Mixed Phenotype Acute Leukemia Patients Research Articles

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.

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.

A Phase I/II Study to Investigate the Addition of Venetoclax to Dasatinib and Steroids in Patients with Newly Diagnosed or Relapsed Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia (Ph+ ALL): The Venda Trial, in Progress

Background: Acute Lymphoblastic Leukemia (ALL) is a hematologic malignancy of immature lymphocytes of either B cell or T cell origin. One particularly high -risk subset is Ph+ ALL, characterized by a translocation of chromosome 9 and 22, which generates the constitutively active BCR-ABL1 fusion tyrosine kinase capable of driving proliferation and survival of leukemic cells. Currently treatment options include multi-agent chemotherapy along with an ABL-specific tyrosine kinase inhibitor (TKI), though these regimens can lead to significant toxicity, particularly in older adults. Venetoclax is a potent and selective small molecule inhibitor of the B-cell lymphoma 2 (BCL-2) anti-apoptotic protein. Strong mechanistic and preclinical data have demonstrated strong synergistic activity between dasatinib and venetoclax I xenograft models and ex vivo patient samples, and provide an impetus to further explore the combination of BCR-ABL and BCL-2 inhibition in vivo. Based on these data, we designed a clinical trial to investigate the safety and preliminary efficacy of venetoclax in combination with the second generation TKI dasatinib in Ph+ ALL. Study Design and Methods This is an open label, Phase I/II investigator initiated trial evaluating the addition of venetoclax to dasatinib and steroids (with Rituximab in CD20+ patients). Phase I is the safety and dose-finding portion of the trial combining the BCL2 inhibitor venetoclax with dasatinib and steroids during the first 28 days of induction followed by dasatinib and venetoclax for the final 48 days of induction in newly diagnosed and relapsed Ph+ ALL and mixed phenotype acute leukemia (MPAL) patients, including patients with molecular only relapse. Primary inclusion criteria include a histologically confirmed diagnosis of Ph+ ALL or MPAL. Newly diagnosed Ph+ ALL or MPAL patients may not have received leukemia-directed therapy other than for steroids or hydrea, and relapsed patients must have not had prior dasatinib exposure. Key exclusion criteria include subjects with CML in myeloid blast crisis or Ph+ AML, ECOG < 2 and patients with a history of pleural effusions. n order to determine the maximum tolerated dose (MTD) and/or recommended phase 2 dose (RP2D), a Bayesian toxicity probability interval design applied to a pre-specified targeted dose limiting toxicity rate of 30%. Three dose cohorts investigating increasing doses of venetoclax will be enrolled. Once the RP2D dose has been identified, a Phase II expansion will examine preliminary efficacy of the combination. The primary endpoint is the rate of measureable residual disease (MRD) negative remission rate as assessed by both BCR-ABL RT-PCR and next generation sequencing (NGS) after induction. Secondary endpoints include relapse-free survival (RFS) and overall survival (OS). Correlative studies include BH3 profiling (measure of apoptosis) and ex vivo screening to dasatinib and venetoclax alone and in combination. Consolidation phase with blinatumomab plus dasatinib and venetoclax is planned for the cohort expansion group. Enrollment began in September 2022 and is expected to be complete by September 2025. This trial is registered at clinicaltrials.gov (NCT04872790).

Identification and Functional Validation of Neoantigen-Specific T Cells in Pediatric Patients with Fusion-Derived Acute Leukemias

Leukemia emerges from the accumulation of genetic mutations in hematopoietic cells, resulting in formation of altered proteins. Some of these mutant proteins (neoantigens) are consequently processed and presented as neoepitopes through major histocompatibility complex (MHC) molecules expressed on the surface of leukemia cells, permitting tumor-specific immune targeting. While adoptive T cell therapies against high mutation burden tumors have shown promising results, less is known about leveraging tumor-specific T cells as an effective approach in pediatric patients, including high-risk leukemia. In this study, peripheral blood or bone marrow samples were obtained from 34 leukemia patients aged 0 -14 years old. The patient tumors had founder fusion genes, including 15 patients with acute lymphoblastic leukemia (ALL), 15 patients with acute myeloid leukemia (AML), and 4 patients with mixed phenotype acute leukemia (MPAL). Samples were collected at the time of diagnosis or relapse. While leukemia blasts were the most abundant cellular component in these samples, the frequency of lymphocytes ranged between 1%- 25%, with a median of 3.5%. KMT2A rearrangements were expressed with either AFF1, MLLT1, MLLT3, MLLT4, MLLT6, or MLLT10 fusion partner in 73.5% of the patients' samples in this study. This included (14/15) ALL, (10/15) AML, and (1/4) MPAL patients . T cells were enriched and expanded for a total of 14 days using a rapid expansion protocol culture system. This approach was successful on all tested samples irrespective of their mutation backgrounds or initial lymphocyte count, with a range of 133-4957fold increases in total cell number and average frequencies of 59.7% and 26.1% for CD4+ and CD8+ T cell, respectively. We further screened the T cell response by examining the expression of 4-1BB, OX40, and IFN-g upon coculture with their autologous leukemia cells, and additionally validated the T cell killing activity against the blasts by a cell killing assay using standard protocol. We found that the expanded T cells exhibited a robust immune activation when cocultured with the autologous leukemia cells from ALL patients with KMT2A rearrangements, indicating an endogenous cell response in all patients in the ALL cohort (100%). However, we observed a considerably reduced reactivity in the samples from the AML and MPAL patients. Interestingly, we observed that such diminished reactivity was associated with a marked downregulation in the expression of MHC class I and II molecules, and that pretreating the leukemia blasts with IFN-γ prior to coculturing with the expanded T cells was sufficient to induce the expression of MHC molecules and subsequent activation of the expanded T cells. Single-cell TCR sequencing of the sorted, tumor-reactive T cells identified the major clonotypes among these reactive T cell clones and the top 4 - 6 clonotypes were selected from 5 patients to evaluate reactivity against the leukemia blasts and examine their specificity against the corresponding driving fusions. We further generated TCR-expressing clones and subsequently cocultured each clone with leukemia blasts to validate their antigen recognition and reactivity by cytotoxicity assay. Interestingly, and in line with the findings from investigations on solid tumors, the leukemia- reactive T cells were consistently characterized by higher expression of both PD1 and CD39 (PD1 hi CD39 +), indicating the capability of utilizing both markers for the enrichment of T cell prior to expansion and subsequent implementation. Despite the high-risk prognosis and low mutation burden in our patient cohort, our findings demonstrate that patient T cells contain endogenous neoantigen-specific clones which can be therapeutically leveraged for targeting leukemia blasts. These results support the utilization of adoptive T cell therapy in the context of hematological malignancies especially given the less restrictive spatial segregation between tumor-specific T cells and leukemia cells compared to solid tumors, where T cell infiltration is a prerequisite for antitumor immunity. Overall, these findings suggest the possibility of leveraging leukemia-associated lymphocytes, fusion oncogene vaccines, and TCR-based therapy as novel immunotherapeutic approaches for mitigating pediatric leukemia, particularly in high-risk patients.

Clinical and Molecular Characteristics of Patients with Mixed Phenotype Acute Leukemia.

Mixed phenotype acute leukemia (MPAL) is a rare heterogeneous disease with a poor prognosis. This study analyzed the clinical, immunophenotypic, molecular, and cytogenetic characteristics of a group of patients with MPAL. This prospective study included 75 patients diagnosed with MPAL according to the World Health Organization (WHO)-2016 diagnostic criteria, using cytochemistry, conventional cytogenetics, and molecular studies. Screening of BCR::ABL1 fusion gene was performed by Fluorescent in-situ hybridization (FISH) and polymerase chain reaction (PCR). Children represented 49.3% of MPAL patients. The main phenotype was B-lymphoid/myeloid (80%). Molecular alterations were detected in 17 patients (22.7%). The BCR::ABL1 fusion gene was detected in 10 patients (13.3%).. Myeloid protocols were used to treat 58 patients (77.3%), and lymphatic protocols in 17. By the end of the follow-up, 57 patients (76%) achieved complete remission (CR). There was no association between BCR::ABL1 and response to treatment. The cumulative overall survival (OS) at 12 months was 47.8%. The bone marrow transplantation (BMT) was associated with better OS (p = 0.027). The disease-free survival (DFS) was not affected by all tested prognostic factors. MPAL is a complex entity with heterogeneous features. BCR::ABL1 is a common abnormality. BMT is associated with better OS.

Integrative Genomic and Transcriptomic Profiling Reveals Distinct Molecular Subsets in Adult Mixed Phenotype Acute Leukemia

Background: Mixed phenotype acute leukemia (MPAL) is a subtype of leukemia with co-expression of lymphoid and myeloid markers. Genetic alterations are critical for the understanding of pathogenesis, diagnosis, risk stratification and treatment of hematological malignancies. Hitherto, in addition to t(9;22)(q34;q22)/BCR::ABL1 (Ph+) and KMT2A-rearrangements (KMT2A-r), BCL11B and ZNF384 rearrangements have been incorporated into the diagnosis and classification of MPAL. The genetic features in the remaining MPAL merit further investigation. This study aims to apply an integrated genomic and transcriptomic approach to explore the molecular characteristics of adult patients with MPAL. Methods: 176 consecutive cases diagnosed with MPAL at the First Affiliated Hospital of Soochow University between January 2006 and November 2019 were included. Patients’ median age was 48 years old (range:16-87). The diagnoses were made according to the WHO 2016 criteria, 86 (48.9%) cases fulfilled T/My MPAL, NOS, 42 (23.9%) cases fulfilled Ph+ MPAL, 36 (20.5%) cases fulfilled B/My MPAL, NOS, 4 (2.3%) cases fulfilled MPAL with KMT2A-r, and 8 cases (4.5%) fulfilled MPAL, NOS, rare types. We performed targeted sequencing in 104 patients, RNA sequencing in 89 patients and whole genome sequencing in 4 MPAL patients. Gene expression profile data were compared to data from 133 AML, 53 B-ALL and 41 T-ALL cases. 120 of the patients who were treated in our hospital were applied for analysis of treatment response and survival. Results: In total, 74 genes were found mutated with variant allele frequency more than 2% in 94 of 104 (90.4%) MPAL patients, with a total of 239 SNVs and 135 indels. Distinct mutational patterns were observed among different MPAL subtypes. Mutations of CEBPA (41.5% vs. 2.0%, P< 0.0001), DNMT3A (34.0% vs. 0%, P< 0.0001) and NOTCH1 (26.4% vs. 9.8%, P= 0.028) were mostly associated with T/My MPAL compared with in other MPALs. RUNX1 mutation was more common in Ph+ MPAL (51.9% vs. 7.8%, P< 0.0001). NOTCH1 mutation associated with B/T MPAL (60.0% vs. 16.2%, P= 0.041). In comparison to other subtypes of leukemia, 33 of the 89 (37.1%) MPAL cases clustered with B-ALL, 30 (33.7%) clustered with T-ALL, and 26 (29.2%) clustered with AML. By integrating targeted sequencing and RNA sequencing data in 89 of the MPAL patients, we defined 8 molecular subgroups (G1-G8) with distinct mutational and gene expression characteristics (Figure 1A). 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 features. With a median follow-up of 49.3 months, the estimated 5-year overall survival (OS) and event-free survival (EFS) in the whole cohort were 37.1 ± 5.1% and 27.9 ± 4.5%, respectively. There was no statistically significant difference in 5-year OS and EFS among patients with T/My, Ph+ and B/My MPAL. Of note, patients receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT) had longer 5-year OS (69.1 ± 7.4% vs. 13.8 ± 5.6%, P< 0.0001, Figure 1B) and EFS (56.2 ± 7.5% vs. 7.4 ± 3.9%, P< 0.0001) than those receiving chemotherapy alone. Moreover, patients receiving allo-HSCT at first complete remission (CR1) had longer 5-year OS than those transplanted at non-CR1 (68.4 ± 11.3% vs. 52.7 ± 14.1%, P= 0.042). There was no significantly difference in the 5-year OS and EFS among the 8 subgroups as a whole (Figure 1C), which might due to the limited number of patients in each subgroup. Conclusions: In this study, we identify 8 molecular subgroups with distinct mutational and gene expression characteristics in adult MPAL. These results indicate that integrative genomic and transcriptomic profiling will provide informative implications on a more precise diagnosis, risk stratification and treatment options for MPAL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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.

Immunophenotypic Features and Clinical Prognosis of Patients with Mixed Phenotype Acute Leukemia

To retrospectively analyze the laborotary test results and clinical data of 31 patients with mixed phenotype acute leukemia (MPAL) in order to summarize and discuss the biological characteristics, curative effect, and prognosis of each subtype of MPAL based on immunophenotype results. MPAL patients diagnosed and treated in our hospital from July 2013 to January 2019 were selected to analyze the data of cell morphology, immunophenotyping, cytogenetics, molecular biology (MICM), and routine blood at initial diagnosis. Follow-up was carried out until the last discharge time. Among 31 patients, there were 19 males and 12 females, with a median age of 41(12-76) years old. According to the results of immunophenotyping and EGIL score, there were 16 cases of myeloid-T lymphoid mixed phenotype (myeloid-T group), 9 cases of myeloid-B lymphoid mixed phenotype (myeloid-B group), 5 cases of T-B lymphoid mixed phenotype (T-B group), and 1 case of myeloid-T-B lymphoid mixed phenotype. Compared between different subtypes, the antigen expression characteristics were the highest positive rate and expression rate of HLA-DR in myeloid-B group, and the positive rate of CD2 in T-B group was significantly higher than that in the myeloid-T group. Meanwhile, the expression rates of CD7 and cCD3 (cytoplasmic CD3) in T-B group were higher than those in myeloid-T group, and cCD79a was positive in all cases of myeloid-B group and T-B group. The median WBC of T-B group was 81.92×109/L, which was significantly higher than that of the other two groups (P<0.05). The quantitative results of WT1 were higher than 10-4 in 92.6% of the patients, and the WT1 expression level in myeloid-B group was significantly lower than the other two groups (P<0.01). Among the 9 patients with myeloid-B mixed phenotype, 5 cases showed BCR-ABL positive. Among 28 patients followed up, 21 cases achieved complete remission (CR), the median time to first obtain CR was 32.5(9-75) days, and the median follow-up time was 16 months (range from 21 days to 6 years). The CR rate and median overall survival (OS) time in myeloid-B group were 88.9% and 40 months, which were higher than the other two groups. The CR rate and 3-year OS rate in T-B group were relatively lower (50.0%, 0). WT1 gene is highly expressed in patients with MPAL, and each subgroup of MPAL based on immuophenotype has its unique antigen expression characteristics. Compared with myeloid-T group and T-B group, myeloid-B group can acquire higher remission rate and have better prognosis.

Zinc finger protein 384 (ZNF384) impact on childhood mixed phenotype acute leukemia and B-cell precursor acute lymphoblastic leukemia

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a heterogeneous malignancy and consists of several genetic abnormalities. Some of these abnormalities are used in clinics for risk calculation and treatment decisions. Patients with ZNF384 rearrangements had a distinct expression profile regardless of their diagnosis, BCP-ALL or mixed phenotype acute leukemia (MPAL) and defined as a new subtype of ALL. In this study, we screened 42 MPAL and 91 BCP-ALL patients for the most common ZNF384 fusions; ZNF384::TCF3, ZNF384::EP300 and ZNF384::TAF15 by using PCR. We identified ZNF384 fusions in 9.5% of MPAL and 7.6% of BCP-ALL. A novel breakpoint was identified in ZNF384::TCF3 fusion in one BCP-ALL patient. T-myeloid MPAL patients showed significantly lower ZNF384 expression compared to lymphoid groups. Patients with ZNF384r had intermediate survival rates based on other subtypes. Prognostic and patient-specific treatment evaluation of ZNF384 fusions in both ALL and MPAL might help to improve risk characterization of patients.

Treatment Resistance in ETP-ALL Is Associated with Progenitor-like Arrest State

Early T-Cell Precursor Acute Lymphoblastic Leukemia (ETP-ALL) is a treatment resistant type of pediatric leukemia whose biology is poorly understood, and new therapies are badly needed. While there are ongoing efforts to profile ETP-ALL using bulk and targeted genomics methods, these technologies fail to capture sub-clonal patterns in ETP-ALL, which may be pertinent to understanding its biology.To synergize with current efforts to use bulk-genomics to characterize ETP-ALL, we performed single cell transcriptomics (scRNA-seq) and single cell chromatin accessibility (scATAC-seq) profiling on 30 ETP-ALL patients: 10 of which had excellent response to induction therapy (Day 29 minimal residual disease (MRD) <0.1%), 10 of which had poor response to induction therapy (Day 29 MRD >20%), and 10 of which responded to initial therapy but relapsed later. As additional comparator groups, we also profiled 10 typical T-ALL patients, and 10 Mixed Phenotype Acute Leukemia (MPAL) patients.We mapped leukemic cells back to a healthy transcriptomic and epigenetic references, which we assembled using scRNA-seq and scATAC-seq of healthy pediatric bone marrow and thymus. We found that leukemic cells from different subtypes of ALL projected to different stages in healthy hematopoietic development. Non-ETP T-ALL patients had the highest proportions of cells projecting to more mature T-cell states, such as ab T cells, and MPAL patients had the highest proportions of cells projecting to myeloid progenitor cells (GMPs). However, the dominant projected population of ETP-ALL and MPAL were both lymphoid (CLP/LMPP) progenitors, supporting a high degree of overlap between these two treatment resistant cancers. Despite clear overall patterns, we found that there was both a large amount of inter-tumoral and intra-tumoral heterogeneity, with leukemic blasts from different patients and different populations within any one patient projecting to different cell states.We then asked if any cell types were enriched in ETP-ALL patients that responded poorly to induction therapy (Day 29 MRD > 20%). We found that non-responders have a higher proportion of LMPP/HSPC (multi-potent) progenitor-like cells and that progenitor-like populations are transcriptomically and epigenetically different between non-responders and responders, with 100+ differentially expressed genes (DEGs) and 50+ differentially accessible chromatin regions. DEGs and enriched chromatin regions are enriched for transcription factor motifs known to regulate hematopoietic differentiation, such as BCL11A/B, MEF2A/B/C (non-responders enriched), TCF7, IRF9 (responders enriched).We established patient derived xenograft (PDX) models for the patients in our dataset (28 of 50 have successfully engrafted thus far) by injection of primary blasts into immunodeficient (NSG) mice. We profiled 12 PDX models using scRNA-seq and found that blasts have a subtle, patient-specific transcriptomic shift upon engraftment, with 25-150 differentially expressed genes between patient and PDX. Interestingly, we found that cells in S/G2M cell cycle phase from PDX and patient are similar, while G1 cells have the largest transcriptomic shift, suggesting that a pool of cycling cells engrafts before settling in a subtly shifted transcriptomic state. Despite these observations, we found that the overall transcriptomic hierarchy is maintained in PDX, with engrafted cells from 5/6 PDX projecting to near-identical stages of arrest along our healthy hematopoietic trajectory, with preserved expression of key differentially expressed genes and transcription factors between non-responders and responders.Our single cell multi-omics analysis reveals a large degree of intra and inter-tumoral transcriptomic and epigenetic heterogeneity in T-ALL, in which non-responding ETP-ALL patients have a higher proportion of blasts arrested in a HSPC/LMPP-like state. On-going mechanistic analyses are being performed using PDX models to assess the role of the progenitor-like cell populations in resistant disease and these analyses will be presented at ASH.*CC and JX, as well as, DTT and KT contributed equally to this work [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

First-in-Human Clinical Study of a Novel CD7-Targeted Chimeric Antigen Receptor (CAR)-T Cell Therapy for Refractory/Relapsed Mixed Phenotype Acute Leukemia (MPAL)

IntroductionMixed phenotype acute leukemia (MPAL) is a rare subtype of acute leukemia with features of both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). The blast cells of MPAL express multilineage immunophenotypic markers and may have a shared B/T/myeloid phenotype. CD7 is expressed by the leukemic blasts and malignant progenitor cells of approximately 30% of AML patients. MPAL is usually associated with a relatively poor prognosis which is even worse compared to either AML or ALL, and even among patients that undergo allogeneic hematopoietic stem cell transplantation (allo-HSCT), underscoring the need for new therapies for these patients. CAR-T cell therapy has shown to be efficacious with a tolerable safety profile in a subset of hematological malignancies. Here, we explored the safety and efficacy of CAR-T therapy in treating refractory/relapsed (r/r) MPAL patients with CD7-positive in a phase I dose-escalation clinical study (https://clinicaltrials.gov NCT04938115).MethodsPeripheral blood (PB) mononuclear cells were obtained from patient themselves or patient transplant donors for those who relapsed post-transplant by leukapheresis. T-cells were purified using CD3+ magnetic beads or CD4+ and CD8+ magnetic beads. The second-generation CD7 CAR with a 4-1BB costimulatory domain was manufactured per the manufacturer's protocol. This phase I dose escalation study was initiated to explore the safety and efficacy of CD7 CAR-T in treating patients with MPAL. Prior to the CAR-T cells infusion, patients received systemic bridging chemotherapy due to rapid disease progression and then all patients received intravenous fludarabine (30mg/m 2/d) and cyclophosphamide (300mg/m 2/d) (FC) lymphodepleting chemotherapy for 3 consecutive days (Day -5 to Day -3). The median time from leukapheresis to CAR-T cell infusion was 14 days.ResultsFrom Nov. 2020 to June 2021, 5 adult acute leukemia patients with CD7-positive (4 MPAL, 1 FLT3-mutated AML) were enrolled and infused with CD7 CAR T-cells, one with a low-dose (1.5×10 5/kg), 2 with a medium dose (5.0×10 5/kg), and 3 with a high dose infusion (1.0×10 6/kg). The median transduction efficiency of products was 70.4% (range: 32.0%-96.9%). As of July 30, 2021 cut-off date, the median observation time was 37 days (range: 35-232 days). Characteristics of enrolled patients are shown in Table 1. The median age was 33 years (range: 21-37 years), and the median bone marrow (BM) blasts percentage by morphology was 28.0% (range: 2.5-51.5%) at enrollment. Four patients had prior allo-HSCT history including one who had extramedullary disease (EMD) who relapsed following a 2 nd transplant and had also received CD19 CAR-T cell therapy prior to enrollment in the current trial. The median interval period from prior transplant to CD7 CAR-T cell infusion was 16 months (range: 8-23 months). Following infusion, the median peak of circulating CD7 CAR-T cell was 1.17×10 5copies/μg (0.651~7.04×10 5copies/μg) genomic DNA which occurred around Day 21 (Day14 - Day21) and 59.2% (4.28%~74.58%) occurring on Day18 (Day11-Day21) by q-PCR and flow cytometry respectively (Fig.1).Four weeks post CD7 CAR-T cell infusion, 4/5 patients achieved complete remission (CR) or CR with incomplete blood recovery (CRi) in BM and all achieved minimal residual disease (MRD)-negative CR. One patient who relapsed post 2 nd transplant, also with EMD, achieved CR in BM, however, remission was not achieved in EMD through PET-CT evaluation on Day 35.One MRD-negative CR patient subsequently underwent consolidative allo-HSCT 120 days post CD7 CAR T-cell infusion and has remained in MRD-negative CR. Other 3 patients without consolidative allo-HSCT remained in MRD-negative CR till the last follow-up. All 5 patients developed cytokine release syndrome (CRS)-three of 5 patients had grade I CRS, one patient had grade II and one patient had grade III CRS. None of the patients had neurotoxicity. Among the 4 patients who had prior allo-HSCT, 1 developed grade I graft-versus-host disease post CAR-T therapy.ConclusionThis study demonstrated that CD7-targeted CAR-T therapy offers an opportunity to achieve CR for CD7-positive MPAL patients even for those who relapsed post-transplant. Safety was manageable, however, more data on additional patients and longer observation times are needed to further evaluate the efficacy of CD7 CAR-T products. [Display omitted] DisclosuresBa: SenlangBio: Current Employment.

PHF6 Mutations Defines a Subgroup of Mixed Phenotype of Acute Leukemia with Aberrant T-Cell Differentiation

Mixed phenotype acute leukemia (MPAL) is a rare form of acute leukemia, and comprises 2% of all acute leukemia diagnoses. With the exception of a small subset harboring BCR/ABL1 or MLL translocations (around 15% in combination), the genetic aberrations of MPAL are largely unknown. PHF6 gene is located at Xq26-27 and encodes plant homeodomain finger 6, a zinc finger containing protein. Recurrent somatic PHF6 mutations are frequent in T-ALL (up to 30%) and rarely in AML (~3%). Here we report recurrent PHF6 mutations in approximately 20% of MPAL patients, making PHF6 the most common genetic aberrations identified in MPAL.From 2013 to 2016, a total of 1329 patients with myeloid neoplasms were sequenced at the MSKCC with a targeted next generation sequencing panel containing 30 genes that are frequently mutated in acute leukemia. We identified 38 PHF6 mutations with predicted pathologic significance in 33 patients (2.5%) spanning the whole coding sequence: 10 frameshift, 11 nonsense, 1 splicing site, and 16 missense (Figure 1). The most common co-mutant gene observed in cases with PHF6 mutations was RUNX1 (30%, 11/37). No TP53 mutations are detected within the PHF6-mutant cohort(Figure 2). The 33 patients with PHF6 mutations included patients with a diagnosis of MPAL (n=5), AML (n=19), MDS (n=5), and MPN (n=4).The disproportional distribution of PHF6 mutations in MPAL prompted us to further analyze these patients. During the same period of time, 24 MPAL patients were identified and confirmed based on WHO 2016 classification (excluding AML-MRC and t-AML). Within this cohort, 3 (12%) had BCR-ABL1 (p190) fusion, and 1 (4%) had MLL translocation, two distinct genetically defined subgroups. Importantly, 5 (21%) harbored PHF6 mutations, these mutations were mutually exclusive with BCR-ABL1 or MLL. Four of the 5 patients showed T-cell lineage differentiation, in contrast to MPAL with BCR-ABL1 or MLL translocations which were characterized by B/myeloid differentiation. The 2-year overall survival for the PHF6 mutation positive MPAL was markedly reduced compared to non-mutated cases (40% vs. 100% in PHF6 unmutated cases (p=0.005)).In the larger subset of AML cases, PHF6 mutations were nearly exclusively present in secondary AML (11 AML-MRC, 6 t-AML and only 1 de novo AML). In contrast to their rarity in de novo AML, PHF6 mutations were identified in 11% (11/88) of AML-MRC and 7.1% (6/84) of t-AML. Interestingly, T-cell markers (cytoplasmic CD3, CD2, CD5, CD7) were often expressed on the blasts of secondary AML cases regardless of PHF6 mutational status (56%, 9/16 vs. unmutated 51%, 36/70). In comparison to unmutated cases, the blasts were often negative for CD33 (69%, 11/16, vs 28%, 20/70, p=0.004 by Fisher's exact test) and CD38 (44%, 7/16, vs. 17%, 12/70, p=0.04 by Fisher's exact test) in PHF6 mutated AML, suggestive of a stemness phenotype with aberrant T-cell differentiation.In conclusion, PHF6 mutations are the most common somatic alteration identified in MPAL and define a subgroup of MPAL with altered T-cell differentiation and inferior outcome. Amongst AML, PHF6 mutations are almost exclusively seen in secondary AML and are also associated with T-cell antigen expression and a more primitive stem/progenitor immunophenotype. PHF6 mutations co-exist with RUNX1 mutations but are mutually exclusive to TP53 mutations. Notably, PHF6 mutations may provide a genetic and functional link between MPALs with poor outcome and secondary AML and provide insights into shared mechanisms of transformation. RNA sequencing studies on the sorted blast population are ongoing and will be presented at the ASH meeting. [Display omitted] DisclosuresArcila:Invivoscribe: Honoraria; Archer: Honoraria; Raindance Tecnologies: Honoraria. Levine:Celgene: Research Funding; Roche: Research Funding; Qiagen: Equity Ownership; Roche: Research Funding; Celgene: Research Funding; Qiagen: Equity Ownership.

Isolated Myeloperoxidase Expression in Pediatric B/Myeloid Mixed Phenotype Acute Leukemia Is Associated with Improved Survival Using ALL-Only Regimens

Background: Mixed phenotype acute leukemia (MPAL) represents a heterogeneous group of leukemias accounting for 2-5% of all newly diagnosed acute leukemias. Diagnostic criteria for mixed phenotype MPAL have evolved over time, with the World Health Organization (WHO) 2016 criteria now being recognized as the more definitive standard. According to WHO classification, isolated expression of myeloperoxidase (MPO) allows for myeloid lineage assignment in patients with an otherwise typical B-acute lymphoblastic leukemia (B-ALL) phenotype, thus meeting criteria for MPAL B/myeloid. Our goal was to determine if there were differences in treatment and survival between this group of patients, classified as MPAL B/myeloid isoMPO, and other MPAL patients.Methods: Data was obtained through a retrospective chart review of pediatric MPAL patients treated between 2001 and 2016 at Children's Healthcare of Atlanta. Cases were identified through our institutional tumor registry and pathology department flow cytometry database. Cases were eligible if the patients were between ages of 1 and 21 years at diagnosis and the leukemia met the WHO 2016 MPAL criteria. We excluded therapy related cases, non-myeloid cases (rare B/T subtype), and cases with a t(9;22) translocation. Data were extracted from clinical records for patient, disease and treatment related variables. All flow cytometry reports were reviewed by study hematopathologists. Cases were divided into two groups - MPAL B/Myeloid isoMPO and other MPAL cases. MPAL B/myeloid cases were classified as MPAL B/Myeloid isoMPO if there was a single B-lineage blast population expressing MPO as the only WHO MPAL myeloid marker. An event was defined as treatment failure requiring therapy change prior to end of induction (EOI), induction failure, induction death, remission death, or relapse. Event-free survival (EFS) and overall survival (OS) were calculated using Kaplan-Meier survival curves. Groups were compared using the log rank test. Chi-squared test was used for sampling distribution analyses.Results: Of the 35 MPAL cases included in our study, 21 (60%) met criteria for MPAL B/myeloid isoMPO. 20 of these patients were treated with ALL chemotherapy alone. One patient underwent bone marrow transplantation (BMT) due to a low DNA index. None of these patients were switched to myeloid therapy during their treatment course. The other 14 MPAL cases consisted of 5 MPAL B/myeloid (non-isolated MPO), 7 MPAL T/myeloid and 2 MPAL patients with t(v;11q23); MLL rearranged. Of these 14 cases, 10 were treated at least in part with myeloid chemotherapy and 7 proceeded to BMT. 6 of 10 patients in this group who started with ALL induction were switched over to AML therapy, with 3 being changed prior to EOI due to treatment failure. Despite these treatment differences, those with MPAL B/myeloid isoMPO had better outcomes. 3-year EFS was significantly higher at 84% for the MPAL B/myeloid isoMPO group compared to 57% in the other MPAL group (p=0.027 log rank test, Fig 1A). 3-year overall survival was 100% and 68%, respectively (p=0.083 log rank test, Fig 1B).Conclusion: There continues to be no consensus as to how to treat MPAL and the changing definition of MPAL has contributed to the difficulty in treating this rare and heterogeneous disease. While the WHO 2016 criteria have simplified the definition of MPAL, it has created a group of patients as shown here, that may be falsely categorized as MPAL. Our results suggest that MPAL B/myeloid isoMPO patients behave clinically like typical B-ALL patients, and should be treated as such. Consideration should be given to redefining the current MPAL WHO criteria to explicitly exclude these cases. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Comprehensive Analysis of Mixed Phenotype Acute Leukemia (MPAL) By Whole Genome Sequencing

Background: Mixed phenotype acute leukemias (MPAL) comprise a rare subgroup of acute leukemias with significant expression of lineage-specific antigens of more than one cell lineage not allowing assignment to a single cell lineage. While two MPAL subgroups are genetically defined according to the WHO 2016 classification, i.e. MPAL with BCR-ABL1 and MPAL with KMT2A rearrangement, the majority of cases are grouped as MPAL, B/myeloid, NOS or MPAL, T/myeloid, NOS and lack any specific or characteristic genetics so far.Aims: Molecular characterization of MPAL by whole genome sequencing (WGS).Patient cohort and methods: Diagnostic peripheral blood or bone marrow samples of 12 MPAL patients (median age 64 years, range 38-96; 5 male, 7 female) were selected for WGS with 90x coverage. One had MPAL with BCR-ABL1, 5 had MPAL, B/myeloid, NOS and 6 had MPAL, T/myeloid, NOS. Library preparation was performed using Truseq DNA PCR-Free HT sample preparation kit (Illumina, San Diego, CA). Sequencing was performed on NovaSeq instruments (Illumina). Tumor-only data was aligned by BaseSpace (BS) WGS app with default parameters and (structural) variant calling was performed with the Tumor/Normal app using an unmatched reference DNA (Promega, Fitchburg, WI). Data was subsequently loaded into BS Variant Interpreter to filter and prioritize variants of interest. For the structural variants we looked at all high confidence variant calls passing filters with at least 5 paired reads and 3 split reads with a somatic QScore of >30. In addition, all calls were eliminated with a population frequency of >1% according to the DGV (Database of Genomic variants). We performed mutation signature analysis by looking at the distribution of base substitutions across the sample with a 3mer context (including the base before and after the substitution) to decompose somatic mutation patterns and compared the results to the COSMIC signature database. For mutation analysis variants with a read frequency of >0.2% and ExAC population frequency of <1% were considered. For the first set of analysis we filtered down the variant list to the coding region of 65 genes described mutated in hematologic malignancies. For the second set of analysis we considered also genes beyond these 65 genes but required that variants had an entry in the COSMIC database and were classified with a “damaging” score by PolyPhen-2 and SIFT data bases.Results: Regarding the first set of analysis we found a median of 6 (range 2-7) mutations per patient, 63 overall, involving 35 of the 65 investigated genes. The majority were missense mutations (n=31), frameshift mutations (n=14) and splice-site mutations (n=13). The genes listed in table 1 were found most frequently mutated. Except for RUNX1 none of the frequently encountered mutations affected the case with MPAL with BCR-ABL1 while there was no restriction of mutations to either of the two other MPAL subgroups. 11 of these 63 mutations had been previously analyzed by routine diagnostics applying next generation sequencing (NGS) yielding identical results.Regarding the second set of analysis without limitation on the above mentioned 65 genes we found a median of 83 (range 63-129) mutations per patient, 1079 overall, involving 736 genes. Specifically, four candidate genes were found mutated in at least five patients (table 2). NBPF1 has been described as tumor suppressor, PRAMEF2 belongs to the PRAME family of genes associated with various malignancies and as a prognostic factor in B-ALL, MUC12 is a highly polymorphous gene involved in a variety of cancer types and described to be epigenetically regulated and thus a marker for early cancer detection, ARSD has been described overexpressed and prognostic in CLL. Again, mutations were not restricted to one MPAL subgroup.We subsequently conducted a context-sensitive mutation signature analysis with the SNV data, using 96 substitution patterns rather than only 6 to increase the complexity. In all instances we found a highly similar pattern across all 12 samples with a strong bias towards C>T substitutions (figure 1).Conclusion: WGS is capable of reproducing routine NGS diagnostics and of identifying mutations not yet described for MPAL which might be used to better understand and categorize this disease. [Display omitted] DisclosuresKern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Walter:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Integrative Genomic Analysis of Adult Mixed Phenotype Acute Leukemia (MPAL)

IntroductionMixed phenotype acute leukemia (MPAL) is a rare subgroup of acute leukemia characterized by blasts presenting both myeloid and lymphoid lineages. Standard of care for MPAL is not defined and prognosis of MPAL is poor. Genetic basis for mixed immunophynotype and distinct molecular characteristics from other leukemia is not well understood.MethodsWe studied 31 bone marrow samples from previously untreated MPAL patients (median age 53) meeting WHO criteria. Genomic DNA/RNA from bone marrow samples were analyzed by comprehensive genomic analysis including targeted capture sequencing of 295 genes (median 393x coverage), RNA sequencing, and Infinium methylation EPIC array (Illumina). Copy number alterations (CNA) were analyzed from methylation array data using ChAMP algorithm. Mutational landscape of MPAL was compared with194 AML, 71 B-ALL, and 14 T-ALL cases sequenced with the same platform. CpG methylation pattern of MPAL was also compared with 194 AML (TCGA), 505 B-ALL and 101 T-ALL cases (data shared by Nordlund et al. Genome Biology. 2013).ResultsOf the 31 MPAL cases, 18 (58%) had myeloid-T and 13 (42%) had myeloid-B phenotype. Four (13%) cases had Philadelphia chromosome, one (3%) had 11q23 rearrangement, and 8 (26%) had complex karyotypes. Eighteen patients (62%) were treated with ALL regimen, 8 (28%) received AML regimen, and 3 (10%) received other therapy.Targeted sequencing revealed 86 driver mutations (55 SNVs and 31 indels) in 35 genes in 29 samples (94%). MPAL carried median 2 (range 0-6) mutations per case that was similar to AML (P = 0.79) and T-ALL (P = 0.92) but was significantly higher than B-ALL(P < 0.001). The most frequently mutated genes were DNMT3A , IDH2 , NOTCH1 , and RUNX1 in 7 (23%) cases each (Figure 1). Myeloid-T and myeloid-B showed distinct patterns of mutations, in which mutations in NOTCH pathway genes had strong trend of enrichment in myeloid-T (P = 0.06), whereas RUNX1 mutations were significantly enriched in myeloid-B (P = 0.02). When mutation landscape was compared among AML, B-ALL, T-ALL and MPAL, TP53 , NRAS , and DNMT3A mutations were shared across all leukemia. In contrast, NPM1 mutation was specific to AML and mutations in NOTCH1 , FBXW7 , and IL7R were specific to myeloid-T MPAL and T-ALL. CNA analysis showed that CDKN2B as a significantly deleted gene in MPAL.Unsupervised hierarchical clustering of promoter methylation revelaed two distinct clusters. This methylation cluster had significant correlation with immunophenotype (73% of cluster 1 was myeloid-B and 88% of cluster 2 was myeloid-T, P < 0.001). Overall, myeloid-T had more hypermethylated CpG loci than myeloid-B (mean beta value 0.28 vs. 0.52 in myeloid-B vs. myeloid-T, P = 0.002). This difference in methylation was independent from IDH mutation. Molecular pathway analysis of differentially methylated probes (DMP) showed that T-cell receptor pathway, T cell differentiation, and activation pathway genes (CD3D , CD247 , LCK , PRF1 , RHOH , and PRKCQ ) were differentially methylated and were expressed between myeloid-B and myeloid-T. Furthermore, motif enrichment analysis of promoter DMP revealed that IRF8 and IRF4 binding motifs were significantly hyper-methylated in myeloid-T compared to myeloid-B. Consistent with this, RAG1 , RAG2 , CXCR4 and PAX5 , downstream targets of IRF4/IRF8 andessential genes in B-cell development, were differentially expressed between two phenotypes. RNA sequencing revealed two known translocations (NSD1-NUP98 , KMT2A-MLLT4 ) in addition to the novel translocations such as FOXP1-DNAJC15 , TNKS - LYZ , and NOP14 - PLEC in MPAL. We did not detect ZN384 fusions that have been reported in pediatric MPAL (Alexander et al. ASH 2016). Unsupervised hierarchical clustering of methylation pattern among MPAL, AML, B-ALL and T-ALL revealed that 41% and 59% of MPAL had AML-like and ALL-like methylation pattern, respectively (Figure 2). Patients who received therapy that matched with their methylation pattern had significantly better response to treatment than patients who received unmatched therapy (CR rate: 77% vs. 31%, P = 0.024).ConclusionMyeloid-T and myeloid-B MPAL showed distinct patterns of mutation, methylation and gene expressions. Methylation data could identify AML-like and ALL-like MPAL which corresponded to better response to therapy when matched therapy was given. These data suggest that molecularly guided therapy may be beneficial in the treatment of MPAL. [Display omitted] DisclosuresTakahashi:Symbio Pharmaceuticals: Consultancy. DiNardo:Agios: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Daiichi-Sankyo: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding. Jabbour:Bristol-Myers Squibb: Consultancy. Andreeff:Daiichi Sankyo: Consultancy. Kantarjian:Bristol-Meyers Squibb: Research Funding; Amgen: Research Funding; ARIAD: Research Funding; Pfizer: Research Funding; Delta-Fly Pharma: Research Funding; Novartis: Research Funding.

Mixed phenotype acute leukemia with PML-RAR\u03b1 positive: a case report and literature review

Mixed phenotype acute leukemia (MPAL) is an uncommon type of leukemia. It is one kind of malignant clonal diseases that expresses more than one genealogical specific antigen simultaneously. Most MPAL patients are associated with clonal chromosomal abnormalities and molecular genetic changes, such as t(9;22) (q34;q11) and KMT2A (MLL) rearrangement. These specific abnormalities usually have important guiding significance in MPAL diagnosis, targeted therapy and prognosis judgment. In this paper, we reported a case of MPAL, T/myeloid (M5) with an unfrequent combination of PML-RARα positivity and t(15;17). The treatment was successful with chemotherapy for both AML and ALL with daunorubicin, cytarabine (DA) and vincristine, prednisone (VP). We reported here this suggestive MPAL case of rare disease condition and effective treatment, in order to provide experience for the early diagnosis and treatment of similar patients.

Prognostic impact of Philadelphia chromosome in mixed phenotype acute leukemia (MPAL): A cancer registry analysis on real-world outcome.

Mixed phenotype acute leukemia (MPAL) is thought to have poor outcome, and presence of the Philadelphia chromosome (Ph+) has been considered to be an adverse prognostic marker. However, most of these reports were in the pre-tyrosine kinase inhibitors (TKIs) era. Recent limited reports indicate improved outcomes for MPAL with the addition of TKIs. We examined the outcomes of 241 cases of MPAL according to the 2008 WHO classification from the Surveillance, Epidemiology, and End Results registry. The MLL+ patients had a median age of 6 years while other subtypes occurred mostly in adults and had comparable age. On multivariate analyses and after adjustment for age, year of diagnosis and chemotherapy status, Ph+ MPAL patients had reduced risk of death in comparison to Ph(-) MPAL patients (hazard ratio [HR] = 0.28, P = .002). So, MLL+ MPAL had the worst outcome with a 10-fold increased risk of death in comparison to Ph+ MPAL patients (HR = 10.2, P < .001). Importantly, the outcome of Ph+ MPAL was comparable to Ph+ acute lymphoblastic leukemia in a 1:1 matched case-control analysis. In conclusion, this is the largest registry study which examines the outcomes of MPAL subtypes. We confirm that MPAL is a heterogenous disease. Note, Ph+ MPAL nowadays has a better OS in comparison to other subtypes and is comparable to Ph+ ALL patients. This is most likely secondary to changes in practice and more utilization of TKIs. On the other hand, MLL rearrangement is associated with infantile MPAL and has a dismal prognosis.

Characteristics of FLT3 gene mutation and clinical efficacy of tyrosine kinase inhibitor in patients with mixed phenotype acute leukemia

Objective To analyze the incidence and mutation characteristics of FLT3 gene mutation and clinical efficacy of tyrosine kinase inhibitor (TKI) in patients with mixed phenotype acute leukemia (MPAL). Methods A total of 48 patients with MPAL who were admitted to Hebei Yanda Lu Daopei Hospital from June 2015 to February 2018 were retrospectively analyzed. The common mutated 58 genes in hematologic malignancies were detected by using amplicon-targeted next generation sequencing, of which internal tandem duplication (ITD) and point mutation occurred in the hotspot region of exon 14, 15 and 20 in FLT3 gene. Multiplex polymerase chain reaction (PCR) analysis was used to detect 35 gene fusions in hematological neoplams. Results There were 7 cases of FLT3 mutation in 48 MPAL patients, which were all ITD mutations. The median length of the inserts of FLT3-ITD was 48 bp, and one MPAL patient carried 2 multiple length inserts simultaneously, and the median variant allele frequency (VAF) was 40.5% (7.9%-84.7%). There were no statistically significant differences in clinical and genetic characteristics between FLT3 mutation-positive and FLT3 mutation-negative MPAL patients (both P > 0.05). Among 7 FLT3 mutation-positive MPAL patients, 4 cases were often accompanied with RUNX1 mutation. A total of 4 MPAL patients with FLT3-ITD-positive received sorafenib or sunitinib combined chemotherapy, and 3 of them achieved complete remission. Conclusions ITD mutation is the main part in the FLT3 mutation of MPAL patients. FLT3-ITD-positive MPAL patients are often accompanied with RUNX1 mutation, which may benefit from targeted therapy with FLT3 kinase inhibitor. Key words: Leukemia, biphenotypic, acute; Mutation; Tyrosine kinase inhibitor

Allogeneic Stem Cell Transplantation Has Limited Benefit in Older Patients with Mixed Phenotype Acute Leukemia

Mixed phenotype acute leukemia (MPAL) is a high risk leukemia that has markers of both myeloid and lymphoid lineage. The role of hematopoietic stem cell transplantation (alloHSCT) remains uncertain especially in the older population. We sought to evaluate the outcomes of our cohort of MPAL patients who were transplanted, including older adults unfit for MAC compared with the outcomes of patients who were consolidated with chemotherapy only. <b>Methods:</b> 74 patients aged 18 years or older, at the Princess Margaret Cancer Centre between January 1, 2000 and December 31, 2018 were evaluated. <b>Results:</b> Among the 74 MPAL patients included in this study, 59 (79%) received induction. Complete remission (CR) was achieved in 47 (79%) treated patients. 25 of 36(80%) achieved a CR using ALL protocols, while 9 of 23 (39%) achieved a CR using AML protocols. Consolidation treatment post CR was split, with 24(51%) receiving chemotherapy followed by an alloHSCT and 23 (49%) receiving chemotherapy only. In the alloHSCT group, RIC was used in 20 (81%) patients while MAC was used in four younger patients. The median age of the transplant cohort was 50 years with a median OS of 21 months. In the chemotherapy only group, the median age was 57 with a median OS of 18 months. Univariate analysis demonstrated no difference in outcomes in terms of age, induction chemotherapy, donor source, and conditioning intensity. The sole predictor of poor OS on univariate analysis was acute graft versus host disease (aGVHD) with a hazard ratio of 3.3 (95% CI 0.9-11). In total, 12 patients (50%) had aGVHD with a median OS of 16 months. However, when Grade 1/2 and Grade 3/4 aGVHD were compared, median OS was not reached in patients with G1/2, compared with an OS of 11 months in those patients with G3/4 aGVHD. Post-transplant relapses occurred solely in the RIC group and occured early. Although, there were no relapses in the MAC group, the non-relapse mortality (NRM) was 75% compared to 18% in the RIC group. The 3 year OS and relapse free survival (RFS) for the entire cohort was 30% and 32%, respectively. When comparing those who had undergone alloSCT and those who received chemo alone, survival in the two groups were similar with a 3 year OS of 29% vs 32% and the 3 year RFS at 29% and 34%, respectively. Subgroup analysis between the two groups was performed for patients with specific poor prognostic factors (older age, complex cytogenetics, higher WCC, conditioning regimen used), but results showed no significant survival differences. <b>Conclusions:</b> Older patients with MPAL have an inferior prognosis and worse outcomes with alloHSCT compared with those of younger adults or children. Despite the increase in access to alloHSCT and an expanding pool of alternative donors, outcomes with RIC remain similar to consolidation with chemotherapy alone. There may be some evidence to suggest that the graft versus leukemia effect can be harnessed to improve outcomes in selected patients.