Measurable/minimal residual disease (MRD) status has been suggested as a powerful indicator of clinical-outcome in T-cell lymphoblastic leukemia/lymphoma (T-ALL). Multicolor flow cytometric (MFC)-based T-ALL MRD reports are limited and traditionally based on the utilization of markers-of-immaturity like TdT and CD99. Moreover, studies demonstrating the multicolor flow cytometric (MFC) approach for the assessment of T-ALL MRD are sparse. Herein, we describe an 11-marker, 10-color MFC-based T-ALL MRD method using an "approach of exclusion." Methods: The study included 269 childhood T-ALL patients treated with a modified-MCP841 protocol. An 11-marker, 10-color MFC-based MRD was performed in bone marrow (BM) samples at the end-of-induction (EOI) and end-of-consolidation (EOC) time-points using Kaluza-version-1.3 software.Results: We studied EOI-MRD in 269 and EOC-MRD in 105 childhood T-ALL patients. EOI-MRD was detectable in 125 (46.5%) samples (median, 0.3%; range, 0.0007-66.3%), and EOC-MRD was detectable in 34/105 (32.4%) samples (median, 0.055%; range, 0.0008-27.6%). Leukemia-associated immunophenotypes (LAIPs) found useful for MRD assessment were dual-negative CD4/CD8 (40.9%), dualpositive CD4/CD8 (23.3%) and only CD4 or CD8 expression (35.8%); dim/subset/ dim-negative surface-CD3 (39%), dim/subset/dim-negative/negative CD5 (28.3%), dim/dim-negative/negative/heterogeneous CD45 (44.7%) and co-expression of CD5/CD56 (7.5%). EOI-MRD-positive status was found to be the most-relevant independent factor in the prediction of inferior relapse-free and overall survival. Conclusion:We described an 11-marker 10-color MFC-based highly sensitive MRD

BackgroundMeasurable residual disease (MRD) assessment using multicolor flow cytometry (MFC) has become the center point of pediatric B‐cell precursor acute lymphoblastic leukemia (BCP‐ALL) risk stratification and therapeutic management. The addition of new markers can improve the accuracy and applicability of MFC‐based MRD assay further. Herein, we evaluated the utility of a new marker, CD304/neuropilin‐1, in the assessment of MFC‐based MRD.MethodsExpression patterns of CD304 were studied in leukemic blasts from BCP‐ALL patients and in normal precursor B cells (NPBC) from uninvolved non‐BCP‐ALL bone marrow samples using 10‐color MFC. MRD was monitored at end‐of‐induction (EOI; Days 35–40) and end‐of‐consolidation (Day 78–80) time points.ResultsWe studied CD304 expression in 300 pediatric BCP‐ALL patients and found it positive in BCP‐ALL blasts in 41.7% of diagnostic samples. It was significantly associated with ETV6‐RUNX1 (p < .001) as well as BCR‐ABL1 (p = .019) and inversely associated with TCF3‐PBX1 fusion gene (p = .0012). It was found clearly negative in NPBC. EOI‐MRD was detectable in 152/300 (50.7%; ≥0.01% in 35.33% and <0.01% in 15.33%) samples, in which CD304 was positive in 72/152 (47.4%) diagnostic and 63/152 (41.4%) MRD samples. It was positive in 45.7% (21/46) of low‐level (<0.01%) MRD samples. In comparison with diagnostic samples, its expression was retained in 68.06% (49/72), lost in 31.94% (23/72), and gained in 14/80 (17.5%) of EOI‐MRD samples.ConclusionsCD304 is commonly expressed in leukemic blasts of BCP‐ALL. It is very useful in distinguishing residual disease from hematogones and is a fairly dependable marker. Hence, it is a valuable addition for enhancing the sensitivity and applicability of MFC‐based MRD assay in BCP‐ALL.

Background: Measurable/minimal residual disease (MRD) status is suggested as a powerful indicator of clinical-outcome in T-cell lymphoblastic leukemia/lymphoma (T-ALL). Contrary to B-cell ALL, reports on TALL MRD are limited and mostly based on molecular methods, mainly from developed countries. Multicolor flow cytometry (MFC)-based TALL studies are very few. Clinically relevant cutoff levels and ideal time-point for MRD assessment are still inconclusive. In view of lack of TALL MRD data from the developing world, we evaluated the prognostic value of MFC-based post-induction (PI)-MRD assessment in TALL in the context of standard practice. Methods: We included 256 childhood-TALL patients (age < 15 years) treated with a modified-MCP841 protocol, which uses high-dose cytarabine during consolidation, as a part of standard hospital practice. MRD was studied using 10-color 11-antibody MFC with any level of detectable disease being considered positive. Post-induction (PI)-MRD was available in all patients, and post-consolidation (PC) MRD was available mostly in PI-MRD-positive patients (n = 88). Results: Three years cumulative-incidence-of-relapse (3years-CIR) in PI-MRD-positive patients was inferior to negative patients (46.3% vs. 18.4%). The median relapse-free-survival (RFS), event-free-survival (EFS) and overall-survival (OS) with hazard ratio (HR) of PI-MRD-positive patients were 21.4 months vs not reached (p < 0.0001, HR-4.7), 21.6 months vs. not-reached (p = 0.0003, HR-2.01) and 37.3 months vs. not reached (p = 0.026, HR-1.64) respectively. RFS, EFS and OS of patients with PI-MRD<0.01% (n = 17) were as inferior as PI-MRD ≥ 0.01% in comparison Tembhare et al. Post-induction MRD & WBC Count in TALL with MRD-negative patients with HR of 4.7 (p < 0.0001), 2.45 (p = 0.0003), and 2.5 (p = 0.029), respectively. Three-years-CIR of patients with hyperleukocytosis (≥100 × 109/L) was also higher (50.5 vs. 27.6%) with inferior RFS, EFS, and OS. Among PI-MRDpositive patients, 3years-CIR, RFS, EFS, and OS of PC-MRD-positive were also inferior to that of negative patients. On multivariate analysis any-level detectable PI-MRD and hyperleukocytosis remained independently associated with inferior RFS, EFS, and OS. A combination of PI-MRD-positive status and hyperleukocytosis identified the patients with the worst clinical outcomes. Conclusion: Detectable PI-MRD using MFC was found to be the strong predictive factor of inferior clinical outcome in TALL patients. The combination of PI-MRD status and hyperleukocytosis provides the most influential tool for the management of TALL in resource constrained settings from developing world.

Introduction: In 2016, Children Oncology Group (COG) described a new high-risk subtype of acute myeloid leukemia (AML) with a distinct immunophenotypicsignature, RAM-phenotype (RAM-AML). Data on clinical and laboratory features of RAM-AML are still limited to COG report only. Herein, we report the clinicopathological characteristics and detailed immunophenotypic features of RAM-AML patients. In COG report, 38% of RAM-AML belonged to acute megakaryoblastic leukemia (AMKL)-subtype. Hence, we further compared the immunophenotypic features RAM-AML with non-RAM-AMKL diagnosed during the same study period. Methods: We included RAM-AML and non-RAM AMKL patients diagnosed between January 2017 and December 2019. We studied their morphological, cytochemical, immunophenotyping, cytogenetic, and molecular characteristics. Mean fluorescent intensity (MFI) and expression-pattern of immunophenotypic markers of RAM-AML were compared with non-RAM AMKLs patients. Results: We identified 11 RAM-AML (1%) and 21 non-RAM AMKL (1.9%) patients in 1102 pediatric-AML patients. Seven of 11 (63.64%) patients belonged to FAB-M7subtype. CD56, CD117, and CD33 demonstrated overexpression, whereas CD45 and CD38 showed under-expression in RAM-AML patients. CD36 was consistently negative in RAM-AML, whereas moderate-bright positive in non-RAM AMKLs patients (p < 0.0001). On principle component analysis, addition of CD36 enhanced the visual-separation between RAM-AML and non-RAM AMKL clusters. Cytogenetic and molecular studies did not show any recurrent abnormality; however, RNAsequencing study revealed CBFA2T3-GLIS2-fusion in three of seven (42.8%) RAM-AML patients. Conclusion: We report the clinicopathological characteristics and the detailed immunophenotypic profile in the world's second series of RAM-AML patients. We further report a novel finding of CD36-negative expression as an additional parameter to the multidimensional immunophenotypic signature of this entity.

BackgroundRecently, anti-CD38 monoclonal antibody (Mab) therapy has become a focus of attention as an additional/alternative option for many hematological neoplasms including T-cell acute lymphoblastic leukemia (T-ALL). It has been shown that antitumor efficacy of anti-CD38-Mab depends on the level of CD38 expression on tumor cells. Reports on CD38 expression in T-ALL are scarce, and data on the effect of cytotoxic chemotherapy on CD38 expression are limited to very few samples. Moreover, it lacks entirely in refractory disease and in adult T-ALL. We report the flow cytometric evaluation of CD38 expression in T-ALL blasts at diagnosis and the effect of cytotoxic chemotherapy on its expression in measurable residual disease (MRD), refractory disease (MRD≥5%), and relapsed disease in a large cohort of T-ALL.MethodsThe study included 347 samples (188 diagnostic, 100 MRD, 24 refractory and 35 relapse samples) from 196 (children: 85; adolescents/adults: 111) patients with T-ALL. CD38-positive blasts percentages (CD38-PBPs) and expression-intensity (mean fluorescent intensity, CD38-MFI) were studied using multicolor flow cytometry (MFC). MFC-based MRD was performed at the end-of-induction (EOI-MRD, day 30–35) and end-of-consolidation (EOC-MRD, day 78–85) subsequent follow-up (SFU-MRD) points.ResultsPatients were classified into early thymic precursor subtype of T-ALL (ETPALL, 54/188, 28.7%), and non-ETPALL (134/188, 71.3%). Of 188, EOI-MRD assessment was available in 152, EOC-MRD was available in 96 and SFU-MRD was available in 14 patients. CD38 was found positive in 97.9% (184/188) of diagnostic, 88.7% (110/124) MRD (including 24-refractory) and 82.9% (29/35) relapsed samples. Median (95% CI) of CD38-PBPs/MFI in diagnostic, MRD, refractory, and relapsed T-ALL samples were, respectively, 85.9% (82.10%–89.91%)/4.2 (3.88–4.47), 74.0% (58.87%–83.88%)/4.6 (3.67–6.81), 79.6% (65.25%–96.11%)/4.6 (3.33–8.47) and 85.2% (74.48%–93.01%)/5.6 (4.14–8.99). No significant difference was noted in CD38 expression between pediatric versus adult and patients with ETPALL versus non-ETPALL. No change was observed in CD38-MFI between diagnostic versus MRD and diagnostic versus relapsed paired samples. However, we noticed a mild drop in the CD38-PBPs in MRD samples compared with the diagnostic samples (p=0.016).ConclusionWe report an in-depth analysis of CD38 expression in a large cohort of T-ALL at diagnosis, during chemotherapy, and at relapse. Our data demonstrated that CD38 is robustly expressed in T-ALL blasts with a little effect of cytotoxic chemotherapy making it a potentially effective target for antiCD38-Mab therapy.

Accurate knowledge of expression patterns/levels of commonly used MRD markers in regenerative normal-B-cell-precursors (BCP) is highly desirable to distinguish leukemicblasts from regenerative-BCP for multicolor flow cytometry (MFC)-based measurable residual disease (MRD) assessment in B-lymphoblastic leukemia (B-ALL). However, the data highlighting therapy-related immunophenotypic-shift in regenerative-BCPs is scarce and limited to small cohort. Herein, we report the in-depth evaluation of immunophenotypic shift in regenerative-BCPs from a large cohort of BALL-MRD samples. Ten-color MFC-MRD analysis was performed in pediatric-BALL at the end-of-induction (EOI), end-of-consolidation (EOC), and subsequent-follow-up (SFU) time-points. We studied normalized-mean fluorescent intensity (nMFI) and coefficientof-variation of immunofluorescence (CVIF) of CD10, CD19, CD20, CD34, CD38, and CD45 expression in regenerative-BCP (early, BCP1 and late, BCP2) from 200 BALL-MRD samples, and compared them with BCP from 15 regenerating control (RC) TALL-MRD samples and 20 treatment-naïve bone-marrow control (TNSC) samples.Regenerative-BCP1 showed downregulation in CD10 and CD34 expression with increased CVIF and reduced nMFI (p < 0.001), upregulation of CD20 with increased nMFI (p = 0.014) and heterogeneous CD45 expression with increased CVIF (p < 0.001). Immunophenotypic shift was less pronounced in the BCP2 compared to BCP1 compartment with increased CVIF in all but CD45 (p < 0.05) and reduced nMFI only in CD45 expression (p = 0.005). Downregulation of CD10/CD34 and upregulation of CD20 was higher at EOI than EOC and SFU time-points (p < 0.001). Regenerative-BCPs are characterized by the significant immunophenotypic shift in commonly used B-ALL-MRD markers, especially CD10 and CD34 expression, as compared to treatment-naïve BCPs. Therefore, the templates/database for BMRD analysis must be developed using regenerative-BCP.

High-sensitivity multicolour flow cytometry (MFC)-based B-lymphoblastic leukaemia (B-ALL) measurable residual disease (BMRD) assay is increasingly being used in clinical practice. Herein, we describe six consistently present low-level populations immunophenotypically mimicking abnormal B-ALL blasts in 441 BMRD samples from 301 children. These included CD19 + CD123 + plasmacytoid dendritic cells differentiating from lymphoid precursors, CD10 + transitional B cells with CD10 + / CD38dim-to-negative/CD20bright/CD45bright phenotype, CD19 + natural killer (NK) cells, CD73bright/CD10 + mesenchymal stromal/stem cells, CD73bright/CD34 + endothelial cells, and a CD34 + CD38dim-to-negative/CD10 À /CD20bright/CD45bright subset of mature B cells. We provide the proportions, comprehensive immunophenotype, and practical clues for proper identification of these low-level populations. Knowledge regarding the presence and immunophenotype of these mimics is essential for accurate interpretation in high-sensitivity MFC-BMRD analysis.

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