NT5C2 Mutations Research Articles

Mechanisms of NT5C2 mutations Driving Thiopurine Resistance in Acute Lymphoblastic Leukemia

▪Relapse acute lymphoblastic leukemia (ALL) is associated with chemotherapy resistance and poor prognosis. Persistent leukemia initiating cells with increased self-renewal capacity, clonal heterogeneity and selection of resistance-driving genetic alterations have been proposed as drivers of leukemia relapse. Gain of function mutations in the 5'-Nucleotidase, Cytosolic II (NT5C2) gene are selectively present in relapsed ALL and confer resistance to chemotherapy with 6-mercaptopurine (6-MP). Yet, the specific mechanisms mediating constitutive activation of NT5C2 remain unknown. Here we performed detailed genetic, enzymatic, structural and functional analyses of relapsed leukemia-associated NT5C2 mutant alleles uncovering previously unrecognized functional heterogeneity in their mechanisms of action. Specifically, detailed nucleotidase assays in the presence of increased concentration of allosteric activators across 10 relapse-associated NT5C2 mutant proteins and analyses of the crystal structures of 7 distinct mutant proteins and wild type NT5C2 in the presence and absence of allosteric factors defined three classes of NT5C2 mutants with distinct mechanisms of action. Class I NT5C2 mutations force an active configuration of the NT5C2 catalytic center in the absence of allosteric activator. In contrast, class II NT5C2 mutations disrupt a novel switch-off mechanism mediated by the dynamic mobilization of the tip region of the arm segment responsible for returning the enzyme to its basal inactive configuration following allosteric activation. Finally, increased allosteric response in the class III p.Gln523* truncating mutation results from loss of the C-terminal acidic tail of NT5C2, which functions as a lock stabilizing the enzyme in its inactive basal configuration. The proposed role of the arm domain region and the C-terminal segment were verified in enzymatic assays incubating wild type NT5C2 with antibodies recognizing these regulatory elements, which phenocopied the increased enzymatic activity and response to allosteric effector of NT5C2 mutations involving these domains. In all, these results identify distinct molecular mechanisms mediating NT5C2 regulation targeted by activating NT5C2 mutant alleles and serve as a framework for the functional annotation of clinically-relevant NT5C2 mutations in relapsed ALL. Finally, the prominent role of the allosteric binding site as critical mediator of the mechanisms mediating thhe increased nucleotidase activity of NT5C2 mutant proteins point to this region as a preferred target for the design of NT5C2 inhibitors for the reversal of chemotherapy resistance in relapsed ALL.Figure 1 Relapse associated NT5C2 mutations and crystal structure of basal and activated wild type human NT5C2. (a) Graphical representation of relapse associated mutations in B-ALL (black) and T-ALL (red).(b-d) In vitro nucleotidase assays assessing the enzymatic activity of (b)class I, (c) class II, and (d)class IIINT5C2 mutations in the presence of increased concentrations of ATP. (e) A Ribbon diagram of the basal structure of wild type NT5C2-X537, displaying the HAD domain (cyan) and its three extensions: N-terminal segment (orange), arm (pink), and C-terminal segment (marine). The mutation sites are depicted as red solid spheres for Cα of each mutated amino acid. The phosphate (yellow for phosphorus) and Mg ions (dark green) are depicted as stick models and solid spheres respectively. (f) A Ribbon diagram of the activated structure of the full-length wild type NT5C2, in which the allosteric helix A (αA) is shown in dark purple. (g,h) Ribbon and surface (for subunit B) depictions of basal and activated dimers of (g) NT5C2-X537 and the (h) full-length, respectively. (i,j) Ribbon diagrams of the basal (i) NT5C2-X537 and activated (j) full-length wild type tetramers, respectively. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Novel homozygous missense mutation in NT5C2 underlying hereditary spastic paraplegia SPG45.

SPG45 is a rare form of autosomal recessive spastic paraplegia associated with mental retardation. Detailed phenotyping and mutation analysis was undertaken in three individuals with SPG45 from a consanguineous family of Arab Muslim origin. Using whole-exome sequencing, we identified a novel homozygous missense mutation in NT5C2 (c.1379T>C; p.Leu460Pro). Our data expand the molecular basis of SPG45, adding the first missense mutation to the current database of nonsense, frameshift, and splice site mutations. NT5C2 mutations seem to have a broad clinical spectrum and should be sought in patients manifesting either as uncomplicated or complicated HSP.

Abstract 4870: Mutational landscape and timing of resistant clone emergence in 104 Chinese pediatric patients with relapsed acute lymphoblastic leukemia

Abstract Relapsed pediatric acute lympoblastic leukemia (ALL), occurring in 15-20% of cases, is one of the leading causes of cancer-related death in children, conferring a dismal survival rate of less than 40% primarily due to drug resistance. We performed whole-genome sequencing (WGS) and RNA-seq of matched diagnosis and relapse tumors as well as WGS of remission DNA from 104 pediatric ALL patients treated at Shanghai Children’s Medical Center (n=89), Institute of Hematology & Blood Disease Hospital in Tianjin (n=14) and the Second Hospital of Anhui Medical University (n=1) between 2003 and 2014. The median age at diagnosis was 6.29 (range, 0.23-16.05) years. Relapse occurred during chemotherapy in 85 patients and after completion of therapy in 19 patients. Somatic mutation rate in ALL increased significantly from an average of 0.23 per Mb at diagnosis to 0.65 per Mb at relapse. We identified 34 potential driver genes at diagnosis and 45 at relapse based on significance of mutation recurrence and pathogenicity. The four most significant relapse-specific driver genes harbor known point mutations on purine metabolism genes NT5C2 (n=13) and PRPS1/PRPS2 (n=7), novel alterations affecting glucocorticoids receptor NR3C1 (n=13) and folylpolyglutamate synthetase FPGS (n=8). Functional characterization showed that all NR3C1 mutations resulted in significant loss of transcription activator function and subcellular mislocalization. Ectopic expression of mutant NR3C1 in ALL cell lines consistently led to prednisone resistance. Five driver genes (TP53, CREBBP, SMARCA4, WHSC1 and NIPBL) showed the highest increase (2-5 fold) of mutation frequency from diagnosis to relapse with mutations collectively present in 28% of relapsed ALL. Truncation mutations in DNA mismatch repair genes MLH1, MSH2, MSH6 and PMS2 occurred exclusively at relapse in four ALL with the highest mutation burden. Targeted ultra-deep sequencing at 5,000X of 268 somatic alterations was performed on 214 serial bone-marrow samples collected during ALL therapy for 17 cases. The resulting mutant allele fraction (MAF) values showed high concordance with minimum residual disease (MRD) measured by flow cytometry, indicating suitability for quantifying leukemia burden. Temporal change of MAF in the serial samples also allows us to track clonal identity and emergence of drug-resistant clones during and after therapy. For example, one case had deep sequencing performed on 11 somatic lesions from 10 bone barrow samples collected during the 15 months between diagnosis and relapse. We found that a minor clone present in 0.5-1% of ALL blasts at diagnosis emerged five months prior to relapse, from which two new subclones arose harboring NT5C2 mutations L406>SSF and D407>DRD. Preliminary data generated from serial bone marrow samples provides the first insight into the use of somatic alterations for monitoring leukemia progress during relapse. Citation Format: Benshang Li, Yongjin Li, Shuhong Shen, Xiaofan Zhu, Xiaotu Ma, Ningling Wang, Yanling Liu, Yu Liu, Hui Zhang, Ting-Nien Lin, Michael Rusch, Michael Edmonson, John Easton, Yingchi Zhang, Jingliao Zhang, Cheng Cheng, Jingyan Tang, James R. Downing, Ching-Hon Pui, Jun J. Yang, Jinghui Zhang. Mutational landscape and timing of resistant clone emergence in 104 Chinese pediatric patients with relapsed acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4870. doi:10.1158/1538-7445.AM2017-4870

Mutational Landscape of Pediatric Acute Lymphoblastic Leukemia Relapsing after Allogeneic Stem Cell Transplantation

The prognosis of children with acute lymphoblastic leukemia (ALL) relapsing following allogeneic hematopoietic stem cell transplantation (allo-SCT) is still dismal. Within the framework of the ALL-REZ BFM 2002/ALL-SZT BFM 2003 trials, we performed whole-exome sequencing (WES) of ten patients relapsing after allo-SCT with the aim to thoroughly characterize the spectrum of acquired mutations and to identify potentially druggable targets, thus laying the ground for future personalized treatment strategies.Patients’ age at initial diagnosis ranged from <12 months to 10 years. Two patients were diagnosed with T-ALL and 8 with pre-B-ALL. Time from initial disease to relapse ranged from 13 to 40 months. Patients underwent allo-SCT 3 to 6 months after diagnosis of relapse, donors were either HLA-matched unrelated volunteers (n=6), HLA-identical siblings (n=1) or HLA-haploidentical family donors (n=1). Time from SCT to relapse ranged from 3 to 21 months. Treatment following the post allo-SCT relapse varied considerably (from palliative care to a second allo-SCT). Only 2/10 patients are still alive, with one recently having been diagnosed with subsequent relapse following a second SCT.To investigate the mutational landscape of relapsed ALL following high-dose chemotherapeutic and immunologic attack both provided by an allo-SCT, five samples per patient were analyzed: initial leukemia (INIT), remission (REMI) after front-line chemotherapy representing patient germline, first relapse (RLPS), full donor-chimeric remission post allo-SCT (TREMI) representing donor germline, and relapse post allo-SCT (TRLPS). For comparative analyses, we defined the following three "oncogenomes" (OGs): OG1 (initial leukemia, SNVs in INIT minus REMI)), OG2 (first relapse, SNVs in RLPS minus REMI), OG3 (post allo-SCT relapse, SNVs in TRPLS minus REMI or TREMI).Median numbers of leukemia-specific SNVs in OG1-3 were 8.5, 34 and 37.5. We detected a median of 0.18 mutations per megabase (MB) in OG1, 0.67 mutations/MB in OG2 and 0.74 mutations/MB in OG3 (p= 0.005 for OG2/3 vs. OG1 [Wilcoxon test]).Although the mutational spectrum was highly diverse between individual patients and within the oncogenomes, we also identified several recurrent alterations: in OG1, five genes had recurrent SNVs (IGSF3, TTN, NOTCH1, CTBP2, NRAS). Seven genes were recurrently affected in three OG2s, including IKZF1, NOTCH1, NRAS, NT5C2. In OG3 we detected eleven recurrently mutated genes in three patients, among which were NRAS, FLT4, and TP53.Notably, TP53 was mutated in 5/10 patients. One patient carried a TP53 germline mutation, one patient had one unique SNV each in OG2 as well as OG3 and three patients had TP53 mutations only present in their OG3s. All but one SNV (resulting in a premature stop codon) were non-synonymously coding and were predicted to be deleterious for protein function.Of particular note, leukemic blasts showed profound plasticity concerning the mutational status of the nucleoside exporter NT5C2, mutations of which were previously shown to drive chemotherapy resistance in relapsed childhood ALL. While NT5C2 alterations were completely absent in OG1, 4 SNVs were detected in the OG2s of 3/10 patients. However, these NT5C2 mutations disappeared again in the OG3s once selection pressure of maintenance chemotherapy employing nucleoside analogues had been withdrawn.To identify novel treatment options in the desperate clinical scenario of post allo-SCT relapse, we searched OG3 for genetic lesions in genes known to be either targeted directly (a certain gene) or indirectly (a certain pathway) by currently approved therapeutic agents. To our surprise, nine out of ten patients exhibited such SNVs, for which additional targeted therapies are already available. Those therapeutic agents comprise small molecules inhibitors as well as antibodies such as Dasatinib, Erlotinib, Ibrutinib, Pazopanib, Tocilizumab, and Trastuzumab.Conclusion:Our comprehensive genetic analysis of ten children with ALL relapsing post allo-SCT revealed profound leukemic cell plasticity (e.g. loss of acquired NT5C2 mutations) as well as identified several recurrent genetic alterations (e.g. NOTCH1, NRAS, IKZF1) including TP53 (5/10 patients). Most importantly, we identified alterations in genes amenable to targeted treatment approaches in 9/10 patients thus potentially opening new therapeutic avenues. DisclosuresBader:Novartis: Consultancy, Honoraria; Servier: Consultancy, Honoraria; Neovii Biotech: Research Funding; Riemser: Research Funding; Medac: Consultancy, Research Funding. Peters:Novartis: Consultancy; Jazz: Speakers Bureau; Amgen: Consultancy; Pfizer: Consultancy; Medac: Consultancy.

Identification of an Ultra High-Risk and Targetable Molecular Signature in Relapsed Pediatric T-ALL

▪Relapsed T-cell acute lymphoblastic leukemia (T-ALL) represents one of the major challenges of pediatric oncology as it is often resistant to treatment and fatal. In the search for genes that define critical steps of relapse and can serve as prognostic markers we compared 67 relapses (REL) and 147 samples collected at initial diagnosis (INI) by targeted sequencing of 313 leukemia-related genes. In addition to the analysis of single nucleotide variants (SNV) and small insertions and deletions (InDels), we made use of the available coverage data for profiling of copy number alterations (CNA).Of the 147 INI patients, 31 were treated according to the ALL-BFM 2000 and 116 according to the AIEOP-BFM ALL 2009 protocol. All REL patients were recruited from the ALL-REZ BFM 2002 trial. We analyzed bone marrow DNA by targeted capture of 313 genes (5964 exons) using the Haloplex Target Enrichment Kit (Agilent). We used Varscan to detect both SNV and InDels. In the absence of available remission samples, we subtracted known SNPs (dbSNP, 1000 gp) and variants present in at least one of 20 non-leukemic samples that we sequenced in parallel to the patients’ samples. Only mutations with an AF >10% were considered. Copy number analysis based on read-depth data was validated by MLPA analysis of 14 genes showing a sensitivity of 99% and by low coverage WGS. In total, we have SNV data on all 147 INI and 67 REL patients and CNA data on 144 and 58 patients, respectively.Altogether, we identified relapse specific genetic events in 32 of 67 RELs. Our results confirm that NT5C2 mutations are highly enriched in relapse (REL: 17/67 vs. INI: 1/147; p=0.0001). Although activation of NT5C2 was associated with the occurrence of early first relapse (p=0.02), it did not correlate with induction failure and therefore has no prognostic impact. Similarly, amplifications of chromosome 17 q11.2-24.3, a region that contains genes of the STAT and ABCA families were significantly more frequent in REL than in INI (REL 7/58 vs. INI 3/144, p=0.0068), but also had no prognostic implications. By contrast, TP53 mutations were highly predictive of a second event: all 8 patients who carried a total of 9 TP53 mutations and deletions died within 9 months after first relapse (p=0.002), whereas 17 of the other 58 (29%) survived. Inactivation of TP53 was significantly correlated with higher mutation rates in other genes compared to those with wild-type TP53 (ttest= p<0.0001). TP53 alterations combined with other recurrent mutations in genes responsible for surveillance of DNA integrity (MSH6 and USP7) recognize 15 of the 49 patients with a fatal post-relapse outcome. This group appeared to be particularly resistant to induction therapy as only 2/14 (no data available for 1 pt) patients compared to 17/52 without TP53/MSH6/USP7 mutations achieved second remission and could receive stem cell transplantation (p=0.0006). Activating mutations of KRAS or NRAS thus activating the Ras/Raf/MEK/ERK pathway were found in 8 of these 49 REL patients, 6 of whom died within 3 months, indicating that these mutations cause treatment resistance (p<0.05). Further, likely inactivating mutations of CNOT3 and known activating insertions of IL7R have been exclusively found in 7 REL patients who failed induction. Four of these 7 RELs also carried TP53 or RAS mutations. This combination of mutations predicts an exquisitely poor outcome and results in an ultra-high risk prognostic signature that identifies 24/49 REL patients, who failed induction treatment and died.In conclusion, targeted sequencing of relapsed T-ALL identified a molecular signature predicting an exquisitely poor outcome in 50% of relapses, who failed salvage treatment. This group of patients does not benefit from current treatment strategies thus identifying a subgroup with a dire clinical need for experimental therapy. Notably, approx. 25% REL patients who failed induction carried RAS and IL7R mutations. These patients may be considered for personalized treatment with either MEK- or JAK/STAT-inhibitors. Another 4 patients carry TP53 missense mutations and may benefit from treatment with p53 refolding compounds. DisclosuresNo relevant conflicts of interest to declare.

Mutational Profiling of Acute Lymphoblastic Leukemia with Testicular Relapse

Relapse acute lymphoblastic leukemia (ALL) is the leading cause of childhood cancer deaths. Although relapse usually occurs in the bone marrow (medullary), extramedullary relapse occasionally occurs. Currently, the clonal origin and evolution of extramedullary relapse remain elusive. We selected two pediatric B-ALL patients who experienced testicular ALL relapse and interrogated their leukemic cells (diagnosis, remission, bone marrow relapse and testicular relapse) with whole exome sequencing.Case D483 (5.6 years old at diagnosis of ALL) developed bone marrow and testicular relapse 5 years after diagnosis of B-ALL. At diagnosis he was treated as an intermediate risk with hyperdiploid-ALL with the absence of any well-known ALL fusion-oncogene. Mutations of KRAS (G12D) and CREBBP (S1436C) were found in the founding leukemic clone at diagnosis and persisted in the bone marrow and testis at relapse). Mutation of CREBBP has been frequently found in ALL (particularly in hyperdiploid subtype) and is correlated with increased incidence of relapsed ALL. A MEF2B mutation (R17Q) was found in the bone marrow and testicular relapse sample. Missense mutation of this gene is frequently found in diffuse large B cell lymphoma (DLBCL); this protein regulates the expression of the proto-oncogene BCL6 and contributes to malignant transformation. Second child, case D727 (1.3 years old at diagnosis) harbored a MLL-AF9 fusion and was assigned as a high risk-ALL at diagnosis. Two NT5C2 mutations occurred at relapse, being present at different VAF in bone marrow and testicle: missense mutation R367Q was present with a VAF of 33.5% in bone marrow and 4.5% in testicle; while D407V was present with a VAF of 6.5% in bone marrow and 35.5% in the testicular relapse. NT5C2 encodes a 5'-nucleotidase involved in purine metabolism. The missense mutations (R367Q and D407V) identified here, have been reported as recurrent mutational hotspots of NT5C2 in relapse ALL and have been functionally validated. These mutations increase the 5'-IMP nucleotidase activity of NT5C2 protein leading to resistance to 6-mercaptopurine, a drug that was a component of the treatment regime of this patient.To understand the evolutionary trajectories of these two ALL cases, we analyzed clonal evolution based on their sequencing data. In patient D483, the relapse leukemia was directly evolved from the diagnosis leukemia clone: all of the mutations at diagnosis were persisted at relapse, and four mutated genes (MEF2B, KCNG1, AIM1, OTUD5) were acquired at both bone marrow and testicular relapse with different variant allele frequency (VAF). In patient D727, however, a faction of mutations present at diagnosis were subsequently lost at relapse, suggesting that relapsed leukemia arose from an ancestral subclone that developed before the overt leukemia at diagnosis. The mutational pattern and VAF cluster analysis results suggest that relapse in the patients' testicle represents an independently subclones from the relapse in their bone marrows. Taken together, our sequencing results suggest that relapse of patient D483 was directly evolved from the diagnosis leukemic clone; while the relapse leukemia cells (both bone marrow and testicle) of patient D727 was likely derived from a common ancestral clone, and the testicular relapse arose independently from the bone marrow relapse leukemia. DisclosuresLill:Sanofi: Speakers Bureau; California Cord Blood Services: Consultancy; Kite: Research Funding.

NT5C2 As a Major Contributor to Thiopurine Resistance at ALL Relapse Via Multiple Mechanisms

Background Relapse remains as a formidable challenge for acute lymphoblastic leukemia (ALL), particularly because it is associated with dramatic drug resistance and thus dismal prognosis. It is crucial to understand the genetic basis of relapsed ALL to develop new therapeutic strategies. Recently, somatic mutations in NT5C2 were identified as one of the most common genomic lesions specific to relapsed ALL and were directly linked to thiopurine resistance (Nat Genet 2013, Nat Med 2013, Nat Commun 2015). A nucleotidase, NT5C2 dephosphorylates purine monophosphate nucleotides and is important for cellular nucleic acid homeostasis. However, the exact mechanisms by which NT5C2 mutations influence thiopurine resistance in ALL are not completely understood.Methods Wildtype and mutant human NT5C2 (p.R39Q, p.R238W, and p.R367Q) were expressed in E. Coli and purified by affinity chromatography followed by gel filtration. Nucleotidase activity was measured by quantifying the release of free phosphate with the Malachite Green assay. NT5C2 enzyme kinetics (Vmax and Km) were determined for endogenous purine nucleotide substrates (inosine monophosphate [IMP] and guanosine monophosphate [GMP]) and thiopurine drug metabolite substrates (thioinosine monophosphate [TIMP] and thioguanosine monophosphate [TGMP]). Human ALL cell lines (Nalm6 and REH) stably overexpressing wildtype or mutant NT5C2 were established by lentiviral transduction. Thiopurine transport was characterized in vitro by quantifying the influx and efflux of 14 C labeled mercaptopurine (MP) and its metabolites.Results To characterize NT5C2 substrate specificity, we first determined the nucleotidase activity of wildtype and mutant proteins with endogenous purine nucleotides (IMP and GMP) and thiopurine metabolites (TIMP and TGMP). Wildtype NT5C2 exhibited nucleotidase activity against all 4 nucleotide metabolites, but IMP and TIMP were clearly the preferred substrates with up to 4-fold higher reaction velocities compared to GMP and TGMP, respectively. All leukemia-derived NT5C2 mutations resulted in substantial increase in nucleotidase activity across substrates, with the p.R367Q mutant consistently exhibiting the greatest gain of activity. By comparison, mutant NT5C2 proteins showed similar substrate preference of IMP over GMP (e.g., reaction rate of 26.6 pmol/min and 10.4 pmol/min for IMP and GMP, respectively, with the p.R367Q NT5C2). However, this inosine over guanosine bias in substrate specificity was reversed with thiopurine metabolites: leukemia-derived mutant NT5C2s were substantially more efficient to dephosphorylate TGMP than TIMP (e.g., reaction rate of 27.8 pmol/min and 41.5 pmol/min for TIMP and TGMP, respectively, with the p.R367Q NT5C2). The "mutant to wildtype" ratio of NT5C2 nucleotidase activity (e.g., the relative increase of Vmax) was significantly greater for thiopurine metabolites than what was observed when endogenous purine nucleotides were used as substrates (e.g., 15.2- and 1.6-fold increase in Vmax for TIMP and IMP with the p.R367Q NT5C2). These results suggest that ALL relapse-associated NT5C2 mutations likely confer not only increase in endogenous nucleotidase activity but also novel enzymatic functions that specially affect thiopurine metabolism.In addition to the increase in thiopurine resistance, human ALL cell lines expressing mutant NT5C2 also showed significant reduction in intracellular levels of thiopurine metabolites after drug treatment, pointing to influence of NT5C2 mutations on thiopurine drug transport. Using 14 C labeled MP, we traced both the influx and efflux of drug metabolites in isogenic cell lines expressing wildtype or mutant NT5C2 s. In both Nalm6 and REH cells, intracellular uptake of MP was reduced by 51% in mutant cells compared to cells with wildtype protein. In contrast, the level of MP metabolites in the extracellular medium was 3 times higher with mutant cells, reflecting increased drug efflux. Therefore, expression of mutant NT5C2 resulted in a net reduction of intracellular exposure to thiopurine, suggesting disturbance of the drug transport pathway as a distinct mechanism for NT5C2-mediated thiopurine resistance.ConclusionsNT5C2 mutations specifically influence thiopurine metabolism and transport, and therefore are critical determinants for drug resistance in relapsed ALL. DisclosuresNo relevant conflicts of interest to declare.

Genomic Landscape of Relapsed Acute Lymphoblastic Leukemia

Introduction: Despite risk stratification according to presenting clinical and genetic features, 10-25% of children with acute lymphoblastic leukemia (ALL) relapse, which is associated with a poor prognosis. Here, we sought to provide a comprehensive overview of the genetic alterations associated with relapse in ALL.Methods: We studied 93 children (27 female, 43 male) diagnosed with ALL (62 B-progenitor, 25 T-lineage) between 1987 and 2008 and treated on total therapy studies XI-XVI who experienced relapse and/or a second tumor. Age at diagnosis ranged from 3 months to 18 years. Median time to relapse was 3 years (range 3 months to 10 years). Seventy patients had a single relapse, 15 cases had 2 relapses, and 8 cases developed a second tumor of different lineage (B-cell lymphoma, chronic myeloid leukemia (n=1 each) and acute myeloid leukemia (n=6)). Diagnosis, relapse and matched normal samples (n=299) were studied using Affymetrix SNP 6.0 microarrays and whole genome or whole exome sequencing.Results: We found 2692 copy number aberrations (CNAs) with a median of 9 (range 0-109) in the diagnosis samples (n=91) compared to a median of 10 (range 0-112) in the relapse samples (n=89) and 12 (range 0-70) in subsequent samples (n=20). The number of CNAs did not differ significantly between diagnosis, relapse or subsequent samples.We identified a 7286 non-silent single nucleotide variants (SNVs) and small insertions or deletions (indels) in 5002 genes, 1392 of which were recurrent. The median number of variants was 12 (range 0-70) at diagnosis (n=91), 21 (range 0-858) at relapse (n=91; P=0.0029 v. diagnosis) and 60 (range 10-650) in subsequent samples (n=20; P<0.0001 v. diagnosis). A subset of samples revealed very high (variant range 658-1703; 7 cases, 9 samples: all relapse or subsequent samples), or high mutation burden (variant range 104-290; 12 cases, 16 samples: 2 diagnosis, 10 relapse, and 4 subsequent samples). Genes encoding mediators of DNA repair were affected in all cases with very high mutation burden, compared to 7 of the high burden cases and 18 out of 72 other cases (most commonly genes TP53, MSH2, and MUTYH; P<0.0001).The most frequently mutated genes were NOTCH1 (n=33), NRAS (n=24), CREBBP (n=20) and KRAS (n=16). Of the recurrently altered genes, only 87 genes were known to be affected in cancer (Cancer Gene Census, COSMIC database), of which 59 were affected in leukemia and lymphoma tissues, indicating that we have identified 1306 novel recurrently affected genes, most commonly C13orf40 and MKI67. Mutations in epigenetic regulators were particularly frequent, with genes mutated in at least 3 cases altered in over 60% of the cohort (e.g. CREBBP, EP300, MLL2, MLL3, KDM6A/B, CTCF, SETD2, TET2/3, and EZH2).Clonal evolution analyses showed multiple patterns of evolution, with relapses sharing either few or many variants with the diagnosis sample in a frequency that reflects both predominant clones and minor subclones propagating relapse. Variants in NOTCH1, NRAS, and CREBBP were preserved from a major clone at diagnosis in 4, 6, and 5 cases respectively, but acquired at relapse or grown out from a minor subclone at diagnosis in 3, 5, and 8 cases respectively. In contrast, variants in USH2A (n=4), FOXA1 (n=3), and purine/pyrimidine synthesis pathway genes NT5C2 (n=3), PRPS1 (n=3) and NT5C1B (n=1) were exclusively found in relapse samples. Notably, the NT5C2 mutations, which are thought to confer resistance to thiopurines, were subclonal at relapse in the majority of cases.We identified 13 cases (10 B-lineage, 3 T-lineage) in which the diagnosis and relapse were fully discordant for all CNAs and sequence mutations, only 4 of which showed a prolonged remission time (>5 years). This suggests that these patients developed a second primary malignancy and may be predisposed to leukemia development. Indeed, one case revealed focal amplifications on chromosome 1q21.1 encompassing the neuroblastoma breakpoint family genes, which are implicated in cancer development. Comprehensive germline analyses are underway.Conclusion: This study has provided detailed insight into the genetic basis of relapse, implicating multiple new genes and pathways involved in treatment resistance, demonstrating multiple patterns of clonal evolution, and revealing an unexpectedly high frequency of genetically discordant second malignancy in relapse in ALL. DisclosuresEvans:Prometheus Labs: Patents & Royalties: Royalties from licensing TPMT genotyping. Mullighan:Amgen: Honoraria, Speakers Bureau; Cancer Science Institute: Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Honoraria; Loxo Oncology: Research Funding.

Clinical Significance of NT5C2 Mutations in Children with First Relapse of B-Cell Precursor Acute Lymphoblastic Leukemia

The ubiquitous cytosolic 5´nucleotidase II (NT5C2) dephosphorylates purine nucleotide monophosphates and has an important role in cellular purine metabolism. Increased levels of nucleotidase activity have been correlated with resistance to nucleoside analog drugs that are commonly used in the treatment of children with acute lymphoblastic leukemia (ALL). Recently, activating mutations of NT5C2 have been identified in relapsed childhood ALL. NT5C2 mutations were present in 20% and 10% of children with relapsed T-cell ALL and with relapsed B-cell precursor ALL, respectively. In vitro studies showed that NT5C2 mutations conferred an increased resistance to purine analog drugs to ALL cell lines. However, the predictive and prognostic value of NT5C2 mutations for response and outcome of patients has remained elusive and systematic studies are warranted.Therefore, we studied the presence of NT5C2 mutations in 259 children with first relapse of B-cell precursor ALL. The patient cohort was a representative subset the German relapse trial ALL-REZ BFM 2002. NT5C2 exons 9, 13, 15 and 16 were sequenced according to Sanger in leukemic samples taken at diagnosis of first relapse. The clinical significance of NT5C2 mutations was assessed by comparing clinical parameters and survival differences between patients with and without NT5C2 mutation, and by multivariate Cox regression modelling.We identified NT5C2 mutations in 5.8% (15/259) of patients with first relapse of B-cell precursor ALL. This confirms the overall lower NT5C2 mutation rate in children with relapsed B-cell precursor ALL compared to those with relapsed T-cell ALL. Sixty percent (9/15) of the patients with NT5C2 mutation showed a G to A transition in codon 367 in exon 13. This mutation substitutes arginine with glutamine (p.R367Q) and represents a mutation hot spot in relapsed T-cell ALL. Our study reveals that p.R367Q is the predominant site of mutation also in relapsed ALL of B-cell precursor lineage. Patients with NT5C2 mutation significantly more often presented with a very early relapse within 18 months after initial diagnosis (P<0.001) compared to patients with wildtype NT5C2. Likewise, the median time of first remission was significantly shorter in patients with NT5C2 mutation (1.5 years versus 2.83 years, P<0.001). Regarding outcome after relapse treatment, patients with NT5C2 mutation showed a significantly reduced event-free (0.143±0.094 vs. 0.483±0.032; P=0.003) and overall survival rate (0.284±0.121 vs. 0.569±0.033; P=0.007) compared to patients with wildtype NT5C2. The predominant second event in relapse patients with NT5C2 mutation was the occurrence of a second relapse. Accordingly, the cumulative incidence of second relapse was significantly increased in patients with NT5C2 mutations compared to patients with wildtype NT5C2 (0.643±0.140 vs. 0.302±0.030; P=0.001). Multivariate analysis including time of relapse and site of relapse as established risk stratification factors in relapsed ALL revealed that NT5C2 mutation is an independent predictor for the occurrence of a second relapse (P=0.002). Surprisingly, mutation of NT5C2 was not associated with response to relapse treatment. Response was assessed by histological examination and by PCR-based sensitive detection of minimal residual disease at different time points during treatment. However, patients with NT5C2 mutation showed a similar proportion of responding and non-responding patients than relapsed children with wildtype NT5C2.We conclude that mutation of NT5C2 can serve as predictor for the occurrence of a second relapse independent of response to relapse treatment in children with relapsed B-cell precursor ALL. DisclosuresNo relevant conflicts of interest to declare.

Targeted Deep Sequencing of Genetic Alterations Identified By Whole Exome Sequencing Reveals Clonal Evolution in Pediatric T-Lymphoblastic Leukemia

Precursor T-cell acute lymphoblastic leukemia (T-ALL) represents one of the major challenges of pediatric oncology, because relapses are frequently refractory to treatment and fatal. The molecular understanding of progression to relapse in T-ALL is limited. We aimed at identifying patterns of clonal evolution and at describing mechanisms of relapse by comparing the genetic and epigenetic alterations in primary and in relapsed pediatric T-ALL.DNA from bone marrow of 13 patients with T-ALL at primary disease, remission and relapse was analyzed by a combination of multiplex ligation- dependent probe amplification (MLPA), Illumina 450k array, whole exome sequencing (WES) and targeted deep sequencing. Targeted deep sequencing was performed after target capture with Agilent HaloPlex. In the target capture design, all loci that showed somatic mutations in WES were included. Deep sequencing was done on all primary disease and relapse samples and on a subset of remission samples. Allele frequencies by HaloPlex were highly reproducible, corresponded well to allele frequencies of loci that were well covered in WES and were consistent after serial dilutions.Analysis of DNA methylation using the Illumina 450k array showed that methylation of relapse samples does not differ significantly from the methylation of the matching primary disease samples, with the variability between different patients being much larger than the variability within samples from the same patient.WES identified on average 10 single nucleotide variants (SNVs) and 1.8 small insertions and deletions (indels) in primary T-ALL and 23.2 SNVs and 2.6 indels in the corresponding relapse samples. Only about 30% of SNVs and indels identified in relapse were already detected in primary disease by WES, while most amplifications and deletions that had been detected by the combination of MLPA and read depth analysis of WES data were conserved from primary disease to relapse. Recurrently, we identified known and novel drivers of T-ALL (NOTCH1, FBXW7, PHF6, WT1, PTEN, NRAS, STAT5B).Targeted resequencing of mutated genes at high depth (median coverage 6233, 90% of targets covered >1000x) identified rare subclonal alleles with a sensitivity in the range of 10-2 to 10-4, depending on the coverage of each individual locus. This allowed us to distinguish de novo mutations that were acquired during treatment from mutations that had already been present at initial diagnosis and were selected for in relapse. Depending on the contribution of clonal selection or de novo mutations, at least two different patterns of relapse could be identified: In a smaller proportion of leukemias, all mutations present at first diagnosis were again detected in relapse, with some additional mutations that were specific for relapse. In most leukemias, the major clone at relapse had arisen from a minor subclone at primary disease and has acquired additional mutations, indicating that clonal selection was the main contributor to the evolution of relapse. In all cases, at least one genetic alteration was detected in samples from both time points.The example of activating mutations in the nucleotidase NT5C2, which have previously been proposed to contribute to resistance against nucleoside analogues, illustrates the genetic plasticity of T-ALL: Activating NT5C2 mutations were identified in 4 out of 13 relapse samples. The only activating NT5C2 mutation that was already detected in a primary disease sample at low allele frequency was not present in the corresponding relapse sample but was replaced by another activating NT5C2 mutation. This indicates that mutations acquired during treatment may outcompete subclonal mutations that were present in the primary leukemia. In at least two relapse samples, subclonal NT5C2 mutations were detected, compatible with the notion that acquisition of resistance towards chemotherapy by mutation of NT5C2 is a late event on the way to relapse.Conclusion: The acquisition of novel genetic alterations and selection of treatment resistant subclones are main contributors to T-ALL relapse. We now aim at identifying molecular signatures that characterize treatment resistant subclones, which may be included in risk stratification algorithms of primary T-ALL. DisclosuresNo relevant conflicts of interest to declare.

Comparison Of Mutational Profiles Of Diagnosis and Relapsed Pediatric B-Acute Lymphoblastic Leukemia: A Report From The COG ALL Target Project

Characterization of the genetic landscape of relapsed pediatric acute lymphoblastic leukemia (ALL) and changes that occur with disease progression provides insight into the molecular basis of relapse and may identify new therapeutic targets. We analyzed 20 diagnosis-remission (germline)-relapse trio samples of pediatric B-ALL by high-coverage (>200x) whole-exome sequencing. Included patients were originally NCI high risk (HR) by either age (≥10 years) or white blood count (≥50,000/microliter), enrolled on a Children's Oncology Group B-ALL trial, and experienced bone marrow relapse. Samples were selected based upon availability of sufficient high quality material from all three time points. We identified recurrent relapse-specific somatic mutations in 5 genes with significant frequency found in genes encoding the purine 5' nucleotidase NT5C2 (n=7, 35%) and the histone acetyltransferase CREBBP (n=2, 10%). Furthermore, we discovered novel recurrent somatic mutations that were highly enriched in relapsed ALL (20%) compared with diagnosis (5%) in WHSC1, USH2A and NT5C1B, another enzyme involved in purine metabolism. Three of the four WHSC1 mutations cause the same amino acid change E1099K in the highly conserved SET domain in which structural modeling predicts perturbed WHSC1-substrate interactions resulting in increased WHSC1 activity. The WHSC1 and NT5C2 mutations are mutually exclusive with a combined prevalence of 55% in relapsed tumors. Analysis of a validation cohort of 63 independent trios from both NCI standard risk and HR cohorts replicated the findings in NT5C2 (n=8, 13%) and WHSC1 E1099K (n=6, 10%). Five pathways were significantly mutated at relapse with high-frequency somatic mutations present at diagnosis and/or relapse of the 20 ALL trio samples: the Ras signaling pathway (NRAS, KRAS, PTPN11, FLT3; 65%), genes involved in histone modification (MLL2, WHSC1, SETD2, CREBBP; 50%), purine metabolism (NT5C2 and NT5C1B; 45%), tyrosine kinase signaling (JAK2, CRLF2; 25%) and genes regulating B-cell development (PAX5, IKZF1; 15%). The median number of non-silent coding region sequence mutations in diagnostic samples was 10 (range 4-25) while that of the relapse samples was 25 (range 7-506) including 3 hypermutated samples with >100 non-silent mutations accompanied by a dominance of C(G)>T(A) substitution in relapse-specific mutations, suggesting a possibility that these mutations may be induced by a specific mutagen. Most of the diagnostic and relapse tumors were polyclonal based on diagnosis-relapse comparison of mutant allele fraction (MAF). Inter-tumor MAF of a recurrently mutated gene was highly heterogeneous despite an estimated >70% leukemia involvement for most specimens, suggesting presence of subclonal mutations. For example, NT5C2 MAF in the relapsed specimens ranges from a low of 0.08 to a high of 0.93. Ten relapsed specimens demonstrated evolution from a minor subclone (<10%) in the diagnostic specimen; 8 of the subclones have oncogenic mutations in NRAS, KRAS, JAK2, WHSC1 or CRLF2. A notable finding was the lack of preservation of specific clonal RAS pathway mutations from diagnosis to relapse as subclonal mutations in KRAS, PTPN11 and FLT3 present in diagnosis were replaced by a dominant NRAS mutation in relapse. We were also able to identify structural alterations present in both relapse and diagnosis tumors from exome sequencing data, including IGH@-CRLF2 fusion (n=2), BTG1 deletion (n=2), ETV6-RUNX1 fusion (n=1), intragenic deletion of RUNX1 (n=1) and MAP3K2 (n=1), focal amplification in the last exon of MYC (n=1) and a t(1;14)(p36;q32) translocation resulting in truncation of SLC2A5 and BTBD7. These results provide new insights into the genetic events contributing to the relapse of pediatric B-ALL, and suggest new potential therapeutic targets. Disclosures:No relevant conflicts of interest to declare.

Genetic Landscapes Of Childhood T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) accounts for 10% to 15% of newly diagnosed cases of childhood acute lymphoblastic leukemia (ALL). Generally, childhood T-ALL patients have a worse prognosis than B cell precursor ALL patients. Recent studies have identified a subtype of T-ALL termed “early T-cell precursor” (ETP) ALL, which is associated with a high risk of treatment failure. In spite of recent improvements of risk stratified multiagent chemotherapy, relapsed patients have a poor prognosis even if they were non-ETP ALL.Recent genome-wide approach revealed frequent NOTCH1 and FBXW7 oncogenic mutations mutations in T-ALL. In addition, previous whole-exome sequencing disclosed novel CNOT3 mutations in approximately 10% of adult T-ALL cases, and thus, CNOT3 was thought to be one of the novel tumor suppressor gene for adult T-ALL. CNOT3 is part of the CCR4-NOT complex that is the major deadenylase of mRNA. NT5C2, encoding a 5ʹ-nucleotidase was identified as relapse specific mutation, of which mutation is associated with the outgrowth of drug-resistant clones in ALL. However, these mutations have been found in a fraction of childhood T-ALL suggests that the existence of other genetic pathogenesis.To discover new oncogenic gene mutations which involved in the pathogenesis of relapsed T-ALL and to identify novel prognostic markers of childhood T-ALL, we performed genome-wide analysis using whole-exome sequencing and 250K SNP array analyses in 8 cases with relapsed T-ALL and 16 cases with non-relapsed T-ALL.The mean coverage in the whole-exome sequencing of tumor and germline samples was 108× and 100× for the 50-Mb target regions, respectively, by which more than 90% of the coding sequences were represented by more than 20 independent reads on average. A mean of nonsilent mutations per sample at presentation was 18, and sample at 1st relapsed was 19. There were only 16 recurrent mutations in 24 cases; however no shared mutation in 8 relapsed cases other than NOTCH1 and FBXW7. NOTCH1 mutations were found in 50% (12/24), and were frequently identified in relapsed cases (6/8). FBXW7 mutations were also frequently found in 6/24 cases, and 60 % (3/6) were compound heterozygous mutations. In those 6 cases, only one case with FBWX7 mutation had a NOTCH1 mutation. CNOT3 mutations were reported to be frequent in adult T-ALL, however we found only two cases with CNOT3 mutations (8.3%). In addition, PHF6 mutation, which is known as X-linked tumor suppressor gene in T-ALL, was recurrent in 3 cases. Other recurrent mutations were shared between 2 cases, respectively. NT5C2 mutation has been reported to a relapse-specific mutation, and we also found NT5C2 mutations in 2 relapsed cases, which detected in only relapsed samples. RPL5 and RPL10 mutations were reported to be found in 10 % of pediatric T-ALL; however there was one mutation in RPL related genes in our study. Furthermore, we found common mutations of acute myeloid leukemia such as TCF7, STAT5A, KIT, RUNX1, and EP300 mutations in a single case. On the other hand, although pediatric T-ALL showed largely normal genomic copy number profiles, homozygous deletions at chromosome 9p21 harboring CDKN2A were frequently detected in our study (17/24 71%). Especially, 9p21 deletions were found in all relapsed cases, suggesting that loss of CDKN2A locus was a critical genetic mechanism of relapsed T-ALL.In conclusion, our results revealed mutations in several known genes, but overall frequency of recurrent somatic mutations in childhood T-ALL is low, even in relapsed samples. Although loss of CDKN2A locus was detected in all relapsed cases, recurrent relapse-specific mutations could not be identified other than NT5C2. These findings suggest that the majority of relapsed T-ALL may be driven by aberrations of CDKN2A and minor clone variants and/or epigenetic modifications during tumor evolution. Disclosures:No relevant conflicts of interest to declare.

Screening for gene mutations: will identification of NT5C2 mutations help predict the chance of relapse in acute lymphoblastic leukemia?

Screening for gene mutations: will identification of NT5C2 mutations help predict the chance of relapse in acute lymphoblastic leukemia?

Activating NT5C2 Mutations Drive ALL Relapse

Abstract Activating NT5C2 mutations occur in 10% to 16% of patients with relapsed ALL.

Relapse-specific mutations in NT5C2 in childhood acute lymphoblastic leukemia

Relapsed childhood acute lymphoblastic leukemia (ALL) carries a poor prognosis despite intensive retreatment, due to intrinsic drug resistance1-2. The biological pathways that mediate resistance are unknown. Here we report the transcriptome profiles of matched diagnosis and relapse bone marrow specimens from ten pediatric B lymphoblastic leukemia patients using RNA-sequencing. Transcriptome sequencing identified 20 newly acquired novel non-synonymous mutations not present at initial diagnosis, of which two patients harbored relapse specific mutations in the same gene, NT5C2, a 5′-nucleotidase. Full exon sequencing of NT5C2 was completed in 61 additional relapse specimens, identifying five additional cases. Enzymatic analysis of mutant proteins revealed that base substitutions conferred increased enzymatic activity and resistance to treatment with nucleoside analogue therapies. Clinically, all patients who harbored NT5C2 mutations relapsed early, or within 36 months of initial diagnosis (p=0.03). These results suggest that mutations in NT5C2 are associated with the outgrowth of drug resistant clones in ALL.

Relapse-specific mutations in cytosolic 5’-nucleotidase II in childhood acute lymphoblastic leukemia.

9507 Background: Relapsed childhood acute lymphoblastic leukemia (ALL) carries a very poor prognosis despite intensive retreatment that often includes allogeneic stem cell transplantation, due to intrinsic drug resistance. Novel therapeutic approaches are urgently needed and identification of the biological pathways that mediate resistance might provide targets for the effective prevention and treatment of relapsed ALL. However, the spectrum of somatic mutations responsible for ALL relapse is not yet known. Methods: We profiled the transcriptome of matched diagnosis and relapse bone marrow specimens from 10 pediatric B lymphoblastic leukemia patients using massively parallel sequencing technology (RNA-seq) to identify novel mutations specific at disease recurrence. Results: Transcriptome sequencing identified 20 newly acquired novel non-synonymous mutations in 10 relapse specimens not present at initial diagnosis. Two patients harbored relapsed specific mutations in the same gene, NT5C2, which codes for the Cytosolic 5’-nucleotidase II protein at different sites in the protein sequence. Mutations were validated as relapse specific after Sanger resequencing of germline, diagnosis and relapse DNA. RNA-seq coverage of each mutation was &gt;100X, indicating that if a relapsed clone were present at diagnosis it made up &lt; 1% of the bulk leukemia. Full exon sequencing of NT5C2 was completed in 61 additional relapse specimens, identifying 5 additional mutations which were also confirmed as relapse specific. Structural modeling of the relapse-associated mutations in the encoded protein Cytosolic 5’-nucleotidase II suggests alteration of enzyme subunit association/dissociation. Clinically, patients who harbored NT5C2 mutations were more likely to relapse earlier following initial diagnosis than those patients without mutations (p=0.03). Conclusions: Mutations in NT5C2 are associated with the outgrowth of drug resistant cells in childhood ALL.