were MRD negative at the time of count recovery. Eight of the forty-four patients, all KMT2Ar, underwent stem cell transplant in first remission due to persistent MRD positivity at the end of consolidation. The 3-year EFS and OS probabilities (SE) of all forty-four DL3 patients were 56.5% (8.0) and 70.5% (7.3), respectively. The 3-year cumulative incidence of relapse (CIR) with death considered a competing event was 29.3% (6.9) and the cumulative incidence of death (CID) as a first event was 8.9% (4.5). Patients lacking a KMT2Ar had superior outcomes with 85.7% (9.4) EFS and 92.9% (6.9) OS. The EFS and OS of KMT2Ar patients were 44.8% (9.5) and 62% (9.1) respectively. CIR and CID for KMT2Ar patients was 43.5% (9.6) and 7.2% (5) respectively. KMT2Ar patients ≥90 days at diagnosis had superior outcomes compared to their younger counterparts with 59.1% (10.5) EFS and 77.3% (8.9) OS. End of induction MRD was predictive of an event in this subgroup, with 85.6% (9.4) EFS for MRD negative patients and 14.3% (13.2) EFS for MRD positive patients. In contrast, KMT2Ar patients &amp;lt; 90 days of age at diagnosis had poor outcomes irrespective of MRD with 12.5% (11.7) EFS and 25% (15.3) OS. Notably KMT2Ar patients &amp;lt; 90 days who were MRD negative at the end of induction had a median time to relapse of 600 days (range 282-969 days), a late time point for this malignancy that typically relapses within a year from initial diagnosis. This suggests the regimen effectively reduced tumor burden leading to an extended duration of remission but failed to sufficiently eradicate disease to prevent relapse. We conclude that the incorporation of bortezomib and vorinostat for treatment of infants with ALL was well tolerated. MRD responses in KMT2Ar patients were robust. Although the study was not powered to evaluate outcomes, EFS and OS in KMT2Ar patients suggest a clinical signal worth pursuing given the low proportion of patients transplanted in first remission. The successor study, TINI 2 (NCT05848687), will build upon the bortezomib/vorinostat backbone by incorporating 2 cycles of blinatumomab and the menin inhibitor ziftomenib in combination with chemotherapy during reinduction.

Results of Cynk-001-AML-001: A Phase I Multi-Dose Study Evaluating the Safety, Tolerability, and Persistence of Cynk-001 in Adults with De Novo or Secondary Acute Myeloid Leukemia in Morphologic Complete Remission with Minimal Residual Disease or Relapsed/Refractory AML

Introduction: Acute myeloid leukemia (AML) is a clinically and genetically heterogeneous disease often with suboptimal outcomes. While progress has been achieved in the treatment of AML, relapse remains a significant challenge. Novel therapeutic options aimed at attaining minimal residual disease (MRD) negative complete remission (CR) are expected to reduce the incidence of relapse and prolong survival. Evidence shows that natural killer (NK) cells can exhibit potent anti-leukemia activity against AML. CYNK-001 is a CD56+CD3- enriched, off-the-shelf, unmodified allogeneic NK cells expanded from placental CD34+ cells. Aim CYNK-001-AML-001 is a phase I multi-dose study evaluating the safety, tolerability, and persistence of CYNK-001 with or without recombinant human interleukin-2 (rhIL-2) in adults with de novo or secondary acute myeloid leukemia (AML) in morphologic complete remission (CR) with minimal residual disease (MRD) or relapsed/refractory (R/R) AML. Methods This Phase I, single-arm, dose escalation clinical trial (NCT04310592) enrolled patients 18-80 years with R/R AML or patient in MRD positive CR. Two lymphodepletion regimens were assessed. Patients in cohorts 1-3 underwent standard lymphodepletion with fludarabine (25 mg/m2/day) and cyclophosphamide (300 mg/m2/day) on days -5, -4 and -3. Subsequent cohorts received enhanced lymphodepletion with fludarabine (30 mg/m2/day) and cyclophosphamide (900 mg/m2/day) on days -6, -5, -4 and -3. Escalating doses of NK cells were administered following a standard 3+3 design, with 2 separate arms (MRD+ arm and R/R arm) starting with cohort 4. A starting dose of 600 million cells on days 0, 7, and 14 was escalated to a maximum dose of 1.8 billion cells on days 0, 7, 14 and 21. The primary objective of the study was to determine the maximum tolerated dose (MTD) or maximum planned dose (MPD) of CYNK-001 and to assess the safety of multiple infusions of CYNK-001 administered using a flat, non-weight-based dose as assessed by the frequency and severity of adverse events (AE). The secondary objective was to assess the clinical efficacy of CYNK-001 in AML subjects. Results 28 patients (17 with R/R AML and 11 with MRD-positive AML) were enrolled during dose escalation, and 27 received CYNK-001. The highest dose of NK cells administered was 1.8 billion cells X 4 in cohort 6b (patients with R/R AML). Treatment was well tolerated with no dose limiting toxicities (DLTs) even at the highest dose. No cases of neurotoxicity or GvHD were reported. Four cases of grade 2 CRS were reported but there were no cases of ≥ grade 3 CRS. Two incidences of grade 4 thrombocytopenia and one grade 4 decreased white blood cell count were noted in patients who had received enhanced lymphodepletion. In R/R AML patients treated with the highest dose of CYNK-001, 2 of 4 patients achieved an objective response of Morphologic Leukemia-Free State (MLFS) on Day 28. One of these patients has maintained a response to D120 and is continuing follow up. One of 3 patients with MRD +ve AML treated with 3 doses of 1.8 billion CYNK-001 cells per dose achieved MRD negativity until day 120 at which time a blast count of 1.6% was observed. Conclusion Treatment with CYNK-001 showed biological activity as evidenced by achievement of MLFS in R/R AML patients and eradication of AML MRD positivity in AML patients in morphologic CR. Therapy was generally well-tolerated, with no dose-limiting toxicities observed with even the highest dose of CYNK-001. This trial yielded important insights into the optimal approach to lymphodepletion and the potential clinical efficacy of allogeneic NK cells in R/R AML. Based on these results, this trial is now closed to further enrollment and future development activities will focus on next generation NK cell therapies edited to optimize persistence and efficacy and limit the requirement for enhanced lymphodepletion.

Euroflow-Based Ultrasensitive NGF for the Evaluation of Bone Marrow Minimal Residual Disease in Multiple Myeloma

Background: Euroflow next-generation flow cytometry sensitively assesses minimal residual disease (MRD) in multiple myeloma (MM). It is widely adopted in labs worldwide for tracking disease progression, therapy efficacy, and decision-making in MM trials. Euroflow MRD significantly enhances MM patient outcomes, alongside advancements in anti-cancer approaches like BiTEs, CAR-T cells, and ADCs, resulting in increased overall survival rates. However, blood-based methods do not always align with bone marrow PC analysis, requiring heightened sensitivity to detect residual malignant cells post-therapy. The recent BloodFlow method, achieves remarkable sensitivity up to 10 -8 by enriching CD138 + PCs from 50 mL peripheral blood. In this study, we compare conventional Euroflow BM-MRD with the ultrasensitive method using CD138 enrichment of BM cells before analysis. Methods: BM-MRD followed Euroflow group guidelines. For ultrasensitive assessment, CD138 + plasma cells were isolated using CD138 MicroBeads. Data analysis was performed with Infinicyt software. Automatic gate and identification tool was used for conventional MRD, while data was manually analyzed for ultrasensitive MRD assessment. Results: We compared conventional and ultrasensitive MRD in the bone marrow of 70 MM patients. All positive MRDs using conventional NGF remained positive with ultrasensitive MRD. Notably, among 40 patients with negative conventional NGF-MRD, 5 cases turned positive after ultrasensitive MRD, by detecting aPCs from 0,0003 to 0,002% in all nucleated cells in the bone marrow. Patients 1, 2, and 3, diagnosed between 2020 and 2022, were in CR during assessment. Patients 4 and 5, diagnosed in 2016 and 2014 respectively, underwent multiple therapies, two autologous hematopoietic cell transplants, and patient 5 also had an allogeneic hematopoietic cell transplant. Ultrasensitive MRD for patients 4 and 5 was done after anti-BCMA CAR T cell therapy. The CD138 isolation approach allowed evaluating BM-MRD at detection levels of 10 -8 cells. Further studies using ultrasensitive MRD will prospectively evaluate the predictive value of the assay in patients with NDMM, especially with high and ultra-high-risk disease. Conclusions: This study demonstrates the significant advantages of direct CD138 enrichment over bulk lysis for detecting residual malignant PCs in MM. Ultrasensitive MRD offers superior sensitivity compared to conventional MRD, a critical factor in the era of more effective therapies leading to deeper responses. Currently, we are prospectively assessing the clinical benefits in a larger patient cohort.

Pediatric Patients with High-Risk B-Cell ALL in First Complete Remission May Benefit from Less Toxic Immunotherapy with Blinatumomab - Results from Randomized Controlled Phase 3 Trial AIEOP-BFM ALL 2017

Background: One of the key questions in trial AIEOP-BFM ALL 2017 focused on the reduction of treatment-related complications by replacing parts of the highly intensive consolidation phase by two courses of Blinatumomab (Blina) in a prospective randomized trial. Herein, we report the toxicity profile of B-cell acute lymphoblastic leukemia (B-ALL) patients (pts) with high-risk (HR) characteristics pending final outcome data. Patients and Methods: 728 pts with HR B-ALL were enrolled from July 15, 2018 to October 31, 2022 in this multicenter trial run in 8 different countries. Patients were included in the HR group in view of the presence of at least one of the following criteria: induction failure; high levels of minimal residual disease (MRD) at day 15 of induction, or at the end of induction (EOI), or at the end of consolidation (EOC); presence of KMT2A::AFF1, of IKZF1+ and positive MRD at EOI (Stanulla et al, JCO 2018), TCF3::HLF, or hypodiploidy. B-ALL pts with one or several of these characteristics represented 20.8% of all pts aged 0-18 years of age with B-ALL. After a 4-drug induction phase, and two weeks of consolidation treatment, patients were randomized to receive Bortezomib in addition to standard consolidation (not part of this report). Before treatment continued with intensive consolidation block HR-1', MRD was analyzed in order to identify patients eligible for subsequent hematopoietic stem cell transplantation (HSCT) based on MRD results. After block HR-1', all B-ALL HR pts were randomized to receive either two courses of consolidation (blocks HR-2', and HR-3'), or two 28-day courses of Blina at 15µg/m 2/d administered by continuous IV infusion (with a 2-week treatment-free interval). Two intrathecal injections of methotrexate (MTX) for CNS prophylaxis were given on days 1 and 29 of each Blina course, respectively. Out of 728 HR B-ALL pts, 619 pts were eligible for randomization [reasons for non-eligibility were: Event (death or relapse) before randomization was due (26), Down syndrome (25; scheduled for a non-randomized intervention with Blina), presence of TCF3::HLF (3; could receive any alternative therapy including Blina), discontinuation/substantial change of preceding therapy (23) or other protocol exclusion criteria (32)]. 572 pts were randomized (92.4% of those eligible). One pt assigned to the experimental arm (EA) and 4 pts assigned to the control arm (CA) received the other arms, respectively. Results: Toxicity was evaluated in randomized pts according to treatment administered during the randomized phase, respectively, until next element started: 268 pts were treated in the control and 281 in the EA (see Table). 16 of them switched in/after the first Blina cycle to the HR blocks due to toxicity or poor response to Blina (due to increasing MRD, &amp;gt;=1x10 -4). Medically relevant adverse reactions of special interest (ARSI) according to MedDRA (specified in the protocol) were evaluated in the randomized treatment phases (CA: HR-2‘/HR-3‘ block, EA: 2 Blina cycles). ARSI were recorded in 61 of 268 pts in the CA (22.8%, 71 ARSI) and in 29 of 281 pts in the EA (10.3% [p&amp;lt;0.001], 33 ARSI); 3 of the 33 ARSI in the EA were related to HR blocks and were observed in 3 of the 16 pts that switched to HR blocks. Life-threatening SAR were seen in 14 pts of the control group (5.2%) and in no patient in the EA (p&amp;lt;0.001). Conclusions: Taken together, these results show for the first time in newly diagnosed pts with HR B ALL, the favorable toxicity profile previously reported with Blinatumomab in pediatric relapsed ALL (Locatelli F et al, JAMA 2021; Brown P et al, JAMA 2021). We have demonstrated that the toxicity profile of Blinatumomab is much more favorable as compared to the intensive chemotherapy approach using HR-blocks. If upcoming analyses of outcome data will show no inferiority of the EA, blinatumomab replacement of some of the intensive chemotherapy blocks will become the new standard of care for treatment if newly diagnosed patients with HR B-ALL.

Recommendations on Minimal Residual Disease Monitoring in Acute Promyelocytic Leukemia Treated with Combination of All- Trans Retinoic Acid and Arsenic Trioxide

Background All- trans retinoic acid (ATRA)-arsenic trioxide (ATO) combination therapy significantly reduces acute promyelocytic leukemia (APL) relapse compared to ATRA combined with chemotherapy, leading to a cure rate of 80-90% in APL patients. The ideal frequency and duration of minimal residual disease (MRD) monitoring needs to be identified in the era of ATO. Methods A total of 1072 APL patients were enrolled in this study, comprising 855 from the APL2012 trial (NCT01987297) and 217 from our center between January 2018 to June 2022 (APL2018 study). The APL2018 protocol was adjusted for consolidation and maintenance therapy based on APL2012 (Chen et. al. PNAS 2021): In consolidation therapy, low-, intermediate-, and high-risk patients were performed following the ATO-group scheme of corresponding risk stratification in the APL2012 study. From December 2019, chemotherapy was no longer administered to high-risk group during consolidation phase. In maintenance treatment, ATO was replaced with oral arsenic realgar-indigo naturalis formula, and methotrexate was removed. RT-PCR or RQ-PCR was routinely used for MRD monitoring, detecting PML::RARA fusion genes in bone marrow before each consolidation and maintenance therapy. MRD testing was conducted every 3 months within the first year, every 6 months within the second year, and annually from 3 to 5 years after treatment. Results A total of 993 patients achieved complete remission (CR) after induction therapy. The negative MRD rates were observed in 61.7% of patients before 1 st consolidation , 95.4% before 2 nd consolidation, 99.3% before 3 rd consolidation, and 99.5% before maintenance therapy , respectively. The median time to achieve negative MRD was 57 (22-301) days with no significant difference between relapsed and non-relapsed patients (p=0.166). A total of 34 patients (30 in the APL2012 trial and 4 in the APL2018 study) relapsed, including 5 at low risk, 9 at intermediate risk, and 20 at high risk. The median recurrence time was 13.5 (3.5-49.0) months after achieving CR, with 94% (32/34) relapsing within 3 years of achieving CR. After induction remission, the risk of relapse decreased with time (44% occurred within the first year, 29% within the second year, 21% within the third year, and only 6% after three years, Figure 1). The median follow-up for the 179 evaluable patients after achieving CR was 39.7 (3.5 -64.6) months in the APL2018 group. The estimated 5-year disease-free survival (DFS) rates were 95.8% (95% CI 92.8-98.9). Prior to the 1 st, 2 nd, and 3 rd consolidation treatment, and before maintenance therapy, the estimated 5-year DFS rates for MRD positive and negative patients were 92.4% (95% CI 86.1-99.2) versus 97.8% (95% CI 94.8-100), p=0.100; 57.1% (95% CI 30.1-100) versus 97.3% (95% CI 94.7-100), p&amp;lt;0.001; 0 versus 96.5% (95% CI 93.1-100), p&amp;lt;0.001; 0 versus 96.8% (95% CI 94.0-99.6), p&amp;lt;0.001, respectively. The results of the APL2012 study were similar to those of APL2018. In the APL2018 study, the estimated 5-year cumulative incidence of relapse (CIR) was 2.51% (95% CI 2.48-2.54), which was 0%, 1.16% (95% CI 1.13-1.19), and 7.83% (95% CI 7.44-8.22) in the low-risk, intermediate-risk, and high-risk patients, respectively (p=0.057). The estimated 5-year CIR for the low to intermediate risk group was 0.74% (95% CI 0.73-0.75), which was statistically different from high-risk patients (p=0.019). The estimated 5-year CIR for MRD positive and negative patients prior to the 1 st and 2 nd consolidation treatments was 4.92% (95% CI 4.76-5.08) versus 1.10% (95% CI 1.08-1.12), p=0.194; 14.29% (95% CI 10.30-18.28) versus 2.08% (95% CI 2.05-2.11), p=0.010, respectively. Conclusion Our results show that MRD after one course of consolidation treatment, rather than before, is a crucial predictive factor for APL relapse. Patients with positive MRD after 1 st consolidation therapy have a high risk of relapse. ATRA-ATO based treatment has significantly reduced APL relapse, so the value of frequent MRD monitoring may be limited. It is recommended to implement an optimized MRD monitoring for risk stratification. For low to intermediate-risk patients and those with negative MRD after a consolidation treatment, the frequency and duration of MRD testing can be minimized. For high-risk patients with sustained MRD negativity for more than 3 years, MRD testing can be considered for discontinuation.

Experience with Blinatumomab in Pediatric Patients in Argentina. Multi-Center Study

Introduction. Very high-risk and relapsed pediatric acute lymphoblastic leukemia (ALL) require high-dose chemotherapy and hematopoietic stem cell transplant (HSCT) as consolidation of treatment. Patients with positive minimal residual disease (MRD) previous to HSCT have lower survival rates. Immunotherapy is thus useful for management of MRD previous to HSCT with less chemotherapy-associated morbidity. Blinatumomab (Blina) is a bi specific monoclonal antibody against CD19 and CD3ε. Objectives. To report the experience with Blina in children with B-ALL in Argentina and assess its toxicity, effectiveness and overall survival (OS). Patients and methods. Retrospective analytical study. The main Argentinian pediatric hospitals participated. Patients younger than 17 years old with B-ALL in 1 st, 2 nd or 3 rd CR who had received 1 or 2 cycles of Blina were included. Those with negative MRD and those who didn´t complete a cycle were excluded. Toxicity was evaluated according to CTC 4.0 and effectiveness according to pre and post-infusion MRD. OS was calculated using Kaplan Meier with GraphPad Prism V5.0. Results. Patients. Thirty patients received Blina and 6 were excluded: 3 due to negative MRD, 2 didn´t complete 1 cycle and 1 for both reasons.Eligible: 24 patients. The median age was 5.5 (range 0.8-17) years. Patients under 1 year: 3. Distribution by sex F/M= 1/1.8. Patients with High Risk ALL represented 42% (n=10) and relapsed ALL 58% (n=14), 10 in 1 st relapse and 4 in 2 nd relapse. Sixteen patients received 1 cycle and 8 received 2. Median MRD previous to Blina was 0.71 (range 0.023-49)%. Thirteen patients received it in CR (MRD &amp;lt;1%): 7 in 1 st CR, 4 in 2 nd CR, and 2 in 3 rd CR. MRD was 1-5% in 6 patients and &amp;gt;5% in 5. Toxicity. During the 1 st and 2 nd weeks, 25% (n=6) and 16% (n=4) presented GII-III neurological toxicity respectively. Blina infusion was temporarily withdrawn for 3 hours to 12 days in 6 patients but all completed the cycle. During the 1 st week, 41.5% (n=10) presented fever. During the entire 1 st cycle 25% (n=6) presented GIII-IV hematological toxicity, 2 patients presented GIV and 1 GIII infectious toxicity. Regarding the 8 patients who received a 2 nd cycle, 1 patient presented febrile neutropenia and 2 presented GIII infectious toxicity without evidence of neurological toxicity. Systemic inflammatory response syndrome was not evidenced. There were no fatal events related to Blina administration. Efficacy. After the 1 st cycle of Blina 58% became MRD negative (n=14), 17% remained in CR (n=4) and 25% didn´t show any response. All patients who received a 2 nd cycle were in CR; however, 5 of them became MRD negative and 3 relapsed later. Ten of the 13 patients who received Blina in CR with MRD+ became negative and 3 relapsed. Of the 5 with MRD 1-5%, 4 became negative, 1 achieved CR with MRD+ and 1 progressed. Of the 5 patients with MRD&amp;gt;5%, 2 achieved CR but none became MRD negative. Fifty percent of the patients received HSCT (n=12): 6 haploidentical, 2 related allogeneic and 4 unrelated. With a median follow-up of 3.5 (1.7-56.9) months, OS at 36 months was 20.3%. Nine patients are alive and in CR (37.5%), 6 post-HSCT and 3 post-Blina still receiving treatment. Five died in CR (4 due to HSCT complications) and 10 died due to disease relapse/progression (3 post HSCT and 7 post-Blina). Conclusions. Blina infusion was safe with the previously described toxicities. The highest response rate was evidenced in patients in CR or with MRD 1-5% pre-Blina. The low survival rate in this cohort may be due to the high rate of relapse/progression and toxicity associated with HSCT, although HSCT is essential to consolidate Blina´s response. Blina therapy is effective in patients with very high risk or relapsed ALL with +MRD. The strategy should focus on the precise indication of patients with MRD+ as a bridge to HSCT.

Breaking Barriers in B-Acute Lymphoblastic Leukemia Treatment: Experience of a Tertiary Hospital with the Use of a Tandem Dual CAR-T 19/22 in Pediatric & AYA Patients

INTRODUCTION Anti-CD19 chimeric antigen receptor (CAR) autologous T-cell therapy tisagenlecleucel (CTL019) has shown efficacy in pivotal trials and real-world data in relapsed/refractory pediatric B-cell precursor acute lymphoblastic leukemia (r/r B-ALL). However, 30-50% of patients relapse, because of CD19 loss in leukemic cells, or persistence/function reduction of CTL019. To improve relapse free survival, multitargeting CAR-T cells, such as bicistronic anti-CD22 and anti-CD19 or single sequential infusion are being developed. We report the safety and efficacy of an in-house manufactured tandem anti-CD19/22 dual CAR-T cells in pediatric, adolescents and young adults (AYA) diagnosed with r/r B-ALL. MATERIAL AND METHODS Eight batches of the tandem anti-CD19/22 dual CAR-T cells were manufactured under Good Manufacturing Practices (GMP) and authorized under a compassionate use program by the Spanish Agency for Medicines and Health (AEMPS). The starting material in all cases was patient´s non mobilized apheresis. After, CD4 + and CD8 + T cells were immune selected, activated with CD3/CD28 beads and transduced with a CD19/CD22 lentiviral vector (from Miltenyi Biotec®). Cell expansion was performed in a CliniMACS Prodigy® device with TexMACs® culture medium supplemented with IL-7 and IL-15. During manufacturing, process and quality controls were carried out and the finished product was infused fresh or cryopreserved. RESULTS A total of 8 patients with a median age of 10.5 (range 6-25) years old received a median of 1 (range 1-3) doses of tandem anti-CD19/22 dual CAR-T cells containing a median of 1.13x106 (range 7.5x105-3x106) cells/kg infused. First 3 patients (37.5%) received cryopreserved product. Two patients (25%) were also diagnosed with Down syndrome (DS). The median time between apheresis and infusion was 10.5 days (range 9-14 ). Five patients (62.5%) received previously tisagenlecleucel and four of them (50%) had also received a hematopoietic stem cell transplantation (HSCT). Leukemia blasts did not express CD19 in 2 patients (25%). Both received tisagenlecleucel previously, and one of them also expressed low levels of CD22. Median pre-infusion minimal residual disease (MRD) was 4.91% (range 0.01-87). Bridging therapy was low dose chemotherapy in 7 patients (87.5%), using inotuzumab in 1 patient. Lymphodepletion regimen was individualized, in 5 patients (62.5%) consisted on fludarabine 30 mg/m2/day for 4 days and cyclophosphamide 600 mg/m2/day for 2 days. Regarding CAR cell product, mean transduction was 41.4 %. Similar CD4 +/CD8 + distribution was seen in most products generated (7/8, 88%), and central memory cells were the main subpopulation found, followed by naïve cells. Most CAR-T cell expansions (88%, median 7, range 7-28) were seen between day +7 and day +21 post-infusion. Regarding toxicity, 7 patients (87.5%) presented cytokine release syndrome (CRS). Three patients (37.5 %) with high pre CAR-T tumor burden (TB) (range 75-87 %) developed CRS grade 3-4 (ASBMT guidelines) followed by ICANS grade 3. On day +28 after infusion, 6 patients (75%) were in complete remission (CR), one of them with positive minimal residual disease (MRD). Two patients died because of disease progression (range 2-4.5 months) and a third one (with DS) because of toxicity before day +28 after CAR-T infusion. Consolidative HSCT was performed in 3 of 6 patients that achieved CR (50%) during the first 90 days after CAR-T cell infusion. During follow up of these transplanted patients (median 8.4 months, range 2.3-12.3), 1 patient (33.3%) relapsed after 11 months. One patient presented central nervous system relapse 1 month after CAR-T infusion and HSCT was performed and with 35 months of follow up, continues alive in CR. The last patient (with DS) relapsed 3 months after infusion and immunotherapy (inotuzumab) has been given in order to perform a second HSCT if CR is achieved. CONCLUSIONS In-house manufacturing of tandem anti-CD19/22 dual CAR-T cells is feasible and reproducible under GMP conditions in an academic institution. Product infusion seems to be well tolerated in most patients, although patients with high TB developed high grade toxicities. Setting bridging therapy, reducing TB and manufacturing time remains a challenge. Our experience suggests the efficacy of its use as a bridge to consolidative HSCT. Nevertheless, a larger number of patients in the context of a clinical trial would be necessary.

Outcomes and Toxicities after Allogeneic Hematopoietic Cell Transplant for Remission Consolidation or Relapse Therapy after CD19 Chimeric Antigen Receptor T Cell Therapy in Children and Young Adults with Acute Lymphoblastic Leukemia

Introduction: CD19-directed chimeric antigen receptor-modified T cells (CART19) induce remarkably high remission rates in relapsed/refractory B-lymphoblastic leukemia (B-ALL), but 50% of children and young adults experience subsequent relapse. Allogeneic hematopoietic cell transplant (HCT) is frequently used after CART19, either for relapse prevention or to treat post-CART19 relapse. However, there is a paucity of safety and outcome data after HCT in this population. This gap is especially relevant because patients often undergo HCT post-CART19 later in their relapsed disease courses (i.e., CR3 or later) than historical patients undergoing HCT for B-ALL. Methods: We conducted a retrospective review of patients aged &amp;lt;30 years with B-ALL who underwent a first HCT for remission consolidation (preemptive cohort) or relapse therapy (relapse cohort) after CART19 at the Children's Hospital of Philadelphia between 2014-2022. The primary outcome was disease-free survival (DFS) from HCT, stratified by cohort. Secondary outcomes included overall survival (OS) and frequency of transplant-related mortality (TRM), graft-versus-host disease (GVHD), sinusoidal obstruction syndrome (SOS), and IgG replacement dependence. Results: The analysis included 41 patients: 19 in the preemptive cohort (pre-planned consolidative HCT, n=5; early loss of B cell aplasia, n=13; positive next-generation sequencing minimal residual disease [MRD], n=1) and 22 in the relapse cohort (morphologic relapse, n=18; detectable flow-based MRD, n=4) (Table 1). With one exception of a patient treated with a CD19/CD28-based product, all patients received investigational or commercial CD19/4-1BB CAR T cells. Median time from CART19 infusion to HCT was 5 months (range, 2-10) for the preemptive and 14 months (range, 5-40) for the relapse cohort. Between CART19 and HCT, 6/19 (32%) preemptive patients received bridging therapy (chemotherapy, n=3; blinatumomab, n=1; inotuzumab, n=2). In the relapse cohort, salvage therapies that successfully induced HCT-acceptable remissions (MRD &amp;lt;0.1%) included inotuzumab (n=11, 50%), cytotoxic chemotherapy (n=5, 23%), blinatumomab (n=2, 9%), CART22 (n=2, 9%), CART19 reinfusion (n=1, 5%), or pembrolizumab (n=1, 5%). At HCT, the majority (79%) of the preemptive cohort was in CR1 or CR2; whereas, the majority (77%) of the relapse cohort was in ≥CR3. All patients received myeloablative conditioning, either total body irradiation-based (n=39) or clofarabine-based (n=2), and 59% received a T cell depleted graft. In the preemptive cohort, with a median follow-up of 36 months, DFS at 1, 2, and 3 years after HCT was 100%, 93% (95% CI, 61-99), and 85% (95% CI, 51-96), respectively (Fig. 1). OS was 100% through 3 years. In the relapse cohort, with a median follow-up of 34 months, DFS was 58% (95% CI, 35-76) and OS was 68% (95% CI, 44-83) by 1 year, and remained the same through 3 years (Fig. 1). When stratified by CD19 immunophenotype at post-CART19 relapse, DFS by 1 year was 67% (95% CI, 34-86) for CD19-positive and 44% (95% CI, 14-72) for CD19-negative disease (p=0.235). Frequently observed toxicities included GVHD and SOS. In the preemptive cohort, 10 patients (53%) developed acute GVHD (3 [16%] were grade ≥3) and 7 (37%) developed chronic GVHD. In the relapse cohort, 11 patients (50%) developed acute GVHD (4 [18%] were grade ≥3) and 6 (27%) developed chronic GVHD. SOS was observed in 1 (5%) preemptive patient and 7 (32%) relapse patients. Four patients (18%), all in the relapse cohort, died of TRM between days +47-65 after HCT. Three had multisystem organ failure in the setting of multiple complications, including severe SOS. One had respiratory failure due to presumed adenoviral pneumonitis. At 1 year post-HCT, 9/18 (50%) preemptive and 8/12 (67%) relapse patients who remained event-free still required IgG replacement. Conclusion: In children receiving their first HCT for post-CART19 remission consolidation, DFS and OS were exceptionally good, without any observed TRM and with comparable rates of GVHD and other major complications to general pediatric HCT populations. For children who were able to undergo HCT following a post-CART19 relapse, of which 77% were in CR3 or later, DFS and OS were also favorable. The majority became long term survivors and all events occurred within 1 year after HCT. However, higher rates of SOS (likely associated with pre-HCT inotuzumab) and TRM were also observed in this cohort.