A hybrid protocol CLAG-M, a possible player for the first-line therapy of patients with mixed phenotype acute leukemia. A Polish Adult Leukemia Group experience.

Introduction: Mixed-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 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.

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.

Genomic heterogeneity within B/T mixed phenotype acute leukemia in a context of an immunophenotype.

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.

Outcomes with high dose cytarabine and mitoxantrone induction for adults with mixed phenotype acute leukemia.

The optimal induction strategy for mixed phenotype acute leukemia (MPAL) is unknown, though retrospective data has shown improved remission rates and overall survival with acute lymphoblastic leukemia (ALL)-based regimens. At Memorial Sloan Kettering Cancer Center (MSKCC), the most utilized induction regimen for MPAL is high dose cytarabine plus mitoxantrone ("ALL-2"), though outcomes with this regimen are not well described. In this study, outcomes to first-line induction chemotherapy in 24 patients at MSKCC with MPAL classified by 2016 World Health Organization criteria are reported. The overall response rate was 94 % (16 of 17) in patients receiving ALL-2, including 86 % (6 of 7) in patients with extramedullary disease. Thirteen patients who received ALL-2 induction proceeded to allogeneic hematopoietic cell transplant (allo-HCT). The most common toxicity associated with ALL-2 was febrile neutropenia, documented in 12 patients. With a median follow-up of 37 months, median overall survival was not reached in the ALL-2 cohort, and 3-year overall survival was 62 %. In multivariate analysis, age ≥ 60 years and MPAL with isolated extramedullary disease were associated with significantly worse overall survival (P = .009 and P = .01, respectively). These results support further prospective investigation of ALL-2 as a front-line induction regimen for adults with MPAL.

An unusual presentation of a pediatric patient with mixed phenotypic acute leukemia with PICALM::MLLT10 gene rearrangement.

Mixed phenotype leukemia (MPAL) is a rare type of acute leukemia with blasts that co-express antigens of more than one lineage on the same cell or that have separate populations of blasts of different lineages. Here, we report a five-year-old male with inguinal lymphadenopathy diagnosed with MPAL-T/Myeloid MPAL-T/M. The clone demonstrated lineage and immunophenotypically distinct blast populations in the bone marrow and lymph nodes. Bone marrow cytogenetic studies confirmed a rare PICALM::MLLT10 gene fusion. Patients with this fusion gene have been found to have high risk features and poor survival rates in several small case series. Our case report highlights an unusual presentation in medullary and extramedullary sites, within a pediatric patient. At the time of submission of this case report, the patient has shown good response to chemotherapy and continues to be in remission.

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

INTRODUCTION: 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. METHODS: 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). RESULTS: 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. CONCLUSION: MPAL is a complex entity with heterogeneous features. BCR::ABL1 is a common abnormality. BMT is associated with better OS.

PHF6 functions as a tumor suppressor by recruiting methyltransferase SUV39H1 to nucleolar region and offers a novel therapeutic target for PHF6-muntant leukemia.

Mutations in the plant homeodomain-like finger protein 6 (PHF6) gene are strongly associated with acute myeloid (AML) and T-cell acute lymphoblastic leukemia (T-ALL). In this study, we demonstrated that PHF6 can bind to H3K9me3 and H3K27me1 on the nucleolar chromatin and recruit histone methyltransferase SUV39H1 to the rDNA locus. The deletion of PHF6 caused a decrease in the recruitment of SUV39H1 to rDNA gene loci, resulting in a reduction in the level of H3K9me3 and the promotion of rDNA transcription. The knockdown of either SUV39H1 or PHF6 significantly attenuated the effects of increase in H3K9me3 and suppressed the transcription of rDNA induced by the overexpression of the other interacting partner, thereby establishing an interdependent relationship between PHF6 and SUV39H1 in their control of rRNA transcription. The PHF6 clinical mutants significantly impaired the ability to bind and recruit SUV39H1 to the rDNA loci, resulting in an increase in rDNA transcription activity, the proliferation of in vitro leukemia cells, and the growth of in vivo mouse xenografts. Importantly, significantly elevated levels of pre-rRNA were observed in clinical AML patients who possessed a mutated version of PHF6. The specific rDNA transcription inhibitor CX5461 significantly reduced the resistance of U937 AML cells deficient in PHF6 to cytarabine, the drug that is most commonly used to treat AML. Collectively, we revealed a novel molecular mechanism by which PHF6 recruits methyltransferase SUV39H1 to the nucleolar region in leukemia and provided a potential therapeutic target for PHF6-mutant leukemia.

Multiple cells of origin in common with various types of mouse N-Myc acute leukemia.

Little is known regarding whether the cell of origin differs among different leukemia types. To address this fundamental issue, we determined the cell of origin in five distinct types of acute leukemia induced by N-Myc overexpression in mice. CD150+CD48-CD41-CD34-c-Kit+Sca-1+Lin- (KSL) (HSC1) cells, CD150-CD48-CD41-CD34-KSL (HSC2) cells, CD150+CD41+CD34-KSL (HPC1) cells, CD150+CD41+CD34+KSL (HPC2) cells, and CD150-CD41-CD34+KSL (HPC3) cells were purified from the bone marrow of adult C57BL/6 mice, transduced with the N-Myc retrovirus vector, and transplanted into lethally irradiated mice. B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML), acute undifferentiated leukemia (AUL), and mixed phenotype acute leukemia (MPAL) developed from five populations. RNA sequencing data supported the phenotypical diagnoses of leukemia, except that AUL appeared transcriptionally close to T-ALL. Whole-genome sequencing revealed that retroviral integration sites were irrelevant to the leukemia types and that T-ALL and AML of MPAL shared the same integration site and many gene mutations, suggesting their common origin. Additionally, leukemic stem cells were identified in the KSL cell population, suggesting that the phenotypes of leukemic stem cells are irrelevant to leukemia types. This study provides experimental evidence for the similar and multiple cells of origin in acute leukemia.

Repeated Lineage Switches in an Elderly Case of Refractory B-Cell Acute Lymphoblastic Leukemia With MLL Gene Amplification: A Case Report and Literature Review.

Lineage switches in acute leukemia occur rarely, and the underlying mechanisms are poorly understood. Herein, we report the case of an elderly patient with leukemia in which the leukemia started as B-cell acute lymphoblastic leukemia (B-ALL) and later changed to B- and T-cell mixed phenotype acute leukemia (MPAL) and acute myeloid leukemia (AML) during consecutive induction chemotherapy treatments. A 65-year-old woman was initially diagnosed with Philadelphia chromosome-negative B-ALL primarily expressing TdT/CD34/HLA-DR; more than 20% of the blasts were positive for CD19/CD20/cytoplasmic CD79a/cytoplasmic CD22/CD13/CD71.The blasts were negative for T-lineage markers and myeloperoxidase (MPO). Induction chemotherapy with the standard regimen for B-ALL resulted in primary induction failure. After the second induction chemotherapy regimen, the blasts were found to be B/T bi-phenotypic with additional expression of cytoplasmic CD3. A single course of clofarabine (the fourth induction chemotherapy regimen) dramatically reduced lymphoid marker levels. However, the myeloid markers (e.g., MPO) eventually showed positivity and the leukemia completely changed its lineage to AML. Despite subsequent intensive chemotherapy regimens designed for AML, the patient's leukemia was uncontrollable and a new monoblastic population emerged. The patient died approximately 8 months after the initial diagnosis without experiencing stable remission. Several cytogenetic and genetic features were commonly identified in the initial diagnostic B-ALL and in the following AML, suggesting that this case should be classified as lineage switching leukemia rather than multiple simultaneous cancers (i.e., de novo B-ALL and de novo AML, or primary B-ALL and therapy-related myeloid neoplasm). A complex karyotype was persistently observed with a hemi-allelic loss of chromosome 17 (the location of the TP53 tumor suppressor gene). As the leukemia progressed, the karyotype became more complex, with the additional abnormalities. Sequential target sequencing revealed an increased variant allele frequency of TP53 mutation. Fluorescent in situ hybridization (FISH) revealed an increased number of mixed-lineage leukemia (MLL) genes, both before and after lineage conversion. In contrast, FISH revealed negativity for MLL rearrangements, which are well-known abnormalities associated with lineage switching leukemia and MPAL. To our best knowledge, this is the first reported case of acute leukemia presenting with lineage ambiguity and MLL gene amplification.

Mixed Phenotype/Lineage Leukemia: Has Anything Changed for 2021 on Diagnosis, Classification, and Treatment?

PURPOSE OF REVIEW: Recent advances in the small field of the rare mixed phenotype acute leukemias (MPAL) are presented focusing on a better understanding of their pathophysiology and search for better therapeutic approaches. RECENT FINDINGS: Three aspects of respective classification, therapy, and immunophenotype of MPAL are reviewed. New proposals have been made to segregate MPAL subtypes based on their genomic landscape. In parallel, it was found that a large array of therapeutic approaches has been tested in the past few years with increasingly good results. Finally, we explored the use of unsupervised flow cytometry analysis to dissect subtle variations in markers expression to better characterize the variegating aspect of MPALs. Genomic and immunophenotypic aspects more clearly link MPAL subtypes with bona fide acute myeloblastic of lymphoblastic leukemias. This is likely to impact therapeutic strategies, towards a better management and outcome.

Pediatric Mixed-Phenotype Acute Leukemia: What's New?

Mixed-phenotype acute leukemias (MPAL) are rare in children and often lack consensus on optimal management. This review examines the current controversies and emerging paradigms in the management of pediatric MPAL. We examine risk stratification, outcomes of recent retrospective and prospective collaborative trials, and the role of transplantation and precision genomics, and outline emerging targets and concepts in this rare entity.

Case Report: The Formation of a Truncated PAX5 Transcript in a Case of Ph-Positive Mixed Phenotype Acute Leukemia With dic(7;9)(p11-p13;p13).

PAX5 plays a critical role in B-cell precursor development and is involved in various chromosomal translocations that involve the fusion of a portion of PAX5 to at least 49 different partners reported to date. Here, we identified a novel PAX5 fusion transcript in a Ph-positive mixed phenotype acute leukemia case with dic(7;9)(q13;q13), in which a translocation juxtaposes the 5' region of PAX5 and the ubiquitin-conjugating enzyme E2D4 (UBE2D4) to generate a PAX5-UBE2D4 fusion gene. To further explore the general characteristics and function of PAX5-UBE2D4, we cloned the full-length cDNA, which was amplified from the bone marrow of the patient. Interestingly, the fusion was located in the nucleus and negatively affected PAX5 transcription activity. Importantly, the fusion promoted tumor growth in nude mice and the proliferation of NIH3T3 cells in vitro. In conclusion, the fusion resulted in partial oncogenic activity, in contrast to the tumor suppressor activity of wild-type PAX5.

Leukemia With TCF3-ZNF384 Rearrangement as a Distinct Subtype of Disease With Distinct Treatments: Perspectives From A Case Report and Literature Review.

BACKGROUND: ZNF384 rearrangements are found in 5-10% of B-cell acute lymphoblastic leukemia (B-ALL) and 48% of B cell/myeloid mixed phenotype acute leukemia (B/M MPAL). ZNF384-rearranged B-ALL is prone to lineage conversion after chemotherapy. TCF3 is the second most common rearrangement partner of ZNF384 in B-ALL (27.5%) and the most common partner in B/M MPAL (53.3%). TCF3-ZNF384 fusion is related to a poor steroid response and a high frequency of relapse. It is mostly reported in children and adolescents but rarely seen in adults. PATIENTS AND METHODS: Here, we report a rare case of adult common B-ALL with TCF3-ZNF384 fusion in which the patient relapsed after one cycle of consolidation chemotherapy. Relapsed leukemia cells from the bone marrow were cultured for 72 hours ex vivo, and a panel of 156 kinds of cytotoxic drugs, targeted therapy drugs, combination chemotherapy drugs, etc., was used for sensitivity screening. The literature on TCF3-ZNF384 fusion was reviewed, and reported cases with TCF3-ZNF384 fusion were summarized. Clinical characteristics were compared between B-ALL and MPAL with TCF3-ZNF384 fusion. RESULTS: The relapsed lymphoblasts showed moderate sensitivity to both acute myelocytic leukemia (AML) - and acute lymphoblastic leukemia (ALL)-directed combination chemotherapy schemes, as well as to multiple targeted therapeutic drugs. The hyper-CVAD (B) scheme showed synergistic effects with multiple targeted compounds and had the highest sensitivity. The patient chose the hyper-CVAD (B) scheme combined with sorafenib and achieved complete remission (CR), then consolidated with myeloid-directed homoharringtonine+cytarabine+daunorubicin (HAD) scheme and gained molecular CR. By reviewing the literature, we found that both the genomic landscapes and gene expression profiles of ZNF384-rearranged B-ALL and MPAL are similar and that both diseases have lineage plasticity. The gene expression profile in TCF3-ZNF384-positive patients shows enrichment of hematopoietic stem cell features. No significant differences in clinical characteristics were found between TCF3-ZNF384-positive ALL and MPAL. CONCLUSION: TCF3-ZNF384-positive leukemia may be a distinct subtype of leukemia regardless of immunophenotype. Considering the frequent lineage switches and sensitivity to both ALL- and AML-directed schemes, a uniform strategy directed at both lymphoid and myeloid lineages or at hematopoietic stem cells may be better for TCF3-ZNF384-positive leukemia. Small molecule targeted therapies may be promising treatment options and deserve further investigation.

Update on Acute Leukemias of Ambiguous Lineage.

Acute leukemias of ambiguous lineage are a heterogenous group of diseases that include acute undifferentiated leukemias and mixed-phenotype acute leukemias (MPALs). These leukemias pose a challenge for pathologists and clinicians alike in diagnosis, treatment, and further management. Recent genetic characterization has provided insights into their underlying biology and classification, and has offered potential for targeted therapies. This article addresses diagnosis of MPALs with examples of the most common pitfalls, recent comprehensive molecular studies, and advancement in treatment and follow-up modalities.

Efficacy and safety of low-dose imatinib in an elderly patient with mixed phenotype acute leukemia with t(9;22)(q34;q11.2);BCR-ABL1.

Low-dose imatinib with monitoring of drug concentrations in blood may successfully control Philadelphia chromosome-positive mixed phenotype acute leukemia (Ph+MPAL), particularly in elderly patients with comorbidities.

Mixed phenotype acute leukemia in a child associated with a NUP98-NSD1 fusion and NRAS p.Gly61Arg mutation.

BACKGROUND: Mixed phenotype acute leukemia (MPAL) is a rare subset of acute leukemia in the pediatric population associated with genetic alterations seen in both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). CASE: We describe a patient with MPAL with a NUP98 (nucleoporin 98)-NSD1 gene fusion (nuclear receptor binding SET domain protein1) and NRAS (neuroblastoma RAS viral oncogene homolog mutation) p.Gly61Arg mutation who was treated with upfront AML-based chemotherapy, received hematopoietic stem cell transplant (HSCT), but unfortunately died from relapsed disease. CONCLUSION: This case highlights the challenges faced in choosing treatment options in MPAL patients with complex genomics, with predominant myeloid features.

Hybrid chemotherapy regimen (FLAG-IDA-vincristine-prednisone) for acute leukemia with mixed-phenotype blasts.

BACKGROUND: Acute leukemia with mixed-phenotype blasts is associated with poor outcomes. There are no standard treatment regimens. Due to disease heterogeneity, controversy exists over whether an AML-based, ALL-based, or a combined (hybrid) AML/ALL-based regimen is most appropriate. MATERIALS AND METHODS: We conducted a single-center, retrospective case series review of patients with acute leukemia with mixed phenotype blasts as described by the European Group for Immunological Characterization of Leukemia (EGIL) or the 2008 WHO classification. Patients were treated from November 2014 and December 2019 with the combination chemotherapy regimen FLAG-idarubicin-vincristine-prednisone with or without rituximab. Outcomes included induction response, time to transplant, time to relapse, overall survival, time to neutrophil or platelet recovery, infection, and duration of hospitalization. RESULTS: The median age was 68 years (range 21-77). Six patients (87.5 %) had unfavorable/complex cytogenetics. All patients achieved a complete remission (CR) or complete remission with incomplete hematologic recovery (CRi). Estimated 1-year overall survival was 85.7 %. There were no deaths during induction, with a 22 day median duration of hospitalization for induction. CONCLUSION: The combination of FLAG, idarubicin, vincristine, and prednisone (FLAG-VIPR) demonstrated favorable induction responses in a disease state with historically poor outcomes and should be studied in a prospective clinical trial.

Acute lymphoblastic leukemia-like treatment regimen provides better response in mixed phenotype acute leukemia: a comparative study between adults and pediatric MPAL patients.

Mixed phenotype acute leukemia (MPAL) is a rare type of leukemia with a limited number of studies conducted to characterize its clinical spectrum and most importantly the best treatment modality. MPAL blasts show more than one phenotype either myeloid/monocytic with T- or B-lymphoid or extremely rare triple lineage associated phenotypic markers. This study aimed to characterize MPAL cases with special emphasis on comparing adult and pediatric age groups, exploring treatment regimens, and clinical outcome. Among 2571 acute leukemia patients, 102 MPAL cases fulfilling the 2008/2016 WHO diagnostic criteria of MPAL were recruited in the study. The incidence of MPAL was 4% of acute leukemia patients. Pediatric cases were 54 (53%) while adults were 48/102 (47%). Myeloid/B-lymphoid phenotype was found in 86/102 (84%), with BCR-ABL fusion gene transcript detected in 14/102(13.7%) patients. ALL-like treatment showed better response rates as compared with the myeloid based regimen (p = 0.001). MPAL behaves in a manner that resembles in clinical features, their lymphoid progenitor counterpart leukemias both in adults and pediatric patients with superior treatment response to ALL-like regimen, especially in adults.