Gaurav Chatterjee

PURPOSE: WHO5 (2022) classification of B-lymphoblastic leukemia (B-ALL) incorporates several novel entities requiring high-throughput sequencing for their accurate characterization. The clinical relevance of this classification in the context of contemporary measurable residual disease (MRD)–directed therapy is unclear. METHODS: We analyzed 533 pediatric B-ALL uniformly treated with Indian Collaborative Childhood Leukaemia group (ICiCLe)-ALL-14 protocol as defined by WHO-2016 and reclassified them as per WHO5 using targeted sequencing, FISH, and cytogenetics. RESULTS: Subtype-defining genomic abnormalities were identified in 81.2% of the cohort as per the WHO5 classification. Among the new subtypes, PAX5 ^alt and MEF2D -r were associated with a trend toward an inferior 3-year event-free survival (EFS) of 32.8% ( P = .003) and 33.7% ( P = .091), respectively. We developed a three-tier genomic risk stratification model incorporating 15 genomic subtypes and the IKZF1 deletion. Children with standard (SGR), intermediate (IGR), and high genomic risk (HGR) demonstrated 3-year EFS of 80.4%, 59.3%, and 45.8% ( P < .0001), and 3-year overall survival of 89.6%, 75.3%, and 62.3% ( P < .0001), respectively. Genomic risk further identified heterogeneous outcomes among ICiCLe risk groups ( P < .0001). SGR was associated with superior EFS irrespective of MRD status (3-year EFS 80.5% in postinduction [PI] MRD–negative v 80.8% PI-MRD–positive patients, P = .530). On multivariable analysis, genomic risk (hazard ratio [HR], 1.7 [95% CI, 1.41 to 2.01]; P < .0001), initial ICiCLe risk (HR, 1.3 [95% CI, 1.06 to 1.49]; P = .009), and PI-MRD (HR, 2.2 [95% CI, 1.66 to 2.90]; P < .0001) independently predicted EFS. CONCLUSION: The study demonstrates the potential role of genomic risk stratification, in conjunction with MRD, in stratifying patients into clinically relevant risk categories.

Published data on flow cytometric assessment of Classic Hodgkin lymphoma (CHL) are largely limited to lymph node biopsies, where histopathological and immunophenotypic findings are already available, thereby restricting its practical applicability. Real‐world evidence demonstrating the additional diagnostic role of flow cytometric immunophenotyping (FCI) in CHL is limited. This study evaluated the utility of FCI in body fluids (BF) and bone marrow samples submitted for the diagnostic evaluation and staging of CHL in routine clinical practice. We studied 126 samples from 120 patients evaluated for CHL diagnosis or staging, including fine‐needle aspirates (FNAs, n = 15), tissue biopsies ( n = 6), bone marrow aspirates (BMAs, n = 76), and BF ( n = 29). Samples were processed using a lyse–stain–wash protocol, acquired on BD‐LSRFortessa, and analyzed using Kaluza software. A single‐tube 16‐color antibody panel (CD3, CD11b, CD13, CD15, CD19, CD20, CD30, CD33, CD38, CD40, CD45, CD64, CD71, CD73, CD95, and CD271) was used. The findings were correlated with the clinical, histopathological, and immunohistochemical data. CHL involvement was detected in 13 samples (5 FNAs, 5 fluids, 2 BMAs, 1 lymph node). The level of Hodgkin and Reed–Sternberg cells (HRS) ranged from 0.03% to 2.86% (median, 0.43%) in positive samples. HRS cells consistently expressed CD30, CD40, CD71, and CD95. FCI demonstrated excellent sensitivity and specificity in fluids, FNA, and core needle biopsy; however, it exhibited poor sensitivity in BMA, likely due to hemodilution or non‐representative sampling. FCI complements histopathology by enabling the rapid and accurate detection of CHL involvement, particularly in BF and limited tissue samples such as FNA and small biopsies.

Measurable residual disease (MRD) assessment is a key determinant of risk stratification and treatment modification in B‐cell acute lymphoblastic leukemia (B‐ALL). Multicolour flow cytometry (MFC) is a globally adopted rapid and sensitive MRD technique. However, technical practices remain significantly variable across laboratories, compromising assay sensitivity, reproducibility, and comparability. Currently, systemic recommendations providing guidelines on the technical aspects of MFC‐MRD in B‐ALL are unavailable. The Cytometry Society (TCS)‐India undertook a structured consensus process to standardize technical aspects of MFC‐MRD testing. A two‐round Delphi survey was conducted among laboratory professionals and clinicians across India to capture the prevailing practices and priorities. Consensus recommendations were prepared through survey findings and further refined during an in‐person consensus congress with national and international experts from Australia, Egypt, Spain, and Russia. This document provides comprehensive recommendations across pre‐analytic, analytic, and post‐analytic phases of MFC‐MRD including minimal antibody panel, best practices for sample collection, transport, and processing, Instrument setup, quality control, acquisition targets, gating strategies, approaches for LOD/LLOQ definition, hemodilution assessment, etc. A structured MRD report format with quality caveats is also provided. The recommendations are categorized into “Must,” “Preferred,” and “Acceptable” levels to suit diverse resource settings while promoting harmonization. Implementation of these standards will enhance assay sensitivity, reproducibility, and comparability, thereby strengthening MRD‐guided clinical decision‐making in B‐ALL.

Assessment of T‐cell clonality is critical for diagnosing T‐cell non‐Hodgkin lymphomas (T‐NHL). Flow cytometric T‐cell receptor (TCR)‐Vβ‐repertoire analysis is specific but technically complex, multi‐tube, and often limited in paucicellular specimens. T‐cell receptor‐beta‐constant‐region‐1 (TRBC1) has emerged as a simpler alternative, but normal distribution ranges in clinically relevant T‐cell subsets remain insufficiently characterized and data on direct comparison with TCR‐Vβ restriction is limited. We prospectively analyzed 70 blood samples from healthy donors and 58 samples (bone marrow, peripheral blood, fine‐needle aspirates, body fluids) from T‐NHL patients. Multiparameter flow cytometry (MFC) was used for diagnostic immunophenotyping in T‐NHL cases and for TRBC1 assessment in T‐NHL cases and 28 immunophenotypic T‐cell subsets in healthy individuals. In T‐NHL, clonality was determined by TRBC1 restriction in aberrant T‐cell populations and, where sufficient material was available, corroborated by TCR‐Vβ repertoire analysis (IOTest Beta Mark).In healthy donors, median TRBC1‐positive fractions across T‐cell subsets ranged from 21.39% to 78.61%, with narrow 99% confidence intervals in major CD4 ⁺ and CD8 ⁺ compartments. Minor subsets occasionally showed extreme values (7%–93%) but remained polyclonal by TCR‐Vβ analysis, leading to thresholds of ≤6% or ≥94% TRBC1 positivity as indicative of clonality in our cohort. Among 57 evaluable T‐NHL cases, all CD3‐positive neoplastic populations showed monotypic TRBC1 expression, with 100% concordance with TCR‐Vβ results in 18 tested cases. TRBC1 was non‐informative on surface‐CD3‐negative T‐NHL, although cytoplasmic staining was sometimes helpful. Flow cytometric TRBC1 analysis is a rapid, cost‐effective, single‐tube method for T‐cell clonality assessment, with applicability to small T‐cell subsets such as paucicellular and measurable residual disease samples.

PURPOSE: The 2022 European LeukemiaNet (ELN22) genetic risk stratification for AML makes critical changes, including removal of FLT3- internal tandem duplications (ITD) allelic ratio and inclusion of mutations in myelodysplasia-related (MR) genes and in-frame bZIP CEBPA mutations. We evaluated the applicability of ELN22 in a uniformly treated younger AML cohort and explored refinements to improve risk prediction. METHODS: We retrospectively analyzed 473 adult patients with AML treated with intensive therapy. A combination of cytogenetics and next-generation sequencing was used to stratify patients into the ELN17 and ELN22 risk. In addition, we also evaluated leukemic stem cell (LSC) burden at diagnosis and postinduction measurable residual disease by multiparametric flow cytometry. RESULTS: A total of 77 cases (16.3%) were reclassified from ELN17, primarily because of changes associated with FLT3 -ITD, CEBPA , and MR gene mutations. As per ELN22, 56.7% of patients were classified as favorable, 28.3% intermediate, and 15.0% adverse risk. ELN22 adverse-risk patients had significantly inferior overall survival and relapse-free survival compared with intermediate- and favorable-risk patients. High LSC burden at diagnosis, presence of WT1 mutations, and DNMT3A-FLT3 -ITD comutated NPM 1 dichotomized the ELN22 intermediate-risk group. CONCLUSION: We conclude that ELN22 is prognostically valid in intensively treated younger patients with AML. Incorporation of WT1 mutation status, triple-mutated NPM1 , and LSC burden may improve risk stratification, particularly within the heterogeneous intermediate-risk category.

Pediatric chronic myeloid leukemia (pCML) is a rare childhood malignancy, representing 2%–3% of all childhood leukemia. Tyrosine kinase inhibitors (TKIs) have greatly improved survival but pose challenges due to their long‐term effects on growth and bone health in children. We prospectively studied treatment‐free remission (TFR) in 45 children with pCML in chronic phase on imatinib. Eligibility criteria were as per current NCCN guidelines, with a less stringent qPCR monitoring scheduled every 3 months. TFR was successful in 71.1% (32 out of 45) of patients after a median follow‐up of 25 (range: 6–42) months. The TFR rates at 12 and 24 months were 70% and 66%, respectively. Children under 5 years had a TFR rate of 88.9%, compared to 61.8% in those over 5 years (p = 0.18). Eleven of the 13 patients who lost MMR did so within 6 months of discontinuation. The cumulative incidence of loss in MMR at 6, 12, and 24 months was 26.4%, 27%, and 33%, respectively. Ten out of 13 (76.9%) patients with discontinuation failure (DF) regained MMR within 3 (2–20) months of restarting imatinib. A significant correlation was found between higher T‐regulatory cell levels at baseline and DF (p = 0.005). More than half patients showed improved bone mineral density after 2 years of TFR. Our findings suggest that high TFR rates can be attained in pCML, with added benefits for bone health. Less frequent molecular monitoring was not associated with adverse outcomes and there seems to be a role of the immune system in sustaining TFR. The study is registered in the Clinical Trials Registry‐India (CTRI/2020/11/029199).

WHO5-2022 classification of B-lymphoblastic leukemia (B-ALL) incorporates several novel entities requiring high-throughput sequencing for their accurate characterization. The clinical relevance of this classification in the context of contemporary MRD-directed therapy is unclear. We analyzed 533 pediatric B-ALL uniformly treated with ICiCLe-ALL-14 protocol as defined by WHO2016 and reclassified them as per WHO5-2022 using targeted sequencing, FISH, and cytogenetics. Subtype-defining genetic abnormalities were identified in 81.2% of the cohort as per the WHO5 classification. Among the new subtypes, PAX5^alt, MEF2D-r, and BCR::ABL1-like(ABL-class) were associated with an inferior 2-year event-free survival (EFS) of 39.1% (p < 0.0001), 53.8% (p = 0.024) and 60.6% (p = 0.043), respectively. We developed a 3-tier genetic risk stratification model incorporating 15 genetic subtypes and the IKZF1 deletion. Children with standard, intermediate, and high genetic risk demonstrated 2-year EFS of 92.6%, 71.0%, and 50.7% (p < 0.0001), and 2-year overall survival of 94.3%, 81.9%, and 71.6% (p < 0.0001), respectively. Genetic risk further identified heterogeneous outcomes among ICiCLe risk groups (p < 0.0001). Standard genetic risk was associated with superior OS and EFS irrespective of MRD status. We demonstrate the applicability of the WHO5 classification in routine practice and create a general framework for incorporating the WHO5 classification in risk-adapted therapy for childhood B-ALL.

Here we review the ‘what and how’ of molecular techniques used in the context of haematopathological diagnostics of both lymphoid and myeloid neoplasms. Keeping in mind that the required resources for molecular testing are not universally available, we will not only discuss novel and emerging techniques that allow more high‐throughput and sophisticated analyses of lymphoid and myeloid neoplasms, but also the more classical, low‐cost alternatives and even some workarounds for molecular testing approaches. In this review we also address other key aspects around molecular techniques for haematopatholgy diagnostics, including preanalytics, data interpretation, and data management, bioinformatics, and interlaboratory precision and performance evaluation.