Nikhil Patkar

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.

Aims: Although acute myeloid leukaemia (AML), myelodysplasia-related (AML-MR), can be defined solely by molecular abnormalities, legacy studies did not analyse the KMT2A gene. The KMT2A -partial tandem duplication ( KMT2A -PTD) is described in myelodysplastic neoplasms (MDS) as well as AML. We describe the clinical, morphological, immunophenotypic and molecular features of AML harbouring KMT2A- PTD. Methods: We studied 802 adult AML patients, for whom next-generation sequencing- based mutation and copy number analysis were available. For the patients harbouring KMT2A -PTD, the immunophenotypic analysis including measurable residual disease (MRD), mutational landscape and the patient outcomes were analysed. Results: We identified 45 patients of de novo and secondary AML harbouring KMT2A -PTD. Morphological dysplasia and immunophenotypic abnormalities, described in MDS, were observed in 35.6% and 40% of de novo cases respectively. Furthermore, 44% of AML with KMT2A -PTD could be diagnosed as AML-MR based on cytogenetics or genomics. We observed progenitor abnormalities, commonly aberrant CD7 (33%) and CD15 (59%), and granulocytic abnormalities like loss of side scatter (50%), asynchronous maturation patterns and loss of CD177 in mature granulocytes. Frequent mutations involving IDH2 (30% of which were R172), FLT3 (32% each), RUNX1 (29%), DNMT3A and U2AF1 (24% each) were noted. We also found that more than 25% of patients did not achieve morphological remission, and the remaining 60% were MRD positive. The outcomes of AML with KMT2A -PTD with or without MR-associated abnormalities were similar. Conclusions: AML harbouring KMT2A -PTD frequently displays MR immunophenotypic abnormalities and is frequently associated with AML-MR type mutations. In addition, they have similar outcomes as compared with AML-MR.

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.

Introduction: Accurate alignment is critical for metagenomic profiling of plasma cell-free DNA (cfDNA), where microbial fragments are short, low in abundance, and often obscured by abundant human DNA. Yet, no unified guideline exists for selecting alignment algorithms in cfDNA studies. Results: We benchmarked six alignment methods (BWA-MEM2, Minimap2, Bowtie2-sensitive, Bowtie2-very-sensitive, Bowtie2-very-sensitive-local, and Kraken2) using in silico simulated datasets and cfDNA from 102 acute leukemia samples with sepsis. Evaluation criteria included bacterial read retention after human read filtering, minimization of false positives in real cfDNA datasets, and detection sensitivity for antimicrobial resistance (AMR) genes. BWA-MEM2 emerged as the most effective for removing stringent human reads and bacterial classification, while bowtie2(very_sensitive_local mode) was optimal for AMR gene detection. Post-filtering, cfDNA profiles exhibited marked heterogeneity in bacterial and AMR gene signatures, reflecting expected clinical variability. Conclusions: BWA-MEM2 provides optimal human read removal and bacterial taxa classification, minimizing false positives, though some true reads may be lost. For AMR gene detection, Bowtie2 (very_sensitive_local mode) demonstrated superior sensitivity in simulated datasets. This study presents the first systematic evaluation of read-based and k-mer–based aligners in plasma cfDNA, offering practical guidance for balancing human read removal with bacterial and AMR gene profiling to achieve robust and clinically meaningful results.

Chimeric Antigen Receptor-T (CAR-T) cell therapy is effective for relapsed/refractory B-acute lymphoblastic leukemia (r/r B-ALL) but is not universally available. We developed a novel humanized CD19-directed CAR-T (HCAR19) approved for Phase 1/1b/2 trials. Patients aged 3–25 years were enrolled with r/r B-ALL and ineligible for allogeneic stem cell transplant. Lymphodepletion utilized standard-dose fludarabine and cyclophosphamide. A 3 + 3 design testing 3 dose-ranges was used to determine Phase-2 Dose (P2D): Dose-A, 1 × 10⁶ HCAR19 cells/kg, Dose-B, 3–5 × 10⁶/kg, and Dose-C, 10–15 × 10⁶/kg. Primary endpoint was overall response rate (ORR) at day-30 on bone-marrow flow-cytometry. From May-2021 to September-2023 12 patients [median age-14 (range: 5–24) years] were enrolled with median bone marrow blasts 19.5% at screening. Cytokine release syndrome occurred in 10 (83%) patients, predominantly Grades 1–2, and Grade-2 immune-cell associated neurotoxicity (ICANS) in 1. All patients had Grade-3 cytopenia. ORR was 91.7% (11/12), complete response (CR) in 8 (66.7%) and partial response in 3 (25%). Seven of 8 CRs were at Dose-levels B and C, all of which were sustained till 12 months follow-up. Patients who received dose levels below 3 × 10⁶/kg, or did not achieve CR, had early loss of response or rapid progression. HCAR19 demonstrated safety, manageable toxicity, and durable remissions. and P2D was determined as 5–10 × 10⁶ HCAR19-cells/kg. Clinical trial registration: The study is registered in the Clinical Trials Registry- India (CTRI/2021/05/033348 and CTRI/2023/03/050689).

Acute promyelocytic leukemia (APL), distinguished by the presence of PML::RARA fusion transcript, is a medical emergency because of its high early death rate, which is preventable when diagnosed early. Current diagnostic methods are precise and reliable but are time intensive, require sophisticated instruments, and analytical expertise. This study has redefined APL identification by CRISPR system (RAPID-CRISPR) to rapidly (<3 hours) detect PML::RARA. APL cell lines (NB4 and UF-1) and bone marrow/peripheral blood samples from 74 patients with APL (66/8, retrospective/prospective) and 48 controls were included in the study. We used a DETECTR (DNA endonuclease-targeted CRISPR transreporter) assay to identify the bcr1, bcr2, and bcr3 PML::RARA isoforms. To ensure high specificity, we used PML::RARA–specific loop-mediated isothermal amplification (LAMP) primers, synthetic protospacer-adjacent motif sites, and isoform-specific CRISPR RNAs. RAPID-CRISPR recognized APL with 100% sensitivity and 100% specificity in an ambispective cohort of patient samples. Furthermore, our blinded validation approach to detect PML::RARA in an unbiased manner provides an additional layer in the diagnostic precision of APL. RAPID-CRISPR demonstrated superior sensitivity, detecting as few as 1 copy of PML::RARA compared with 10 copies by the gold-standard reverse transcriptase qualitative and quantitative polymerase chain reaction. The nucleic acid extraction–free protocol combined with the 1-step reverse transcriptase LAMP–based DETECTR followed by lateral flow readout makes the RAPID-CRISPR assay suitable for diagnosing APL in point-of-care settings. This simple, cost-effective tool, with its easy-to-read format, is particularly valuable in underresourced regions. The assay facilitates timely diagnosis and prompt administration of lifesaving therapies such as all-trans retinoic acid and arsenic trioxide in APL.