Immunophenotyping Of Leukemia Research Articles

Characteristics and Prognosis of "Acute Promyelocytic Leukemia-like" Nucleophosmin-1-Mutated Acute Myeloid Leukemia in a Retrospective Patient Cohort.

Background: AML with NPM1 mutation is the largest subcategory of AML, representing about 35% of AML cases. It is characterized by CD34 negativity, which suggests a relatively differentiated state of the bulk of leukemic blasts. Notably, a significant subset of NPM1-mutated AML cases also exhibit HLA-DR negativity, classifying them as "double-negative", and mimicking, therefore, the CD34- HLA-DR- immunophenotype of acute promyelocytic leukemia (APL). Objectives: This study focuses on the "acute promyelocytic leukemia-like" ("APL-like") subset of NPM1-mutated AML, which can be challenging to distinguish from APL at presentation, prior to confirming RARa translocations. We aim to investigate the hematologic and immunophenotypic parameters that may aid to its distinction from APL. Additionally, we explore differences in genetic profile and prognosis between "APL-like" and "non-APL-like" NPM1-mutated AML cases. Methods: We conducted a retrospective evaluation of 77 NPM1-mutated AML cases and 28 APL cases. Results: Morphological characteristics, hematologic parameters (such as DD/WBC and PT/WBC), and specific immunophenotypic markers (including SSC, CD64, and CD4) can assist in the early distinction of "APL-like" NPM1-mutated AML from APL. Regarding differences in genetic profiles and outcomes between "APL-like" and non-"APL-like" NPM1-mutated AML cases, we observed a significantly higher incidence of IDH1/2 /TET2 mutations, along with a significantly lower incidence of DNMT3A mutations in the "APL-like" subset compared to the non-"APL-like" subset. The frequency of Ras-pathway and FLT3 mutations did not differ between these last two groups, nor did their prognoses. Conclusions: Our findings contribute to a comprehensive characterization of NPM1-mutated AML, enhancing diagnostic accuracy and aiding in the detailed classification of the disease. This information may potentially guide targeted therapies or differentiation-based treatment strategies.

Integrative immunophenotypic and genetic characterization of acute myeloid leukemia with CBFB rearrangement.

We sought to characterize the immunophenotype of acute myeloid leukemia (AML) with CBFB rearrangement and correlate the results with cytogenetic and molecular data. Sixty-one cases of AML with CBFB rearrangement were evaluated. The sample population consisted of 33 men and 28 women, with a median age of 49 years. Flow cytometry immunophenotypic analysis showed that myeloblasts were positive for CD34 and CD117 in all cases, and myeloperoxidase was positive in 52 of 55 (95%) cases. The most common abnormalities included decreased CD38 in 90%, increased CD13 in 85%, increased CD123 in 84%, and decreased HLA-DR in 84% of cases. Monocytes were increased, with a mature immunophenotype, and accounted for 23.7% of total cells. Among 60 cases with available karyotype, inv(16)(p13.1q22) was most common in 50 (83%) cases, followed by t(16;16) (p13.1;q22) in 6 (10%). Type A CBFB::MYH11 transcript was most common, detected in 84% of cases. Mutational analysis showed mutations of NRAS in 37%, FLT3 in 25%, and KIT in 24% of cases. Comparing cases with type A vs non-type A transcripts, blasts in type A cases more frequently exhibited CD64 positivity and increased CD13 levels while showing a lower frequency of CD7 and CD56 expression. Trisomy 22 and mutations in KIT, NF1, and TET2 were identified only in cases with type A transcript. Myeloblasts of AML with CBFB rearrangement are positive for CD34, CD117, and myeloperoxidase. These neoplasms most frequently carry inv(16)(p13.1q22) and type A fusion transcript. NRAS mutation was the most common mutation. Some immunophenotypic and genetic correlations occurred with different types of transcripts.

Redefining hyperviscosity in acute leukemia: Potential implications for red cell transfusions in the microvasculature.

Hyperleukocytosis is an emergency of acute leukemia leading to blood hyperviscosity, potentially resulting in life-threatening microvascular obstruction, or leukostasis. Due to the high number of red cells in the circulation, hematocrit/hemoglobin levels (Hct/Hgb) are major drivers of blood viscosity, but how Hct/Hgb mediates hyperviscosity in acute leukemia remains unknown. In vivo hemorheological studies are difficult to conduct and interpret due to issues related to visualizing and manipulating the microvasculature. To that end, a multi-vessel microfluidic device recapitulating the size-scale and geometry of the microvasculature was designed to investigate how Hct/Hgb interacts with acute leukemia to induce "in vitro" leukostasis. Using patient samples and cell lines, the degree of leukostasis was different among leukemia immunophenotypes with respect to white blood cell (WBC) count and Hct/Hgb. Among lymphoid immunophenotypes, severe anemia is protective against in vitro leukostasis and Hct/Hgb thresholds became apparent above which in vitro leukostasis significantly increased, to a greater extent with B-cell acute lymphoblastic leukemia (ALL) versus T-cell ALL. In vitro leukostasis in acute myeloid leukemia was primarily driven by WBC with little interaction with Hct/Hgb. This sets the stage for prospective clinical studies assessing how red cell transfusion may affect leukostasis risk in immunophenotypically different acute leukemia patients.

CD371-positive pediatric B-cell acute lymphoblastic leukemia: propensity to lineage switch and slow early response to treatment

AbstractIn the effort to improve immunophenotyping and minimal residual disease (MRD) assessment in acute lymphoblastic leukemia (ALL), the international Berlin-Frankfurt-Münster (iBFM) Flow Network introduced the myelomonocytic marker CD371 for a large prospective characterization with a long follow-up. In the present study, we aimed to investigate the clinical and biological features of CD371-positive (CD371pos) pediatric B-cell precursor ALL (BCP-ALL). From June 2014 to February 2017, 1812 pediatric patients with newly diagnosed BCP-ALLs enrolled in trial AIEOP-BFM ALL 2009 were evaluated as part of either a screening (n = 843, Italian centers) or validation cohort (n = 969, other iBFM centers). Laboratory assessment at diagnosis consisted of morphological, immunophenotypic, and genetic analysis. Response assessment relied on morphology, multiparametric flow cytometry (MFC), and polymerase chain reaction (PCR)-MRD. At diagnosis, 160 of 1812 (8.8%) BCP-ALLs were CD371pos. This correlated with older age, lower ETV6::RUNX1 frequency, immunophenotypic immaturity (all P < .001), and strong expression of CD34 and of CD45 (P < .05). During induction therapy, CD371pos BCP-ALLs showed a transient myelomonocytic switch (mm-SW: up to 65.4% of samples at day 15) and an inferior response to chemotherapy (slow early response, P < .001). However, the 5-year event-free survival was 88.3%. Among 420 patients from the validation cohort, 27 of 28 (96.4%) cases positive for DUX4-fusions were CD371pos. In conclusion, in the largest pediatric cohort, CD371 is the most sensitive marker of transient mm-SW, whose recognition is essential for proper MFC MRD assessment. CD371pos is associated to poor early treatment response, although a good outcome can be reached after MRD-based ALL-related therapies.

Clinical Characteristics and Diagnosis of Ph-Positive Mixed Phenotype Acute Leukemia.

The goal was to improve the clinical cognition of Ph-positive mixed phenotype acute leukemia and avoid misdiagnosis or delayed diagnosis. The clinical manifestations and laboratory results (bone marrow cell morphology, multiparameter flow cytometry, and cytogenetics) of a case of Ph-positive mixed phenotype acute leukemia were analyzed, and related literature was reviewed. Blood routine: WBC 386.35 x 109/L, HGB 117.00 g/L, PLT 31 x 109/L; 80% of the original cells can be seen by artificial classification. Morphological examination of bone marrow cells showed that the proliferation of nucleated cells was obviously active, and the original cells accounted for 76%. The size of the original cells was somewhat uniform, most of the cells had less mass, were stained light grayish blue, the cytoplasm particles were not obvious, the nuclei were mostly round or quasi-round, some of them showed distortion and nuclear notch, and the chromatin was coarse. Some of the cells were rich in mass, small azurin granules were seen, the nuclei were regular, most of them were round, the chromatin was fine, the myeloperoxidase and esterase staining were negative, the eosinophils accounted for 2.5%, and the basophils accounted for 0.5%. Flow cytometry immunotyping: Two groups of abnormal cells were seen in the bone marrow. 1. A group included 12.32% of nuclear cells and showed abnormal myeloid primitive cell phenotype. Main expression: CD117, CD34, CD38, HLA-DR, CD33, CD64, CD123, weak expression: CD13, CD19. 2. The other group included 45.61% of the nuclear cells and had a B-lymphoblastic phenotype. Main expression: CD34, CD38, HLA-DR, CD123, CD19, CD10, CD9, cCD79a, TDT, weak expression of CD13, CD22. Mixed phenotype acute leukemia (M/B) immunophenotype was considered. Chromosome: 46,XY,t(9; 22)(q34;q11.2) [20]. BCR-ABL (P210) fusion gene was positive. Mixed phenotype acute leukemia (MPAL) is a rare type of malignant hematologic disease. Its diagnosis is based on the comprehensive evaluation of bone marrow cell morphology, immunophenotype, molecular and cytogenetic features.

IMMUNOPHENOTYPE OF LEUKEMIC CELLS IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS WITH NOTCH1 AND SF3B1 GENE MUTATIONS.

The typical chronic lymphocytic leukemia (CLL) immunophenotype is vital for diagnosis, but the expression of some antigens varies and has prognostic value. There are data that reduced CD20 expression is associated with NOTCH1 and SF3B1 gene mutations. To determine a high-risk group of CLL patients for prediction of unfavorable NOTCH1 and SF3B1 gene mutations based on immunophenotyping of leukemic cells. Flow cytometric and molecular-genetic analysis (mutations of NOTCH1, SF3B1, and TP53 genes using the polymerase chain reaction followed by direct sequencing) was performed in a group of 86 previously untreated CLL patients. The immunophenotype of leukemic cells of all examined patients met the criteria of CLL diagnosis. NOTCH1 gene mutations were found in 21 patients (24.4%), and SF3B1 gene mutations - in 7 patients (8.1%). There were no TP53 gene mutations among the examined patients. A decreased number of CD20+CD5+ cells and a downward trend in the relative index of mean fluorescence intensity (iMFI) of CD20+ cells were found in patients with NOTCH1 and SF3B1 gene mutations. Based on the iMFI level (higher and/or lower than 3.0) and the number of CD20+CD5+ cells among all B-cells (higher and/or lower than 50%), we distinguished CLL cases with low and relatively high levels of CD20 antigen expression. Using ROC analysis and the parameter of low CD20 antigen expression, we could predict the presence of NOTCH1 and SF3B1 gene mutations in 73.3 ± 0.06% of patients (p = 0.001). The risk of NOTCH1 and SF3B1 gene mutations in cases with low CD20 antigen expression was 6.96 (95% CI = 2.53-19.18; p = 0.0001). The revealed regularities were statistically significant for patients in whom the diagnosis was established in all Binet - Rai stages except A0-AI. Our data confirmed a reduced CD20 expression in CLL patients with NOTCH1 and SF3B1 mutations. In addition, an approach was proposed to identify high-risk CLL patients for prediction of such mutations: previously untreated CLL patients at advanced Binet - Rai stages (BII, CIII, CIV) with a reduced number of double-positive CD20+CD5+ cells in peripheral blood and/or low iMFI of CD20+ cells.

Guidelines for the use of flow cell sorting in diagnosis and monitoring of acute leukemia

Flow cell sorting is an advanced laboratory technique that combines the analytical capabilities of flow cytometry with the ability to isolate pure cell populations from heterogeneous samples. It has tremendous potential both for fundamental research and laboratory diagnosis. For example, the combination of cell sorting and molecular genetic studies can be used to clarify ambiguous results of acute leukemia immunophenotyping obtained both at diagnosis and during minimal residual disease monitoring. These guidelines are based on years of experience in incorporating cell sorting into the diagnostic and monitoring processes at the Leukemia Immunophenotyping Laboratory of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. They include methods used for the confirmation of flow cytometry data depending on the type of leukemia, the stage of a flow cytometry assay and previous therapy. They also describe cell sorting algorithms for disease diagnosis and the specifics of sample preparation for cell sorting in different molecular genetic studies.

Immunophenotyping of Acute Leukemia in Pediatric Patients: Tertiary Care Centre Experience from Lahore

Acute Leukemia is the most prevalent malignancy of childhood, globally. Immunophenotyping by flowcytometry has developed as an important technique for its outstanding contributions in diagnosis and management of leukemia patients. Objective: To determine the frequency and immunophenotyping by flowcytometry of Acute leukemia in pediatric age group. Methods: The study was conducted at The University of Child health sciences and Children’s Hospital, Lahore from July-2021 to Feb-2022. Data were gathered from 101 consecutive patients, of age from 0.5 to 15 years, that had acute leukemia immunophenotyping using flowcytometry. Results: Flowcytometric immunophenotyping of 101 leukemia patients over 8-month period showed that 67.3% and 33.6% male were females respectively. AML, T-ALL, and B-ALL prevalence was highest in patients with 5 to 10 years of age. Out of 101 leukemia patients, 16 (15.8%) and 85 (84.1%) had AML and ALL diagnosis respectively. Of these 85 ALL cases, 72 (84.7%) were B-ALL and 13 (15.2%) were T-ALL. Study founded that most frequent CD markers in B-ALL, were CD79 and CD19 (100%) whereas CD3 and CD5 in T-ALL and CD13 (93.8%) and CD34 (87.5%) in AML. CD34 was a common marker among B-ALL, T-ALL and AML. Among B-ALL and T-ALL, AntiTdt was a common CD marker whereas HLA-DR was common among AML and B-ALL. Conclusions: Results of Acute leukemia immunophenotyping were homologous to worldwide published research. For accurate leukemia lineage, immunophenotyping of AML and ALL is essential since, if therapy is started based solely on morphological diagnosis, approximately 25% of patients may not respond or recure.

Abstract 3924: Cooperation between mutant IL7R and TLX3 generates mixed myeloid lymphoid leukemia from thymocyte progenitors

Abstract Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Despite the current pediatric treatment protocols have resulted in 5-year overall survival rates of up to 90%, treatments have substantial side effects. Therefore, it is important to identify more specific therapeutic targets and develop more effective and less toxic drugs and drug combinations. We have shown that overexpression of TLX3 in association with mutant IL7R (mutIL7R) is sufficient to generate aggressive leukemia from transduced CD4-CD8- (DN) thymocyte progenitors. To better understand mutIL7R/TLX3-mouse leukemia, we combined single cell RNA sequencing (scRNAseq), Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq), DNA Next Generation Sequencing (NGS) Chromatin Immunoprecipitation Sequencing (ChIP-Seq) and flow cytometry to explore the cooperation of mutIL7R and TLX3 in orchestrating leukemogenesis. We found that mutIL-7R+TLX3 induced murine mixed T/myeloid leukemia. Myeloid cell clones (CD11b+) expressed identical TCR rearrangements as the T cell clones (CD8+), indicating that mutant IL-7R+TLX3 generated mixed leukemia from T cell progenitors. Moreover, the passaged leukemia lost the T-cell phenotype and acquired a pro-B-like phenotype, suggesting preferential expansion of pro-B cells (CD19+, B220+ IgM-), which were not detected in the founder leukemia presumably due to its low frequency. We next sought to determine whether the oncogene combination is sufficient to drive a similar immunophenotype profile in vitro. We showed that ectopic expression of mutIL7R and TLX3 in DN primary thymocytes growing on OP9-DL4 pushed the cells to myeloid (CD11b+, Gr1+) and B-cell (B220) phenotype over a time course of 4, 8 and 20 days, while T-cell development was blocked, suggesting that TLX3 expression prevented differentiation into CD4+/CD8+ T-cells. To better understand the genes regulated by TLX3 on driving a mixed phenotype leukemia we performed ChiP-seq of D1 cell line ectopically expressing mutIL7R and TLX3. We identified RUNX as a possible key mediator of the mixed-phenotype-leukemia induced by TLX3 in combination with mutIL7R. Additionally, scRNAseq showed upregulation of PU.1 in mice leukemia, suggesting PU.1 as a potential therapeutic target. To assess whether targeted therapy could successfully treat mutIL7R/TLX3-mouse leukemia, we treated mice that had been injected with the thymocytes transduced with mutIL-7R+TLX3 genes. We found that PU.1 inhibition reduced spleen weights, indicating decreasing of leukemia engraftment. Taken together, we generated a high-resolution dataset with detailed information about the transcriptional landscape and immunophenotype of leukemia generated by mutIL-7R+TLX3, a combination that is observed in patient samples, and suggests new therapeutic drug strategies. Citation Format: Gisele Rodrigues. Cooperation between mutant IL7R and TLX3 generates mixed myeloid lymphoid leukemia from thymocyte progenitors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3924.

Molecular Cytogenetics and Immunophenotypic Characterization of Childhood Acute Leukaemia Patients in Tertiary Care Center of Bangladesh – An observative study

Background: Childhood leukemia is genetically a heterogeneous disease. Various types of cytogenetic abnormalities and immunophenotypic character are present in leukaemia which are important for risk stratification, treatment and play as significant prognostic factor. Pediatric acute leukaemia presents with varying clinical, morphological, immunological and molecular characteristics. It is very highly curable if diagnosed and treated properly. For detail typing and subtyping of acute leukemia immunophenotyping and cytogenetics are crucial. The aim of this study was to find out the genetic abnormalities and immunophenotypic characterization of childhood acute leukaemia patients in Bangladesh. Material &amp; Methods: This was a retrospective observational study and was conducted in the Department of pediatric hemato-oncology of Combined Military Hospital, Dhaka and Ahsania Mission Cancer Hospital, Mirpur, Dhaka, Bangladesh during the period from February,2014 to March, 2022. There was total 98 cases of acute leukaemia. Results: In total 98 patients completed the study. We found that 79.59% patients were ALL and 20.41% patients were AML. Among ALL 80.64% were B cell type, 6.40% were T cell Type ; 12.82 % had TEL/AML1 or ETV6/RUNX1 t(12;21)(p13;q22), 5.13% patient had TCF3/PBX1 or E2A/PBX1 t(1;19)(q23;p13). In AML30.00% patient had PML/RARAt(15;17)(q22;q21), 10.00% patient had AML1/ETO or RUNX1/CBFA2T1 t(8;21) (q22;q22), 5.00% patient had FLT3/ ITD. In case of B-ALL highest expression of antigen was CD19 (91.64%) followed by CD10 (80.58%), HLADR (67.94%), CD22 (72.68%), CD79a (72.68%), TdT (52.14%) and CD34 (48.98%). In 44.24% cases there was co-expression of CD10 and CD19 and there was 11.6% expression of myeloid marker CD13 and 1.58% expression of T cell marker CD5. In case of T-ALL there was 100% expression of CD3. Expression of other antigen CD4, CD5, CD7, CD8, CD4/8 co-expression, TdT was 60%. There was 40% expression of CD1a and CD2. There was 20% expression of CD10, CD34 and TCRab also. In case of AML highest expression was MPO (93.75%) followed by CD33 (87.50%), CD13 (81.25%), CD117 (75%), HLADR (43.75%) and CD64 (50%). There was 6.25% aberrant expression of B-ALL marker CD19 and T-ALL marker CD3, CD4, CD5, CD7 also. Conclusion: Depending on this study we can say that except few variations distribution of immunophenotypical subtypes and genetic abnormalities of childhood acute leukaemia are almost similar to other literature published from neighboring countries.This study will serve as a guideline for future study in our country in this aspect.

Immunophenotyping of Childhood Acute Lymphoblastic Leukemia in Qazvin City, Iran: A Cross-Sectional Study

Background: Acute lymphoblastic leukemia (ALL) is the most prevalent childhood cancer. ALL is a heterogeneous type of malignancy and treatment protocols vary based on the immunological classification of ALL. The critical step for treating ALL is to identify immunological subgroups by flow cytometry findings. Objective: In this study, immunophenotypic information was evaluated for the first time in children with ALL in Qazvin City, Iran. Methods: This cross-sectional study reviewed the clinical and laboratory data of children with ALL during 2019-2020. Next, children with ALL were immunophenotyped by flow cytometry applying a panel of the specific monoclonal antibodies for some clusters of differentiation (CD) molecules, including CD20, CD21, CD10, CD34, CD38, and terminal deoxynucleotidyl transferase (TdT). The data were separately analyzed using SPSS software, version 24. Findings: Of 52 children with ALL in the age range of 6 months to 15 years, 23 children (44.23%) had B-ALL-ProB (pro-B) cell immunotyping features, 26 (50%) had B-ALL-PreB (pre-B) cell immunotyping features, and 3 (5.7%) had T-cell immunotyping features. The ages of T-cell group children were higher than those of B-cell group children. The most common clinical and laboratory findings were fever (26 cases, 55.31%). In 55% of children, periodic acid-schiff (PAS) staining was positive. The presence of the terminal deoxynucleotidyl transferase (TdT) enzyme was higher in B-cell patients than in T-cell cases. Children with CD34+ were higher in the pro-B group than in the pre-B group. Conclusion: our study shows that the immunophenotypic characteristics of children with ALL are more similar to previous reports and can be used for monitoring and prognosis of children with ALL in Qazvin City.

Immunophenotype of Acute Lymphoblastic Leukemia: The Experience of University Hospital Centre Casablanca - Morocco.

Acute lymphoblastic leukemia (ALL) encompasses a group of lymphoid neoplasms that morphologically and immunophenotypically resemble B-lineage and T-lineage precursor cells. Our objective is to describe the immunophenotypic aspects of acute lymphoblastic leukemia (All) diagnosed by flow cytometry at the hematology laboratory of IBN ROCHD University Hospital Center and to compare them with those reported in other series. This is a descriptive study over a period from August 2016 to October 2021, during a 5 year-and-2-month period. Immunophenotyping was performed at the flow cytometry unit on a Beckman-Coulter with 6 colors and 2 lasers in the hematology laboratory of the same hospital and data was collected retrospectively from the patients' files, their medical prescription files, kalisil software, and a data collection form we have established. The 440 patients had ages ranging from 1 month to 76 years, with a median of 9.5 years and the overall male-to-female ratio was 1.44. The immunological subtyping revealed that 82.5% of cases were B-ALL and 17.5% were T-ALL; of these B-ALL cases 230 (63.36%) were children (range: 0.1 - 15 years) and 134 (36.91%) were adults (range: 16 - 76 years); T-ALL were distributed in both age groups, 49 cases (37.7%) were children (range: 2 - 15 years) and 28 (21.56%) were adults (range: 18 - 63 years). All patients presented at least one abnormal blood count; thrombocytopenia has been observed in 89.4% of cases, anemia in 86.5% of cases, hyperleukocytosis in 79.8% of cases, leukopenia in 10.6% of cases, and pancytopenia in 4.8% of cases. The frequency of B-cell markers in B-ALL was found to be 363 (100%) for CD19, 323 (88.94%) for CD10, 290 (80%) for CD79a, and 73 (20%) for CD20. CD34 expression was found in 73 (20%) cases of B-ALL. HLA-DR was found in 54 (15%) cases, while TdT was found in 43 (13%) cases. Aberrant expression of myeloid antigens was found in 94 (26%) cases of B-ALL. Among T-ALL, the positivity of CD3 and CD7 was 100% (77 cases), while CD5 was positive in 58 (75%) cases. CD34 expression was found in only 19 cases of T-ALL. CD4 and CD8 expression was checked in only 9 adult patients and 4 pediatric cases. Out of them, 77.82% of cases were negative for both CD4 and CD8. CD4 and CD8 double positivity was seen in only 11.1% of cases, and 22.4% of cases showed either CD4 or CD8 positivity. We concluded that our study was similar to reports in the Americas and Europe, and it was the first large one that describes the immunophenotypic profile of ALL in the Moroccan population.

ALL-REZ BFM 2002 Is Associated with Improved Outcome as Compared to ALL-R3 Strategy in Children with Standard Risk Isolated CNS Relapse of Acute Lymphoblastic Leukemia, while Maintaining Comparable Efficacy in Patients with Bone Marrow Relapse. Results of the Multi-National, Multi-Center Trial IntReALL SR 2010

Background: Around 15% of children with acute lymphoblastic leukemia (ALL) experience disease relapse. The prognosis of these patients largely depends on the site of recurrence, time to relapse and leukemia immunophenotype. The ALL-REZ BFM 2002 and the ALL R3 trials have proved to be effective in previous trials. The IntReALL SR 2010 trial, conducted under the umbrella of the I-BFM SG, aimed at investigating feasibility, toxicity and efficacy of both treatment strategies in a large multi-national, multi-center prospective randomized trial. Methods: Patients with a first standard-risk (SR) ALL relapse aged 1 - 17 years were recruited to the trial from 23/Oct/2014 to 31/Jul/2020 in 17 countries (210 centers). SR relapse criteria were: late (≥ 6 months after end of initial therapy) isolated bone marrow (BM) relapse, late or early (< 6 months after end of initial therapy and ≥ 18 months after initial diagnosis) combined BCP-ALL BM relapse, any late/early isolated extramedullary (IEM) relapse. They were randomized at a 1:1 rate to receive either arm A (ALL-REZ BFM 2002) or B (ALL R3). During consolidation, patients were randomized to receive the CD22-directed monoclonal antibody epratuzumab in addition to backbone chemotherapy. Results of this 2nd randomization will be communicated separately. Patients with late BM relapse were allocated to consolidation and maintenance chemotherapy or to allogeneic hematopoietic stem-cell transplantation (HSCT), depending on their minimal residual disease (MRD, cut-off arm A 10e-3 and arm B 10e-4) assessed after induction therapy. Patients with early either combined BM or IEM relapse were allocated to receive allogeneic HSCT. Results: Overall, 620 patients were randomized to receive arm A (n=304) or Arm B (n=316). Patient's characteristics were equally distributed among both groups (table 1). Induction death, 2nd remission rate, death in remission (5-years cumulative incidence (CI) 4.1 vs 7.4%, p=0.10), and subsequent relapse rates by arm A and B (CI 23.8 vs 22.8, p=0.80) did not differ between both groups. The MRD response rate with < 10e-4 post induction was significantly higher in Arm B (p=0.015), but not significantly different based on the arm specific cut-off, leading to comparable rates of allogeneic HSCT. The probability of 5-years event-free survival (pEFS) with arm A versus arm B was 70.2±3.1 SE versus 68.5±3.1 (p=0.61, figure 1) and that of overall survival (pOS) was 86.7±2.3 versus 87.1±2.0 (p=0.8). Subgroup analyses revealed a significantly higher pEFS in patients with isolated CNS relapse when treated with arm A (81.6±6.3, n=40) compared to arm B (43.3±8.3, n=45, p=0.001), whereas in patients with BM involvement or those with non-CNS IEM relapse, no significant difference was found (p=0.41). However, patients with isolated BM relapse experienced a higher CI of subsequent relapse with arm A (12.5±2.1, n=146) as compared to arm B (6.5±1.6, n=153, p=0.008). Patients with late BM relapse and MRD poor response after induction had comparable pDFS (arm A 65.8±6.4, n=74; arm B 68.8±6.2, n=75; p=0.71), which also did not differ from that of patients with MRD good response (arm A 67.7±5.6, n=100; arm B 68.9±5.6, n=113; p=0.77). Patients with late IEM relapse achieved a pEFS of 86.7±4.6 whereas those with early relapse and HSCT indication achieved a pEFS 61.8±5.4 (p=0.0004). Rates of reported severe adverse events (SAEs) per treatment phase did not differ between arms except nervous system disorders occurring more frequently in arm A (p=0.03, table 1). Conclusion: Both arm A and B were effective in this large multi-national trial. EFS and OS probabilities compare favorably to results recently published by other cooperative groups. In particular, the outcome of patients with early IEM relapse or with late BM relapse and MRD poor response is encouraging confirming the benefit of allo HSCT in these subgroups. Due to superior outcome of isolated CNS relapse with arm A, patients without BM involvement will receive that arm as backbone in future trials. Since arm B showed a better MRD response rate after induction and a lower relapse rate in patients with BM relapse, it will be implemented as backbone for future trials in patients with BM involvement. Toxicity (TRM and SAE rates) is substantial in both arms demonstrating the urgent medical need of replacing toxic chemotherapy elements with equally effective, but less toxic targeted immunotherapy strategies in future trials. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CD72 Is a Pan-Tumor Antigen Associated with Childhood Acute Leukemia

Immunotherapy has recently gained increasing attention as a new paradigm shift in cancer and has revolutionized the course of cancer treatment. The identification of ideal tumor-associated antigens (TAA) is a challenging process, since antigens have to be specific for the target cells and assure no off-target effects. We exploited a gene expression dataset of acute leukemias previously generated in our lab to identify potentially interesting TAAs. Among the most interesting hyper- and over-expressed genes with respect to normal bone marrow (BM) and with a surface protein localization, the CD72 was selected because of its specific association with childhood B-cell precursor acute lymphoblastic leukemias (BCP-ALL) and with a large proportion of pediatric acute myeloid leukemias (AML) cases. Based on the findings described below, we here propose CD72 as a novel and robust pan-acute leukemia TAA. Even though CD72 is known to be expressed by B-lineage cells, we profiled its expression in a variety of cell lines and primary samples, providing a map of its expression. CD72 was expressed by BCP-ALL cell lines (NALM-6, SEM, RS-4;11, RHC-ACV) and various AML cell lines (SHI-1, SKNO-1, MV4;11, MOML-13). Conversely, T-ALL cell lines (DND-41, TALL-1, RPMI-8402, ALL-SIL, JURKAT) and non-hematopoietic tumor cell lines (DAOY, NB-1, HT-29, A549, HeLa, MCF7) did not express the antigen. This supports the idea that CD72 is specific for hematopoietic tumors. Furthermore, we tested CD72 expression on major populations within normal BM specimens and on other healthy tissues: skin fibroblasts, mesenchymal stromal cells, skeletal myoblasts, endothelial cells. We confirmed that the antigen is expressed on B-lineage cells, whereas in other cell types we did not detect any signal. Then, to assess the interest of CD72 for the development of chimeric antigen receptor (CAR)-based immunotherapy approaches, we documented that CD72 was not expressed on CD34-positive/CD38-negative hematopoietic stem and progenitor cells (HSPCs) from both mobilized peripheral blood cells and healthy-regenerating BM samples (Figure 1). We profiled CD72 expression by flow-cytometry on 289 pathological pediatric BM samples, including: 93 BCP-ALL at diagnosis and 44 at relapse; 38 T-ALL at diagnosis and 3 at relapse; 68 AML at diagnosis and 12 at relapse; blast cells from 6 myelodysplastic syndromes; 14 non-hematopoietic solid tumors invading BM (4 rhabdomyosarcomas, 6 neuroblastomas, 4 Ewing's sarcomas); blast cells from 3 T-cell Lymphoblastic Lymphomas and 8 B-lineage Non-Hodgkin Lymphomas (NHL) with BM involvement. Data from clinical samples confirmed the in silico and in vitro prediction. CD72 was positive in 134/137 (98%) of the examined BCP-ALL cases and in all B-lineage NHL and 52/80 (65%) of AML cases. Conversely, CD72 was expressed only in 2/44 (4%) of T-ALL cases and was absent in T-cell NHL and in non-hematopoietic solid tumors. Notably, CD72 was expressed by all 12 BCP-ALL patients that were previously treated with CD19-directed immunotherapies, including four patients who lost CD19 expression. Moreover, CD72 was positive on two rare cases of CD19-negative BCP-ALL at diagnosis (Figure 2). Within our AML cohort, we did not detect any difference in CD72 expression across AML genetic subgroups. Taken together, the preliminary data here reported highlight the potential clinical usefulness of CD72 for advanced diagnostics, including the ability to identify blast cells in case of CD19 loss, and provide support for developing novel immunotherapy approach directed at CD72 that may be endowed with the unique potential of targeting both acute lymphoid and myeloid leukemia. Figure 1. CD72 expression by flow-cytometry on healthy CD34+ and CD34+/CD38- cells from healthy-regenerating bone marrow. CD72 expression was evaluated according to AIEOP-BFM Consensus guidelines, comparing the fluorescence shift and distribution pattern of the cells to the appropriate negative control. Figure 2. CD72 expression on CD19-negative BCP-ALL by flow-cytometry at first diagnosis and at time of relapse after CD19-directed immunotherapy. B-lineage was assigned with the contribution of other antigens relevant in immunophenotyping of acute leukemia. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Cooperation between Mutant IL7R and TLX3 Generates Mixed Myeloid/Lymphoid Leukemia from Thymocyte Progenitors

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Despite current pediatric treatment protocols that have resulted in 5-year overall survival rates up to 90%, treatments have substantial side effects. Therefore, it is important to identify more specific therapeutic targets and develop more effective and less toxic drugs and drug combinations. We have shown that overexpression of TLX3 in association with mutant IL7R (mutIL7R) is sufficient to generate aggressive leukemia from transduced CD4-CD8- (DN) thymocyte progenitors. To better understand mutIL7R/TLX3-mouse leukemia, we combined single cell RNA sequencing (scRNAseq), Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq), DNA Next Generation Sequencing (NGS) and flow cytometry to explore the cooperation of mutIL7R and TLX3 in orchestrating leukemogenesis. We found that mutIL-7R+TLX3 induced murine mixed T/myeloid leukemia. Myeloid cell clones (CD11b+) expressed identical TCR rearrangements with T cell clones (CD8+), indicating that mutant IL-7R+TLX3 generates mixed leukemia from T cell progenitors. Moreover, the passaged leukemia lost the T-cell phenotype and acquired a pro-B-like phenotype, suggesting preferential expansion of pro-B cells (CD19+, B220+ IgM-), which were not detected in the founder leukemia presumably due to its low frequency. We next sought to determine whether the oncogene combination is sufficient to drive a similar immunophenotype profile in vitro. We showed that ectopic expression of mutIL7R and TLX3 in DN primary thymocytes pushed the cells to myeloid (CD11b+, Gr1+) and B-cell (B220) phenotype over a time course of 4, 8 and 20 days, while T-cell development was blocked, suggesting that TLX3 expression prevented differentiation into CD4+/CD8+ T-cells. To further characterize the mixed T/myeloid leukemia at the molecular level, we compared the leukemia expressed genes to a TLX-bound genes public dataset. We found that 42 TLX3 target genes are expressed in mutIL7R+TLX3 leukemia. Interaction network analysis suggested significant Myc stimulation. We then confirmed Myc upregulation at the protein level in a thymocyte cell line, indicating that mutIL7R alone induces Myc but requires TLX-3 cooperation to induce maximal Myc expression levels. Taken together, we generated a high-resolution dataset with detailed information about the transcriptional landscape and immunophenotype of leukemia generated by mutIL-7R+TLX3, a combination that is observed in patient samples, and suggests new therapeutic drug strategies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Recommendations for laboratory testing of UK patients with acute myeloid leukaemia.

Recommendations for laboratory testing of UK patients with acute myeloid leukaemia.

Flow cytometry immunophenotypic features of pure erythroid leukemia and the distinction from reactive erythroid precursors.

The immunophenotype of pure erythroid leukemia (PEL) as determined by flow cytometry immunophenotypic analysis is not well characterized. The immunophenotypic difference between PEL and reactive conditions is under-explored. We assessed and compared the immunophenotype of 24 PEL cases and 28 reactive cases containing early erythroid precursors by flow cytometry. The neoplastic erythroid cells in all PEL cases were positive for CD36 and CD71. CD45 was also positive in all cases, but the expression level was often dimmer than granulocytes. CD117 expression ranged from partial to uniform, and CD235a was often only positive in the CD117-dim to negative cells, corresponding to more differentiated subset. PEL cases frequently (87%) showed decreased or negative CD38 expression, contrasting to reactive early erythroid precursors that showed bright CD38 (p < 0.0001). CD7 (25%) and CD13 (29%) aberrant expressions were only observed in PEL but not in the reactive erythroid cells. Normal early erythroid precursors in all reactive bone marrows showed partial expression of CD4; In contrast, aberrant CD4 expression was detected in 71% PEL cases, either uniformly positive (50%) or completely negative (21%). While normal/reactive bone marrows almost always contained a small subset of CD34-positive early erythroid precursors, the neoplastic pronormoblasts in all PEL cases were CD34 negative. Although not increased in number, CD34-positive myeloblasts were frequently detected in PEL and demonstrated an aberrant immunophenotype in 90% PEL cases. PEL shows a distinctive immunophenotype which can be distinguished from reactive erythroid precursors by flow cytometry immunophenotyping.

P328: DYNAMIC EVOLUTION OF TCF3-PBX1 LEUKEMIAS AT THE SINGLE-CELL LEVEL UNDER CHEMOTHERAPY PRESSURE

Background: Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. While research has been focused on high-risk patients, the biology of low-to-intermediate-risk patients has so far been inadequately investigated and can lead to overtreatment with severe side effects and under treatment with risk of relapse. The translocation t(1;19) codes for chimeric fusion protein TCF3-PBX1, which is associated with intermediate risk ALL. Using our previous generated TCF3-PBX1 conditional knock-in mice, we established a model to study in vivo chemotherapy resistance. Aims: We hypothesize that chemotherapy and microenvironment play a crucial role in development of resistance in TCF3-PBX1 leukemias by influencing transcriptional regulation, activation of signaling pathways and the hierarchical structure of leukemic cells. In this project we aim to characterize dynamic changes of TCF3-PBX1 leukemia cells in different tissues under chemotherapy pressure. Methods: Recipient C57/BL6 healthy mice were sub-lethally irradiated, transplanted with mouse TCF3-PBX1 leukemia cells and treated with vehicle (n=15), prednisolone (n=13) and daunorubicin (n=15) for 20 days. Mice were monitored for signs of disease regularly and circulating GFP+ TCF3-PBX1 leukemia cells were assessed by flow cytometry. Sick mice were sacrificed, leukemia cells were isolated from five different organs (bone marrow, spleen, lymph nodes, spinal cord and brain) and characterized by immunophenotyping, sanger sequencing, bulk RNA sequencing (bulk RNAseq) of GFP+ sorted cells, single cell RNA sequencing and mass cytometry (CYTOF). Results: All mice transplanted with TCF3-PBX1 leukemia cells and treated with vehicle succumbed to disease with a median survival of about 70 days. We optimized chemotherapy drug concentration and transplanted cell dose, so 60% of mice treated with prednisolone or daunorubicin survived at least 150 days. Leukemic infiltration was showed by histological stainings in analyzed tissues including central nervous system (CNS) (spinal cord, brain) and GFP+ leukemia cells were quantified by flow cytometry. No major differences were observed in immunophenotype of TCF3-PBX1 leukemia and variant allele frequency (VAF) of the known PTPN11 mutation in analyzed tissues or depending on in vivo treatments. Bulk RNAseq of FACS-sorted GFP+ TCF3-PBX1 leukemia cells were clustered and CNS cells separated from other tissues. scRNAseq revealed additional heterogeneity within each tissue based on signaling pathway and cell cycle activity. Interleukin signaling, regulation of apoptosis pathways, and signaling by the B cell receptor (BCR) were regulated based on pathway analysis in CNS compared to other tissues. Hence, we elucidated the hierarchical structure of hematopoietic cells, interaction of leukemic cells with the microenvironment, and changes in signaling response to chemotherapy in vivo treatment by mass spectrometry (CyTOF) to validate the identified altered signaling pathway activities at protein level. Summary/Conclusion: We have developed a mouse model in order to characterize in vivo chemotherapy resistance depending on niche. Global transcriptomics and phospho-proteomics at the single-cell level might elucidate novel mechanisms of chemotherapy resistance suitable for pharmacological therapies.

Germline CHEK2 and ATM Variants in Myeloid and Other Hematopoietic Malignancies

An intact DNA damage response is crucial to preventing cancer development, including in myeloid and lymphoid malignancies. Deficiencies in the homologous recombination (HR) pathway can lead to defective DNA damage responses, and this can occur through inherited germline mutations in HR pathway genes, such as CHEK2 and ATM. We now understand that germline mutations can be identified frequently (~ 5-10%) in patients with myeloid and lymphoid malignancies, and among the most common of these are CHEK2 and ATM. We review the role that deleterious germline CHEK2 and ATM variants play in the development of hematopoietic malignancies, and how this influences clinical practice, including cancer screening, hematopoietic stem cell transplantation, and therapy choice. In recent large cohorts of patients diagnosed with myeloid or lymphoid malignancies, deleterious germline loss of function variants in CHEK2 and ATM are among the most common identified. Germline CHEK2 variants predispose to a range of myeloid malignancies, most prominently myeloproliferative neoplasms and myelodysplastic syndromes (odds ratio range: 2.1-12.3), and chronic lymphocytic leukemia (odds ratio 14.83). Deleterious germline ATM variants have been shown to predispose to chronic lymphocytic leukemia (odds ratio range: 1.7-10.1), although additional studies are needed to demonstrate the risk they confer for myeloid malignancies. Early studies suggest there may also be associations between deleterious germline CHEK2 and ATM variants and development of clonal hematopoiesis. Identifying CHEK2 and ATM variants is crucial for the optimal management of patients and families affected by hematopoietic malignancies. OPENING CLINICAL CASE: "A 45year-old woman presents to your clinic with a history of triple-negative breast cancer diagnosed five years ago, treated with surgery, radiation, and chemotherapy. About six months ago, she developed cervical lymphadenopathy, and a biopsy demonstrated small lymphocytic leukemia. Peripheral blood shows a small population of lymphocytes with a chronic lymphocytic leukemia immunophenotype, and FISH demonstrates a complex karyotype: gain of one to two copies of IGH and FGFR3; gain of two copies of CDKN2C at 1p32.3; gain of two copies of CKS1B at 1q21; tetrasomy for chromosome 3; trisomy and tetrasomy for chromosome 7; tetrasomy for chromosome 9; tetrasomy for chromosome 12; gain of one to twocopies of ATM at 11q22.3; deletion of chromosome 13 deletion positive; gain of one to two copies of TP53 at 17p13.1). Given her history of two cancers, you arrange for germline genetic testing using DNA from cultured skin fibroblasts, which demonstrates pathogenic variants in ATM [c.1898 + 2T > G] and CHEK2 [p.T367Metfs]. Her family history is significant for multiple cancers. (Fig.1)." Fig. 1 Representative pedigree from a patient with germline pathogenic ATM and CHEK2 variants who was affected by early onset breast cancer and chronic lymphocytic leukemia. Arrow indicates proband. Colors indicate cancer type/disease: purple, breast cancer; blue, lymphoma; brown, melanoma; yellow, colon cancer; and green, autoimmune disease.

Diagnosis of Acute Leukemia by Multiparameter Flow Cytometry with the Assistance of Artificial Intelligence.

We developed an artificial intelligence (AI) model that evaluates the feasibility of AI-assisted multiparameter flow cytometry (MFC) diagnosis of acute leukemia. Two hundred acute leukemia patients and 94 patients with cytopenia(s) or hematocytosis were selected to study the AI application in MFC diagnosis of acute leukemia. The kappa test analyzed the consistency of the diagnostic results and the immunophenotype of acute leukemia. Bland–Altman and Pearson analyses evaluated the consistency and correlation of the abnormal cell proportion between the AI and manual methods. The AI analysis time for each case (83.72 ± 23.90 s, mean ± SD) was significantly shorter than the average time for manual analysis (15.64 ± 7.16 min, mean ± SD). The total consistency of diagnostic results was 0.976 (kappa (κ) = 0.963). The Bland–Altman evaluation of the abnormal cell proportion between the AI analysis and manual analysis showed that the bias ± SD was 0.752 ± 6.646, and the 95% limit of agreement was from −12.775 to 13.779 (p = 0.1225). The total consistency of the AI immunophenotypic diagnosis and the manual results was 0.889 (kappa, 0.775). The consistency and speedup of the AI-assisted workflow indicate its promising clinical application.