Acute Mixed Lineage Leukemia Research Articles

Early Results of the Phase I/II Study Investigating the All-Oral Combination of the Menin Inhibitor Revumenib (SNDX-5613) with Decitabine/Cedazuridine (ASTX727) and Venetoclax in Acute Myeloid Leukemia (SAVE)

Background: The interaction of menin with lysine methyltransferase 2A (KMT2A) is a dependency in acute leukemia caused by either rearrangement of the KMT2A (KMT2Ar) or Nucleoporin 98 ( NUP98r) genes, or mutation of the Nucleophosmin 1 gene ( NPM1mt). The menin inhibitor revumenib (previously SNDX-5613), is a potent, oral, selective inhibitor of the menin-KMT2A interaction with demonstrated safety and clinical activity in highly refractory acute leukemias (Issa GC, Nature 2023). KMT2Ar or NPM1mt leukemias are highly susceptible to induction of apoptosis through BCL2 inhibition, and dual Bcl-2 and menin inhibition led to synergistic activity in KMT2Ar or NPM1mt leukemia models (Carter BZ, Blood 2021). Therefore, we designed a phase I/II, investigator-initiated trial of the all-oral combination of revumenib, venetoclax and the hypomethylating agent ASTX727 in children and adults with relapsed/refractory (R/R) acute myeloid leukemia (AML) (NCT05360160). Methods: Patients (pts) with R/R AML or myeloid mixed-lineage acute leukemia (MPAL) aged 12 years and older were eligible.Dose escalation followed a 3+3 design. ASTX727 (decitabine/ cedazuridine) was administered at 35 mg/100 mg PO daily days 1-5, venetoclax at 400 mg (target dose) PO daily days 1-14, and revumenib 113 mg PO Q12h (dose level [DL] 0) or 163 mg PO Q12h (DL 1, used in phase II monotherapy), days 1-28 with either posaconazole or voriconazole (strong CYP3A4 inhibitors, for antifungal prophylaxis). Maintenance with revumenib monotherapy is planned following hematopoietic stem cell transplant (HSCT) for 1 year. For the first pt cohort, bone marrow examination was performed on cycle 1 day 14 and at the end of the cycle to assess for early morphologic remission and to inform future dose adjustments. Results: As of 7/20/2023, 8 pts were enrolled, 6 at DL0, and 2 at DL1. The median age was 27 years (range, 12-62 years), including 2 children (ages 12 and 16), 5 with KMT2Ar, 2 with NUP98r and 1 with NPM1mt. One pt had MPAL, and one had bone marrow and extramedullary disease. The median prior lines of therapy was 2.5 (range 1-4), 5 pts (63%) had prior venetoclax, 5 pts (63%) had a prior hypomethylating agent, 5 pts (63%) had prior HSCT, and one had prior menin inhibitor. The most common all-grade treatment-related adverse events (TRAEs) in ≥25% of pts were febrile neutropenia (63%), hyperphosphatemia (63%), nausea (63%), and AST/ALT elevation (25%). Grade ≥ 3 TRAEs were febrile neutropenia (63%), decreased platelets count (25%), and decreased neutrophil count (25%). There was 1 dose-limiting toxicity (DLT), grade 4 prolonged thrombocytopenia and neutropenia, at DL0 (resolved after dose hold), prompting enrollment of 3 additional pts, and escalation to DL1 when no additional DLTs were noted (1/6 pts with DLT). Pts at DL1 (N=2) have not completed the DLT period. There were no deaths due to TRAEs. No grade 3 or higher QTc prolongation occurred. Two pts had grade 2 differentiation syndrome (bone pain only), resolved with steroids. One pt ( NUP98r) had asymptomatic leukocytosis at the end of cycle 1 with neutrophilia, monocytosis, decrease in peripheral blasts, decrease in bone marrow blasts from 66% to 8%, and persistence of NUP98r by fluorescence in situ hybridization at 90% indicating differentiation. Seven of 8 pts are response-evaluable, and all 7 attained a morphologic remission (overall response rate of 100%) (Figure 1). Best responses achieved included a complete remission (CR) in 1 pt (including resolution of extramedullary disease), CR with partial hematologic recovery (CRh) in 1 pt, CR with incomplete platelet count recovery (CRp) in 3 pts, partial response (PR) in 1 pt, and 1 pt with morphologic leukemia free state (MLFS). Measurable residual disease was undetectable by flow (sensitivity at 10 -4) in 3 of 7 pts (43%). Three pts transitioned to HSCT following response, 2 are in continued remission, 1 has started maintenance and 1 pt died of transplant complications prior to starting maintenance. Enrollment is ongoing and updated data will be presented. Conclusions: Early results indicate acceptable safety and high efficacy of this combination in R/R myeloid leukemias with either KMT2Ar or NPM1mt or NUP98r. This study is ongoing with plans to establish the recommended phase 2 dose and optimize delivery of this combination.

Targeting matrix metallopeptidase 2 by hydroxyurea selectively kills acute myeloid mixed-lineage leukemia

Oncogene-induced tumorigenesis results in the variation of epigenetic modifications, and in addition to promoting cell immortalization, cancer cells undergo more intense cellular stress than normal cells and depend on other support genes for survival. Chromosomal translocations of mixed-lineage leukemia (MLL) induce aggressive leukemias with an inferior prognosis. Unfortunately, most MLL-rearranged (MLL-r) leukemias are resistant to conventional chemotherapies. Here, we showed that hydroxyurea (HU) could kill MLL-r acute myeloid leukemia (AML) cells through the necroptosis process. HU target these cells by matrix metallopeptidase 2 (MMP2) deficiency rather than subordinate ribonucleotide reductase regulatory subunit M2 (RRM2) inhibition, where MLL directly regulates MMP2 expression and is decreased in most MLL-r AMLs. Moreover, iron chelation of HU is also indispensable for inducing cell stress, and MMP2 is the support factor to protect cells from death. Our preliminary study indicates that MMP2 might play a role in the nonsense-mediated mRNA decay pathway that prevents activation of unfolding protein response under innocuous endoplasmic reticulum stress. Hence, these results reveal a possible strategy of HU application in MLL-r AML treatment and shed new light upon HU repurposing.

Acute undifferentiated/unclassifiable leukemia or disseminated non-hematologic neoplasm? When treatment is the compelling decision

The rapid collection of exact diagnostic and prognostic data is essential to guide therapy in patients diagnosed with acute leukemia. Basic diagnostic steps confirm the hematopoietic nature of the illness and allow distinction between Acute Lymphoid And Myeloid Leukemia (ALL and AML) throughout morphology, immunophenotype and cytogenetic/molecular analysis. However, because the complete results from the latter assays are not readily available, only morphology and Flow Cytometry (FC) permit to define quickly the cell lineage of the disease, thus orientating treatment. A small group of cases is referred to as Acute Undifferentiated Leukemia (AUL), because unclassifiable by current diagnostic markers [1]. AUL makes up 2-7% of Myeloperoxidase (MPO)-negative leukemias and poses a difficult treatment challenge [1,2]. The importance of a correct diagnostic input is further emphasized by the different results obtained with AML or ALL regimens in mixed lineage acute leukemia, another rare subset with debated diagnosis to treatment relationships [3]. Moreover, although rare in the modern era of improved diagnostic methodology, a pure leukemic presentation of an unrecognized metastatic carcinoma may need to be differentiated from AUL, AML or ALL, an issue which may be aggravated if FC results are inconclusive but treatment cannot be deferred until the availability of the other time-consuming diagnostic procedures.

DETECTION OF HEMATOPOIETIC NEOPLASMS IN CEREBROSPINAL FLUID BY IMMUNOPHENOTYPING: SINGLE-CENTER DATA ANALYSIS

Cerebrospinal fluid (CSF) infiltration can be identified at diagnosis and during the course of many hematopoietic malignancies, mainly acute leukemia and aggressive non-Hodgking lymphomas (NHL). The accurate diagnosis of central nervous system (CNS) infiltration allows patients with infiltrated CSF to be properly treated, avoiding the use of unnecessary CNS therapy for patients who do not have compromised CSF. Cytomorphology is the classic method for diagnosing CSF neoplastic infiltration, but it presents variability in sensitivity and specificity according to the experience of the morphologist and cell abnormalities resulting from sample preparation. Currently, multiparametric flow cytometry (MFC) has great sensitivity and specificity to detect cells of hematopoietic neoplasms in the CSF. This is a retrospective study to assess the frequency of CSF infiltration in a cohort of patients with hematologic malignancies treated at our institution. to evaluate the casuistry and frequency of CSF infiltration by hematologic neoplasms diagnosed by immunophenotyping in our institution. the results of CSF immunophenotyping were collected using the flow cytometry laboratory database. Assess period: from March 2008 to June 2021. Patients were grouped by type of disease and the frequency with which each one was represented in the cohort was verified. a total of 1572 CSF samples were analyzed by the laboratory during the study period. The frequency of diagnoses of patients whose samples were sent for evaluation were, in decreasing order: 956(60.8%), B-cell precursor acute lymphoblastic leukemia (BCP ALL) samples 238(15.1%) T-cell acute lymphoblastic leukemia (T-ALL) samples, 168(10.6%) AML samples, 120 (7.6%) B and T NHL, 42 (2.6%) mixed lineage acute leukemia (MPAL), 34(2.1%) blastic crisis of chronic myeloid leukemia (CML), 14(0.8%) other mature lymphoid neoplasms. Immunophenotyping showed 311(19.78%) positive results for CSF infiltration. The diagnoses of these patients and their respective frequency were: 156 (50.1%) BCP-ALL, 49(15.7%) AML, 41(31.1%) T-ALL, 37(11.8%) NHL, 13 (4.1%) blastic crisis of CML, 8 (2.5%) MPAL, 7(2.2%) other mature lymphoid neoplasms. The frequencies of NHL subtypes observed in this cohort were: 16 (1%) Large B Cell Lymphoma, 7(0.44%) Burkitt Lymphoma, 6 (0.39%) Mantle Cell Lymphoma, 5(0.31%) Adult T-Cell Leukemia-Lymphoma, 1 (0.06%) Marginal Zone Lymphoma, 1 (0.06%) Folicular Lymphoma and 1 (0.06%) Sezary Syndrome. It should be emphasized that among the samples from patients with other mature lymphoid neoplasms, 4 (0.25%) were from patients with Chronic Lymphocytic Leukemia (CLL), which is an unusual finding. this study observed that 91% of the samples with CSF infiltration were from patients diagnosed with acute leukemia and blastic crisis of CML. Mature lymphoid neoplasms have a lower frequency of CNS infiltration than acute leukemia, as also reflected in our series. These findings show the importance of an accurate assessment of the CSF for these diseases, because the results influence the choice of the therapeutic modality and intensity to be adopted in the different treatment protocols. we demonstrate the ability of immunophenotyping as a robust method for evaluating CSF samples from patients diagnosed with hematologic malignancies.

Prognostički značaj vrednosti serumskog feritina pri inicijalnoj dijagnozi kod bolesnika sa akutnom mijeloidnom leukemijom

Introduction/Aim: Acute myeloid leukemia (AML) is a heterogenous malignant disease whose course and outcome are influenced by a number of prognostic factors. Serum ferritin (SF) is often elevated in oncology patients, and it has been shown that it strongly influences an unfavorable outcome in various malignancies. The aim of this study is to assess the effect of high SF values on overall survival and disease-free survival, as well as to assess the correlation of SF values with other prognostic markers, such as clinical and laboratory parameters. Methods: Retrospective analysis included 108 patients diagnosed with AML at the Clinic for Hematology of the Clinical Center of Serbia (CCS), in Belgrade, in the period 2017 - 2019. Patients with acute promyelocytic leukemia, acute mixed lineage leukemia, secondary AML and patients treated with palliative therapy were excluded from the study. Patients were grouped based on the SF cutoff value of 800 µg/L. Results: Patients with higher SF values had a significantly higher incidence of early death (p = 0.020), sepsis in the induction phase of therapy (p < 0.010), and significantly lower initial hemoglobin levels (p = 0.040), as compared to patients with lower SF values. SF at diagnosis appeared to be a significant independent predictive factor of overall survival (p = 0.019) and of disease-free survival (p = 0.040). Conclusion: Our study showed a significant association of high SF values with sepsis in induction, early death, mean hemoglobin, overall survival, and disease-free survival. Identification of SF as an independent prognostic factor and a potential target site of the action of new drugs could contribute to a better prognosis of AML patients.

Program and Abstracts for 2020 Annual Meeting of the Society for Glycobiology.

Mixed-lineage Acute Leukemia (MLL) is one of the most high-risk forms of pediatric cancer. Although the long-term survival rates of pediatric acute lymphoblastic leukemia have increased over the past 40 years, the current chemotherapeutic treatment schemes often fail in the treatment of MLL. The biological mechanisms resulting in drug resistance, however, are still not fully understood and there is an urgent need to identify novel diagnostic and therapeutical targets. We have established the first integrated multi-omics investigation of primary patient MLL samples and control precursor B bonemarrow cells from healthy donors, mapping their proteome, transcriptome and glycome. 4–6 million cells from 3 normal bone marrow (BM) and 2 MLL samples were used for analysis on our multi-omics platform. Porous-Graphitised Carbon (PGC) nanoLC-ESIMS/MS was used for Glycomics analyses after the enzymatic and chemical release of N- and O-glycans, respectively. The proteome was explored using RP-LC-ESI-MS/MS analyses, performed after offline high-pH fractionation, in addition to RNA-seq analyses. Overall, 4225 proteins were identified across the patient MLL and control BM cells, of which 216 were overexpressed in MLL (p 2). Preliminary analyses of the RNA-seq data matched well with the proteomics findings, revealing significant alterations in expression of glycoprotein signalling receptors and extracellular matrix proteins as well as various transcription factors. High-pH fractionation also allowed us to identify numerous important glycosyltransferases and expression changes thereof on protein and RNA– seq level. N- and O-glycomics revealed overall lower levels of α2–6 sialylated N- and O-glycans in MLL cells (correlating with ST6Gal1 transcript and ST6Gal1 protein levels) as well as an increase in Core 2 type O-glycans (correlating with GCNT1 transcript level). Patient MLL cells exhibit distinct N- and O-glycan fingerprints, along with specific alterations of receptor tyrosine kinase signalling networks. In addition to wellknown MLL glycoprotein markers, our integrated multiomics workflow identified a number of diagnostic/therapeutic protein candidates that have not previously been described.

Long Term Analysis of a Monocentric Cohort of Therapy-Related Acute Lymphoblastic Leukemia

Therapy related Acute Lymphoblastic Leukemia (t-ALL) refers to ALL developed in patients who have received prior cytotoxic therapies, including chemotherapies (CT) and/or radiotherapy (RT). Contrary to therapy-related myeloid neoplasms, t-ALL has not been fully recognized and its clinical and biological features remain poorly described. We reviewed all consecutive cases of adult t-ALL treated at our institution and described their clinical, phenotypic, and genetic characteristics. Between 1998 and 2019, 36 patients were admitted at Institut Paoli-Calmettes for t-ALL which represents 3.5% of all ALL admitted during the same period (n=1036). Median age was 67.5 years (ranges, 20-83). Twenty-eight (78%) patients were females. Thirty-eight (78%) patients of the t-ALL were secondary to a solid cancer. Among the most frequent solid tumors, we found 14 (39%) breast cancers, 5 (14%) pulmonary cancers and 4 (11%) thyroid cancers. Eight patients had a t-ALL secondary to a hematological malignancy (4 Non-Hodgkin lymphomas, 3 Hodgkin lymphomas and one myeloma). Three (8%) patients presented with more than one primary cancer. Primary malignancies were treated with CT in 7 cases (19%), RT in 11 cases (31%) and CT+RT in 16 cases (44%), in association or not with surgery of the primary tumor. Median time between the start of first malignancy treatment and t-ALL diagnosis was 5.4 years (ranges, 0.3-35). Median white blood cell count (WBC) was 15.9 (ranges, 0.6-386). Phenotypic characteristics at diagnosis were consistent with B-ALL for 31 patients (86%), and T-ALL or acute mixed-lineage leukemia for 2 (6%) and 3 (8%) patients, respectively. 9 (22%) cases were Philadelphia chromosome-positive by karyotype or Fluorescent in situ hybridization, 5 (14%) cases had MLL rearrangement, 5 (14%) cases had hypodiploidy, 3 (8%) cases had hyperdiploidy and 3 (8%) cases had normal karyotype. The 11 (30%) other cases had other genetic abnormalities. Thirty-one (86%) patients received intensive CT and 5 (14%) patients received a palliative treatment. For one patient, treatment modalities were missing. Twenty-eight (78%) were in complete remission (CR) after CT and 7 (19%) patients underwent an allogenic hematopoietic stem cell transplantation in first CR. Median follow-up was 52 months. Median overall survival calculated from the time of t-ALL diagnosis was 16 months and median leukemia-free survival was 14.3 months. Multivariate analyses taking age, WBC, MLL alteration, complex karyotype and BCR-ABL rearrangement into account, found that BCR-ABL alteration was associated with a higher OS (HR=4.9; ranges, 1.1-22.6). In conclusion, our results confirm that t-ALL is a rare event. Besides an older age at diagnosis, t-ALL does not display specific phenotypic or genetic characteristics. In our series, the patient outcome was poor. Further genetic studies are needed to better understand the pathogenesis of this ALL subgroup. Disclosures No relevant conflicts of interest to declare.

Poor Prognosis Biomolecular Factors Are Highly Frequent in Childhood Acute Leukemias From Oaxaca, Mexico.

Objective:To investigate the cellular and molecular epidemiology of acute leukemias in vulnerable populations of children and adolescents in Oaxaca de Juarez, Mexico.Material and Methods:Descriptive, cross-sectional and retrospective study, conducted from 2014 to 2018 in which profiles of molecular and immunophenotypic aberrations were investigated in children and adolescents diagnosed with acute leukemia, by evaluating 28 molecular abnormalities by HemaVision-Q28 multiplex RT-PCR kit and standardized EuroFlow Immunophenotyping of bone marrow cells.Results:We included 218 patients, with 82.5% younger than 14 years and 17.5% adolescents. The median age was 9 years and a main peak of incidence was recorded at age of 4 to 5 years. B-cell acute lymphoblastic leukemia was diagnosed in 70.64% of all cases, acute myeloid leukemia was in 22.48%, T-cell acute lymphoblastic leukemia in 6.42%, and mixed lineage acute leukemia in 0.46% of cases. Overall, chromosomal translocations were positive in 29.82% of cases. While 65.31% of patients with acute myeloid leukemia reported aberrancies, only in 18.83% of B-cell acute lymphoblastic leukemia cases genetic abnormalities were obvious. Surprisingly, most prevalent translocations in B-cell acute lymphoblastic leukemia were t(9;22) in 20.7%, followed by t(4;11) in 17.2% and t(6;11) in 13.8%, whereas patients with acute myeloid leukemia showed t(15;17) in 40.6% and t(8;21) in 21.9%. In contrast, an homogeneous expression of t(3;21) and t(6;11) was recorded for T-cell acute lymphoblastic leukemia and mixed lineage acute leukemia cases, respectively. Except for t(1;19), expressed only by pre-B cells, there was no association of any of the studied translocations with differentiation stages of the B-leukemic developmental pathway.Conclusion:Our findings identify near 50% of patients with acute lymphoblastic leukemia at debut with high-risk translocations and poor prognosis in B-cell acute lymphoblastic leukemia as well as an unexpected increase of acute myeloid leukemia cases in young children, suggesting a molecular shift that support a higher incidence of poor prognosis cases in Oaxaca.

Outcome of Hematopoietic Stem Cell Transplantation in the Patients with Pediatric Acute Lymphoblastic Leukemia Enrolled in the Japan Association of Childhood Leukemia Study (JACLS) ALL-02

Background: Though the outcome of patients with acute lymphoblastic leukemia (ALL) has been greatly improved, some high-risk patients still need hematopoietic stem cell transplantation (HSCT). In this study, we evaluated clinical characteristics of children with ALL who were treated according to Japan Association of Childhood Leukemia Study (JACLS) ALL-02 trial and received HSCT to determine prognostic factors for the outcome of HSCT. Methods: Between April 1, 2002 and March 31, 2008, 1,252 patients aged 1-18 years with newly diagnosed ALL were enrolled in JACLS ALL-02 trial. Ph+ALL, mature B-ALL, and NK-leukemia were excluded. Of all the 1,252 patients, 211 (16.8%) patients were reported to receive HSCT, of which 206 patients with adequate information of 1st HSCT were subjected to further analysis. In this study, HSCT in 1st complete remission (CR) in JACLS ALL-02 trial was indicated for the patients with extremely high risk (ER: BCP-ALL with PPR and/or evidence of t(4;11) (or KMT2A-AFF1 positive), hypodiploidy (≤ 44) and/or acute mixed lineage leukemia/ acute unclassified leukemia). As a precaution, in ER patients, the timing of HSCT differs depending on whether they have a matched related donor. Results: Of all the 206 patients, 83 patients received HSCT in first CR (CR1), 68 patients in CR2, 54 patients in non-CR and 1 patient in induction failure (IF). 5-year overall survival (5y-OS) for 206 patients was 50% (95% CI 42.7-56.8). In detail, 73.8% (95% CI 62.6-82.1) for CR1 patients, 49.4% (95% CI 35.9-61.5) for CR2 patients, and 14.8% (95% CI 6.9-25.5) for non-CR patients, respectively. In univariate analysis, JACLS risk classification, disease status at HSCT (CR or non-CR or IF), stem cell source were significant prognostic factors. In multivariate analysis, only disease status retained as prognostic factor (p<0.01, HR=1.77). For CR1 patients, stem cell source was significant prognostic factor on multivariate analysis (5y-OS = 41.7%, 66.7%, and 79.3% for peripheral blood, cord blood, bone marrow; p = 0.023). When we focused on immunophenotype, stem cell source retained their effect only for patients with T-ALL. For CR2 patients, central nerves system involvement at diagnosis (p=0.02, HR=7.2), S classification (p=0.03, HR=1.2), and NCI risk (p=0.04, HR=2.5) were significant prognostic factors on multivariate analysis. S classification retained its prognostic impact only for patients with BCP-ALL (p=0.01, HR=1.5). For non-CR patients, NCI or JACLS risk classification at first diagnosis, S classification, frequency of relapse before HSCT, stem cell source, and conditioning for HSCT did not affect treatment outcome. Interestingly, non-CR patients with high-hyperdiploid (HHD) (n=5) had a significantly better 5y-OS (80%) than the other subtypes. In multivariate analysis, age at diagnosis (<10 years, p=0.04, HR=2.0)) and HHD (p=0.006, HR=1.74) contribute to the better outcome of HSCT in non-CR. Conclusion: The current study showed that the prognostic factors for HSCT varied according to the disease status or disease immunophenotype. Also, it is noteworthy that HHD patients may be rescued even in non-CR status by HSCT. Disclosures No relevant conflicts of interest to declare.

Single Cell Sequencing Reveals Heterogeneity of Gene Expression in KMT2A Rearranged Infant ALL at Relapse Compared to Diagnosis

Introduction Infant acute lymphoblastic leukemia (ALL) with KMT2A rearrangement (KMT2A-r) is associated with a very poor prognosis. Disease free survival from the date of diagnosis is approximately 20% to 40%, depending on age, white blood cell count, and response to induction therapy. Refractory and relapsed infant ALL is often resistant to attempts at re-induction, and second remission is difficult to both achieve and maintain. Genomic sequencing studies of infant KMT2A-r ALL clinical samples have demonstrated an average of fewer than 3 additional non-silent somatic mutations per case at diagnosis, most commonly sub-clonal variants in RAS pathway genes. We previously reported relapse-associated gains in somatic variants associated with signaling, adhesion, and B-cell development pathways (Blood 2016 128:1735). We hypothesized that relapsed infant ALL is characterized by recurrent, altered patterns of gene expression. In this analysis, we utilized single cell RNA sequencing (scRNAseq) to identify candidate genes with differential expression in diagnostic vs. relapse leukemia specimens from 3 infants with KMT2A-r ALL. Methods Cryopreserved blood or bone marrow specimens from 3 infants enrolled in the Children's Oncology Group AALL0631 trial were selected for analysis. Samples from both diagnosis (DX) and relapse (RL) time points were thawed and checked for viability (>90% of cells viable) using trypan blue staining. Samples were multiplexed and processed for single cell RNA sequencing using the Chromium Single Cell 3' Library Kit (v2) and 10x Genomics Chromium controller per manufacturer's instructions (10x Genomics, Pleasanton, CA). Single cell libraries were converted to cDNA, amplified, and sequenced on an Illumina NovaSeq instrument. Two technical replicates were performed. Samples were de-multiplexed using genotype information acquired from previous whole exome sequencing (WES) and demuxlet software. Transcript alignment and counting were performed using the Cell Ranger pipeline (10x Genomics, default settings, Version 2.2.0, GRCh37 reference). Quality control, normalization, gene expression analysis, and unsupervised clustering were performed using the Seurat R package (Version 3.0). Dimensionality reduction and visualization were performed with the UMAP algorithm. Analyses were restricted to leukemia blasts with CD19 expression by scRNAseq. Results The clinical features for each case are shown in Table 1. Cells from the 3 infant ALL samples clustered together, distinct from cells of non-infant B-ALL, T-ALL, and mixed lineage acute leukemia biospecimens in the Children's Mercy scRNAseq database, but largely did not overlap with one another. For each of the 3 infant cases, cells from DX and RL time points could be distinguished by differential patterns of gene expression (Figure 1). Individual genes with statistically significant (p<0.05) log-fold change values were examined. Figure 2 summarizes the number of genes with up-regulation of expression by scRNAseq at RL compared to DX. Only 6 genes, DYNLL1, HMGB2, HMGN2, JUN, STMN1, and TUBA1B, were significantly increased at RL across all 3 cases. We repeated this analysis, restricting to leukemia blasts with CD79A expression, and identified these same 6 genes, and 4 additional genes: H2AFZ, NUCKS1, PRDX1, and TUBB, as consistently up-regulated in RL clusters. We examined the expression of candidate genes of interest, including clinically targetable genes, to compare the distribution of expression at DX and RL (Table 2). Conclusion Genomic factors underlying the aggressive, refractory clinical phenotype of relapsed infant ALL have yet to be defined. Each of these 3 cases demonstrates unique expression patterns at relapse, readily distinguishable from both the paired diagnostic sample and the other 2 relapse samples. Thus, scRNAseq is a powerful tool to identify heterogeneity in gene expression, with the potential to discover recurrent genomic drivers within resistant disease sub-clones. Ongoing analyses include scRNAseq in additional infant ALL samples, relative quantification of transcript expression in single cells, and comparison with bulk RNAseq data. Disclosures No relevant conflicts of interest to declare.

Does Mixed Chimerism after Allogeneic Hematopoietic Cell Transplantation in Patients with Fanconi Anemia Impact on Outcome?

Fanconi anemia (FA) cells are characterized by genomic instability which places FA patients (pts) at risk for malignancies; leukemia and oropharyngeal/urogenital cancers. The risk of development of leukemia is theoretically eliminated after hematopoietic cell transplantation (HCT).Simultaneous presence of both host- and donor-derived cells in the recipient often referred to as mixed chimerism (MC) is observed in a large proportion of patients after HCT with non-malignant disorders. In FA patients however, MC might have a unique implication and the persistent existence of FA cells represents a management dilemma as the lingering FA cells may theoretically evolve into a malignant clone. We have studied a large population of FA patients who underwent allogeneic HCT at our institution and report here the outcome of those with MC.From January 1995 until December 2017, 163 FA pts underwent allogeneic HCT at our center; chimerism data at last contact were available on 100 pts who are the subject of this analysis. Females (51) had a mean age at HCT of 8.7 (SD:3.0, Min-Max: 2.0-14.1) years whereas in males (49) it was 8.1 (SD:3.4; Min-Max: 1.4-17.4) years (P-Value: 0.415). Donor was HLA matched family member in 78 pts, Haplo-identical family member in 17 (92 of them were bone marrow and 3 peripheral stem cells); 3 had unrelated cord blood. Total body irradiation was used in 24 pts.Median time to ANC and platelets recovery was 14 and 22 days respectively. Cumulative incidence of overall aGVHD Grade III or above was 4%. Median follow up time was 67.2±14.6 months (95% CI: 38.7-95.7) from HCT date (Min: 7.3, Max:188.1). Chimerism analysis at last follow up showed full chimerism (100%; Myeloid/lymphoid) in 46 pts; 54 had MC defined as the presence of any residual recipient cells. No statistically significant association was noted between full and MC pts in the incidence of aGvHD (P-Value: 0.331). The 10-year cumulative probability of Overall Survival (OS) was 0.904±0.042. No significant difference was observed in OS between full and MC pts (0.954±0.032 vs. 0.883±0.059, P-value: 0.943) with 4 deaths in each group. New malignancy occurred in 4 pts; 2 in each group. In full Chimerism pts: Acute Mixed Lineage Leukemia and carcinoma and in MC pts: acute myeloid leukemia and acute lymphocytic leukemia, (P-Value=1.00). Graft failure occurred in 2 pts in the mixed chimerism group vs none in the full chimerism group (P-Value=1.0).We conclude that mixed chimerism in FA pts does not appear to have an adverse effect on outcome in our follow-up period; longer follow-up time however is needed to confirm the validity of this statement. DisclosuresNo relevant conflicts of interest to declare.

A Novel Inducible Mouse Model of MLL-ENL-driven Mixed-lineage Acute Leukemia.

Previous retroviral and knock-in approaches to model human t(11;19)+ acute mixed-lineage leukemia in mice resulted in myeloproliferation and acute myeloid leukemia not fully recapitulating the human disease. The authors established a doxycycline (DOX)-inducible transgenic mouse model "iMLL-ENL" in which induction in long-term hematopoietic stem cells, lymphoid primed multipotent progenitor cells, multipotent progenitors (MPP4) but not in more committed myeloid granulocyte-macrophage progenitors led to a fully reversible acute leukemia expressing myeloid and B-cell markers. iMLL-ENL leukemic cells generally expressed lower MLL-ENL mRNA than those obtained after retroviral transduction. Disease induction was associated with iMLL-ENL levels exceeding the endogenous Mll1 at mRNA and protein levels. In leukemic cells from t(11;19)+ leukemia patients, MLL-ENL mRNA also exceeded the endogenous MLL1 levels suggesting a critical threshold for transformation. Expression profiling of iMLL-ENL acute leukemia revealed gene signatures that segregated t(11;19)+ leukemia patients from those without an MLL translocation. Importantly, B220+ iMLL-ENL leukemic cells showed a higher in vivo leukemia initiation potential than coexisting B220- cells. Collectively, characterization of a novel transgenic mouse model indicates that the cell-of-origin and the fusion gene expression levels are both critical determinants for MLL-ENL-driven acute leukemia.

Acute mixed lineage leukemia with plasmacytoid dendritic cell neoplasm: report of one case and review of literature

目的 提高对急性混合细胞白血病（MAL）合并母细胞性浆细胞样树突细胞肿瘤（BPDCN）的认识。 方法 通过报道1例MAL合并BPDCN患者并复习相关文献，分析BPDCN的临床特点、治疗方案及预后。 结果 患者为中年女性，临床表现脾大、淋巴结肿大，骨髓涂片可见肿瘤细胞呈母细胞形态，形态单一，核不规则，染色质细，可见数个小核仁，胞质量少，无颗粒状且嗜碱性，骨髓免疫分型可见30.79%的细胞CD56-、CD4-、CD123st+、HLA-DRst+、BDCA2+，为异常浆细胞样树突细胞，根据相关评分系统，表达CD123及BDCA2，总分为3分，可诊断为BPDCN。患者有长期服用硫唑嘌呤以及血小板减少的病史，染色体核型检测发现存在7号染色体缺失，提示该患者有可能存在骨髓增生异常综合征病史，骨髓细胞免疫分型提示同时存在髓系幼稚细胞及异常单核细胞的表达，急性粒-单核细胞白血病诊断明确。 结论 MAL合并BPDCN的诊断需综合临床表现、形态学、免疫学、分子遗传学等，应争取对疾病尽早诊断及治疗。

Acute mixed-lineage leukemia with t(9;22) (q34;q11) complicated with nasal cerebral type of mucormycosis: report of one case and review of literature

目的 探讨伴t (9; 22）（q34；q11）急性混合细胞白血病（AMLL）合并鼻脑型毛霉菌感染的临床及生物学特征。 方法 报道1例伴t (9; 22）（q34; q11）AMLL合并鼻脑型毛霉菌感染患者的诊治过程，并进行相关文献复习。 结果 该患者的临床表现和实验室检查均提示为伴t (9; 22）（q34；q11）AMLL合并鼻脑型毛霉菌感染，予环磷酰胺及激素诱导治疗后行VPDA方案化疗以及亚胺培南／西司他丁钠盐联合伏康唑脂质体两性霉素B治疗，病情好转。 结论 伴t (9; 22）（q34；q11) AMLL合并毛霉菌感染患者预后差，早发现、早诊断是提高治愈率的关键。

Risk-Adjusted Therapy of Acute Lymphoblastic Leukemia Can Optimize the Indication of Stem Cell Transplantation and Cranial Irradiation: Results of Japan Association Childhood Leukemia Study Group (JACLS) Protocol ALL-02

BACKGROUNDThe trial JACLS ALL-02 for treatment of childhood acute lymphoblastic leukemia (ALL) was designed to reduce acute and long-term toxicity in selected patient groups with favorable prognosis and to improve outcome in unfavorable-risk groups by treatment intensification. These aims were pursued through a refined stratification strategy using white blood cell count, age, immunophenotype, unfavorable genetic aberrations, and treatment response providing an excellent discrimination of risk groups. PATIENTS AND METHODSBetween April 2002 and March 2008, 1252 children with newly diagnosed de novo ALL with 1-18 years of age were enrolled to JACLS ALL-02 trial. Ph+ALL, mature B-ALL, and NK-leukemia were excluded. Patients with BCP-ALL were stratified into 3 groups: standard-risk (SR), high-risk (HR), extremely high-risk (ER), based on initial prednisolone (PSL) response and the modified National Cancer Institute(NCI) workshop criteria. Prednisolone poor response (PPR) was determined after 7 days of monotherapy with prednisone and one intrathecal dose of methotrexate. PSL good responders (PGR; < 1,000 blasts/microL) were divided into SR and HR according to the modified NCI workshop criteria by WBC 10K and age 10, and received conventional therapy. BCP-ALL with PPR (≥ 1,000 blasts/microL) or t(4;11), and acute mixed lineage leukemia/ acute unclassified leukemia were assigned to ER and received intensified post-induction therapy . Patients with T-ALL were treated by a specific protocol that was different from the protocol used for BCP-ALL. Bone marrow response was also evaluated in aspiration smears on day 15 and 33 of induction treatment, and those who had slow early bone marrow response, defined as an M3 marrow on day 15 or M2/3 marrow on day33, shifted to the higher risk and received augmented post-induction therapy. Cranial irradiation was restricted to patients with initial central nerve system involvement or T-ALL with high WBC (≥10K) at diagnosis. Alternatively, protracted TIT was given during induction, intensification and maintenance depending on the risk group (12 doses in SR/T and 15 in HR/ER). Slow early responders who had an M3 marrow on day 15 in ER/T or M2/3 marrow on day33 in any risk, underwent to stem cell transplantation (SCT). PPRs without slow early bone marrow response underwent to SCT only if a matched sibling donor was available. The probability of event-free survival (EFS) and overall survival were constructed using the Kaplan-Meier method. Events in the analysis of EFS included induction failure, death, relapse and secondary malignant neoplasm. All statistical analyses were done according to intent-to treat methods. RESULTSEstimated 4-year EFS and OS for all 1252 patients was 83.7% (SE=1.1) and 90.3% (SE=0.89), slightly better in EFS than the former study (ALL-97; 79.3%, SE=1.7, p=0.054). The SR group (N=457, 37%) achieved excellent 4-year EFS of 90.4% (95%CI: 87.2, 92.7) and 4-year OS 97.3% (95.3, 98.5); in SR without slow early bone marrow response, patients with hyperdiploid and triple trisomy showed excellent 4-year EFS/OS of 100%. In the HR group (N=542, 43%), the estimated 4-year EFS and 4-year OS were 84.9% (81.5, 87.7) and 89.3% (86.3, 91.6), respectively; older age (≥ 6 years) was predictive for inferior OS (hazard ratio=1.14, p<0.0001), and ETV6-RUNX1 was predictive for favorable OS (HR=0.182, p<0.03). In the ER group (N=139, 11%), intensification of consolidation treatment using high-dose dexamethasone and AML-oriented agents led to considerable improvement yielding a 4-year EFS of 74.1% (66.0, 80.6) and a 4-year OS of 87.0 (80.2, 91.6). In T group (N=107, 8%), the dose reduction of L-asparaginase and anthracyclines compared with the ALL-97 resulted in inferior outcome in 4-year EFS 65.1% (54.9, 73.6), but not in 4-year OS 83.2% (74.6, 89.1), although the incidence of pancreatitis decreased. In non-T-ALL, The 5-year cumulative risk of an isolated and total CNS relapse was lower in ALL-02 than in ALL-97 (isolated CNS relapse: 0.8% vs 2.7%, p=0.017, total CNS relapse: 1.4% vs 4.5%, p=0.01). The proportion of CRT and SCT were 2%, 8% in ALL-02 respectively, whereas 31%, 11% in ALL-97 respectively. CONCLUSIONSRefined stratification and risk-adjusted therapy in the JACLS ALL-02 trial can bring about the abolition of prophylactic CRT in non-T ALL and the optimized indication of SCT without compromising the treatment results. DisclosuresNo relevant conflicts of interest to declare.

Effect of the microtransplantation of allogeneic hematopoietic stem cells as maintenance therapy for elderly patients with acute leukemia.

The incidence of acute myeloid leukemia (AML) increases with age. Elderly patients with AML are less tolerant to high-intensity consolidation therapy than younger patients, with significantly worse prognoses. Induction and consolidation therapy combined with allogeneic hematopoietic stem cell microtransplantation may improve the prognosis of elderly patients with AML. The present study reports the effect of maintenance therapy with low-dose chemotherapy treatment combined with microtransplantation in elderly patients with AML following consolidation. Between January 2011 and April 2014, three elderly patients (>55 years old), including one 58-year-old patient with acute mixed lineage leukemia (AMLL) and two patients with AML aged 59 years and 62 years, underwent microtransplantation maintenance therapy. Following a complete response to induction chemotherapy and consolidation chemotherapy with two to four cycles of medium dose Ara-c (auto transplantation was performed in the patient with AMLL), maintenance therapy was performed using low-dose Ara-c combined with human leukocyte antigen haploidentical allogeneic hematopoietic stem cell microtransplantation, which was repeated every 3 to 6 months. The patients were followed up for 27, 20 and 16 months, respectively, and all three patients achieved disease-free survival. The bone marrow Wilms' tumor suppression gene (WT1) level of the case with AMLL was dynamically monitored. The results showed that the WT1 level was abnormally high prior to microtransplantation and gradually declined to normal levels subsequent to the process. None of the patients suffered from graft versus host disease during the microtransplantation process. In conclusion, microtransplantation maintenance therapy following consolidation therapy is feasible in elderly patients with AML, and is expected to be able to further remove residual lesions and improve treatment efficacy. A large-scale clinical trial is required to confirm the effect of maintenance therapy in elderly patients with AML.

Flow Cytometric Detection of Asparagine Synthetase Protein in Leukemia Cells; Indication for L-Asparaginase Therapy

Modern clinical treatments of childhood acute lymphoblastic leukemia (ALL) employ enzyme-based methods for depletion of blood asparagine in combination with standard chemotherapeutic agents. L-asparaginase (L-asp) therapy causes depletion of plasma asparagine followed by the loss of intracellular asparagine. Due to the lack of a rapid up-regulation of asparagine synthetase (ASNS) protein content in ALL cells, they are preferentially killed by L-asp. Elevated expression of ASNS within the leukemia cells causes decreased sensitivity to L-asp. The proof of ASNS deficiency in leukemia cells is considered to predictive for effectiveness of L-asp even in acute myeloid leukemia (AML) other than ALL patients. The establishment of quantitative estimation of ASNS protein content would be useful for the L-asp treatment in leukemia therapy.Objective: Our aim was to set up a flow cytometry system to check ASNS deficiency in leukemia cells and to investigate the sensitivity to L-Asp and the ASNS expression in AML leukemia cells.Methods: AML (KG-1, HL-60, U937) and ALL (MOLT-4, RS4;11) and CML (K562) cell lines were grown in RPMI1640 medium with 10% FCS. Primary leukemic cells from the peripheral blood or bone marrow of 20 AML patients were harvested on EDTA and isolated by Ficoll density gradient within 72h. ASNS expression was evaluated by cytosolic flow cytometry with Z5808 McAb (Hybridoma 31: 325-332.2012) and expressed as a ΔMFI(Difference of Mean Fluorescence Intensity(MFI) between by Z5808 and isotypic control) or MFI ratio(MFI by Z5808/MFI by isotypic control). When a sufficient amount of leukemic cells was available, sensitivity to L-asp (expressed as an IC50 - concentration inhibiting 50% of cell viability) was evaluated in vitro by incubating various concentrations of E. coliL-asp with the cells and by measuring the cell viability with a counting kit (WST1 viability assay) at day 3.Results: Determination of IC50 for the HL-60 (⊿MFI 48 ± 8.01, MFI ratio 1.77 ± 0.03) and U937 (⊿MFI 16.7 ± 0.47, MFI ratio 1.19 ± 0.02) demonstrated that these cells were equally sensitive to L-asp than the ALL cell line MOLT-4 in vitro (0.37 and 0.02IU/mL versus 0.15 IU/mL, respectively). K562 and KG-1 (⊿MFI 135.7 ± 5.66, MFI ratio 2.48 ± 0.09) cells with the highest ASNS expression exhibited resistance to L-asp (>10 IU/ml). Both of ASNS Expression by ⊿MFI and MFI ratio was inversely correlated with L-asp sensitivity judging from cell line studies. Judging from cell line study, the threshold for ASNS protein expression effective for L-asp treatment was considered to be <25 for ⊿MFI and <1.8 for MFI ratio respectively. Fresh leukemia cases contained three ALLs, Ph1ALL, and 13 AML; M0, 1; M1, 3; M2, 2; M4, 1; M5, 3; M7, 3; and Acute Mixed lineage leukemia, 2 cases. IC50 determination was possible on 11/20 patients. Four displayed a high sensitivity to L-asp (IC50 < 0.01 IU/mL) whereas two displayed resistant to L-asp (IC50 >10 IU/mL) Remaining 5 patients were a moderate sensitivity (IC50 < 0.5 IU/mL). ASNS Expression in ALL was almost near zero. ASNS expression in fresh AML was low in all cases except for one M2 and one M4 cases. Indeed, at high ASNS expression, these cases were resistant in vitro to L-asp. The patients with blasts sensitive to L-asp had mainly M1, M5 or M7 AML. A good inverse correlation between ASNS expression and sensitivity to L-asp was observed also in primary leukemic cells from AML patients.Conclusions: We demonstrated here that some of AML cell lines with low ASNS expression are more sensitive to L-asp than leukemia cells with high ASNS expression. Also, fresh AML cells with low ASNS expression are more sensitive to L-asp than with high ASNS expression. 11/13AML or two AMLL cases were supposed to be effective for L-asp. Plasma asparagine depletion by L-asp in selected patients having low ASNS may be a promising therapeutic approach even for AML.This work was supported by Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (C) KAKENHI Grant Number 24590713. DisclosuresNo relevant conflicts of interest to declare.

The DOT1L Inhibitor EPZ-5676: Safety and Activity in Relapsed/Refractory Patients with MLL-Rearranged Leukemia

The DOT1L Inhibitor EPZ-5676: Safety and Activity in Relapsed/Refractory Patients with MLL-Rearranged Leukemia

Absolute Lymphocyte Counts at the End of Induction Is a Prognostic Indicator in Childhood Acute Lymphoblastic Leukemia

BACKGROUND: Recently, several studies have demonstrated that absolute lymphocyte counts (ALC) after induction therapy predicted treatment outcome. To address this issue, we here assessed the impact of the ALC at the end of induction therapy on outcomes in childhood ALL.METHODS: We reviewed 141 cases of pediatric ALL with 1-21 years of age treated on the Japan Association Childhood Leukemia Study group ALL-02 series of treatment trials between 2002 and 2013. Patients with Philadelphia chromosome-positive ALL were excluded. Variables retrospectively analyzed included ALC at several time points during remission induction, age at diagnosis, gender, initial white blood cell count (WBC), cytogenetics, immunological phenotype, stratified risk, treatment response for bone marrow (the percentage of blasts at day 15), and outcome. Events in the analysis of event-free survival (EFS) included induction failure, death, relapse and secondary malignant neoplasm. The comparison of categorical variables between groups was performed by chi-square test. The probability of EFS and overall survival (OS) were analyzed with the use of the Kaplan–Meier method and a stratified log-rank test. A multivariate analysis of survival was performed with the use of a Cox proportional-hazard model to evaluate the treatment effect with adjustment for stratification factors.RESULTS: The subjects included 121 of B-precursor ALL, 10 of T cell ALL, and 4 of acute mixed lineage leukemia/ acute unclassified leukemia. We found high WBC count at diagnosis (>100K/microL) and slow early responder for bone marrow at day 15 to be an unfavorable prognostic indicator, and also the ALC at the end of induction (day29) to be a statistically significant predictor of improved OS and EFS in our cohort. Patients with ALC ≥ 800/microL had a superior 5-year overall survival (100 ± 1.7% vs 88.1 ± 4.3 %, p=0.0001) and EFS (98.3 ± 1.7% vs 81.8 ± 5.0 %, p=0.0001). Multivariate analysis demonstrated that ALC at day29 was an independent, clinically significant predictor of improved EFS and OS after controlling WBC at diagnosis, gender, age at diagnosis, and cytogenetics. Multiple regression analysis adjusting for initial WBC count, peripheral blast counts at day8, and cytogenetics, also revealed an independent relationship (p=0.005) between treatment response (the percentage of blasts at day 15) and ALC at day29.CONCLUSIONS: ALC is a simple, statistically significant prognostic factor in childhood ALL that may refine current risk stratification algorithms. DisclosuresNo relevant conflicts of interest to declare.

Salvaged Cord Blood Transplantation For 19 Patients With Progressive Hematologic Malignancies

Few clinical studies have investigated the role of salvaged unrelated cord blood transplantation (CBT) for progressive hematologic malignancies. The aim of this report is to identify the potential benefits of unrelated CBT in progressive hematologic malignancies.19 consecutive patients with progressive myeloid and lymphoid malignancies who received salvaged CBT following myeloablative conditioning regimens (12 TBI/CY/Ara-C, 6 Bu / CY / Ara-C, and 1 Bu /CY) from July 2005 to December 2012 were analyzed retrospectively. Of the 19 patients, 6 suffered from acute myeloid leukemia(AML), 5 acute lymphoid leukemia(ALL),：1 acute mixed lineage leukemia (AMLL), 1 myelodysplastic syndrome-refractory anemia with excess blasts(MDS-RAEB), 2 acute myeloid leukemia transformed from myelodysplastic syndrome, and 4 lymphoma, all of them in non-remission (NR) before transplantation. Median age of them were 13（range 6-32）years and median body weight were 45 (range 18–73 ) kg. All patients received 1 CBT unit ≤2 loci HLA-mismatched with the recipient. Infused TNC was 4.07（range 2.76-6.02）×107/kg and CD34+ stem cell 2.08（range 0.99-8.65）×105/kg. All patients were engrafted with neutrophil exceeded 0.5×109/L on median day +17（range 14-37d）and plt counts of＞20×109/L on median day +35 (range 17-70d). 10 patients (52.6%) experienced pre-engraftment syndrome (PES) and 3 (15.8%) patients progressed to acute GVHD. The incidence of Ⅱ-Ⅳ aGVHD and cGVHD were 10.5% and 21.1%. With a median follow up of 10(range 1-58) months，10 cases survived and 2 relapsed．The estimated 2 year overall survival (OS), disease-free survival (DFS) and non-relapse mortality (NRM) rate was 45.6%, 34.2% and 39.8%. Salvaged CBT might be a promising modality for treatment of progressive hematologic malignancies, even with high leukemia burden. Disclosures:No relevant conflicts of interest to declare.