Molecular Minimal Residual Disease Research Articles

Abstract CT245: Circulating tumor DNA and nivolumab maintenance: A pilot study in diffuse large B cell lymphoma

Abstract Introduction: Molecular minimal residual disease (MRD) status correlates with disease relapse in patients (pts) with diffuse large B cell lymphoma (DLBCL). However, the clinical impact of early treatment intervention a time of detectable MRD (dMRD) prior to clinical relapse is unknown. Checkpoint inhibitor (CPI) therapy has activity in some pts with DLBCL. We hypothesized that early CPI therapy in patients with dMRD without clinical relapse may lower relapse rate. We conducted a pilot study in high risk pts in remission after most recent line of therapy (LOT) in whom maintenance nivolumab (MN) was offered at time of dMRD to assess effect on MRD clearance and relapse rate. Methods: Pts >18 years (yrs) with high-risk DLBCL (ABC-subtype, high grade B cell lymphoma (HGBCL), MYC translocation, relapsed/refractory disease, or Ki67 >90%) who had achieved CR on most recent LOT were eligible. Patients were monitored for detectable MRD (dMRD) based on the clonoSEQ assay for circulating tumor DNA (ctDNA). Pts had monthly MRD assessment and every 4 month imaging for 2 yrs. If conversion from uMRD to dMRD without clinical relapse, pts received MN (240 mg IV q2 weeks) for up 2 yrs. Pts with overt relapse at time of dMRD or dMRD at initial MRD assessment were ineligible for MN. The primary endpoint was rate of conversion from dMRD to undetectable MRD (uMRD). Results: 20 pts were screened and 15 pts enrolled between June 2018 and March 2022. 4 had transformed DLBCL (2 from FL, 1 from MZL, and 1 Richter’s transformation from CLL), 2 pts had HGBCL (one of which was also transformed). All pts with dMRD had radiographic relapse within 4 months of molecular relapse. No pts with uMRD by ctDNA had clinical relapse on study during 2 yrs follow up. Of the 8 pts who enrolled after first LOT, none had molecular or clinical relapse. Of the 7 pts with 2+ prior LOT, 3 had dMRD: two had clinical relapse at same time as dMRD and were taken off study (one had dMRD at baseline MRD assessment and the other 135 days post enrollment after initial period of uMRD). In the single patient eligible for MN, dMRD preceded radiographic relapse by 105 days, 410 days after enrollment. The pt received 2 cycles of MN before clinical progression and was taken off study. This pt failed to achieve conversion of dMRD to uMRD. Conclusion: Due to small sample size and lower than expected relapse, we were unable to demonstrate conversion of dMRD to uMRD using MN. However, this pilot study did confirm high correlation of uMRD and dMRD status with disease free survival and clinical relapse within 4 months, respectively. If strong negative predictive value is confirmed in a larger study, ctDNA MRD testing may be used to spare pts from imaging or to guide additional work up in pts with inconclusive findings on scans. Citation Format: Shazia Nakhoda, Tamarrah Sklarz, Cheyenne Pagan, Matthew Matasar, Zachary A. Frosch, Marcus Messmer, Asya Varshavsky Yanovsky, Rashmi Khanal, Carlyn Tan, Eli Mikkelsen, Henry Fung, Eric Ross, Mark Roschewski, Allison Jacob, Nadia Khan. Circulating tumor DNA and nivolumab maintenance: A pilot study in diffuse large B cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT245.

Clinical efficacy and safety of flumatinib versus dasatinib combined with multi-drug chemotherapy in adults with Philadelphia-positive acute lymphoblastic leukemia

Introduction: Flumatinib, a highly selective ABL kinase inhibitor, exhibits stronger inhibition of intracellular BCR-ABL tyrosine kinase activity, compared to Imatinib. However, there is limited research comparing the real-world efficacy and safety of flumatinib and dasatinib in patients with Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL). Objective: Investigating the differences in therapeutic efficacy and safety between flumatinib and dasatinib in combination with multi-drug chemotherapy for the treatment of newly diagnosed Ph+ ALL. Method: In this study, we assessed 43 patients with newly diagnosed Ph+ ALL (20 in the flumatinib group, 23 in the dasatinib group). Results: There were no significant differences in gender, age, fusion gene type, initial blood routine, bone marrow blast cell ratio or chromosome karyotype between the two groups. Within 1 month, there were no significant differences in the complete response (CR), major molecular response (MMR) or minimal residual disease (MRD) negativity rate between the flumatinib and dasatinib groups. Similarly, within 3 months, there were no significant differences in CR or MMR rates between the two groups. However, the rates of complete molecular response (CMR) and MRD negativity within 3 months were significantly higher in the flumatinib group, compared to the dasatinib group (P < 0.05). Additionally, the flumatinib group exhibited fewer adverse reactions compared to the dasatinib group. Conclusion: These findings suggest that flumatinib is a safe and effective tyrosine kinase inhibitor (TKI) for achieving CMR and MRD negativity in patients with Ph+ ALL, as supported by this small series of patients.

A new genomic framework to categorize pediatric acute myeloid leukemia.

Recent studies on pediatric acute myeloid leukemia (pAML) have revealed pediatric-specific driver alterations, many of which are underrepresented in the current classification schemas. To comprehensively define the genomic landscape of pAML, we systematically categorized 887 pAML into 23 mutually distinct molecular categories, including new major entities such as UBTF or BCL11B, covering 91.4% of the cohort. These molecular categories were associated with unique expression profiles and mutational patterns. For instance, molecular categories characterized by specific HOXA or HOXB expression signatures showed distinct mutation patterns of RAS pathway genes, FLT3 or WT1, suggesting shared biological mechanisms. We show that molecular categories were strongly associated with clinical outcomes using two independent cohorts, leading to the establishment of a new prognostic framework for pAML based on these updated molecular categories and minimal residual disease. Together, this comprehensive diagnostic and prognostic framework forms the basis for future classification of pAML and treatment strategies.

How to Manage Philadelphia-Positive Acute Lymphoblastic Leukemia in Resource-Constrained Settings.

Recent studies have indicated that more than half of adult patients newly diagnosed with Ph+ ALL can now achieve a cure. However, determining the most suitable protocol for less-resourced settings can be challenging. In these situations, we must consider the potential for treatment toxicity and limited access to newer agents and alloSCT facilities. Currently, it is advisable to use less intensive induction regimens for Ph+ ALL. These regimens can achieve high rates of complete remission while causing fewer induction deaths. For consolidation therapy, chemotherapy should remain relatively intensive, with careful monitoring of the BCR-ABL1 molecular transcript and minimal residual disease. AlloSCT may be considered, especially for patients who do not achieve complete molecular remission or have high-risk genetic abnormalities, such as IKZF1-plus. If there is a loss of molecular response, it is essential to screen patients for ABL mutations and, ideally, change the TKI therapy. The T315I mutation is the most common mechanism for disease resistance, being targetable to ponatinib. Blinatumomab, a bispecific antibody, has shown significant synergy with TKIs in treating this disease. It serves as an excellent salvage therapy, aside from achieving outstanding results when incorporated into the frontline.

Cladribine, High-Dose AraC, Plus Gemtuzumab Ozogamicin (CLAG-GO) As Frontline Intensive Therapy for Fit Patients with Core-Binding Factor Acute Myeloid Leukemia: Preliminary Results

Background: Core-binding factor (CBF) acute myeloid leukemias (AML) are associated with favorable outcomes when treated with intensive therapy regimens, particularly based on high-dose cytarabine. In addition, gemtuzumab-ozogamicin (GO) added to frontline chemotherapy showed improved survival in patients with CBF-AML in a meta-analysis of five randomized trials (Hills RK, et all. 2014). Since then, GO has been incorporated to the intensive regimen [traditional ‘3+7‘ or fludarabine-cytarabine and GCSF (FLAG)] and has become the standard approach for CBF-AML. Methods: Fit patients with newly diagnosed AML with inv(16) or t(16;16), CBFB::MYH11 [AML -inv(16)] and AML with t(8;21), RUNX1::RUNX1T1 [AML -t(8;21)] were eligible to receive cladribine, cytarabine, GCSF and GO (CLAG-GO) regimen as frontline intensive chemotherapy. We evaluated baseline patient's characteristics, disease features, and early molecular-response dynamics, including qPCR for each CBF-AML, along with count recovery after cycle 1 (C1) and 2 (C2). Response criteria were standard as defined by the European Leukemia Net (ELN) 2022. Results: Fifteen patients (pts) diagnose with CBF-AML were included, with a median age 47 years (range, 20 - 66 years), 53% were males. Median WBC were 15.5x10^9/L (range, 2.4 - 103). Eleven pts (73%) had AML -inv(16) and 4 pts (27%) had AML -t(8;21). Nine of 11 AML -inv(16) had a CBFB::MYH11 fusion transcript variant A and 2 of 11 pts had a CBFB::MYH11 fusion transcript variant non-A. Median CBF transcript level by qPCR at diagnose was 54.5% (range, 29.5 - 63.6) for AML -inv(16) and median qPCR of 100% (range, 100 - 100) for AML -t(8;21). Median number of co-mutations was 2 (range, 0 - 5), commonly kinase signaling mutations in components of the RAS/MAPK pathway (Figure 1). All pts received induction therapy with cladribine 5mg/m2 (D1-D5), cytarabine 2g/m2 (D1-D5), GCSF (D1 and D5) and GO 3mg/m2 (D1 or D2). If responses were achieved, subsequent consolidation cycles (up to 6 cycles) could be given with CLAG-GO (CLAG on D1-D3 and GO on D1 of C3 and C5). Thirteen pts (87%) were evaluable after induction therapy. Of the 2 non-evaluable pts, one died during induction, and 1 patient is still receiving induction therapy at the last cut off. All pts (13/13) achieved a complete remission (CR) with full count recovery (complete remission -CR- 100%). Twelve of them had minimal residual disease (MRD) assessed by flow cytometry (FC), and 11/12 (91%) pts achieved MRD negative (MRD-ve). One patient had MRD positive (0.7%) after C1. In one patient, MRD is unavailable. Twelve of 15 pts were monitored using qPCR for variant A CBFB::MYH11 or RUNX1::RUNX1T1 in different times points throughout C1. One patient died and 2 pts carried non-A variant CBFB::MYH11 fusion transcript hence qPCR was not available. Molecular MRD at D14 of C1 showed that 6/7 (86%) pts achieved qPCR &amp;lt;1, and by the end-of-induction 8/12 (67%) achieved qPCR &amp;lt;0.1 (Figure 2). There was 1 death (4-week mortality = 6%) during induction in a patient with intracranial bleeding secondary to thrombocytopenia and platelet-refractoriness. Thirteen of the 13 recovered their counts following C1, with median time to absolute neutrophil count (ANC) ≥1.0x10^9/L of 20 days (range, 13 - 23) and platelets ≥100x10^9/L of 21 days (range, 17 - 28). Ten pts received C2 of therapy in our institution. One patient was transferred to another hospital, one patient just finished induction therapy and 1 patient started consolidation regimen with FLAG-GO per physician discretion. Nine of 10 pts were evaluable after C2, and all them (100%) sustained a CR, all were MRD-ve by FC (including a patient that previously was positive) and 7 of 8 pts achieved qPCR &amp;lt;0.05. After C2, median days to ANC ≥1.0x10^9/L of 16 days (range, 0 - 27) and platelets ≥100x10^9/L of 21 days (0 - 37). In general, CLAG-GO was well tolerated in induction with only 1 patient had grade 3 increased alanine transferase and 1 patient had grade 3 increased aspartate transferase (both related to GO), which resolved spontaneously; and 9 pts of 15 have had an episode of neutropenic fever (3 of them infection was proved). After a median follow-up of 5 months, overall survival is 92%. Conclusions: CLAG-GO is highly effective and safe when treating CBF-AML as frontline therapy and seems encouraging treatment. More patients need to be treated and longer follow up is needed to confirm these preliminary results.

Phenotypic Drug Response Profiling Identifies Asparaginase-Based Synergistic Combinations for Very High Risk Acute Lymphoblastic Leukaemia

In acute lymphoblastic leukaemia (ALL), empirical multi-drug chemotherapy combinations lead to &amp;gt;85% cure rates in children. But 10-15% of patients show suboptimal response and as drugs are dosed to tolerance, further intensification is not possible. An exception is the key antileukaemic drug, asparaginase (ASNase), which is not associated with dose-related toxicity. We evaluated the use of ex-vivo co-culture based drug response profiling (DRP) to identify novel synergistic and effective drug combinations with ASNase for patients with suboptimal responses to standard chemotherapy in ALL. Patient-derived xenograft (PDX) ALL cells from 5 patients with high-risk B-ALL (3 B-other, 2 IKZF plus) were treated with 120 drugs in 5-point serial dilutions in combination with or without an IC 50 dose of ASNase, in co-cultures with hTERT-immortalized primary bone marrow stromal cells. Eight clinically relevant agents with promising sensitizing activity as identified by decreased area-under-the-curve with addition of ASNase were identified (SEL, selinexor; ELTA, eltanexor; VEN, venetoclax; BZM, bortezomib; navitoclax; carfilzomib; mitoxantrone, birinapant). We validated combinations of ASNase with the 8 selected drugs plus 4 drugs used in induction therapy (prednisolone, dexamethasone, vincristine and daunorubicin) using a 4x4 drug matrix in 15 high-risk ALL PDX samples. Synergy scores were calculated using the zero-interaction potency (ZIP) model. Synergy (ZIP score ≥10) or additivity (ZIP score &amp;gt;0 and &amp;lt;10) with ASNase was identified for exportin-1 inhibitors (XPO1i; selinexor, eltanexor), venetoclax and proteosomal inhibitors (bortezomib, carfilzomib) in 100% (15/15), 86.6% (13/15) and 80-86.6% (12/15 and 13/15) samples respectively. Antagonism (ZIP score &amp;lt;0) was noted in more than one-third (6/15) of samples with ASNase in combination with steroids or vincristine. Three-drug combinations were evaluated ex-vivo in these 15 PDX samples. Synergy was identified with addition of low doses of BZM or VEN to the ASNase-SEL combination (ASNase-SEL-BZM and ASNase-SEL-VEN). Next, 4-6 mice per treatment arm were transplanted intravenously with 1 million ALL cells from 4 high-risk B-ALL patients. Randomized cohorts were treated with vehicle, ASNase-BZM-ELTA (ABE) and ASNase-dexamethasone-vincristine (ADV). Leukemic burden was assessed weekly using flowcytometry. The mice tolerated both treatment arms well. Platelet counts recovered within one week after an initial drop in the ABE arm. Both treatment groups had significant improvement in event free survival (EFS) as compared to the control group (ABE p-value 0.0082; ADV p-value 0.0325) though no significant differences were noted between the 2 treatment arms (p-value 0.8971). Three patients with high-risk medullary relapsed/refractory BCP-ALL, 2 not in morphological remission and 1 at time of very early relapse, received a combination of ASNase-BZM-VEN-SEL (ABVS) after consent. One patient for whom DRP suggested sensitivity to both SEL and VEN achieved molecular remission (molecular minimal residual disease &amp;lt; 10 -5) after one course of therapy remaining in complete remission 11 months later. Two patients with sensitivity to SEL but not to VEN as assessed by DRP, had progressive disease. ABVS was well tolerated in these patients with no grade 3-4 adverse events. Our results identify XPO1 inhibitors as a new class of drugs with promising anti-leukaemic activity in BCP-ALL. Co-culture based DRP shows promise in identifying alternative novel sensitive synergistic combinations ex-vivo with decreased toxicity for patients who have an inadequate response to standard therapy.

Machine Learning Enhancement of Flow Cytometry Data Accelerates the Identification of Minimal Residual Acute Myeloid Leukemia

Background: Identification of minimal residual disease (MRD) in acute myeloid leukemia (AML) is essential for assessing risk of relapse and guiding therapeutic management. While molecular assays have streamlined AML MRD detection, flow cytometric immunophenotyping (FCI) remains the only available test modality for approximately 50% of patients lacking traceable molecular MRD targets. Unfortunately, due to the high level of expertise required, long manual analysis time and complexity of AML MRD FCI data, only a few reference laboratories offer this as an orderable test. Methods: To facilitate the analysis of AML MRD by FCI, we devised an unsupervised machine learning (ML) pipeline that intakes raw FCI data, eliminates acquisition errors (FlowCut), performs state-of-the-art clustering (PARC) and dimensionality reduction (UMAP), estimates how aberrant or different-from-normal each cluster is compared to a cohort of controls (15 donor and 15 reactive marrow aspirates), downsamples clusters with high event counts without significant loss of low-event clusters, and corrects for downsampling using an upsampling factor. This CCADDAS (Clinically-intended Clustering of All events, Dimensionality reduction, Downsampling and Aberrancy Scaling) pipeline was containerized, deployed in a cloud environment (Google Cloud Vertex AI), and used to process raw FCI files from 50 AML MRD FCI assays (3-tube/10-color panel) with 0.1% to &amp;lt;5% leukemic subsets, and 20 assays with no detectable leukemic subsets by prior conventional analysis. Machine learning-enhanced data files were analyzed using a commercially available FCI analysis software (Kaluza v 2.1, Beckman Coulter). Results: Cluster-informed downsampling reduced the total number of events needed to be analyzed from 1 million cells to 179,531 cells per tube on average (82% data reduction), while preserving low event subsets (clusters smaller than 5000 cells) and allowing for accurate quantitative estimates for any gated population [# events x mean (upsampling factor)]. MRD was detected in all positive cases, and absent on all negative cases when analyzed using the enhanced downsampled data (100% correlation with original data), with accurate quantitative estimates (Spearman r = 0.85). Analysis time was dramatically reduced from 73 minutes to 8 minutes per case on average (89% reduction of manual analysis time). Dimensionality reduction parameters simplified gating of lineage- and maturation-defined subsets. Cluster comparison against control samples facilitated the rapid identification of MRD subsets, based on a calculated aberrancy scale parameter that helped distinguish MRD blasts (mean ± 2SD: 0.85 ± 0.3) from benign blasts (mean ± 2SD: 0.26 ± 0.3) (p&amp;lt;0.0001). Conclusion: We introduce CCADDAS, an unsupervised containerized cloud ML pipeline that enhances raw AML-MRD FCI data with ML annotations, cluster-informed downsampling and comparison to normal controls to create markedly smaller and software-agnostic FCI files. CCADDAS simplifies and accelerates detection of AML-MRD in clinical diagnostics, reducing number of cells analyzed by 82% and manual analysis time by 89%, without impacting test performance. Moreover, CCADDAS can be deployed on any laboratory-developed AML-MRD assay, and its small-sized export is compatible with any clinical FCI software, computing platform and analysis strategy. Adoption of CCADDAS is likely to facilitate the implementation of AML MRD FCI analysis by more clinical laboratories.

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

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

Cell-Free DNA Extracted from CSF for the Molecular Diagnosis of Pediatric Embryonal Brain Tumors

Background: Liquid biopsies are revolutionary tools used to detect tumor-specific genetic alterations in body fluids, including the use of cell-free DNA (cfDNA) for molecular diagnosis in cancer patients. In brain tumors, cerebrospinal fluid (CSF) cfDNA might be more informative than plasma cfDNA. Here, we assess the use of CSF cfDNA in pediatric embryonal brain tumors (EBT) for molecular diagnosis. Methods: The CSF cfDNA of pediatric patients with medulloblastoma (n = 18), ATRT (n = 3), ETMR (n = 1), CNS NB FOXR2 (n = 2) and pediatric EBT NOS (n = 1) (mean cfDNA concentration 48 ng/mL; range 4–442 ng/mL) and matched tumor genomic DNA were sequenced by WES and/or a targeted sequencing approach to determine single-nucleotide variations (SNVs) and copy number alterations (CNA). A specific capture covering transcription start sites (TSS) of genes of interest was also used for nucleosome footprinting in CSF cfDNA. Results: 15/25 CSF cfDNA samples yielded informative results, with informative CNA and SNVs in 11 and 15 cases, respectively. For cases with paired tumor and CSF cfDNA WES (n = 15), a mean of 83 (range 1–160) shared SNVs were observed, including SNVs in classical medulloblastoma genes such as SMO and KMT2D. Interestingly, tumor-specific SNVs (mean 18; range 1–62) or CSF-specific SNVs (mean 5; range 0–25) were also observed, suggesting clonal heterogeneity. The TSS panel resulted in differential coverage profiles across all 112 studied genes in 7 cases, indicating distinct promoter accessibility. Conclusion: CSF cfDNA sequencing yielded informative results in 60% (15/25) of all cases, with informative results in 83% (15/18) of all cases analyzed by WES. These results pave the way for the implementation of these novel approaches for molecular diagnosis and minimal residual disease monitoring.

Abstract 3374: Circulating cell-free tumor DNA dynamics capture minimal residual disease with neoadjuvant immune checkpoint blockade plus chemoradiotherapy for patients with operable esophageal/gastroesophageal junction cancer

Abstract Introduction: There is a critical need to incorporate molecular assessments of minimal residual disease (MRD) during neoadjuvant immunotherapy, in order to identify individuals at high risk for disease recurrence based on analyses of circulating cell-free tumor DNA (ctDNA) landscapes. Here we employed longitudinal liquid biopsies to dynamically assess clinical outcomes with neoadjuvant immuno-chemoradiotherapy in patients with esophageal/gastroesophageal junction (E/GEJ) cancer. Methods: We utilized targeted error-correction sequencing to perform high-depth ctDNA next-generation sequencing for 141 serial plasma and 32 matched white blood cell (WBC) DNA samples from 32 patients with operable stage II/III E/GEJ cancer that received neoadjuvant immune checkpoint blockade (ICB) with chemoradiotherapy prior to surgery (NCT03044613). ctDNA analyses were performed at baseline, post-ICB induction, after completion of chemoradiotherapy (pre-op), and post-operatively (post-op). Using a tumor-agnostic WBC DNA-informed panel NGS approach we determined the cellular origin of plasma variants, filtering out germline and clonal hematopoiesis (CH) variants and evaluated ctDNA clonal dynamics over time. Molecular MRD was evaluated post-ICB, pre-op and post-op and correlated with recurrence-free (RFS) and overall survival (OS). Results: Twenty out of 32 patients had detectable ctDNA at any timepoint. Of the 12 patients with undetectable ctDNA, 9 had only CH- and/or germline-derived variants, while 3 patients had no detectable variants of any origin. ctDNA clearance post-ICB was correlated with tumor regression &amp;gt;80% at the time of resection (Fischer’s exact p=0.04). The subset of patients that did not attain complete pathologic response was heterogeneous with respect to ctDNA dynamics; such that ctDNA clearance pre-op identified patients with longer OS despite residual tumor of &amp;gt;0% at the time of resection (log rank p=0.06). Patients with undetectable ctDNA or ctDNA clearance pre-op had a longer RFS (log rank p=0.007) and OS (log rank p=0.03). Molecular MRD was associated with RFS and OS such that patients with ctDNA clearance post-op had longer RFS (log-rank p=0.007) and OS (log-rank p=0.017). Conclusion: ctDNA clearance post-ICB, pre-op and post-op reflects differential clinical outcomes for patients with E/GEJ cancer receiving neoadjuvant immuno-chemoradiotherapy. Understanding ctDNA dynamics and their relationship with pathological response and long-term outcomes can help identify patients at higher risk for recurrence and open a therapeutic window for future intervention. Citation Format: Blair V. Landon, Ronan J. Kelly, Ali H. Zaidi, Archana Balan, Jenna V. Canzoniero, Gavin Pereira, Zineb Belcaid, Russell K. Hales, K Ranh Voong, Richard J. Battafarano, Blair A. Jobe, Stephen C. Yang, Stephen Broderick, Jinny Ha, Kellie N. Smith, Elizabeth Thompson, Fyza Y. Shaikh, James R. White, Cynthia L. Sears, Eun J. Shin, Ali I. Amjad, Benny Weksler, Josephine L. Feliciano, Chen Hu, Vincent K. Lam, Valsamo Anagnostou. Circulating cell-free tumor DNA dynamics capture minimal residual disease with neoadjuvant immune checkpoint blockade plus chemoradiotherapy for patients with operable esophageal/gastroesophageal junction cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3374.

Optimizing Molecular Minimal Residual Disease Analysis in Adult Acute Lymphoblastic Leukemia.

Minimal/measurable residual disease (MRD) evaluation has resulted in a fundamental instrument to guide patient management in acute lymphoblastic leukemia (ALL). From a methodological standpoint, MRD is defined as any approach aimed at detecting and possibly quantifying residual neoplastic cells beyond the sensitivity level of cytomorphology. The molecular methods to study MRD in ALL are polymerase chain reaction (PCR) amplification-based approaches and are the most standardized techniques. However, there are some limitations, and emerging technologies, such as digital droplet PCR (ddPCR) and next-generation sequencing (NGS), seem to have advantages that could improve MRD analysis in ALL patients. Furthermore, other blood components, namely cell-free DNA (cfDNA), appear promising and are also being investigated for their potential role in monitoring tumor burden and response to treatment in hematologic malignancies. Based on the review of the literature and on our own data, we hereby discuss how emerging molecular technologies are helping to refine the molecular monitoring of MRD in ALL and may help to overcome some of the limitations of standard approaches, providing a benefit for the care of patients.

Prognostic impact of postoperative circulating tumor DNA as a molecular minimal residual disease marker in patients with pancreatic cancer undergoing surgical resection.

We aimed to clarify the prognostic impact of postoperative circulating tumor DNA (ctDNA) shortly after pancreatectomy in patients with pancreatic ductal adenocarcinoma (PDAC). Preoperative and paired postoperative blood samples were obtained from 66 patients in patients with PDAC. Cell-free DNA was extracted from the plasma, and KRAS mutations, as a benchmark of ctDNA, were examined using droplet digital PCR. Disease-free survival (DFS) and overall survival (OS) were compared between patients with presence and absence of ctDNA. In univariate analysis, patients with detectable postoperative ctDNA showed worse survival than those with undetectable in both DFS (P=.034) and OS (P=.022). Multivariate analysis also revealed that the presence of postoperative ctDNA was an independent risk factor for recurrence (hazard ratio: 2.677, P=.011). In contrast, preoperative ctDNA detection did not affect long-term outcomes. These trends persisted in 34 patients with resectable PDAC who underwent resection after neoadjuvant chemotherapy. Patients with detectable postoperative ctDNA were more prone to developing hepatic recurrence than those with undetectable postoperative ctDNA (P=.039). Postoperative ctDNA, as a minimal residual marker, can be useful for predicting the risk of recurrence in patients with PDAC even after curative resection.

Prophylactic Donor Lymphocyte Infusion in Patients after Allogeneic Stem Cell Transplantation with Post-Transplant Cyclophosphamide Is Associated with Low Relapse Risk and Excellent Survival in Patients below 65 Years with Acute Myeloid Leukemia and High-Risk Myelodysplasia

BackgroundPost-transplant cyclophosphamide (PTCy) effectively prevents graft-versus-host-disease (GVHD) in allogeneic stem cell transplantation (alloSCT) with matched related and matched unrelated donors resulting in low risk of non-relapse mortality (NRM) (<10%). However, PTCy-alloSCT is associated with high relapse rates of approximately 20-30% after 1 year and 30-40% after 2 years (Gooptu et al. Blood 2021, Ruggeri et al. J Hemat Oncol 2018). Prophylactic donor lymphocyte infusion (DLI) may reduce relapse, although it may increase the risk of GVHD. AimTo provide evidence for reduction of relapse and improvement of survival by combining allogeneic SCT with PTCy and subsequent standardized prophylactic donor lymphocyte infusions (DLI) in a cohort of patients with acute myeloid leukemia (AML) and high risk myelodysplasia (MDS). Patients and MethodsSixty-five patients with AML (n=50) or high risk MDS (n=15) received myeloablative (n=15) or reduced-intensity (n=50) alloSCT with a matched related (n=8) or 9/10 (n=19) or 10/10 (n=38) matched unrelated donor. Based on modified ELN criteria, 10 AML patients were intermediate risk and 40 poor risk. GVHD prophylaxis consisted of PTCy, mycophenolate until day +30, and tacrolimus tapered from +70 (related or 10/10 unrelated) or day +90 (9/10 unrelated). All patients were planned to receive DLI at 6 months after transplantation at a dose of 3 x 10^6 CD3+ cells/kg in related and 1.5 x 10^6 CD3+ cells/kg in unrelated alloSCT. In poor risk patients (poor risk AML and high risk MDS) additional low dose DLI (0.3 or 0.15 x 10^6 CD3+ cells/kg in related and unrelated alloSCT, respectively) was planned one month after tacrolimus tapering (i.e. 3-4 months after alloSCT). In poor risk patients with a sensitive molecular or immunophenotypical minimal residual disease (MRD) marker (<0.1%) low dose DLI was not administered but MRD monitored; only in case of MRD positivity at 2 or 4 months additional low dose DLI was given. ResultsOf the 65 patients, 49 received standard dose DLI at 6 months after alloSCT (75%), either as a first dose at 6 months (40%), or as a second DLI after a low dose DLI at 3-4 months after alloSCT (35%). Reasons for not receiving standard dose DLI were NRM (5%), GVHD (9%), relapse (9%) and donor-related (2%). Of the 22 poor risk patients with a sensitive MRD marker, 15 received the first DLI at 6 months; 6 received both low dose DLI and 6 month DLI because of MRD positivity at 2 or 4 months after alloSCT; one patient did not receive prophylactic DLI because of systemic relapse at day 105. With a median follow up of 400 days (range 183-814), 12 patients developed systemic relapse. Most relapses (67%) occurred very early after transplant (median 110 days, range 63 to 165 days), before low dose DLI could be effectively given. These early relapses occurred significantly more in patients >= 65 years (88%) than <65 years (p=0.01 Fisher's exact). Overall survival at 1 year was 95% (95% CI 89-100%) in patients <65 (n=42), significantly higher than the 68% (95% CI 49-95%) in patients >=65 (n=23) (p=0.01). Relapse rate at 1 year was 5% (95% CI 1-15%) in patients <65, significantly lower than the 37% (95% CI 25-58%) in patients >=65 (p=0.001). Grade 2-4 acute and chronic GVHD incidence at 1 year was low (6% (95% CI 2-14%) and 11% (2.5-25%), respectively). Survival at 1 year was similar (p=0.35) in poor risk patients without MRD marker (80%, 95% CI 67-96%), poor risk with MRD marker (95%, 95% CI 87-100%) and intermediate risk (86%, 95% CI 63-100%). For these groups relapse risk was also similar: 18% (95% CI 7-33%), 16% (95% CI 3-36%) and 11% (95% CI 5-41%), respectively. ConclusionsThis study is the first to demonstrate feasibility of prophylactic DLI after PTCy-alloSCT with a low associated risk of GVHD. The observed low incidence of relapse at 1 year after transplantation (5%) of patients aged <65 years appears to be substantially less than the reported risk without pre-planned DLI (20-30%). Although the value of early DLI administration (around 3-4 months) is still unclear, standard dose DLI administration at 6 months appears feasible in intermediate risk patients and poor risk patients with sensitive MRD markers. Early relapses after alloSCT, at a time point before early DLI could be effective, caused the inferior survival of patients aged >= 65 years. The results of this study can serve as a reference for designing future prophylactic cellular interventions in the PTCy-alloSCT setting. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Detection of Early Molecular Response (EMR) and Minimal Residual Disease (MRD) in Patients with Diffuse Large B-Cell Lymphoma (DLBCL) Using a Validated Next Generation Sequencing (NGS) Assay for the Detection of Tumor Variants in Circulating Tumor (ct)DNA

DLBCL is the most common type of non-Hodgkin lymphoma in adults. Although frontline standard of care treatment is effective in 60-70% of patients, 10-15% have primary resistance and 20-30% will relapse within 18 months. There is an unmet need to predict treatment refractoriness and relapse in order to intensify treatment approaches early during induction therapy. Early molecular response (EMR), defined as the dynamics of ctDNA after one cycle of immunochemotherapy, was shown to be prognostic in a small cohort of first-line DLBCL patients (Kurtz et. al. J. Clin. Oncol 2018), and recently validated using samples from the POLARIX study (NCT03274492) in previously untreated DLBCL. To support clinical studies, the Roche Molecular CAP/CLIA Laboratory has developed and validated the AVENIO Oncology Assay Non-Hodgkin Lymphoma Test (AOA NHL Test) that uses next generation sequencing of plasma cell-free (cf)DNA and of matched genomic (g)DNA to identify tumor-specific single nucleotide variants (reporter SNVs) in a panel of > 400 genomic regions and to monitor the dynamics of the reporter SNVs on treatment using a Monte-Carlo p-value statistical algorithm (Newman et. al. Nat Biotechnol 2016). The AOA NHL Test determines ctDNA presence or absence as an indicator of MRD, and assigns high-risk or low-risk EMR status in patients with DLBCL by measuring changes in ctDNA burden on treatment and applying a previously established cutoff. The analytical validation studies for the AOA NHL Test were designed to address accuracy, sensitivity, specificity, linearity, and precision of the following 1) variant detection in plasma samples; 2) ctDNA detection (measured as mean allele fraction (AF) of reporter SNVs using a Monte-Carlo p-value statistical algorithm); and 3) classification accuracy of the log-fold change (LFC) of mean mutant molecules per mL of plasma (MMPM), using clinically relevant cutoffs. Validation samples consisted of Genome in a Bottle blended gDNAs, serial dilution of healthy donor cfDNA blends, and serial dilution of DLBCL ctDNA into healthy donor cfDNA. Key results of the analytical validation are shown in Table 1. In summary, analytical validation of the AOA NHL Test demonstrated high sensitivity for detection of individual variants and ctDNA, and the capability of accurately detecting changes in ctDNA levels as measured by LFC in MMPM. The assay sensitivity improves with the increase in cfDNA input mass as well as the number of reporter variants while specificity (i.e. ability to detect signal over noise) can be maintained from sample to sample by pre-selecting a Monte-Carlo p-value cutoff. The final recommended sample acceptance criteria as well as sequencing QC metrics will be presented. Of 858 cases of DLBCL patients examined using the initial assay prototype and the validated AOA NHL Test, including samples from the POLARIX biomarker validation study, 846 (98.6%) had identifiable reporter variants in pre-treatment plasma samples with a median number of reporter SNVs of 137 per patient (range 1-1039), demonstrating that almost 100% of patients can be monitored with this assay. Of the 12 patients with no detectable reporters, 11 had less than 10 ng cfDNA available, highlighting the importance of sufficient sample input criteria established by analytical validation. The test is currently being used in a global prospective Phase 2 clinical trial (NCT04980222) to test the safety and efficacy of glofitamab in combination with R-CHOP in patients with High Risk EMR status. Acknowledgements: Alex Lovejoy for advice on the analytical validation design, Suzanne Cheng, Jennifer Holmstrom and Jill Ray for program management, and Bowdoin Su and Yanwen Jiang for abstract preparation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Persistence of Mutations in Complete Remission Including DNMT3A, TET2 and ASXL1 Mutations Is Associated with Worse Prognosis in Patients with Acute Myeloid Leukemia Treated in ALFA 0702 Study

Background: Only few studies have validated the use of molecular minimal measurable residual disease evaluation using error-corrected NGS (NGS-MRD). The prognostic relevance of detection of isolated DNMT3A, TET2 or ASXL1 (DTA) mutations post-induction remains controversial. Benchmarking of NGS-MRD with respect to other molecular MRD tools (such as WT1 expression and NPM1 mutation) has yet to be conducted. Material and methods: All patients included in the ALFA-0702 study who reached cytological complete remission (CR) or CR with incomplete platelets recovery after one induction course and with available material were included (n=189). Standard NGS sequencing of 67 genes was performed at diagnosis. Error-corrected NGS was performed on post-induction bone marrow samples using a custom panel including all mutations identified at diagnosis (n=735 mutations in 51 genes). Median sequencing depth in CR was 24695x after UMI deduplication. A threshold of 0.1% VAF was set as cut-off value for NGS-MRD detection for all targets. WT1 expression data in CR were available in 100 patients with initial WT1 over-expression. NPM1-MRD was evaluated in 67 patients by error-corrected NGS. For this evaluation, one alternative read was considered sufficient to define positivity. Leukemia free survival (LFS) and overall survival (OS) probabilities were evaluated since CR. Results: Median age at diagnosis was 46 years. ELN2017 risk was favorable in 52 (27%) patients, intermediate in 75 (40%) and unfavorable in 60 (32%). With a median follow-up from CR of 47 months, median LFS and OS was 35 and 41 months respectively. At diagnosis, gene mutations were detected in 181 (95%) patients. The median number of mutations per patient was 4 (range 1-10). Ninety-one patients had no target detected (NGS-MRD neg) and ninety had at least one target detected (NGS-MRD pos) including 42 patients with detection of DTA mutations only (DTA-MRD). NGS-MRD pos patients were significantly older (p=0.0018) than NGS-MRD neg patients and harbored a higher median number of mutations at diagnosis (4 vs 3, p=0.0021). There were no significant differences in terms of demographics and clinical presentation (age, WBC, number of mutation) between patients with DTA-MRD and non-DTA-MRD patients. In univariable analyses, NGS-MRD pos was associated with a lower probability of LFS (4-years LFS: 44% vs 68%, p= 6e-04) and OS (4-years OS: 57% vs 81%, p= 9e-04). There was no impact of the initial number of mutations on LFS nor OS. There were no significant differences in LFS and OS between DTA-MRD and non-DTA-MRD patients (figure 1). When adjusting analysis on ELN2017 status, MRD-NGS pos remained associated with shorter LFS (HR 2.15[1.34-3.44] , p=0.001) and OS (HR 2.41[1.38-4.20] p=0.002). As a continuous variable, the number of mutations detected in CR was associated with shorter LFS and OS. Specifically, detection of ≥2 mutations predicted poorer LFS (HR, p=3e-05) and OS (HR, p=4 e-05), compared with detection of only 1 mutation (figure 2). Sixteen patients were positive for WT1 MRD evaluation, and 84 were negative (WT1 neg). Among the WT1 neg patients 37 were NGS-MRD pos and 47 were NGS-MRD neg. When adjusting for both ELN17 and WT1 expression in CR, there was no significant impact of MRD-NGS positivity on LFS (HR=1.62 [0.85 - 3.09]) nor OS (HR=1.28 [0.59 - 2.74]) In the 67 patients with NPM1 mutation, 41 patients had NPM1 undetectable MRD (including 21 NGS-MRD pos and 20 NGS-MRD neg) and 26 had detectable NPM1-MRD (including 16 NGS-MRD pos and 10 NGS-MRD neg). When adjusting for both ELN status and NPM1-MRD, NGS-MRD pos was associated with a non-significant trend towards lower LFS (HR=2.34[0.96-5.71] p=0.063). Discussion and conclusions: In the homogeneously treated ALFA-0702 population, MRD-NGS positivity after the first induction course predicts shorter LFS and OS independently of ELN17 risk stratification. Detection of a higher number of mutations in CR is associated with a worse prognosis. Contrary to previous studies accruing older patients treated less intensively persistence of DTA mutations has similar prognostic relevance than non-DTA mutations. Future studies will be necessary to determine the superiority or addivitity of NGS-MRD with respect to available molecular MRD tools. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

From Generic RNA-Based MRD Assessment Towards Patient-Specific Genomic (DNA-based) MRD in AML and Other Non-B Cell Acute Leukemia

Pediatric acute myeloid (AML) is considered as a heterogeneous disease affecting 15% of children with acute leukemia. Outcome is still unsatisfactory with an overall survival around 70-80% and a relapse rate of 30-40% after primary treatment. The most important factors determining clinical outcome are genetic aberrations, and early response to treatment. Many studies have demonstrated the prognostic significance of minimal residual disease (MRD) monitoring. Although MRD monitoring is now an integral part in the management (including MRD-guided stratification) of Acute Lymphoblastic Leukemia (ALL) in children (generally done by ASO-qPCR on tumor specific IG/TCR targets), its clinical utility in childhood non-lymphoblastic leukemia (such as AML) is currently unknown/not used. Contemporary approaches to MRD detection in AML rely on PCR amplification of usually fixed genetic abnormalities (MRD-PCR) or on flowcytometric profiling of leukemia-associated immunophenotypes (LAIP). Molecular MRD detection is in general more sensitive than flow-based MRD, but the applicability is lower since recurrent mutational targets (e.g. fusion genes) using conventional technology, are present in only 40-50% of pediatric AML patients. This RNA-based MRD is common practice for CBFb-MYH11, RUNX1-RUNXT1, KMT2A-AFF1, -MLLT1, -MLLT3, and NPM1 exon 12 mutations and standard curves necessary for MRD quantification are available for all mentioned genes. Since the introduction of Whole Transcriptome Sequencing (WTS; RNAseq) as standard diagnostics in our laboratory, we identified various additional fusion genes in AML (increasing available molecular targets up to 75%), but also in other non-B cell malignancies such as SCLL, CEL, HES and BPDCN. By designing patient specific primers/probe sets surrounding the breakpoint sequences, we were able to generate 36 RNA-based MRD assays (including FOP-FGFR1, RANBP2-ALK, DEK-NUP214, ETV6-PDGFRB, KMT2A-AFDN) and monitor MRD during/after therapy. Although RNA-based MRD approach allows additional molecular monitoring in these patients, this type of MRD displays several disadvantages: sensitivity of a certain MRD assay can be low due to low expression levels of the fusion gene or low RNA yield and/or quality. Furthermore, quantification can be hampered by lack of available standard curves. Therefore DNA-based MRD is favorable over RNA-based MRD and Whole Exome Sequencing (WES) or Whole Genomic Sequencing (WGS) can identify the genomic breakpoint sequence in respectively genomic exons or introns. In addition, other genomic aberrations (such as insertions, deletions and point mutations) can be identified by these techniques and used for development of DNA-based MRD. By using this approach we were able to generate 17 additional DNA-based MRD assays (including FOP-FGFR1, RANBP2-ALK, RUNX1-CBFA2T3 NPM1-MLF1 UBTF-TD, p53 deletion), all with very high sensitivity (e.g. around 10-4 to 5x10-5) and patient-derived standard curves to quantify MRD. Figure 1 shows an example for DNA-based MRD monitoring of 3 AML patients harboring the recently described poor-outcome UBTF-TD aberration showing no/slow decrease of MRD in all patients. In conclusion, we show that fusion gene detection by WTS, preferably in combination with mutation analysis by WES/WGS, is providing comprehensive diagnostic results to improve the development of highly sensitive patient specific MRD targets to allow better molecular monitoring (and prediction of relapse), eventually leading to treatment stratification of patients in the future. We will present an update of our results and interesting cases at the upcoming conference. Figure 1: By WES, 3 patients with tandem duplications (TD) in exon 13 of the UBTF gene were identified, and patient specific primers and probes were designed for MRD on follow-up DNA samples. MRD was quantified by using a standard curve derived from serial dilutions from the diagnostic DNA. Quantitative range, sensitivity and background were determined according to the ESG-MRD-ALL guidelines. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Recommendations for laboratory testing of UK patients with acute myeloid leukaemia.

Recommendations for laboratory testing of UK patients with acute myeloid leukaemia.

Allogeneic hematopoietic stem cell transplantation for pediatric acute myeloid leukemia in first complete remission: a meta-analysis

Identification of pediatric patients with acute myeloid leukemia (AML) candidates to receive allogeneic hematopoietic stem cell transplantation (allo-HSCT) in first complete remission (CR1) is still a matter of debate. Currently, transplantation is reserved to patients considered at high risk of relapse based on cytogenetics, molecular biology, and minimal residual disease (MRD) assessment. However, no randomized clinical trial exists in the literature comparing transplantation with other types of consolidation therapy. Here, we provide an up-to-date meta-analysis of studies comparing allo-HSCT in CR1 with chemotherapy alone as a post-remission treatment in high-risk pediatric AML. The literature search strategy identified 10 cohorts from 9 studies performing as-treated analysis. The quantitative synthesis showed improved overall survival (OS) (relative risk, 1.15; 95% confidence interval [CI], 1.06–1.24; P = 0.0006) and disease-free survival (relative risk, 1.31; 95% CI, 1.17–1.47; P = 0.0001) in the allo-HSCT group, with increased relapse rate in the chemotherapy group (relative risk, 1.26; 95% CI, 1.07–1.49; P = 0.006). Sensitivity analysis including prospective studies alone and excluding studies that reported the comparison only on intermediate-risk patients confirmed the benefit of allo-HSCT on OS. Further research should focus on individualizing allo-HSCT indications based on molecular stratification and MRD monitoring.

Molecular Measurable Residual Disease Assessment before Hematopoietic Stem Cell Transplantation in Pediatric Acute Myeloid Leukemia Patients: A Retrospective Study by the I-BFM Study Group.

Hematopoietic stem cell transplantation (HSCT) is a curative post-remission treatment in patients with acute myeloid leukemia (AML), but relapse after transplant is still a challenging event. In recent year, several studies have investigated the molecular minimal residual disease (qPCR-MRD) as a predictor of relapse, but the lack of standardized protocols, cut-offs, and timepoints, especially in the pediatric setting, has prevented its use in several settings, including before HSCT. Here, we propose the first collaborative retrospective I-BFM-AML study assessing qPCR-MRD values in pretransplant bone marrow samples of 112 patients with a diagnosis of AML harboring t(8;21)(q22; q22)RUNX1::RUNX1T1, or inv(16)(p13q22)CBFB::MYH11, or t(9;11)(p21;q23)KMT2A::MLLT3, or FLT3-ITD genetic markers. We calculated an ROC cut-off of 2.1 × 10−4 that revealed significantly increased OS (83.7% versus 57.1%) and EFS (80.2% versus 52.9%) for those patients with lower qPCR-MRD values. Then, we partitioned patients into three qPCR-MRD groups by combining two different thresholds, 2.1 × 10−4 and one lower cut-off of 1 × 10−2, and stratified patients into low-, intermediate-, and high-risk groups. We found that the 5-year OS (83.7%, 68.6%, and 39.2%, respectively) and relapse-free survival (89.2%, 73.9%, and 67.9%, respectively) were significantly different independent of the genetic lesion, conditioning regimen, donor, and stem cell source. These data support the PCR-based approach playing a clinical relevance in AML transplant management.

PB1760: AZACITIDINE AS TREATMENT OF KMT2A INFANT ACUTE LYMPHOBLASTIC LEUKEMIA: SINGLE CENTER EXPERIENCE

Background: KMT2A rearranged (r) infant acute lymphoblastic leukemia (ALL) is characterized by a very poor prognosis, particularly after relapse. Intensive chemotherapy does not improve outcome and novel therapies are under investigation for this dismal disease. Recent studies reported an hypermethylated DNA profile in KMT2Ar ALL, leading to repression and silencing of tumor suppressor genes. Hypomethylating agents (HMA) have been hypothesized to exert an antileukemic activity restoring normal gene expression. Aims: Here we report the off-label administration of Azacitidine (AZA) in two little patients aged 1 month old and 26 days old respectively, diagnosed with KMT2Ar infant ALL. Methods: Both patients were treated according to the high risk arm of the AIEOP-BFM ALL 2017 protocol as front-line therapy. AZA was administered every 21 days (d) for 4 cycles (cy), with the following schedule: 55 mg/m2 for 5 d, 55 mg/m2 for 7 d, 75 mg/m2 for 5 d, 75 mg/m2 for 7 d, respectively; prednisone 1 mg/m2 was associated to each cycle. Results: Patient 1 was diagnosed with infant B ALL t(11;19)/KMT2A-MLLT1r, high-risk (HR) for age, hyperleucocytosis and KMT2Ar. At the end of induction (EOI) he was in hematologic remission with molecular minimal residual disease (MRD) and t(11;19) still detectable. The child received consolidation chemotherapy followed by 2 cy of bispecific CD3-CD19 antibody (ab) Blinatumomab and underwent allogeneic hematopoietic stem cell transplant (HSCT) from matched unrelated donor (MUD) with detectable MRD. Two months after transplant patient relapsed with 68% CD19 CD22 negative blasts in bone marrow. Due to early relapse and no target therapy available, AZA was administered. Grade 4 cytopenia and pneumonia were main adverse events. Patient achieved MRD complete remission (CR) and underwent to haploidentical HSCT from mother, followed by 3 courses of 40 mg/m2 AZA maintenance for 5 d, every 21 d. Patient is alive, in MRD negative CR3, 3 months after SCT. Patient 2 was diagnosed with infant B ALL t(4;11)/KMT2A-AFF1, HR for age, hyperleucocytosis and KMT2Ar. At the EOI bone marrow showed blasts 25% detected by flow cytometry. Patient received one courses of chemotherapy according to the HR protocol, followed by 2 cy of Blinatumomab. The child achieved MRD negative CR and underwent HSCT from MUD. Thirty two days after HSCT, patient relapsed with 60% BM blasts detected by flow cytometry. Due to early relapse after HSCT, he received 2 cy of anti-CD22 ab Inotuzumab, achieving CR without platelet recovery (CRi). Nevertheless, t(4;11) was positive again at the end of the 2nd cy. Patient then received AZA as 3th-line therapy achieving CRi after 2nd cy. However, due to severe cytopenia and gastrointestinal toxicity, patient received 3 out of 5 days treatment of the 3th cy and the 4th cy was delayed. Patient relapsed after the 4thcourse. Summary/Conclusion: In our experience, single agent AZA alone was effective and well tolerated in heavily pretreated patients. To our knowledge, single agent HMA in KMT2Ar ALL has been reported only in one patient. Preclinical studies have revealed heterogeneous results when HMA are combined with cytotoxic drugs; results of two pilot trials of AZA combined with chemotherapy are still ongoing. Further clinical trials could clarify the optimal schedule, timings and dosage of HMA.