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Background: The apparent lack of additional missense mutations data on mixed-phenotype leukemia is noteworthy. Single amino acid substitution by these non-synonymous single nucleotide variations can be related to many pathological conditions and may influence susceptibility to disease. This case-control study aimed to unravel whether the ZAP70 missense variant (rs104893674 (C > A)) underpinning mixed-phenotype leukemia. Methods: The rs104893674 was genotyped in clients who were mixed-phenotype acute leukemia-, acute lymphoblastic leukemia- and acute myeloid leukemia-positive and matched healthy controls, which have been referred to all major urban hospitals from multiple provinces of country- wide, IRAN, from February 11' 2019 to June 10' 2023, by amplification refractory mutation system-polymerase chain reaction method. Direct sequencing for rs104893674 of the ZAP70 gene was performed in a 3130 Genetic Analyzer. Results: We found that the AC genotype of individuals with A allele at this polymorphic site (heterozygous variant-type) contribute to the genetic susceptibility to acute leukemia of both forms, acute myeloid leukemia and acute lymphoblastic leukemia as well as with a mixed phenotype. In other words, the ZAP70 missense variant (rs104893674 (C > A)) increases susceptibility of distinct cell populations of different (myeloid and lymphoid) lineages to exhibiting cancer phenotype. The results were all consistent with genotype data obtained using a direct DNA sequencing technique. Conclusion: Of special interest are pathogenic missense mutations, since they generate variants that cause specific molecular phenotypes through protein destabilization. Overall, we discovered that the rs104893674 (C > A) variant chance in causing mixed-phenotype leukemia is relatively high. [ABSTRACT FROM AUTHOR]

Abstract Background The apparent lack of additional missense mutations data on mixed-phenotype leukemia is noteworthy. Single amino acid substitution by these non-synonymous single nucleotide variations can be related to many pathological conditions and may influence susceptibility to disease. This case-control study aimed to unravel whether the ZAP70 missense variant (rs104893674 (C > A)) underpinning mixed-phenotype leukemia. Methods The rs104893674 was genotyped in clients who were mixed-phenotype acute leukemia-, acute lymphoblastic leukemia- and acute myeloid leukemia-positive and matched healthy controls, which have been referred to all major urban hospitals from multiple provinces of country- wide, IRAN, from February 11’ 2019 to June 10’ 2023, by amplification refractory mutation system-polymerase chain reaction method. Direct sequencing for rs104893674 of the ZAP70 gene was performed in a 3130 Genetic Analyzer. Results We found that the AC genotype of individuals with A allele at this polymorphic site (heterozygous variant-type) contribute to the genetic susceptibility to acute leukemia of both forms, acute myeloid leukemia and acute lymphoblastic leukemia as well as with a mixed phenotype. In other words, the ZAP70 missense variant (rs104893674 (C > A)) increases susceptibility of distinct cell populations of different (myeloid and lymphoid) lineages to exhibiting cancer phenotype. The results were all consistent with genotype data obtained using a direct DNA sequencing technique. Conclusion Of special interest are pathogenic missense mutations, since they generate variants that cause specific molecular phenotypes through protein destabilization. Overall, we discovered that the rs104893674 (C > A) variant chance in causing mixed-phenotype leukemia is relatively high.

Introduction: Mixed-phenotype acute leukemia (MPAL) is a rare disease with poor prognosis. So far, no standard approach has been established as the "knowhow" of MPAL is based only on retrospective analyses performed on small groups of patients. Materials and methods: In this study, a retrospective analysis of the outcomes of adult MPAL patients included in the PALG registry between 2005 and 2024 who received the CLAG-M hybrid protocol as induction or salvage therapy was performed. Results: Sixteen of 98 MPAL patients received CLAG-M: eight as first-line and eight as salvage therapy. In the first line, two patients achieved partial response (PR), and six achieved complete remission (CR), of whom four successfully underwent allogeneic hematopoietic stem cell transplantation (alloHSCT). Two patients who did not undergo alloHSCT promptly relapsed. Within the whole group, the overall response rate (ORR) was 75% (n = 12/16). With the median follow-up of 13 months, six out of eight patients remain in CR, however, two of them died due to acute graft versus host disease. Out of eight patients who received CLAG-M in the second line, four patients (50%) obtained CR. AlloHSCT was conducted in seven cases, six of which were in CR. Only two patients remained in CR at the time of the last follow-up. Tolerance to treatment was good. The median times for severe neutropenia and thrombocytopenia were 22 days (range, 16-24) and 17 days (range, 12-24), respectively. Overall, grade 3-4 infections were observed in 12 cases, and all infections presented successful outcomes. Conclusions: CLAG-M is an effective first-line salvage regimen for MPAL with an acceptable safety profile. Early achievement of CR with prompt alloHSCT allows for satisfactory disease control. [ABSTRACT FROM AUTHOR]

While our understanding of the molecular basis of mixed phenotype acute leukemia (MPAL) has progressed over the decades, our knowledge is limited and the prognosis remains poor. Investigating cases of familial leukemia can provide insights into the role of genetic and environmental factors in leukemogenesis. Although familial cases and associated mutations have been identified in some leukemias, familial occurrence of MPAL has never been reported. Here, we report the first cases of MPAL in a family. A 68-year-old woman was diagnosed with MPAL and received haploidentical stem cell transplantation from her 44-year-old son. In four years, the son himself developed MPAL. Both cases exhibited similar characteristics such as biphenotypic leukemia with B/myeloid cell antigens, Philadelphia translocation (BCR-ABL1 mutation), and response to acute lymphoblastic leukemia-type chemotherapy. These similarities suggest the presence of hereditary factors contributing to the development of MPAL. Targeted sequencing identified shared germline variants in these cases; however, in silico analyses did not strongly support their pathogenicity. Intriguingly, when the son developed MPAL, the mother did not develop donor-derived leukemia and remained in remission. Our cases provide valuable insights to guide future research on familial MPAL. [ABSTRACT FROM AUTHOR]

IntroductionMixed-phenotype acute leukemia (MPAL) is a rare disease with poor prognosis. So far, no standard approach has been established as the “know-how” of MPAL is based only on retrospective analyses performed on small groups of patients.Materials and methodsIn this study, a retrospective analysis of the outcomes of adult MPAL patients included in the PALG registry between 2005 and 2024 who received the CLAG-M hybrid protocol as induction or salvage therapy was performed.ResultsSixteen of 98 MPAL patients received CLAG-M: eight as first-line and eight as salvage therapy. In the first line, two patients achieved partial response (PR), and six achieved complete remission (CR), of whom four successfully underwent allogeneic hematopoietic stem cell transplantation (alloHSCT). Two patients who did not undergo alloHSCT promptly relapsed. Within the whole group, the overall response rate (ORR) was 75% (n = 12/16). With the median follow-up of 13 months, six out of eight patients remain in CR, however, two of them died due to acute graft versus host disease. Out of eight patients who received CLAG-M in the second line, four patients (50%) obtained CR. AlloHSCT was conducted in seven cases, six of which were in CR. Only two patients remained in CR at the time of the last follow-up. Tolerance to treatment was good. The median times for severe neutropenia and thrombocytopenia were 22 days (range, 16–24) and 17 days (range, 12–24), respectively. Overall, grade 3-4 infections were observed in 12 cases, and all infections presented successful outcomes.ConclusionsCLAG-M is an effective first-line salvage regimen for MPAL with an acceptable safety profile. Early achievement of CR with prompt alloHSCT allows for satisfactory disease control.

Abstract Background Mixed phenotype acute leukemia (MPAL), a rare subgroup of leukemia characterized by blast cells with myeloid and lymphoid lineage features, is difficult to diagnose and treat. A better characterization of MPAL is essential to understand the subtype heterogeneity and how it compares with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Therefore, we performed single-cell RNA sequencing (scRNAseq) on pediatric MPAL bone marrow (BM) samples to develop a granular map of the MPAL blasts and microenvironment landscape. Methods We analyzed over 40,000 cells from nine pediatric MPAL BM samples to generate a single-cell transcriptomic landscape of B/myeloid (B/My) and T/myeloid (T/My) MPAL. Cells were clustered using unsupervised single-cell methods, and malignant blast and immune clusters were annotated. Differential expression analysis was performed to identify B/My and T/My MPAL blast-specific signatures by comparing transcriptome profiles of MPAL with normal BM, AML, and ALL. Gene set enrichment analysis (GSEA) was performed, and significantly enriched pathways were compared in MPAL subtypes. Results B/My and T/My MPAL blasts displayed distinct blast signatures. Transcriptomic analysis revealed that B/My MPAL profile overlaps with B-ALL and AML samples. Similarly, T/My MPAL exhibited overlap with T-ALL and AML samples. Genes overexpressed in both MPAL subtypes’ blast cells compared to AML, ALL, and healthy BM included MAP2K2 and CD81. Subtype-specific genes included HBEGF for B/My and PTEN for T/My. These marker sets segregated bulk RNA-seq AML, ALL, and MPAL samples based on expression profiles. Analysis comparing T/My MPAL to ETP, near-ETP, and non-ETP T-ALL, showed that T/My MPAL had greater overlap with ETP-ALL cases. Comparisons among MPAL subtypes between adult and pediatric samples showed analogous transcriptomic landscapes of corresponding subtypes. Transcriptomic differences were observed in the MPAL samples based on response to induction chemotherapy, including selective upregulation of the IL-16 pathway in relapsed samples. Conclusions We have for the first time described the single-cell transcriptomic landscape of pediatric MPAL and demonstrated that B/My and T/My MPAL have distinct scRNAseq profiles from each other, AML, and ALL. Differences in transcriptomic profiles were seen based on response to therapy, but larger studies will be needed to validate these findings.

Background: Mixed phenotype acute leukemia (MPAL), a rare subgroup of leukemia characterized by blast cells with myeloid and lymphoid lineage features, is difficult to diagnose and treat. A better characterization of MPAL is essential to understand the subtype heterogeneity and how it compares with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Therefore, we performed single-cell RNA sequencing (scRNAseq) on pediatric MPAL bone marrow (BM) samples to develop a granular map of the MPAL blasts and microenvironment landscape. Methods: We analyzed over 40,000 cells from nine pediatric MPAL BM samples to generate a single-cell transcriptomic landscape of B/myeloid (B/My) and T/myeloid (T/My) MPAL. Cells were clustered using unsupervised single-cell methods, and malignant blast and immune clusters were annotated. Differential expression analysis was performed to identify B/My and T/My MPAL blast-specific signatures by comparing transcriptome profiles of MPAL with normal BM, AML, and ALL. Gene set enrichment analysis (GSEA) was performed, and significantly enriched pathways were compared in MPAL subtypes. Results: B/My and T/My MPAL blasts displayed distinct blast signatures. Transcriptomic analysis revealed that B/My MPAL profile overlaps with B-ALL and AML samples. Similarly, T/My MPAL exhibited overlap with T-ALL and AML samples. Genes overexpressed in both MPAL subtypes' blast cells compared to AML, ALL, and healthy BM included MAP2K2 and CD81. Subtype-specific genes included HBEGF for B/My and PTEN for T/My. These marker sets segregated bulk RNA-seq AML, ALL, and MPAL samples based on expression profiles. Analysis comparing T/My MPAL to ETP, near-ETP, and non-ETP T-ALL, showed that T/My MPAL had greater overlap with ETP-ALL cases. Comparisons among MPAL subtypes between adult and pediatric samples showed analogous transcriptomic landscapes of corresponding subtypes. Transcriptomic differences were observed in the MPAL samples based on response to induction chemotherapy, including selective upregulation of the IL-16 pathway in relapsed samples. Conclusions: We have for the first time described the single-cell transcriptomic landscape of pediatric MPAL and demonstrated that B/My and T/My MPAL have distinct scRNAseq profiles from each other, AML, and ALL. Differences in transcriptomic profiles were seen based on response to therapy, but larger studies will be needed to validate these findings. [ABSTRACT FROM AUTHOR]

B/T mixed phenotype acute leukemia (MPAL) is a rare aggressive leukemia. Three cases of B/T MPAL were identified with comprehensive immunophenotypic, cytogenetic, and molecular studies. T-lineage predominant B/T MPAL shares a genetic signature with T-ALL whereas B/T lineage co-dominant B/T MPAL lacks such a T-ALL signature. All three patients were treated with lineage-matched-ALL therapy and alive at the last follow-up. Our study is the first to demonstrate molecular heterogeneity within B/T MPAL in a context of an immunophenotype of T-lineage versus B-lineage predominance. The implication of such a phenotype-genotype association on diagnostic classification is briefly discussed.

Abstract Herein, we present a rare case of acute myeloid leukemia (AML) with CBFA2T3‐rearrangement and the expression of megakaryocytic and lymphoid markers, highlighting the need for a high suspicion index in differential diagnosis and applying adequate workup to avoid misdiagnosing this entity. CBFA2T3::GLIS2‐positive AML is primarily found in infants with non‐down syndrome acute megakaryoblastic leukemia (non‐DSAMKL). Flow cytometry immunophenotyping plays an important role in recognizing the unique immunophenotype of bright CD56 expression with dim/negative expression of HLA‐DR, CD38, and CD45 termed the RAM immunophenotype in this entity. Still, CBFA2T3::GLIS2‐positive acute leukemia with T/megakaryocytic markers could be misdiagnosed as T‐lymphoblastic leukemia/lymphoma, early T‐cell precursor acute lymphoblastic leukemia/lymphoma, NK lymphoblastic leukemia, AML with minimal differentiation, or AML with myelodysplasia‐related changes.

Adult B-lineage acute lymphoblastic leukemia (B-ALL) with t(4;11)(q21;q23) is very rare. It is characterized by mixed-lineage leukemia and has the potential for lineage switching during the treatment course. We report the disease course of a patient with B-ALL with t(4;11)(q21;q23) to demonstrate that close monitoring of cell morphology and immunophenotyping is necessary to capture the lineage switch at an early stage. Cell morphology, immunophenotyping, and cytogenetics were used to evaluate the patient's disease status. A 36-year-old woman was diagnosed with B-ALL with t(4;11)(q21;q23), which encodes the KMT2A::AFF1 fusion. After the initial induction chemotherapy, her disease remained refractory, and the patient received salvage immunotherapy with blinatumomab and inotuzumab ozogamicin. However, the ALL did not respond. Repeated bone marrow examinations unexpectedly revealed the emergence of a major population of monoblasts, in addition to a minor population of the original B lymphoblasts. The patient was diagnosed with disease evolution from B-ALL to mixed-phenotype acute leukemia (MPAL, B/myeloid). We present this case to highlight the potential of KMT2A-rearranged B-ALL to undergo lineage switch following B-cell targeted therapy. Patients with this kind of B-ALL should therefore be closely monitored to capture potential changes in the nature of the disease and prompt appropriate treatment. [ABSTRACT FROM AUTHOR]

Introduction: 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. Methods: 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. Results: 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. Conclusion: 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. [ABSTRACT FROM AUTHOR]

Abstract Background The diagnosis of mixed phenotype acute leukemia (MPAL) with T/megakaryocyte or T/myeloid lineages accompanied by t(3;3) is always a challenge. Therefore, multiple experimental methods are usually required to avoid misdiagnosis. In this report, we presented a rare case of MPAL with T/myeloid lineages accompanied by t(3;3) and discussed the experience of differential diagnosis and our appreciation of the MPAL with T/megakaryocyte and T/myeloid lineages accompanied by t(3;3). Case presentation: A 31-year-old woman was admitted to our hospital due to recurrent fever for 20 days. Two distinct blast populations were detected by flow cytometry analysis: one population fulfills the immunophenotypic criteria for T-lymphoblastic leukemia, while the other population is highly suggestive of megakaryoblasts. These immunophenotypic features support the diagnosis of MPAL (T/megakaryocyte), which is rarely reported​. Interestingly, a complex karyotype was detected afterward by cytogenetics with t(3;3)(q21;q26.2), indicating a diagnosis of AML with t(3;3), a subset of which is also characterized by megakaryocytic markers such as CD41 and CD61. It seems that the second blast population detected by flow cytometry could not be classified into either diagnosis based on the morphology, immunophenotyping, and even cytogenetic findings, posing a real diagnostic problem because of the lack of clear-cut cytogenetic morphological defined criteria to distinguish between acute megakaryocytic leukemia and AML with t(3;3). Combining all of the examination data, this case was ultimately diagnosed as MPAL (T + My)-NOS with t(3;3) through differential diagnosis. Before the cytogenetic results were available, the patient received an acute lymphoblastic leukemia (ALL) regimen for MPAL treatment, but the effect was unsatisfactory. After the diagnosis was clear, she received an AML-like regimen with azacitidine for 7 days and venetoclax for 14 days, and achieved complete morphological remission. Conclusion MPAL with either T/megakaryocyte or T/myeloid lineages accompanied by t(3;3) is rare, and it is difficult to make a clear diagnosis. Thus, comprehensive examinations, including bone marrow cell morphology, flow cytometry analysis, cytogenetics, and molecular analysis are recommended to avoid misdiagnosis. AML-like regimen including azacitidine and venetoclax may be effective for treating MPAL (T + My)-NOS with t(3;3).

Keywords: Acute lymphoblastic leukemia; Mixed phenotype acute leukemia; Daratumumab; Venetoclax EN Acute lymphoblastic leukemia Mixed phenotype acute leukemia Daratumumab Venetoclax 669 672 4 03/03/23 20230301 NES 230301 Dear Editor: Here, we report on a twelve-year-old boy diagnosed with mixed phenotype acute leukemia (T cell ALL/acute myeloid leukemia, AML) bearing a I KMT2A::MLLT4 i fusion in June 2018. In the further course, the patient received low-dose cytarabine, two additional DLI doses, and mistletoe therapy. 1 Treatment course of a patient with with KMT2A:MLLT4-positive mixed phenotype acute leukemia (T cell ALL/AML). [Extracted from the article]

Background: The goal was to improve the understanding of mixed phenotypic acute leukemia (MPAL) complicated with plasmacytoid dendritic cell (PDC) proliferation. Methods: A case of mixed phenotype acute leukemia with plasmacytoid dendritic cell hyperplasia was reported. The clinical characteristics, treatment, and prognosis were analyzed by reviewing relevant literature. Results: The patient was a young female with clinical manifestations of splenomegaly and lymph node enlargement. The bone marrow smear showed hyperactive proliferation, 95% of protoblastic cells. The protoblastic cell body is large, more cell mass, stained gray blue, a small amount of azurophilicgranule can be seen in some cytoplasm, pseudopodia, drag tail, and other phenomena. The nucleus was twisted and folded. Chromatin is fine with nucleoli and Auer rods seen in the cytoplasm. Immune typing: Abnormal primordial cells accounted for 44.75%, and the primordial cells expressed both myeloid markers (CD33, CD13, MPO) and T-series markers (CD7, CD5, Ccd3), which were considered MPAL (M/T) according to WHO diagnostic criteria. In addition, a group of plasmoid dendritic cells occupied an increased proportion of 10.31% of nuclear cells. No obvious phenotypic abnormalities were observed. BCR/ABL fusion genes P190/P210 were negative. NRAS, NOTCH1, and DNMT3A mutations were detected by polymerase chain reaction. Combined with the above results, acute mixed cell leukemia (M/T) with plasmacytoid dendritic cell proliferation was diagnosed. Conclusions: The diagnosis of mixed phenotype acute leukemia with plasmacytoid dendritic cell proliferation needs to be integrated with clinical manifestations, cytomorphology, immunology, cytogenetics, and molecular biology, etc. Disease should be diagnosed and treated as early as possible. [ABSTRACT FROM AUTHOR]