Children with central nervous system (CNS) involvement by B-lymphoblastic leukemia (B-ALL) at diagnosis have higher risk features and greater rates of relapse than those without CNS involvement. This study sought to elucidate the relationship between cerebrospinal fluid (CSF) blast clearance rate with overall survival (OS) and relapse in pediatric B-ALL. Patients with CNS involvement of newly diagnosed B-ALL were stratified into 3 CSF clearance groups by days after initial lumbar puncture: fast (1 to 7 d), intermediate (8 to 15 d), and slow (15+ d) clearers. Among 246 new patients, 28% (69) were CSF+ at diagnosis (87% [60] CNS2, 13% [9] CNS3); 55% (38) fast, 35% (24) intermediate, and 10% (7) slow clearers. The hazard ratio for OS was 5.8 (95% CI: 1.2- 27.9, P =0.029) times higher in slow clearers compared with fast clearers, and 17.4 times higher compared with intermediate clearers (95% CI: 1.7-178, P =0.016). There were no significant differences in risk group ( P =0.3) or relapse rates ( P =0.5). These data suggest that among pediatric patients with CNS+ B-ALL disease at diagnosis, those who cleared CSF faster had better OS compared with patients who were slower at clearing the CSF of blasts. A study of the functional dynamics of underlying differences in CSF clearance rates may inform therapeutic modification.

additional and extended data from stuying constituitive and gated CAR-T cells.

Classic Hodgkin lymphoma (cHL) shares mutations with primary mediastinal B cell lymphoma (PMBL) but differs in histology, clinical behavior, and phenotype. To define transcriptional programs underlying these differences, we performed flow cytometric cell sorting and low-input RNA sequencing of Hodgkin and Reed-Sternberg (HRS) cells from eighteen primary tumors, paired intra-tumoral B cells, and four cHL cell lines, and compared them with RNA-sequencing data from 40 PMBL cases. Transcriptomic profiling revealed that HRS cells undergo abortive plasma cell differentiation with robust activation of the unfolded protein response (UPR), a feature shared with multiple myeloma but absent in diffuse large B cell lymphoma and PMBL. HRS cells also demonstrated profound immune evasion, including suppression of B cell identity genes and loss of natural killer cell recognition through downregulation of SLAM family ligands such as CD48. Comparative analysis with PMBL highlighted shared oncogenic programs and key distinctions: HRS cells exhibited greater loss of B cell identity, absence of GCB- and plasma cell markers, and unique upregulation of cytoskeletal and mitotic pathways consistent with their multinucleated morphology. These findings establish HRS cells as aberrantly differentiated GCB cells with partial plasmacytic features, UPR activation and distinct immune evasion strategies.

Single cell data of the clusters, gene expresion, biomarker and called annotations

Relapsed acute leukemia can be difficult to salvage. An uncommon but increasingly recognized and aggressive mechanism of relapse involves lineage switch. In lineage switch, the immunophenotype of the leukemia at relapse differs from the immunophenotype at initial diagnosis, with the underlying genetic driver(s) conserved, confirming a clonal relationship. Lineage switch is most common—and was first recognized—in B-cell acute lymphoblastic leukemia with KMT2A rearrangement, which often relapses as acute myeloid leukemia. In an era where antigen-targeted therapies, including chimeric antigen receptor T-cells and bispecific T-cell engagers, are increasingly utilized and thus apply selective antigen pressure, this may increase the incidence of lineage switch across different leukemia subtypes. Patients with lineage switch have dismal outcomes and optimal therapies remain unknown, thus there is a large unmet need to better understand the biology, define the diagnosis, and determine the therapeutic approaches to lineage switch. Here, we address these needs providing a review of the current biology of lineage switch, the relationship to different genetic subtypes and present definitions and recommendations for immunophenotypic and molecular monitoring.

Classic Hodgkin lymphoma (cHL) associated with Epstein-Barr virus (EBV) positivity as well as non-nodular sclerosis (non-NS) histologic subtypes have demonstrated poorer outcomes compared to EBV-negative and nodular sclerosis cases. We report a prespecified subset analysis of patients enrolled in the phase III SWOG S1826 trial to evaluate outcomes based on EBV status and histologic subtype in patients treated with nivolumab-AVD (N-AVD) or brentuximab vedotin (BV)-AVD. In the phase III SWOG S1826 trial, patients with stage III-IV cHL were randomized to N-AVD or BV-AVD. Of 970 eligible patients, 522 had known EBV status. N-AVD improved 3-year progression-free survival (PFS) in EBV-positive (91% v 70%; HR, 0.30; P = 0.01) and EBV-negative patients (90% v 84%; HR, 0.64; P = 0.09). Among 664 patients with slides available for histology review, 102 (15.4%) had non-NS subtypes. N-AVD prolonged 3-year PFS in patients with non-NS (86% v 63%; HR, 0.33; P = 0.006) and NS histologic subtype (93% v 86%; HR, 0.53; P = 0.01). Non-NS histology independently was associated with inferior outcomes (HR, 2.54; P < 0.0001) after adjusting for treatment in the entire cohort. However, N-AVD treatment still had favorable PFS within this high-risk group. In addition, patients with EBV positivity or non-NS histology cHL treated with BV-AVD had significantly worse PFS (HR, 1.97; P = 0.03 for EBV; and HR, 3.08; P < 0.0001 for histology). N-AVD substantially abrogated the historically poor prognosis associated with EBV positivity and non-NS histology in advanced-stage cHL. These results support N-AVD as frontline standard of care, particularly in high-risk biologic subgroups. (NCT03907488).

Peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS) rarely occurs in pediatric and young adult populations, where little is known about its clinicopathologic and molecular features. We characterized 16 cases of PTCL, NOS diagnosed in patients ≤21 years old. Seven (44%) cases demonstrated SMARCB1/INI1 loss by immunohistochemistry and SMARCB1/INI1 alterations by next-generation sequencing, including biallelic SMARCB1/INI1 deletion (n = 4), stop codon variant with 1-copy deletion (n = 1) or copy-neutral loss-of-heterozygosity (n = 1), and frameshift variant (n = 1). One case demonstrated biallelic SMARCE1 alterations (a nonsense variant and copy-neutral loss-of-heterozygosity), which, to our knowledge, has not been previously described in a hematopoietic neoplasm. The SWI/SNF-intact group harbored pathogenic TET2, PTEN, EZH2, or TP53 variants. CDKN2A deletions were present in 3 of 7 SWI/SNF-deficient and 0 of 4 SWI/SNF-intact cases. Chromosome 22q11.2 alterations were present on karyotype in 2 of 3 SMARCB1/INI1-deficient cases. SWI/SNF deficiency was associated with intermediate-to-large cell cytomorphology, frequent mitotic (88%; P = .041) and apoptotic (88%) activity, Reed-Sternberg-like cells (63%), necrosis (50%), and fibrosis (50%). All cases expressed CD45 and CD43 at initial diagnosis. SWI/SNF-deficient cases predominantly demonstrated a CD4+/CD8-, TCRab, and PTCL-GATA3 phenotype, whereas SWI/SNF-intact cases predominantly demonstrated a CD8+/CD4-, TCRgd, and PTCL-TBX21 phenotype. A PTCL-TBX21 phenotype was more common in SWI/SNF-intact than in SWI/SNF-deficient cases (P = .041). Cytotoxic markers were expressed in 71% of SWI/SNF-deficient and 88% of SWI/SNF-intact cases. Decreased or absent CD3 expression characterized 88% of SWI/SNF-deficient and 13% of SWI/SNF-intact cases (P = .01). Moreover, 63% of SWI/SNF-deficient cases demonstrated decreased or absent expression of ≥3 pan-T-cell antigens, compared with 13% of SWI/SNF-intact cases. Treatment was heterogeneous. Primary treatment failure or relapse occurred in 4 of 8 SWI/SNF-deficient and 4 of 7 SWI/SNF-intact cases. The median overall survival and event-free survival were 48.7 and 47.5 months for the SWI/SNF-deficient group, and 16.4 and 8.9 months for the SWI/SNF-intact group (P = nonsignificant). Death due to disease or therapy-related complications occurred in 4 of 8 (50%) cases in the SWI/SNF-deficient group and 6 of 7 (86%) of cases in the SWI/SNF-intact group. Our findings expand the knowledge of the clinicopathologic and molecular features of pediatric PTCL and highlight SWI/SNF-deficient T-cell lymphoma as a biologically distinct type of PTCL that is associated with characteristic clinicopathologic features.

Measurable/minimal residual disease (MRD) monitoring is an essential component of modern treatment protocols for ALL and AML. Early response to therapy, as measured by MRD levels at defined time points during treatment, serves as an important prognostic indicator and enables risk-adapted stratification with intensification of treatment for patients with poor response while potentially reducing therapy-related toxicity in those demonstrating excellent clearance of leukemic cells. MRD assays must be highly sensitive and specific; multidimensional/parameter flow cytometry (MFC), quantitative polymerase chain reaction (qPCR), and next generation sequencing (NGS) are most used, while morphology is no longer the gold standard of response. MRD by MFC is used globally for risk and response in ALL and AML and is applicable to nearly 100% of patients with a sensitivity of 10-4. For ALL, immunoglobulin/T-cell receptor rearrangements detected by qPCR or NGS offer a nearly universal target applicable to more than 90% of patients with B-ALL and up to 70% of patients with T-ALL with a sensitivity of 10-5. For AML, molecular testing is more frequently implemented in Europe and Asia than in the United States; however, the utilization of qPCR and/or digital droplet PCR (dPCR) methodologies for high-risk fusions is evolving. NGS for somatic mutations are not yet routinely implemented for risk or response in pediatric AML, but continued advances in NGS technologies will continue to transform the MRD landscape of pediatric leukemias.

OBJECTIVES:In healthy individuals, CD34+ stem cells are detectable in peripheral blood in relatively low and stable numbers. The clinical utility of peripheral blood CD34+ stem cell quantitation in the evaluation of pancytopenia has not been systematically studied. METHODS:We used a large archival clinical cohort (n = 154) of pediatric patients from our pediatric-only institution without a history of hematologic disorder or circulating blasts on morphology who underwent peripheral blood flow cytometric screening for any reason. Circulating CD34+ stem cells and total progenitors were quantified and correlated with clinical characteristics and outcomes. RESULTS:Patients ultimately diagnosed with aplastic anemia (n = 15) showed significantly decreased circulating progenitors compared with those with cytopenias of alternative causes, whereas patients with infectious, inflammatory, or other presumed reactive conditions often demonstrated larger CD34+ progenitor populations, consistent with preserved or responsive marrow function. Using receiver operating characteristic analysis, the optimal cutoff for %CD34+ progenitors was 0.0124%, which was rounded to 0.01% for clinical interpretability. CONCLUSIONS:These findings support the potential clinical utility of peripheral blood CD34+ quantitation in the diagnostic workup of pediatric pancytopenia.

Introduction The BCR::ABL1-like subtype of B-lymphoblastic leukemia/lymphoma (B-ALL) presents unique challenges in clinical diagnosis and therapeutic decision-making due to its genomic complexity and the diverse methodologies available for detecting its oncogenic drivers and associated variants. Therefore, consensus guidelines for variant interpretation in BCR::ABL1-like B-ALL are essential, as these could directly impact clinical trial eligibility and/or standard-of-care therapy selection. Methods ClinGen assembled a Somatic Cancer Variant Curation Expert Panel (SC-VCEP) to establish criteria for classifying the oncogenicity of variants in BCR::ABL1-like B-ALL based upon those previously drafted by the Somatic Cancer Clinical Domain Working Group and the NTRK SC-VCEP. The SC-VCEP evaluated ABL1 fusions involving 5 gene partners individually, excluding BCR::ABL1, and determined guidelines for oncogenicity based on fusion architecture, cancer association, and functional evidence. Results Per the SC-VCEP’s criteria, the establishment of oncogenicity for ABL1 fusions required expected gene orientations, a breakpoint preserving the ABL1 tyrosine kinase domain (TKD) and its reading frame, and detection of the fusion in three or more cases of BCR::ABL1-like B-ALL described in peer-reviewed medical literature. The presence of supportive fusion functional data, together with evidence of response to tyrosine kinase inhibitor (TKI)-based therapy in at least one patient, could replace the necessity for sufficient case reports. AMP/ASCO/CAP categorization guidelines were separately developed by the SC-VCEP and regarded fusions as Tier 1A for the diagnosis of BCR::ABL1-like B-ALL if a previously established diagnosis of B-ALL was present and an oncogenic/likely oncogenic BCR::ABL1-like variant was detected. Tier 1A designation for a fusion being predictive of response to therapy was not applicable, as no TKIs are currently approved by the U.S. Food and Drug Administration for treatment of patients with BCR::ABL1-like B-ALL, and clinical trial options should be considered. Discussion and Conclusion The work completed by the SC-VCEP has ultimately received Step 2 approval by ClinGen and has led to current efforts whereby the criteria for oncogenicity and AMP/ASCO/CAP categorization are applied to a set of pilot fusions detected in ABL-class genes. The pilot analysis includes 15 fusion partners with ABL1, ABL2, CSF1R, PDGFRA, and PDGFRB. This cohort features both well-described and less common BCR::ABL1-like genomic rearrangements in the medical literature. Appropriate classification of oncogenicity and AMP/ASCO/CAP tier for the pilot fusions will substantiate the SC-VCEP-derived guidelines, allowing them ultimately to be shared publicly with an overarching goal of unifying the diagnostic, prognostic, and therapeutic implications of BCR::ABL1-like B-ALL genetic variants.

R code of our analysis for driver mutation discovery in Hodgkin lymphoma WGS and WES.

R code of our analysis clock like mutational signatures in Hodgkin lymphoma WGS and WES.

Abstract Chimeric antigen receptor (CAR) T-cell therapy has remarkably succeeded in treating lymphoblastic leukemia. However, its success in acute myeloid leukemia (AML) remains elusive because of the risk of on-target off-tumor toxicity to hematopoietic stem/progenitor cells (HSPC) and insufficient T-cell persistence and longevity. Using a SynNotch circuit, we generated a high-precision “IF-THEN” gated logical circuit against the combination of CD33 and CD123 AML antigens and demonstrated antitumor efficacy against AML cell lines and patient-derived xenografts. Unlike constitutively expressed CD123 CAR-T cells, those expressed through the CD33 SynNotch circuit could preserve HSPCs and lower the risk of on-target off-tumor hematopoietic toxicity. These gated CAR-T cells exhibited lower expression of exhaustion markers (PD-1, TIM-3, LAG-3, and CD39), higher frequency of memory T cells (CD62L+CD45RA+), and enhanced expansion. Although targeting AML, the moderated circuit CAR signal also helped mitigate cytokine release syndrome, potentially addressing one of the ongoing challenges in CAR-T immunotherapy. Significance: Our study demonstrates the use of “IF-THEN” SynNotch-gated CAR-T cells targeting CD33 and CD123 in AML reduces off-tumor toxicity. This strategy enhances T-cell phenotype, improves expansion, preserves HSPCs, and mitigates cytokine release syndrome—addressing critical limitations of existing AML CAR-T therapies.

Lineage switch (LS), defined as the immunophenotypic transformation of acute leukemia, has emerged as a mechanism of relapse after antigen-targeted immunotherapy, which is associated with dismal outcomes. Through an international collaborative effort, we identified cases of LS after a host of antigen-targeted therapies (eg, CD19, CD22, CD38, and CD7), described how LS was diagnosed, reviewed treatment approaches, and analyzed overall outcomes for this form of postimmunotherapy relapse. Collectively, 75 cases of LS were evaluated, including 53 (70.7%) cases of B-cell acute lymphoblastic leukemia (B-ALL) transforming to acute myeloid leukemia (AML), 17 (22.7%) cases of B-ALL transforming to mixed phenotypic acute leukemia (MPAL)/acute leukemias of ambiguous lineage (ALAL), and 5 (6.7%) cases of rare LS presentation (ie, T-cell ALL to AML). An additional 10 cases with incomplete changes in immunophenotype, referred to as "lineage drift" were also described. With a primary focus on the 70 cases of LS from B-ALL to AML or MPAL/ALAL, LS emerged at a median of 1.5 months (range, 0-36.5) after immunotherapy, with 81.4% presenting with LS within the first 6 months from the most proximal immunotherapy. Although most involved KMT2A rearrangements (n = 45, 64.3%), other rare cytogenetic and/or molecular alterations were uniquely observed. Treatment outcomes were generally poor, with remission rates of <40%. The median overall survival after LS diagnosis was 4.8 months. Outcomes were similarly poor for those with rare immunophenotypes of LS or lineage drift. This global initiative robustly categorizes lineage changes after immunotherapy and, through enhanced understanding, establishes a foundation for improving outcomes of LS.