Minimal Residual Disease Levels Research Articles

Abstract 3637: Ultrasensitive assays for combined detection of CEA+ extracellular vesicles and soluble CEA

Abstract Colorectal cancer (CRC) is the third most common cancer worldwide and is the second leading cause of cancer death in the United States. Carcinoembryonic antigen (CEA) is a biomarker commonly used in the management of CRC. It is typically present at low levels in the blood of healthy adults but is elevated in CRC samples and some other cancers. CEA is most commonly used to monitor treatment efficacy and as a critical part of post-treatment surveillance for residual disease, recurrence, and/or progression/metastasis. The CRC recurrence rate averages >25%, and liver metastasis occurs in 25-30% of cases. Improved CEA assays could enable earlier determination of treatment failure, detection of lower levels of minimal residual disease (MRD) after treatment, or earlier detection of recurrence. CEA is a GPI-linked membrane protein secreted from CRC cells mainly by enzymatic cleavage of its GPI anchor. However, we have identified alternative secretion of CEA in association with extracellular vesicles (EVs). We hypothesized that the soluble CEA produced by canonical shedding and the alternatively secreted CEA may provide distinct information about the tumor or its microenvironment and serve as independent but complementary biomarkers of CRC. We developed ultrasensitive, multi-marker electrochemiluminescence (ECL) immunoassays for measuring intact EVs presenting CEA, CD73, or both in human plasma. We observed that plasma levels of CEA+EVs and CD73+EVs were elevated in some late-stage CRC samples compared to controls. CEA and CD73 double-positive EV (CEA+CD73+EVs) levels were also elevated in the same samples, exhibiting a 3-fold improved separation of these samples with CRC from controls, compared to detection of CEA+EVs. Additionally, approximately 25% of stage IV CRC samples had elevated CEA+EV and CEA+CD73+EV levels while exhibiting low soluble CEA. We confirmed this result in an independent sample set that included samples across all CRC stages, and also identified a few earlier-stage samples that exhibited high CEA+EVs and CEA+CD73+EVs without elevated soluble CEA. Since CEA+CD73+EVs and soluble CEA assays identified different subsets of samples, we combined the two assays into a single classifier for late stage CRC and controls, yielding improved receiver operator characteristic (ROC) with area under the curve (AUC) of 0.975 versus 0.927 for the standard soluble CEA assay or 0.788 for CEA+CD73+EVs alone. These results suggest that assaying EVs presenting multiple surface markers can increase specificity for the detection of tumor-derived EVs relative to a single marker and that assaying both soluble and EV-associated forms of CEA in plasma may improve the value of CEA measurement in CRC. Citation Format: Lucie Hebert, Misk Al-Ameen, Talis Spriggs, Collin Nelson, Evan A. Gizzie, Jeffrey L. Franklin, Robert J. Coffey, David A. Routenberg, Jacob N. Wohlstadter. Ultrasensitive assays for combined detection of CEA+ extracellular vesicles and soluble CEA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3637.

Abstract 5071: Minimal residual disease (MRD) status by peripheral blood mononuclear cells (PBMCs) and circulating tumor DNA (ctDNA) demonstrates rapid, deep, and sustained response in patients (Pts) with relapsed/refractory follicular lymphoma (R/R FL) treated with subcutaneous (SC) epcoritamab monotherapy in the pivotal phase 1/2 EPCORE™ NHL-1 trial

Abstract Introduction: Outcomes in high-risk R/R FL are poor. Epcoritamab SC, a CD3xCD20 bispecific antibody, demonstrated promising efficacy with a manageable safety profile in pts with R/R FL after ≥2 prior lines of therapy (phase 1/2 EPCORE NHL-1; NCT03625037). Here we evaluated MRD with PBMCs and ctDNA in pts with R/R FL and correlated the results with clinical findings. Methods: Adults with R/R FL 1-3A enrolled in EPCORE NHL-1 received epcoritamab SC in 28-day cycles. MRD analysis was performed on longitudinal plasma and PBMC samples collected at prespecified timepoints (clonoSEQ assay, Adaptive Biotechnologies). Screening tumor biopsies were used to identify trackable tumor clones; samples were quantified as tumor clones detected per mL plasma for ctDNA and per one million nucleated cells for PBMC. Overall response and progression-free survival (PFS) were assessed using Lugano criteria by independent review committee. Results: Of 128 pts in the R/R FL 1-3A cohort, 91 with PBMC data and 100 with ctDNA data were MRD evaluable. Overall MRD negativity was achieved in 67.0% [95% CI: 56.4-76.5] of pts per PBMC and 68.0% [95% CI: 57.9-77.0] of pts per ctDNA. Concordance of subject-level MRD status was high (79 of 89 with PBMC and ctDNA data; 88.8%). In most pts, MRD negativity was reached prior to radiographic complete response (PBMC, 76.4%; ctDNA 58.5%). MRD negativity by PBMC preceded ctDNA (median days to MRD negativity, 28 [PBMC] vs 57 [ctDNA]), suggesting a difference in methodology in detecting tumor DNA. Pts who became MRD negative had prolonged PFS (median not reached). Baseline MRD levels were not associated with clinical outcomes (PBMC; Wilcoxon P=0.59, ctDNA P=0.39). PFS was similar in pts achieving MRD negative status (median not reached, PBMC and ctDNA), independent of high-risk/difficult-to-treat disease (≥4 prior lines, Ann Arbor stage III-IV, bulky disease, double refractory, FLIPI 3-5, and POD24). The overall MRD negativity rate was also similar in high-risk pts, except those with ≥4 prior lines of therapy. Conclusions: Based on the EPCORE-NHL-1 study, one of the first pivotal studies to report MRD in pts with R/R FL, epcoritamab drives rapid, deep, and sustained responses as supported by MRD assessment by both PBMC and ctDNA. MRD results correlate with clinical outcome, and achieving MRD negativity is associated with longer PFS. PFS was similar among all pts who achieved MRD negativity, including those with high-risk disease. Citation Format: David Soong, Işıl Altıntaş, John Karavitis, Kim M. Linton, Wojciech Jurczak, Catherine Thieblemont, Kevin Zhao, Edith Szafer-Glusman, Daniela Hoehn, Elena Favaro, Mariana Sacchi, Maria Jure-Kunkel. Minimal residual disease (MRD) status by peripheral blood mononuclear cells (PBMCs) and circulating tumor DNA (ctDNA) demonstrates rapid, deep, and sustained response in patients (Pts) with relapsed/refractory follicular lymphoma (R/R FL) treated with subcutaneous (SC) epcoritamab monotherapy in the pivotal phase 1/2 EPCORE™ NHL-1 trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5071.

Relationship between subtype-specific minimal residual disease level and long-term prognosis in children with acute lymphoblastic leukemia.

Minimal residual disease (MRD) based risk stratification criteria for specific genetic subtypes remained unclear in childhood acute lymphoblastic leukemia (ALL). Among 723 children with newly diagnosed ALL treated with the Chinese Children Leukemia Group CCLG-2008 protocol, MRD was assessed at time point 1 (TP1, at the end of induction) and TP2 (before consolidation treatment) and the MRD levels significantly differed in patients with different fusion genes or immunophenotypes (P all < 0.001). Moreover, the prognostic impact of MRD varied by distinct molecular subtypes. We stratified patients in each molecular subtype into two MRD groups based on the results. For patients carrying BCR::ABL1 or KMT2A rearrangements, we classified patients with MRD < 10-2 at both TP1 and TP2 as the low MRD group and the others as the high MRD group. ETV6::RUNX1+ patients with TP1 MRD < 10-3 and TP2 MRD-negative were classified as the low MRD group and the others as the high MRD group. For T-ALL, We defined children with TP1 MRD ≥ 10-3 as the high MRD group and the others as the low MRD group. The 10-year relapse-free survival of low MRD group was significantly better than that of high MRD group. We verified the prognostic impact of the subtype-specific MRD-based stratification in patients treated with the BCH-ALL2003 protocol. In conclusion, the subtype-specific MRD risk stratification may contribute to the precise treatment of childhood ALL.

Minor population of NK lymphocytes with CD19 coexpression

Single reports were published concerning a minor subpopulation of NK cells with weak coexpression of the B cell antigen CD19 in the patients’ blood and bone marrow. The frequency and relative number of CD56+CD19+dim cells is virtually not assessed, and there is no data on their phenotypic characteristics, as well as the connection of this subpopulation with any disease state. The purpose of the present study was to assess the frequency, relative quantity and phenotypic characteristics of CD56+CD19+dim lymphocytes in blood of patients referred for assessment of the lymphocyte subpopulation profile. Peripheral blood of immunocompromised individuals was taken, and subpopulation composition of lymphocytes was determined using eight-color flow cytometry (markers: CD3, CD4, CD8, CD19, CD25, CD45, CD56, HLA-DR). To estimate incidence of the CD56+CD19+dim subpopulation, we have carried out a retrospective analysis of LMD files on 1210 studies for 935 patients (average age, 39.8±14.7 years old) including 84 children under 18 years old. The study was performed repeatedly for some patients. Phenotyping of CD56+CD19+dim cells was performed using a panel of antibodies to B cell, T/NK cell antigens. The occurrence of blood samples containing CD56+CD19+dim was 1.2%, with a relative content of 2.1±1.9% among total lymphocyte population (0.8±0.6% of leukocytes). Long-term persistence of the subpopulation was noted in the patients throughout the entire observation period. The comparison of specific marker expression by NK CD56+CD19+dim, and CD56+CD19- cells revealed high expression of CD2, CD57, reduced expression density of CD7, CD16, CD38. The phenotype of the studied NK cell subpopulation was as follows: CD56+dimCD19+dimCD2+brightCD7+dimCD11c+CD16+dimCD38+dimCD45RA+CD57+CD94+dimNKG2D+CD3-CD4-CD5-CD20-CD21-CD25-CD45R0-CD62L-CD79b-CD117-, with variable expression of CD8 and HLA-DR. The phenotype is consistent with activated terminally differentiated adaptive NK associated with cytomegalovirus infection. The individuals with CD56+CD19+dim had a history of CMV-infection and reactivation of chronic EBV-infection at the time of the study. A probable cause of CD19 coexpression may be trogocytosis of B cell membrane fragments by natural killer cells during active EBV-infection. CD56+CD19+dim lymphocytes can reach noticeable values thus altering the results of studies performed by flow cytometry. The errors are most likely to occur upon assessing the minimal residual disease levels in acute B cell leukemias. The minor CD56+CD19+dimNK subpopulation may be detected in routine immunological analysis. Its functional features and association with certain disorders require further studies.

Clinical features and outcomes of patients with acute myeloid leukemia: the single-center experience of 668 patients in China

ABSTRACT Objective To investigate efficacy and prognostic factors in the treatment of adult newly-diagnosed acute myeloid leukemia (AML) with or without allogeneic hematopoietic stem cell transplantation (Allo-HSCT). Methods We retrospectively analyzed 668 patients with newly-diagnosed AML (non-M3 type) in the Department of Hematology at Shanghai Changhai Hospital from January 2012 to December 2021. Based on different induction chemotherapy regimens, patients were categorized into an IA (idarubicin, IDA + cytarabine, Ara-C) (3 + 7, regimen) group (n = 303) and a DA (daunorubicin, DNR + cytarabine, Ara-C) (3 + 7, regimen) group (n = 365) with or without allo-HSCT. Minimal residual disease (MRD), complete response (CR), overall response rate (ORR), progression-free survival (PFS), overall survival (OS), and adverse effects (AE) were analyzed and compared. Characteristics significantly associated with overall or progression-free survival (OS or PFS) upon univariate analysis were subsequently included in a Cox proportional hazard model. Results This study used data from 668 AML patients. After induction therapy, the CR rate in the IA group was 70.63% and ORR was 79.87%, which were significantly higher than those in the DA group (with a CR rate of 56.99% and an ORR of 70.14%) (P = 0.0002 and P = 0.0035, respectively). There were no significant differences in drug safety between the two chemotherapy regimens used in IA and DA (P > 0.05). The recurrence rate was lower in patients with an MRD < 0.001 than in patients with an MRD ≥ 0.001. A continuous negative MRD during the period is significant because it is associated with prolonged OS and PFS of AML patients. Data from 100 patients in the two groups who underwent allo-HSCT were analyzed using univariate analysis and the Cox proportional hazards model. From the multivariate analysis, MRD was found to be the only independent predictor of OS (P = 0.042; HR 1; 95%CI 0.00–0.76). Conclusion In the treatment of adult AML patients, IA regimen is associated with a high CR rate and ORR rate and does not increase treatment-related toxicity. IA regimen prolongs OS and PFS in AML patients and reduces the likelihood of leukemia cells’ subsequent infiltration into the central nervous system. There is a high correlation between the level of MRD after treatment and the patient's bone marrow recurrence. To obtain superior treatment effects for patients undergoing allo-HSCT, the MRD should be reduced to less than 0.001 before pretreatment. A negative MRD before allo-HSCT can prolong OS in patients with AML. We examined the clinical characteristics and outcomes of AML patients in China, finding novel information on prognostic factors and primary treatment of AML that may be applicable in routine clinical practice.

[Clinical Analysis of Philadelphia Chromosome-Like Acute Lymphoblastic Leukemia in Children].

To explore the clinical characteristics, molecular characteristics, treatment and prognosis of pediatric Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) with a therapeutic target. A total of 27 patients of Ph-like ALL with targeted drug target were initially diagnosed in Children's Hospital of Soochow University from December 2017 to June 2021. The data of age, gender, white blood cell (WBC) count at initial diagnosis, genetic characteristics, molecular biological changes, chemotherapy regimen, different targeted drugs were given, and minimal residual disease (MRD) on day 19, MRD on day 46, whether hematopoietic stem cell transplantation (HSCT) were retrospective analyed, and the clinical characteristics and treatment effect were summarized. Survival analysis was performed by Kaplan-Meier method. The intensity of chemotherapy was adjusted according to the MRD level during induced remission therapy in 27 patients, 10 patients were treated with targeted drugs during treatment, and 3 patients were bridged with HSCT, 1 patient died and 2 patients survived. Among the 24 patients who did not receive HSCT, 1 patient developed relapse, and achieved complete remission (CR) after treatment with chimeric antigen receptors T cells (CAR-T). The 3-year overall survival, 3-year relapse-free survival and 3-year event-free survival rate of 27 patients were (95.5±4.4)%, (95.0±4.9)% and (90.7±6.3)% respectively. Risk stratification chemotherapy based on MRD monitoring can improve the prognosis of Ph-like ALL in children, combined with targeted drugs can achieve complete remission as soon as possible in children whose chemotherapy response is poor, and sequential CAR-T and HSCT can significantly improve the therapeutic effect of Ph-like ALL in children whose MRD is continuously positive during induced remission therapy.

Crebbp Mutations Are Associated with Slow Minimal Residual Response to Upfront Induction Chemotherapy in Pediatric High-Hyperdiploid B-Cell Precursor Acute Lymphoblastic Leukemia Treated in the AIEOP-BFM ALL 2009 Protocol

Introduction High-hyperdiploidy (HHD) represents the most common cytogenetic abnormality found in B-cell precursor acute lymphoblastic leukemia (B-ALL). Despite the overall good outcome of this entity, up to 15% of patients suffer from disease relapse. Genome-wide ALL studies with next-generation sequencing (NGS) approaches identified a distinct pattern of somatic mutations in HHD ALL, enriched in mutations occurring in genes activating the RAS pathway and in CREBBP gene, especially in relapsed patients. Although several studies have attempted to explore the prognostic significance of these mutations that are potentially susceptible of targeted therapies, their impact in uniformly treated patients with newly diagnosed B-ALL is not well characterized, and there is no consensus on their clinical significance. In this study, we aimed to investigate the frequency of recurrent mutations and to define their prognostic role in the setting of newly diagnosed HHD B-ALL, by using end of induction minimal residual disease (MRD) and event-free survival (EFS). Methods We included 101 consecutive patients, aged between 1-17 years, with newly diagnosed HHD B-ALL and enrolled in the AIEOP-BFM ALL 2009 protocol (NCT01117441). HHD was defined by the presence of a DNA index ≥1.16 and lack of ETV6::RUNX1, BCR::ABL1, KMT2A::AFF1 translocations. Diagnostic leukemia samples of 89 patients were sequenced using a validated clinical NGS panel profiling 39 genes involved in leukemia pathogenesis. Mutations affecting RAS pathway ( NRAS, KRAS, PTPN11, NF1, BRAF) and mutations affecting CREBBP gene were included. In addition, digital multiplex ligation-dependent probe amplification (digital MLPA) analysis for the identification of IKZF1 deletion and IKZF1plus phenotype was evaluable for 67 patients. A Fisher exact test and Wilcoxon rank sum test were used for categorical and continuous variables. MRD was assessed by an IG/TR PCR assay at day +33 of induction (timepoint 1, TP1) and at day +78 (timepoint 2, TP2). EFS was estimated by using Kaplan and Meier method and differences between groups were tested using the log-rank test. Resistance to treatment, relapse, death or second malignant neoplasm were considered as events, whichever occurred first. Results Patients' characteristics according to the presence of studied mutations are shown in Table 1. Mutations affecting RAS pathway and CREBBP occurred in 57.3% and 12.3% of HHD B-ALL patients, respectively. Clonal RAS pathway mutations, defined by variant allele frequency (VAF) ≥25%, were found in 16.8% of cases. Among the 11 patients with mutations of CREBBP, 7 had also a mutation affecting KRAS and the association was significant (p=0.03). Seven patients (10.4%) had deletion of IKZF1, of whom 5 (7.4%) were IKZF1plus. There was no association between any mutations or IKZF1 status and patient presenting characteristics, as including sex, age and WBC counts. MRD was evaluable in 84 patients at the end of induction (TP1). MRD level ≥5x10 -4 was found in 36.4% of patients with CREBBP mutations and in 9.6% of those without, and this association was statistically significant (p=0.02). Additionally, MRD negativity was found in 9.1% and 38.4%, respectively. At TP2, MRD was still detectable in 45.5% of patients with CREBBP mutations compared to 20.6% of those with CREBBP wildtype (p=0.08). Similarly, we observed an association between RAS pathway mutations and MRD at TP1 (p=0.01) with a marked difference in MRD negative patients (21.3% vs 51.4% in patients with and without mutations). No association was observed between MRD level and clonal RAS pathway mutations or IKZF1 status. Despite the association between CREBBP mutations and high MRD at TP1, no significant difference in final high-risk group was found (27.3% vs 11.5%, p=0.17). A total of 8 events were observed, of which 6 were relapses. No impact of any mutations or IKZF1 status in term of 5-years EFS was found in our cohort. Conclusion Our study shows that CREBBP mutations in HHD B-ALL are associated with inferior early response to treatment in terms of MRD level at the end of induction (TP1) but not with worse EFS with our whole current chemotherapy backbone. Therefore, the inclusion of CREBBP mutated patients in very low risk protocols should be carefully evaluated. The validation of these findings is ongoing through the analysis of patients treated by other international study groups adopting different chemotherapy backbones.

Minimal Residual Disease-Negative Complete Remission at the End of Induction Is a Prognostic Indicator of Long-Term Survival in Adult Patients with Ph+ Acute Lymphoblastic Leukemia Receiving First-Line Therapy

Background: Minimal residual disease (MRD) is an established, prognostic indicator for patients with acute lymphoblastic leukemia (ALL), including Philadelphia chromosome-positive (Ph+) ALL. The objective of this study was to evaluate the association between MRD and long-term outcomes (event-free survival [EFS] and overall survival [OS]) in adult patients with Ph+ ALL receiving first-line therapy. Methods: A systematic literature review (SLR) was performed to identify eligible studies, including interventional and observational studies reporting survival outcomes by MRD status in adults ≥18 years of age with Ph+ ALL receiving first-line therapy. Key outcomes and data elements of interest were EFS, OS, and MRD or MRD-negative complete remission (CR) at end of induction (EOI). Aggregate study-level and individual patient data (IPD) meta-analyses were conducted. The IPD approach included a log-level MRD analysis to evaluate the concordance of deepening levels of molecular response with long-term EFS or OS in patients with CR and an analysis evaluating the utility of MRD-negative CR over CR alone. The analyses were conducted using frequentist and Bayesian approaches with fixed-effects and random-effects models. Kaplan-Meier method with log-rank tests was used for survival analysis. Where applicable, Cox regression method with and without adjustment for prognostic factors were applied. Results: The SLR identified 19 publications (18 unique studies), 10 (9 unique studies) of which had sufficient MRD and EFS/OS data. Nine studies (n=704) were included in the aggregate study-level analysis; 3 of the 9 studies (Phase II AP24534-11-001 [Jabbour et al. 2015], Phase II GIMEMAINCB 84344-201 [Martinelli et al. 2017], and Phase III GRAAPH-2005 RCT [Chalandon et al. 2015]) had IPD available. MRD was assessed by polymerase chain reaction in all 9 studies. Findings from the aggregate study-level meta-analysis demonstrated a significant long-term survival benefit (EFS and OS) in favor of patients with MRD-negative CR. This significant benefit was generally consistent across analysis types (base-case or sensitivity analyses), analytic models (fixed- or random-effects), and approaches (frequentist or Bayesian). Evidence from the IPD analysis, based on patients with CR, was mostly consistent with the aggregate study-level meta-analysis, supporting MRD-negative CR at the EOI as being predictive of EFS or OS. Log-level MRD analysis showed that patients who are in CR with deeper molecular response ( BCR::ABL1/ABL1 ≤0.01%) at EOI have significantly better long-term EFS and OS compared with patients with other levels of MRD (ie, BCR::ABL1/ABL1 &amp;gt;0.01%). This significant long-term survival benefit was also demonstrated in patients with MRD-negative CR ( BCR::ABL1/ABL1 ≤0.01%) at EOI compared with patients who were MRD-positive ( BCR::ABL1/ABL1 &amp;gt;0.01%) ( Table 1) or based on achievement of CR alone (regardless of BCR::ABL1/ABL1 values). Results from the IPD analysis standardized for outcome definition, with and without adjustment for prognostic factors (age, gender, white blood cell count at baseline, and stem cell transplant), were consistent with the findings from the log-level MRD analysis. The analysis showed a significant association between MRD-negative/MRD-negative CR status (ie, BCR::ABL1/ABL1 ≤0.01% threshold) at EOI with improved long-term EFS and OS. The results were significant for EFS in both the adjusted and unadjusted models. The OS results were significant only in the unadjusted model. Conclusions: These analyses indicate that a deeper molecular response at EOI results in better long-term EFS and OS; MRD-negative CR has a greater prognostic value than CR and is strongly associated with long-term EFS and OS in patients with Ph+ ALL. Acknowledgement: The authors acknowledge APHP, who provided the individualized patients data for the GRAAPH study.

RADAR Trial: MRD Response Adapted Trial for Newly Diagnosed Transplant Eligible Myeloma Patients

Background: Achieving and sustaining minimal disease negativity improves clinical outcomes in newly diagnosed transplant eligible myeloma (NDMM). Outcomes in NDMM are influenced by levels of minimal residual disease (MRD) post therapy and genetic risk by FISH or molecular profiling prior to therapy. Trials exploring response adapted post transplant strategies using MRD as a prognostic tool are going. We provide an update of the current UK-MRA RADAR study. Study Design/Methods: The UK-MRA RADAR study is a national multicentre risk adapted response guided multiarm multistage (MAMS) phase II/III trial which will recruit 1400 patients with NDMM eligible for ASCT. Eligible patients receive induction with 4 cycles of RCyBorD (lenalidomide, cyclophosphamide, bortezomib, dexamethasone), followed by high-dose melphalan and stem cell rescue. Patients with high-risk disease receive Isatuximab in addition to RCyBorD during induction. Genetic risk classification is performed using standard of care FISH testing at local sites. Participants enter the standard or high risk pathway based on presence of atleast two markers: t(4;14), t(14;16), t(14;20), del(17p), del(1p) and gain (1q21). MRD analysis in RADAR uses a multiparametric flow cytometry panel with CD319 and Cd229 used to augment post-isatuximab sample analysis at a central laboratory, with negativity defined at threshold of 10 -5(Figure 1). For standard risk patients, the study addresses two pivotal MRD-related questions: 1) For those with sustained MRD negativity after 12 months of isatuximab maintenance, can treatment be stopped safely to allow patients a treatment-free interval thereby reducing treatment burden? 2) For patients with persistent detectable disease after ASCT, does intensification of consolidation and maintenance therapy augment conversion to MRD negativity compared to lenalidomide maintenance? High risk participants receive intensified consolidation and maintenance regimens post_ASCT aligned to OPTIMUM/MUKnine ( NCT03188172). A further amendment is planned to explore the activity of a BCMA bispecific antibody in high risk patients pre and post transplant. It is hypothesised this will improve both depth (MRD negativity) and ability to sustain response (PFS). Feasibility and benefits of multimodal MRD incorporating imaging and blood-based testing will be tested in the new high risk pathway. In RADAR, we will also gather information on how patients respond to receiving information about their genetic risk assignment and treatment response (MRD). This is a unique opportunity to understand the patients perspective on personalised medicine, which has implications for implementation in clinical care, and future trial design. Trial Progress. RADAR opened to recruitment in May 2021 and is currently recruiting across 87 hospital sites with a further 16 sites in set up. 504 patients have been recruited; 87 are classified as high risk on FISH analysis ( 375 standard risk, 15 unable to determine, 19TBC, 8 withdrawn before FISH analysis). 220 have reached D+100 MRD assessment. Trackable phenotypes were available in 98.2% of patients. Discussion: The RADAR study simultaneously tests escalation and de-escalation strategies, according to disease response, and using MRD to guide post transplant therapy in NDMM patients. Use of central flow cytometry laboratory for MRD has proven feasible in patients to date. Recruitment is on target and outcomes will provide RCT-level evidence for the adaptive use of MRD in treatment allocation and intensification post transplant. The platform design enables adaptation with ability to test new strategies for high risk patients and for standard risk patients who are MRD positive post-transplant. Funding This trial is funded by Cancer Research UK (C9203/A24078), Sanofi Genzyme and Celgene : A BMS company. Sanofi Genzyme and Celgene: A BMS company provide study drug and unrestricted educational grants to support study coordination. This work is also supported by Core Clinical trials unit infrastructure from Cancer Research UK ( C7852/A25447). The funder of the study had no role in study design, data collection, data analysis, data interpretation or writing of the abstract

Exploring the Efficacy and Feasibility of Outpatient Blinatumomab Short Cycles in Pediatric Patients with Relapsed/Refractory B-ALL in LMIC: Preliminary Results

CONCLUSIONS In low- and middle-income countries (LMIC) like Mexico, the treatment of patients with refractory or relapsed acute lymphoblastic leukemia (r/r ALL) represents a significant challenge due to limited access to immunotherapy and hematopoietic stem cell transplantation (HSCT). As a result, the one-year survival rate is reported to be less than 20%. The use of intensive chemotherapy has not improved survival in this group due to its high toxicity. Blinatumomab uses the patient's own cytotoxic T-cells to induce lysis of the leukemia cells expressing CD19 and has been established as a bridge therapy for transplant in patients with r/r B-ALL. The usual pattern of administration is a dose scaled up to complete a total of 28 days of infusion. According to various studies, it is considered appropriate to use repeated cycles of this antibody to achieve the desired response. However, there are also reports of patients achieving complete responses within the first 15 days of the cycle. The high costs of Blinatumomab's administration present a significant barrier to its use in LMIC. As a consequence, the application of prolonged or multiple cycles of the drug becomes unaffordable for a substantial portion of the global population. Demonstrate the safety and efficacy of utilizing short cycles of Blinatumomab (21 days) as a bridge therapy to HSCT in pediatric patients with r/r ALL to explore the potential cost-reduction benefits and increased access to this therapy in resource-limited settings. A pilot clinical trial was conducted. Pediatric patients (&amp;lt;16 y) diagnosed with r/r B- ALL between the years 2020-2022 were included. The usual Blinatumomab dosing regimen was adopted, but the total duration of the cycle was reduced. Continuous infusion was initiated at a dose of 5 mcg/m2/day for the first 7 days, followed by an increase to 15 mcg/m2/day for 14 days (21-day total treatment duration). To mitigate cytokine release syndrome (CRS) a single dose of dexamethasone was administered at the start of the infusion and after the dose escalation. The entire regimen was administered on an outpatient basis, with an infusion pump at home. Caregivers were instructed on the operation of the pump and detection of any signs of toxicity, with 24/7 contact with the medical team and the option to hospitalize if necessary. A total of 7 patients were included, and all of them received Blinatumomab in a single 21-day cycle. The median age was 5 years (15 mo- 13 y). The majority of patients were stratified as high-risk at the time of diagnosis (85.7%). Among the patients, 4 had relapsed ALL, while 3 had refractory disease. The median time from the initial diagnosis to the use of Blinatumomab was 16 months (ranging 7-29 mo), and the median number of treatment lines before Blinatumomab administration was 3 (ranging 2-4). The highest level of minimal residual disease (MRD) detected before the Blinatumomab treatment was 4.2% (by flow cytometry). Remarkably, all patients (100%, n=7) achieved a complete response, with MRD being negative for flow cytometry after the 21-day cycle of Blinatumomab. In 5 out of 7 patients, the administration of the drug was completely ambulatory. Only 2 patients experienced grade I CRS which was successfully managed with steroids. No adverse effects greater than grade 2 were reported, and no patient required support with transfusion therapy during or after the infusion. There were no reports of profound neutropenia, and no treatment-related deaths occurred. HSCT was conducted in all patients to consolidate the response, with 6 patients undergoing haploidentical and 1 patient undergoing identical allogeneic HSCT. At present, 5 out of 7 patients are alive and in complete remission. One patient died on day +51 post-HSCT due to pulmonary thromboembolism, and the second patient experienced relapse on day +395 post-HSCT and subsequently died on day +640 due to disease progression. The median overall survival at 3 years was 66.7%, with a median follow-up period of 21 months (4 -30mo). An alternative outpatient, low-toxicity and cost-effective treatment approach was implemented. The findings from this study could shed light on a promising strategy to improve the outcomes for patients with ALL patients with limited therapeutic options, particularly in the context of resource-constrained settings.

Evaluation of Hematopoiesis and Disease Status in Patients with Hematologic Malignancies Using Whole-Body Bone Marrow Imaging with 18 F-FLT PET/MRI

Introduction Bone marrow examination is primarily focused on a limited region of the ilium, thereby inadequately assessing systemic hematopoiesis or residual tumor cells after therapy. Magnetic resonance imaging (MRI) of the spine can examine the fat marrow, but does not provide a direct assessment of hematopoietic cells. The use of the radiopharmaceutical 3‘-deoxy-3‘- 18F-fluorothymidine ( 18F-FLT) positron emission tomography (PET), which measures nucleic acid synthesis, can provide a quantifiable means of assessing hematopoietic cell proliferation. The combination of whole-body MRI and 18F-FLT PET may provide a more accurate assessment of whole-body bone marrow hematopoiesis in hematologic malignancies. Methods A prospective, open-label, exploratory study (UMIN000041491) was conducted to evaluate hematopoiesis using 18F-FLT PET/MRI imaging in patients planned for allogeneic hematopoietic stem cell transplantation. Subjects were patients over 20 years of age with hematologic malignancies who underwent 18F-FLT PET/MRI imaging before transplantation and at 50 and 180 days after transplantation. After an intravenous injection of 185 MBq 18F-FLT, patients were transferred to a simultaneous 3.0 T PET/MR scanner (Signa PET/MR; GE Healthcare, Waukesha, WI). The PET acquisition was performed in 3D mode with a 5.5 min/bed position (89 slices/bed) and a 24-slice overlap in 5 to 6 beds. The percentage of fat marrow was determined using MRI proton density fat fraction (PDFF) with a 1 cm 2 region of interest at 26 sites: cervical spine (C2-C7), thoracic spine (Th1-Th12), lumbar spine (L1-L5), and superior posterior iliac spine (two sites on each side). Similarly, the standardized uptake value (SUV) of 18F-FLT was measured. Results Thirteen patients with hematologic malignancies (8 with acute myeloid leukemia, 2 with acute lymphoblastic leukemia, and 3 with malignant lymphoma) were evaluated between August 2020 and April 2023. The median age of the patients was 46 years (range: 23 ~ 65). Seven patients underwent cord blood transplantation, while the remainder received peripheral blood stem cell transplantation. Prior to transplantation, all 13 patients underwent 18F-FLT PET/MRI scans. After transplantation, due to treatment-related death or worsening of the primary disease, 11 patients underwent scans on day 50, while 8 patients underwent scans on day 180, resulting in a total of 32 evaluable scans. In the 28 cases in hematologic remission at imaging, mean PDFF values were higher in the iliac (63.3% ± standard deviation 13.9) and lumbar (60.4% ± 14.9) than in the thoracic (47.2% ± 17.1) and cervical (37.5% ± 14.8) spines. Conversely, mean 18F-FLT SUV was higher in the thoracic (6.98 ± 2.31) and cervical (6.82 ± 2.03) than in the lumbar (5.06 ± 2.39) and iliac (3.95 ± 1.88) spine. This site-specific disparity in PDFF and 18F-FLT SUV was consistent both before and after transplantation. In all 32 cases, the median correlation coefficient between PDFF and 18F-FLT SUV was -0.60 (range: -0.92 ~ 0.17, Pearson's cumulative correlation coefficient), and 25 cases (78.1%) had a negative correlation coefficient below -0.4. Comparing the 12 cases with residual disease (4 cases with hematologic non-remission and 8 cases with hematologic remission but abnormal G-banding or quantitative polymerase chain reaction) with the 20 cases in complete remission, the correlation between PDFF and 18F-FLT SUV was significantly weaker in the residual disease group (-0.37 vs. -0.75, p&amp;lt;0.001, Mann-Whitney U test). When the correlation coefficient threshold was set at -0.57, the ROC curve for determining residual disease had an AUC of 0.86, a sensitivity of 83.3%, and a specificity of 85.0%. Discussion It is important to understand that the fat marrow and hematopoiesis degree in the iliac bone differs from that in the thoracic and cervical spine throughout the transplantation process, thereby influencing the disease status evaluation in hematological malignancies. The correlation between PDFF and 18F-FLT SUV, which is strong in cases of remission, weakens in cases of residual disease, even at the level of minimal residual disease. This discrepancy may reflect aberrant tumorigenic cell proliferation that differs from the norm. Thus, 18F-FLT PET/MRI was useful in understanding the hematopoietic and disease state of the systemic bone marrow in patients with hematologic malignancies.

Treatment of Molecular Relapses in Pediatric AML Saves Toxicities Prior to Hematopoietic Stem Cell Transplantation (HSCT) - Results of AMoRe2017 Trial

Introduction The long-term survival for children with AML has increased to 70 % - but relapsed AML patients still have a poor prognosis (about 30 %). Recent data suggest that in AML residual leukemic cells below the 5 % limit required for a morphologic CR can adversely affect outcome and that increasing levels of Minimal Residual Disease (MRD) are almost always followed by clinical relapse. Early MRD detection provides an opportunity to implement pre-emptive therapies as a bridge-to-transplant approach, to potentially prevent hematological relapse and increase the probability of successful HSCT. A promising approach is the use of Azacitidine (Aza) for treatment of molecular relapse which we evaluated within the clinical trial “AMoRe2017”. Aza has been shown to reverse epigenetic changes in malignant cells, restoring the normal function of genes critical for differentiation and cell cycle control. The trials' primary objective was the evaluation of the effect of Aza treatment in AML subjects at molecular relapse after CR1 regarding molecular response prior to further treatment (reinduction/HSCT). Patients and Methods AMoRe2017 enrolled children with AML (N=20), &amp;gt;3 months &amp;lt; 21 yrs., of which n=1 dropped out due to laboratory measurement error. The median age was 10.9 yrs. (range 1.7-18.9 yrs.). N=10 (53 %) had a CBFB-AML, n=5 (27 %) KMT2A-rearrangement, n=1 (5 %) NUP98::NSD1, n=1 (5 %) FLT3-ITD, n=1(5 %) PICALM::MLLT10 and n=1 (5 %) NPM1. N=8 (42 %) were classified to subgroup M2, n=4 (21 %) to M5, n=3 (16 %) into M1 and n=4 (21 %) to M4/M4 Eo. At baseline, the white blood cell count averaged 3.8x10 9/l (range 0.1-6.6x10 9/l), without leukemic blasts in PB or BM. Each patient had at least one quantifiable genetic marker (rt-PCR), achieved CR with molecular remission, following initial therapy, and subsequently had a molecular relapse (defined as an increase of the MRD level by at least 1 log, less than 5 % BM myeloblasts and absence of proven extramedullary disease). Patients were treated for up to 6 cycles with 75 mg/m 2 i.v. Aza on days 1 to 7 in a 28-day cycle. Molecular response was categorized as molecular stabilization (&amp;lt; 1 log decrease or increase from baseline), molecular progression (≥ 1 log increase from baseline without hematological relapse), molecular improvement (≥ 1 log decrease from baseline), and hematological relapse (at least 5 % blasts in PB and/or BM and/or proven histological extramedullary relapse). Results During Aza treatment, 10 patients (53 %) had an open relapse, which occurred in n=9 after 1 st or 2 nd cycle. These patients were monitored for KMT2A::SEPTIN6 (n=1), KMT2A::MLLT3 (n=2), PICALM::MLLT10 (n=1), FLT3-ITD (n=1), NUP98::NSD1 (n=1), CBFB::MYH11 (n=1) and RUNX1::RUNX1T1 (n=3). 9 patients (47 %; RUNX1::RUNX1T1 n=5, CBFB::MYH11 n=1, NPM1 n=1, KMT2A::ELL n=2) showed a molecular response to Aza and proceeded to HSCT without receiving intensive re-induction treatment. 16 out of the 19 patients are alive (overall survival 84 %), 2 died from HSCT-associated side effects (VOD, infection), and 1 from disease progression. Considering genetics, 7 out of 11 patients with favorable genetics (core-binding leukemia n=10 or NPM1 n=1) responded to the Aza therapy ( RUNX1::RUNX1T1 n=5, CBFB::MYH11 n=1, NPM1 n=1). By contrast, in cases with KMT2A-rearrangements, NUP98::NSD1 or FLT3-ITD, 2 out of 8 patients showed improvement or stabilization. In total 152 adverse events (AEs) associated to Aza were reported, the majority classified as grade 1 or 2 (n= 114; 75 %). Mainly affected was the hematopoietic system (40 %), the gastrointestinal tract (15 %), kidney and liver (14 %). Cardiotoxicity was not reported. Discussion The use of Aza as pre-emptive “bridge-to-transplant” approach treating the molecular relapse showed efficacy in patients with RUNX1::RUNX1T1, CBFB::MYH11, NPM1, and KMT2A::ELL, who could be transplanted directly. This pilot provided proof of principle that molecular relapses could be treated, and MRD levels be reversed, at least in subgroups. In addition, avoiding intensive re-induction therapy prior to HSCT is feasible. Conversely, the relatively slow-acting mechanism of epigenetic therapy seems to be ineffective in highly proliferative subgroups of AML with KMT2A-rearrangements or FLT3-ITD. Further studies are mandatory to define treatment options for molecular relapses of non-favorable pediatric AML and to confirm potential improvement of outcome post-HSCT.

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.

Independent Prognostic Value of DNA-Based Minimal Residual Disease Assessment in Unselected Population-Based Cohort of Pediatric Acute Myeloid Leukemia

Monitoring of minimal residual disease (MRD) in childhood acute myeloid leukemia (AML) can assess response to treatment and predict relapse. Current MRD monitoring methods in childhood AML - flow cytometry and quantitation of fusion transcripts - have significant shortcomings and do not cover the entire spectrum of patients. We demonstrated (Lukes et al, Hemasphere 2020) the feasibility of MRD monitoring using DNA-based primary targets in selected subgroups of AML. Now we extended this approach to the unselected consecutive population of pediatric AML. We included 133 children (0-18) out of 135 consecutively diagnosed with AML in Czechia (2012-2022), all treated on non-MRD-based protocols. For genetic characterization, we used algorithm consisting of screening for recurrent gene fusions and mutations by PCR and targeted-NGS followed by transcriptome sequencing (WTS). Targeted-NGS (or PCR) was performed to identify genomic fusion sequences. 130 patients were eligible for MRD monitoring (3 patients died early). MRD was preferentially monitored by DNA-based qPCR (alternatively by amplicon NGS or qRT-PCR). MRD levels were expressed relative to diagnosis. Primary genetic aberration was found by targeted screening in 102 children; remaining 31 were further investigated using WTS. In 27 children, WTS identified rare, novel or atypical primary genetic aberrations not covered by the targeted screening. Primary genetic aberration was found in 97% of cases (129/133). Majority of AMLs (81%) were classified into common subtypes: AML with KMT2Ar, PML::RARA, RUNX1::RUNX1T1, CBFB::MYH11, mutation (m) of GATA1 , CEBPA or NPM1. Recurrent but rare aberrations in pediatric AML were found in 16 patients: UBTFm, RUNX1m, HOXA10 translocation, KAT6A::CREBBP, KAT6A::LEUTX, DEK::NUP214, BCR::ABL1, NUP98::NSD1 and CBFA2T3::GLIS2. In 5 patients, fusion genes were found, previously described sporadically (SPFQ::ZFP36L216, XPO1::TNRC1817) or not at all in AML (ETV6::CTNNB1, FUS::FEV, ZEB2::RUNX1). Only secondary aberrations were identified in remaining 4 patients. In 82 patients (of 84 examined), genomic fusion sequence was found and qPCR designed and used. Of 41 children carrying genetic aberrations other than fusions, in 29 quantification system was successfully implemented. Altogether, quantification system for MRD monitoring was established in 122 patients, with primary aberrations as DNA targets in 116 patients, reaching sensitivity of 10 -4 in 112 and 5x10 -4 in 4 patients. Four and two children were monitored with sensitivity of 10 -4 using secondary aberrations as DNA targets or by qRT-PCR-based quantification of fusion transcripts, respectively. MRD clearance significantly differed among genetic subtypes: while GATA1m AML had the fastest response, in contrast to patients with prognostically favorable genetic subtypes - CBFB::MYH11, RUNX1::RUNX1T1, CEBPAm and NPM1m - who were treated predominantly on the standard risk (SR) arm of the AML-BFM 2012 Registry Protocol and none achieved molecular remission (mREM) at D28 (70% vs 0%, p &amp;lt; 0.0001). Significant proportion of patients (25-89% within individual subtypes) did not achieve mREM even after the last block of chemotherapy. The response of patients with KMT2Ar AML and AML classified into remaining subtypes (predominantly intermediate- and high-risk) was overall faster compared to prognostically favorable subtypes on SR arm (KMT2Ar D28 mREM 33% vs 0% at D28, p &amp;lt; 0.0001). KMT2A::MLLT10/3 had faster clearance then other KMT2Ar (mREM D28 44% vs. 0% in other KMT2Ar, p = 0.02). In a multivariate analysis including (cyto)genetic risk and treatment, D28 MRD was the only significant predictor of outcome using both the 10 -3 (p = 0.006 for EFS and 0.012 for OS) and 10 -2 levels (p = 0.004 for EFS and 0.01 for OS) for stratification. Similarly, in an alternative multivariate analysis model, D56 MRD (positive at any level versus negative) was also the only significant predictor of outcome independent of risk and treatment (p = 0.005 for EFS and 0.01 for OS) In summary, we present a strategy for MRD monitoring in pediatric AML that is technologically feasible, real-life applicable to vast majority of all patients, and has clear prognostic significance. Supported by National Institute of Cancer Research No. LX22NPO5102 funded by the European Union-Next Generation EU, and AZV grant NU20-07-00322.

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.

Prognostic Utility of Minimal Residual Disease (MRD) after Curative Intent Induction Therapy for DLBCL: A Prospective Real-World Ctdna Study

Background: The prognostic value of MRD using ultra-sensitive ctDNA assays after 1L DLBCL immunochemotherapy was recently reported (Roschewski, ASH 2022). However, real-world evidence (RWE) for the utility of ctDNA MRD assays to predict survival outcomes after standard-of-care (SOC) 1L treatment for DLBCL remains less clear. Here, we report longitudinally measured ctDNA levels, without use of tumor biopsies, in a real-world population of DLBCL patients treated with curative intent using current anthracycline-based induction regimens. Methods: We prospectively enrolled patients with DLBCLs from Samsung Medical Center undergoing 1L therapy for ctDNA profiling at 3 pre-defined milestones (Baseline, Interim after 3 cycles (C4D1), and end of treatment [EOT]). A total of 99 patients had baseline pretreatment plasma available with sufficient tumor burden for inclusion in this study. 364 samples were profiled in a blinded manner by Phased Variant Enrichment &amp; Detection Sequencing (PhasED-Seq, Foresight Diagnostics). Tumor-derived Phased Variants (PVs) were identified directly from baseline plasma (median 4 mL) without the use of tumor tissue, with matched constitutional DNA from paired PBMCs used to censor germline variants and CHIP. PVs were used to longitudinally assess ctDNA-MRD after therapy, including at the C4D1 and EOT timepoints. MRD levels were compared to standardized responses by PET/CT (Lugano 2014), PFS, and OS. Patients/samples were reported as MRD positive when ctDNA levels exceeded an analytical detection threshold (~1:10 6 cfDNA molecules) corresponding to 98% specificity. Results: Among the 99 evaluable patients, the median age was 58 years and 38% were women. 67% had stage III-IV disease, 53% had IPI scores ≥3, and 6% had MYC and BCL2 translocations. All patients were treated with anthracycline based therapy, with 94% receiving RCHOP and 6% receiving DA-EPOCH-R. The median follow-up was 44 months. We tested ctDNA performance at each landmark for predicting outcomes. ctDNA-MRD levels by PhasED-Seq at each landmark were prognostic for both PFS and OS. Specifically, when dividing pretreatment ctDNA levels at the median, ctDNA significantly predicted outcomes (PFS: P=0.004, HR=2.7; OS: P=0.0004, HR=5.1). At the C4D1 and EOT landmarks, we divided patients into those with detectable versus undetectable ctDNA-MRD. Both timepoints significantly stratified patients for outcomes including at C4D1 (PFS: P=0.0002, HR=5.0; OS: P=0.0046, HR=4.1) and at EOT (PFS: P&amp;lt;0.0001, HR=7.8; OS: P&amp;lt;0.0001, HR=10.8). As the current standard for assessing response in DLBCL is imaging, we also examined the prognostic value of PET/CT scans at the same time-point as ctDNA. While interim PET/CT scans in our cohort were prognostic for PFS, they were not significantly prognostic for OS (PFS: P=0.02, HR=2.5; OS: P=0.06, HR=2.4). At EOT, PET/CT scans were prognostic for both PFS and OS (PFS: P=0.011, HR=3.4; OS: P=0.011, HR=4.0). At both milestones, PET/CT appeared less discriminatory than ctDNA-MRD. Exemplifying this, the overall survival of patients at 24 months was better separated at EOT by ctDNA-MRD than it was by PET/CT scan (OS24 ctDNA-MRD+: 37%, ctDNA-MRD-: 94%, compared with PET/CT PR/SD/PD: 50%, PET/CT CR: 86%). Finally, to further compare the prognostic value of ctDNA-MRD in the context of the current standard PET/CT scan, we performed a Cox proportional hazards model to predict PFS and OS, using both PET/CT and ctDNA-MRD as univariate predictors, as well as a multivariate model considering both PET/CT and ctDNA. As expected, interim and EOT ctDNA-MRD and PET/CT scans were predictive of outcomes when considered in univariate analysis. However, when considered in combination, while the prognostic value of ctDNA-MRD for OS remained at both interim and EOT timepoints, PET/CT scan was no longer significantly prognostic of outcomes (Fig 1). Conclusions: These data both demonstrate the feasibility and the prognostic utility of ctDNA-MRD during and after SOC induction therapy for DLBCL in a real-world population using an ultrasensitive ctDNA-MRD assay. The higher predictive value and accuracy of detectable ctDNA-MRD as compared with PET/CT suggest opportunities for integration of such assays in lymphoma response criteria, to potentially inform future clinical decision making.

Combination Chemotherapy in Patients with Newly Diagnosed Blastic Plasmacytoid Dendritic Cell Neoplasms (BPDCN): First Results of a Prospective French Trial (LpDessai)

Introduction Blastic plasmacytoid dendritic cell neoplasms (BPDCN) are now well-characterized diseases clearly referenced in the 5 th edition of the World Health Organization (WHO) Classification of Tumors (2022) 1 but treatment remains a real challenge. There is no consensus on the first line treatment even if CD123 targeted therapies are available in the USA and some European countries. Standard chemotherapy remains largely used as first line treatment with better results of leukemia-based regimens despite a high toxicity rate in this frail population 2,3. To establish a reference chemotherapy scheme, we prospectively evaluated the efficacy and toxicity of a combination chemotherapy (idarubicin, methotrexate, L-asparaginase, and dexamethasone) in newly diagnosed BPDCN patients (pts) in France. Patients and methods This single stage phase II study enrolled consecutive adult pts with suspected BPDCN in participating French centers. BPDCN diagnosis was centrally reviewed for cytology and immunophenotype (IF) (Pr Garnache Ottou F, UMR RIGHT BESANCON) and if needed for histology (Dr Petrella T, Montréal, Canada) according to published recommendations 1,4. After giving their informed consent, patients were evaluated for tumoral involvement by PET-scan, systematic lumbar puncture, and the mSWAT score was calculated for skin extension. Patients then received three 21 days-cycles of Ida/Metho/L-asp/Dex combination, before evaluation. Eligible patients with complete response (CR), complete response with incomplete bone marrow recovery (CRi) or partial response (PR) underwent allogeneic hematopoietic stem cell transplantation (HCT). Those not eligible received consolidation chemotherapy with 28 days cycles of Metho/L-asp/Dex (Figure 1). The primary objective was the proportion of patients with CR after 3 cycles of chemotherapy. Secondary objectives were the proportion of patients with an objective response (ORR) defined as CR, CRi or PR, the minimal residual disease (MRD) in responding patients evaluated by the presence of plasmacytoid dendritic cell blast measured by flow cytometry in the bone marrow, the incidence of severe adverse events and overall survival (OS). The competent ethics committee (Comité de Protection des Personnes Île de France 8) approved the study. The Besançon University hospital promoted the trial, financed by a grant of the French National Cancer Institute (INCa-DGOS_11093). Results Twenty-eight pts, originating from 16 centers, were screened between May 2019 and March 2023. Two patients with a misdiagnosis were screen failures. Two patients did not receive the planned chemotherapy due to clinical deterioration; 24 patients (21 male, 3 female) received at least one chemotherapy cycle and 23 are analyzed with a current end-point on June 30, 2023. Median age was 65y (21-79y). ECOG status was 0-1 in 20 cases, and 2 in 3 cases. A cutaneous involvement was identified in 16 pts (70%) and an extra medullary involvement was present in 13 (62%) pts: spleen, n= 6 (25%); lymph nodes, n= 7 (30%). Twenty-three pts (90%) had a bone marrow infiltration (identified only on IF in 4 cases), and 3 cases (14%) a documented central nervous system involvement. Nine pts had to stop planned treatment due to severe adverse events (3 deaths, 4 acute renal failure and 2 grade 4 febrile neutropenia) and in 4 pts, at least one cycle had to be delayed. Among the 15 pts receiving at least 3 cycles, 12 (80% and 50% of the whole cohort) were in ORR (CR = 8, CRi = 2, PR = 2) after the 3rd cycle of chemotherapy, 2 pts did not respond. Eight of ten (80%) CR/CRi pts had a MRD &amp;lt; 10 -4 without any further relapse. Nine of 12 responding patients (75%) received an allogeneic HCT 1-2 months after the end of the 3 rd cycle. Global OS at 6 months for responding or failing pts were 100% and 37% respectively (Figure 2). Conclusion In selected pts, an adapted chemotherapy could offer high CR rate in pts able to follow the planned treatment but remains toxic in frail pts, with only half of the them achieving a 3 rd cycle of chemotherapy. We confirm that pts achieving ORR and receiving HCT obtain prolonged CR. MRD level seems to correlate with OS and could be a useful tool to manage treatment toxicity in BPDCN frail pts.

Immune Profiling of Newly Diagnosed Multiple Myeloma (NDMM) Treated with Quadruplet Induction and Autologous Stem Cell Transplantation (ASCT) and Comparison with Achievement of Minimal Residual Disease (MRD) Negativity

Background Incorporation of anti-CD38 monoclonal antibodies (mAb) to the triplet backbone of proteasome inhibitor, immunomodulatory agent and corticosteroid has resulted in unprecedented depth of response including MRD negativity in transplant eligible NDMM. While numerous immunological mechanisms of modern quadruplet combinations are proposed, the modulation of the immunoprofiles bone marrow (BM) and peripheral blood (PB) with quadruplet therapy and ASCT are unknown. The baseline as well as longitudinal study of the composition and functionality of the immune microenvironment with treatment may have implications on disease response. Methods We prospectively collected clinically annotated patient (pt) samples from a phase II single institution clinical trial in transplant eligible NDMM (NCT04991103). Pts received quadruplet combination with daratumumab, bortezomib, lenalidomide and dexamethasone for six cycles followed by MRD adapted deferral of ASCT for pts who achieved MRD&amp;lt;10 -5. Pts deferring ASCT received three additional cycles of consolidation followed by maintenance. Pts who did not meet threshold for ASCT deferral received ASCT followed by maintenance therapy per protocol. We collected paired PB and BM samples at the time of screening (SCN), post-induction (I), and post-consolidation/ASCT (C). We purified and biobanked viable PB mononuclear cells (PBMCs). We thawed PBMCs, treated with FcR blocking Ab, stained with fluorochrome-labeled antibodies and a live/dead fixable stain, and then analyzed using the FACSymphony A5 SE flow cytometer. We identified immune cell subsets in PBMCs using FlowJo v10.2. We report data as percentage of each population within total live PBMCs. Results We collected 48 samples from 8 pts at three timepoints (SCN, I and C) and performed initial immunophenotyping analysis on the PBMCs. We observed no significant changes over time in the frequencies of peripheral CD45 +, CD3 +, NK T cells, monocytes or dendritic cells (not shown). We noted decreased frequencies of multiple B cell subsets, including naïve, transitional, memory and double negative cells (not shown), as well as CD56 +CD16 + NK cells (Fig. panel A) post-induction with no recovery following consolidation . While the frequencies of CD3 + T cells did not change with time, we observed a shift in the CD4:CD8 ratio, with an increase in the proportion of CD8 + T cells post-consolidation (Fig. panel B). Within the post-consolidation CD8 + T cell compartment, we noted increased frequencies of cells that presented with senescent (PD1 negCD57 +), exhausted (PD1 +CD57 neg) or anergic (PD1 +CD57 +) profiles. Consistent with this, the frequencies of terminally differentiated effector memory cells re-expressing CD45RA (TEMRA) CD8 + T cells increased at consolidation (Fig. panel C) and many of these cells exhibited an anergic profile (CD57 +PD1 +, Fig. panel D). Comparison of the baseline immune profiles and MRD levels post-induction revealed that the pt attaining MRD&amp;lt;10 -5 had the highest frequency of-NK cells as well as proportionally higher T CM/T EM and lower TEMRA/T Naive cells at diagnosis. This subject was able to defer ASCT per protocol and subsequently exhibited the lowest frequency of CD8 + TEMRA with an anergic phenotype (PD1 +CD57 +) post quadruplet consolidation. The subject with the least response to quadruplet induction and highest MRD post-induction had a high proportion of CD8 + T cells with an exhausted phenotype (PD1 +CD57 neg) at screening. Conclusion This is the first longitudinal analysis comparing MRD levels to the immune profiles of PB and BM cells derived from a cohort of NDMM patients prospectively treated with quadruplet induction and ASCT. We conclude that quadruplet induction negatively affects the NK cell and B cell compartment during induction and these compartments do not rapidly recover in the immediate post-transplant period. ASCT post quadruplet therapy is associated with preferential expansion of the effector CD8 + T cell compartments, including terminally differentiated TEMRA cells, which express markers associated with T cell exhaustion and anergy. Additional studies evaluating BM immune profile and T cell exhaustion are ongoing and shown at the meeting.

CRLF2 Expression in Mexicans with Acute Lymphoblastic Leukemia across the Age Spectrum

Introduction Acute lymphoblastic leukemia (ALL) is a hematological malignancy that primarily affects children also occurs in adults. Certain subtypes of ALL, such as Philadelphia chromosome-like ALL (Ph-like ALL), present unique challenges and are associated with poor outcomes. Alterations in the CRLF2 (Cytokine Receptor-like Factor 2) gene have emerged as critical contributors to the pathogenesis of Ph-like ALL. CRLF2 gene rearrangements result in the overexpression of CRLF2 protein, leading to dysregulated signaling pathways and enhanced leukemogenesis. The prevalence and clinical implications of CRLF2 overexpression in Mexicans with ALL remain underexplored. Hence, this study aimed to investigate the prevalence of CRLF2 overexpression in Mexican patients with ALL across different age groups. Patients and Methods: We conducted a prospective study on a cohort of ALL patients from a single institution in Mexico that cares for patients across the age spectrum. Consecutive patients with a diagnosis of ALL were enrolled in the study and were assessed for CRLF2 overexpression using flow cytometry and fluorescent in situ hybridization (FISH). Patients were treated with a local BFM-based chemotherapy regimen according to age and risk stratification. In adolescents and young adults ≥16, a pediatric-inspired regimen was used and in patients with positive measurable residual disease (MRD) allogeneic hematopoietic cell transplantation was sought. To determine the impact of CRLF2 overexpression on clinical characteristics and treatment response, we analyzed various parameters, including response rates, MRD, and survival. Statistical analyses were performed using SPSS 25, encompassing descriptive analysis, chi-squared tests, and survival analysis with the Kaplan-Meier method. Results: A total of 79 patients were enrolled with a median age of 17 years (2-70 years), 34 were female and 45 males with a median white blood cell count of 10.1x103/L (.63-649.57). Our findings revealed that 21 out of 79 patients (26.6%) exhibited CRLF2 overexpression. The proportion of CRLF positive ALL increased with age; it was 20.5% in patients aged 0-16 years, 34.6% in 17-39 and 40% in &amp;gt;60 years. There were no statistically significant differences observed in white blood cell counts, central nervous system involvement, response to treatment, or minimal residual disease levels in CRLF2 positive or negative patients (Table 1). In the Kaplan-Meier survival analysis, it was observed that positive CRLF2 expression was significantly associated with lower overall survival (OS) with a median of 24.9 months (0.2-65.4 months); with 85.8% OS at 2 years in CRLF2 negative vs 41.2% in CRLF2 positive patients (Figure 1). Event-free survival rate at 2 years was also lower with 70.8% vs 35.3% at 2 years in CLRF2 positive patients. Moreover, this association was found to be consistent across all age subgroups, including patients aged 0-16, 17-39, and over 40 years (P &amp;lt; 0.05). Conclusion: Our study highlights the significance of CRLF2 overexpression as a cost-effective biomarker indicative of an unfavorable prognosis in ALL across the age spectrum. Early detection of this alteration through flow cytometry plays a vital role in risk stratification and should prompt the implementation of tailored therapeutic interventions to improve clinical outcomes. Additionally, our study underscores the importance of conducting population-specific research to gain comprehensive insights into the genetic landscape and disease characteristics of ALL in different ethnic groups.

Improved MRD Negativity Rates in Adverse Genomic Risk B-ALL Patients with Chemotherapy / Blinatumomab Induction: Experience from the Australasian Leukaemia Lymphoma Group (ALLG) ALL06/09 Studies

Introduction: Despite the benefits conferred by pediatric treatment protocols, outcomes in adolescent and young adult (AYA) acute lymphoblastic leukemia (ALL) remain inferior to children. Poor outcomes in AYA ALL have been associated with reduced tolerability of intensive cytotoxics and higher incidence of adverse risk disease. Our earlier ALLG ALL06 study demonstrated intensive pediatric induction/consolidation could be delivered in a similar time frame as in children suggesting tolerability was not a major contributor to poor outcome in AYA ALL. ALL06 established post consolidation (TP2=d79) minimal residual disease (MRD) positivity (MRD+) and high BMI was predictive of inferior disease free- and overall survival (OS). In order to improve TP2 MRD negativity (MRD-) in B-cell disease, the ALLG ALL09 study substituted standard consolidation for blinatumomab (blin) in phase I and II of a BFM-style protocol . We present the impact of blin on outcomes in genomic cohorts from ALL09 and compare these to similar cohorts from ALL06 treated with standard chemotherapy. Methods: Between Sep 2020 and Apr 2022, 55 patientswere enrolled to ALL09. Of these, 45 had appropriate samples for genomic analyses and 39/45 had an MRD marker identified. These 39 patients formed the genomic cohort presented below. Genomic subtyping was performed using whole transcriptome sequencing, MLPA, karyotype and FISH analyses, and disease was classified into genomic adverse (AR) vs standard (SR) risk based on contemporary literature classifiers. Bone marrow MRD was assessed using ASO-PCR for IGH and TCR rearrangements, at d33 post induction (TP1), and TP2 post blin consolidation. Differences in TP2 MRD were assessed, with estimated 3 yr relapse free survival (RFS) and OS calculated using the Kaplan-Meier method. Results: In the ALL09 cohort, median follow up was 629 days with 3 yr RFS 84.5% and OS 87.1%. In the genomic cohort, median follow up was 602 days with 3 yr RFS 88.8% and OS 93.2%, respectively. These compared favorably to 3 yr RFS and OS (both 67.5%) in the B-cell genomic cohort from ALL06. Between ALL09 and ALL06 there were similar proportions of AR patients (73% and 62.5%, respectively). In ALL09, the most frequent subtypes were PAX5alt (18%) and PAX5 p.P80R (11%) versus KMT2Ar and DUX4r (both 17.5%) in ALL06. In ALL09, Ph-like disease represented 13% vs 12.5% in ALL06. In the ALL09 genomic cohort, the MRD+ rate reduced between TP1 and TP2 (p&amp;lt; 0.001), with the reduction in the rate of positivity being more pronounced in the AR cohort (AR MRD+ TP1=85% vs AR TP2=39%; p&amp;lt; 0.001). In contrast, in ALL06 MRD+ in the AR cohort reduced by only 28% between TP1 and TP2 (p &amp;gt;0.05). This suggests a positive impact of blin consolidation in AR patients in ALL09. Despite improved AR MRD+ rates overall, not all genomic subtypes in ALL09 followed this trend (Table 1). To date, there are 5 relapses in the ALL09 cohort, 4 from the genomic cohort. Of these, 1 was hypodiploid and strikingly the remaining 3 patients harbored a TCF3 rearrangement ( TCF3r). All 4 relapses were significantly associated with MRD+ at TP2 (RFS: MRD-, 100% (n=14) vs MRD+, 71% (n=25); p= 0.013), and all were observed in the AR genomic group. Of interest, the fifth case, outside of the genomic cohort, was classified as PAX5alt. In ALL09, high-risk (HR) patients proceeded to intensive HR block chemotherapy with the aim of eliminating MRD prior to allogeneic transplant (SCT). In the genomics cohort 15 patients proceeded to HR therapy, n=12 were considered AR. Sustained MRD+ through HR blocks 1 and 2 was noted in patients harboring PAX5 p.P80R (3/3), KMT2Ar (2/2) and TCF3r (2/3). Of the 9 patients who proceeded to SCT, 3 had PAX5 p.P80R 1 had PAX5alt, 2 were KMT2Ar and there was 1 each of DUX4r, hypodiploid and TCF3r. The 3 patients who remained MRD+ in the SR cohort had PAX5 p.P80R and all 3 proceeded to SCT. Conclusion: This is the first report of the impact of blin consolidation on genomic subsets of de novo AYA ALL. Our study suggests blin sensitivity as assessed by TP2 MRD response may be genomic subtype dependent, with TCF3r associated with sustained MRD+, leading to eventual relapse (3/3 patients). In addition, the PAX5 p.P80R subset was also associated with sustained high levels of TP2 MRD+ (3/4 with an MRD marker) and SCT (3/5 patients). These results may assist with identification of AYA ALL subsets that could benefit from alternative approaches as blin becomes increasingly incorporated into front-line protocols.