CD19-directed chimeric antigen receptor (CAR) T-cell therapy has transformed outcomes for patients with relapsed or refractory (r/r) mantle cell lymphoma (MCL), yet more than 40% relapse within one year. Early identification of patients at risk for progression could inform post CAR-T surveillance and consolidation strategies. Measurable residual disease (MRD) has emerged as a powerful prognostic biomarker in frontline MCL, but its role after CAR T-cell therapy remains incompletely defined. We retrospectively analyzed 37 patients with r/r MCL treated with brexucabtagene autoleucel (brexu-cel). MRD was assessed using next-generation immunoglobulin high-throughput sequencing (Ig-HTS) of peripheral blood mononuclear cells obtained before lymphodepletion, at 1- and 3-months post-infusion, and every 3 months thereafter. Clonotype identification was successful in 36 of 37 patients. Pre-lymphodepletion MRD levels were lower in patients receiving bridging therapy (337 [0-198 449] vs. 21 213 [1-788 251]; p = 0.04), and MRD undetectability trended toward improved progression-free survival (PFS; unreached vs. 28.5 months; HR 5.2; p = 0.07). Post-infusion, patients with detectable Day 28 MRD had inferior PFS compared with those with undetectable MRD (10.9 vs. 51.5 months; HR 3.99; p = 0.002), whereas Day 28 PET-CT response did not correlate with PFS (p = 0.35; HR 1.8). Longitudinal MRD monitoring identified relapse a median of 6.5 months before PET/CT in most relapsing patients (15 out of 18). Early and serial MRD monitoring is thus a sensitive prognostic and surveillance tool in brexu-cel treated MCL, with Day 28 MRD serving as an early predictor of long-term outcomes.

Determination of measurable residual disease (MRD) in pediatric acute lymphoblastic leukemia (ALL) using next generation immunosequencing (clonoSEQ; Adaptive Biotechnologies) has emerged as an essential and sensitive tool. The clonoSEQ assay identifies and tracks all the B-cell and T-cell receptor rearrangements present in a sample. With widening clinical use of immunosequencing for MRD monitoring (NGS-MRD), clinicians are increasingly encountering cases with results that are considered to be inconclusive; where immunosequencing yields detectable clonotypic sequences, yet it is unclear whether they indicate ALL, normal background, or an independent etiology. There is a need for guidance in the clinical interpretation of immunosequencing results. In this manuscript we describe various features of immunosequencing reporting and their clinical implications through a series of illustrative clinical cases. Each case presents a distinct scenario encountered in the clinical setting where NGS-MRD had varying clinical implications. Through aggregate evaluation of the various components of the results, such as the sequence locus, sequence abundance, clonal tracking, and levels of detection and blank, we formulate systematic interpretative tools that can be leveraged to inform clinical decision making. The goal of this manuscript is to serve as a useful guide for clinicians as they interpret immunosequencing results for pediatric ALL.

Monitoring B-cell depletion is essential for tracking functional persistence of B-cell directed chimeric antigen receptor (CAR) T-cells. While flow cytometry remains the standard-of-care, high-throughput immunosequencing, widely used for measurable residual disease (MRD) determination, quantifies every B-cell receptor in a sample and provides untapped data on B-cell depletion and reconstitution. We describe a set of immunosequencing-based metrics and newly reframe their use as clinical tools for tracking post-CAR B-cell depletion and reconstitution. We performed a single-institution retrospective analysis of children and young adults with B-cell acute lymphoblastic leukemia (B-ALL) treated with CAR T-cells. The ratio of total immunoglobulin heavy chain sequences to total nucleated cells (total IgH/TNC) quantified B-cell abundance, while additional metrics were leveraged to interrogate the B-cell repertoire’s developmental stage. We analyzed 276 samples (BM: n=77, PB: n=199). Patients maintaining post-CAR B-cell depletion exhibited low B-cell abundance (IgH/TNC) and a relatively immature B-cell repertoire; those who lost B-cell depletion (evidenced by rising IgH/TNC) showed a more mature population. We observed a strong correlation between immunosequencing and CD19 flow cytometry metrics in both blood and bone marrow. We also identified that all patients with flow cytometry-determined B-cell aplasia retained non-zero immunoglobulin levels detectable by immunosequencing. We investigated and suggest possible constituents of this “residual repertoire floor” in the B-cell depleted state. These results demonstrate that immunosequencing provides a dual-platform for both MRD determination and quantitative assessment of B-cell depletion and reconstitution. This approach provides a sensitive immunosequencing-based framework for longitudinal monitoring of CAR activity and B-cell ontogeny post-infusion.

High-throughput sequencing (HTS) of clonal immunoglobulin (Ig) gene rearrangements has become a mainstay of B-lymphoblastic leukemia (B-ALL) measurable residual disease (MRD) monitoring; Ig HTS also provides insight into the widely documented leukemic clonal heterogeneity in Ig heavy chain (IgH) variable (V), diversity (D), and joining (J) gene rearrangements in B-ALL. B-ALL IgH clonal composition is canonically presumed to reflect the transformed cell's rearrangement state: B cells develop along a pathway with IgH D-J followed by IgH V gene rearrangement; thus, subclones comprised of a common DNJ sequence, but distinct alternative V gene involvement, would be assumed to derive from a cell stage at which only D-J recombination had taken place with persisting recombination machinery for subsequent V-DJ rearrangement. We and others have observed that this ‘V-DJ diversity’ may be associated with prognostic cytogenetic features, but its relevance as a supposed reflection of B cell developmental state has not been confirmed. Here, we define the biologic significance of IgH variable gene diversity in B-ALL and reveal its direct implications on HTS MRD and its potential impact on clinical outcome. We analyzed 148 B-ALL samples from patients enrolled on former Children's Oncology Group (COG) trials (N=99) and an institutional tissue bank (N=49) to test the functional implications of IgH clonal composition in B-ALL. We defined 3 cohorts: 1) No clonal IgH (N=7; 4.7%); 2) Diverse, with V-DJ ‘subclones’ containing unique V genes but a common DNJ stem (N=53; 35.8%), and 3) Non-diverse, with single VDJ rearrangement(s) per allele (N=88; 59.5%). We confirmed that the extent of V-DJ diversity in each B-ALL – quantified by Shannon entropy index – is inherently preserved across tissue sites (bone marrow (BM) vs. peripheral blood (PB) R2=0.99; P<.0001) and early induction therapy timepoints rather than merely a stochastic phenomenon. Chromosome microarray demonstrated no association between prognostic cytogenetics, V-DJ diversity, and other non-IgH breakpoint accumulation across 4 biologically distinct, prognostic cytogenetic categories. Using CyTOF, we found that regardless of the pattern of IgH rearrangement(s), all cases were enriched in pro-BII cell populations. However, by Gene Ontology and Hallmark gene set enrichment analysis (GSEA) and CyTOF, cases with V-DJ diversity had distinct transcriptional and intracellular proteomic features – including enriched gene and protein expression of pentose phosphate and mitochondrial metabolism genes and proteins, as well as activated mTOR signaling – compared to cases with non-diverse VDJ rearrangements. Further, by ATACseq, we observed distinct chromatin accessibility between diverse and non-diverse cohorts, including increased accessibility at the IgH locus in diverse cases. Integrated single-cell and clinical Ig HTS clonality data revealed that distinct V-DJ subclone sequences (derived from a similar progenitor population sharing a DNJ stem) comprise discrete cell populations over a variable range of abundances; therefore, all or some such sequences will not necessarily meet clinical HTS clonality/MRD assay criteria to be defined as ‘dominant/trackable’ for MRD determination. Both via single-cell RNA seq and custom Tapestri sequencing, we found that diverse V-DJ subclone sequences distinguish discrete cell subpopulations. Thus, we tested the impact of IgH rearrangement diversity on MRD detection and outcome using a dataset from 307 subjects with high risk (HR) pediatric B-ALL. Among the prognostically inferior cohort from COG AALL0232 which lacked any designated ‘trackable’ sequences (N=31/307) (Fries et al. Haematologica 2023), we found that 7 (of 31; 2% of the total 307) had subclonal IgH rearrangements with unique V/common DJ sequences of similar abundance such that none were designated 'trackable.' Despite treatment intensification based on positive end of induction (EOI) flow cytometry MRD on AALL0232, these patients had inferior 5-year EFS of 71.4% (95%CI 44.7-100%; P=.001). In 5 (of 7) cases, a subset of V-DJ 'subclones' were still detectable at the EOI timepoint, suggesting treatment resistance. In conclusion, IgH V-DJ diversity reflects distinct metabolic/transcriptional pathway activation in proB-like leukemia cells and defines discrete B-ALL cell populations with possibly distinct treatment responses, potentially impacting clinical outcome.

We report on a series of three patients diagnosed with secondary acute lymphoblastic leukemia (sALL) following treatment for multiple myeloma (MM) where serial, commercially available, next-generation sequencing (NGS) based tracking of measurable residual disease (MRD) using the clonoSEQ assay provided valuable clinical insight. Each of the patients in this series had been treated for their MM with regimens that had included autologous stem cell transplantation following high dose melphalan chemotherapy and had been on maintenance treatment with the immunomodulatory drug lenalidomide for at least one year.

BACKGROUND B-cell acute lymphoblastic leukemia (B-cell ALL) is the most common childhood cancer. Despite a high overall cure rate, relapsed B-cell ALL remains a leading cause of cancer-related death among children. The addition of the bispecific T-cell engager molecule blinatumomab (an anti-CD19 and anti-CD3 single-chain molecule) to therapy for newly diagnosed standard-risk (as defined by the National Cancer Institute) B-cell ALL in children may improve outcomes. METHODS We conducted a phase 3 trial involving children with newly diagnosed standard-risk B-cell ALL who had an average or high risk of relapse. Patients were randomly assigned to receive chemotherapy alone or chemotherapy plus two nonsequential 28-day cycles of blinatumomab. The primary end point was disease-free survival. RESULTS The data and safety monitoring committee reviewed the results from the first interim efficacy analysis, which included 1440 patients who had undergone randomization (722 to chemotherapy alone and 718 to blinatumomab and chemotherapy) and recommended early termination of randomization. At a median follow-up of 2.5 years, the estimated 3-year disease-free survival (SE) was 96.01.2% with blinatumomab and chemotherapy and 87.9 +/- 2.1% with chemotherapy alone (difference in restricted mean survival time, 72 days; 95% confidence interval, 36 to 108; P<0.001 by stratified log-rank test). The estimated 3-year disease-free survival among patients with an average relapse risk was 97.5 +/- 1.3% with blinatumomab and chemotherapy and 90.2 +/- 2.3% with chemotherapy alone; among those with a high relapse risk, the corresponding values were 94.1 +/- 2.5% and 84.8 +/- 3.8%. Cytokine release syndrome, seizures, and sepsis of grade 3 or higher were rare during blinatumomab cycles, but the overall incidence of nonfatal sepsis and catheter-related infections was significantly higher among patients with an average relapse risk who had been assigned to receive blinatumomab and chemotherapy than among those assigned to receive chemotherapy alone. CONCLUSIONS Adding blinatumomab to combination chemotherapy in patients with newly diagnosed childhood standard-risk B-cell ALL of average or high risk of relapse significantly improved disease-free survival.

COG study AALL1731 established that adding two cycles of the bispecific T-cell engager blinatumomab to risk-adapted chemotherapy (chemo) significantly improved 3-year disease-free survival (DFS) of children with National Cancer Institute (NCI) standard risk (SR) B-acute lymphoblastic leukemia (ALL) at average or higher risk of relapse (Gupta et al, NEJM 2025). Longer-term follow-up is critical to ensure the benefit persists. Further work is also needed to determine prognostic features in patients (pts) whose therapy now includes blinatumomab. AALL1731 enrolled newly diagnosed NCI SR [age 1-<10 years with initial white blood cell count <50,000/µL] B-ALL pts, without BCR::ABL1 fusion, testicular or central nervous system (CNS)3 disease. After 3-drug induction, pts were risk stratified based on leukemia genetics, CNS status, day 8 blood flow cytometry-defined minimal residual disease (fcMRD), and end of induction (EOI) bone marrow (BM) fcMRD and EOI clonoSEQ assessment. Pts at average risk of relapse (SR-Avg) with detectable or indeterminate EOI BM ClonoSEQ MRD were randomized to standard chemo (Arm A) or chemo plus 2 cycles of blinatumomab (Arm B). Pts at high risk of relapse (SR-High) with end of consolidation (EOC) BM mpFC MRD <0.1% were randomized to higher intensity chemo (Arm C) or chemo plus 2 cycles of blinatumomab (Arm D). Interim analyses (June 2024) showed that addition of blinatumomab was associated with improved DFS; enrollment was terminated and Arm A/C pts not yet in Maintenance offered crossover and blinatumomab. Using current data to 6/25, we compared DFS between Arms A/C and B/D, censoring pre-Maintenance pts when crossover was offered. We also determined the effect of blinatumomab on the impact of traditional prognosticators by comparing the associated risk within Arm A/C pts vs within Arm B/D pts. Median follow-up was 3.5 years (IQR=2.5-4.3). Of 1,444 randomized pts, 133 were censored. In intent-to-treat analyses, 4-year DFS (± standard error) was 94.8±1.5% for pts randomized to blinatumomab arms (B/D) vs 86.9±2.2% control arms (A/C). Adding blinatumomab significantly improved DFS [Hazard Ratio (HR) 0.41, 95% confidence interval (CI) 0.27-0.62, 1-sided p<0.0001]. The 4-year cumulative incidence of relapse (CIR) for those randomized to blinatumomab vs control arms was 4.4±0.9% vs 12.6±1.5% (p<0.0001). While BM involved relapses were significantly reduced (4-year CIR 3.1±0.8% vs. 9.4±1.3%, p<0.0001), isolated CNS relapses were unchanged (1.4±0.5% vs 2.2±0.6%, p=0.41). In Arms A/C (control) traditional variables used in COG retained adverse prognostic significance, in contrast to Arms B/D (blinatumomab). For example, in control arms, Hispanic ethnicity was associated with a two-fold increase in risk (HR 2.1, 95CI 1.3-3.4; p=0.004) while in blinatumomab arms the HR for Hispanic ethnicity was 1.3 (95CI 0.6-2.7; p=0.51). In control arm pts, EOI fcMRD≥0.01% had a 4-year DFS of 82.3±2.7% vs 89.7±1.7% for EOI fcMRD<0.01% (p=0.008). In blinatumomab arm pts however, EOI fcMRD≥0.01% 4-year DFS was 92.8±1.9% vs. 95.9±1.1% for MRD <0.01% (p=0.08). Notably, blinatumomab arm pts with EOI fcMRD ≥0.01% had superior outcomes to control arm pts with EOI fcMRD <0.01%. Dramatic impact was seen in pts with EOC BM fcMRD 0.01%-<0.1% vs EOI MRD ≥0.01% but EOC fcMRD <0.01% (control:HR 5.9, 95CI 2.5-14.0; p<0.0001; blinatumomab:HR 2.6, 95CI 0.6-11.8; p=0.21). Indeed, EOC MRD 0.01%-<0.1% pts on Arm C experienced 4-year DFS of 30.8±16.8% (N=16) vs 81.8±11.6% (N=13) on Arm D. CNS2 pts, who in previous COG trials had inferior outcomes, had 4-year DFS of 96.8±2.3% on blinatumomab arms. Unfavorable genetics [iAMP21, KMT2A rearrangement, t(17;19), hypodiploidy] retained adverse impact (Arm C: HR 3.5 vs favorable, 95CI 1.6-7.6; p=0.002; Arm D: HR 4.1, 95CI 1.1-15.2; p=0.04). However, addition of blinatumomab was still associated with improved outcomes in pts with unfavorable genetics (4-year DFS Arm C 77.1±5.9% vs Arm D 91.7±3.6%).The benefit associated with adding blinatumomab to the treatment of NCI SR B-ALL is maintained with further follow-up. While some adverse prognosticators retain significance in the context of blinatumomab-containing backbones, the addition of blinatumomab is of benefit for all examined subgroups, with most prognosticators losing significance. These results confirm this new standard therapy and have important implications for which subgroups could be considered for a reduction in traditional chemo in the future.

Introduction: Current protocols treating acute lymphoblastic leukemia (ALL) in children, adolescents, and young adults utilize bone marrow (BM) measurable (minimal) residual disease (MRD) to assess early treatment response and risk stratify post-induction therapy. Measuring MRD in peripheral blood (PB) may provide a less invasive way to quantitate leukemia burden facilitating more frequent and nuanced assessment of disease status and may provide additional insight on tumor biology. Methods: Next-generation sequencing of immunoglobulin and T-cell receptor loci by the clonoSEQ Assay (Adaptive Biotechnologies Corporation, Seattle, USA) was used to evaluate paired BM and PB samples from participants enrolled on a front line ALL clinical trial DFCI 16-001 (NCT03020030). Participants were selected by having paired samples available. Participants on 16-001 were stratified into initial risk groups by age, presenting white blood cell count, immunophenotype, CNS status, and adverse leukemia biology. MRD was evaluated at End-induction IA, 4 weeks after starting therapy (TP1, 138 pairs after QC) and if the TP1 result was greater than 100 per million cells (10^-4) MRD was evaluated again at end-induction IB, 10 weeks after the start of treatment (TP2, 42 pairs after QC). MRD was assessed in patients classified as very high risk at a third timepoint approximately 19 weeks after the start of therapy (TP3, 26 pairs after QC). BM MRD value by clonoSEQ if available determined final risk group; PB MRD was not used for clinical decision-making. Scatterplots were created and Spearman correlation coefficients were calculated to assess correlation between BM and PB MRD. Results: This analysis includes 213 paired BM and PB samples from 149 participants with median age at diagnosis 6.1 years (1-19.7), 54% male, 74% White, 77% Non-Hispanic, and 87% with B-cell immunophenotype. In this cohort, 50% were categorized as initial low risk, 29% initial high risk, and 21% initial very high risk. Of the 213 paired samples, 7 PB samples failed quality control (QC) leaving 206 for the correlation analysis. BM and PB MRD were highly correlated across all samples (R=0.78, p<2.2x10-16) and at each time point including TP1 (R=0.79, p<2.2x10-16), TP2 (R=0.71, p=1.7x10-7), and TP3 (R=.83, p=1.2x10-7). BM and PB MRD were highly correlated for both B-ALL (N=184, R=0.76, p<2.2x10-16) and T-ALL samples (N=29, R=0.88, p=6.1x10-10). Concordance was similar to the whole cohort for B-ALL samples at TP1(R=0.76, p<2.2x10-16), TP2 (R=0.7, p=3.2x10-6), and TP3 (R=0.89, p=8.9X10-9). For 44 paired samples with protocol defined high BM MRD (more than 100 per million cells for TP1, more than 1000 per million cells for TP2 or TP3), correlation with PB was also strong (R=0.50, p=5.0x10-4) however there is a discordance rate of 50% with 22 having a PB value below the protocol cut-off value. Using the whole cohort, samples with BM count in the range that may have effected management on 16-001 between 100 and 3000 per million cells were not correlated with PB (R=0.056, p=0.76). In subgroup analysis by initial risk group (low, high, and very high) and favorable tumor biology (presence of double trisomies 4 and 10 or ETV6::RUNX1) correlation remained strong. Conclusions: In this analysis, BM and PB MRD during treatment for pediatric ALL were highly correlated, particularly at high and low levels of detectable BM disease, across protocol defined risk groups, immunophenotype, and with favorable leukemia biology. At moderate levels of BM detectable disease, correlation with PB detectable disease was less strong limiting immediate direct replacement of BM sampling with PB. PB represents a desirable sample for assessing MRD allowing more frequent and less invasive evaluation of ALL disease status particularly for children, allowing for less painful modality to monitor disease status and less exposure to sedation/anesthesia. High correlation between BM and PB suggests that PB may represent a valuable adjunct for patient monitoring. With regard to specific levels for risk stratification, more work is needed. Correlation of PB MRD with ALL outcomes including event free and overall survival in the future will be essential in determining utility of PB MRD assessments.

Background: Several biomarkers are utilized to monitor for B-ALL relapse. For minimal residual disease (MRD), these include flow cytometery (FC) and next-generation sequencing (NGS). NGS is increasingly being used due to higher sensitivity than FC. For patients receiving CAR T-cells, CD19+ peripheral blood (PB) FC-BCA, and less often, bone marrow (BM) FC-BCA are utilized to assess persistence. How these assays cross-compare with outcomes is unknown. Objective: To cross-compare BM NGS-MRD, BM FC-BCA and PB FC-BCA in CAYA patients with B-ALL who achieved a BM FC-MRDneg complete remission (CR) on D+28 following CAR T-cells. Additionally, we compare these to the use of NGS to assess BCA (NGS-BCA) in the BM. Secondary objectives included analysis of the association of these biomarkers with relapse risk and CAR T-cell persistence in the BM. Methods: A multi-institution retrospective study of patients who received investigational CD22 or CD19/22 CAR T-cell constructs at the National Cancer Institute or commercial CD19 CAR T-cells at Johns Hopkins Children's Center from 2/2013-4/2025 was conducted. All patients had disease restaging at approximately D+28 post CAR T-cell infusion and were included for analysis if they achieved an FC-MRDneg remission, defined as no disease detected at a level of >0.01% of mononuclear cells. PB BCA was defined as <10 B-cells/uL. Patients with Adaptive clonoSEQ® reports from bone marrow specimens at D+28 were assessed for BM NGS-MRD and BM NGS-BCA. Those with any NGS identified disease-associated clone >0 were considered as NGS-MRD+. BM BCA was defined as <1% B-cells. BM FC-BCA was based on the percentage of CD19+ B-cells in relationship to total cells or total lymphocytes. BM NGS-BCA was calculated by dividing total immunoglobulin heavy chain sequence(s) by total nucleated cells. CAR T-cell persistence was evaluated by FC in the BM for investigational constructs and was defined as >0% CAR T-cells. Results: There were 43 patients who were FC-MRDneg with concurrent PB FC-BCA at D+28. The median age at CAR infusion was 13.6 years (range 0.8-38.1). Twenty-four subjects (55.8%) were male. Thirty-two patients (74.4%) received an investigational CAR-T cell construct and 11 (25.6%) received tisagenlecleucel. Sixteen of these 43 patients (37.2%) were NGS-MRD+, of whom 14 (87.5%) had NGS-BCA. Across 27 patients with NGS-MRD negativity, 23 (85.2%) had NGS-BCA. Thus, 37 (86.0%) patients had NGS-BCA regardless of NGS-MRD status. Forty-two patients had BM FC-BCA assessed, of which 36 (85.7%) had FC-BCA. BM FC-BCA and NGS-BCA were fully concordant. Of the 6 patients who did not have BM FC-BCA or BM NGS-BCA, 4 had non-CNS extramedullary disease (EMD). Of the 29 patients with available CAR T-cell persistence data, 22 (75.9%) had detectable BM CAR of which 16 (72.7%) were also NGS-MRDneg. All patients with detectable CAR T-cells had both PB FC-BCA and BM FC-BCA. Eighteen patients (41.9%) relapsed, of which 9 (52.9%) were NGS-MRD+ and 13 (76.5%) had NGS-BCA at D+28. Out of 16 patients who were NGS-MRD+ at D+28, 9 (56.3%) relapsed. However, of the 7 NGS-MRD+ patients who did not relapse, six received a post-CAR consolidative stem cell transplant. Out of 27 who were NGS-MRDneg, 9 (33.3%) relapsed. Across 16 patients with CD19+ disease pre-CAR and who experienced relapse, 12 had available immunophenotypic analysis across whom 2 were confirmed to have CD19 negative (CD19-neg) relapse. Both patients were NGS-MRD+ at D+28; and only one had BM BCA at D+28. The median time to relapse for patients who were NGS-MRD+ at D+28 was 4.8 months (range 1.5-11.6). This compared to 9.6 months (range 4.5-20.9) for those who were NGS-MRDneg (p = 0.02). Of 36 patients with BM FC-BCA and BM NGS-BCA, 13 (36.1%) relapsed; of 6 patients without BM FC-BCA and BM NGS-BCA, 5 (83.3%) relapsed. Nine of 12 patients (75%) with non-CNS EMD relapsed. Conclusion: We identified several discrepancies amongst BM NGS-MRD status and BM BCA by either FC or NGS. Interestingly, >85% of patients who were NGS-MRD+ still had BCA, by either FC or NGS. Regardless of NGS-MRD status, most patients had NGS-BCA. BM FC-BCA and BM NGS-BCA were fully concordant. While additional analysis of post-CAR T-cell surveillance metrics is underway, these results raise awareness of discrepencies in disease detection methodologies. Further investigation utilizing these biomarkers in B-ALL surveillance is warranted.

Background: T-cell receptor (TCR) sequencing is increasingly incorporated into the diagnosis and monitoring of T-cell lymphomas, including cutaneous T-cell lymphomas (CTCL). However, interpretation of TCR sequencing results is often confounded by the emergence of multiple dominant TCR clonotypes, whose etiology remains poorly understood. We aimed to determine the incidence of multiple dominant TCR clonotypes through serial TCR high-throughput sequencing (TCR-HTS), to immunophenotype and genetically characterize these dominant T-cell populations, and to assess their potential clonal relationships to CTCL. Methods: We reviewed results from 1677 samples from 235 patients with CTCL (MF n=163, SS n=67, Other n= 23) sequenced using the clonoSEQ® platform (median 6 samples per patient) from 2012-2023. Dominant TCR clonotypes were defined per clonoSEQ® criteria, and only productive rearrangements were included. Clonotypes with identical VDJ rearrangements (e.g., single-nucleotide variants) were excluded. Among patients with an emerging second dominant productive TCRβ clonotype, 11 had cryopreserved PBMCs available for analysis. These PBMCs, along with those from 10 age-matched healthy donors, underwent TCR-HTS using the LymphoTrack® assay. Multiparametric spectral flow cytometry (MFC), including antibodies to TCRBC1 and/or TRBV regions, enabled identification and characterization of clonal T-cell populations. Clonal and polyclonal T-cell populations were sorted by FACS and underwent whole-exome sequencing (WES) and hybrid-capture TCR sequencing. Sorted monocytes were used as germline for WES variant calling. To provide a reference for the mutational profile of benign T-cells, normal T-cells from one patient were singly sorting, ex-vivo expanded into colonies, and underwent WES. Results: Among 235 CTCL patients, 109 (46%) had two or more unique dominant, productive TCRβ clonotypes identified in one or more specimens, and 41 (17%) had three or more. TCR-HTS using an alternative platform confirmed a high-frequency clonotype (>3%) in all 11 available CTCL PBMC samples, and in none of the 10 healthy donors tested. MFC identified one or more clonal T-cell populations per patient. These variably expressed CD4, CD8, or CD4-CD8- (double-negative), and most commonly displayed a terminal effector phenotype (CD45RA+, CD56+, and CD57+). These immunophenotypes were most consistent with T-cell clones of uncertain significance (T-CUS) and were distinct from typical CTCL profiles. FACS-sorted T-CUS populations underwent TCR sequencing to confirm their clonal identity. In 10/11 patients, the dominant TCRβ clonotype matched that of TCR-HTS (median 91% TCR clonotype fraction, range 41-100%). WES was performed on 10 sorted T-CUS populations, 9 patient-matched CTCL biopsies, 8 polyclonal T-cell populations, and 15 single T-cell derived colonies. T-CUS harbored significantly fewer somatic variants than CTCL biopsies (mean 68 versus 781, p=0.004), but more than polyclonal T cells (mean 17, p=0.01). Single-cell-derived colonies of normal T-cells from a CTCL patient had a mutational burden (mean 41) comparable to T-CUS (p=0.24). To evaluate whether T-CUS populations in CTCL patients shared a common lineage with the primary CTCL clone, we compared somatic variants in matched T-CUS and CTCL samples. No shared variants were found, indicating no common clonal origin. However, T-CUS harbored nonsynonymous variants involving CTCL-associated and/or cancer-associated genes including ARID1A, ATRX, FSIP2, GATA3, PTPRR, PTPRK, STAT1, TCF3, TP63, and TRRAP. STAT3 and STAT5B variants were not detected in the clonal sorted populations, and there were no unexplained cytopenias, arguing against T-cell large granular lymphocytic leukemia. Unlike CTCL, T-CUS lacked UV-associated mutational signatures. Conclusions: Emergence of new dominant TCR clonotypes is common in CTCL and often arise within T-CUS populations that fluctuate in frequency in the blood and skin. While T-CUS harbor variants in genes associated with T-cell malignancies, there was no evidence of evolution of these populations into secondary malignancies, and T-CUS appear clonally unrelated to CTCL. The clinical significance of emerging dominant T-cell clonotypes in CTCL should be interpreted in the context of a high prevalence of T-CUS.

Introduction Lower baseline disease burden associates with improved survival in children and young adults with B-cell acute lymphoblastic leukemia (B-ALL) treated with chimeric antigen receptor T cells (CAR-T). Conversely, low total CD19+antigen load (an aggregate measure of malignant and physiologic B cells) at pre-CAR baseline resulted in decreased CAR-T response durability in early clinical trials. While these data used flow cytometry measures, limited data exists characterizing the role of pre-CAR next-generation sequencing (NGS) on CAR-T outcomes. NGS is an alternative minimal residual disease (MRD) measure that can detect B-ALL at 1 clonal cell/million nucleated cells. Inherent to the assay (clonoSEQ, Adaptive Biotechnologies) is a complementary measure of immunoglobulin heavy chain (IgH)/total nucleated cell (TNC) which represents the total B cell number represented in the repertoire. We conducted a retrospective single-institutional study to evaluate the role of baseline NGS on post-CAR-T outcomes and hypothesized that while lower pre-CAR NGS MRD would result in improved survival, lower IgH/TNC may represent a low aggregate pre-CAR B cell load and result in inferior outcomes. Methods We conducted a retrospective analysis of patients ≤26 years who received CD19, CD22 or CD19/22 CAR at Stanford between August 2017 and August 2024. Univariate and multivariate Cox regression analyses were used to assess the impact of demographic and disease variables on overall survival (OS) and event-free survival (EFS). These variables included age, gender, race, cytogenetic risk, CAR-T indication, type of CAR-T received, and prior therapies, as well as our primary variables of interest: pre-CAR disease burden (NGS; log peak clonal cells), and pre-CAR B cell load (NGS; total immunoglobulin heavy chain (IgH)/total nucleated cell (TNC) ratio), where total IgH/TNC of <0.5% was defined as a low aggregate B cell load. We aimed to assess the role of baseline NGS MRD (Aim 1) and baseline total IgH/TNC (Aim 2) on survival (OS/EFS) in our overall patient cohort. To account for the fact that all high disease burden patients would be expected to have ≥0.5% IgH/TNC, as an exploratory aim, we studied the impact of these measures in a limited cohort of those with low pre-CAR disease burden (<5% bone marrow blasts). Results The dataset included 76 CAR-T infusions across 60 patients, with a median age at infusion of 13 years (range 1-25). The cohort comprised 53% male and 47% female patients, with 57% receiving commercial CD19 CAR-T (tisagenlecleucel), 7% investigational CD22 CAR-T, and 26% investigational CD19/22 bispecific CAR-T. Only the first CAR-T infusion per patient was included in the analyses. Univariate and multivariate analyses of the entire cohort revealed that only pre-CAR log peak leukemic clonal cells significantly associated with OS (Hazard ratio (HR)=1.15, p=0.0038) and EFS (HR=1.15, p<0.0001). When restricting analysis to patients with <5% baseline blasts by flow cytometry (N=36), log peak leukemic cells did not significantly associate with OS or EFS (OS: HR=1.17, p=0.086; EFS: HR=1.09, p=0.10). However, in this low tumor burden cohort, univariate analysis demonstrated that baseline pre-CAR bone marrow total IgH/TNC of <0.5% associated with lower EFS (HR=0.27 for patients with >0.5% total IgH/TNC compared to <0.5%, (p=0.046)). While the sub-stratified sample size of the low-burden cohort did not support multivariate analysis, expanded multisite validation efforts are underway. Conclusion In our overall cohort, increased log peak leukemic MRD clonal cells by NGS at pre-CAR baseline associated with inferior OS and EFS. While the NGS MRD measure of disease burden did not maintain significance in the already low-burden B-ALL cohort, measures of low pre-CAR aggregate B cell load (<0.5% total IgH/TNC) associated with inferior EFS in this cohort. While multi-site validation efforts are underway, this first study of NGS as a measure of pre-CAR B cell load supports that CAR-T cells targeting B cells may require threshold levels of aggregate B-cell load for effective expansion and durable B-ALL surveillance.

Introduction: Circulating tumor DNA (ctDNA)-based measurable residual disease (MRD) assessment is a promising noninvasive biomarker to guide treatment response and surveillance in Large B-cell lymphoma (LBCL). While PET-CT surveillance remains standard of care following anti-CD19 CAR T-cell therapy (CAR19), its limited positive predictive value, radiation exposure, and reduced accuracy due to post-treatment inflammation highlight the need for more specific and dynamic monitoring tools. ctDNA enables early relapse reduction, risk stratification, and real-time monitoring. Prior studies have shown its additive prognostic value to PET CT following third-line axi-cel (clonoSEQ, Frank et al., JCO 2021) and liso-cel (PhasED-Seq, Stepan et al., Blood 2023) administration. Here, we evaluate prospective ctDNA monitoring using clonoSEQ in patients receiving axi-cel or liso-cel in second line era, and explore ctDNA-guided surveillance to refine or reduce conventional use of PET-CT. Method: In this prospective, observational study of real-world patients receiving CD19 CAR T-cell therapy, pre-planned plasma collection at pre-lymphodepletion (PLD), days 14, 28, 90 and 180 post CAR19 resulted in 1105 samples and (VDJ) clonotype was identified from archival paraffin-embedded tissue to track MRD. PET-CT scans were performed before and at 1, 3, 6, and 12 mo post CAR19; responses assessed per Lugano criteria (Deauville 1-3 was considered PET-negative). Results: 142 patients were included (axi-cel, n=116; liso-cel, n=26). The median age was 66 years (23–83) for axi-cel and 79 years (55–88) for liso-cel; 61% vs 42% were male, and 77% vs 84% were stage III/IV. Both groups had a median of 1 prior line of therapy (range 1–4) with 61% vs 58% receiving treatment in second line, and 64% vs 80% had an R-IPI score of 3–5. Median follow-up was 17.2 months (range 0.9–36.5) for axi-cel and 11.5 months (2.2–28.1) for liso-cel. Best ORR and CR rates were 85% and 80% for axi-cel; 88% and 69% for liso-cel. Progression occurred in 45(38.7%) axi-cel patients and in 14(53.8%) liso-cel patients. For axi-cel, ctDNA levels were prognostic at all timepoints (PLD, Days 14, 28, and 90). High pre-treatment ctDNA burden (>1000 lymphoma genomes/mL plasma) was associated with inferior progression-free survival (PFS) compared to those <1000 LG/mL (median PFS: 1.87 vs 31.4 months). When excluding those who progressed at or prior to each timepoint, those with undetectable ctDNA was associated with significantly improved PFS at Day 14 (HR 4.2, 95% CI 2.2–7.9; p<0.001), Day 28 (HR 2.4, 1.1–5.8; p=0.009), and Day 90 (HR 2.5, 0.5–11.4; p=0.02). For liso-cel, undetectable ctDNA at Day 14 (HR 1.58, 95% CI 0.49–5.05; p=0.41) and Day 28 (HR 1.24, 0.24–6.39; p=0.77) was not significantly associated with PFS, with Day 90 being significant (HR 7.17, 0.46–111.2; p=0.05), consistent with prior PhasED-Seq data. These findings likely reflect differences in T-cell tumor killing kinetics between CAR19; we observed axi-cel more rapidly cleared ctDNA compared to liso-cel. This highlights the potential need for product-specific ctDNA surveillance strategies. For axi-cel patients with remission >12 months (n=54), ctDNA was undetectable in 41 of 52 patients (79%), 40 of 48 (83%), and 40 of 42 (95%) at Day 14, 28 and 90, respectively. By Day 180, ctDNA was undetectable all patients. Among the 59 relapses observed in this entire cohort (45 axi-cel, 14 liso-cel), 55 occurred within a year. Importantly, detectable ctDNA consistently emerged at or prior to clinical relapse, in 94% patients (n=52 of 55), regardless of CAR19 given. Three false-negative MRD cases occurred, one with a single 0.8 cm cervical lesion, CSF only relapse, and single 0.6 cm skin lesion in leg-type DLBLCL; the latter two cases were also a false negative by PET-CT. Overall, the negative predictive value (NPV) of undetectable ctDNA for concurrent PET-detectable disease, was 100% at Day 28 and 95.6% at Day 90. Conclusion: ctDNA-based MRD monitoring allows for early prognostication following CAR19, particularly by day 14 in axi-cel-treated patients. While its prognostic value in liso-cel emerges later at day 90, ctDNA detectability preceded or coincided with clinical relapse across products. These findings support the integration of ctDNA into post–CAR T surveillance with potential for reducing routine PET-CT imaging, particularly in undetectable MRD patients or to guide additional workup in patients with inconclusive results.

10014 Background: Minimal (measurable) residual disease (MRD) at end of induction (EOI) therapy is a strong predictor of outcome in pediatric B-ALL. Currently, EOI MRD is assessed in bone marrow (BM). We hypothesized that the highly sensitive assay, high-throughput sequencing (HTS) of immunoglobulin loci, can effectively monitor MRD in peripheral blood (PB) and may provide a less invasive way to track therapy response. Methods: We conducted HTS MRD on paired EOI BM and PB samples from 808 NCI standard risk (SR) pediatric B-ALL patients enrolled on Children’s Oncology Group study AALL1731 (NCT03914625). We determined the correlation between BM and PB HTS MRD via Spearman’s rank correlations. We calculated the BM/PB MRD ratio and compared these by subgroup using Kruskal-Wallis tests. We defined subgroups by cytogenetics (cyto) ( ETV6::RUNX1, double trisomies of chromosome 4 and 10 (DT), Unfavorable (hypodiploidy, iAMP21, or KMT2A-rearranged), or Neutral (lacking ETV6::RUNX1, DT, or unfavorable)), and risk group (SR-average (AVG) and SR-High). Flow cytometry-defined EOI BM MRD was < 0.01% for all SR-AVG patients (N = 623) and ≥0.01% for selected SR-High patients (N = 185). Results: There was strong correlation between PB and BM HTS MRD with an overall correlation coefficient of 0.75 (P < 0.001). Correlation was similar by cytogenetics: ETV6::RUNX1, 0.69 (N = 63; P < 0.001), DT, 0.75 (N = 147; P < 0.001), Neutral, 0.74 (N = 580; P < 0.001), and Unfavorable, 0.66(N = 18; P = 0.003). For risk groups, correlation for SR-AVG was 0.67 (p < 0.001) and SR-High, 0.64 (p < 0.001). Of the 591 SR-AVG patients with detectable BM HTS MRD, PB HTS MRD was detectable in 474 (80.2%), undetectable in 94 (15.9%) and indeterminate (no leukemic cell detected and < 500,000 total cells in sample) in 23 (3.9%). Among 182 SR-High patients with detectable BM HTS MRD, 175 (96.2%) had detectable PB HTS MRD. Disease burden was higher in the BM than PB with a significantly higher BM/PB ratio in SR-High compared to SR-AVG patients (median 16.5 vs 2.6, P < 0.001). The median BM/PB ratio also varied by cytogenetics with those with Unfavorable cyto having the highest ratio (15.3 vs 6.3 in DT, 3.8 in ETV6::RUNX1, 3.1 in Neutral; P = 0.013). Conclusions: This is the largest analysis of paired B-ALL BM/PB HTS MRD to date. We show strong correlation between PB and BM across risk and cytogenetic groups. The ratio of BM/PB MRD varied and was highest among patients with Unfavorable cyto suggesting BM tropism. Importantly, PB MRD was detectable in nearly all patients with flow EOI BM MRD ≥0.01%, a threshold warranting therapy intensification. However, most patients with EOI BM flow MRD < 0.01% also had detectable PB HTS MRD. Thus, PB HTS MRD may provide a useful adjunct for screening and clinical management of B-ALL patients. Defining a PB HTS MRD threshold useful for risk stratification will require correlation with outcome.

Background: Molecular characterization of B-lymphoblastic lymphoma (B-LLy) has yet to identify prognostic biomarkers for use in treatment stratification or driver mutations to explain its extramedullary clinical phenotype distinct from B-lymphoblastic leukemia (B-ALL). Patients with B-LLy (with morphologically identical lymphoblasts but <25% bone marrow (BM) involvement) are empirically treated on B-ALL protocols based on clinical stage (Murphy I/II: localized/standard risk (SR); III/IV: disseminated/high risk (HR)), although debate remains as to whether B-LLy represents a separate entity. The unique clinical characteristics of B cell cancers are often presumed to reflect their stage of development at malignant transformation. In normal B cells, ordered rearrangement of immunoglobulin (Ig) heavy (IgH) and light chain genes is linked to stage of development. Upon malignant transformation, distinctive clonal Ig genomic rearrangements expand, and thus afford disease tracking. We applied Ig high-throughput sequencing (HTS) to compare the clonal composition of B-LLy with B-ALL to test whether Ig rearrangement state indicates distinct biology between these entities, and to test whether minimal disseminated disease (MDD) measured by HTS at diagnosis can refine currently limited B-LLy clinical staging. Methods: We performed Ig HTS (Adaptive ClonoSEQ) to define and compare the Ig rearrangement state of B-LLy with that of B-ALL using extracted gDNA from paraffin-embedded tumor slide preparations from 34 patients with B-LLy (N=15 SR; N=17 HR; N=2 stage unknown) from Children's Oncology Group (COG) protocols APEC14B1 and AALL0932, and diagnostic BM from 283 patients with B-ALL from COG protocols AALL0331 (N=141; SR) and AALL0232 (N=142; HR). We also measured B-LLy MDD in 31 patients by tracking tumor-associated Ig rearrangements in pre-treatment BM and peripheral blood (PB). Results: All 34 B-LLy and 281/283 B-ALL samples had dominant IgH and/or Ig light chain clone(s). We detected IgH clones in 91.1% of B-LLy, 86.7% of which reflected complete V(D)J rather than partial diversity (D)-joining (J) rearrangements (vs. 74.8% complete V(D)J in B-ALL). In contrast, patients with B-ALL had more incomplete DJ rearrangements (P=0.04) indicative of a transformation state prior to variable (V) gene recombination. B-ALL V(D)J clones were enriched for usage of the most D-proximal V genes, IGHV06-01 and IGHV01-02, consistent with data indicating preferential D-proximal V gene usage in immature lymphoid progenitors and leukemia. B-LLy did not share this preferential V gene usage and lacked representation of IGHV06-01, distinguishing it from B-ALL. Further, 82.4% of B-LLy (vs. 59.7% of B-ALL; P=0.017) had dominant Ig light chain clone(s), indicative of a more mature rearrangement state. Ig HTS data from the BM and/or PB were available for 31 patients: 80.6% were MDD+, including 69.2% (N=9 of 13) with localized/SR B-LLy. BM/PB MDD level did not significantly vary between patients with localized/SR (median 0.012%; range 0-88.13%) vs. disseminated/HR (median 0.052%; range 0-24.31%) clinical staging. There was also no significant difference in MDD level between the BM and PB among 16 patients with evaluable samples from both sites. While a low event rate limited association with outcome, 0 of 6 patients without measurable MDD experienced relapse/progression/death (vs. 3 of 25 MDD+ patients). Conclusions: B-LLy Ig clonal composition reflects a more mature developmental state than B-ALL and a spectrum of BM/PB MDD across clinical stages. Developmentally ordered Ig rearrangement first involves IgH D-J recombination, followed by V gene rearrangement, and finally Ig light chain V-J rearrangement at kappa and then lambda loci. Thus, Ig clonal composition provides biologically informative data relevant to B cell maturation stage, which here supports classification of B-LLy as a distinct entity from B-ALL. Further, the spectrum of MDD observed in this cohort agnostic of clinical stage suggests that Ig HTS may have novel utility in refining currently limited B-LLy risk stratification by distinguishing select patients who lack measurable disease dissemination or for tracking treatment response via PB HTS. In sum, Ig HTS may have a role in informing therapeutic strategies based on the unique features of B-LLy and in characterizing it as a biologically distinct entity from B-ALL.

Lineage switch (LS) refers to the immunophenotypic transformation of one leukemia lineage to another (ie, lymphoid to myeloid) with retention of baseline genetics. This phenomenon was originally observed in infants with B-lymphoblastic leukemia (B-ALL) withKMT2Arearrangements following chemotherapy, but is now increasingly being observed as a form of immune escape following targeted therapies among children and adults with B-ALL with and withoutKMT2Arearrangements. In this report, we present two cases of adolescents with B-ALL harboringCRLF2rearrangements (Philadelphia-like phenotype) who developed LS to acute myeloid leukemia following CD19 targeted therapy. To our knowledge, these are the first cases of LS to be reported in patients withCRLF2rearranged acute lymphoblastic leukemia. In addition to raising awareness that this genetic mutation may associate with lineage plasticity, our cases illustrate the importance of multi-modal disease surveillance in the diagnosis of LS.

Aims Characterise T-cell receptor gene (TR) repertoires of small intestinal T cells of patients with newly diagnosed (active) coeliac disease (ACD), refractory CD type I (RCD I) and patients with CD on a gluten-free diet (GFD). Methods Next-generation sequencing of complementarity-determining region 3 (CDR3) of rearranged T cell receptor β (TRB) and γ (TRG) genes was performed using DNA extracted from intraepithelial cell (IEC) and lamina propria cell (LPC) fractions and a small subset of peripheral blood mononuclear cell (PBMC) samples obtained from CD and non-CD (control) patients. Several parameters were assessed, including relative abundance and enrichment. Results TRB and TRG repertoires of CD IEC and LPC samples demonstrated lower clonality but higher frequency of rearranged TRs compared with controls. No CD-related differences were detected in the limited number of PBMC samples. Previously published LP gliadin-specific TRB sequences were more frequently detected in LPC samples from patients with CD compared with non-CD controls. TRG repertoires of IECs from both ACD and GFD patients demonstrated increased abundance of certain CDR3 amino acid (AA) motifs compared with controls, which were encoded by multiple nucleotide variants, including one motif that was enriched in duodenal IECs versus the PBMCs of CD patients. Conclusions Small intestinal TRB and TRG repertoires of patients with CD are more diverse than individuals without CD, likely due to mucosal recruitment and accumulation of T cells because of protracted inflammation. Enrichment of the unique TRG CDR3 AA sequence in the mucosa of patients with CD may suggest disease-associated changes in the TCRγδ IE lymphocyte (IEL) landscape.