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Rarely, immunophenotypically immature B-cell precursor acute lymphoblastic leukemia (BCP-ALL) carries an immunoglobulin- MYC rearrangement (IG-MYC-r). This can result in diagnostic confusion with Burkitt lymphoma/leukemia and use of individualized treatment schedules of unproven efficacy. Here we compare the molecular characteristics of these conditions and investigate historic clinical outcome data. We identified 90 cases registered in a national BCP-ALL clinical trial/registry. When present, diagnostic material underwent cytogenetic, exome, methylome and transcriptome analyses. The outcomes analyzed were 3-year event-free survival and overall survival. IG-MYC-r was identified in diverse cytogenetic backgrounds, co-existing with either established BCP-ALL-specific abnormalities (high hyperdiploidy, n=3; KMT2A-rearrangement, n=6; iAMP21, n=1; BCR-ABL1, n=1); BCL2/BCL6-rearrangements (n=15); or, most commonly, as the only defining feature (n=64). Within this final group, precursor-like V(D)J breakpoints predominated (8/9) and KRAS mutations were common (5/11). DNA methylation identified a cluster of V(D)J-rearranged cases, clearly distinct from Burkitt leukemia/lymphoma. Children with IG-MYC-r within that subgroup had a 3-year event-free survival of 47% and overall survival of 60%, representing a high-risk BCP-ALL. To develop effective management strategies this group of patients must be allowed access to contemporary, minimal residual disease-adapted, prospective clinical trial protocols.

Hypodiploid acute lymphoblastic leukemia (ALL) is an aggressive blood cancer with a poor prognosis despite intensive chemotherapy or stem cell transplant. Children and adolescents with positive end-of-induction minimal residual disease have an overall survival lower than 30%. However, data regarding therapeutic alternatives for this disease is nearly nonexistent, emphasizing the critical need for new or adjunctive therapies that can improve outcomes. We previously reported on the therapeutic efficacy of venetoclax (ABT-199) in hypodiploid B-lineage ALL but with limitations as monotherapy. In this study, we set out to identify drugs enhancing the anti-leukemic effect of venetoclax in hypodiploid ALL. Using a highthroughput drug screen, we identified dinaciclib, a cyclin-dependent kinase inhibitor that worked synergistically with venetoclax to induce cell death in hypodiploid cell lines. This combination eradicated leukemic blasts within hypodiploid ALL patient-derived xenografts mice with low off-target toxicity. Our findings suggest that dual inhibition of BCL-2 (venetoclax) and CDK9/MCL-1 (dinaciclib) is a promising therapeutic approach in hypodiploid ALL, warranting further investigation to inform clinical trials in this high-risk patient population.

PURPOSE To improve the outcomes of patients with T-cell acute lymphoblastic leukemia (T-ALL) and lymphoblastic lymphoma (T-LL), the proteasome inhibitor bortezomib was examined in the Children's Oncology Group phase III clinical trial AALL1231, which also attempted to reduce the use of prophylactic cranial radiation (CRT) in newly diagnosed T-ALL. PATIENTS AND METHODS Children and young adults with T-ALL/T-LL were randomly assigned to a modified augmented Berlin-Frankfurt-Münster chemotherapy regimen with/without bortezomib during induction and delayed intensification. Multiple modifications were made to the augmented Berlin-Frankfurt-Münster backbone used in the predecessor trial, AALL0434, including using dexamethasone instead of prednisone and adding two extra doses of pegaspargase in an attempt to eliminate CRT in most patients. RESULTS AALL1231 accrued 824 eligible and evaluable patients from 2014 to 2017. The 4-year event-free survival (EFS) and overall survival (OS) for arm A (no bortezomib) versus arm B (bortezomib) were 80.1% ± 2.3% versus 83.8% ± 2.1% (EFS, P = .131) and 85.7% ± 2.0% versus 88.3% ± 1.8% (OS, P = .085). Patients with T-LL had improved EFS and OS with bortezomib: 4-year EFS (76.5% ± 5.1% v 86.4% ± 4.0%; P = .041); and 4-year OS (78.3% ± 4.9% v 89.5% ± 3.6%; P = .009). No excess toxicity was seen with bortezomib. In AALL0434, 90.8% of patients with T-ALL received CRT. In AALL1231, 9.5% of patients were scheduled to receive CRT. Evaluation of comparable AALL0434 patients who received CRT and AALL1231 patients who did not receive CRT demonstrated no statistical differences in EFS ( P = .412) and OS ( P = .600). CONCLUSION Patients with T-LL had significantly improved EFS and OS with bortezomib on the AALL1231 backbone. Systemic therapy intensification allowed elimination of CRT in more than 90% of patients with T-ALL without excess relapse.

PURPOSE Blinatumomab, a bispecific T-cell engager immunotherapy, is efficacious in relapsed/refractory B-cell ALL (B-ALL) and has a favorable toxicity profile. One aim of the Children's Oncology Group AALL1331 study was to compare survival of patients with low-risk (LR) first relapse of B-ALL treated with chemotherapy alone or chemotherapy plus blinatumomab. PATIENTS AND METHODS After block 1 reinduction, patients age 1-30 years with LR first relapse of B-ALL were randomly assigned to block 2/block 3/two continuation chemotherapy cycles/maintenance (arm C) or block 2/two cycles of continuation chemotherapy intercalated with three blinatumomab blocks/maintenance (arm D). Patients with CNS leukemia received 18 Gy cranial radiation during maintenance and intensified intrathecal chemotherapy. The primary and secondary end points were disease-free survival (DFS) and overall survival (OS). RESULTS The 4-year DFS/OS for the 255 LR patients accrued between December 2014 and September 2019 were 61.2% ± 5.0%/90.4% ± 3.0% for blinatumomab versus 49.5% ± 5.2%/79.6% ± 4.3% for chemotherapy ( P = .089/ P = .11). For bone marrow (BM) ± extramedullary (EM) (BM ± EM; n = 174) relapses, 4-year DFS/OS were 72.7% ± 5.8%/97.1% ± 2.1% for blinatumomab versus 53.7% ± 6.7%/84.8% ± 4.8% for chemotherapy ( P = .015/ P = .020). For isolated EM (IEM; n = 81) relapses, 4-year DFS/OS were 36.6% ± 8.2%/76.5% ± 7.5% for blinatumomab versus 38.8% ± 8.0%/68.8% ± 8.6% for chemotherapy ( P = .62/ P = .53). Blinatumomab was well tolerated and patients had low adverse event rates. CONCLUSION For children, adolescents, and young adults with B-ALL in LR first relapse, there was no statistically significant difference in DFS or OS between the blinatumomab and standard chemotherapy arms overall. However, blinatumomab significantly improved DFS and OS for the two thirds of patients with BM ± EM relapse, establishing a new standard of care for this population. By contrast, similar outcomes and poor DFS for both arms were observed in the one third of patients with IEM; new treatment approaches are needed for these patients (ClinicalTrials.gov identifier: NCT02101853 ).

Background There is growing evidence for the inherited basis of susceptibility to childhood acute lymphoblastic leukemia (ALL). Genome-wide association studies have identified non-coding ALL risk variants at the ARID5B gene locus, but their exact functional effects and the molecular mechanism linking ARID5B to B-cell ALL leukemogenesis remain largely unknown. Methods We performed targeted sequencing of ARID5B in germline DNA of 5008 children with ALL. Variants were evaluated for association with ALL susceptibility using 3644 patients from the UK10K cohort as non-ALL controls, under an additive model. Cis-regulatory elements in ARID5B were systematically identified using dCas9-KRAB–mediated enhancer interference system enhancer screen in ALL cells. Disruption of transcription factor binding by ARID5B variant was predicted informatically and then confirmed using chromatin immunoprecipitation and coimmunoprecipitation. ARID5B variant association with hematological traits was examined using UK Biobank dataset. All statistical tests were 2-sided. Results We identified 54 common variants in ARID5B statistically significantly associated with leukemia risk, all of which were noncoding. Six cis-regulatory elements at the ARID5B locus were discovered using CRISPR-based high-throughput enhancer screening. Strikingly, the top ALL risk variant (rs7090445, P = 5.57 × 10–45) is located precisely within the strongest enhancer element, which is also distally tethered to the ARID5B promoter. The variant allele disrupts the MEF2C binding motif sequence, resulting in reduced MEF2C affinity and decreased local chromosome accessibility. MEF2C influences ARID5B expression in ALL, likely via a transcription factor complex with RUNX1. Using the UK Biobank dataset (n = 349 861), we showed that rs7090445 was also associated with lymphocyte percentage and count in the general population (P = 8.6 × 10–22 and 2.1 × 10–18, respectively). Conclusions Our results indicate that ALL risk variants in ARID5B function by modulating cis-regulatory elements at this locus.

Resistance to mitochondrial apoptosis predicts inferior treatment outcomes in patients with diverse tumor types, including T-cell acute lymphoblastic leukemia (T-ALL). However, the genetic basis for variability in this mitochondrial apoptotic phenotype is poorly understood, preventing its rational therapeutic targeting. Using BH3 profiling and exon sequencing analysis of childhood T-ALL clinical specimens, we found that mitochondrial apoptosis resistance was most strongly associated with activating mutations of JAK3. Mutant JAK3 directly repressed apoptosis in leukemia cells, because its inhibition with mechanistically distinct pharmacologic inhibitors resulted in reversal of mitochondrial apoptotic blockade. Inhibition of JAK3 led to loss of MEK, ERK and BCL2 phosphorylation, and BH3 profiling revealed that JAK3-mutant primary T-ALL patient samples were characterized by a dependence on BCL2. Treatment of JAK3-mutant T-ALL cells with the JAK3 inhibitor tofacitinib in combination with a spectrum of conventional chemotherapeutics revealed synergy with glucocorticoids, in vitro and in vivo. These findings thus provide key insights into the molecular genetics of mitochondrial apoptosis resistance in childhood T-ALL, and a compelling rationale for a clinical trial of JAK3 inhibitors in combination with glucocorticoids for patients with JAK3-mutant T-ALL.

PURPOSE Children's Oncology Group trial AALL1621 was conducted to prospectively determine the safety and efficacy of inotuzumab ozogamicin (InO) in pediatric and adolescent patients with relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL). PATIENTS AND METHODS This single-arm phase II trial enrolled patients age 1-21 years with R/R CD22-positive B-ALL. In cycle 1, InO dosing was 0.8 mg/m2 intravenously on day 1 and 0.5 mg/m2 on days 8 and 15 of a 28-day cycle with response evaluation at day 28. Using a two-stage design, the trial was continuously monitored for dose-limiting toxicities and sinusoidal obstruction syndrome (SOS). CD22 expression was retrospectively evaluated by central flow cytometry. RESULTS Forty-eight patients were evaluable for response and toxicity; 19 had complete response (CR) and nine CR with incomplete count recovery (CRi) after cycle 1 (CR/CRi rate: 58.3%; two-sided 90% CI, 46.5 to 69.3). Twenty-seven of 28 patients with CR or CRi had minimal residual disease measured by flow cytometry; 18 (66.7%) had minimal residual disease < 0.01%. Seven of 28 patients (25%) with CR or CRi had delayed count recovery past day 42 in cycle 1. Three (6.3%) patients had grade 3 ALT elevation and one patient had grade 3 hyperbilirubinemia in cycle 1. Of 21 patients undergoing hematopoietic stem-cell transplantation after InO, 6 (28.6%) developed grade 3 SOS. Partial CD22 expression and lower CD22 site density were associated with lower likelihood of response to InO. CONCLUSION InO is effective and well tolerated in heavily pretreated children and adolescents with R/R CD22-positive B-ALL. SOS after hematopoietic stem-cell transplantation and prolonged cytopenias were notable. CD22 modulation was identified as a mechanism of resistance. Expanded study of InO combined with chemotherapy is underway.

Low-intensity maintenance therapy with 6-mercaptopurine (6-MP) limits the occurrence of acute lymphoblastic leukemia (ALL) relapse and is central to the success of multiagent chemotherapy protocols. Activating mutations in the 5′-nucleotidase cytosolic II (NT5C2) gene drive resistance to 6-MP in over 35% of early relapse ALL cases. Here we identify CRCD2 as a first-in-class small-molecule NT5C2 nucleotidase inhibitor broadly active against leukemias bearing highly prevalent relapse-associated mutant forms of NT5C2 in vitro and in vivo. Importantly, CRCD2 treatment also enhanced the cytotoxic activity of 6-MP in NT5C2 wild-type leukemias, leading to the identification of NT5C2 Ser502 phosphorylation as a novel NT5C2-mediated mechanism of 6-MP resistance in this disease. These results uncover an unanticipated role of nongenetic NT5C2 activation as a driver of 6-MP resistance in ALL and demonstrate the potential of NT5C2 inhibitor therapy for enhancing the efficacy of thiopurine maintenance therapy and overcoming resistance at relapse.Significance:Relapse-associated NT5C2 mutations directly contribute to relapse in ALL by driving resistance to chemotherapy with 6-MP. Pharmacologic inhibition of NT5C2 with CRCD2, a first-in-class nucleotidase inhibitor, enhances the cytotoxic effects of 6-MP and effectively reverses thiopurine resistance mediated by genetic and nongenetic mechanisms of NT5C2 activation in ALL.This article is highlighted in the In This Issue feature, p. 2483

Supplementary Figure from Pharmacologic Inhibition of NT5C2 Reverses Genetic and Nongenetic Drivers of 6-MP Resistance in Acute Lymphoblastic Leukemia

Supplementary Table from Pharmacologic Inhibition of NT5C2 Reverses Genetic and Nongenetic Drivers of 6-MP Resistance in Acute Lymphoblastic Leukemia

PDF file - 80KB, Identification of active compounds that inhibited the phosphatase activity of SHP2 with various efficiencies.

RNAseq Gene Lists

S1. Schematic diagram of NSD2 isoform indicating domains, the location of amino acid E1099 within the SET domain, and the targets of the shRNAs. S2. NSD2 Overexpression in B-ALL Cell Lines Does Not Impart Clonal Advantage. S3. Overexpression of REIIBP in B-ALL Cell Lines Does Not Impart Clonal Advantage. S4. Knockdown of WT NSD2 in B-ALL Cell Lines Does Not Lead to Phenotypic Differences. S5. Knockdown of NSD2 in RS4;11 and RCH-ACV lines leads to increased chemosensitivity. S6. Sensitivity to 6-MP Upon Knockdown of NSD2 in RS4;11 is Not Depedent of Level of Knockdown. S7. Knockdown of NSD2 in WT B-ALL Cell Lines Does Not Lead to Increased Sensitivity. S8. NSD2 WT and EK Overexpression in B-ALL Cell Lines Leads to Distinct Genetic Signature Unique to Each Cell Line. S9. NSD2 WT and EK Overexpression in B-ALL Cell Lines Leads to Distinct Epigenetic Signature Resulting in Gene Expression Changes. S10. NSD2 Knockdown in Mutant B-ALL Cell Lines Leads to Distinct Genetic Signature Unique to Each Cell Line. S11. ATAC peaks significantly correlate with gene expression for RS4;11 and RCH-ACV cell lines.

VAF concordance

Cell line IC50 values

NSG mice were injected in the tail with either ETP-ALL (TALL-x-11) or typical T-ALL (TALL-x-2) 1X106 cells and following engraftment to 65% the mice were randomized to receive vehicle, ABT-199 or ABT-263 treatment for two-weeks. The spleen weights (A) and absolute human CD45+ counts (B) along with % human CD45+ cell in the blood (C) were measured for the ETP-ALL primagraft following treatment. The leg bones were harvested and representative images are shown for the different treatments (D). Similar experiments were performed for the typical T-ALL primagraft spleen weights (E), absolute human CD45+ count in the spleen (F) with % human CD45+ cells in the blood were measured (G). Similarly the legs bones were harvested and representative images are shown (H).

Supplementary Figures 1-8

Lineage-ambiguous leukemias are high-risk malignancies of poorly understood genetic basis. Here, we describe a distinct subgroup of acute leukemia with expression of myeloid, T lymphoid, and stem cell markers driven by aberrant allele-specific deregulation of BCL11B, a master transcription factor responsible for thymic T-lineage commitment and specification. Mechanistically, this deregulation was driven by chromosomal rearrangements that juxtapose BCL11B to superenhancers active in hematopoietic progenitors, or focal amplifications that generate a superenhancer from a noncoding element distal to BCL11B. Chromatin conformation analyses demonstrated long-range interactions of rearranged enhancers with the expressed BCL11B allele and association of BCL11B with activated hematopoietic progenitor cell cis-regulatory elements, suggesting BCL11B is aberrantly co-opted into a gene regulatory network that drives transformation by maintaining a progenitor state. These data support a role for ectopic BCL11B expression in primitive hematopoietic cells mediated by enhancer hijacking as an oncogenic driver of human lineage-ambiguous leukemia.Significance:Lineage-ambiguous leukemias pose significant diagnostic and therapeutic challenges due to a poorly understood molecular and cellular basis. We identify oncogenic deregulation of BCL11B driven by diverse structural alterations, including de novo superenhancer generation, as the driving feature of a subset of lineage-ambiguous leukemias that transcend current diagnostic boundaries.This article is highlighted in the In This Issue feature, p. 2659

Supplementary Data file including supplementary methods and clinical case vignettes.

This file contains Supplementary Tables 1-15 detailing clinical and genomic information about each patient analyzed in this study, along with the results of RNA-seq and gene set enrichment analyses.

PDF file - 111 KB, Predicted binding pose of #220-324 with SHP2 phosphatase.

Reasons for not using recommended targeted therapy.

This file contains Supplementary Figures 1-23 with corresponding supplementary figure legends, as well as the legends for Supplementary Tables 1-15.

Supplementary materials and methods and supplementary figure legends.

PDF file - 111 KB, #220-324 inhibited SHP2-mediated cell signaling.

PDF file - 65 KB, Effect of Triton X-100 on the function of #220-324 in inhibiting SHP2 activity.

The plant homeodomain 6 gene (PHF6) is frequently mutated in human T-cell acute lymphoblastic leukemia (T-ALL); however, its specific functional role in leukemia development remains to be established. Here, we show that loss of PHF6 is an early mutational event in leukemia transformation. Mechanistically, genetic inactivation of Phf6 in the hematopoietic system enhances hematopoietic stem cell (HSC) long-term self-renewal and hematopoietic recovery after chemotherapy by rendering Phf6 knockout HSCs more quiescent and less prone to stress-induced activation. Consistent with a leukemia-initiating tumor suppressor role, inactivation of Phf6 in hematopoietic progenitors lowers the threshold for the development of NOTCH1-induced T-ALL. Moreover, loss of Phf6 in leukemia lymphoblasts activates a leukemia stem cell transcriptional program and drives enhanced T-ALL leukemia-initiating cell activity. These results implicate Phf6 in the control of HSC homeostasis and long-term self-renewal and support a role for PHF6 loss as a driver of leukemia-initiating cell activity in T-ALL.Significance:Phf6 controls HSC homeostasis, leukemia initiation, and T-ALL leukemia-initiating cell self-renewal. These results substantiate a role for PHF6 mutations as early events and drivers of leukemia stem cell activity in the pathogenesis of T-ALL.This article is highlighted in the In This Issue feature, p. 305

Supplementary Materials and Methods. Supplementary Table S1. QC of Affymetrix U133_Plus_2.0 Data. Supplementary Table S2. Summary of the Objective Response Measure (ORM) scoring methodology. Supplementary Table S3. Correlation of xenograft with parent U133_Plus_2.0 expression. Supplementary Table S4. Probe sets where parent expression is always greater than xenograft. Supplementary Table S5. Probe sets where xenograft expression is always greater than parent. Supplementary Table S6. Complete summary of in vivo AZD1480 single agent responses for individual mice. Supplementary Table S7. In vitro Combination Indices (CIs) for AZD1480 and selumetinib. Supplementary Table S8. In vivo responses of JAK-mutated ALL xenografts to AZD1480/selumetinib combination treatment. Supplementary Table S9. Complete summary of in vivo AZD1480 single agent and AZD1480/selumetinib combination responses for individual mice. Supplementary Figure S1. Chemical structures of (A) AZD1480 and (B) selumetinib. Supplementary Figure S2. Relationship between high CRLF2 expression and engraftment potential of the P9906 cohort. Supplementary Figure S3. Probe set expression by range. Supplementary Figure S4. Heatmap of microarray gene expression data corresponding to the qRT-PCR data shown in Figure 1. Supplementary Figure S5. In vitro cytotoxicity of AZD1480 against ALL xenograft cells. Supplementary Figure S6. In vivo efficacy of single agent AZD1480 against ALL xenografts. Supplementary Figure S7. Time of drug exposure affects synergistic interactions of AZD1480 and selumetinib against JAK-mutated ALL xenografts. Supplementary Figure S8. The extent of inhibition of the JAK/STAT and MAPK signaling pathways depends on the length of exposure to AZD1480/selumetinib.

Patient sequencing data, by patient number and diagnosis.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. The highest rates of treatment failure occur in specific genetic subsets of ALL, including hypodiploid B-cell ALL (B-ALL), for which effective alternative therapies to current intensive chemotherapy treatments have yet to be developed. Here, we integrated biochemical and genomic profiling with functional drug assays to select effective agents with therapeutic potential against hypodiploid B-ALL. ABT-199, a selective Bcl-2 inhibitor, was effective in reducing leukemic burden in vitro and in vivo in patient-derived xenograft models of hypodiploid B-ALL. Daily oral treatment with ABT-199 significantly increased survival in xenografted mice. The unexpected efficacy of ABT-199 observed in hypodiploid leukemias lacking BIM expression (the major reported mediator of ABT-199–induced apoptosis) led us to investigate the mechanism of action of ABT-199 in the absence of BIM. Treatment with ABT-199 elicited responses in a dose-dependent manner, from cell-cycle arrest at low nanomolar concentrations to cell death at concentrations above 100 nmol/L. Collectively, these results demonstrate the efficacy of Bcl-2 inhibition and potential therapeutic strategy in hypodiploid B-ALL.Significance:These results demonstrate the efficacy of ABT-199 in vivo and provide encouraging preclinical data of Bcl-2 as a potential target for the treatment of hypodiploid B-ALL.

Purpose: High-throughput sequencing (HTS) of immunoglobulin heavy-chain genes (IGH) in unselected clinical samples for minimal residual disease (MRD) in B lymphoblastic leukemia (B-ALL) has not been tested. As current MRD-detecting methods such as flow cytometry or patient-specific qPCR are complex or difficult to standardize in the clinical laboratory, sequencing may enhance clinical prognostication.Experimental Design: We sequenced IGH in paired pretreatment and day 29 post-treatment samples using residual material from consecutive, unselected samples from the Children's Oncology Group AALL0932 trial to measure MRD as compared with flow cytometry. We assessed the impact of ongoing recombination at IGH on MRD detection in post-treatment samples. Finally, we evaluated a subset of cases with discordant MRD results between flow cytometry and sequencing.Results: We found clonal IGH rearrangements in 92 of 98 pretreatment patient samples. Furthermore, while ongoing recombination of IGH was evident, index clones typically prevailed in MRD-positive post-treatment samples, suggesting that clonal evolution at IGH does not contribute substantively to tumor fitness. MRD was detected by sequencing in all flow cytometry–positive cases with no false-negative results. In addition, in a subset of patients, MRD was detected by sequencing, but not by flow cytometry, including a fraction with MRD levels within the sensitivity of flow cytometry. We provide data that suggest that this discordance in some patients may be due to the phenotypic maturation of the transformed cell.Conclusion: Our results provide strong support for HTS of IGH to enhance clinical prognostication in B-ALL. Clin Cancer Res; 20(17); 4540–8. ©2014 AACR.

Legends for each supplementary figure

Supplemental Figure 1. Protein and gene expression profiling of hypodiploid ALL. Supplemental Figure 2. Pairing biochemical characterization with viability/survival assays in hypodiploid B-ALL. Supplemental Figure 3. Primagraft samples used for ex vivo and in vivo experiments. Supplemental Figure 4. In vivo engraftment of hypodiploid ALL xenografts and response to ABT-199 during trial. Supplemental Figure 5. Clonal characterization of hypodiploid tumors at the beginning and end of trial. Supplemental Figure 6. Genome editing assays. Supplemental Figure 7. Genomic, karyotypic characterization and mechanistic experiments on Beck-1732 cells. Supplementary Tables of samples used in the in vivo studies.

Supplementary Figure 6. Pre-treatment flow cytometry with post-treatment MRD by HTS identified in a triple flow cytometry-sorted fraction of mature B cells. To determine if mpFC was missing MRD as compared to HTS, we evaluated 10 additional samples by sequencing IGH repertoire in triple flow cytometry-sorted fraction of mature B cells (defined by uniform bright CD20 expression, bright CD45, and absence of CD10), and correlated the detected IGH sequences with the paired, pre-treatment index IGH clone. 2 samples had insufficient DNA post-sorting for sequencing analysis at a sensitivity of 1 in 10,000. Of the remaining 8 patients, one case had a clonal IGH rearrangement identified in the post-treatment sample by sequencing at a level of 0.01%, matching the index clone, but no abnormal lymphoblast population detected by flow cytometry. Flow cytometry analyses of the pre-treatment (A) and post-treatment (B) samples are shown. Orange = neoplastic lymphoblast population; blue = mature, reactive B cells; grey = background hematopoietic events (grey), including predominantly maturing elytroid and myeloid forms, are also shown in first two plots for each row.