Mixed Phenotype Acute Leukemia Research Articles

Association between Direct Acting Agents used for chronic hepatitis C virus infection and the occurrence of acute leukaemia – A disproportionality analysis

BACKGROUND AND AIMSA recent single-center study reported a significant increase in acute myeloid leukaemia (AML) cases, including mixed-phenotype acute leukaemia (MPAL), after exposure to direct acting agents (DAA). We investigated whether DAA use increased the risk of AML in patients with chronic hepatitis C virus (HCV) infection. METHODSWe conducted a disproportionality analysis of the WHO Pharmacovigilance database Vigibase up to 2020. Queries focused on all DAAs, subclasses, combinations or each DAA separately as well as interferon and ribavirin as negative controls. The primary outcome was AML. Secondary outcomes were AML without MPAL, MPAL, acute lymphoid leukemia (ALL) and acute leukemia (AL, high-level term encompassing AML, ALL, MPAL and unspecified acute leukemia [UAL]). The information component (IC0.25) and proportional reporting ratio (PRR0.25) were computed to assess a potential pharmacovigilance signal. RESULTSWe identified 49 notifications reporting any AL occurrence after anti-HCV treatments from June 1997 to December 2020: 23 (47%) involved a DAA, 24 (49%) interferon and 12 (24%) ribavirin. The DAAs sofosbuvir and ledipasvir were suspected in 74% (n=17) and 39% (n=9) of cases. The events reported were AML (n=22), ALL (n=11), AML and ALL (n=1) and UAL (n=15) and no MPAL. DAA, interferon or ribavirin were not significantly associated with AML, ALL or AL. CONCLUSIONThis study did not find any association between DAA exposure and the occurrence of AML. Nevertheless, vigilance should remain, particularly for MPAL, which may not have been well captured in our study because of its rareness and high risk of misclassification.

Clonotypic VDJ rearrangements in Mixed Phenotype Acute Leukemia can be Successfully Utilized to Track Minimal Residual Disease (MRD).

Multiplex polymerase chain reaction (PCR) and next-generation sequencing (NGS) can both be used to identify a neoplastic clonotype by targeting CDR3 and assessing rearrangements in IgH, IgK, IgL, TCR-β, and TCR-gamma loci. The clonotypic sequence can be robustly used to track minimal residual disease (MRD). The ability to track MRD by NGS in mixed phenotype acute leukemia (MPAL) is unknown and warrants investigation. Institutional Review Board (IRB) approval was obtained. Central Moffitt Cancer Center (MCC) database was searched to locate any patients with MPAL from over 600,000 entries. Patient charts were manually curated to identify those with clonoSEQ data, and clinical data was procured from the electronic medical record (EMR). Twenty-nine patients with MPAL were identified. Only 2 patients with clonoSEQ testing were found. Both demonstrated a B/myeloid phenotype, and both were bilineal. NGS (clonoSEQ) identified 4 dominant (IGH) (patient A; 8/2019) and 2 dominant sequences (patient B; 10/2019), respectively. In both patients, clonoSEQ testing successfully tracked minimal residual disease and mirrored clinical disease burden. This report is the first to confirm the utility of NGS-based MRD tracking in patients with MPAL and shows increased sensitivity of NGS over MRD flow cytometry.

Clinical Characteristics and Diagnosis of Ph-Positive Mixed Phenotype Acute Leukemia.

The goal was to improve the clinical cognition of Ph-positive mixed phenotype acute leukemia and avoid misdiagnosis or delayed diagnosis. The clinical manifestations and laboratory results (bone marrow cell morphology, multiparameter flow cytometry, and cytogenetics) of a case of Ph-positive mixed phenotype acute leukemia were analyzed, and related literature was reviewed. Blood routine: WBC 386.35 x 109/L, HGB 117.00 g/L, PLT 31 x 109/L; 80% of the original cells can be seen by artificial classification. Morphological examination of bone marrow cells showed that the proliferation of nucleated cells was obviously active, and the original cells accounted for 76%. The size of the original cells was somewhat uniform, most of the cells had less mass, were stained light grayish blue, the cytoplasm particles were not obvious, the nuclei were mostly round or quasi-round, some of them showed distortion and nuclear notch, and the chromatin was coarse. Some of the cells were rich in mass, small azurin granules were seen, the nuclei were regular, most of them were round, the chromatin was fine, the myeloperoxidase and esterase staining were negative, the eosinophils accounted for 2.5%, and the basophils accounted for 0.5%. Flow cytometry immunotyping: Two groups of abnormal cells were seen in the bone marrow. 1. A group included 12.32% of nuclear cells and showed abnormal myeloid primitive cell phenotype. Main expression: CD117, CD34, CD38, HLA-DR, CD33, CD64, CD123, weak expression: CD13, CD19. 2. The other group included 45.61% of the nuclear cells and had a B-lymphoblastic phenotype. Main expression: CD34, CD38, HLA-DR, CD123, CD19, CD10, CD9, cCD79a, TDT, weak expression of CD13, CD22. Mixed phenotype acute leukemia (M/B) immunophenotype was considered. Chromosome: 46,XY,t(9; 22)(q34;q11.2) [20]. BCR-ABL (P210) fusion gene was positive. Mixed phenotype acute leukemia (MPAL) is a rare type of malignant hematologic disease. Its diagnosis is based on the comprehensive evaluation of bone marrow cell morphology, immunophenotype, molecular and cytogenetic features.

Clinical features of immature leukemias in children.

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL), mixed phenotypic acute leukemia (MPAL), and acute myeloid leukemia with minimal differentiation (AML-M0) all originate from immature hematopoietic progenitor cells and have a poor prognosis. We investigated the clinical characteristics of these immature leukemias in 17 children (ETP-ALL: 8, MPAL: 5, AML-M0: 4) at seven institutions. Clinical and laboratory findings were comparable across disease types. Eleven and six patients received ALL- and AML-oriented induction chemotherapy, with six and four achieving complete remission (CR), respectively. Five additional patients achieved CR after salvage with the other type of chemotherapy. Eight patients received hematopoietic cell transplantation (HCT) in first CR, and six survived without relapse. However, six of seven patients who did not receive HCT during first CR relapsed; all underwent HCT later, and only three survived. The 5-year event-free survival (EFS) and overall survival (OS) rate were 37% and 69%, respectively. Patients who achieved CR after induction chemotherapy and received HCT in first CR had favorable EFS and OS. Notably, all patients who received HCT in first CR survived 5years after diagnosis. Appropriate induction chemotherapy and HCT in first CR could improve the outcome of immature leukemias.

Allogeneic hematopoietic cell transplantation for acute myeloid leukemia with BCR::ABL1 fusion.

BCR::ABL1 fusion is found in<1% of de novo acute myeloid leukemia (AML) cases and confers a poor prognosis. This Japanese nationwide survey analyzed patients with AML (n=22) and mixed phenotype acute leukemia (MPAL) (n=10) with t(9;22) or BCR::ABL1 who underwent allogeneic hematopoietic cell transplantation (allo-HCT) between 2002 and 2018. The 3-year overall survival (OS) rates were 81.3% and 56.0%, respectively (p=0.15), and leukemia-free survival (LFS) rates were 76.2% and 42.0%, respectively (p=0.10) in patients with AML and MPAL. The relapse rates were 9.5% and 14.0% (p=0.93), and the non-relapse mortality (NRM) rates were 14.3% and 44.0%, respectively (p=0.10) in patients with AML and MPAL. One in 17 patients with AML, with pre-transplant tyrosine kinase inhibitors (TKI), and three in five patients with AML, without pre-transplant TKI, did not achieve complete remission (CR) before allo-HCT (p=0.024). Among the 20 patients with known disease status after allo-HCT, 95.0% were in hematological or molecular CR. None of the four patients who received post-transplant TKI for prophylaxis or measurable residual disease relapse experienced hematological relapse. In conclusion, our results suggest that pre-transplant TKI could improve disease status before allo-HCT. Moreover, allo-HCT resulted in high OS, high LFS, low relapse, and low NRM rates in patients with AML with BCR::ABL1.

The application of targeted RNA sequencing for the analysis of fusion genes, gene mutations, IKZF1 intragenic deletion, and CRLF2 overexpression in acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is characterized by highly genetic heterogeneity, owing to recurrent fusion genes, gene mutations, intragenic deletion, and gene overexpression, which poses significant challenges in clinical detection. RNA sequencing (RNA-seq) is a powerful tool for detecting multiple genetic abnormalities, especially cryptic gene rearrangements, in a single test. Sixty samples (B-ALL, n = 49; T-ALL, n = 9; mixed phenotype acute leukemia (MPAL), n = 2) and 20 controls were analyzed by targeted RNA-seq panel of 507 genes developed by our lab. Of these, 16 patients were simultaneously analyzed for gene mutations at the DNA level using a next-generation sequencing panel of 51 genes. Fusion genes, CRLF2 expression, and IKZF1 intragenic deletion were also detected by reverse transcription-polymerase chain reaction (RT-PCR). Karyotype analysis was performed using the R-banding and G-banding technique on bone marrow cells after 24 hours of culture. Partial fusion genes were analyzed using fluorescence in situ hybridization (FISH). Compared with the results of Karyotype analysis, FISH, and RT-PCR, the detection rate of fusion genes by targeted RNA-seq increased from 48.3% to 58.3%, and six unexpected fusion genes were discovered, along with one rare isoform of IKZF1 intragenic deletion (IK10). The DNA sequencing analysis of 16 ALL patients revealed that 96.2% (25/26) of gene mutations identified at the DNA level were also detectable at the RNA level, except for one mutation with a low variant allele fraction. The detection of CRLF2 overexpression exhibited complete concordance between RT-PCR and RNA-seq. The utilization of RNA-seq enables the identification of clinically significant genetic abnormalities that may go undetected through conventional detection methods. Its robust analytical performance might bring great application value for clinical diagnosis, prognosis, and therapy in ALL.

Laboratory characterization of the pediatric B/T subtype of mixed-phenotype acute leukemia: Report of a case series.

Mixed-phenotype acute leukemia (MPAL) is a rare disease associated with difficulties in the correct lineage assignment of leukemic cells. One of the least common subtypes within this category is characterized by the simultaneous presence of B- and T-lineage-defining antigens. Each case of suspected B/T MPAL should be considered in light of all available laboratory and clinical data to avoid misdiagnosis. In this study, we describe 6 pediatric patients who presented with leukemic blasts bearing B- and T-lineage antigens at diagnosis, including their clinical, immunophenotypic, morphologic, and cytogenetic characteristics. In 3 patients, more or less distinct populations of B- and T-lymphoid origin were found; the other 3 patients had a single mixed-phenotype blast population. All cases fulfilled the World Health Organization criteria, but not all of them turned out to be bona fide cases of B/T MPAL according to the available clinical and laboratory data. Found genetic lesions were helpful for the confirmation of MPAL instead of 2 concomitant tumors, but for a general B/T MPAL diagnosis, genetic studies provided the only descriptive data. The accurate diagnosis of B/T MPAL requires a multidisciplinary approach combining high-tech laboratory methods and close cooperation between treating physicians and pathologists.

Genomic and global gene expression profiling in pediatric and young adult acute leukemia with PICALM::MLLT10 Fusion.

MLLT10 fusion is a rare but recurrent genetic driver in acute leukemias. To better understand the genomic landscape of PICALM::MLLT10 (PM) positive acute leukemia, we performed genomic profiling and gene expression profiling in twenty PM-positive patients, including AML (n = 10), T-ALL/LLy (n = 8), Mixed-phenotype acute leukemia (MPAL), T/B (n = 1) and acute undifferentiated leukemia (AUL) (n = 1). Besides confirming the known activation of HOXA, differential gene expression analysis compared to hematopoietic stem cells demonstrated the enrichment of genes associated with cell proliferation-related pathways and relatively high expression of XPO1 in PM-AML and PM-T-ALL/LLy. Our study also suggested PHF6 disruption as a key cooperating event in PICALM::MLLT10-positive leukemias. In addition, we demonstrated differences in gene expression profiles as well as remarkably different spectra of co-occurring mutations between PM-AML and PM-T-ALL/LLy. Alterations affecting TP53 and NF1, hallmarks of PM-AML, are strongly associated with disease progression and relapse, whereas EZH2 alterations are highly enriched in PM-T-ALL/LLy. This comprehensive genomic and transcriptomic profiling provides insights into the pathogenesis and development of PICALM::MLLT10 positive acute leukemia.

T/myeloid mixed phenotype acute leukaemia harbouring TLX3::BCL11B with TLX3 activation.

T/myeloid mixed phenotype acute leukaemia (MPAL) is a rare aggressive acute leukaemia with poorly understood pathogenesis. Herein, we report two cases of T/myeloid MPAL harbouring BCL11B-associated structural variants that activate TLX3 (TLX3::BCL11B-TLX3-activation) by genome sequencing and transcriptomic analyses. Both patients were young males with extramedullary involvement. Cooperative gene alterations characteristic of T/myeloid MPAL and T-lymphoblastic leukaemia (T-ALL) were detected. Both patients achieved initial remission following lineage-matched ALL-based therapy with one patient requiring a lineage-switched myeloid-based therapy. Our study is the first to demonstrate the clinicopathological and genomic features of TLX3::BCL11B-TLX3-activated T/myeloid MPAL and provide insights into leukaemogenesis.

Targeting TNF/IL-17/MAPK pathway in hE2A-PBX1 leukemia: effects of OUL35, KJ-Pyr-9, and CID44216842.

t(1;19)(q23;p13) is one of the most common translocation genes in childhood acute lymphoblastic leukemia (ALL) and is also present in acute myeloid leukemia (AML) and mixed-phenotype acute leukemia (MPAL). This translocation results in the formation of the oncogenic E2A-PBX1 fusion protein, which contains a trans-activating domain from E2A and a DNA-binding homologous domain from PBX1. Despite its clear oncogenic potential, the pathogenesis of E2A-PBX1 fusion protein is not fully understood (especially in leukemias other than ALL), and effective targeted clinical therapies have not been developed. To address this, we established a stable and heritable zebrafish line expressing human E2A-PBX1 (hE2APBX1) for high-throughput drug screening. Blood phenotype analysis showed that hE2APBX1 expression induced myeloid hyperplasia by increasing myeloid differentiation propensity of hematopoietic stem cells (HSPCs) and myeloid proliferation in larvae, and progressed to AML in adults. Mechanistic studies revealed that hE2A-PBX1 activated the TNF/IL-17/MAPK signaling pathway in blood cells and induced myeloid hyperplasia by upregulating the expression of the runx1. Interestingly, through high-throughput drug screening, three small molecules targeting the TNF/IL-17/MAPK signaling pathway were identified, including OUL35, KJ-Pyr-9, and CID44216842, which not only alleviated the hE2A-PBX1- induced myeloid hyperplasia in zebrafish but also inhibited the growth and oncogenicity of human pre-B ALL cells with E2A-PBX1. Overall, this study provides a novel hE2A-PBX1 transgenic zebrafish leukemia model and identifies potential targeted therapeutic drugs, which may offer new insights into the treatment of E2A-PBX1 leukemia.

Lineage Switching from Acute Myeloid Leukemia associated with Systemic Mastocytosis to B-Acute Lymphoblastic Leukemia: A Diagnostic Dilemma

Relapse acute leukemia is defined as the reappearance of more than 5% blast in the bone marrow. In most instances, blast expresses the specific lineage (myeloid or lymphoid), similar to a diagnosis. However, it rarely converts to a different lineage, either myeloid shifting to lymphoid or vice versa, during relapse. Thus, it is labelled as switching lineage acute leukemia, after excluding the criteria for mixed phenotypic acute leukemia, which can be a challenge to diagnose. We describe a 24-year-old gentleman with a known case of acute myeloid leukemia associated with systemic mastocytosis (SM); eight months later and after two complete cycles of chemotherapy, relapse occurred with a switched lineage to B-acute lymphoblastic leukemia. The laboratory investigation approaches and the challenges in the diagnosis are also discussed in this case report

Successful treatment of a B/T MPAL patient by chemo-free treatment with venetoclax, azacitidine, and blinatumomab

B/T mixed phenotype acute leukemia (MPAL), which represents only 2–3% of all MPAL cases, is classified as a high-risk leukemia subtype. Adults diagnosed with B/T MPAL have a notably low 3-year survival rate, estimated at 20–40%. The rarity and undercharacterization of B/T MPAL present substantial challenges in identifying an optimal treatment protocol. This report aims to shed light on this issue by presenting a case in which a patient with a complex karyotype was treated using a combination of venetoclax, azacitidine, and blinatumomab. This novel, chemo-free regimen resulted in the patient achieving both hematologic and molecular complete remission, with no severe organ or hematological toxicity observed. Notably, the patient continued to maintain molecular remission for 1 year following the transplantation. Based on these findings, the combination of venetoclax, azacitidine, and blinatumomab could be considered a potential therapeutic approach for B/T MPAL patients, meriting further investigation.

A case report of paediatric mixed phenotype acute leukaemia (MPAL) with T and megakaryocytic differentiation – a possible new classification

A case report of paediatric mixed phenotype acute leukaemia (MPAL) with T and megakaryocytic differentiation – a possible new classification

Genetic Characterization of Pediatric Mixed Phenotype Acute Leukemia (MPAL).

Mixed phenotype acute leukemia (MPAL) is a rare hematologic malignancy in which the leukemic cells cannot be assigned to any specific lineage. The lack of well-defined, pathogenetically relevant diagnostic criteria makes the clinical handling of MPAL patients challenging. We herein report the genetic findings in bone marrow cells from two pediatric MPAL patients. Bone marrow cells were examined using G-banding, array comparative genomic hybridization, RNA sequencing, reverse transcription polymerase chain reaction, Sanger sequencing, and fluorescence in situ hybridization. In the first patient, the genetic analyses revealed structural aberrations of chromosomal bands 8p11, 10p11, 11q21, and 17p11, the chimeras MLLT10::PICALM and PICALM::MLLT10, and imbalances (gains/losses) on chromosomes 2, 4, 8, 13, and 21. A submicroscopic deletion in 21q was also found including the RUNX1 locus. In the second patient, there were structural aberrations of chromosome bands 1p32, 8p11, 12p13, 20p13, and 20q11, the chimeras ETV6::LEXM and NCOA6::ETV6, and imbalances on chromosomes 2, 8, 11, 12, 16, 19, X, and Y. The leukemic cells from both MPAL patients carried chromosome aberrations resulting in fusion genes as well as genomic imbalances resulting in gain and losses of many gene loci. The detected fusion genes probably represent the main leukemogenic events, although the gains and losses are also likely to play a role in leukemogenesis.

Myeloid/natural killer (NK) cell precursor acute leukemia as a distinct leukemia type

Myeloid/natural killer (NK) cell precursor acute leukemia (MNKPL) has been described on the basis of its unique immunophenotype and clinical phenotype. However, there is no consensus on the characteristics for identifying this disease type because of its rarity and lack of defined distinctive molecular characteristics. In this study, multiomics analysis revealed that MNKPL is distinct from acute myeloid leukemia, T cell acute lymphoblastic leukemia, and mixed-phenotype acute leukemia (MPAL), and NOTCH1 and RUNX3 activation and BCL11B down-regulation are hallmarks of MNKPL. Although NK cells have been classically considered to be lymphoid lineage–derived, the results of our single-cell analysis using MNKPL cells suggest that NK cells and myeloid cells share common progenitor cells. Treatment outcomes for MNKPL are unsatisfactory, even when hematopoietic cell transplantation is performed. Multiomics analysis and in vitro drug sensitivity assays revealed increased sensitivity to l -asparaginase and reduced levels of asparagine synthetase (ASNS), supporting the clinically observed effectiveness of l -asparaginase.

Chronic Myeloid Leukemia Presenting with Mixed Phenotype B/Myeloid Blast Phase: A Diagnostic Challenge

Abstract Introduction/Objective Chronic myeloid leukemia (CML) is typically identified in chronic phase, however about 5% of cases present in a more advanced phase. In blast phase (BP), the blasts are most commonly of myeloid lineage, less commonly lymphoid, and rarely mixed lineage cases are encountered. A mixed phenotype at presentation raises the possibility of a de novo mixed phenotype acute leukemia (MPAL) with BCR::ABL1 fusion and requires careful correlation with ancillary studies to confirm the diagnosis. Methods/Case Report The patient is a 60-year-old man who presented with leukocytosis, anemia, normal platelet count, and blasts identified on peripheral blood smear examination. A bone marrow biopsy demonstrated hypercellular marrow and a dimorphic population of large and small blasts, comprising nearly 70% of cellularity. Flow cytometry demonstrated distinct myeloid and B-lymphoid blast populations, both of which expressed CD13 and TdT. Interphase fluorescence in situ hybridization (FISH) showed the BCR::ABL1 fusion in more than 90% of nuclei, including segmented nuclei, and conventional cytogenetic studies showed t(9;22)(q34.1;q11.2) in 19 of 20 metaphases. Molecular studies demonstrated the minor BCR::ABL1 p190 transcript, as well as ASXL1 and dual RUNX1 mutations. The discrepant number of BCR::ABL1 positive cells by FISH and conventional cytogenetic studies compared to the proportion of blasts, and its presence in segmented nuclei, enabled a diagnosis of CML with mixed phenotype blast phase despite absence of features such as basophilia or significant granulocytic left shift. Results (if a Case Study enter NA) NA Conclusion We share this case of CML in mixed phenotype blast phase to highlight the importance of careful evaluation in distinguishing this rare presentation from de novo MPAL, particularly with BCR::ABL1 fusion. Features such as a disproportionate number of nuclei or metaphases carrying the t(9;22) translocation on FISH or conventional cytogenetic studies and the presence of BCR::ABL1 in mature granulocytes can be helpful when other morphologic features of CML are absent.

Real-World Performance of 2022 Revised World Health Organization (WHO) Criteria for Mixed Phenotype Acute Leukemia (MPAL) B/Myeloid Defined Only By Isolated MPO Expression (isoMPO)

Mixed phenotype acute leukemia (MPAL) is biologically and immunophenotypically heterogeneous, and optimal diagnostic criteria remain unclear. The two current pathologic classifications [2022 World Health Organization (WHO) 5 th Ed. and International Consensus Classification (ICC)] group MPAL by cell lineages and, when present, cytogenetic drivers. In both, myeloperoxidase (MPO) is a key determinant of myeloid lineage. However, the diagnosis of MPAL has been inconsistently applied to cases of acute leukemia expressing MPO alone to define myeloid lineage. To address this, the 2022 WHO revised prior cell lineage definitions to emphasize antigen intensity (≥50%) relative to a positive control population, attempting to standardize lineage assignment across flow cytometer settings and pathologist interpreters. Distinction between pediatric B-lymphoblastic leukemia (B-ALL) and MPAL B/Myeloid with isolated MPO is crucial for therapeutic decision-making. For example, on Children's Oncology Group (COG) regimens, patients with MPAL B/Myeloid receive an anthracycline during induction and intensified chemotherapy post-induction, whereas standard-risk B-ALL patients do not. We hypothesized that the 2022 WHO criteria applied to MPO expression in children with acute leukemia would identify leukemic subtypes (B-ALL vs. MPAL B/Myeloid with isolated MPO) with differences in clinical characteristics and/or outcome. We applied 2022 WHO criteria to flow cytometry data from a multicenter pediatric cohort of leukemias originally classified as MPAL B/Myeloid or B-ALL (Oberley AJCP 2017; Raikar Blood 2018; Oberley Leukemia 2020). Maximal antigen intensity on the leukemic population was measured in 0.5 log increments and normalized to maximal intensity of a control (neutrophil) population. MPO expression was scored as none, low (&amp;lt;50% intensity of mature neutrophils, the typical level in B-ALL with isolated MPO expression) or high (≥50% intensity of neutrophils, meeting 2022 WHO criteria for myeloid lineage assignment). Since the current study focused on MPAL B/Myeloid cases with isolated MPO expression, cases of MPAL T/Myeloid and of MPAL B/Myeloid with myeloid lineage assignment based on monocytic markers were excluded. Clinicopathologic data were compared with MPO expression scores by univariate and multivariate analyses. Primary endpoint was categorical difference in presenting features. Secondary endpoints were end of induction minimal residual disease (EOI MRD) ≥0.01%, event-free survival (EFS) and overall survival (OS). All analyses were two-sided; p&amp;lt;0.05 was the threshold for statistical significance. Among all patients at diagnosis (n=176), median age was 6.14 years (range 0.3-21), 68% were &amp;lt;10 years old, 59% were male, 63% were Hispanic, 76% had initial white blood cell (WBC) count &amp;lt;50K/µL, and 81% were CNS1. MPO was absent, low (&amp;lt;50%) or high (≥50%) in 48%, 29% and 23% of the cohort, respectively. Prevalence of favorable cytogenetics ( ETV6:: RUNX1 or double trisomy) was inversely proportional to MPO intensity [no MPO (50%), low MPO (27%), high MPO (17%); p&amp;lt;0.001). MPO expression (low or high) was more prevalent in patients &amp;gt;10 years of age (p&amp;lt;0.001). There was no difference in MPO by presenting WBC count (p=0.40). Among patients treated with ALL induction with known EOI MRD results (n=166), MPO intensity was not significantly associated with EOI MRD positivity, EFS or OS (multivariate p&amp;gt;0.40 for all three endpoints after accounting for presenting features and initial therapy). Our study demonstrates that 2022 WHO criteria for interpreting MPO intensity can be consistently applied to flow cytometry data from a variety of laboratories. Based on our analysis, only 44% of previous MPAL B/Myeloid cases with isolated MPO were now classified as MPAL. However, the clinical significance of this distinction remains unclear. After accounting for differences in presenting features, MPO expression was not additionally predictive of MRD positivity, EFS or OS. Further investigation is required to support the proposed threshold of 50% MPO intensity of a neutrophil control population as clinically relevant to assign myeloid lineage. Prospective standardized application of 2022 WHO cell lineage assignment criteria across centers will facilitate detailed understanding of the biological underpinnings and optimal therapeutic strategies for this heterogeneous disease.

Precision Lineage Deconvolution in Mixed Phenotype Acute Leukemia Using Cite-Seq Derived Hematopoietic Stages Identifies Lineage Dynamics Associated with Treatment Response

Introduction: Mixed phenotype Acute Leukemia (MPAL) is challenging due to ambiguous biology and lack of formal therapeutic guidelines. Although MPAL itself is rare, mixed phenotype lineage expression in secondary AML (sAML-MP) occurs with greater frequency and is associated with poor clinical outcomes. Multimodal single cell sequencing with cellular indexing of transcriptomes and epitopes (CITE-seq) offers more precise lineage characterization and assessment of lineage dynamics. Here we demonstrate a novel, unbiased approach to quantitative multistage hematopoietic lineage assessment in MPAL and sAML-MP using CITE-seq data. In addition, we demonstrate that this approach can be translated from single cell to bulk analysis. Methods: Sample preparation: Four MPAL and 6 sAML-MP samples displaying T-cell and myeloid subsets were identified and flow sorted into T and myeloid subgroups. RNA-seq was performed on each subset and analyzed using DESeq2. ComBat was used for batch correction when integrating with disparate data sources. Single cell lineage deconvolution: CITE-seq samples were processed using Seurat. A single cell deconvolution library was constructed using previously published hematopoietic stage clustering and labeling, and applied to CITE-seq data from five MPAL samples in the same dataset. Deconvolution algorithm parameters were optimized through a bootstrapping approach on in-silico sample mixtures. Bulk and translated lineage deconvolution: Bulk lineage deconvolution was performed using a published library of 13 stages of hematopoiesis derived from healthy donors.This was applied to bulk RNA-seq data from a cohort of published MPAL samples as well as the 6 sAML-MP and 4 MPAL samples described above. A pseudobulk was created for each CITE-seq lineage stage. Bulk lineage deconvolution was then re-run on the MPAL and sAML-MP samples using this library. Clinical Outcomes: A Wilcoxon signed-rank test was used to analyze differences among deconvolution-derived patient clusters, including response to induction therapy, specific gene mutations, and likelihood of transplant. Results: Lineage evolution with treatment: Deconvolution was applied on 17,848 MPAL cells and 35,038 control PBMC/BMMC cells. Antibody Derived Tag (ADT) features were consistent with lineage deconvolution stage assignments. CD38 was negatively correlated with HSC (-0.34, p &amp;lt; 0.001) and LMPP (-0.18, p &amp;lt; 0.001) while CD34 was positively correlated with both HSC (0.11, p &amp;lt; 0.001) and LMPP (0.27, p &amp;lt; 0.001) stages. MPAL 5 in particular was assessed at diagnosis and at two later time points. Unsupervised hierarchical clustering of all timepoints resulted in 10 clusters ( Figure A). The diagnostic time point was largely contained in clusters 4 (LMPP character, N = 1545 cells, 37.13%) and 9 (HSC character, N = 756 cells, 18.16%). The majority of both relapse timepoints were within cluster 4, with 76.77% (N=357 cells) of T1 and 73.92% (N=1100 cells) of T2 located in it. Bulk Unsupervised Analysis: Unsupervised analysis of sorted MPAL (N = 8), sorted AML-MP (N = 12), and unsorted MPAL (N = 24) bulk samples resulted in six distinct clusters, each with a distinct lineage signature ( Figure B). Some were enriched for more differentiated stages such as monocyte or GMP (clusters 6, 5), while others were enriched earlier stages such as LMPP (cluster 2). Secondary AML-MP samples were notably enriched in cluster 4. We found significant associations between lineage clusters and mutations in RUNX1 (p &amp;lt; 0.01), FLT3 (p = 0.004). Clinical Outcomes: Bulk RNA deconvolution cluster was significantly associated with complete remission (p = 0.0009). On average, individuals with an incomplete/no response to induction chemotherapy had a higher NK signature (p = 0.001), while individuals assigned to transplant had decreased LMPP character (p = 0.0495). Among the single-cell stages, decreased CLP 2 levels were associated with a poor response (p = 0.0224) while CMP / LMPP (p = 0.040) and CD4 N2 (p = 0.022) levels were decreased in patients who were able to undergo HSCT. Conclusions: Precise identification of lineage signatures in mixed phenotype leukemias shows promise in identifying clinically meaningful biological subsets of these diseases. Prospective analysis of lineage-derived biomarkers should be performed to undertake identification of formal risk stratification and treatment schemas.

Treatment Outcomes for Adults with Mixed Phenotypic Acute Leukemia: A Large Single-Center Retrospective Study

Introduction: Mixed phenotype acute leukemia (MPAL) is a rare subtype of acute leukemia characterized by differentiation along more than one lineage. In the absence of eosinophilia and/or stereotypical tyrosine kinase gene fusions, there is no established optimal therapy for adults with MPAL. Typical treatments include acute myeloid leukemia (AML)-type, acute lymphoblastic leukemia (ALL)-type or hybrid-type therapy approaches. We conducted a retrospective comparison of different induction chemotherapy strategies at our institution to help clarify the optimal initial chemotherapy for patients with MPAL. Methods: Patients evaluated at the University of Washington/Fred Hutchinson Cancer Center diagnosed with MPAL from 1/2005 to 9/2022 were identified through our IRB-approved institutional repository. MPAL diagnosis was confirmed by independent pathology review (AE and XC). Lineage assignment used the 2022 World Health Organization criteria based on initial diagnostic immunophenotype or genetic features. Treatment was classified as either AML-type (anthracycline + cytarabine with or without purine analogs, e.g. 7+3, CLAG-M or FLAG-IDA), ALL-type (anthracycline + cyclophosphamide + steroid + vincristine, e.g. hyperCVAD, or CALGB 10403) or hybrid-type regimens (most often CLAG-M + vincristine + dexamethasone). Log-rank survival and Cox proportional hazards were used to compare survival. Results: We identified a total of 49 patients with MPAL who received induction chemotherapy at our institution. These included 14 patients (28.6%) treated with ALL-type therapies, 9 patients (18.4%) treated with AML-type therapies and 26 patients (53.1%) treated with hybrid-type therapies. The proportions of B/myeloid, T/myeloid and rare B/T phenotypes were similar among the 3 treatment groups, though patients treated with ALL-type regimens were significantly younger (Table 1). The overall proportion of patients who achieved complete remission (CR) was high (67%) with a non-statistically significant trend for non-AML-type treatments (ALL-type = 10/14, 71.4%; AML-type = 5/9, 55.6%; hybrid-type = 21/26, 80.8%; p = 0.22). Overall, patients had similar survival regardless of induction therapy type with 43.8% of patients treated with ALL-type therapy, 50.0% of patients treated with AML-type therapy, and 39.0% of patients treated with hybrid-type therapy alive 48 months post-induction treatment (p=0.68) (Figure 1). Compared to patients treated with ALL-type therapy, the Cox proportional hazard for death among patients treated with AML-type therapy was 1.5 (95% CI 0.4 - 5.3, p = 0.5) and for those who received hybrid-type therapy was 1.5 (95% CI 0.6 - 39, p = 0.4). Among 36 patients who achieved complete response after induction chemotherapy, there was a trend toward longer survival among patients who were found to be measurable residual disease (MRD) negative by flow cytometry compared to MRD+ with 63.1% in the MRD- group alive at 48 months compared to 33.3% in the MRD+ group (p = 0.07). Compared to the MRD- group, the MRD+ Cox proportion hazard ratio for survival was 2.6 (95% CI 0.9 - 7.9, p = 0.07). The proportion of patients subsequently transplanted was similar in each treatment group (28 patients total; Table 1). Patients who received HSCT lived longer. In a landmark analysis of patients who survived a minimum of 100 days following initiation of induction therapy, 57.8% of HSCT recipients were still alive at 48 months compared to 20.2% of patients who did not undergo HSCT (p = 0.001). Compared to patients who did not receive a HSCT, the Cox proportion hazard ratio for survival for those receiving HSCT was 0.25 (95% CI 0.1 - 0.61, p &amp;lt; 0.003). Conclusion: Although our data showed a non-statistically significant trend toward a higher CR rate with ALL-type regimens for patients with MPAL regardless of immunophenotype, it failed to show a survival benefit at 48 months based on choice of ALL-, AML- or hybrid-type induction regimens. Despite being one of the largest cohorts reported of patients with MPAL, this study was limited by sample size and its retrospective nature. Clinical trials are needed to better determine optimal induction regimens. A survival benefit was seen for patients receiving HSCT consolidation; therefore, HSCT should be encouraged for all eligible patients with MPAL.

Mixed Phenotypic Acute Leukemia -Clinical Presentation, Prognosis and Outcomes of Patients Treated with Acute Lymphoblastic Leukemia like Protocols a Multi-Institutional Study from India

Mixed phenotypic acute leukemia (MPAL) is a rare and difficult to treat type of acute leukemia. Broad consensus favour use of acute lymphoblastic leukemia (ALL) like treatment protocols in the management of MPAL. Data regarding this rare diagnosis is limited mainly to retrospective studies and limited data available from low middle income countries (LMIC). We report epidemiology and real-world outcome data of MPAL patients treated with ALL like protocols among the multi-institutional data base set up by hematology cancer consortium (HCC) in India. In this retrospective multicentric study of a prospectively maintained data set, we collected data from eleven member centers of HCC using an electronic database. Patients who fulfilled the WHO 2016 classification criteria for MPAL were included. Patients diagnosed between January 2018 and March 2022 were included in the study. The data included demographic details, performance status (ECOG), comorbidities, CNS involvement, testicular involvement, presence of mediastinal mass, phenotype of MPAL (B+M, T+M, T+B), karyotype, FISH, PCR and NGS abnormalities. If treatment was not received by patients the reasons were also captured. We also evaluated the treatment protocol used and use of tyrosine kinase inhibitors. The primary objective was to evaluate event-free survival (EFS) and overall survival (OS) at 1year. The impact of the treatment protocol used, age, baseline WBC count, LDH, performance status, CNS involvement on EFS and OS were studied. A total of 114 patients were diagnosed to have MPAL during the study period. The baseline epidemiological features are tabulated in table-1. Sixty-six (58%) patients were treated with ALL like treatment protocols and 48(42%) patients did not receive any treatment due to various reasons at HCC centers. 49(74%) patients received paediatric inspired protocols and 17(26%) patients received adult-type of protocols. Forty three (65%) patients achieved a complete remission after induction. Ten(15%) patients died during induction. Six(9%) patients died after achieving CR of which 3 died due to infection,3 died due to progressive disease and rest were lost to follow up. Minimal residual disease (MRD) done using either flowcytometry or PCR was available in 37 patients. MRD negative status was achieved in 17(46%) patients post phase II of induction. After a median follow up of 12 months, EFS was 61% and OS was 67%(figure1). Estimated EFS and OS at 24 months were 42% and 55% respectively. There were no significant differences in EFS and OS between different MPAL phenotypes. Allogeneic stem cell transplant (ASCT) was performed only in 3 patients. Negative MRD status at the end of second phase of induction was significantly associated with improved EFS with a Hazard ratio of 0.349(CI-0.117-1.036). Age, cytogenetic profile, LDH, baseline WBC count and CNS involvement were analyzed by univariate analysis for effect on treatment outcomes. There was no significant effect of these variables on both EFS and OS. Our study is one of the largest studies conducted on MPAL patients in a resource limited setting. If patients received treatment, the outcomes were encouraging despite poor access to allogeneic stem cell transplant in first complete remission, when compared with similar real world multi-institutional analysis. Induction mortality of 10% was comparable to other similar studies. However, the worse 2-year EFS and OS may be explained by poor rates of allogeneic stem cell transplant (Allo-HSCT). The abandonment of treatment and limited use of Allo-HSCT in first complete remission were major limitations in improving overall outcomes in our patients.