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Supplementary Figure 1 from Expression of Late Cell Cycle Genes and an Increased Proliferative Capacity Characterize Very Early Relapse of Childhood Acute Lymphoblastic Leukemia

Supplementary Figure 2 from Expression of Late Cell Cycle Genes and an Increased Proliferative Capacity Characterize Very Early Relapse of Childhood Acute Lymphoblastic Leukemia

Supplementary Table 1 from Expression of Late Cell Cycle Genes and an Increased Proliferative Capacity Characterize Very Early Relapse of Childhood Acute Lymphoblastic Leukemia

Purpose: In childhood acute lymphoblastic leukemia (ALL), ∼25% of patients suffer from relapse. In recurrent disease, despite intensified therapy, overall cure rates of 40% remain unsatisfactory and survival rates are particularly poor in certain subgroups. The probability of long-term survival after relapse is predicted from well-established prognostic factors (i.e., time and site of relapse, immunophenotype, and minimal residual disease). However, the underlying biological determinants of these prognostic factors remain poorly understood.Experimental Design: Aiming at identifying molecular pathways associated with these clinically well-defined prognostic factors, we did gene expression profiling on 60 prospectively collected samples of first relapse patients enrolled on the relapse trial ALL-REZ BFM 2002 of the Berlin-Frankfurt-Münster study group.Results: We show here that patients with very early relapse of ALL are characterized by a distinctive gene expression pattern. We identified a set of 83 genes differentially expressed in very early relapsed ALL compared with late relapsed disease. The vast majority of genes were up-regulated and many were late cell cycle genes with a function in mitosis. In addition, samples from patients with very early relapse showed a significant increase in the percentage of S and G2-M phase cells and this correlated well with the expression level of cell cycle genes.Conclusions: Very early relapse of ALL is characterized by an increased proliferative capacity of leukemic blasts and up-regulated mitotic genes. The latter suggests that novel drugs, targeting late cell cycle proteins, might be beneficial for these patients that typically face a dismal prognosis.

It has been the hypothesis of this study that clonal or subclonal variants of TP53 and KRAS that signify ultra-high risk in relapsed pediatric T-ALL indicate a poor prognosis at initial disease. We analyzed two cohorts of 160 and 226 BFM-patients, one consisting of 81 patients who later relapsed and 79 matched non-relapsing controls. Cohort-2 consisted of consecutive patients of whom 30 relapsed. In cohort-1 targeted sequencing revealed TP53 clonal and subclonal variants in 6/81 relapsing but in none of the non-relapsing patients (p=0.014). KRAS alterations occurred in 9/81 relapsing and 2/79 non-relapsing patients (p=0.032). No relapsing patient with TP53 and/or KRAS alterations survived, whereas 19/67 relapsing patients without such variants did (p=0.023). In cohort-2, 196 patients without relapse included 10 with TP53 or KRAS variants, all of whom were non-high risk based on treatment response. However, in the entire group of 386 patients the 188 patients with poor treatment response included 9 with such a variant of whom 8 relapsed and died. Of these, 5 had not undergone stem-cell-transplantation. In conclusion, these data show that subclonal and clonal TP53 and KRAS alterations at initial diagnosis indicate a dismal prognosis in children with T-ALL and poor treatment response.

Chemotherapy is a standard treatment for pediatric acute lymphoblastic leukemia (ALL), which sometimes relapses with chemoresistant features. However, whether acquired drug-resistance mutations in relapsed ALL pre-exist or are induced by treatment remains unknown. Here we provide direct evidence of a specific mechanism by which chemotherapy induces drug-resistance-associated mutations leading to relapse. Using genomic and functional analysis of relapsed ALL we show that thiopurine treatment in mismatch repair (MMR)-deficient leukemias induces hotspot TP53 R248Q mutations through a specific mutational signature (thio-dMMR). Clonal evolution analysis reveals sequential MMR inactivation followed by TP53 mutation in some patients with ALL. Acquired TP53 R248Q mutations are associated with on-treatment relapse, poor treatment response and resistance to multiple chemotherapeutic agents, which could be reversed by pharmacological p53 reactivation. Our findings indicate that TP53 R248Q in relapsed ALL originates through synergistic mutagenesis from thiopurine treatment and MMR deficiency and suggest strategies to prevent or treat TP53-mutant relapse. Zhou and colleagues demonstrate that thiopurine chemotherapy in mismatch repair-deficient ALL cells leads to R248Q TP53 mutations and clonal selection that favors on-treatment ALL relapse and chemoresistance.

Activating mutations in cytosolic 5′-nucleotidase II (NT5C2) are considered to drive relapse formation in acute lymphoblastic leukemia (ALL) by conferring purine analog resistance. To examine the clinical effects of NT5C2 mutations in relapsed ALL, we analyzed NT5C2 in 455 relapsed B-cell precursor ALL patients treated within the ALL-REZ BFM 2002 relapse trial using sequencing and sensitive allele-specific real-time polymerase chain reaction. We detected 110 NT5C2 mutations in 75 (16.5%) of 455 B-cell precursor ALL relapses. Two-thirds of relapses harbored subclonal mutations and only one-third harbored clonal mutations. Event-free survival after relapse was inferior in patients with relapses with clonal and subclonal NT5C2 mutations compared with those without (19% and 25% vs 53%, P < .001). However, subclonal, but not clonal, NT5C2 mutations were associated with reduced event-free survival in multivariable analysis (hazard ratio, 1.89; 95% confidence interval, 1.28-2.69; P = .001) and with an increased rate of nonresponse to relapse treatment (subclonal 32%, clonal 12%, wild type 9%, P < .001). Nevertheless, 27 (82%) of 33 subclonal NT5C2 mutations became undetectable at the time of nonresponse or second relapse, and in 10 (71%) of 14 patients subclonal NT5C2 mutations were undetectable already after relapse induction treatment. These results show that subclonal NT5C2 mutations define relapses associated with high risk of treatment failure in patients and at the same time emphasize that their role in outcome is complex and goes beyond mutant NT5C2 acting as a targetable driver during relapse progression. Sensitive, prospective identification of NT5C2 mutations is warranted to improve the understanding and treatment of this aggressive ALL relapse subtype.

PURPOSE Minimal residual disease (MRD) helps to accurately assess when children with late bone marrow relapses of B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) will benefit from allogeneic hematopoietic stem-cell transplantation (allo-HSCT). More detailed dissection of MRD response heterogeneity and the specific genetic aberrations could improve current practice. PATIENTS AND METHODS MRD was assessed after induction treatment and at different times during relapse treatment until allo-HSCT (indicated in poor responders to induction; MRD ≥ 10−3) for patients being treated for late BCP-ALL bone marrow relapses (n = 413; median follow-up, 9.4 years) in the ALL-REZ BFM 2002 trial/registry (ClinicalTrials.gov identifier: NCT00114348 ). RESULTS Patients with both good (MRD < 10−3) and poor responses to induction treatment reached excellent event-free survival (EFS; 72% v 65%) and overall survival (OS; 82% v 74%). Patients with MRD of 10−2 or greater after induction had reduced EFS (56%), and their MRD persisted until allo-HSCT more frequently than it did in patients with MRD of 10−3 or greater to less than 10−2 ( P = .037). Patients with 25% or more leukemic blasts after induction (early nonresponders) had the poorest prognosis (EFS, 22%). Interestingly, patients with MRD of 10−3 or greater before allo-HSCT (late nonresponders) still had an EFS of 50% and OS of 63%, which in principle justifies allo-HSCT in these patients. From a panel of selected candidate genes, TP53 alterations (frequency, 8%) were the only genetic alteration with independent prognostic value in any MRD-based response subgroup. CONCLUSION After induction treatment, MRD-based treatment stratification resulted in excellent survival in patients with late relapsed BCP-ALL. Prognosis could be further improved in very poor responders by intensifying treatment directly after induction. TP53 alterations can be defined as a novel genetic high-risk marker in all MRD response groups in late relapsed BCP-ALL. Here we identified early and late nonresponders to be considered as events in future trials.

Aim: Outcomes of children with high-risk (HR) relapsed acute lymphoblastic leukaemia (ALL) (N = 393), recruited to ALLR3 and ALL-REZ BFM 2002 trials, were analysed. Minimal residual disease (MRD) was assessed after induction and at predetermined time points until haematopoietic stem cell transplantation (SCT). Methods: Genetic analyses included karyotype, copy-number alterations and mutation analyses. Ten-year survivals were analysed using Kaplan-Meier and Cox models for multivariable analyses. Results: Outcomes of patients were comparable in ALLR3 and ALL-REZ BFM 2002. The event-free survival of B-cell precursor (BCP) and T-cell ALL (T-ALL) was 22.6% and 26.2% (P = 0.94), respectively, and the overall survival (OS) was 32.6% and 28.2% (P = 0.11), respectively. Induction failures (38%) were associated with deletions of NR3C1 (P = 0.002) and BTG1 (P = 0.03) in BCP-ALL. The disease-free survival (DFS) and OS in patients with good vs poor MRD responses were 57.4% vs 22.6% (P < 0.0001) and 57.8% vs 32.0% (P = 0.0004), respectively. For BCP- and T-ALL, the post-SCT DFS and OS were 42.1% and 56.8% (P = 0.26) and 51.6% and 55.4% (P = 0.67), respectively. The cumulative incidences of post-SCT relapse for BCP- and T-ALL were 36.9% and 17.8% (P = 0.012) and of death were 10.7% and 25.5% (P = 0.013), respectively. Determinants of outcomes after SCT were acute graft versus host disease, pre-SCT MRD (≥10 −3), HR cytogenetics and TP53 alterations in BCP-ALL. Conclusion: Improvements in outcomes for HR ALL relapses require novel compounds in induction therapy to improve remission rates and immune targeted therapy after induction to maintain remission after SCT. Trial registration: ALLR3: NCT00967057; ALL REZ-BFM 2002: NCT00114348

Acute lymphoblastic leukemia (ALL) is the most frequent malignancy in childhood. Minimal residual disease (MRD) monitoring is an important prognostic factor for ALL treatment response and patient stratification. MRD monitoring uses personalized real-time PCR to measure the amount of cancer cells among normal cells. Due to clonal tumor evolution or secondary rearrangement processes, MRD markers can disappear during treatment, leading to false-negative MRD results and wrong decision-making in personalized treatments. Therefore, monitoring of multiple MRD markers per patient is required. For the first time, the authors present personalized multiplex mediator probe PCR (MP PCR) for MRD monitoring in ALL. These assays can precisely quantify more MRD markers in less sample material. Therefore, clinical outcomes will be less affected by clonal tumor evolution. Personalized duplex MP PCR assays were developed for different genomic MRD markers, including immunoglobulin/T-cell receptor gene rearrangements, gene fusions, and gene deletions. One duplex assay was successfully applied in a prospective patient case and compared with hydrolysis probes. Moreover, the authors increased the multiplex level from duplex to 4-plex and still met the EuroMRD requirements for reliable quantification. In addition, the authors' MRD-MP design guidelines and multiplex workflow facilitate and accelerate MP PCR assay development. This helps the standardization of personal diagnostics.

Chemotherapy is a standard treatment for pediatric acute lymphoblastic leukemia (ALL), which sometimes relapses with chemoresistant features. However, whether acquired drug-resistance mutations in relapsed ALL pre-exist or are induced by treatment remains unknown. Here we provide direct evidence of a specific mechanism by which chemotherapy induces drug-resistance-associated mutations leading to relapse. Using genomic and functional analysis of relapsed ALL we show that thiopurine treatment in mismatch repair (MMR)-deficient leukemias induces hotspot TP53 R248Q mutations through a specific mutational signature (thio-dMMR). Clonal evolution analysis reveals sequential MMR inactivation followed by TP53 mutation in some patients with ALL. Acquired TP53 R248Q mutations are associated with on-treatment relapse, poor treatment response and resistance to multiple chemotherapeutic agents, which could be reversed by pharmacological p53 reactivation. Our findings indicate that TP53 R248Q in relapsed ALL originates through synergistic mutagenesis from thiopurine treatment and MMR deficiency and suggest strategies to prevent or treat TP53-mutant relapse.

We aimed at identifying the developmental stage at which leukemic cells of pediatric T‐ALLs are arrested and at defining leukemogenic mechanisms based on ATAC‐Seq. Chromatin accessibility maps of seven developmental stages of human healthy T cells revealed progressive chromatin condensation during T‐cell maturation. Developmental stages were distinguished by 2,823 signature chromatin regions with 95% accuracy. Open chromatin surrounding SAE1 was identified to best distinguish thymic developmental stages suggesting a potential role of SUMOylation in T‐cell development. Deconvolution using signature regions revealed that T‐ALLs, including those with mature immunophenotypes, resemble the most immature populations, which was confirmed by TF‐binding motif profiles. We integrated ATAC‐Seq and RNA‐Seq and found DAB1, a gene not related to leukemia previously, to be overexpressed, abnormally spliced and hyper‐accessible in T‐ALLs. DAB1‐negative patients formed a distinct subgroup with particularly immature chromatin profiles and hyper‐accessible binding sites for SPI1 (PU.1), a TF crucial for normal T‐cell maturation. In conclusion, our analyses of chromatin accessibility and TF‐binding motifs showed that pediatric T‐ALL cells are most similar to immature thymic precursors, indicating an early developmental arrest., Analysis of chromatin accessibility of human T‐cell precursors revealed progressive chromatin condensation during maturation. Pediatric T‐ALLs resemble the most immature populations indicating that the epigenetic landscape of this type of leukemia is most similar to the earliest thymic precursors.

Aneuploidy is common in paediatric B-cell precursor acute lymphoblastic leukaemia (ALL). Specific subgroups, such as high hyperdiploidy (>50 chromosomes or DNA Index ≥1·16) and hypodiploidy (

Relapsed acute lymphoblastic leukaemia (ALL) is associated with resistance to chemotherapy and poor prognosis. Gain-of-function mutations in the 5'-nucleotidase, cytosolic II (NT5C2) gene induce resistance to 6-mercaptopurine and are selectively present in relapsed ALL. Yet, the mechanisms involved in NT5C2 mutation-driven clonal evolution during the initiation of leukaemia, disease progression and relapse remain unknown. Here we use a conditional-and-inducible leukaemia model to demonstrate that expression of NT5C2(R367Q), a highly prevalent relapsed-ALL NT5C2 mutation, induces resistance to chemotherapy with 6-mercaptopurine at the cost of impaired leukaemia cell growth and leukaemia-initiating cell activity. The loss-of-fitness phenotype of NT5C2+/R367Q mutant cells is associated with excess export of purines to the extracellular space and depletion of the intracellular purine-nucleotide pool. Consequently, blocking guanosine synthesis by inhibition of inosine-5'-monophosphate dehydrogenase (IMPDH) induced increased cytotoxicity against NT5C2-mutant leukaemia lymphoblasts. These results identify the fitness cost of NT5C2 mutation and resistance to chemotherapy as key evolutionary drivers that shape clonal evolution in relapsed ALL and support a role for IMPDH inhibition in the treatment of ALL.

Introduction Patients who suffer a relapse of pediatric T-cell acute lymphoblastic leukemia (T-ALL) face a dismal prognosis. Prognostic molecular biomarkers that reliably predict the risk of relapse at the time of first diagnosis are not available. Inactivating mutations in TP53 were previously detected in approximately 10% of relapsed patients (Hof et al. J Clin Oncol. 2011) and are invariably associated with fatal outcome (Richter-Pechanska et al. Blood Cancer J. 2017). Mutations in other genes were identified to be either specific for relapse (NT5C2 and CCDC88A) or to be associated with a poor prognosis in relapse (IL7R, KRAS, NRAS, USP7, CNOT3 and MSH6) (Meyer et al. Nat Genet. 2013; Richter-Pechanska et al. Blood Cancer J. 2017). We hypothesized that subclones bearing such mutations can give rise to relapse and analyzed these 9 genes at initial diagnosis of T-ALL with targeted ultra-deep sequencing. Methods Leukemia samples collected at initial diagnosis of 81 children with T-ALL who later relapsed were analyzed. As a control group, we selected 79 children with T-ALL who remained in first remission for at least three years and were matched with regard to treatment response, treatment, age and sex. Targeted deep sequencing was performed by using the Agilent Haloplex High Sensitivity kit with unique molecular identifiers for reliable detection of mutations with very low allele frequencies (average read depth: 1,012x). Results Overall, we detected 75 mutations among 7 targeted genes in 33 / 81 relapsing and 21 / 79 non-relapsing patients. The average allele frequency (AF) of the identified mutations was 25% (0.8% - 83%; SD ± 18%). More than half of the variants (43/75) showed AFs below 30% and were thus classified as subclonal. Interestingly, 7 pathogenic TP53 mutations (subclonal: n=5, clonal: n=2) with AFs of 4.4% - 49.4% were exclusively discovered in 6 patients who experienced a relapse. While 2 of these patients received an allogeneic stem cell transplantation in first remission because of poor treatment response, the remaining 4 patients were treated by chemotherapy in the high-risk (n=1) or medium-risk (n=3) arm. None of the 79 non-relapsing control patients carried TP53 mutations. Consistent with the hypothesis of clonal evolution as a mechanism of relapse in T-ALL, Sanger Sequencing of the relapse sample of one TP53-positive patient confirmed that the subclone harboring the TP53 mutation A159D at initial diagnosis (AF 5.4%) expanded to a major clone (AF 42%) in relapse. The presence of TP53 mutations in two further TP53-positive patients in at least one available post-remission sample is also compatible with clonal selection. However, in a fourth patient the low allele frequency of the TP53 mutation at relapse indicates that the TP53 subclone persisted but did not expand during the development of relapse. In addition to TP53, we identified pathogenic KRAS mutations to be significantly enriched in relapsing patients (9 / 81) compared to non-relapsing patients (2 / 79) at the time of initial diagnosis (chi-squared test, p= 0.032; Table 1). Conclusion Subclonal and clonal mutations in TP53 and KRAS at initial diagnosis were enriched in T-ALL patients who later relapsed and identified approximately 17% of patients suffering a relapse. We thus propose that (subclonal) mutations of TP53 and KRAS may define a subgroup of high-risk T-ALL patients already at the time of first diagnosis. The identification of such mutations may complement the current risk stratification which depends on treatment response and may determine a new molecularly defined subgroup of T-ALLs that may benefit from intensified treatment strategies. Figure 1 Figure 1. Disclosures Schrappe: SigmaTau: Other: research support; Amgen: Other: research support; Servier: Honoraria; Novartis: Honoraria; JazzPharma: Honoraria; Servier: Honoraria, Other: research support; JazzPharma: Honoraria, Other: research support; SHIRE: Other: research support; Novartis: Honoraria, Other: research support. Cario: Novartis: Other: Lecture Fee. Muckenthaler: Silence Therapeutics: Research Funding. Kulozik: Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BioMedX: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; bluebird bio, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Consultancy, Honoraria.

Chemotherapy is curative for most children with acute lymphoblastic leukemia (ALL). Here we provide direct evidence that thiopurine chemotherapeutics can also directly induce drug resistance mutations leading to relapse. Using a large relapsed ALL cohort assembled from Chinese, US and German patients, we found that TP53 R248Q mutations were highly enriched at relapse compared to diagnosis. Relapse-specific TP53 R248Q was associated with the acquisition of MMR deficiency mutations in MSH2, MSH6, or PMS2 and a novel relapse-specific mutational signature. Using isogenic MCF10A cells with or without engineered MSH2 knockout, and the Nalm6 ALL cell line which has native MMR deficiency, we found that this novel signature was caused by a synergistic mutagenic interaction between thiopurine treatment and mismatch repair (MMR) deficiency (called the thio-dMMR signature) that contributes to a hypermutator phenotype and acquisition of TP53 R248Q in residual ALL during remission. Treatment-induced TP53-mutant clones then expand due to broad chemoresistance, leading to eventual relapse. Indeed, thiopurines preferentially induced C>T mutations at the center of NCG trinucleotides, which can lead to TP53 R248Q, and the thiopurine mutation rate was accelerated 2- to 10-fold in MMR-deficient ALL and cell lines. Thiopurine treatment induced C>T mutations preferentially on the transcribed strand, rather than the untranscribed strand, of mRNAs, which further increased the likelihood of TP53 R248Q induction. Further, experimental thiopurine treatment was able to directly induce TP53 R248Q variants in MMR-deficient cultured cells, including Nalm6 and MCF10A MSH2-/-, by activating the thio-dMMR mutational signature, while MMR-proficient MCF10A cells did not experience R248Q induction. The sequential acquisition of MMR deficiency mutations, followed by TP53 mutations, during post-diagnosis ALL evolution was supported by clonal evolution analysis of serial patient samples. p53 R248Q promoted resistance to multiple ALL chemotherapeutic agents, and was associated with on-treatment relapse and poor relapse-treatment response. Our findings indicate that the enrichment of TP53 R248Q in relapsed ALL is due to synergistic mutagenesis from thiopurine treatment and MMR deficiency, followed by selection for TP53 R248Q's chemoresistance phenotype. This suggests that cancer drug resistance mutations may not always pre-exist subclonally at diagnosis, but may be therapy-induced in some patients. Additionally, the qualitative and quantitative mutational signature output of a mutagen (e.g., thiopurines) can vary based on the genetic background. Finally, our findings suggest potential therapeutic strategies, including avoiding thiopurine treatment in MMR-deficient relapses, and therapeutic p53 mutant reactivation, to deal with this genetically-unstable, chemoresistant disease. Citation Format: Fan Yang, Samuel W. Brady, Huiying Sun, Chao Tang, Lijuan Du, Malwine Barz, Xiaotu Ma, Yao Chen, Houshun Fang, Xiaomeng Li, Pandurang Kolekar, Omkar Pathak, Jiaoyang Cai, Lixia Ding, Tianyi Wang, Arend von Stackelberg, Shuhong Shen, Caiwen Duan, Cornelia Eckert, Hongzhuan Chen, Yu Liu, Jeffery M. Klco, Hui Li, Benshang Li, Jinghui Zhang, Renate Kirschner-Schwabe, Bin-Bing S. Zhou. Thiopurines and mismatch repair deficiency cooperate to fuel TP53 mutagenesis and ALL relapse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 633.

Background: Minimal residual disease (MRD) supports a more accurate assessment when children and adolescents with late bone marrow relapses of B-cell precursor (BCP) acute lymphoblastic leukaemia (ALL) will benefit from allogeneic haematopoietic stem cell transplantation (alloHSCT). A more detailed dissection of MRD heterogeneity, dynamics and the specific genetic aberrations involved promises to further improve treatment stratification. Methods: Patients treated according to the ALL-REZ BFM 2002 trial/registry protocol with late BCP ALL bone marrow relapses (n=413) were included. AlloHSCT was indicated for patients with MRD≥10-3 after induction treatment (poor response). Findings: Patients with good (MRD

We compared 24 primary pediatric T‐cell acute lymphoblastic leukemias (T‐ALL) collected at the time of initial diagnosis and relapse from 12 patients and 24 matched patient‐derived xenografts (PDXs). DNA methylation profile was preserved in PDX mice in 97.5% of the promoters (ρ = 0.99). Similarly, the genome‐wide chromatin accessibility (ATAC‐Seq) was preserved remarkably well (ρ = 0.96). Interestingly, both the ATAC regions, which showed a significant decrease in accessibility in PDXs and the regions hypermethylated in PDXs, were associated with immune response, which might reflect the immune deficiency of the mice and potentially the incomplete interaction between murine cytokines and human receptors. The longitudinal approach of this study allowed an observation that samples collected from patients who developed a type 1 relapse (clonal mutations maintained at relapse) preserved their genomic composition; whereas in patients who developed a type 2 relapse (subset of clonal mutations lost at relapse), the preservation of the leukemia's composition was more variable. In sum, this study underlines the remarkable genomic stability, and for the first time documents the preservation of the epigenomic landscape in T‐ALL‐derived PDX models.

Summary Activating mutations in the cytosolic 5′-nucleotidase II gene NT5C2 drive resistance to 6-mercaptopurine in acute lymphoblastic leukemia. Here we demonstrate that constitutively active NT5C2 mutations K359Q and L375F reconfigure the catalytic center for substrate access and catalysis in the absence of allosteric activator. In contrast, most relapse-associated mutations, which involve the arm segment and residues along the surface of the inter-monomeric cavity, disrupt a built-in switch-off mechanism responsible for turning off NT5C2. In addition, we show that the C-terminal acidic tail lost in the Q523X mutation functions to restrain NT5C2 activation. These results uncover dynamic mechanisms of enzyme regulation targeted by chemotherapy resistance-driving NT5C2 mutations, with important implications for the development of NT5C2 inhibitor therapies.

In this Article, author Benedikt Brors was erroneously associated with affiliation number '8' (Department of Developmental Neurobiology, St Jude Children's Research Hospital, Memphis, Tennessee, USA); the author's two other affiliations (affiliations '3' and '7', both at the German Cancer Research Center (DKFZ)) were correct. This has been corrected online.

Key Points Intragenic PAX5 amplification defines a novel, relapse-prone subtype of B-cell precursor acute lymphoblastic leukemia with a poor outcome.

Relapse of T-cell acute lymphoblastic leukemia (T-ALL) has a dismal prognosis, with only 20% of afflicted children surviving.[1][1] Children with relapsed T-ALL are commonly treated within high-risk arms of second-line treatment protocols that include mandatory hematopoietic stem cell

Intragenic PAX5 amplification defines a novel, relapse-prone subtype of B-cell precursor acute lymphoblastic leukemia with a poor outcome.

Acute lymphoblastic leukemia (ALL) is an aggressive hematological tumor resulting from the malignant transformation of lymphoid progenitors. Despite intensive chemotherapy, 20% of pediatric patients and over 50% of adult patients with ALL do not achieve a complete remission or relapse after intensified chemotherapy, making disease relapse and resistance to therapy the most substantial challenge in the treatment of this disease. Using whole-exome sequencing, we identify mutations in the cytosolic 5'-nucleotidase II gene (NT5C2), which encodes a 5'-nucleotidase enzyme that is responsible for the inactivation of nucleoside-analog chemotherapy drugs, in 20/103 (19%) relapse T cell ALLs and 1/35 (3%) relapse B-precursor ALLs. NT5C2 mutant proteins show increased nucleotidase activity in vitro and conferred resistance to chemotherapy with 6-mercaptopurine and 6-thioguanine when expressed in ALL lymphoblasts. These results support a prominent role for activating mutations in NT5C2 and increased nucleoside-analog metabolism in disease progression and chemotherapy resistance in ALL.

Purpose In the clinical management of children with relapsed acute lymphoblastic leukemia (ALL), treatment resistance remains a major challenge. Alterations of the TP53 gene are frequently associated with resistance to chemotherapy, but their significance in relapsed childhood ALL has remained controversial because of small studies. Patients and Methods Therefore, we systematically studied 265 first-relapse patients enrolled in the German Acute Lymphoblastic Leukemia Relapse Berlin-Frankfurt-Mü nster 2002 (ALL-REZ BFM 2002) trial for sequence and copy number alterations of the TP53 gene by using direct sequencing and multiplex ligation-dependent probe amplification. Results We observed copy number and sequence alterations of TP53 in 12.4% (27 of 218) of patients with B-cell precursor ALL and 6.4% (three of 47) of patients with T-cell ALL relapse. Backtracking to initial ALL in 23 matched samples revealed that 54% of all TP53 alterations were gained at relapse. Within B-cell precursor ALL, TP53 alterations were consistently associated with nonresponse to chemotherapy (P < .001) and poor event-free survival (P < .001) and overall survival rates (P = .002). TP53 alterations also had a significant impact on survival within intermediate-risk (S2) and high-risk (S3/S4) relapse patients (P = .007 and P = .019, respectively). This prognostic significance of TP53 alterations was confirmed in multivariate analysis. Besides their clinical impact, TP53 alterations were associated with a higher fraction of leukemic cells in S/G2-M phase of the cell cycle at relapse diagnosis. Conclusion Alterations of the TP53 gene are of particular importance in the relapse stage of childhood ALL, in which they independently predict high risk of treatment failure in a significant number of patients. Therefore, they will aid in future risk assessment of children with ALL relapse.

Minimal residual disease (MRD) quantified after induction treatment of childhood acute lymphoblastic leukemia (ALL) predicts risk of relapse. It has been assumed that early relapses derive from a residual population of leukemic cells, which is still present after induction and that relapsed disease will consequently be more resistant to treatment. To test these hypotheses, we performed a prospective study on patients treated according to the frontline-trial ALL-BFM 2000, which used MRD response for risk-group stratification. Patients (n=45) showed a median time to relapse of 1.5 years. In 89% of patients at least one T-cell-receptor/immunoglobulin gene rearrangement chosen for initial MRD quantification remained stable; however, at least one of the preferred markers for MRD stratification at relapse was different to diagnosis in 50% of patients. A similar proportion of very early, early and late relapses appeared to gain a marker at relapse although backtracking-analysis revealed that in 77% of cases, the gained markers were present as small sub-clones at initial diagnosis. Comparing initial and relapse MRD response to induction, 38% of patients showed a similar, 38% a better and 25% a poorer response after relapse. These data demonstrate an unexpectedly high clonal heterogeneity among very early/early relapses and challenge some current assumptions about relapsed ALL.

We aimed at understanding the relapse-driving processes in pediatric T-ALL and performed an integrated longitudinal multi-level omics analysis of 13 T-ALL patients at initial diagnosis (INI) and relapse (REL). We compared the mutation (SNV/InDels) and copy number alteration (CNA) patterns as well as gene expression, methylation levels and chromatin accessibility by ATAC-Seq. Aberrant expression of T-ALL transcription factors (TAL1, TAL2,LMO2, TLX1, TLX3, NKX2.4 and NKX2.5) was preserved from initial presentation to relapse in all patients. These leukemia-driving events defined the expression patterns, methylation profiles and the chromatin accessibility landscapes. A global differential analysis of the RNA-Seq data (DESeq2, padj We then focused our analysis on the 2 types of relapse in pediatric T-ALL, which we have previously defined on the basis of subclonal mutation profiles (Kunz et al., 2015). These types of relapse are characterized by either clonal evolution of cells derived from the major clone at initial presentation (type 1) or emergence and evolution of a minor initial clone showing a molecular profile that is distinct from the predominant initial clone (type 2). When considering type 1 and type 2 relapses separately we identified a strong trend for type 2 relapses to acquire more mutations (p=0.0879, ttest) than type 1 relapses. Further to the known activating mutations in NT5C2 acquired at relapse by 8/13 patients no other mutations or CNAs were recurrently acquired in the relapses of this group of patients. However, mutations in proto-oncogenes or genes involved in DNA surveillance were acquired by 7/8 type 2 relapse patients in our series. Changes of CNAs also occurred more frequently in type 2 than in type 1 relapses (pval= 0.0267, ttest). This increased complexity on the genetic level was also apparent on the epigenetic level, with an increase of changes in the methylation pattern (mean difference in β value between INI and REL: type 1 - 0.00034; type2 - 0.002 (pval< 0.0001, chi2)), chromatin accessibility (number of differentially accessible ATAC-peaks: type 1 - 4 (0.006%) ; type 2 - 1018 (1.3%); (pval< 0.0001, chi2)) and on the expression level (number of differentially expressed genes: type 1 - 11; type 2 - 111, pval When considering differences between leukemias at the time of initial diagnosis, which later develop either type 1 or type 2 relapses we found 1.016 genes to be differentially expressed (524: up; 492: down in type 1; DE-Seq2: padj In sum, the multilevel omic comparison of pediatric T-ALL that develop either a type 1 or a type 2 relapse show remarkably more complex changes of the genetic and epigenetic profiles during the transition from initial to relapsing disease. Notably, pediatric T-ALLs, who later develop a type 1 relapse display an epigenetic and transcriptomic landscape predicting an upregulation of DNA repair functions, which we suggest to potentially play a role in developing resistance to DNA damaging agents in this type of relapse. Disclosures Muckenthaler: Novartis: Research Funding. Bourquin:Amgen: Other: Travel Support. Kulozik:bluebird bio: Consultancy, Honoraria.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy that is classified according to surface marker expression. In order to reveal the cells of origin in pediatric T-ALL and to understand mechanisms of relapse we used ATAC-Seq (Assay for Transposase-Accessible Chromatin Sequencing) to compare chromatin accessibility landscapes of healthy T-cell precursors to those of T-ALL cells obtained at initial diagnosis (INI) and relapse (REL). We have FACS sorted 7 differentiation stages of normal T-cell precursors contained in the thymus of infants undergoing cardiac surgery (DN2, DN3, ISP, DPCD3-, DPCD3+, CD4+ and CD8+) and subjected these to ATAC-Seq. Unsupervised learning by principal component analysis (PCA) clustered sorted populations according to the maturation stage, demonstrating that regulatory chromatin signatures of thymocytes are highly stage-specific and re-shaped during T-cell differentiation. We next compared normal T-cell precursors at different stages of maturation to pediatric T-ALLs and found fundamental differences with 30% of open chromatin regions to be more and 28% being less accessible in T-ALL (DESeq2, padj We then subjected the ATAC-seq data of all matched leukemia samples obtained at initial disease and at relapse to PCA. INI and REL samples derived from the same patient always clustered in close proximity and were separated according to the T-ALL driving fusion genes. A global analysis of differential accessibility revealed only 0.26% of ATAC-regions to be less- or more-accessible at relapse when compared to the matched initial samples (DESeq2, padj Moreover, we trained the deconvolution algorithm CIBERSORT to recognize particular T-cell differentiation stages using ATAC-profiles of the 7 FACS-sorted healthy T-cell populations. We used regulatory chromatin landscape of non-sorted (total) thymus to assess the accuracy of deconvolution. Comparison of predicted fractions in total thymus to FACS measurements revealed highly accurate identification of the maturation stages (r2 = 0.95). CIBERSORT analysis confirmed that the profiles were largely preserved between INI and REL of each sample pair. Notably, however, while in T-ALLs that later developed into a type 1 relapse only one type of early T-cell progenitor dominated the deconvolution profile, T-ALLs that developed into a type 2 relapse showed heterogeneous profiles with contributions of progenitors at different maturation stages. In sum, these epigenomic analyses revealed that the chromatin landscape of normal T-cell precursors evolves in the course of thymic maturation and that the early maturation stages are the likely origin of T-ALL cells. Remarkably, pediatric T-ALLs that later develop a type 2 relapse consist of subclones with a variable profile of chromatin accessibility that define different stages of maturation. These data indicate that T-ALLs with the propensity to develop a type 2 relapse differ from type 1 in that they originate from early precursors that carry the potential of further development into different stages of maturation before the leukemia becomes apparent with a highly subclonal pattern. Disclosures Muckenthaler: Novartis: Research Funding. Bourquin:Amgen: Other: Travel Support. Kulozik:bluebird bio: Consultancy, Honoraria.

In childhood acute lymphoblastic leukemia (ALL), persistence of leukemic blasts during therapy is of crucial prognostic significance. In the present study, we address molecular and cell biologic features of blasts persisting after 1 week of induction glucocorticoid therapy. Genome-wide gene expression analysis of leukemic samples from precursor B-cell ALL patients (n=18) identified a set of genes differentially expressed in blasts at diagnosis day 0 (d0) and persisting on day 8 (d8). Expression changes indicate a shift towards mature B cells, inhibition of cell cycling and increased expression of adhesion (CD11b/ITGAM) and cytokine (CD119/IFNGR1) receptors. A direct comparison with normal B cells, which are largely therapy resistant, confirmed the differentiation shift at the mRNA (n=10) and protein (n=109) levels. Flow cytometric analysis in independent cohorts of patients confirmed both a decreased proliferative activity (n=13) and the upregulation of CD11b and CD119 (n=29) in d8 blasts. The differentiation shift and low proliferative activity in d8 blasts may account for the persistence of blasts during therapy and affect their sensitivity to further therapeutic treatment. CD11b and CD119 are potential specific markers for d8 blast persistence and detection of minimal residual disease, which warrant further investigation.

Purpose: In childhood acute lymphoblastic leukemia (ALL), ∼25% of patients suffer from relapse. In recurrent disease, despite intensified therapy, overall cure rates of 40% remain unsatisfactory and survival rates are particularly poor in certain subgroups. The probability of long-term survival after relapse is predicted from well-established prognostic factors (i.e., time and site of relapse, immunophenotype, and minimal residual disease). However, the underlying biological determinants of these prognostic factors remain poorly understood. Experimental Design: Aiming at identifying molecular pathways associated with these clinically well-defined prognostic factors, we did gene expression profiling on 60 prospectively collected samples of first relapse patients enrolled on the relapse trial ALL-REZ BFM 2002 of the Berlin-Frankfurt-Münster study group. Results: We show here that patients with very early relapse of ALL are characterized by a distinctive gene expression pattern. We identified a set of 83 genes differentially expressed in very early relapsed ALL compared with late relapsed disease. The vast majority of genes were up-regulated and many were late cell cycle genes with a function in mitosis. In addition, samples from patients with very early relapse showed a significant increase in the percentage of S and G2-M phase cells and this correlated well with the expression level of cell cycle genes. Conclusions: Very early relapse of ALL is characterized by an increased proliferative capacity of leukemic blasts and up-regulated mitotic genes. The latter suggests that novel drugs, targeting late cell cycle proteins, might be beneficial for these patients that typically face a dismal prognosis.

Relapsed precursor T-cell acute lymphoblastic leukemia is characterized by resistance against chemotherapy and is frequently fatal. We aimed at understanding the molecular mechanisms resulting in relapse of T-cell acute lymphoblastic leukemia and analyzed 13 patients at first diagnosis, remission and relapse by whole exome sequencing, targeted ultra-deep sequencing, multiplex ligation dependent probe amplification and DNA methylation array. Compared to primary T-cell acute lymphoblastic leukemia, in relapse the number of single nucleotide variants and small insertions and deletions approximately doubled from 11.5 to 26. Targeted ultra-deep sequencing sensitively detected subclones that were selected for in relapse. The mutational pattern defined two types of relapses. While both are characterized by selection of subclones and acquisition of novel mutations, 'type 1' relapse derives from the primary leukemia whereas 'type 2' relapse originates from a common pre-leukemic ancestor. Relapse-specific changes included activation of the nucleotidase NT5C2 resulting in resistance to chemotherapy and mutations of epigenetic modulators, exemplified by SUZ12, WHSC1 and SMARCA4. While mutations present in primary leukemia and in relapse were enriched for known drivers of leukemia, relapse-specific changes revealed an association with general cancer-promoting mechanisms. This study thus identifies mechanisms that drive progression of pediatric T-cell acute lymphoblastic leukemia to relapse and may explain the characteristic treatment resistance of this condition.

Activating mutations in cytosolic 5'-nucleotidase II (NT5C2) drive resistance to 6-mercaptopurine (6-MP) in relapsed acute lymphoblastic leukemia (ALL). These gain-of function alleles encode proteins with increased nucleotidase activity, yet their specific molecular mechanisms are poorly understood. Allosteric activation of NT5C2 is primarily mediated by a marked conformational change in the helix A region, which reconfigures the catalytic center for substrate access and catalysis. Here we demonstrate that constitutively active NT5C2 mutant proteins (K359Q and L375F) adopt active helix A region structures in the absence of allosteric activator. Moreover, crystallographic, structural modeling, genetic, and functional antibody analyses identify a switch-off mechanism mediated by the dynamic mobilization of Asp407 in the tip region of the arm segment which, guided by positively charged residues located in the surface of a basic inter-monomeric cavity exposed upon allosteric activation, turns off NT5C2 by interacting with and disrupting the helical conformation of helix A. Loss of this regulatory mechanism in NT5C2 mutants involving the tip region of the arm segment and the inter-monomeric cavity drives increased nucleotidase activity and allosteric response. Finally, increased allosteric activation by the Q523X mutation results from loss of the NT5C2 C-terminal acidic tail segment, which promotes a closed inactive state in basal conditions. These results uncover dynamic mechanisms responsible for NT5C2 regulation targeted by chemotherapy resistance-driving NT5C2 mutations, with important implications for the development of NT5C2 inhibitor therapies. This abstract is also being presented as Poster 05. Citation Format: Chelsea L. Dieck, Gannie Tzoneva, Farhad Forouhar, Zachary Carpenter, Marta Sanchez-Martin, Alberto Ambesi-Impiombato, Renate Kirschner-Schwabe, Scott Lew, Jayaraman Seetharaman, Liang Tong, Adolfo Ferrando. Mechanisms of NT5C2 activating mutations driving thiopurine resistance in relapsed lymphoblastic leukemia [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr PR02.

For most children who relapse with acute lymphoblastic leukemia (ALL), the prognosis is poor, and there is a need for novel therapies to improve outcome. We screened samples from children with B-lineage ALL entered into the ALL-REZ BFM 2002 clinical trial (www.clinicaltrials.gov, #NCT00114348) for somatic mutations activating the Ras pathway (KRAS, NRAS, FLT3, and PTPN11) and showed mutation to be highly prevalent (76 from 206). Clinically, they were associated with high-risk features including early relapse, central nervous system (CNS) involvement, and specifically for NRAS/KRAS mutations, chemoresistance. KRAS mutations were associated with a reduced overall survival. Mutation screening of the matched diagnostic samples found many to be wild type (WT); however, by using more sensitive allelic-specific assays, low-level mutated subpopulations were found in many cases, suggesting that they survived up-front therapy and subsequently emerged at relapse. Preclinical evaluation of the mitogen-activated protein kinase kinase 1/2 inhibitor selumetinib (AZD6244, ARRY-142886) showed significant differential sensitivity in Ras pathway-mutated ALL compared with WT cells both in vitro and in an orthotopic xenograft model engrafted with primary ALL; in the latter, reduced RAS-mutated CNS leukemia. Given these data, clinical evaluation of selumetinib may be warranted for Ras pathway-mutated relapsed ALL.

Acute lymphoblastic leukemia (ALL) accounts for ∼25% of pediatric malignancies. Of interest, the incidence of ALL is observed ∼20% higher in males relative to females. The mechanism behind the phenomenon of sex-specific differences is presently not understood. Employing genome-wide genetic aberration screening in 19 ALL samples, one of the most recurrent lesions identified was monoallelic deletion of the 5' region of SLX4IP. We characterized this deletion by conventional molecular genetic techniques and analyzed its interrelationships with biological and clinical characteristics using specimens and data from 993 pediatric patients enrolled into trial AIEOP-BFM ALL 2000. Deletion of SLX4IP was detected in ∼30% of patients. Breakpoints within SLX4IP were defined to recurrent positions and revealed junctions with typical characteristics of illegitimate V(D)J-mediated recombination. In initial and validation analyses, SLX4IP deletions were significantly associated with male gender and ETV6/RUNX1-rearranged ALL (both overall P < 0.0001). For mechanistic validation, a second recurrent deletion affecting TAL1 and caused by the same molecular mechanism was analyzed in 1149 T-cell ALL patients. Validating a differential role by sex of illegitimate V(D)J-mediated recombination at the TAL1 locus, 128 out of 1149 T-cell ALL samples bore a deletion and males were significantly more often affected (P = 0.002). The repeatedly detected association of SLX4IP deletion with male sex and the extension of the sex bias to deletion of the TAL1 locus suggest that differential illegitimate V(D)J-mediated recombination events at specific loci may contribute to the consistent observation of higher incidence rates of childhood ALL in boys compared with girls.

In the search for genes that define critical steps of relapse in pediatric T-cell acute lymphoblastic leukemia (T-ALL) and can serve as prognostic markers, we performed targeted sequencing of 313 leukemia-related genes in 214 patients: 67 samples collected at the time of relapse and 147 at initial diagnosis. As relapse-specific genetic events, we identified activating mutations in NT5C2 (P=0.0001, Fisher’s exact test), inactivation of TP53 (P=0.0007, Fisher’s exact test) and duplication of chr17:q11.2-24.3 (P=0.0068, Fisher’s exact test) in 32/67 of T-ALL relapse samples. Alterations of TP53 were frequently homozygous events, which significantly correlated with higher rates of copy number alterations in other genes compared with wild-type TP53 (P=0.0004, Mann–Whitney’s test). We subsequently focused on mutations with prognostic impact and identified genes governing DNA integrity (TP53, n=8; USP7, n=4; MSH6, n=4), having key roles in the RAS signaling pathway (KRAS, NRAS, n=8), as well as IL7R (n=4) and CNOT3 (n=4) to be exclusively mutated in fatal relapses. These markers recognize 24/49 patients with a second event. In 17 of these patients with mostly refractory relapse and dire need for efficient treatment, we identified candidate targets for personalized therapy with p53 reactivating compounds, MEK inhibitors or JAK/STAT-inhibitors that may be incorporated in future treatment strategies.

Acute Lymphoblastic Leukemia (ALL) is the most common malignancy in children. Altogether 90% of pediatric ALL patients achieve a complete hematologic remission with current high dose combination chemotherapy and 80% of them remain leukemia free. However, the outcome for patients showing refractory disease or those whose leukemia relapses after an initial transient response remains disappointingly poor with cure rates of less than 40%. To investigate genetic drivers of relapse and resistance and explore the specific roles of clonal evolution in disease progression and relapse here we performed whole-exome sequence analysis of matched diagnosis, germline (remission) and relapse DNA samples in a panel of 55 pediatric ALL patients including 33 T-cell ALLs and 22 B-cell precursor ALLs. These analyses identified an average of 9 mutations present in diagnostic samples and 17 mutations in relapsed leukemia DNAs. Phylogenetic tree analysis for each of the 48 cases with optimal variant call parameters analyzing their clonal evolution dynamics during disease progression, combined with whole genome sequencing of targeted samples with low exonic mutation input, showed that branched evolution in which relapse clones contain some, but not all genetic lesions present in the major clone at diagnosis as the primary mechanism driving tumor progression and relapse present in 45/48 (94%) cases. In addition, and consistent with previous reports we identified the presence of chemotherapy associated mutations in NT5C2 (10/55), TP53 (3/55), CREBBP (4/55) and the NR3C1 glucocorticoid receptor gene (2/55). However, and most strikingly, 23/27 (85%) recurrently mutated genes in this series with mutations preferentially selected or retained at the time of relapse (mutation never lost in the relapse clone) were not implicated in relapse ALL before (HTR3A, MED12, USP9X, CACNA1H, ODZ3, AACS, SAMD4A, ANO5, PAPPA, NAALADL2, HIST3H2A, FZD7, TBX15, NEB, GREB1L, PLXNA4, SGK223, TSC1, PTPRG, FGF10, SYCP2, TRPM3 and EYS). A branched pattern of genetic evolution and the presence of recurrent mutations selected at relapse support that chemotherapy imposes a strong Darwinian genetic selection in leukemic cell populations. In this context it is worth noting that RAS-MAPK pathway activating mutations in NRAS, KRAS and PTPN11 were present in 24/55 (44%) cases in our series. Interestingly, some leukemias showed retention or emergence of RAS mutant clones at relapse, while in others, RAS mutant clones present at diagnosis were replaced by RAS wild type populations, supporting a role for both positive and negative selection evolutionary pressures in clonal evolution of RAS-mutant leukemia. Most notably, and in agreement with this hypothesis, inducible expression of mutant KRAS in human ALL lines demonstrate that oncogenic KRAS G12D induces methotrexate resistance, but also improves leukemia response to vincristine; a phenotype perfectly recapitulated in a isogenic ALL leukemia model generated from a conditional inducible Kras G12D knockin mice. Mechanistically, KRAS G12 expression induces MAPK dependent abrogation of methotrexate induced apoptosis. Moreover, Kras mutant tumors show enhanced G2/M cell cycle arrest and apoptosis upon spindle poisoning with vincristine, a phenotype linked with increased PLK phosphorylation and transcriptional down-regulation of mitotic genes. Finally clonal competition assays demonstrate that the differential response to methotrexate and vincristine in isogenic Kras wild type and Kras mutant ALL cells results in clonal dominance of Kras G12D populations in cultures treated with methotrexate, while Kras wild type cells are selected the context of vincristine treatment. In all these results show novel insight on the genetics and mechanisms of clonal selection, disease progression and relapse in ALL and demonstrate a previously unrecognized dual role of RAS mutations in chemotherapy response. Disclosures Loh: Abbvie: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.