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

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

Unusual Presentation of SET::NUP214-Associated Concomitant Hematological Neoplasm in a Child-Diagnostic and Treatment Struggle.

Simultaneous multilineage hematologic malignancies are uncommon and associated with poorer prognosis than single-lineage leukemia or lymphoma. Here, we describe a concomitant malignant neoplasm in a 4-year-old boy. The child presented with massive lymphoproliferative syndrome, nasal breathing difficulties, and snoring. Morphological, immunocytochemical, and flow cytometry diagnostics showed coexistence of acute myeloid leukemia (AML) and peripheral T-cell lymphoma (PTCL). Molecular examination revealed a rare t(9;9)(q34;q34)/SET::NUP214 translocation as well as common TCR clonal rearrangements in both the bone marrow and lymph nodes. The disease showed primary refractoriness to both lymphoid and myeloid high-dose chemotherapy as well as combined targeted therapy (trametinib + ruxolitinib). Hence, HSCT was performed, and the patient has since been in complete remission for over a year. This observation highlights the importance of molecular techniques for determining the united nature of complex SET::NUP214-positive malignant neoplasms arising from precursor cells with high lineage plasticity.

TCF3 gene rearrangements in pediatric B-cell acute lymphoblastic leukemia-A single center experience.

INTRODUCTION: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common neoplasm in children. One of the long known recurrent rearrangements in BCP-ALL is t(1;19)(q23;p13.3)/TCF3::PBX1. However, other TCF3 gene rearrangements were also described that are associated with significant difference in ALL prognosis. METHODS: The current study aimed to analyze the spectrum of TCF3 gene rearrangements in children in Russian Federation. A cohort of 203 patients with BCP-ALL was selected based on FISH screening and was studied by karyotyping, FISH, RT-PCR and high throughput sequencing. RESULTS: T(1;19)(q23;p13.3)/TCF3::PBX1 is the most common aberration in TCF3-positive pediatric BCP-ALL (87.7%), with its unbalanced form prevailing. It resulted from TCF3::PBX1 exon 16-exon 3 fusion junction (86.2%) or unconventional exon 16-exon 4 junction (1.5%). Rarer events included t(12;19)(p13;p13.3)/TCF3::ZNF384 (6.4%) and t(17;19)(q21-q22;p13.3)/TCF3::HLF (1.5%). The latter translocations demonstrated high molecular heterogeneity and complex structure-four distinct transcripts were shown for TCF3::ZNF384 and each patient with TCF3::HLF had a unique transcript. These features hamper TCF3 rearrangement primary detection by molecular methods and brings FISH screening to the fore. A case of novel TCF3::TLX1 fusion in a patient with t(10;19)(q24;p13) was also discovered. Survival analysis within the national pediatric ALL treatment protocol demonstrated the severe prognosis of TCF3::HLF compared to both TCF3::PBX1 and TCF3::ZNF384. CONCLUSION: So, high molecular heterogeneity of TCF3 gene rearrangement in pediatric BCP-ALL was demonstrated and a novel fusion gene TCF3::TLX1 was described.

Recognizing Minor Leukemic Populations with Monocytic Features in Mixed-Phenotype Acute Leukemia by Flow Cell Sorting Followed by Cytogenetic and Molecular Studies: Report of Five Exemplary Cases.

Mixed-phenotype acute leukemia (MPAL), a rare and heterogeneous category of acute leukemia, is characterized by cross-lineage antigen expression. Leukemic blasts in MPAL can be represented either by one population with multiple markers of different lineages or by several single-lineage populations. In some cases, a major blast population may coexist with a smaller population that has minor immunophenotypic abnormalities and may be missed even by an experienced pathologist. To avoid misdiagnosis, we suggest sorting doubtful populations and leukemic blasts and searching for similar genetic aberrations. Using this approach, we examined questionable monocytic populations in five patients with dominant leukemic populations of B-lymphoblastic origin. Cell populations were isolated either for fluorescence in situ hybridization or for clonality assessment by multiplex PCR or next-generation sequencing. In all cases, monocytic cells shared the same gene rearrangements with dominant leukemic populations, unequivocally confirming the same leukemic origin. This approach is able to identify implicit cases of MPAL and therefore leads to the necessary clinical management for patients.

Molecular Heterogeneity of Pediatric AML with Atypical Promyelocytes Accumulation in Children-A Single Center Experience.

Acute promyelocytic leukemia (APL) pathogenesis is based on RARA gene translocations, which are of high importance in the diagnosis of and proper therapy selection for APL. However, in some cases acute myeloid leukemia (AML) demonstrates APL-like morphological features such as atypical promyelocytes accumulation. This type of AML is characterized by the involvement of other RAR family members or completely different genes. In the present study, we used conventional karyotyping, FISH and high-throughput sequencing in a group of 271 de novo AML with atypical promyelocytes accumulation. Of those, 255 cases were shown to carry a typical chromosomal translocation t(15;17)(q24;q21) with PML::RARA chimeric gene formation (94.1%). Other RARA-positive cases exhibited cryptic PML::RARA fusion without t(15;17)(q24;q21) (1.8%, n = 5) and variant t(5;17)(q35;q21) translocation with NPM1::RARA chimeric gene formation (1.5%, n = 4). However, 7 RARA-negative AMLs with atypical promyelocytes accumulation were also discovered. These cases exhibited TBL1XR1::RARB and KMT2A::SEPT6 fusions as well as mutations, e.g., NPM1 insertion and non-recurrent chromosomal aberrations. Our findings demonstrate the genetic diversity of AML with APL-like morphological features, which is of high importance for successful therapy implementation.

Correlation of the surface expression of thymic stromal lymphopoietin receptor with the presence of CRLF2 gene rearrangements in children with B-lineage acute lymphoblastic leukemia.

INTRODUCTION: In this study, we aimed to compare the immunophenotype of tumor cells in children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) harboring rearrangements of the CRLF2 gene with that in children without such aberrations with a specific focus on the surface expression of the related protein thymic stromal lymphopoietin receptor (TSLPR). METHODS: We examined bone marrow samples from 46 patients with primary BCP-ALL who had CRLF2 rearrangements detected by FISH (CRLF2(+) cohort). A total of 140 consecutive patients with intact CRLF2 were included in a control CRLF2(-) cohort. TSLPR expression was studied by flow cytometry. RESULTS: The majority of CRLF2(+) patients were conventionally positive (≥20% positive cells) for TSLPR (33 of 46, 71.7%). Among the remaining children in this group, two were completely TSLPR-negative, seven had less than 10% TSLPR-positive cells, and four had between 10% and 20% TSLPR-positive cells. By contrast, the majority of CRLF2(-) patients had no TSLPR-positive cells (119 of 140, 85.0%), while in 15 cases (10.7%), the percentage of TSLPR-positive cells was below 10%, and in six cases (4.3%), it was between 10% and 20%. Receiver operator characteristic analysis revealed a threshold of only 1.6% TSLPR-positive cells for the effective prediction of the presence of CRLF2 rearrangement. Moreover, this threshold retained its predictive value when only children with low TSLPR positivity were studied. CONCLUSION: When surface TSLPR is detected at the diagnosis of BCP-ALL, close attention should be given to the search for chromosomal aberrations involving CRLF2 at any level of expression.

Genetic characteristics and treatment outcome in infants with KMT2A germline B-cell precursor acute lymphoblastic leukemia: Results of MLL-Baby protocol.

The aim of this study was to present the diagnostic and outcome characteristics of infants with germline status of KMT2A gene (KMT2A-g) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) treated consistently according to the MLL-Baby protocol, a moderate-intensity protocol. Of the 139 patients enrolled in the MLL-Baby study, 100 (71.9%) carried different types of rearranged KMT2A (KMT2A-r), while the remaining 39 infants (28.1%) had KMT2A-g. KMT2A-g patients were generally older (77% older than 6 months), less likely to have a very high white blood cell count (greater than 100 × 109 /L), less likely to be central nervous system (CNS)-positive, and more likely to be CD10-positive. The 6-year event-free survival and overall survival rates for all 39 patients were 0.74 (standard error [SE] 0.07) and 0.80 (SE 0.07), respectively. Relapse was the most common adverse event (n = 5), with a cumulative incidence of relapse (CIR) of 0.13 (SE 0.06), while the incidence of a second malignancy (n = 1) and death in remission (n = 3) was 0.03 (SE 0.04) and 0.08 (SE 0.04), respectively. None of the initial parameters, including genetics and the presence of recently described fusions of NUTM1 and PAX5 genes, was able to distinguish patients with different outcomes. Only rapidity of response, measured as minimal residual disease (MRD) by flow cytometry, showed a statistically significant impact. Moderate-intensity therapy, as used in the MLL-Baby protocol in infants with KMT2A-g BCP-ALL, yields results comparable to other infant studies. Patients with a slow multicolor flow cytometry (MFC)-MRD response should be subjected to advanced therapies, such as targeted or immunotherapies.

Reliable Flow-Cytometric Approach for Minimal Residual Disease Monitoring in Patients with B-Cell Precursor Acute Lymphoblastic Leukemia after CD19-Targeted Therapy.

We aimed to develop an antibody panel and data analysis algorithm for multicolor flow cytometry (MFC), which is a reliable method for minimal residual disease (MRD) detection in patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) treated with CD19-directed therapy. The development of the approach, which was adapted for the case of possible CD19 loss, was based on the additional B-lineage marker expression data obtained from a study of primary BCP-ALL patients, an analysis of the immunophenotypic changes that occur during blinatumomab or CAR-T therapy, and an analysis of very early CD19-negative normal BCPs. We have developed a single-tube 11-color panel for MFC-MRD detection. CD22- and iCD79a-based primary B-lineage gating (preferably consecutive) was recommended. Based on patterns of antigen expression changes and the relative expansion of normal CD19-negative BCPs, guidelines for MFC data analysis and interpretation were established. The suggested approach was tested in comparison with the molecular techniques: IG/TR gene rearrangement detection by next-generation sequencing (NGS) and RQ-PCR for fusion-gene transcripts (FGTs). Qualitative concordance rates of 82.8% and 89.8% were obtained for NGS-MRD and FGT-MRD results, respectively. We have developed a sensitive and reliable approach that allows MFC-MRD monitoring after CD19-directed treatment, even in the case of possible CD19 loss.

Epigenetic regulator genes direct lineage switching in MLL/AF4 leukemia.

The fusion gene MLL/AF4 defines a high-risk subtype of pro-B acute lymphoblastic leukemia. Relapse can be associated with a lineage switch from acute lymphoblastic to acute myeloid leukemia, resulting in poor clinical outcomes caused by resistance to chemotherapies and immunotherapies. In this study, the myeloid relapses shared oncogene fusion breakpoints with their matched lymphoid presentations and originated from various differentiation stages from immature progenitors through to committed B-cell precursors. Lineage switching is linked to substantial changes in chromatin accessibility and rewiring of transcriptional programs, including alternative splicing. These findings indicate that the execution and maintenance of lymphoid lineage differentiation is impaired. The relapsed myeloid phenotype is recurrently associated with the altered expression, splicing, or mutation of chromatin modifiers, including CHD4 coding for the ATPase/helicase of the nucleosome remodelling and deacetylation complex. Perturbation of CHD4 alone or in combination with other mutated epigenetic modifiers induces myeloid gene expression in MLL/AF4+ cell models, indicating that lineage switching in MLL/AF4 leukemia is driven and maintained by disrupted epigenetic regulation.

B-lineage antigens that are useful to substitute CD19 for minimal residual disease monitoring in B cell precursor acute lymphoblastic leukemia after CD19 targeting.

BACKGROUND: The potential loss of CD19 during targeted treatment of B cell precursor acute lymphoblastic leukemia (BCP-ALL) can hamper flow cytometric minimal residual disease (MRD) monitoring. In the current study, we present expression data for antigens that are candidates for CD19 substitution: surface CD22, CD24, CD10, and intracellular (i) CD79a. METHODS: Bone marrow samples from 519 consecutive children (below 18 y.o.) with primary BCP-ALL were studied with a focus on expression of CD19, CD10, CD22, CD24, and iCD79a. As these antigens are planned to be used as substitutions for CD19 for primary B cell gating, only total expression on the leukemic population (≥95% cells) was considered appropriate. RESULTS: It was found that each of these antigens is totally expressed in nearly 90% of patients. For each single marker, a subgroup of patients without complete positivity presented with BCP-ALL harboring diverse cytogenetic and molecular genetic aberrations. Based on expression data, we have developed algorithm of simultaneous application of these antigens for initial B-lineage compartment gating, that is applicable for nearly all patients after CD19 targeting. CONCLUSION: We conclude that the addition of CD22, CD24, and iCD79a to the conventional antibody panel and their application together with CD10 allow for the identification of B-lineage compartment including residual tumor blasts, for MFC-MRD searching in virtually all patients with BCP-ALL after CD19-directed treatment.

Lineage Conversion in Pediatric B-Cell Precursor Acute Leukemia under Blinatumomab Therapy.

We report incidence and deep molecular characteristics of lineage switch in 182 pediatric patients affected by B-cell precursor acute lymphoblastic leukemia (BCP-ALL), who were treated with blinatumomab. We documented six cases of lineage switch that occurred after or during blinatumomab exposure. Therefore, lineage conversion was found in 17.4% of all resistance cases (4/27) and 3.2% of relapses (2/63). Half of patients switched completely from BCP-ALL to CD19-negative acute myeloid leukemia, others retained CD19-positive B-blasts and acquired an additional CD19-negative blast population: myeloid or unclassifiable. Five patients had KMT2A gene rearrangements; one had TCF3::ZNF384 translocation. The presented cases showed consistency of gene rearrangements and fusion transcripts across initially diagnosed leukemia and lineage switch. In two of six patients, the clonal architecture assessed by IG/TR gene rearrangements was stable, while in others, loss of clones or gain of new clones was noted. KMT2A-r patients demonstrated very few additional mutations, while in the TCF3::ZNF384 case, lineage switch was accompanied by a large set of additional mutations. The immunophenotype of an existing leukemia sometimes changes via different mechanisms and with different additional molecular changes. Careful investigation of all BM compartments together with all molecular -minimal residual disease studies can lead to reliable identification of lineage switch.

The Absence of Retroelement Activity Is Characteristic for Childhood Acute Leukemias and Adult Acute Lymphoblastic Leukemia.

Retroelements (RE) have been proposed as important players in cancerogenesis. Different cancer types are characterized by a different level of tumor-specific RE insertions. In previous studies, small cohorts of hematological malignancies, such as acute myeloid leukemia, multiple myeloma, and chronic lymphocytic leukemia have been characterized by a low level of RE insertional activity. Acute lymphoblastic leukemia (ALL) in adults and childhood acute leukemias have not been studied in this context. We performed a search for new RE insertions (Alu and L1) in 44 childhood ALL, 14 childhood acute myeloid leukemia, and 14 adult ALL samples using a highly sensitive NGS-based approach. First, we evaluated the method sensitivity revealing the 1% detection threshold for the proportion of cells with specific RE insertion. Following this result, we did not identify new tumor-specific RE insertions in the tested cohort of acute leukemia samples at the established level of sensitivity. Additionally, we analyzed the transcription levels of active L1 copies and found them increased. Thus, the increased transcription of active L1 copies is not sufficient for overt elevation of L1 retrotranspositional activity in leukemia.

Acute myeloid leukemia with t(X;6)9p11;q23);MYB-GATA1 and female sex: GATA1 insufficiency may be insufficient for pathogenesis.

Pediatric acute myeloid leukemia (AML) is genetically heterogenous (Olsson et al., 2016). t(X;6)(p11;q23) is a rare but recurrent chromosomal translocation in infant AML thought to be associated with male sex and basophilic differentiation (Dastugue et al., 1997). Here we report molecular characterization of AML with t(X;6)(p11;q23);MYB-GATA1 in two female infants and demonstrate preserved GATA1 expression in the sample tested. These findings further debunk a concept that this fusion was restricted to males, in whom it disrupts the only copy of the X-linked GATA1 gene, causing presumable complete loss of GATA1 function. Our data also demonstrate the power and efficiency of RNA sequencing for subclassification of leukemia on a clinically relevant timeline.

BTK, NUTM2A, and PRPF19 Are Novel KMT2A Partner Genes in Childhood Acute Leukemia.

Chromosomal rearrangements of the human KMT2A/MLL gene are associated with acute leukemias, especially in infants. KMT2A is rearranged with a big variety of partner genes and in multiple breakpoint locations. Detection of all types of KMT2A rearrangements is an essential part of acute leukemia initial diagnostics and follow-up, as it has a strong impact on the patients' outcome. Due to their high heterogeneity, KMT2A rearrangements are most effectively uncovered by next-generation sequencing (NGS), which, however, requires a thorough prescreening by cytogenetics. Here, we aimed to characterize uncommon KMT2A rearrangements in childhood acute leukemia by conventional karyotyping, FISH, and targeted NGS on both DNA and RNA level with subsequent validation. As a result of this comprehensive approach, three novel KMT2A rearrangements were discovered: ins(X;11)(q26;q13q25)/KMT2A-BTK, t(10;11)(q22;q23.3)/KMT2A-NUTM2A, and inv(11)(q12.2q23.3)/KMT2A-PRPF19. These novel KMT2A-chimeric genes expand our knowledge of the mechanisms of KMT2A-associated leukemogenesis and allow tracing the dynamics of minimal residual disease in the given patients.

Blinatumomab following haematopoietic stem cell transplantation - a novel approach for the treatment of acute lymphoblastic leukaemia in infants.

Blinatumomab with subsequent haematopoietic stem cell transplantation was applied in 13 infants with acute lymphoblastic leukaemia (ALL). Eight patients were treated in first remission due to slow clearance of minimal residual disease (MRD); one for MRD-reappearance after long MRD negativity, one for primary refractory disease and three during relapse treatment. In slow MRD responders, complete MRD response was achieved prior to transplantation, with an 18-month event-free survival of 75%. In contrast, only one of five patients with relapsed/refractory ALL is still in complete remission. These data provide a basis for future studies of immunotherapy in very high-risk infant ALL.

Relative expansion of CD19-negative very-early normal B-cell precursors in children with acute lymphoblastic leukaemia after CD19 targeting by blinatumomab and CAR-T cell therapy: implications for flow cytometric detection of minimal residual disease.

CD19-directed treatment in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) frequently leads to the downmodulation of targeted antigens. As multicolour flow cytometry (MFC) application for minimal/measurable residual disease (MRD) assessment in BCP-ALL is based on B-cell compartment study, CD19 loss could hamper MFC-MRD monitoring after blinatumomab or chimeric antigen receptor T-cell (CAR-T) therapy. The use of other antigens (CD22, CD10, CD79a, etc.) as B-lineage gating markers allows the identification of CD19-negative leukaemia, but it could also lead to misidentification of normal very-early CD19-negative BCPs as tumour blasts. In the current study, we summarized the results of the investigation of CD19-negative normal BCPs in 106 children with BCP-ALL who underwent CD19 targeting (blinatumomab, n = 64; CAR-T, n = 25; or both, n = 17). It was found that normal CD19-negative BCPs could be found in bone marrow after CD19-directed treatment more frequently than in healthy donors and children with BCP-ALL during chemotherapy or after stem cell transplantation. Analysis of the antigen expression profile revealed that normal CD19-negative BCPs could be mixed up with residual leukaemic blasts, even in bioinformatic analyses of MFC data. The results of our study should help to investigate MFC-MRD more accurately in patients who have undergone CD19-targeted therapy, even in cases with normal CD19-negative BCP expansion.