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Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous disease with poor prognosis and inferior outcome. Although multiple studies have been perform on genomics of T-ALL, data from Indian sub-continent is scarce. Methods: In the current study we aimed to identify the genetic variability of T-ALL in an Indian cohort of pediatric (age ≤ 12 years) T-ALL patients (n = 25) by whole transcriptome sequencing along with whole exome sequencing and correlated the findings with clinical characteristics and disease outcome. Results: The median age was 7 years (range 3 -12 years). RNA sequencing revealed a definitive fusion event in 14 cases (56%) (including a novel fusions) with STIL::TAL1 in 4 (16%), followed by NUP21::ABL1, TCF7::SPI1, ETV6::HDAC8, LMO1::RIC3, DIAPH1::JAK2, SETD2::CCDC12 and RCBTB2::LPAR6 in 1 (4%) case each. Significant aberrant expression was noted in RAG1 (64%), RAG2 (80%), MYCN (52%), NKX3-1 (52%), NKX3-2 (32%), TLX3 (28%), LMO1 (20%) and MYB (16%) genes. WES data showed frequent mutations in NOTCH1 (35%) followed by WT1 (23%), FBXW7 (12%), KRAS (12%), PHF6 (12%) and JAK3 (12%). Nearly 88.2% of cases showed a deletion of CDKN2A/CDKN2B/MTAP genes. Clinically significant association of a better EFS and OS (p=0.01) was noted with RAG2 over-expression at a median follow up of 22 months, while a poor EFS (p=0.041) and high relapse rate (p=0.045) was observed with MYB over-expression. Conclusion: Overall, the present study demonstrates the frequencies of transcriptomic and genetic alterations from Indian cohort of pediatric T-ALL and is a salient addition to current genomics data sets available in T-ALL. [ABSTRACT FROM AUTHOR]

Background: Philadelphia chromosome (Ph)–like B‐acute lymphoblastic leukemia (B‐ALL) is a clinically significant, high‐risk genetic subtype of B‐ALL cases. There are few data on the incidence, characterization, and treatment outcomes of Ph‐like ALL cases from low‐ and middle‐income countries. There is a pressing need to establish a well‐organized/cost‐effective approach for identifying Ph‐like ALL instances. Methods: Multiplex reverse transcriptase polymerase chain reaction, nCounter NanoString, and fluorescence in situ hybridization were used to detect and characterize Ph‐like ALL cases among recurrent genetic abnormalities (RGA)neg B‐ALL cases. At the end of induction therapy, flow cytometry‐minimal residual disease (MRD) assay was used to quantify MRD positivity in Ph‐like ALL cases. Results: Of 130 newly diagnosed B‐ALL cases, 25% (BCR::ABL1), 4% (ETV6::RUNX1), 5% (TCF3::PBX1), 2% (KM2TA::AFF1), and 65% RGAneg B‐ALL cases were revealed by multiplex reverse transcriptase polymerase chain reaction. Among RGAneg B‐ALL cases, 24% Ph‐like ALL cases using nCounter NanoString were identified, with 48% CRLF2high cases with 45% CRLF2::P2RY8 and 18% CRLF2::IGH rearrangements(∼r) revealed by fluorescence in situ hybridization. In 52% of CRLF2low cases, 17% ABL1 and JAK2∼r 8% EPOR::IGH & PDGRFB∼r were identified. Ph‐like ALL cases had higher total leukocyte count (p <.05), male preponderance (p <.05), and high MRD‐positivity/induction failure compared with RGAneg B‐ALL cases. Furthermore, in Ph‐like ALL cases, 11 significant genes using quantitative polymerase chain reaction were identified and validated. CRLF2, IGJ, CEACAM6, MUC4, SPATS2L and NRXN3 genes were overexpressed and show statistical significance (p <.05) in Ph‐like ALL cases. Conclusions: This study showed the high incidence of Ph‐like ALL cases with kinase activating alterations and treatment outcomes from low‐ and middle‐income region. Furthermore, a surrogate cost‐effective multiplex panel of 11 overexpressed genes for the prompt detection of Ph‐like ALL cases is proposed. Plain Language Summary: Identification of recurrent gene abnormalities (RGA)neg B‐acute lymphoblastic leukemia (B‐ALL) cases using multiplex‐reverse transcriptase polymerase chain reaction.Identification and characterization of Philadelphia (Ph)–like ALL cases using nCounter NanoString gene expression profiling and fluorescence in situ hybridization.Furthermore, Ph‐like ALL cases were characterized according to CRLF2 expression and kinase‐activating genomic alterations. Minimal residual disease of Ph‐like ALL cases were quantified using flow cytometry‐minimal residual disease assay.A surrogate molecular approach was established to detect Ph‐like ALL cases from low‐ and middle‐income countries. A surrogate approach for the rapid baseline detection of Philadelphia chromosome (Ph)–like acute lymphoblastic leukemia cases, followed by its kinase gene alterations. Early detection of Ph–like acute lymphoblastic leukemia cases is extremely important to start personalized treatments to improve the overall survival/outcomes of these cases. [ABSTRACT FROM AUTHOR]

Introduction: Pediatric T-cell acute lymphoblastic leukemia (T-ALL) poses significant challenges due to its aggressive nature and resistance to standard treatments. Long non-coding RNAs (lncRNAs) have emerged as potential biomarkers and therapeutic targets in leukemia. This study aims to characterize the lncRNA landscape in pediatric T-ALL, identify specific lncRNAs signatures, and assess their clinical relevance. Methods: RNA sequencing was performed on T-ALL patient and control samples. Differential expression analysis identified dysregulated lncRNAs and mRNAs. Functional enrichment analysis revealed potential roles of these lncRNAs in cancer pathogenesis. Validation of candidate lncRNAs was conducted using real-time PCR. Clinical correlations were assessed, including associations with patients' clinical characteristics and survival outcomes. Results: Analysis identified 674 dysregulated lncRNAs in pediatric T-ALL, with LINC01221 and CRNDE showing the most interactions in cancer progression pathways. Functional enrichment indicated involvement in apoptosis, survival, proliferation, and metastasis. Top 10 lncRNAs based on adjusted p value < 0.05 and Fold Change > 2 were selected for validation. Seven lncRNAs LINC01221, PCAT18, LINC00977, RP11-620J15.3, RP11-472G21.2, CTD-2291D10.4, and CRNDE showed correlation with RNA sequencing data. RP11-472G21.2 and CTD-2291D10.4 were highly expressed in T-ALL patients, with RP11-620J15.3 correlating significantly with better overall survival (p = 0.0007) at a median follow up of 32 months. The identified lncRNAs were further analysed in B-ALL patients. Distinct lncRNAs signatures were noted, distinguishing T-ALL from B-ALL and healthy controls, with lineage-specific overexpression of LINC01221 (p < 0.0001), RP11-472G21.2 (p < 0.001) and CRNDE (p = 0.04) in T-ALL. Conclusion: This study provides insights into the lncRNA landscape of pediatric T-ALL, offering potential diagnostic and prognostic markers. RP11-620J15.3 emerges as a promising prognostic marker, and distinct lncRNAs signatures may aid in the differentiation of T-ALL subtypes. Further research with larger cohorts is warranted to validate these findings and advance personalized treatment strategies for pediatric T-ALL patients. [ABSTRACT FROM AUTHOR]

Background: The gold standard for the identification of Philadelphia (Ph)‐like acute lymphoblastic leukemia (ALL) patients is gene expression profiling. Because of its diverse nature, its identification is extremely difficult and expensive. On the genomic and proteomic landscape of Ph‐like ALL patients, there is a paucity of published literature from developing countries. Methods: The authors used digital barcoded nCounter NanoString gene expression profiling for its detection, followed by molecular and proteomic characterization using fluorescence in situ hybridization and liquid chromatography–tandem mass spectrometry (LC–MS/MS). Results: The authors found 32.05% Ph‐like ALL patients and their median age at presentation was considerably higher than Ph‐negative ALL cases (p =.0306). Furthermore, we identified 20% CRLF2 overexpressed cases having 8.33% CRLF2‐IGH translocation with concomitant R683S mutation and 8.33% CRLF2‐P2RY8 translocation. In 80% of CRLF2 downregulated cases, we identified 10% as having JAK2 rearrangement. Minimal residual disease‐positivity was more common in Ph‐like ALL cases (55.55% vs. 25% in Ph‐negative ALL cases). Immunoglobulin J chain (Jchain), small nuclear ribonucleoprotein SmD1 (SNRPD1), immunoglobulin κ constant (IGKC), NADH dehydrogenase (ubiquinone) 1 α subcomplex subunit 2 (NDUFA2), histone H2AX (H2AFX), charged multivesicular body protein 4b (CHMP4B), and carbonyl reductase (NADPH) (CBR1) proteins were identified to be substantially expressed in Ph‐like ALL patients, using LC–MS/MS. Gene enrichment analysis indicated that involvement of spliceosomal mediated messenger RNA splicing pathway and four microRNAs was statistically significant in Ph‐like ALL patients. Conclusions: For the first time, we have described incidence, molecular, and proteomic characterization of Ph‐like ALL, in developing nations. Plain language summary: In developing countries, detecting Philadelphia (Ph)‐like B‐lineage acute lymphoblastic leukemia is complicated and challenging due to its diverse genetic landscape.There is no well‐defined and cost‐effective methodology for its detection.The incidence of this high‐risk subtype is very high in adult cases, and there is an urgent need for its accurate detection.We have developed an online PHi‐RACE classifier for its rapid detection, followed by delineating the genomic and proteomic landscape of Ph‐like acute lymphoblastic leukemias for the first time in Indian patients. The identification of Philadelphia chromosome (Ph)–like acute lymphoblastic leukemia (ALL) cases with NanoString nCounter digital‐barcoded gene expression profiling was examined in Indian patients. A higher incidence of Ph‐like ALL cases (32.05%) was identified. Furthermore, genomic and proteomic characterization of Ph‐like ALL cases was performed in Indian patients. This is the first report on the incidence and characterization of Ph‐like ALL cases from developing nations. [ABSTRACT FROM AUTHOR]

Methods: Forty pediatric (0–12 years) B-ALL DNA samples (20 paired Diagnosis-Relapse) and an additional six B-ALL DNA samples (without relapse at 3 years post treatment), as the non-relapse arm, were retrieved from the biobank for advanced genomic analysis. Deep sequencing (1050–5000X; mean 1600X) was performed using a custom NGS panel of 74 genes incorporating unique molecular barcodes. Results: A total 47 major clones (>25% VAF) and 188 minor clones were noted in 40 cases after bioinformatic data filtering. Of the forty-seven major clones, eight (17%) were diagnosis-specific, seventeen (36%) were relapse-specific and 11 (23%) were shared. In the control arm, no pathogenic major clone was noted in any of the six samples. The most common clonal evolution pattern observed was therapy-acquired (TA), with 9/20 (45%), followed by M-M, with 5/20 (25%), m-M, with 4/20 (20%) and unclassified (UNC) 2/20 (10%). The TA clonal pattern was predominant in early relapses 7/12 (58%), with 71% (5/7) having major clonal mutations in the NT5C2 or PMS2 gene related to thiopurine-dose response. In addition, 60% (3/5) of these cases were preceded by an initial hit in the epigenetic regulator, KMT2D. Mutations in common relapse-enriched genes comprised 33% of the very early relapses, 50% of the early and 40% of the late relapses. Overall, 14/46 (30%) of the samples showed the hypermutation phenotype, of which the majority (50%) had a TA pattern of relapse. Conclusions: Our study highlights the high frequency of early relapses driven by TA clones, demonstrating the need to identify their early rise during chemotherapy by digital PCR. [ABSTRACT FROM AUTHOR]

60% (6/10) with I BCR-ABL i SP like sp CGFs showed MRD positivity compared to 41.1% (23/56) of I BCR-ABL1 i SP pos sp B-ALLs. Summary of the diagnostic approach used to detect BCR-ABL1-like B-acute lymphoblastic leukemia associated chimeric gene fusions (BCR-ABL-like CGFs). I BCR-ABL1 i -like or Philadelphia chromosome-like B-acute lymphoblastic leukemia/lymphoma ( I BCR-ABL1 i SP like sp B-ALL) initially described based on gene expression profiles (GEP) is characterized by the presence of tyrosine-kinase inhibitor (TKI) targetable chimeric gene fusions (CGF), the spectrum of which is yet to be fully elucidated. The higher frequency of MRD positivity compared to I BCR-ABL1 i SP pos sp group (60% vs. 41%) can be explained by the inclusion of TKIs in treatment of I BCR-ABL1 i SP pos sp cases. [Extracted from the article]

The overexpression of cytokine receptor-like factor-2 (CRLF2) identified by anti-thymic stromal lymphopoietin receptor/TSLPR flow cytometry (FCM) has been reported as a screening tool for the identification of BCR-ABL1-like B-cell acute lymphoblastic leukemia/B-ALL with CRLF2 re-arrangement. TSLPR expression was studied prospectively in consecutive 478 B-ALLs (≤ 12 years (n = 244); 13–25 years (n = 129); > 25 years (n = 105)) and correlated with various hematological parameters and end-of-induction measurable residual disease (day 29; MRD ≥ 0.01% by 10-color FCM). TSLPR positivity in ≥ 10% leukemic cells was detected in 14.6% (n = 70) of B-ALLs. CRLF2 re-arrangement was detected in eight cases (11.4%) including P2RY8-CRLF2 (n = 6), and IgH-CRLF2 (n = 2) with a median TSLPR positivity of 48.8% and 99% leukemic cells, respectively. Recurrent gene fusions/RGF (BCR-ABL1 (17.1%); ETV6-RUNX1 (4.2%), TCF3-PBX1 (1.4%)), other BCR-ABL1-like chimeric gene fusions/CGFs (PDGFRB-rearrangement (2.9%), IgH-EPOR (1.4%)), CRLF2 extra-copies/hyperdiploidy (17.1%), and IgH translocation without a known partner (10%) were also detected in TSLPR-positive patients. CD20 positivity (52.9% vs 38.5%; p = 0.02) as well as iAMP21 (4.3% vs 0.5%; p = 0.004) was significantly more frequent in TSLPR-positive cases. TSLPR-positive patients did not show a significantly higher MRD, compared to TSLPR-negative cases (37% vs 33%). Increasing the threshold cut-off (from ≥ 10 to > 50% or > 74%) increased the specificity to 88% and 100% respectively in identifying CRLF2 translocation. TSLPR expression is not exclusive for CRLF2 translocations and can be seen with various other RGFs, necessitating their testing before its application in diagnostic algorithms. In patients with high TSLPR positivity (> 50%), the testing may be restricted to CRLF2 aberrancies, while patients with 10–50% TSLPR positivity need to be tested for both CRLF2- and non-CRLF2 BCR-ABL1-like CGFs. [ABSTRACT FROM AUTHOR]