First report of familial mixed phenotype acute leukemia: shared clinical characteristics, Philadelphia translocation, and germline variants.

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.

Clinico-hematological and immunophenotypic profile of acute leukemia of ambiguous lineage: A four year experience from a single tertiary care centre of West India.

Background: : Acute leukemia of ambiguous lineage (ALAL) is a heterogeneous group of rare leukemias that lacks definite evidence of differentiation along one lineage. It includes acute undifferentiated leukemia and mixed-phenotype acute Leukaemia (MPAL). Aims: The present study highlighted the clinicohematological and immunophenotypic profile of ALAL cases diagnosed in the tertiary care centre of western India. Study Design: Retrospective observational study. Methods and Materials: Patients diagnosed with acute leukemia, preferably their bone marrow aspirate samples, were collected in ethylene diamine tetra-acetic acid-vial. The immunophenotypic study was done using the Fluorescence Activated Cell Sorting (FACS) Canto Eight color flow cytometer and a broad panel of monoclonal antibodies. The patients were diagnosed for ALAL, based on the World Health Organization (WHO) 2017 classification. Statistical Analysis Used: Descriptive statistics with categorical and continuous variables. Results: Overall incidence of ALAL was about 2.1%, and singly MPAL and AUL were 1.8%, and 0.3%, in frequency, respectively. The age range was 4 to 57 (median age 21 years), of which 11 (57.9%) were adults and eight were children (42.1%). There was male predominance with Male: Female ratio of 5.3:1. On immunophenotyping, B/myeloid MPAL (42.1%) was the commonest among the ALAL. Translocation t(9;22) was the most common cytogenetic abnormality found mainly in B/myeloid MPAL. Two cases were of chronic myeloid leukemia in a mixed phenotypic blast crisis. Over all prognosis of ALAL was poor. Conclusion: ALAL is overall associated with poor prognosis as both their diagnosis and treatment decisions are difficult owing to the lack of optimum treatment data and the rarity of the disease. Hence, A careful diagnosis with the help of immunophenotyping is crucial.

How I Diagnose Acute Leukemia of Ambiguous Lineage.

OBJECTIVES: Classification of acute leukemia involves assigning lineage by resemblance to normal progenitor cells. This approach provides descriptive information about the blast cells that is useful for disease monitoring, provides clues to pathogenesis, and can help clinicians select effective chemotherapeutic regimens. Acute leukemias of ambiguous lineage (ALALs) are those leukemias that either fail to show evidence of myeloid, B-, or T-lymphoid lineage commitment or show evidence of commitment to more than 1 lineage. The different treatment regimens for acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) make ALAL a challenge both diagnostically and therapeutically. METHODS: Current classification criteria have reduced the reported incidence of mixed-lineage leukemias by emphasizing fewer markers and categorizing some biphenotypic leukemias with recurrent cytogenetic abnormalities as other entities. Several recent studies have explored the genomic and epigenetic landscape of mixed-phenotype acute leukemia (MPAL) and have suggested a further refinement of the World Health Organization classification to emphasize the genomic heterogeneity of MPAL. RESULTS: Genomic and expression profile data for MPAL reveal mutations commonly seen in both AML and ALL, with T-/myeloid MPAL showing overlapping features with early T-cell precursor lymphoblastic leukemia. CONCLUSIONS: Our review aimed to discuss the diagnostic challenges, recent genomic studies, and therapeutic strategies in this poorly understood disease.

The Intrinsically Disordered Proteins MLLT3 (AF9) and MLLT1 (ENL) - Multimodal Transcriptional Switches With Roles in Normal Hematopoiesis, MLL Fusion Leukemia, and Kidney Cancer.

AF9 (MLLT3) and ENL (MLLT1) are members of the YEATS family (named after the five proteins first shown to contain this domain: Yaf9, ENL, AF9, Taf14, Sas5) defined by the presence of a YEATS domain. The YEATS domain is an epigenetic reader that binds to acetylated and crotonylated lysines, unlike the bromodomain which can only bind to acetylated lysines. All members of this family have been shown to be components of various complexes with roles in chromatin remodeling, histone modification, histone variant deposition, and transcriptional regulation. MLLT3 is a critical regulator of hematopoiesis with a role in maintaining the hematopoietic stem or progenitor cell (HSPC) population. Approximately 10% of acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL) patients harbor a translocation involving MLL (mixed lineage leukemia). In the context of MLL fusion patients with AML and ALL, MLL-AF9 and MLL-ENL fusions are observed in 34 and 31% of the patients, respectively. The intrinsically disordered C-terminal domain of MLLT3 (AHD, ANC1 homology domain) undergoes coupled binding and folding upon interaction with partner proteins AF4, DOT1L, BCOR, and CBX8. Backbone dynamics studies of the complexes suggest a role for dynamics in function. Inhibitors of the interaction of the intrinsically disordered AHD with partner proteins have been described, highlighting the feasibility of targeting intrinsically disordered regions. MLLT1 undergoes phase separation to enhance recruitment of the super elongation complex (SEC) and drive transcription. Mutations in MLLT1 observed in Wilms tumor patients enhance phase separation and transcription to drive an aberrant gene expression program.

Use of midostaurin in mixed phenotype acute leukemia with FLT3 mutation: A case series.

Mixed phenotype acute leukemia (MPAL) is a rare type of acute leukemia where blasts present phenotypes from more than one lineage. A poor prognostic has been associated with this disease, and limited data are currently available to guide the choice of therapy. Regarding FLT3-positive MPAL, only one case treated with midostaurin has been published to date. Here, we report the successful use of midostaurin to treat three FLT3-positive MPAL T/myeloid and B/myeloid patients. Midostaurin was successfully added to intensive induction (two patients) and consolidation chemotherapy (three patients) without significant adverse events requiring a dose adjustment or discontinuation. The therapy received resulted in complete remission for two patients and complete remission with an incomplete hematologic recovery for the third. All patients proceeded to HSCT and stayed in remission after an extended follow-up respectively at 28, 31, and 11 months later. These results suggest that the addition of midostaurin during induction and consolidation therapy may represent a treatment option for FLT3-positive MPAL.

Glycoproteome remodeling in MLL-rearranged B-cell precursor acute lymphoblastic leukemia.

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) with mixed-lineage leukemia gene rearrangement (MLL-r) is a poor-prognosis subtype for which additional therapeutic targets are urgently needed. Currently no multi-omics data set for primary MLL r patient cells exists that integrates transcriptomics, proteomics and glycomics to gain an inclusive picture of theranostic targets. Methods: We have integrated transcriptomics, proteomics and glycomics to i) obtain the first inclusive picture of primary patient BCP-ALL cells and identify molecular signatures that distinguish leukemic from normal precursor B-cells and ii) better understand the benefits and limitations of the applied technologies to deliver deep molecular sequence data across major cellular biopolymers. Results: MLL-r cells feature an extensive remodeling of their glycocalyx, with increased levels of Core 2-type O-glycans and complex N-glycans as well as significant changes in sialylation and fucosylation. Notably, glycosaminoglycan remodeling from chondroitin sulfate to heparan sulfate was observed. A survival screen, to determine if glycan remodeling enzymes are redundant, identified MGAT1 and NGLY1, essential components of the N-glycosylation/degradation pathway, as highly relevant within this in vitro screening. OGT and OGA, unique enzymes that regulate intracellular O-GlcNAcylation, were also indispensable. Transcriptomics and proteomics further identified Fes and GALNT7-mediated glycosylation as possible therapeutic targets. While there is overall good correlation between transcriptomics and proteomics data, we demonstrate that a systematic combined multi-omics approach delivers important diagnostic information that is missed when applying a single omics technology. Conclusions: Apart from confirming well-known MLL-r BCP-ALL glycoprotein markers, our integrated multi-omics workflow discovered previously unidentified diagnostic/therapeutic protein targets.

A pediatric BAL case with double Ph chromosomes and trisomy 5.

Biphenotypic acute leukemias (BAL) are known as a type of leukemia involving cells with myeloid and along with lymphoid origin, in which genomic changes are detected. It has been stated that the most common genomic changes in BAL are t(9;22) and the translocations of the 11q23 region, these anomalies cause poor prognostic effects. We detected trisomy 5 (+5) in addition to the double Ph chromosome in a case where we investigated the genomic changes using molecular and conventional cytogenetic methods. Bone marrow transplantation was planned due to the poor response to prednisone. According to the information we have obtained, our report will be the first article to discuss the aberrations found in addition to the Ph chromosome in BAL and the effect of these aberrations on prognosis. However, the double observation of the Ph chromosome, which has a poor prognostic effect, is expected to affect the prognosis more negatively, this case will contribute to the literature in terms of trisomy 5. We think that more case reports are needed to reveal the anomalies and their prognostic significance in BAL.

Deubiquitinating enzyme inhibitor alleviates cyclin A1-mediated proteasome inhibitor tolerance in mixed-lineage leukemia.

Drug resistance is a significant obstacle to effective cancer treatment. Drug resistance develops from initially reversible drug-tolerant cancer cells, which offer therapeutic opportunities to impede cancer relapse. The mechanisms of resistance to proteasome inhibitor (PI) therapy have been investigated intensively, however the ways by which drug-tolerant cancer cells orchestrate their adaptive responses to drug challenges remain largely unknown. Here, we demonstrated that cyclin A1 suppression elicited the development of transient PI tolerance in mixed-lineage leukemia (MLL) cells. This adaptive process involved reversible downregulation of cyclin A1, which promoted PI resistance through cell-cycle arrest. PI-tolerant MLL cells acquired cyclin A1 dependency, regulated directly by MLL protein. Loss of cyclin A1 function resulted in the emergence of drug tolerance, which was associated with patient relapse and reduced survival. Combination treatment with PI and deubiquitinating enzyme (DUB) inhibitors overcame this drug resistance by restoring cyclin A1 expression through chromatin crosstalk between histone H2B monoubiquitination and MLL-mediated histone H3 lysine 4 methylation. These results reveal the importance of cyclin A1-engaged cell-cycle regulation in PI resistance in MLL cells, and suggest that cell-cycle re-entry by DUB inhibitors may represent a promising epigenetic therapeutic strategy to prevent acquired drug resistance.

Mixed Phenotype Acute Leukemia, B/Myeloid (Bilineal and Biphenotypic), With t(2;22)(q35;q12);EWSR1-FEV.

BACKGROUND: Ewing sarcoma breakpoint region 1 gene (EWSR1) rearrangements are largely associated with the Ewing sarcoma family of tumors. OBSERVATIONS: We report the first case of infantile, mixed phenotype acute leukemia, B/myeloid (bilineal and biphenotypic [B-lymphoid and B-lymphoid/myeloid]), with a t(2;22)(q35;q12). The EWSR1-fifth Ewing variant gene fusion and nonsense mutation in STAG2 were detected by next-generation sequencing and markedly high expression of fifth Ewing sarcoma variant mRNA detected by quantitative reverse transcription polymerase chain reaction. The patient was treated with a combined myeloid/lymphoid leukemia regimen followed by allogeneic stem cell transplant and was in complete remission at 3.8-year follow-up. CONCLUSIONS: Our case study underscores the importance of a comprehensive evaluation of acute leukemia and provides insights into the phenotype of EWSR1 rearranged neoplasms in the context of partner genes and cell type.

Identification of a novel KMT2A/GIMAP8 gene fusion in a pediatric patient with acute undifferentiated leukemia.

Acute undifferentiated leukemia (AUL) is a very rare hematologic neoplasm that expresses no markers specific for either myeloid or lymphoid lineages. While commonly observed in several acute leukemias, KMT2A rearrangements in AUL have been rarely reported in the literature. We report the third case to our knowledge of AUL harboring a KMT2A rearrangement. Furthermore, the KMT2A/GIMAP8 gene fusion identified in this case represents a novel KMT2A rearrangement.

An Original Complex Rearrangement Involving Chromosomes 9, 11, and 14, Harboring a Complex KMT2A Gene Rearrangement in an Infant With Mixed-phenotype Acute Leukemia.

KMT2A gene rearrangements represent the most frequent group of abnormalities in childhood leukemia (~70% of cases), with over 120 rearrangements described. The investigation of KMT2A rearrangements is still a vast field to be explored. Several studies have been characterizing different outcomes and leukemogenic mechanisms, depending on the translocation partner gene involved in childhood KMT2A-r leukemias. Therefore, the detection of the translocation partner gene, including in the context of complex rearrangements, may help to better delineate the disease. Here, we describe clinical and molecular cytogenetic data of a new complex variant translocation, involving chromosomes 9, 11, and 14, presenting a KMT2A gene extra copy and rearrangements, in an infant with de novo mixed-phenotype acute leukemia.

A transcriptomic continuum of differentiation arrest identifies myeloid interface acute leukemias with poor prognosis.

Classification of acute lymphoblastic and myeloid leukemias (ALL and AML) remains heavily based on phenotypic resemblance to normal hematopoietic precursors. This framework can provide diagnostic challenges for immunophenotypically heterogeneous immature leukemias, and ignores recent advances in understanding of developmental multipotency of diverse normal hematopoietic progenitor populations that are identified by transcriptional signatures. We performed transcriptional analyses of a large series of acute myeloid and lymphoid leukemias and detected significant overlap in gene expression between cases in different diagnostic categories. Bioinformatic classification of leukemias along a continuum of hematopoietic differentiation identified leukemias at the myeloid/T-lymphoid interface, which shared gene expression programs with a series of multi or oligopotent hematopoietic progenitor populations, including the most immature CD34+CD1a-CD7- subset of early thymic precursors. Within these interface acute leukemias (IALs), transcriptional resemblance to early lymphoid progenitor populations and biphenotypic leukemias was more evident in cases originally diagnosed as AML, rather than T-ALL. Further prognostic analyses revealed that expression of IAL transcriptional programs significantly correlated with poor outcome in independent AML patient cohorts. Our results suggest that traditional binary approaches to acute leukemia categorization are reductive, and that identification of IALs could allow better treatment allocation and evaluation of therapeutic options.

A regulatory element in the 3'-untranslated region of CEBPA is associated with myeloid/NK/T-cell leukemia.

OBJECTIVES: CCAAT/enhancer-binding protein α (CEBPA) is an essential transcription factor for myeloid differentiation. Not only mutation of the CEBPA gene, but also promoter methylation, which results in silencing of CEBPA, contributes to the pathogenesis of acute myeloid leukemia (AML). We sought for another differentially methylated region (DMR) that associates with the CEBPA silencing and disease phenotype. METHODS: Using databases, we identified a conserved DMR in the CEBPA 3'-untranslated region (UTR). RESULTS: Methylation-specific PCR analysis of 231 AML cases showed that hypermethylation of the 3'-UTR was associated with AML that had a myeloid/NK/T-cell phenotype and downregulated CEBPA. Most of these cases were of an immature phenotype with CD7/CD56 positivity. These cases were significantly associated with lower hemoglobin levels than the others. Furthermore, we discovered that the CEBPA 3'-UTR DMR can enhance transcription from the CEBPA native promoter. In vitro experiments identified IKZF1-binding sites in the 3'-UTR that are responsible for this increased transcription of CEBPA. CONCLUSIONS: These results indicate that the CEBPA 3'-UTR DMR is a novel regulatory element of CEBPA related to myeloid/NK/T-cell lineage leukemogenesis. Transcriptional regulation of CEBPA by IKZF1 may provide a clue for understanding the fate determination of myeloid vs. NK/T-lymphoid progenitors.

Mixed phenotype acute leukaemia with t(9,22), BCR-ABL1: A case report.

INTRODUCTION: Mixed phenotype acute leukaemia (MPAL) is a rare entity of acute leukaemia. CASE REPORT: Here we report a case of a 39-year-old lady, with an incidental finding of hyperleukocytosis (white blood cells count: 139.2 x 109/L). Her peripheral blood film revealed 36% of blasts and a bone marrow aspiration showed 53% of blasts. Immunophenotyping showed a population of blasts exhibiting positivity of two lineages, myeloid lineage and B-lymphoid lineage with strong positivity of CD34 and terminal deoxynucleotidyl transferase (Tdt). A conventional karyotyping revealed the presence of Philadelphia chromosome. She was diagnosed with MPAL with t(9,22), BCR ABL1, which carried a poor prognosis. She was treated with acute lymphoblastic leukaemia (ALL) chemotherapy protocol coupled with a tyrosine kinase inhibitor and was planned for an allogeneic stem cells transplant. CONCLUSION: This MPAL case was diagnosed incidentally in an asymptomatic patient during medical check-up. We highlight this rare case report to raise the awareness about this rare disease. Understanding the pathogenesis of the disease with the underlying genes responsible for triggering the disease, uniform protocols for diagnosis and targeted treatment will help for proper management of these patients.

Heritable genetic background alters survival and phenotype of Mll-AF9-induced leukemias.

The MLL-AF9 fusion protein occurring as a result of t(9;11) translocation gives rise to pediatric and adult acute leukemias of distinct lineages, including acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and mixed-phenotype acute leukemia (MPAL). The mechanisms underlying how this same fusion protein results in diverse leukemia phenotypes among different individuals are not well understood. Given emerging evidence from genome-wide association studies that genetic risk factors contribute to MLL-rearranged leukemogenesis, here we tested the impact of genetic background on survival and phenotype of a well-characterized Mll-AF9 knockin mouse model. We crossed this model with five distinct inbred strains (129, A/J, C57BL/6, NOD, CAST) and tested their F1 hybrid progeny for dominant genetic effects on Mll-AF9 phenotypes. We discovered that genetic background altered peripheral blood composition, with Mll-AF9 CAST F1 having a significantly increased B-lymphocyte frequency, while the remainder of the strains exhibited myeloid-biased hematopoiesis, similar to the parental line. Genetic background also had an impact on overall survival, with Mll-AF9 A/J F1 and Mll-AF9 129 F1 having significantly shorter survival and Mll-AF9 CAST F1 having longer survival, compared with the parental line. Furthermore, we observed a range of hematologic malignancies, with Mll-AF9 A/J F1, Mll-AF9 129 F1, and Mll-AF9 B6 F1 developing exclusively myeloid cell malignancies (myeloproliferative disorder [MPD] and AML), whereas a subset of Mll-AF9 NOD F1 developed MPAL and Mll-AF9 CAST F1 developed ALL. This study provides a novel in vivo experimental model in which to evaluate the underlying mechanisms by which MLL-AF9 results in diverse leukemia phenotypes and provides definitive experimental evidence that genetic risk factors contribute to survival and phenotype of MLL-rearranged leukemogenesis.

The reciprocal world of MLL fusions: A personal view.

Over the last 15 years the Diagnostic Center of Acute Leukemia (DCAL) at the Frankfurt University has diagnosed and elucidated the Mixed Lineage Leukemia (MLL) recombinome with >100 MLL fusion partners. When analyzing all these different events, balanced chromosomal translocations were found to comprise the majority of these cases (~70%), while other types of genetic rearrangements (3-way-translocations, spliced fusions, 11q inversions, interstitial deletions or insertion of chromosomal fragments into other chromosomes) account for about 30%. In nearly all those complex cases, functional fusion proteins can be produced by transcription, splicing and translation. With a few exceptions (10 out of 102 fusion genes which were per se out-of-frame), all these genetic rearrangements produced a direct MLL fusion gene, and in 94% of cases an additional reciprocal fusion gene. So far, 114 patients (out of 2454 = ~5%) have been diagnosed only with the reciprocal fusion allele, displaying no MLL-X allele. The fact that so many MLL rearrangements bear at least two fusion alleles, but also our findings that several direct MLL fusions were either out-of-frame fusions or missing, raises the question about the function and importance of reciprocal MLL fusions. Recent findings also demonstrate the presence of reciprocal MLL fusions in sarcoma patients. Here, we want to discuss the role of reciprocal MLL fusion proteins for leukemogenesis and beyond.

[Therapeutic Outcomes of 28 ALL Patients with MLL Gene Rearrangement].

OBJECTIVE: To analyze the therapeutic outcomes of acute lymphoblastic leukemia(ALL) patients with MLL gene rearrangement. METHODS: Clinical outcomes of 28 ALL patients aged less than 12 years old with MLL gene rearrangement treated with Chinese Childhood Leukemia Group ALL 2008 (CCLG -ALL 2008) protocol from January 2008 to April 2015 were retrospectively analyzed. RESULTS: Among 28 patients, 14 were boys and 14 were girls, median age was 36(4-144) months and median follow-up period was 12.5 (ranged 2-106) months, and 10 were infants. All patients were given induction chemotherapy and the high-risk project of CCLG-ALL 2008. At the last time of follow-up, five patients gave up and were loss to follow-up. The 5 years OS and EFS were 52.2% and 47.8% respectively for the other 23 patients. The 5 years OS and EFS(28.6% and, 88.9%), (P=0.012), (28.6%, 77.8%), (P=0.048) rate of WBC≥50×109/L and ＜50×109/L groups at first vistit showed a significantly different. Patients with good response to predisone, less than 12 months， sex， CD10- showed low trend of 5 years OS and EFS, however there was no statistical difference (P＞0.05). CONCLUSION: The 5 years OS is 52.2% and 5 years EFS is 47.8% for those ALL patients with MLL gene rearrangement. The WBC≥50×109/L at first visit is adverse prognostic factor.

The molecular functions of common and atypical MLL fusion protein complexes.

Mixed-lineage leukemia (MLL) fuses with a variety of partners to produce a functionally altered MLL complex that is not expressed in normal cells, which transforms normal hematopoietic progenitors into leukemia cells. Because more than 80 fusion partners have been identified to date, the molecular functions of MLL fusion protein complexes appear diverse. However, over the past decade, the common functions utilized for leukemic transformation have begun to be elucidated. It appears that most (if not all) MLL fusion protein complexes utilize the AF4/ENL/P-TEFb and DOT1L complexes to some extent. Based on an understanding of the underlying molecular mechanisms, several molecular targeting drugs are being developed, opening paths to novel therapies. Here, we review the recent progress made in identifying the molecular functions of various MLL fusions and categorize the numerous fusion partners into several functionally-distinct groups to help discern commonalities and differences among various MLL fusion protein complexes.