Mixed Phenotype Acute Leukemia Research Articles

Treatment Outcomes of Childhood Picalm:MLLT10+ Acute Leukemias: An International Retrospective Study

Introduction: Acute leukemia with translocation t(10;11)(p12-13;q14-21) results in generation of the PICALM:MLLT10 fusion gene, and is distinct from t(10;11)(p11-13;q23) which gives rise to KMT2A (formerly MLL) gene rearrangement. Defective PICALM function leads to aberrant hematopoiesis, while defective MLLT10 function impairs chromatin-mediated gene regulation. The PICALM:MLLT10 gene has been reported in patients with Acute Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL) predominantly T-cell, Acute Undifferentiated Leukemia (AUL) and Mixed Phenotype Acute Leukemia (MPAL). MLLT10 interacts directly with DOT1L in a complex that regulates the methylation of H3K79. MLLT10 fusions are thought to misdirect DOT1L to the promoters of HOXA genes, leading to hypermethylation of H3K79. This hypermethylation causes constitutive activation of HOXA activity and prevents cell maturation and differentiation. The clinico-biological characteristics, response to conventional treatments, and survival outcomes of childhood PICALM:MLLT10+ leukemias are poorly understood. Methods: This was an international, multicenter, retrospective cohort study. Data were submitted by ten International-BFM (I-BFM) study Group-AML and ALL committees and by the Children`s Oncology Group (COG)-AML Study committee. For study inclusions, patients were required to be ≤ 21 years of age at diagnosis, diagnosed between January 1995 and December 2019 with de novo ALL, AML, AUL or MPAL and PICALM:MLLT10 fusion-positive. Patients with KMT2A-rearranged leukemia or acute promyelocytic leukemia were excluded. Kaplan Meier curves were employed for survival analysis with Cox proportional hazard modelling to compare effect of AML vs ALL therapy, and receipt of hematopoietic stem cell transplant (HSCT) vs no HSCT, on survival. Pearson's Chi Squared was employed as a non-parametric measure to determine the effect of end of induction-1 (EIO-1) MRD positive vs negative status on incidence of relapse. P value cut-off for data to reach statistical significance was <0.05. Results: Ninety-eight children with acute leukemia were included, 60 were males. Median age at diagnosis was 12 years (IQR:9-14.8 years) and median initial white cell count was 40.5 x 109/L (IQR:13.8-168.9). Fifty-five (55.6%) patients were diagnosed with T-ALL, 39 (39.4%) with AML, while precursor-B ALL, AUL and MPAL were each diagnosed in single patients; 1 subtype diagnosis was unknown. Ten (10.1%) patients presented with CNS disease and 13 (13.1%) presented with non-CNS extramedullary manifestations. Of 66 with cytogenetic/molecular data available, 33 (50%) had additional cytogenetic-molecular aberrations; NOTCH1, WT1 and RAS pathway mutations predominated. Most patients received an initial chemotherapy regimen guided by their disease immunophenotype: 58/98 (58.6%) received an ALL-like chemotherapy regimen upfront, 38 (38.4%) received an AML-like regimen, 1 (1.0%) a hybrid regimen, 1 initial chemotherapy regimen was unknown. Five-year event-free survival (EFS) was 50.4% (CI 39.1-64.9). For AML patients, 5-year EFS was 22.5% (CI 9.75-52.0) and for ALL 67.7% (CI 54.2-84.7), p=<0.001. Five-year overall survival (OS) was 56.3% (CI 46.3-68.5). For AML patients, 5-year OS was 26.7% (CI 13.3-53.6) and for ALL 75.7% (CI 64.6-88.8), p=<0.001. Thirty-eight patients died, 22 (57.9%) deaths were disease-related, 4 (10.5%) treatment-related. Patients who received HSCT in CR1, demonstrated a trend towards an inferior 5-year EFS (23.7% vs 56.5%) and OS (42.6% vs 64.5%) than those who did not; however, analysis was limited by small sample size. Four (22.2%) HSCT recipients had ALL but most, 14 (77.8%), had AML, which may account for the unfavourable outcomes associated with HSCT. There was no statistically significant difference in rate of relapse in the EOI-1 MRD-positive compared with MRD-negative patients (p=0.49). Of those who relapsed, 25 (75.8%) had an isolated bone marrow relapse, 3 (9.1%) isolated CNS, 3 (9.1%) other sites and, for 2 patients, site of relapse was unknown. Conclusions: Children with PICALM:MLLT10+ AML have inferior 5-year EFS and OS outcomes compared to their T-ALL counterparts. MRD status at EOI-1 was not predictive of relapse. Targeted therapies with DOT1L inhibitors or DNA demethylating agents should be explored, particularly for patients with AML.

Evaluation of Analysis Strategy for Cytoplasmic Lineage-Associated Markers in Mixed-Phenotype Acute Leukemia (MPAL) As Devised By Who Classification 2022

Background: Conventional rating of flow cytometrically determined expression of cytoplasmic lineage-associated markers MPO, CD3 and CD79a has defined positivity by ≥10% leukemic cells being positive compared to internal negative controls. WHO classification 2022 has devised a new approach defining positivity by part of leukemic population exceeding 50% of expression level of normal counterpart populations used as positive controls. Aim: Evaluate interpretation of cytoplasmic expression of MPO, CD3 and CD79a in MPAL and other acute leukemias. Methods: 277 acute leukemia cases were analyzed (MPAL-BM, n=18; MPAL-TM, 20; AML M0, 42; AML M1, 40; AML M2, 39; APL, 39; BCP-ALL, 40; T-ALL, 39). Following WHO 2022 positivity for cytoplasmic MPO, CD3 and CD79a was determined setting a cutoff at 50% of mean fluorescence intensity (MFI) of granulocytes, mature T-cells and mature B-cells, respectively. An alternative refined approach was evaluated calculating the MFI ratio between leukemic populations and positive controls. Results of MFI ratios are given as log differences, i.e. 0 representing identical MFIs, -1 meaning 1 log lower expression in leukemic population compared to controls. Log difference of -1.5 was set as cutoff. Results: Applying WHO 2022 majority of cases were rated positive for expected markers, i.e. AML, MPAL-BM and MPAL-TM for MPO, T-ALL and MPAL-TM for CD3 and BCP-ALL and MPAL-BM for CD79a (figure, A). However, there were cases rated positive in other groups although in general only few leukemic cells were causing positive rating, indicating limited specificity of this approach. In detail, MPO was positive in 69% of M0, 95% of M1, 92% of M2, 92% of APL, 61% of MPAL-BM and 70% of MPAL-TM, but also in other groups (BCP-ALL, 35%; T-ALL, 51%).Applying WHO 2022, diagnosis would have changed to BCP-ALL in 39% of MPAL-BM and to T-ALL in 30% of MPAL-TM cases. CD3 was positive in 92% T-ALL and 95% MPAL-TM cases, but also in other groups (M0, 29%; M1, 25%; M2, 15%, APL, 49%; BCP-ALL, 13%; MPAL-BM, 56%). Applying WHO 2022, diagnosis would have changed to AML in 5% of MPAL-TM cases. CD79a was positive in 100% of BCP-ALL and 94% of MPAL-BM, but also in other groups (M0, 55%; M1, 43%; M2, 51%, APL, 49%; T-ALL, 33%; MPAL-TM, 75%). Applying WHO 2022, diagnosis would have changed to AML in 6% of MPAL-BM cases. In order to refine the WHO 2022 approach and to improve specificity MFI ratio between leukemic cells and positive controls was applied. Similarly to the WHO 2022 approach the MFI ratio approach resulted in expected differences but also significant overlaps (figure, B), although to a lower degree for MPO. In detail, MPO was positive in 31% of M0, 75% of M1, 77% of M2, 85% of APL, 72% of MPAL-BM and 50% of MPAL-TM, but also in other groups (BCP-ALL, 13%; T-ALL, 23%). Applying the MFI ratio approach, diagnosis would have changed to BCP-ALL in 28% of MPAL-BM and to T-ALL in 50% of MPAL-TM cases. CD3 was positive in 92% of T-ALL and 100% of MPAL-TM cases, but also in other groups (M0, 14%; M1, 23%; M2, 18%, APL, 85%; BCP-ALL, 13%; MPAL-BM, 72%). Applying the MFI ratio approach there would have been no changes in MPAL-TM diagnosis. CD79a was positive in 98% of BCP-ALL and 94% of MPAL-BM, but also in other groups (M0, 50%; M1, 65%; M2, 46%, APL, 87%; T-ALL, 56%; MPAL-TM, 85%). Applying the MFI ratio approach, diagnosis would have changed to AML in 6% of MPAL-BM. In 6 and 7 cases analyzed by WHO 2022 and MFI ratio, respectively, changes in diagnosis from MPAL to ALL were not supported by NGS yielding mutations known to be associated with myeloid neoplasms while changes were supported by NGS in a minority of cases. Discussion: The WHO 2022 definition, as compared to conventional analysis, changed diagnosis in a significant number of MPAL cases, in particular due to rating MPO negative. At the same time, application of WHO 2022 approach to cases with AML, BCP-ALL and T-ALL suggests limited specificity with significant rates of positivity outside the lineage determined by conventional approach. The MFI ratio approach also changed diagnosis in a significant number of MPAL cases in comparison to the conventional approach, however, data suggests a better specificity for MPO. Further, MFI ratio considers the whole leukemic population and thus may result in higher reproducibility. Overall this data argues in favor of considering multiple lineage-associated markers instead of single markers. In addition, classification based on genetic markers should be maximized also in MPAL.

Clinical Experience with Azacitidine and Venetoclax +/- Gemtuzumab in Pediatric Patients with Relapsed or Refractory Acute Leukemias: A Report from the Children's Hospital of Philadelphia

Background: Children with refractory or relapsed (R/R) leukemia, particularly acute myeloid leukemia (AML), have limited curative options. Allogeneic hematopoietic stem cell transplantation (HSCT) for patients who achieve subsequent complete remission (CR) after relapse is generally required for long-term cure, but many children with multiply-R/R AML are unable to achieve CR with conventional salvage regimens. Dual therapy with the hypomethylating agent azacitidine and BCL-2 inhibitor venetoclax (aza/ven) is well-tolerated in adults with AML and has become the standard of care for elderly patients or those with co-morbid conditions precluding intensive chemotherapy. The safety of venetoclax in combination with cytarabine +/- idarubicin was established in children with R/R AML (Karol Lancet Oncology 2020), but pediatric experience with the aza/ven combination remains limited. We sought to describe our institutional experience with aza/ven-based therapy in children and adolescents/young adults with R/R acute leukemias. Methods: We conducted a retrospective chart review of patients aged 0-21 years at initial leukemia diagnosis who were treated with commercially-available aza/ven at the Children's Hospital of Philadelphia (CHOP) from January 1, 2019 to June 30, 2022. Data were collected for baseline patient demographics, leukemia immunophenoptyping and genetics, therapy administration, clinical response, and post-aza/ven outcomes. Azacitidine 100 mg/m2 daily intravenously (IV) was given on days 1-5 of each 28-day cycle. Venetoclax was given daily as oral tablets with 3-day ramp-up dosing to minimize tumor lysis syndrome (TLS) risk as 200 mg (day 1), 400 mg (day 2), and 800 mg (days 3-28) adult equivalent dosing (Place Future Oncology 2018) with 50% dose reduction for patients receiving concomitant azoles. Results: Thirty-seven pediatric patients with R/R acute leukemia received aza/ven-based therapy at CHOP during the study period; 27 (72.9%) had AML, 7 (18.9%) had B-cell acute lymphoblastic leukemia (B-ALL), 1 had T-cell ALL (2.7%), and 2 (5.4%) had mixed phenotype acute leukemia (MPAL). Six children did not complete a full 28-day cycle of aza/ven due to progressive disease (n=4) or severe TLS (n=2). Within the cohort of patients who completed 1 cycle, 22/31 (71%) had multiply-relapsed leukemia, and 9/31 (29%) had primary chemorefractory disease. The median age at initial leukemia diagnosis was 8 years (range 0.5-21). Patients in this cohort received a median of 2 aza/ven cycles (range 1-6). Eight patients with AML also received one dose of gemtuzumab ozogamicin (GO) 3 mg/m2 IV (given on day 4, 5, or 8) with cycle 1 aza/ven; 3 patients received additional GO in subsequent cycles. Twelve of 31 patients who completed 1 cycle aza/ven had cycle 2 or later chemotherapy held or delayed, most commonly to facilitate resolution of drug-, infection- or leukemia-induced cytopenias. Amongst the 29 patients who underwent bone marrow response assessment after aza/ven cycle 1 (n=25) or cycle 2 (n=4), 14 were in CR with negative measurable residual disease (MRD) by flow cytometry (<0.1% for AML, <0.01% for ALL/MPAL), including young children with high-risk AML genetics such as CBFA2T3-GLIS2 and ETV6-EP300 fusions. One additional patient with residual leukemia after cycle 1 aza/ven achieved MRD-negative CR after cycle 2. The remaining patients had either persistent leukemia detectable in peripheral blood or were MRD+ in bone marrow at first response assessment.After receipt of aza/ven, 11 patients with AML and 1 with MPAL proceeded to HSCT, 2 patients with B-ALL were treated with autologous CD19 chimeric antigen receptor T cells (CD19CART), and 1 patient with AML received allogeneic CD123CART. The study cohort was followed for a median of 7.8 months (range 1-32 months). At last follow up, 39% (12/31) of patients were alive (Figure 1). Conclusions: We report the experience of a single institution with 37 pediatric patients with multiply-R/R AML, ALL, and MPAL treated with aza/ven-based therapy, often in the outpatient clinic with good tolerability. Importantly, MRD-negative CR was achieved in 14/37 children (37.8%), nearly always after one cycle of aza/ven, which enabled consolidative HSCT in most responders and long-term survival of some patients. Formal clinical trial evaluation of aza/ven in children with R/R acute leukemias, ideally at an earlier stage of relapse, is warranted. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Single-Cell Pan-Cancer Analysis Reveals Treatment Resistance Stem/Progenitor-like Subpopulation in the High-Risk Pediatric Leukemia

Leukemic stem cells have long been considered a therapy-resistant population in myeloid leukemia. Several recent publications have also described a population with stem/progenitor-like phenotype in pediatric B-cell acute lymphoblastic leukemia (B-ALL). However, the existence and cell-of-origin of stem/progenitor-like blasts in other subtypes of leukemias have not been studied extensively. Moreover, the relationship of this subpopulation with clinical outcomes and strategies to target them remain to be systematically explored in pediatric leukemia. To investigate these questions, we extensively characterized diagnostic samples of 84 patients with high-risk pediatric leukemias, including AML (10), B-ALL (23), ETP T-ALL (30), non-ETP T-ALL (10), and mixed phenotype acute leukemia (MPAL) (11). We performed scRNA-Seq and scATAC-Seq on all 84 patient samples, generating an atlas of more than one million cells (Figure 1A, UMAP colored by sample). By projecting leukemic cells onto a novel healthy reference atlas composed of pediatric and infant bone marrow and thymus, we identified and characterized a stem/progenitor-like subpopulation with a common immunophenotype CD34+CD44+CD133+ in all five leukemia subtypes (Figure 1B). More importantly, we found the frequency of this subpopulation is highly correlated with the level of minimal residual disease after induction chemotherapy, suggesting therapy resistance of stem/progenitor-like leukemic blasts. We identified several pathways commonly up-regulated in stem/progenitor-like subpopulations across different high-risk leukemias, including previously reported leukemia stem cell (LSC) signatures in AML, quiescent signature, and ribosome biogenesis. To better power analyses relating stem/progenitor-like subpopulations with clinical outcome, we deconvoluted and analyzed >3000 bulk RNA-Seq samples from the TARGET and Children's Oncology Group AALL0434 T-ALL databases using our single-cell molecular signatures and found a universal existence of stem/progenitor-like leukemic blasts that predict poor clinical outcome across different high-risk leukemias. Our single-cell analysis consistently associated chemoresistance with presence of stem/progenitor-like leukemic blasts across 5 subtypes of high-risk pediatric leukemia, leading us to seek novel therapeutic strategies targeting this subpopulation. Using single-cell derived cell signatures, we performed an in silico drug screen using multiple independent drug/target databases. This analysis revealed several pan-leukemia candidate drug targets for stem/progenitor-like leukemic blasts, including FLT3, CD44, CD133, BCL2, and PIK3R1. We also found leukemia-subtype-specific candidates such as BRAF for AML, TGFB1 for B-ALL, IL1B for ETP-ALL, and STAT3 for AML and B-ALL. Both pharmacological and genetic experiments using cell lines and PDX models are underway to validate the candidate drug targets. Finally, we employed ligand-receptor analysis to identify interactions that stem/progenitor-like blasts could have with their surrounding environment. This analysis revealed a set of immunosupressive ligand-receptor pairs between the stem/progenitor-like leukemic blasts and NK cells. Using our predicted ligand-receptor ineractions, we computed patient-specific NK cytotoxic scores and discovered an immunosuppressive role of stem/progenitor-like leukemic blasts towards NK cells in all leukemia subtypes, suggesting a mechanism for immune surveillance escape and potential resistance to immunotherapy in stem/progenitor-like blasts. Taken together, our data reveal the existence of a common stem/progenitor-like blast population in high-risk pediatric leukemias. These cells exhibit characteristics of leukemia stem cells, quiescence signatures, resistance to chemotherapy, and immunosuppressive effect towards NK cells. They are also highly predictive of poor clinical outcomes across different high-risk leukemias. We identified common and leukemia-subtype-specific candidate drug targets. Follow-up studies targeting these genes along with conventional chemotherapy are underway with promise to improve the clinical outcome for high-risk pediatric leukemias. CC & JX, as well as DTT & KT, contributed equally to the work. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Single-Cell Profiling of Acute Myeloid Leukemia Identified ARMH1, a Novel Protein Associated with Proliferation, Migration, and Drug Resistance

Despite advancements in treatments, half of pediatric acute myeloid leukemia (AML) patients relapse (Chen, J. & Glasser, C.L., 2020), making it pertinent to identify novel biomarkers that can be used for better stratification of patients post end of induction (EOI) therapy as well as to develop targeted therapies that could lead to higher clinical remission (CR) rates. Single-cell RNA sequencing (scRNAseq) analysis has revolutionized cancer research by identifying novel cell types, genes, and pathways. Our group used scRNAseq analysis of pediatric AML bone marrow (BM) samples collected at diagnosis (Dx) and EOI to identify the upregulation of multiple genes including a novel gene, Armadillo like helical domain containing 1 (ARMH1)/C1orf228, in cancerous blast cells (Thomas, B.E. et al., 2020). ARMH1 has not been previously associated with AML. Also, ARMH1 expression was observed to be significantly higher at Dx in samples from patients with relapse compared to samples from patients with CR (P<2.2e-16). Independent validation for ARMH1 blast-association using single cell and bulk transcriptome profiling revealed that ARMH1 expression was significantly higher in scRNAseq data of AML Dx blast cells compared to EOI non-blast cells (fold change, FC=1.8, P<2.2e-31) as well as in bulk RNAseq data of Dx samples with high blast% (>90%) compared to EOI samples (FC=1.8, P=.003) (Ulukaya, G. et al., 2021). Further analysis on other leukemias like T cell-acute lymphocytic leukemia (T-ALL) and mixed phenotypic acute leukemia (MPAL) also revealed higher expression of ARMH1 in the blast cells as compared to non-blast cells. Survival analysis on bulk RNAseq data using the survival genie platform (Dwivedi, B. et al., 2022) revealed that higher expression of ARMH1 was significantly associated with poor overall survival (OS) in both AML (P=.003) (Ulukaya, G. et al., 2021) and T-ALL (P=.014) datasets. Comparative gene expression analysis using the the Pediatric Single Cell Cancer Atlas - PedSCAtlas - tool (Mumme, H.L. et al., 2021), revealed negligible expression of ARMH1 in normal lymphoid, myeloid, and erythroid cell lineages, further confirming the leukemic blasts-specific over-expression of ARMH1. ARMH1 encodes a cytosolic protein of yet unknown function. The malignant blast specificity of ARMH1 suggested a role in leukemic cells' survival/maintenance. To ascertain its functional involvement in cancer cells, ARMH1 was silenced using siRNAs in both normal and AML cell lines. Through in vitro assays, we demonstrate that ARMH1 silencing significantly reduced leukemic cells' capacity to proliferate (P=.0041) and migrate (P=.0001). We also observed slightly increased sensitivity to chemotherapy drug, Cytarabine in the ARMH1 silenced cancer cells compared to control cancer cells in which the gene is fully functional. However, suppression of ARMH1 expression had no effect on cell growth in the normal HEK293t cell line (P=0.913). On the other hand, overexpression of ARMH1 via transient transfection of ARMH1 gene into the AML cell line, made the cells more resistant to Cytarabine and significantly increased both migration (P=.041) and cell proliferation (P=.032). In conclusion, we demonstrate for the first time that ARMH1, a still poorly understood protein, contributes to the increased proliferation and migration of malignant AML cells. With specific overexpression in blast cells of several pediatric leukemias and the newly discovered roles in proliferation, migration, and chemotherapy resistance, ARMH1 may be a good candidate for targeted treatment of pediatric leukemias.

A Phase 1b Dose Escalation and Expansion Study of SNDX-5613, Azacitidine (AZA) and Venetoclax (VEN) in Newly Diagnosed, Patients ≥ 60 Years with Untreated NPM1-Mutated/ FLT3-Wild Type AML or KMT2A-Rearranged Acute Myeloid Leukemia (AML)

Background: SNDX-5613 is a potent, orally available, small molecule inhibitor of menin, which binds to MLL1, encoded by the KMT2A gene, and MLL1 fusion proteins. Menin expression leads to aberrant gene transcription through its activation of Homeobox-related genes, leading to maturation arrest and leukemogenesis in KMT2A-rearranged and NPM1-mutated leukemias. Inhibition of menin activity significantly blocks the transcriptional signature and subsequently the proliferative capacity of leukemia cells harboring NPM1 mutations or KMT2A rearrangements. SNDX-5613 and its analogs have demonstrated robust single-agent anti-proliferative activity in NPM1-mutated AML cells and AML patient-derived xenograft models. Currently, SNDX-5613 is being explored as a single agent in a phase 1/2 study in patients with NPM1-mutated or KMT2A-rearranged relapsed/refractory AML, mixed phenotypic acute leukemia and acute lymphoblastic leukemia. The primary objective of this study is to determine the safety and recommended dose of SNDX-5613 combined with AZA and VEN in untreated AML patients age ≥ 60 years who are not candidates for intensive chemotherapy. Study Design and Methods: This study is a sub-study of the BeatAML Master Trial (NCT03013998) in which untreated AML patients age ≥ 60 are assigned an investigational therapy based on cytogenetic and central genomic analysis. Patients must specifically harbor an identifiable NPM1 mutation without a concomitant FLT3 mutation or have a KMT2A rearrangement to be eligible. In this phase 1b study, the primary objective is to determine the safety and recommended dose using a standard 3+3 design based on dose-limiting toxicities with 3 dose levels (DL) of SNDX-5613 to be evaluated in combination with VEN/AZA and an anti-fungal (required during induction): 75mg (DL-1), 113mg (DL1), and 163mg (DL2). In continuation phase with anti-fungals not required, SNDX-5613 will be dosed at 113mg (DL-1), 163mg (DL1), or 226mg (DL2) with appropriate dose reductions added for both SNDX-5613 and VEN if continuing on anti-fungals. The secondary objectives are to assess the overall response rate, measurable residual disease (MRD) status, overall survival, transplant rate and duration of remission. During induction cycles, VEN (D1-28) and AZA (D1-7) are dosed per standard of care every cycle. SNDX-5613 is dosed continuously orally twice daily with dose levels listed above. In patients who achieve a morphologic remission (<5% blasts), SNDX-5613 will be held if absolute neutrophil count (ANC) is < 0.5x109/L and/or platelets (PLT) < 50x109/L. Patients can receive up to 3 induction cycles to achieve a composite complete remission (CRc: CR, CRi, CRh). Patients can receive continuation therapy with AZA, VEN and SNDX-5613 once they achieve CRc and after hematologic recovery. Following the determination of a safe and recommended dose in phase 1b, there will be a dose expansion cohort of 12-20 patients following the same schedule as described above (Figure 1) to further evaluate safety, tolerability and correlative biomarkers. Correlative objectives include assessment of SNDX-5613 pharmacokinetics and its correlation with response and toxicities. Additionally, the study will evaluate the impact of MRD, as measured by central flow cytometry and next-generation sequencing of NPM1 mutations. Additional evaluations and biomarkers of potential interest include menin- KMT2A-specific transcriptomic changes, myeloid differentiation/cell subset changes, mechanisms of resistance, and discovery of baseline features (methylation, mutations, etc.) that can impact outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Acute Lymphoblastic Leukemia in Patients 60 Years and Older Treated with Pediatric-Inspired Protocols, 20 Years of Follow-up

Background Chemotherapy-based approaches remain an essential component of current adult acute lymphoblastic leukemia (ALL) treatment. Poor outcomes in older patients with ALL result from the disease's adverse biology and poor tolerance to chemotherapy. Pediatric-inspired protocols have been used over the past 20 years in our center for patients > 60-year-old, both Philadelphia negative (Ph-), and Philadelphia positive (Ph+). Historically, Ph+ patients have been believed to have inferior outcomes. Our goal was to estimate the outcomes of this subgroup of patients and to compare outcomes between Ph- and Ph+ patients. Methods All newly diagnosed patients with ALL aged > 60 years, and receiving induction chemotherapy with a modified pediatric-inspired regimen between January 2002 and January 2022 were included. The treatment regimen consisted of induction, central nervous system prophylaxis, seven 3-week cycles of intensification, and 24 3-week cycles of maintenance (PM DFCI-based protocols). Only Ph- patients received asparginase. All Ph+ patients received a Tyrosine Kinase Inhibitor (TKI) during all phases of treatment and continued indefinitely after maintenance. There were adjustments in the type of drugs and dosages over the years. Standard descriptive statistics were used. Ninety-five percent confidence intervals were provided for estimates of interest where possible. Fisher's exact test was used to assess the impact of categorical covariates. P < 0.05 were considered to indicate a significantly different result. Kaplan-Meier survival estimate was used to evaluate Leukemia free survival (LFS) and overall survival (OS). Results A total of 122 patients were included: Ph- B-ALL, 50% (N=61); Ph+ B-ALL, 38% (N=46); Mixed Phenotype Acute Leukemia (MPAL), 4% (N=5); and T-ALL, 8% (N=10). Out of the Ph+ patients, only one had an MPAL diagnosis, the rest of them had B-ALL diagnoses. Patient and disease characteristics by Ph status are presented in table 1. For the whole cohort, the complete remission rate after induction (CR) was 83% (N=101), and allogeneic stem cell transplantation (alloSCT) in CR1 was carried out for 10% (N=12). Ten patients (8.2%) died during induction and the cumulative incidence of relapse (CIR) at 1-year was 11.7 % (95% CI 6.7 - 18.2). CR, mortality at induction, alloSCT, and CIR rates were similar in the Ph- and Ph+ groups. With a median follow-up of 52 months (range 4-209 months) among survivors, the median OS was 54 months (95%CI: 28-82). The 1-year and 4-year OS rates were 76% (95%CI: 68-83%) and 51.2% (95%CI: 41.4-60.2%), respectively. Leukemia-free survival (LFS) at years 4 years was 41.9% (95%CI: 32.7-50.9). There were no statistical differences in median OS and LFS between Ph- and Ph+ patients. OS at 4 years for patients with Ph- ALL was 49.7% (95%CI: 37.1-61) vs 53.9% (95%CI:37.9-67.5) in Ph+ ALL patients (p=0.72) (Figure 1). At 4 years LFS was 40.8% (95%CI: 29 - 52) for Ph- patients and, 43.6% (95%CI: 28 - 57) for the Ph+ population (p=0.68). Conclusion Pediatric-inspired chemotherapy protocols with appropriate dose adjustments for age are associated with good outcomes in older ALL patients. Clinical and disease characteristics and outcomes were similar between Ph- and Ph+ ALL patients. We aim to further define the optimal therapy for this group of patients with this challenging disease. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Precision-Engineered Cell Therapy Orca-T Demonstrates High Relapse-Free Survival at 1 Year While Reducing Graft-Versus-Host Disease and Toxicity

BACKGROUND Allogeneic hematopoietic stem cell transplants (alloHSCT) remain the only curative treatment for many advanced hematologic malignancies; however, despite improvements in donor matching and supportive care, these transplants are associated with significant toxicity and reduced quality of life. Strategies to maintain or enhance the graft-vs-tumor and graft-vs-infection effects while eliminating graft-vs-host disease (GVHD) have long been a key goal in the field. Orca-T is a high-precision, immunotherapy consisting of stem and immune cells, derived from allogeneic donors, that leverages highly purified, polyclonal donor regulatory T cells to control alloreactive immune responses. Orca-T has demonstrated a favorable safety profile and promising control of both relapse and GVHD. METHODS As of 15 July 2022, 180 patients have received Orca-T. Of those patients, 127 have ≥180 days of follow-up and a diagnosis of acute leukemia in CR [AML (n=62), ALL (n=42), mixed phenotype acute leukemia(n=4), CML with prior blast crisis (n=4)], or high risk MDS (n=15). Patients received Orca-T as part of a single-center Phase 2 study (n=32, NCT01660607) or a multicenter Phase 1b study (n=95, NCT04013685). Median follow-up was 387 days (range 41-2062); 72 and 36 patients have > 1 year and >18 months of follow-up, respectively. Patients were aged 19-69 (median 48) and 57% male. Donors were HLA-matched related (n=66) or unrelated (n=61). Patients received investigator's choice of myeloablative conditioning regimens (busulfan-based, n=98; TBI-based, n=29) prior to Orca-T, followed by single-agent GVHD prophylaxis with either tacrolimus (n=124) or sirolimus (n=3). For comparison purposes, an independent CIBMTR-based cohort was identified which consisted of patients with AML, ALL, or MDS who received myeloablative alloHSCT with a PBSC source between 2016-2018 followed by tac/methotrexate PPX. Matching for relapse risk was based on minimal residual disease (MRD) status in acute leukemia patients; development of an additional risk matched CIBMTR comparator based on DRI score and MRD status is ongoing. RESULTS Orca-T was successfully manufactured in a single, centralized GMP manufacturing facility, distributed throughout the U.S., and infused for all patients enrolled. The relapse-free survival was 81% at both 1 year and 18 months in Orca-T recipients. MRD status was determined for 77 patients with acute leukemia by multicolor flow cytometry; of these patients, 31% were MRD+ when they received Orca-T. Amongst MRD- patients (n=53), RFS with Orca-T was 90% at both 1 year as compared to 66% in the CIBMTR cohort (n=324 MRD- patients). Amongst MRD+ patients (n=24), RFS was 68% at one year with Orca-T as compared to 48% in the comparator cohort (n=104). Relapse prevention with Orca-T appeared to be enhanced further with a conditioning regimen consisting of busulfan, fludarabine, and thiotepa ("BFT", n=56 patients, median f/u 342 days); RFS was 90% at 12 months in this group. This included patients with MDS (n=6, 100% RFS at 1 yr), MRD+ acute leukemia (n=14, RFS 73% at 1 yr), MRD- acute leukemia (n=26, RFS 96% at 1 year), and acute leukemia with unknown MRD status (n=11, 91% RFS at 1 yr) (Figure 1). As with relapse, severe infections were low following Orca-T with 11% of patients developing Grade 3 infections per the BMT-CTN grading scale. Median times to neutrophil and platelet engraftment were rapid with Orca-T at 13 and 16 days, respectively; graft failure was rare at 1.6%. Grade ≥ 3 aGVHD and mod/severe cGVHD rates were low with Orca-T at 5% and 6%, respectively, through 1 year post-transplant. Non-relapse mortality was low at 5% at 1 year; NRM with BFT was 0%. Overall, Orca-T shows GRFS of 76% and 69% at 1 year and 18 months post-transplant, respectively; OS was 91% and 86% at these time points post-transplant. No formal statistical comparison to the CIBMTR cohort was performed (Table 1). CONCLUSIONS At more than 1 year of median follow-up, outcomes with Orca-T, a high-precision immunotherapy, demonstrate robust graft-vs-leukemia and graft-vs-infection effects while markedly reducing GVHD and NRM despite MAC. These outcomes were accomplished with consistent and reliable cell manufacturing and distribution of Orca-T at a national scale. A multi-center randomized controlled phase 3 trial comparing Orca-T to SOC, utilizing BFT or TBI-based conditioning, is currently enrolling across the US (NCT05316701). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Updated Outcomes of the University of Southern California (USC) ALL Frontline Regimen (based on CCG-1882) after Protocol Modification in the Era of Novel Agents in Ph-Negative ALL Patients

INTRODUCTION: Pediatric inspired regimens in adolescent young adults and adults have improved outcomes in acute lymphoblastic leukemia (ALL). CALGB 10403 was prospectively tested in patients 17-39 years and achieved a 3-year event free survival of 59%. In 2007, we reported outcomes after incorporating peg-asparaginase (PEG) dosed at 2000mg/m in induction for patients aged 17- 55 years. This was well tolerated and achieved a complete remission (CR) rate of 96%. In 2014, we reported experience with the use of multiple doses of PEG (CCG-1882) for newly diagnosed ALL adult patients achieving a 7-year overall survival (OS) of 51%. Since then, the USC ALL regimen consisting of 2 induction phases, was further modified with a goal of maintaining good outcomes and improving compliance and toxicities. Fractionated doses of cytarabine were changed to a single dose, the methotrexate dose of 1g/ m2 (2.5 g/m2 if T cell) given D1,15 in intensification phases was changed to 3g/ m2 (B and T cell) given in single doses, and consolidation was increased to six cycles allowing for PEG holidays. Moreover, the approval and incorporation of novel agents such as blinatumomab and inotuzumab also changed outcomes in ALL. Therefore, this study reports an update of outcomes since further modification of the USC ALL pediatric-based regimen in the era of novel agents. METHODS: This is a retrospective review which included adults aged 18 years with newly diagnosed Philadelphia negative ALL (2016-2020) treated at the USC/Norris Comprehensive Cancer Center and the Los Angeles County Hospital (LAC). Primary objectives were over-all survival (OS), event-free survival (EFS), disease-free survival (DFS) at 3 years. Secondary outcomes were complete remission/complete remission with incomplete recovery (CR/CRi) and minimal residual disease (MRD) by flow cytometry rates. OS, DFS, EFS were reported through Kaplan Meier curves and Log-rank tests. Two-sided p value ≤0.05 was significant. RESULTS: 121 patients with Ph-negative ALL were reviewed (49.6% Ph-negative B-ALL, 23.9% Ph-like B-ALL, 0.8% B cell lymphoblastic leukemia (LBL), 9.1% T cell ALL, 4.1% T cell LBL, 4.1% early T-cell, 5.8% mixed phenotype acute leukemia (MPAL). Median age at diagnosis was 39.3 years. There were 71.9% Hispanic, 15.7% White, 9.9% Asian, 2.5% African Americans. 57.9% males, 42.1% females. In terms of risk stratification, the majority were unfavorable at 54.6% and 37.8% unknown. Median BMI was 29.6 kg/ m2. 52.5% had hepatic steatosis and 5% had CNS disease pre-induction. 68.7% received PEG during induction 1, and 67.9% during induction 2. Post-induction 1, 83.5% patients achieved CR/Cri with 49% MRD negativity by flow. Post-induction 2, 75% achieved CR/Cri with 57.1% MRD negativity by flow. 26.4% received blinatumomab but not allo-HSCT. 20% received blinatumomab and underwent allo-HSCT. 34.7% received blinatumomab for MRD flow positivity, median 2 cycles, only 5.8% received inotuzumab, 35.5% underwent allogenic stem cell transplant. 28.9% and 9.1% had any relapse and refractory disease, respectively. Mortality rate was at 14.9%. 3y OS was at 80.7%, 3y EFS at 54.3% and 3y DFS 61.3%. When stratified by MRD flow negativity after induction 1 and 2, 3y OS was 83.7% vs 91.1% (p=0.55), 3y DFS was 68% vs 64.1%(p=0.98) and 3y EFS was 68% vs 59.5% (p=0.84). When stratified by blinatumomab use, 3y OS 72.5% vs 85.1% (p=0.15), 3y EFS was 31.6% vs 67.5% (p=0.001), 3y DFS was 40.1% vs 73.1% (p=0.001) (Fig 1-2). Use of PEG resulted in grade 3/4 toxicities including hypersensitivity (4.1%), transaminitis (21.5%), pancreatitis (5.4%), hypertriglyceridemia (49.5%), hypofibrinogenemia (45.5%), hyperbilirubinemia (21.5%) and thrombosis (16.5%). CONCLUSIONS: The modified pediatric-based USC ALL induction regimen using PEG maintained a high 3y OS at 85.3% and appreciable 3y EFS at 54.3% and 3y DFS at 61.3%, with tolerable toxicities in an era of novel agents like blinatumomab. This compares favorably with the original USC ALL regimen, CALGB 10407 and GRAALL 2005. Of note, 3y EFS and 3y DFS were significantly lower in those who underwent blinatumomab since these patients were MRD flow positive and lacked deeper remissions, but 3y OS (72.5%) remained high regardless, even though 26.4% who received blinatumomab were not able to go for allo-HSCT. This suggests the beneficial effect of blinatumomab. Further follow-up is necessary to provide a more robust and mature survival data. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Low-Dose Antithymocyte Globulin Plus Low-Dose Post-Transplant Cyclophosphamide for Prevention of Graft-Versus-Host Disease after Haploidentical Peripheral Blood Stem Cell Transplants: A Large Sample, Long-Term Follow-up Retrospective Study

Background: The use of haploidentical hematopoietic stem cell transplantation (haplo-HSCT) to treat hematological diseases is increasing. Two regimens for in vivo T-cell depletion are currently used in haplo-HSCT for prophylaxis of graft-versus-host disease (GVHD), including anti-thymocyte globulin (ATG)-based and posttransplant cyclophosphamide (PTCy)-based regimens. Our previous studies have demonstrated that low-dose ATG plus low-dose PTCy (low-dose ATG/PTCy) combined with cyclosporine (CSA) and mycophenolate mofetil had promising activity for the prevention of GVHD in haploidentical peripheral blood stem cell transplantation (haplo-PBSCT). Now, the efficacy of the low-dose ATG/PTCy regimen for GVHD prevention in haplo-HSCT was evaluated in a large sample with a long-term follow-up. Methods: The patients undergoing haplo-PBSCT between May 2017 and June 2021 in our center were enrolled in the retrospective study. The study included a variety of hematological diseases, such as acute myeloid leukemia (AML, n=113), acute lymphoblastic leukemia (ALL, n=49), mixed phenotype acute leukemia (MPAL, n=4), myelodysplastic syndromes (MDS, n=18), chronic myeloid leukemia (CML, n=10), non-Hodgkin's lymphoma (NHL, 30) and multiple myeloma (MM, n=1). All patients received low-dose ATG/PTCy regimen for prophylaxis of GVHD including ATG 2.5 mg/kg administered on day −2 to −1 and cyclophosphamide (Cy) 50 mg/kg on day +3, cyclosporine A (CsA) and mycophenolate mofetil (MMF) initiating on day +4. The starting infusion dose of CsA was 2 mg/kg, after which the dose was modified to obtain a nadir serum level between 200 and 300 ng/ml, eventually tapering from day +90 to day +180. MMF was administered orally at 15 mg/kg three times daily (maximum dose of 3 g per day) until day +34 and discontinued if no aGvHD. Mycophenolic acid sodium enteric tablets (MPA) correspond to MMF instead. Grafts were from mobilized peripheral blood stem cells. Family members selected as haploidentical donors were defined on the HLA-A, -B, -C, -DRB1, and -DQB1 locus at the high-resolution level of the recipient donor number of human leukocyte antigen (HLA) mismatches >2. Results: Two hundred and twenty-five patients undergoing haplo-PBSCT were enrolled in the study. There were 144 males and 81 females with a median age of 38 years (range, 6 to 69 years). At the time of transplantation, 146 (64.89%, 146/225) patients were in complete response (CR), while 79 (35.11%, 79/225) patients had active disease. One hundred and five patients received myeloablative conditioning (MAC) regimens, while 40 patients received reduced-intensity conditioning (RIC) regimens. All patients received PBSC grafts with the median CD34+ cells of 10.3 (1.64-46.55) × 108/kg, and the median mononuclear cells (MNCs) was 15.35 (3.48-39.74) × 108/kg. Ten patients failed to engraft, including 6 patients with primary graft failure and 4 with secondary graft failure. The median time for neutrophil engraftment was 12 days (range, 9-28 days), while the median time for platelet engraftment was observed in 13 days (range, 9-87 days). The cumulative incidence (CI) of grades I-IV acute GVHD (aGVHD) was 30.7% (24.6-36.9%). The CIs of grades II-IV and III-IV acute GVHD by 100 days post-transplant were 12.4% (95%CI, 8.0-16.8%) and 4.6% (95%CI, 1.8-7.4%), respectively. While chronic GVHD was 31.2% (95%CI, 24.6-37.9%) and moderate/severe chronic GVHD was 16.4% (95%CI, 11.1%-21.7%). A total of 90 patients died. Fifty-nine patients died of non-relapse causes (NRM 59/225, 26.2%). With a median follow-up of 26 months (range, 0-61 months), 50 cases relapsed. The median time of relapse was 15.5 months (range, 1−46 months). The mean OS and DFS were 38.01 months (95%CI, 34.33-41.6%) and 35.45 months (95%CI, 31.75-39.15%). The 2-year disease-free survival (DFS), overall survival (OS), and GVHD-free, relapse-free survival (GRFS) was 54.8% (95%CI, 48.94-62.26%) and 60.9% (95%CI, 54.24-67.56%) and 48.0% (95%CI, 41.14-54.86%), respectively. The 2-year CI of relapse was 26.6% and NRM was 16.6%. All patients were included in immune reconstitution studies and 81 cases were analyzed at each endpoint. On days +120, median CD3+, CD4+, CD8+, CD19+ and CD56/CD16+ counts were 954 (95-4891), 180 (8-743),754 (73-4272), 34 (1-276) and 215 (19-2539)/μl, respectively. Conclusion: The low-dose ATG/PTCy regimen presented a high efficacy for prophylaxis of GVHD in haplo-PBSCT.

A Single Cell Atlas and Interactive Web-Resource of Pediatric Cancers and Healthy Bone Marrow

Single-cell profiling allows for in-depth characterization of the tumor microenvironment and revealing the heterogeneity of pediatric cancers. Ease of access and analysis of the primary tumor single-cell data from studies on solid and blood pediatric tumors is essential for improving diagnostics and therapies. We have developed the Pediatric Single Cell Cancer Atlas (PedSCAtlas), with the goal of highlighting the heterogeneity of malignant and microenvironment cells across pediatric cancers. The atlas allows for quick exploration, comparative analysis, visualization, and prognostic assessment of genes/pathways across pediatric cancers without requiring bioinformatics analysis or computational support. The PedSCAtlas currently contains expression data of >1.2 million single cell (sc) and single nuclei (sn) from 134 pediatric hematological and solid-organ cancer samples and healthy bone marrow (BM). The hematological cancer samples are dominated by acute leukemias containing scRNA-seq data of acute myeloid leukemia (AML), B-cell acute lymphoblastic leukemia (B-ALL), T-cell ALL (T-ALL), and mixed phenotype acute leukemia (MPAL). The scRNA-seq data are generated from the BM samples collected during the clinical diagnosis of the disease. The data for the atlas was either generated at the Bhasin lab or collected from public resources including the GEO database, ScPCA (https://scpca.alexslemonade.org/), and HCA (https://data.humancellatlas.org/). Additionally, the atlas also contains bulk RNA-sequencing data and clinical information retrieved from the TARGET Initiative (https://ocg.cancer.gov/programs/target). The single cell/nuclei datasets are processed using a uniform workflow that includes quality control and filtering, normalization, integration with batch correction (if needed), dimensionality reduction, clustering, and cellular annotations. The data normalization and integration are performed using sctransform (Hao and Hao et al, Cell 2021) and harmony (Korsunsky et al, Nature Methods 2019) algorithms respectively. The visualization of integrated pediatric cancers single cell was achieved using Uniform Manifold Approximations and Projection (UMAP) method (Becht et al, Nature Biotech 2018). The clusters were annotated based on the expression of cell-specific markers. Differential expression analysis was used to identify potential biomarker sets for malignant cells of disease subtypes. To further ascertain that these biomarkers were malignant cell (blast) specific in the acute leukemia subtypes, the genes were filtered to exclude those with consistent expression in healthy BM cell types. To compare the expression profile of genes between single cells and bulk data, the raw counts of bulk RNA-seq datasets were filtered and normalized using the Voom algorithm (Ritchie et al, Nucleic Acids Research 2015). The resource has modules for biomarker analysis, bulk RNA deconvolution, and survival analysis to facilitate easy analysis of single-cell data. Survival analysis is available for the bulkRNA-seq dataset; the survival is calculated using the overall survival values and gene expression/enrichment input by the user. The SingleCell module allows users to visualize the pediatric cancer dataset on a UMAP by selecting the aspect (i.e., cell type) to group the dataset by; it also allows users to enter a gene name or select a pathway of their interest and view its expression or enrichment in a feature or violin plot. The ImmuneCell module allows users to interact with the adult healthy BM dataset by inputting a gene and viewing its expression on a feature plot. The Biomarkers module contains pre-defined biomarker sets for each disease subtype in the selected cancer. The final module, BulkExpression, contains the bulk RNA-seq data for the selected cancer type and allows users to view gene expression or analyze the survival associations of an input gene or gene set. The web resource source code is written in R programming language and the interactive web server has been implemented using the R Shiny and ShinyDashboard packages. The PedScAtlas resource provides a unique and straightforward tool for biomarker identification, analysis of pediatric cancer subtype heterogeneity, and transcriptome profile of the tumor microenvironment. The resource is available online at https://bhasinlab.bmi.emory.edu/PediatricSC/. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

COVID-19 Vaccination Outcomes in Children, Adolescents, and Young Adults with Acute Lymphoblastic Leukemia

Background:While several studies have shown that less than half of adults on active therapy for chronic hematological malignancies develop an immune response to COVID-19 vaccination, little is known about the efficacy of COVID-19 vaccines in the midst of active therapy for acute lymphoblastic leukemia (ALL). Furthermore, COVID-19 vaccine outcomes in pediatric leukemia remain uncharacterized. Given the differences in the intensity of chemotherapy regimens between pediatric ALL and adult chronic hematological malignancies as well as age related immune differences, there is a need to specifically define vaccination outcomes in pediatric ALL. This study aims to define the safety and efficacy of COVID-19 vaccination in children and adolescents and young adults with ALL, the most common pediatric leukemia. Methods: We conducted a prospective single center Institutional Review Board approved study. Eligible patients were ages 5-25 years with a diagnosis of ALL or receiving ALL-like therapy who had received COVID-19 vaccination. Pediatric oncology patients at our center undergo universal surveillance for COVID-19 via nasopharyngeal PCR regardless of symptom status, prior to any sedation or hospitalization. Labs were drawn at 3,6-, and 12-months post-vaccination for anti-COVID-19 spike (S) and nucleocapsid (N) IgG ELISA and anti S neutralizing antibody assays, in vitro COVID-19 peptide stimulation T-cell response studies, and T-cell receptor (TCR) sequencing for COVID specific TCR. These tests were validated in healthy COVID-19 infection convalescent and healthy post-vaccination cohorts. Results: Between April 2021 and April 2022, the study enrolled 21 patients (median age 12.5 years). Diagnoses included high risk B-ALL (n=13), mixed-phenotype acute leukemia (MPAL) (n=2), and standard risk B-ALL (n=6). Three had relapsed ALL. All were in complete remission and receiving chemotherapy at the time of vaccination (17 receiving maintenance therapy and 4 in an intensive chemotherapy phase). Twenty received the BNT162b2 mRNA vaccine (10mcg dose n=11 and 30mcg dose, n=9); one received the Ad26.COV2.S vaccine. One patient received one dose (excluded from efficacy analysis), 17 received 2 doses, and 2 received three doses (i.e. a booster dose) of BNT162b2. Vaccinations were well tolerated with no patient experiencing anaphylaxis or an allergic reaction, including 10 patients with PEG-asparaginase allergy (BNT162b2 contains PEG). Three patients presented to the emergency department with fever; one of those was hospitalized for hypotension. Thirteen of 20 patients (65%) were positive for spike (S) anti-COVID-10 IgG following two doses of BNT162b2 (n=12) or one dose of Ad26.COV2.S (n=1). Eleven of these 13 were negative for N IgG, an antibody profile (S IgG+, N IgG-) consistent with an immune response to vaccination rather than a prior undiagnosed COVID-19 infection. All three patients with ≥ 6 months post-vaccination data continue to be positive for S IgG. Notably, 6 of 12 lymphopenic patients (absolute lymphocyte count <500 at the time of vaccination) and 3 of 4 patients in an intense phase of therapy during vaccination showed an immune response. Six of 8 patients with PEG-allergy had an immune response. All patients with positive S IgG showed presence of neutralizing anti-S antibodies. There was no difference in immune response based on vaccine dose. Five of 20 patients (3 of 13 patients who were S IgG+ following vaccination) developed COVID-19 infection following vaccination; one of these occurred after the third dose. Hospitalization was needed in one of five cases of COVID-19. All infections occurred during the omicron wave and included 3 sequencing confirmed omicron infections (variant testing not done for 2). Conclusions: In our study, two doses of COVID-19 vaccination induced a neutralizing antibody response in over half of the children and AYA actively receiving ALL chemotherapy including those who were receiving intensive chemotherapy or had PEG allergy. Adverse events were uncommon. Omicron infections occurred in our study, a finding consistent with the occurrence of breakthrough omicron variant infections in immunocompetent vaccinated individuals. T-cell response studies for our cohort are ongoing. Additional long-term follow-up and larger studies are needed to investigate durability of immune response and optimal vaccine frequency especially in the setting of omicron variants. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Maintenance Therapy with Combination of Azacitidine, Danazol and Thalidomide after Intensive Chemotherapy in Acute Myeloid Leukemia Patients

INTRODUCTION Despite the efficacy and outcome of AML patients increases in recent decades, the rate of relapse is still high, especially in the patients who are ineligible for allo-HSCT. How to prevent relapse is still a major therapeutic challenge. Recently, several drugs including Azacitidine (AZA), CC-486, Lenalidomide, androgen, Midostaurin, etc, used as maintenance therapy have shown a better EFS or OS, indicating that maintenance therapy is an important aspect of AML treatment. Herein, we reported the data of 11 AML patients who were administered AZA combined with Danazol (DNZ) and Thalidomide (THD) as maintenance therapy after intensive chemotherapy (IC). The purpose of this study was to observe the efficacy and safety of this maintenance therapy. METHODS Between Feb 2017 and Mar 2021, 11 patients diagnosed with AML were treated at the Baiyun Hospital. The induction therapy involved daunorubicin (DNR) 60 mg/m2/day on days 1-3, cytarabine (Ara-c) 100 mg/m2/day on days 1-7. If the patient did not achieve CR, salvage therapy was administered. Post-remission, three cycles of consolidation chemotherapy with high-dose Ara-c (3g/m2/q12h d1-3) and two cycles of reduced dose of chemotherapy (DA/HA/EA) were administered. Patient remained in CR after IC and was treated with 6-8 cycles of AZA combined with DNZ and THD maintenance therapy. AZA subcutaneous injection at 100 mg/day (approximate 50-75 mg/m2/day) d1-7, DNZ 200 mg Bid and THD 100 mg/day po followed by AZA, every four weeks. After completing the 6-8 cycles, patients continued the maintenance therapy with DNZ and THD for at least three years unless relapse or intolerance occurred. Disease evaluation included bone marrow and blood test until the duration of CR over five years. RESULTS Between Feb 2017 and Mar 2021, 11 patients who were enrolled in this study. Baseline patient and disease characteristics are summarized in Table 1. All 11 patients were treated with DA induction therapy, 10 patients achieved CR, 1 patient achieved remission after salvage therapy. 10 of 11 patients completed all consolidation therapies. 1 patient diagnosed AML with t (8;21) refused to continue chemotherapy because of renal failure after CR, and was directly switched to maintenance therapy. 9 0f 10 patients completed all IC and started on maintenance therapy. 1 AML with t (8;21) patient relapsed after stopping chemotherapy for two months and achieved CR2 by FLAG re-induction therapy refused continued chemotherapy and transplantation, and was directly switched to maintenance therapy. All patients received a median of 7 (6-12) courses of AZA combined with DNZ and THD maintenance therapy. Treatment was well tolerated and accepted by the patients. Common adverse events included mild neutropenia, thrombocytopenia, and elevated aminotransferase. No patient discontinued the maintenance therapy due to adverse events. Until June 2022, the median follow-up time was 37 (14-63) months. The RFS at 1-year and 3-years was 100% and 71.1%; the OS at 3-years was 100%, respectively (Figure 1a, 1b). All 11 patients, 3 died and 8 patients were alive with RFS. Of the 3 patients who died, 1 relapsed at 36 months and died due to disease progression at 46 months. The remaining 2 patients, one was diagnosed and died with secondary mixed phenotypic acute leukemia (NOS, T/My), the other died in a road accident. Notably, 2 cases of AML with t (8; 21) (one patient relapsed after IC, another patient developed renal failure) remained in CR by the end of follow-up. The CR duration was 24 and 13 months, respectively. The clinical outcomes of maintenance therapy after IC treatment for 11 patients are summarized in Figure 1c. Conclusion: AZA combined with DNZ and THD maintenance therapy is safe and effective in AML patients who are ineligible for allo-HSCT. Keywords: Azacitidine; Danazol; thalidomide; maintenance therapy; AML Figure 1 Response and long-term outcomes of the entire cohort Figure 1 legend, a. Relapse-free survival of the 11 patients. b. Overall survival of the 11 patients. c. Swimmer plot of the 11 patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Case of Ambiguous Lineage Acute Leukemia Posing Diagnostic Challenge

Abstract Introduction/Objective N/A. Methods/Case Report Acute leukemias with ambiguous lineage encompass those leukemias that show no clear evidence of differentiation along a single lineage and account for less than 4% of all cases of acute leukemias and often poses diagnostic dilemma. We present a challenging case of an ambiguous lineage acute leukemia in a previously healthy 27-year-old male, who presented to the ED with acute fever, dyspnea on exertion, nausea and vomiting. A complete blood count (CBC) showed a white blood cell count of 323 × 109/L, Hemoglobin of 3.3 g/dl, Platelet count of 44 × 109/L and a differential count of Neutrophil: 11, Lymphocyte: 31, Monocyte: 02, Band Neutrophil:1, Myelocyte :1, Blast: 54. Bone marrow aspirate smears revealed a heterogenous population of small to large sized blasts comprising 83% of cellularity. They expressed scant to moderate basophilic cytoplasm, high N/C ratio, round to irregular nuclear contour, finely granular chromatin and some with nucleoli. Other lineages were markedly suppressed. Flow cytometric analysis revealed an abnormal blast population (~80% of total) expressing CD34, HLA-DR, Tdt (subset), CD71 (dim) and CD38. In addition, the blasts expressed myeloid associated antigens CD13, MPO (smaller subset), as well as B-cell associated antigens CD19 (dim), cytoplasmic CD22 and cytoplasmic CD79a. They also expressed T-cell associated antigens, CD2, CD3 (smaller subset), CD5 (subset) and CD7 (subset) but negative for cytoplasmic CD3. This unusual pattern of expression posed a challenge as to what type of mixed phenotype leukemia it should be with variable expressions from all three lineage of myeloid, B- and T-cell. Further work-up showed, scattered blasts are also positive for MPO by cytochemical (on aspirate smear) and immunohistochemical stain (on core). The latter also showed negativity for CD3. Based on all these findings, this mixed phenotype acute leukemia (MPAL) was best categorized as MPAL (B/myeloid) over so called “Triphenotypic (B/T/myeloid) acute leukemia”. We considered expression of myeloid and B cell markers as more lineage defining in this case as compared to small subset expressing T lineage marker CD3. This case exemplifies the importance of multimodal comprehensive analysis of acute leukemia, especially in the setting of MPAL with expression of multi- lineage markers posing a diagnostic challenge. Results (if a Case Study enter NA) N/A. Conclusion N/A.

A Novel Fusion Partner in a Case of Myeloid/Lymphoid Neoplasm with FGFR1 Rearrangement

Abstract Introduction/Objective Myeloid/lymphoid neoplasms with rearrangement of FGFR1 are uncommon and heterogeneous group of hematological malignancies that may present as myeloproliferative neoplasm, acute myeloid leukemia, T or B lymphoblastic lymphoma/leukemia, or mixed phenotypic acute leukemia. Patients can present with lymphadenopathy, mediastinal mass, or systemic symptoms. Eosinophilia is common in peripheral blood or bone marrow. Several chromosomal rearrangements and fusion genes have been described with FGFR1 rearrangement and all of them encode an aberrant tyrosine kinase. Tyrosine kinase inhibitors may offer a prospective therapeutic approach although specific targeted therapies have yet to be established. Methods/Case Report We present a case of a 14-year-old male who presented with rapid neck pain and swelling. Imaging revealed a 15 x 8 x 6 cm neck mass extending into the anterior mediastinum. Laboratory evaluation revealed anemia, thrombocytopenia, and leukocytosis (41.7 K/mm3), with absolute eosinophilia and blasts of 50%. Flow cytometry of peripheral blood demonstrated a population of T-lymphoblasts comprising about 60% of the cellularity with dim CD45, cytoplasmic CD3, CD7, TdT, CD38, CD4, CD5, CD10, and CD15, supporting T-lymphoblastic leukemia/lymphoma. The patient received induction chemotherapy, and subsequent bone marrow biopsy showed morphologic remission. Six weeks from diagnosis, the patient developed a gluteal mass with an atypical T-cell infiltrate that was negative for TdT; and at 15 weeks from diagnosis, developed an enlarging abdominal mass, also with an atypical T-cell infiltrate also negative for TdT and CD1a. At that time molecular diagnostic studies of the abdominal wall mass discovered a previously unpublished ATG16L1-FGFR1 fusion, supporting myeloid/lymphoid neoplasm with FGFR1 rearrangement, T-lymphoblastic leukemia/lymphoma. Results (if a Case Study enter NA) NA. Conclusion Although a variety of translocations have been identified with FGFR1 rearrangement, the novel fusion partner ATG16L1 has not been previously reported. The case also illustrates an unusual extramedullary recurrence of the T-cell lymphoblastic leukemia/lymphoma with significant antigenic shift including loss of TdT and CD1a.

Acute Leukemias of Ambiguous Lineage presenting in Extramedullary Location – Unusual and Challenging Diagnoses: A Report of Two Cases

Abstract Introduction/Objective Acute leukemias of ambiguous lineage are uncommon, accounting for &amp;lt;1% of all leukemias, and extramedullary presentations of these leukemias are rare. Methods/Case Report We report two cases of acute leukemia of ambiguous lineage presenting in extramedullary sites. Case 1 is a 41-year-old female who presented with right cervical lymphadenopathy and fatigue. Laboratory and imaging studies showed leukopenia, anemia, and abnormal cervical, supraclavicular, axillary, mediastinal, and abdominal pelvic lymphadenopathy. Biopsy of the axillary lymph node revealed sheets of morphologic blasts. Flow cytometry showed the blasts were of mixed phenotype, with T-cell differentiation supported by CD3 and TdT, and myeloid subpopulation supported by myeloperoxidase and lysozyme. Both these populations were negative for CD10, CD20, CD4, CD8, CD34, CD138, CD1a, CD79a. The morphology and immunophenote establish the diagnosis of T/myeloid mixed-phenotype acute leukemia, not otherwise specified. Case 2 is a 65-year-old female who presented with bilateral lower extremity weakness. Multiple lytic lesions involving the thoracic and cervical spine were detected by imaging. Biopsy of a T12 vertebral lesion revealed sheets of large malignant cells. Extensive immunohistochemical workup revealed positivity for CD45 (weak), CD4, and CD43, supporting a diagnosis of hematopoietic neoplasm of likely hematopoietic origin. Subsequent evaluation eventually led to discovery of chronic myeloid leukemia in blast phase with undifferentiated blasts, BCR-ABL1 positive, originally presenting as extramedullary acute leukemia with undifferentiated blasts. Results (if a Case Study enter NA) NA. Conclusion These cases highlight the challenges of diagnosing peripheral solid tumors of unknown origin. Extramedullary presentation of acute leukemia of ambiguous lineage are particularly challenging and, although rare, include phenotypes ranging from mixed phenotype to undifferentiated types.

The 8p11 myeloproliferative syndrome: Genotypic and phenotypic classification and targeted therapy

EMS(8p11 myeloproliferative syndrome, EMS) is an aggressive hematological neoplasm with/without eosinophilia caused by a rearrangement of the FGFR1 gene at 8p11-12. It was found that all cases carry chromosome abnormalities at the molecular level, not only the previously reported chromosome translocation and insertion but also a chromosome inversion. These abnormalities produced 17 FGFR1 fusion genes, of which the most common partner genes are ZNF198 on 13q11-12 and BCR of 22q11.2. The clinical manifestations can develop into AML (acute myeloid leukemia), T-LBL (T-cell lymphoblastic lymphoma), CML (chronic myeloid leukemia), CMML (chronic monomyelocytic leukemia), or mixed phenotype acute leukemia (MPAL). Most patients are resistant to traditional chemotherapy, and a minority of patients achieve long-term clinical remission after stem cell transplantation. Recently, the therapeutic effect of targeted tyrosine kinase inhibitors (such as pemigatinib and infigratinib) in 8p11 has been confirmed in vitro and clinical trials. The TKIs may become an 8p11 treatment option as an alternative to hematopoietic stem cell transplantation, which is worthy of further study.

Adult mixed phenotype acute leukemia (MPAL): B/myeloid MPALisoMPO is distinct from other MPAL subtypes.

Myeloperoxidase (MPO) is considered a specific marker of myeloid/non-monocytic lineage in the diagnosis of mixed phenotype acute leukemia (MPAL). However, the clinical significance of isolated dim MPO expression in otherwise typical B lymphoblastic leukemia (B-ALL; referred to as B/myeloid MPALisoMPO ) in adult patients is unknown. We compared flow cytometric immunophenotype and clinicopathological findings among cases of B/myeloid MPALisoMPO (n=13), other MPAL subtypes (n=10, B/myeloid and T/myeloid MPAL), B-ALL (n=64), and acute myeloid leukemia (AML, n=58), using the 2016 WHO classification. For MPAL cases, MPO was reported as the percent of MPO positive blasts and its intensity (dim or moderate/strong). The pattern of heterogenous antigen expression (inversely coordinated expression between myeloid and lymphoid markers and cell size) was assessed. Cases of B/myeloid MPALisoMPO showed a fairly homogenous single B-lineage blast population with dim MPO expression whereas cases of other MPAL subtypes displayed heterogeneous antigen expression and moderate/strong MPO expression. The percent of MPO positive blasts in these two groups was similar. Expressions of CD15, CD117, and monocytic markers were more common in other MPAL than in B/myeloid MPALisoMPO . B/myeloid MPALisoMPO patients had similar overall and leukemia free survivals as B-ALL patients and better than other MPAL patients. This is the first study to investigate the clinical significance of adult B/myeloid MPALisoMPO using the 2016 WHO classification. Our results suggest that B/myeloid MPALisoMPO clinically behaves more similarly to B-ALL than to other MPAL subtypes, supporting the 2016 WHO classification to segregate this entity from other MPAL subtypes.

Integrative genomic and transcriptomic profiling reveals distinct molecular subsets in adult mixed phenotype acute leukemia.

Mixed phenotype acute leukemia (MPAL) is a subtype of leukemia in which lymphoid and myeloid markers are co-expressed. Knowledge regarding the genetic features of MPAL is lacking due to its rarity and heterogeneity. Here, we applied an integrated genomic and transcriptomic approach to explore the molecular characteristics of 176 adult patients with MPAL, including 86 patients with T-lymphoid/myeloid MPAL (T/My MPAL-NOS), 42 with Ph+ MPAL, 36 with B-lymphoid/myeloid MPAL (B/My MPAL-NOS), 4 with t(v;11q23), and 8 with MPAL, NOS, rare types. Genetically, T/My MPAL-NOS was similar to B/T MPAL-NOS but differed from Ph+ MPAL and B/My MPAL-NOS. T/My MPAL-NOS exhibited higher CEBPA, DNMT3A, and NOTCH1 mutations. Ph+ MPAL demonstrated higher RUNX1 mutations. B/T MPAL-NOS showed higher NOTCH1 mutations. By integrating next-generation sequencing and RNA sequencing data of 89 MPAL patients, we defined eight molecular subgroups (G1-G8) with distinct mutational and gene expression characteristics. G1 was associated with CEBPA mutations, G2 and G3 with NOTCH1 mutations, G4 with BCL11B rearrangement and FLT3 mutations, G5 and G8 with BCR::ABL1 fusion, G6 with KMT2A rearrangement/KMT2A rearrangement-like features, and G7 with ZNF384 rearrangement/ZNF384 rearrangement-like characteristics. Subsequently, we analyzed single-cell RNA sequencing data from five patients. Groups G1, G2, G3, and G4 exhibited overexpression of hematopoietic stem cell disease-like and common myeloid progenitor disease-like signatures, G5 and G6 had high expression of granulocyte-monocyte progenitor disease-like and monocyte disease-like signatures, and G7 and G8 had common lymphoid progenitor disease-like signatures. Collectively, our findings indicate that integrative genomic and transcriptomic profiling may facilitate more precise diagnosis and develop better treatment options for MPAL.

Multidisciplinary management of disseminated Exophiala dermatitidis mycosis in an infant with mixed phenotype acute leukemia: a case report

BackgroundExophiala dermatitidis is a dematiaceous fungus isolated from various environmental sources. Systemic E. dermatitidis infections can lead to fatal outcomes, and treatment has not yet been standardized. Although E. dermatitidis is also known to cause cutaneous infection, it has not been previously reported to appear as ecthyma gangrenosum (EG), an uncommon cutaneous lesion in neutropenic patients that is mainly caused by Pseudomonas aeruginosa.Case presentationA 2-month-old male infant with mixed-phenotype acute leukemia presented with prolonged fever unresponsive to antibacterial and antifungal agents during myelosuppression due to remission induction therapy. He also presented with skin lesions on the left wrist and left lower quadrant of the abdomen. The abdominal lesion gradually turned black and necrotic, which was consistent with the findings of the EG. E. dermatitidis was isolated from the blood, stool, wrist skin, and endotracheal aspirate. During hematopoietic recovery, consolidation in both lungs was evident. Multiagent antifungal treatment failed to eliminate E. dermatitidis from blood. In order to salvage the central venous catheter, ethanol lock therapy (ELT) was adopted, following which the blood culture became negative. The abdominal lesion that evolved as a necrotic mass connecting the small intestine and subcutaneous tissue adjacent to the skin was surgically resected. After these interventions, the general condition improved.ConclusionDisseminated E. dermatitidis mycosis in the neutropenic infant was successfully managed with a multidisciplinary treatment consisting of multiagent antifungal treatment, ELT, and surgery.