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Among the recently described subtypes in childhood B-lymphoblastic leukemia (B-ALL) were DUX4- and PAX5-altered (PAX5alt). By using whole transcriptome RNA sequencing in 377 children with B-ALL from the Malaysia-Singapore ALL 2003 (MS2003) and Malaysia-Singapore ALL 2010 (MS2010) studies, we found that, after hyperdiploid and ETV6-RUNX1, the third and fourth most common subtypes were DUX4 (n = 51; 14%) and PAX5alt (n = 36; 10%). DUX4 also formed the largest genetic subtype among patients with poor day-33 minimal residual disease (MRD; n = 12 of 44). But despite the poor MRD, outcome of DUX4 B-ALL was excellent (5-year cumulative risk of relapse [CIR], 8.9%; 95% confidence interval [CI], 2.8%-19.5% and 5-year overall survival, 97.8%; 95% CI, 85.3%-99.7%). In MS2003, 21% of patients with DUX4 B-ALL had poor peripheral blood response to prednisolone at day 8, higher than other subtypes (8%; P = .03). In MS2010, with vincristine at day 1, no day-8 poor peripheral blood response was observed in the DUX4 subtype (P = .03). The PAX5alt group had an intermediate risk of relapse (5-year CIR, 18.1%) but when IKZF1 was not deleted, outcome was excellent with no relapse among 23 patients. Compared with MS2003, outcome of PAX5alt B-ALL with IKZF1 codeletion was improved by treatment intensification in MS2010 (5-year CIR, 80.0% vs 0%; P = .05). In conclusion, despite its poor initial response, DUX4 B-ALL had a favorable overall outcome, and the prognosis of PAX5alt was strongly dependent on IKZF1 codeletion.

Despite the immense genetic heterogeneity of B-lymphoblastic leukemia [or precursor B-cell acute lymphoblastic leukemia (B-ALL)], RNA sequencing (RNA-Seq) could comprehensively interrogate its genetic drivers, assigning a specific molecular subtype in >90% of patients. However, study groups have only started to use RNA-Seq. For broader clinical use, technical, quality control, and appropriate performance validation are needed. We describe the development and validation of an RNA-Seq workflow for subtype classification, TPMT/NUDT15/TP53 variant discovery, and immunoglobulin heavy chain (IGH) disease clone identification for Malaysia-Singapore acute lymphoblastic leukemia (ALL) 2020. We validated this workflow in 377 patients in our preceding Malaysia-Singapore ALL 2003/Malaysia-Singapore ALL 2010 studies and proposed the quality control measures for RNA quality, library size, sequencing, and data analysis using the International Organization for Standardization 15189 quality and competence standard for medical laboratories. Compared with conventional methods, we achieved >95% accuracy in oncogene fusion identification, digital karyotyping, and TPMT and NUDT15 variant discovery. We found seven pathogenic TP53 mutations, confirmed with Sanger sequencing, which conferred a poorer outcome. Applying this workflow prospectively to the first 21 patients in Malaysia-Singapore ALL 2020, we identified the genetic drivers and IGH disease clones in >90% of patients with concordant TPMT, NUDT15, and TP53 variants using PCR-based methods. The median turnaround time was 12 days, which was clinically actionable. In conclusion, RNA-Seq workflow could be used clinically in management of B-cell ALL patients.

Leukemias are routinely sub-typed for risk/outcome prediction and therapy choice using acquired mutations and chromosomal rearrangements. Down syndrome acute lymphoblastic leukemia (DS‐ALL) is characterized by high frequency of CRLF2‐rearrangements, JAK2‐mutations, or RAS‐pathway mutations. Intriguingly, JAK2 and RAS-mutations are mutually exclusive in leukemic sub‐clones, causing dichotomy in therapeutic target choices. We prove in a cell model that elevated CRLF2 in combination with constitutionally active JAK2 is sufficient to activate wtRAS. On primary clinical DS‐ALL samples, we show that wtRAS-activation is an obligatory consequence of mutated/hyperphosphorylated JAK2. We further prove that CRLF2-ligand TSLP boosts the direct binding of active PTPN11 to wtRAS, providing the molecular mechanism for the wtRAS activation. Pre‐inhibition of RAS or PTPN11, but not of PI3K or JAK‐signaling, prevented TSLP‐induced RAS‐GTP boost. Cytotoxicity assays on primary clinical DS‐ALL samples demonstrated that, regardless of mutation status, high-risk leukemic cells could only be killed using RAS‐inhibitor or PTPN11-inhibitor, but not PI3K/JAK‐inhibitors, suggesting a unified treatment target for up to 80% of DS‐ALL. Importantly, protein activities-based principal-component-analysis multivariate clusters analyzed for independent outcome prediction using Cox proportional-hazards model showed that protein‐activity (but not mutation-status) was independently predictive of outcome, demanding a paradigm-shift in patient‐stratification strategy for precision therapy in high-risk ALL.

Identifying patient-specific clonal IGH/TCR junctional sequences is critical for minimal residual disease (MRD) monitoring. Conventionally these junctional sequences are identified using laborious Sanger sequencing of excised heteroduplex bands. We found that the IGH is highly expressed in our diagnostic B-cell acute lymphoblastic leukemia (B-ALL) samples using RNA-Seq. Therefore, we used RNA-Seq to identify IGH disease clone sequences in 258 childhood B-ALL samples for MRD monitoring. The amount of background IGH rearrangements uncovered by RNA-Seq followed the Zipf’s law with IGH disease clones easily identified as outliers. Four hundred and ninety-seven IGH disease clones (median 2, range 0–7 clones/patient) are identified in 90.3% of patients. High hyperdiploid patients have the most IGH disease clones (median 3) while DUX4 subtype has the least (median 1) due to the rearrangements involving the IGH locus. In all, 90.8% of IGH disease clones found by Sanger sequencing are also identified by RNA-Seq. In addition, RNA-Seq identified 43% more IGH disease clones. In 69 patients lacking sensitive IGH targets, targeted NGS IGH MRD showed high correlation (R = 0.93; P = 1.3 × 10−14), better relapse prediction than conventional RQ-PCR MRD using non-IGH targets. In conclusion, RNA-Seq can identify patient-specific clonal IGH junctional sequences for MRD monitoring, adding to its usefulness for molecular diagnosis in childhood B-ALL.

In minimal residual disease (MRD), where there are exceedingly low target copy numbers, digital PCR (dPCR) can improve MRD quantitation. However, standards for dPCR MRD interpretation in acute lymphoblastic leukemia are lacking. Here, for immunoglobulin/T-cell receptor-based MRD, we propose an objective, statistics-based analytic algorithm. In 161 postinduction samples from 79 children with acute lymphoblastic leukemia, MRD was performed by dPCR and real-time quantitative PCR (qPCR) using the same markers and primer-probe sets. The dPCR raw data were analyzed by using an automated algorithm. dPCR and qPCR results were highly concordant (P 0.0001): 98% (50 of 51) of qPCR positive were positive by dPCR, whereas 95% (61 of 64) of qPCR negative results were also negative by dPCR. For MRD quantitation, both qPCR and dPCR were tightly correlated (R

Thiopurines (eg, 6-mercaptopurine [MP]) are highly efficacious antileukemic agents, but they are also associated with dose-limiting toxicities. Recent studies by us and others have identified inherited NUDT15 deficiency as a novel genetic cause of thiopurine toxicity, and there is a strong rationale for NUDT15-guided dose individualization to preemptively mitigate adverse effects of these drugs. Using CRISPR-Cas9 genome editing, we established a Nudt15(−/−) mouse model to evaluate the effectiveness of this strategy in vivo. Across MP dosages, Nudt15(−/−) mice experienced severe leukopenia, rapid weight loss, earlier death resulting from toxicity, and more bone marrow hypocellularity compared with wild-type mice. Nudt15(−/−) mice also showed excessive accumulation of a thiopurine active metabolite (ie, DNA-incorporated thioguanine nucleotides [DNA-TG]) in an MP dose–dependent fashion, as a plausible cause of increased toxicity. MP dose reduction effectively normalized systemic exposure to DNA-TG in Nudt15(−/−) mice and largely eliminated Nudt15 deficiency–mediated toxicity. In 95 children with acute lymphoblastic leukemia, MP dose adjustment also directly led to alteration in DNA-TG levels, the effects of which were proportional to the degree of NUDT15 deficiency. Using leukemia-bearing mice with concordant Nudt15 genotype in leukemia and host, we also confirmed that therapeutic efficacy was preserved in Nudt15(−/−) mice receiving a reduced MP dose compared with Nudt15(+/+) counterparts exposed to a standard dose. In conclusion, we demonstrated that NUDT15 genotype–guided MP dose individualization can preemptively mitigate toxicity without compromising therapeutic efficacy.

Prolonged exposure to thiopurines (eg, mercaptopurine [MP]) is essential for curative therapy in acute lymphoblastic leukemia (ALL), but is also associated with frequent dose-limiting hematopoietic toxicities, which is partly explained by inherited genetic polymorphisms in drug metabolizing enzymes (eg, TPMT). Recently, our group and others identified germ line genetic variants in NUDT15 as another major cause of thiopurine-related myelosuppression, particularly in Asian and Hispanic people. In this article, we describe 3 novel NUDT15 coding variants (p.R34T, p.K35E, and p.G17_V18del) in 5 children with ALL enrolled in frontline protocols in Singapore, Taiwan, and at St. Jude Children's Research Hospital. Patients carrying these variants experienced significant toxicity and reduced tolerance to MP across treatment protocols. Functionally, all 3 variants led to partial to complete loss of NUDT15 nucleotide diphosphatase activity and negatively influenced protein stability. In particular, the p.G17_V18del variant protein showed extremely low thermostability and was completely void of catalytic activity, thus likely to confer a high risk of thiopurine intolerance. This in-frame deletion was only seen in African and European patients, and is the first NUDT15 risk variant identified in non-Asian, non-Hispanic populations. In conclusion, we discovered 3 novel loss-of-function variants in NUDT15 associated with MP toxicity, enabling more comprehensive pharmacogenetics-based thiopurine dose adjustments across diverse populations.

Publisher's Note: There is an [Inside Blood Commentary][1] on this article in this issue. To the editor: Our understanding of acute lymphoblastic leukemia (ALL) has expanded tremendously in the past few years because of large-scale genomic studies.[1][2],[2][3] ALL is characterized by a relatively

BackgroundDown syndrome acute lymphoblastic leukemia (DS-ALL) is characterized by the high frequency of CRLF2-rearrangements, JAK2-mutations, or RAS-pathway mutations. Intriguingly, JAK2 and RAS mutations are mutually exclusive in leukemic sub-clones, causing dichotomy in therapeutic target choices.ResultsHere we show that in primary leukemic cells from DS-ALL, in the absence of RAS-mutations, wild-type (wt)RAS is active, and/or can be induced by the physiological ligand TSLP of the transmembrane-receptor CRLF2. We show active/inducible RAS in 14/20 (70%) of primary DS-ALL samples analyzed, 8 of which had no RAS-mutations, but 75% of those had either mutated or hyperphosphorylated JAK2. No wtRAS cases with mutated/hyperphosphorylated JAK2 were observed that lacked activated RAS protein. We prove in a cell model that elevated CRLF2 in combination with constitutionally active JAK2 is sufficient to activate wtRAS. We show that TSLP boosts the direct binding of active PTPN11 to wtRAS. Pre-inhibition of RAS or PTPN11, but not of PI3K or JAK signaling, prevented TSLP-induced RAS-GTP boost.Using multivariate-clustering based on RAS-activity/inducibility we show significant separation between standard-risk and high-risk DS-ALL groups. Cox proportional-hazards model showed protein-activity (but not mutation status) as independently predictive of outcome.ConclusionsOur data indicate that RAS protein activity levels (and not JAK2/RAS mutation profiles), are predictive of outcome. Importantly, our data suggest that inhibition of RAS and direct RAS-pathway components should be combined with PI3K/mTOR and/or JAK2 inhibitors for high-risk cases. Therapeutically this is relevant for >75% of DS-ALL and our additional data suggest that it warrants further investigation in high-risk non-DS-ALL.

Identifying patient-specific clonal IGH/TCR junctional sequences is critical for minimal residual disease (MRD) monitoring. Conventionally these junctional sequences are identified using laborious Sanger sequencing of excised heteroduplex bands. We found that the IGH is highly expressed in our diagnostic B-cell acute lymphoblastic leukemia (B-ALL) samples using RNA-Seq. Therefore, we used RNA-Seq to identify IGH disease clone sequences in 258 childhood B-ALL samples for MRD monitoring. The amount of background IGH rearrangements uncovered by RNA-Seq followed the Zipf's law with IGH disease clones easily identified as outliers. Four hundred and ninety-seven IGH disease clones (median 2, range 0-7 clones/patient) are identified in 90.3% of patients. High hyperdiploid patients have the most IGH disease clones (median 3) while DUX4 subtype has the least (median 1) due to the rearrangements involving the IGH locus. In all, 90.8% of IGH disease clones found by Sanger sequencing are also identified by RNA-Seq. In addition, RNA-Seq identified 43% more IGH disease clones. In 69 patients lacking sensitive IGH targets, targeted NGS IGH MRD showed high correlation (R = 0.93; P = 1.3 × 10

Acute erythroid leukemia (AEL) is a high-risk leukemia of poorly understood genetic basis, with controversy regarding diagnosisin the spectrum of myelodysplasia and myeloid leukemia. We compared genomic features of 159 childhood and adult AEL cases with non-AEL myeloid disorders and defined five age-related subgroups with distinct transcriptional profiles: adult, TP53mutated; NPM1 mutated; KMT2A mutated/rearranged; adult, DDX41 mutated; and pediatric, NUP98 rearranged. Genomic features influenced outcome, with NPM1 mutations and HOXB9 overexpression being associated with a favorable prognosis andTP53, FLT3 or RB1 alterations associated with poor survival. Targetable signaling mutations were present in 45% of cases and included recurrent mutations of ALK and NTRK1, the latter of which drives erythroid leukemogenesis sensitive to TRK inhibition.This genomic landscape of AEL provides the framework for accurate diagnosis and risk stratification of this disease, and the rationale for testing targeted therapies in this high-risk leukemia. Refereed/Peer-reviewed

Purpose Although IKZF1 deletion ( IKZF1del) confers a higher risk of relapse in childhood B-cell acute lymphoblastic leukemia (B-ALL), it is uncertain whether treatment intensification will reverse this risk and improve outcomes. The Malaysia-Singapore ALL 2010 study (MS2010) prospectively upgraded the risk assignment of patients with IKZF1del to the next highest level and added imatinib to the treatment of all patients with BCR- ABL1 fusion. Patients and Methods In total, 823 patients with B-ALL treated in the Malyasia-Singapore ALL 2003 study (MS2003; n = 507) and MS2010 (n = 316) were screened for IKZF1del using the multiplex ligation-dependent probe amplification assay. The impact of IKZF1del on the 5-year cumulative incidence of relapse (CIR) was compared between the two studies. Results Patient characteristics were similar in both cohorts, including IKZF1del frequencies (59 of 410 [14.4%] v 50 of 275 [18.2%]; P = .2). In MS2003, where IKZF1del was not used in risk assignment, IKZF1del conferred a significantly higher 5-year CIR (30.4% v 8.1%; P = 8.7 × 10−7), particularly in the intermediate-risk group who lacked high-risk features (25.0% v 7.5%; P = .01). For patients with BCR-ABL1–negative disease, IKZF1del conferred a higher 5-year CIR (20.5% v 8.0%; P = .01). In MS2010, the 5-year CIR of patients with IKZF1del significantly decreased to 13.5% ( P = .05) and no longer showed a significant difference in patients with BCR-ABL1-negative disease (11.4% v 4.4%; P = .09). The 5-year overall survival for patients with IKZF1del improved from 69.6% in MS2003 to 91.6% in MS2010 ( P = .007). Conclusion Intensifying therapy for childhood B-ALL with IKZF1del significantly reduced the risk of relapse and improved overall survival. Incorporating IKZF1del screening significantly improved treatment outcomes in contemporary ALL therapy.

Widely used as anticancer and immunosuppressive agents, thiopurines have narrow therapeutic indices owing to frequent toxicities, partly explained by TPMT genetic polymorphisms. Recent studies identified germline NUDT15 variation as another critical determinant of thiopurine intolerance, but the underlying molecular mechanisms and the clinical implications of this pharmacogenetic association remain unknown. In 270 children enrolled in clinical trials for acute lymphoblastic leukemia in Guatemala, Singapore and Japan, we identified four NUDT15 coding variants (p.Arg139Cys, p.Arg139His, p.Val18Ile and p.Val18_Val19insGlyVal) that resulted in 74.4-100% loss of nucleotide diphosphatase activity. Loss-of-function NUDT15 diplotypes were consistently associated with thiopurine intolerance across the three cohorts (P = 0.021, 2.1 × 10(-5) and 0.0054, respectively; meta-analysis P = 4.45 × 10(-8), allelic effect size = -11.5). Mechanistically, NUDT15 inactivated thiopurine metabolites and decreased thiopurine cytotoxicity in vitro, and patients with defective NUDT15 alleles showed excessive levels of thiopurine active metabolites and toxicity. Taken together, these results indicate that a comprehensive pharmacogenetic model integrating NUDT15 variants may inform personalized thiopurine therapy.

Acute myeloid leukemia (AML) with an FLT3 internal tandem duplication (FLT3-ITD) mutation is an aggressive hematologic malignancy with a grave prognosis. To identify the mutational spectrum associated with relapse, whole-exome sequencing was performed on 13 matched diagnosis, relapse, and remission trios followed by targeted sequencing of 299 genes in 67 FLT3-ITD patients. The FLT3-ITD genome has an average of 13 mutations per sample, similar to other AML subtypes, which is a low mutation rate compared with that in solid tumors. Recurrent mutations occur in genes related to DNA methylation, chromatin, histone methylation, myeloid transcription factors, signaling, adhesion, cohesin complex, and the spliceosome. Their pattern of mutual exclusivity and cooperation among mutated genes suggests that these genes have a strong biological relationship. In addition, we identified mutations in previously unappreciated genes such as MLL3, NSD1, FAT1, FAT4, and IDH3B. Mutations in 9 genes were observed in the relapse-specific phase. DNMT3A mutations are the most stable mutations, and this DNMT3A-transformed clone can be present even in morphologic complete remissions. Of note, all AML matched trio samples shared at least 1 genomic alteration at diagnosis and relapse, suggesting common ancestral clones. Two types of clonal evolution occur at relapse: either the founder clone recurs or a subclone of the founder clone escapes from induction chemotherapy and expands at relapse by acquiring new mutations. Relapse-specific mutations displayed an increase in transversions. Functional assays demonstrated that both MLL3 and FAT1 exert tumor-suppressor activity in the FLT3-ITD subtype. An inhibitor of XPO1 synergized with standard AML induction chemotherapy to inhibit FLT3-ITD growth. This study clearly shows that FLT3-ITD AML requires additional driver genetic alterations in addition to FLT3-ITD alone.

The RUNX1/AML1 gene is among the most frequently mutated genes in human leukaemia. However, its association with T-cell acute lymphoblastic leukaemia (T-ALL) remains poorly understood. In order to examine RUNX1 point mutations in T-ALL, we conducted an amplicon-based deep sequencing in 65 Southeast Asian childhood patients and 20 T-ALL cell lines, and detected RUNX1 mutations in 6 patients (9.2%) and 5 cell lines (25%). Interestingly, RUNX1-mutated T-ALL cases seem to constitute a subset of early immature T-ALL that may originate from differentiated T-cells. This result provides a deeper insight into the mechanistic basis for leukaemogenesis.

Background At diagnosis, recurrent genomic aberrations - aneuploidy and oncogene fusion that drive B-lymphoblastic leukemia - best predict treatment response. Currently, karyotyping and RT-PCR can discern only 50% of the genomic aberrations driving childhood B-ALL. RNA sequencing (RNA-Seq) interrogates the whole transcriptome through deep sequencing. Using RNA-Seq to study expressed SNP variants and expression level of each chromosome, we can identify their copy number; this enables digital karyotyping. For each patient, this digital karyotyping together with oncogene fusion sequencing and transcriptome profiling allow full molecular interrogation of the genomic aberrations that drive leukemia. We molecular subtyped and risk assigned 377 children with B-lymphoblastic leukemia using RNA-Seq. We showed that in childhood B-lymphoblastic leukemia, a single platform RNA-seq significantly improved risk assignment. Methods Whole transcriptome RNA-Seq was performed on the diagnostic bone marrow or peripheral blood samples of 377 children enrolled on the Ma-Spore ALL 2003 and Ma-Spore ALL 2010 studies. Oncogene fusions, variant calling, hierarchical clustering and gross chromosomal copy number calling were performed using RNA-Seq. These are combined for molecular assignment. As compared with conventional risk stratification using a combination of cytogenetics, DNA index and oncogene fusion panel, risk assignment including newly discovered molecular subtypes are made using RNA-Seq. Results Patients are classified into 20 molecularly distinct subtypes (Table 1) in 5 categories: 1. Oncogene fusion category with 10 subtypes. 2. Gene expression profiles with 5 subtypes. 3. Aneuploidy defines 3 subtypes, namely high hyperdiploidy, hypodiploidy and near haploid. 4. Biallelic mutation of PAX5 characterized by PAX5 P80R. 5. B-others. The 20 molecular subtypes are then risk assigned to Low, Intermediate or High risk groups (Table 1). Specifically, 1. Low Risk (LR) - 66% (250/377) of the cohort 2. Intermediate Risk (IR) - 15% (57 of 377) of the cohort 3. High risk (HR) - 12% (44/377) of the cohort 4. B-others - 7% (26/377) of the cohort In the patients with available survival data, compared to 39% by conventional methods, RNA-Seq improves risk assignment by assigning 93% of the total cohort into a distinct risk group (Figure 1). The number of LR patients by RNA-Seq nearly doubled (RNA-Seq n=238 vs conventional n=125), with comparable cumulative incidence of relapse (CIR; 6.2% vs 5.2%) and overall survival (OS; 98.2% vs 99.1%). The number of HR patients also increased >2-fold (RNA-Seq n=41 vs conventional n=17), with a slightly lower CIR (41.2% vs 52.9%) and a similar OS (65.4% vs 64.7%). The RNA-Seq defined risk groups have more significantly distinct outcome (P-value comparing CIR: 1×10-8 vs1×10-7; P-value comparing OS: 2×10-12 vs3×10-7; Figure 1). In conventional risk assignment, 67% of the relapses and 68% of the deaths are in B-Others (61% of patients). Using RNA-Seq, HR (11% of patients) accounts for the most relapses (38%) and deaths (59%) while B-Others (7% of patients) account for 7% of relapses and 5% of deaths. In multivariate analysis using competing risk regression on CIR together with age, gender, WBC and Day 33 MRD, only risk stratification by RNA-Seq and Day 33 MRD remained prognostically significant (RNA-Seq subtype P=6.4×10-4 or 5.9×10-3 for HR or IR vs SR; MRD P=1.3×10-5 or 0.020 for HR or IR vs SR). While in Cox proportional hazards regression on OS, Day 33 MRD lost its significance; RNA-Seq risk stratification is the most significant factor that predicts OS (RNA-Seq subtype P=3.4×10-4 or 0.27 for HR or IR vs SR; MRD P=0.19 or 0.24 for HR or IR vs SR). Conclusion When compared to conventional karyotyping and RT-PCR, in B-lymphoblastic leukemia, RNA-Seq significantly improved molecular diagnosis and risk assignment; it assigns 93% of patients into a distinct molecular and risk group. In Cox proportional hazards regression on OS, Day 33 MRD lost its prognostic significance, and RNA-Seq risk stratification assignment is the only significant predictive factor of overall survival. Disclosures No relevant conflicts of interest to declare.

Background As a single platform, RNA sequencing (RNA-Seq) can accurately identify majority of oncogenic driver fusion genes and aneuploidy. This provides refined molecular subtyping and risk stratification; RNA-Seq is increasing used upfront for genetic subtype assignment in on-going contemporary ALL trials. The IGH gene is highly expressed in our diagnostic ALL samples using RNA-Seq. We investigated the usefulness of RNA-Seq in identifying IGH disease sequences for MRD monitoring in 258 childhood B-lymphoblastic leukemia (B-LL) samples. Methods RNA-seq was performed on a total of 259 diagnostic bone marrow or peripheral blood samples from children with B-LL treated in the Ma-Spore ALL 2003 and ALL 2010 studies. The reads were aligned to germline IGH gene V, D and J segments, and reads covered IGH VHDJH or DJH junctional regions are clustered into clones. Overall, the distribution of read count of the IGH background clones follows Zipf's law, i.e. the read count of the IGH background clones is negative linearly correlated with the number of IGH clones with that read count in the log-log scale (Figure 1A). IGH disease clones are outliers with exceedingly high read count. For each sample, a linear model is fitted between log transformed read count (using only read count ≤10) and log transformed number of IGH clones. The expected number of IGH clones (E-value) of a read count can be predicted by the fitted linear model. A clone with E-value Results In 90.3% of the patients, 497 IGH disease clones (median 2, range 0-7 clones/patient) are identified. The number of IGH disease clones significantly correlated with genetic subtype (P=1.9×10-8; Table 1). Specifically, high hyperdiploid patients have the most IGH disease clones (median 3) with 67% (22/33) have >2 IGH disease clones, due to the gain of chromosome 14. In contrast, DUX4 high expressers have the least (median 1) IGH disease clones with 19% (8/43) have no IGH disease clone, due to the rearrangements involving one of the IGH locus. Of the 348 IGH disease clones identified by Sanger sequencing, 90.8% are also identified by RNA-Seq; and in the 199 Sanger sequences that yielded sensitive markers, 191 (96.0%) are also defined as IGH disease clones by RNA-Seq. In addition, RNA-Seq identified 43% more IGH disease clones. In 69 patients lacking sensitive IGH targets by Sanger sequencing, the disease clones identified by RNA-Seq were used collectively for MRD monitoring by targeted deep sequencing of IGH (IGH-Seq) and showed high correlation with conventional RQ-PCR MRD using non-IGH targets (R=0.93; P=1.3x10-14;Figure 1C). In addition, IGH-Seq showed improved sensitivity with quantifiable MRD in 20% more samples compared to RQ-PCR (78% vs 58%; Figure 1C). In the samples that are positive below sensitivity or negative by RQ-PCR, IGH-Seq obtained quantifiable MRD for 60% (9/15) or 25% (5/20) of the samples (Figure 1C). Conclusion In conclusion, IGH disease clone sequences are highly expressed and can be distinctly identified from whole transcriptome RNA-Seq in >90% of children with B-LL. These can be used for molecular MRD monitoring using RQ-PCR or NGS-based IGH-Seq. In addition to identifying the constellation of oncogene fusions and chromosomal aneuploidies that drive B-LL and RNA-Seq can identify IGH MRD sequences for molecular MRD monitoring. Disclosures No relevant conflicts of interest to declare.

Chromosomal translocations are a genomic hallmark of many hematologic malignancies. Often as initiating events, these structural abnormalities result in fusion proteins involving transcription factors important for hematopoietic differentiation and/or signaling molecules regulating cell proliferation and cell cycle. In contrast, epigenetic regulator genes are more frequently targeted by somatic sequence mutations, possibly as secondary events to further potentiate leukemogenesis. Through comprehensive whole-transcriptome sequencing of 231 children with acute lymphoblastic leukemia (ALL), we identified 58 putative functional and predominant fusion genes in 54.1% of patients (n = 125), 31 of which have not been reported previously. In particular, we described a distinct ALL subtype with a characteristic gene expression signature predominantly driven by chromosomal rearrangements of the ZNF384 gene with histone acetyltransferases EP300 and CREBBP. ZNF384-rearranged ALL showed significant up-regulation of CLCF1 and BTLA expression, and ZNF384 fusion proteins consistently showed higher activity to promote transcription of these target genes relative to wild-type ZNF384 in vitro. Ectopic expression of EP300-ZNF384 and CREBBP-ZNF384 fusion altered differentiation of mouse hematopoietic stem and progenitor cells and also potentiated oncogenic transformation in vitro. EP300- and CREBBP-ZNF384 fusions resulted in loss of histone lysine acetyltransferase activity in a dominant-negative fashion, with concomitant global reduction of histone acetylation and increased sensitivity of leukemia cells to histone deacetylase inhibitors. In conclusion, our results indicate that gene fusion is a common class of genomic abnormalities in childhood ALL and that recurrent translocations involving EP300 and CREBBP may cause epigenetic deregulation with potential for therapeutic targeting.

Purpose To improve treatment outcome for childhood acute lymphoblastic leukemia (ALL), we designed the Malaysia-Singapore ALL 2003 study with treatment stratification based on presenting clinical and genetic features and minimal residual disease (MRD) levels measured by polymerase chain reaction targeting a single antigen-receptor gene rearrangement. Patients and Methods Five hundred fifty-six patients received risk-adapted therapy with a modified Berlin-Frankfurt-Münster–ALL treatment. High-risk ALL was defined by MRD ≥ 1 × 10−3 at week 12 and/or poor prednisolone response, BCR-ABL1, MLL gene rearrangements, hypodiploid less than 45 chromosomes, or induction failure; standard-risk ALL was defined by MRD ≤ 1 × 10−4 at weeks 5 and 12 and no extramedullary involvement or high-risk features. Intermediate-risk ALL included all remaining patients. Results Patients who lacked high-risk presenting features (85.7%) received remission induction therapy with dexamethasone, vincristine, and asparaginase, without anthracyclines. Six-year event-free survival (EFS) was 80.6% ± 3.5%; overall survival was 88.4% ± 3.1%. Standard-risk patients (n = 172; 31%) received significantly deintensified subsequent therapy without compromising EFS (93.2% ± 4.1%). High-risk patients (n = 101; 18%) had the worst EFS (51.8% ± 10%); EFS was 83.6% ± 4.9% in intermediate-risk patients (n = 283; 51%). Conclusion Our results demonstrate significant progress over previous trials in the region. Three-drug remission-induction therapy combined with MRD-based risk stratification to identify poor responders is an effective strategy for childhood ALL.

The distribution of thiopurine methyltransferase (TPMT) activity in Asian populations has not been well documented. We studied the TPMT phenotype in three major Asian ethnic groups in Singapore, namely the Chinese (Ch), Malays (Mal) and Indians (Ind), with the aim of carrying out a comprehensive survey of the distribution of TPMT activity in Asians.A radiochemical assay was used to measure the enzymatic activity of TPMT in the red blood cells (RBCs) of 479 healthy adults (Ch=153, Mal=163 and Ind=163). Cut-off points for intermediate TPMT activity were validated using a receiver operating curve (ROC) analysis. PCR-based methods were used to screen for the TPMT*3C, TPMT*3A and TPMT*6 variants.The histogram of the combined population cohort showed a bimodal distribution of TPMT activity, with no subject having low TPMT activity (5 units). In total, TPMT variants were detected in 14 subjects (*1/*3C in 13 subjects; *1/*3A in one subject). We observed significant inter-ethnic differences in terms of TPMT activity (p0.001), with the Malays showing a higher median activity than the Chinese or Indians (17.8 units vs 16.4 units). The Malays also showed a higher methylation rate--with a cut-off point for intermediate TPMT activity of 11.3 units--than the Chinese (9.9 units) or Indians (9.4 units). A high phenotype-genotype correlation of97% was observed in all three races. We also genotyped 418 childhood leukaemias. The combined analysis of subjects participating in this and a previous study--1585 subjects--showed that 4.7% of Chinese (n=30/644), 4.4% of Malays (n=24/540) and 2.7% of Indians (n=11/401) were heterozygous at the TPMT gene locus.This is the first comprehensive TPMT phenotype and genotype study in Asian populations, particularly in the Malays and Indians.

Polymorphisms in drug-metabolizing genes may lead to the production of dysfunctional proteins and consequently affect therapeutic efficacy and toxicity of drugs. Different frequencies of polymorphic alleles among the races have been postulated to account for the observed ethnic variations in drug responses. In the current study, we aimed to estimate the frequencies of 14 polymorphisms in eight genes (TPMT, NQO1, MTHFR, GSTP1, CYP1A1, CYP2D6, ABCB1, and SLC19A1) in the Singapore multiracial populations by screening 371 cord blood samples from healthy newborns. To improve genotyping efficacy, we designed an oligonucleotide array based on the principle of allele-specific primer extension (AsPEX) that was capable of detecting the 14 polymorphisms simultaneously. Cross-validation using conventional polymerase chain reaction–restriction fragment-length polymorphism (PCR–RFLP) assays demonstrated 99% concordant results. Measurements on the fluorescent intensity displayed clear distinctions among different genotypes. Statistical analyses showed significantly different allele distributions in several genes among the three races, namely Chinese, Malays, and Indians. Comparing the allelic frequencies in Chinese with previous studies in Caucasian populations, NQO1 609C > T and SLC19A1 80G > A were distinctly different, whereas close similarity was observed for MTHFR 677C > T. We have demonstrated an array-based methodology for rapid multiplex detection of genetic polymorphisms. The allelic frequencies reported in this study may have important therapeutic and prognostic implications in the clinical use of relevant drugs.

Background L-asparaginase is a crucial drug in childhood acute lymphoblastic leukemia (ALL) induction therapy, but much debate remains regarding the optimal formulation and dosage. As minimal residual disease (MRD) can accurately measure extremely low levels of lymphoblasts, it is a sensitive reflection of leukemia cell kill. We utilized MRD to compare the efficacy of Erwinia-asparaginase (Erwinia-asp) and E. coli-asparaginase (E. coli-asp) during induction therapy for childhood ALL. Procedure Of 116 precursor-B ALL patients, 22 were treated with Erwinia-asp, 90 with E. coli-asp, and 4 were switched from E. coli-asp to Erwinia-asp. MRD levels at the end of induction were analyzed for 90 patients (Erwinia-asp = 16; E. coli-asp = 74). Patients were stratified into MRD ≥10−2, between 10−2–10−4 and ≤10−4. Toxicity information during induction was available for 110 patients. Results MRD was the only significant prognosticator compared to conventional criteria. Patients treated with Erwinia-asp were 6.7 times more likely to have MRD levels ≥10−2 (P = 0.031), reflecting slower lymphoblast clearance. While non-asparaginase related toxicities were similar in both groups, more E. coli-asp patients experienced severe asparaginase-related toxicity. Conclusion E. coli-asp is superior to Erwinia-asp in childhood ALL induction. Although E. coli-asp is more toxic, this is balanced by better response to therapy. Early response to treatment as measured by MRD is a direct reflection of leukemic cell kill and is a significant prognosticator of eventual outcome, making it a good surrogate marker to evaluate the efficacy of induction drugs in childhood ALL. Pediatr Blood Cancer 2006;47:299–304. © 2005 Wiley-Liss, Inc.

Background: β-Thalassemia is endemic to many regions in Southeast Asia and India, and Methods: Gap-PCR was used to simultaneously amplify the β-globin gene from genomic DNA and to detect the Δ619bp deletion mutation. Multiplex minisequencing was then performed on the amplified β-globin fragment to detect an additional 15 common Southeast Asian and Indian β-thalassemia mutations. Site-specific primers of different lengths were subjected to multiple rounds of annealing and single-nucleotide extension in the presence of thermostable DNA polymerase and the four dideoxynucleotides, each labeled with a different fluorophore. Minisequencing products were separated and detected by capillary electrophoresis, followed by automated genotyping. The optimized assay was subjected to a double-blind validation analysis of 89 β-thalassemia and wild-type DNA samples of known genotype. Results: Homozygous wild-type or mutant DNA samples produced electropherograms containing only a single colored peak for each mutation site, whereas samples heterozygous for a specific mutation displayed two different-colored peaks for that mutation site. Samples were automatically genotyped based on color and position of primer peaks in the electropherogram. In the double-blind validation analysis, all 89 DNA samples were genotyped correctly (100% assay specificity). Conclusions: The described semiautomated multiplex minisequencing assay can detect the most common Southeast Asian and Indian β-thalassemia mutations, is amenable to high-throughput scale up, and may bring population-based screening of β-thalassemia in endemic regions a step closer to implementation.

There is growing evidence that non-coding sequences in human genome often function as transcriptional regulatory elements of protein-coding genes. In fact, germline polymorphisms and somatically acquired mutations within regulatory DNA can profoundly alter chromatin structure and modify gene transcription, directly contributing to tumorigenesis. However, there is a paucity of unbiased genome-wide characterization of somatic non-coding mutations in cancer. Using T-cell acute lymphoblastic leukemia (T-ALL) as a model disease, we herein report a systematic interrogation of driver non-coding genomic alterations by paired whole-genome and transcriptome sequencing of 31 children with T-ALL. To identify non-coding mutations with potential regulatory impact in a genome-wide fashion, our analytical pipeline consisted of 3 approaches: 1) the “hotspot analysis” for recurring mutations at the nearby positions, 2) the “regional recurrence analysis” for predefined regulatory regions with significant enrichment of non-coding mutations, 3) the “transcriptional factor analysis” for mutations that potentially result in gain/loss of transcription factor binding sites and alter expression of adjacent genes. Remarkably, T-ALL oncogenes LMO1 and TAL1 emerged as loci with the most significant recurring non-coding mutations. At the LMO1 locus, 3 patients (9.7%) showed an identical single-nucleotide mutation proximal to the transcription start site of the long isoform of LMO1. This recurring mutation resulted in the gain of a canonical Myb binding site (AACGG) and ~120-fold increase in LMO1 transcription compared to patients with wildtype genotype. TAL1 overexpression was observed in 15 patients, of whom 11 had intrachromosomal rearrangement (STIL-TAL1 fusion). The remaining 4 patients had somatic insertion that created a MYB-mediated super enhancer, consistent with recent reports. LMO1 enhancer mutation was further confirmed in an independent validation cohort (N=26), in which we additionally identified a novel intrachromosomal rearrangement between MED17 and LMO1 resulting in transcriptional activation of the latter. In a panel of T-ALL cell lines, LMO1 enhancer mutation was again associated with dramatic overexpression of LMO1, an active enhancer histone mark (H3K27ac), Dnase hypersensitivity, and allele-specific binding of MYB. Interestingly, we also observed robust binding of TAL1, CREBBP, RUNX1, ETS1, ELF1 and RNA Polymerase II at this site. Reporter gene assay confirmed the MYB-mediated transcription activation effects of this LMO1 enhancer mutation in vitro. In this genome-wide investigation of non-coding mutations in T-ALL, we identified novel enhancer mutations with drastic effects on oncogene activation. Our findings expand the understanding of how genomic alterations in regulatory DNA contribute to cancer pathogenesis. Citation Format: Maoxiang Qian, Shaoyan Hu, Hui Zhang, Yu Guo, Jin Yang, Xujie Zhao, Lin Wan, Jun Lu, Jian Pan, Meimei Chang, Shirley K. Kham, Yong Cheng, Chunliang Li, Allen E. Yeoh, Anders Skanderup, Jun J. Yang. Whole-genome sequencing identified novel non-coding mutations causal of oncogene activation in T-cell 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 3005. doi:10.1158/1538-7445.AM2017-3005

Sixth-five 14-thalassaemia genes from 14 unrelated Chinese β-thalassaemia major patients and 37 Chinese β-carriers were analysed by allele-specific oligonucleotide (ASO) hybridization after DNA amplification by the polymerase chain reaction (PGR). Six mutations were studied and are represented by 49.2% of codon 41-42, 30.8% of IVSII #654, 6.2% of 17β 3.1% of IVSI #5 (G→G) and 1.5% of -28 TATA box. The complete mutations responsible for β-thalassaemia major in 13 of our 14 affected families were identified. For these families prenatal diagnosis at 10 weeks gestation using DNA amplification and ASO hybridization will replace the globin chain biosynthesis technique at 19 weeks gestation

Acute lymphoblastic leukaemia (ALL) is the most common paediatric malignancy. Although 90% of patients are now long-term survivors, the remaining 10% have poor outcome predominantly due to drug resistance. In this study, we carried out genome-wide microRNA (miRNA) microarray analysis on diagnostic bone marrow samples to determine miRNA expression profiles associated with poor outcome in ALL. A reduced expression of MIR335 was identified as the most significant miRNA abnormality associated with poor outcome. It is well known that glucocorticoid (GC) resistance is one of the major reasons contributing to poor outcome. We show that exogenous expression of MIR335 in ALL cells increases sensitization to prednisolone-mediated apoptosis. Moreover, we demonstrate that MAPK1 is a novel target of MIR335, and that MEK/ERK inhibitor treatment enhanced prednisolone-induced cell death through the activation of BIM (BCL2L11). These results provide a possible underlying molecular mechanism to explain the association between reduced MIR335 with poor clinical outcome, and suggest that approaches to re-introduce MIR335 expression or override MAPK1 activity may offer promising therapeutic strategies in the treatment of ALL.

Early response to 7 days of prednisolone (PRED) treatment is one of the important prognostic factors in predicting eventual outcome in childhood acute lymphoblastic leukemia (ALL). Using proteomic tools and clinically important leukemia cell lines (REH, 697, Sup-B15, RS4; 11), we have identified potential prognostic protein biomarkers as well as discovered promising regulators of PRED-induced apoptosis. After treatment with PRED, the four cell lines can be separated into resistant (REH) and sensitive (697, Sup-B15, RS4;11). Two dimensional gel electrophoresis (2-DE) and MALDI-TOF/TOF MS identified 77 and 17 significantly differentially expressed protein spots (p

FMS-like tyrosine kinase 3 (FLT3) is critical for normal haematopoiesis and have been reported to be expressed in the majority of acute myeloid and lymphoid malignancies. We correlated the impact of FLT3 mutations and its expression with age, WHO 2008 classification and treatment outcome in 531 childhood acute leukaemias. Of 150 acute myeloid leukaemia (AMLs), 18 (12%) harboured FLT3-ITD while nine (6%) had FLT3-TKD. FLT3-ITD and -TKD were rare in acute megakaryoblastic leukaemia (AMKL; FLT3-ITD 0/26, FLT3-TKD 1/26) and children below 3years (n=2/48). Acute promyelocytic leukaemia (APL) with t(15;17);PML-RARa (n=7/18; 39%) harboured the highest frequency of FLT3 mutations, followed by myelomonocytic (n=4/18; 22%) and AML with t(8;21);RUNX1-RUNX1T1 (n=2/21; 9%). FLT3 expression levels were also lowest in AMKL, both in Down's and non-Down's (p=0.002) followed by patients

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • More than 27 mutations in the TPMT gene causing decreased TPMT activity have been reported in various races. • Decreased TPMT activity is associated with increased myelosuppression from azathioprine or thioguanine treatment. • TPMT*3C is the most common genetic variant found in Asian populations. WHAT THIS STUDY ADDS • TPMT*26 (208FL) is a novel mutation, previously not reported, which is associated with reduced TPMT activity in three siblings in an Asian Chinese family. • This mutation is associated with increased sensitivity to azathioprine clinically. AIMS Azathioprine, mercaptopurine and thioguanine are commonly used to treat autoimmune disorders, leukaemia and solid organ transplantation. However, azathiopurine and its metabolites can also cause adverse reactions such as myelosuppression. These manifestations may be attributed to polymorphisms or mutations in the thiopurine methyltransferase (TPMT) gene that might result in low TPMT enzyme activity. Our aim was to investigate if azathioprine-related myelosuppression is associated with TPMT polymorphism, which in turn affects its enzyme activity. METHODS A 61-year-old Chinese man with severe atopic eczema developed moderate myelosuppression with standard doses of azathioprine. His TPMT activity was measured using radiochemical assay. Genotyping of TPMT *3C, *3A and *6 were screened using polymerase chain reaction-restriction fragment length polymorphism. Novel mutation was detected by sequencing. Family studies of his three other siblings were performed. RESULTS After 4 weeks of azathioprine treatment, the patient's white blood cells and absolute neutrophil count dropped by 40–45%. He was then taken off azathioprine, and blood counts returned to normal. TPMT activity test showed intermediate levels of 9.1 nmol h−1 ml−1 peripheral red blood cells (pRBC). Resequencing of the TPMT gene revealed a missense mutation PheLeu at 208 aa position in exon 9 (ss105107120). Two of his three siblings were heterozygous for 208FL, which accounts for the decreased enzyme activity (brother 8.9 nmol h−1 ml−1 pRBC, sister 8.8 nmol h−1 ml−1 pRBC). The remaining sibling had wild-type allele with normal enzyme activity. Screening of 100 normal healthy Chinese subjects did not reveal any individual with this mutation. CONCLUSION We report a novel mutation TPMT*26 (208FL) associated with a decrease in TPMT enzyme activity.

To study genetic epidemiology of childhood acute lymphoblastic leukemia (ALL) in the Chinese and Malays, we investigated 10 polymorphisms encoding carcinogen- or folate-metabolism and transport. Sex-adjusted analysis showed NQO1 609CT significantly protects against ALL, whilst MTHFR 677CT confers marginal protection. Interestingly, we observed that NQO1 609CT and MTHFR 1298 C-allele have greater genetic impact in boys than in girls. The combination of SLC19A1 80GA heterozygosity and 3'-TYMS -6bp/-6bp homozygous deletion is associated with reduced ALL risk in Malay boys. Our study has suggested the importance of gender and race in modulating ALL susceptibility via the folate metabolic pathway.

Mutation screenings, which were conventionally carried out individually because of different assay conditions, are usually time consuming and not cost effective. Using microarray technology, simultaneous molecular diagnosis of multiple mutations on a single platform is possible. To evaluate this idea, we developed a DNA chip platform to simultaneously detect 23 mutations of the beta-globin gene and 9 mutations of thiopurine methyltransferase (TPMT) gene based on the principle of arrayed primer extension (APEX). A blinded test consisting of 200 DNA samples with known genotypes was performed to validate this strategy. High genotyping accuracy of 97.3% and 100% for beta-globin and TPMT genes, respectively, were achieved. Further analysis on the fluorescent intensity demonstrated clear separation between the real signal and the background noise, which enabled us to set two cutoff values (V(lower) = 4.0 and V(upper) = 12.0) to determine the genotype quantitatively. Our results showed that APEX is a highly reliable genotyping strategy to detect mutations that cause beta-thalassemia or TPMT enzyme deficiency.

The purpose of this study was to determine the frequency of thiopurine methyltransferase (TPMT) polymorphisms in a multiracial Asian population and to assess its relevance in the management of childhood acute lymphoblastic leukemia (ALL). Six hundred unrelated cord blood samples from 200 Chinese, Malay, and Indian healthy newborns were collected at the National University Hospital, Singapore; an additional 100 children with ALL were analyzed for five of the commonly reported TPMT variant alleles using polymerase chain reaction/restriction fragment length polymorphism and allele-specific polymerase chain reaction-based assays. In the cord blood study, the TPMT*3C variant was detected in all three ethnic groups; Chinese, Malays, and Indians had allele frequencies of 3%, 2.3%, and 0.8%, respectively. The TPMT*3A variant was found only among the Indians at a low allele frequency of 0.5%. The TPMT*6 variant was found in one Malay sample. Among the children with ALL, two white and one Chinese were heterozygous for the TPMT*3A variant and showed intermediate sensitivity to 6-mercaptopurine during maintenance therapy. Three Chinese patients and one Malay patient were heterozygous for the TPMT*3C variant. Mercaptopurine sensitivity could be validated in only one out of four TPMT*3C heterozygous patients. The overall allele frequency of the TPMT variants in this multiracial population was 2.5%. The TPMT*3C was the most common variant allele; TPMT*3A and TPMT*6 were rare. These results support the feasibility of performing TPMT genotyping in all children diagnosed with acute leukemia to minimize toxicity from thiopurine chemotherapy.

Despite successful application of risk-adapted treatment for pediatric ALL, about 20% of patients still relapse. And a substantial portion of such patients lack any conventional high-risk (HR) feature. As contemporary chemotherapy involves multiple drugs, treatment failure may attribute to the genetic variations in genes responsible for drug metabolism and transport. In this study, we established a SNP-based predictor to prognosticate patients enrolled on Malaysia-Singapore ALL 2003 (MS2003) and 2010 (MS2010) Studies. A cohort of 310 MS2003 patients was initially genotyped using Affymetrix DMET™ Plus Array. A nested Random Forests was designed to evaluate the association between the genotypes and cumulative incidence of relapse (CIR, including resistance). Shortlisted candidates were confirmed by competing risk regression adjusting for known prognostic factors. Such candidate SNPs were subsequently genotyped in additional 242 MS2003 patients and 106 MS2010 patients for further validation. A scoring metric indicating the number of relapse-associated genotypes in a patient was used to predict outcome. (Fig. 1) Of 1936 markers interrogated by DMET platform, 410 SNPs and InDels were eligible for downstream analyses after quality control. Random Forests identified 16 CIR-associated SNPs, 9 of which were verified in regression model and selected for further genotyping. Subsequent validation on all MS2003 patients (N=519) confirmed 3 risk genotypes that were associated with higher 5-yr CIR: A/A of rs4534 in CYP11B1 (P=3.10x10-3, HR=2.34 (95% CI 1.33-4.12)), C/T and T/T of rs1056837 in CYP1B1 (P=3.30x10-4, HR=2.66 (95% CI 1.56-4.53)), and T/T of rs3803390 in SLC28A1 (P=6.60x10-3, HR=2.18 (95% CI 1.24-3.82)). The number of risk genotypes was shown to strongly correlate with 5-yr CIR after adjusting presenting features including age, WBC and ALL subtype (P=6.70x10-8; Table 1). The risk was nearly 7-fold higher in patients with 2 risk genotypes compared to those without any risk genotype (P=2.00x10-8; Table 1 and Fig. 2a; there was no patient with all 3 risk genotypes). Further adjustment for Day 33 MRD did not alter the significance. Even in non-HR patients (without BCR - ABL1 fusion, MLL rearrangements, hypodiploidy, or Day 33 MRD>=1x10-2), this scoring metric remained significant (P=2.60x10-3 for incremental effect; P=1.20x10-3 for 2 risk genotypes vs. no risk genotype; Table 1 and Fig. 2b). Concordantly, increasing number of risk genotypes was also associated with a higher probability of Day 33 MRD-HR (i.e. MRD>=1x10-2; 6.5% of patients without any risk genotype vs. 9.8% of patients with 1 risk genotype vs. 29.0% of patients with 2 risk genotypes; P

BACKGROUND ALL is the most common form of childhood cancer with >80% cured with contemporary treatment protocols. Accurate risk stratification in childhood ALL is essential to avoid under- and over-treatment. Currently, we use presenting clinical, biological features, and minimal residual disease (MRD) quantitation to risk stratify patients. Although whole genome gene expression profiling (GEP) can accurately classify patients with ALL into various WHO 2008 defined subgroups, its value in predicting relapse remained to be defined. We hypothesized that global time-series GEPs of bone marrow (BM) samples at diagnosis and specific points during initial remission-induction therapy can measure the success of cytoreduction and be used for relapse prediction. METHODS We generated time-series GEPs from 181 children with de novo ALL at diagnosis and Day 8 of remission-induction therapy. We computed the time-series changes from diagnosis to Day 8 of remission-induction, termed Effective Response Metric (ERM-D8; Fig 1), that measures both the magnitude and direction of time-series change in multi-dimensional gene space towards the normal centroid, and we compared its ability to predict relapse against contemporary risk assignment methods including NCI criteria, cytogenetics and MRD. RESULTS Unsupervised hierarchical clustering of the first 96 patients revealed that 30.3% (10 of 33) patients whose diagnosis and day 8 GEP pairs clustered together in the same branch suffered a relapse compared to 7.5% (4 of 53) who clustered to different branches. This suggests that patients with minimal changes in GEP after 8 days of remission-induction therapy have a 4-fold significantly higher risk of relapse (p=5.4x10-3). To test this observation, we extended the study to 181 ERM-D8 in 8 batches. Using leave-one-batch-out cross-validation, we found that ERM-D8 is predictive of relapse; patients with favorable ERM-D8 (n=115) had a significantly lower 14.5% 5-year cumulative risk of relapse (CIR) compared to 44.5% with unfavorable ERM-D8 (n=66; p= 5.1x10-6; Fig 2). In multivariate analysis, ERM-D8 remained significant (Table 1; p=7.9x10-4, HR 3.19 [1.62-6.29]) after controlling for early clinical features of age (p= 0.37), WBC (p=0.05), cytogenetics (p=0.021) and Day 8 peripheral blast response (p=0.96). Even after adjusting for Day 33 MRD (p=5.4x10-3), ERM-D8 remained independently predictive of relapse (Table 1; p=8.5x10-3, HR 2.63[1.28-5.42]). ERM-D8 helped further refine Day 33 MRD risk stratification. Day 33 MRD negative patients with unfavorable ERM-D8 (n=29) had a 5 fold higher risk of relapse compared to patients with favorable ERM-D8 (n=60; 5-year CIR 21.7% vs 4.4; p=0.021). Similarly Day 33 MRD positive patients with unfavorable ERM-D8 (n=33) have double the risk of relapse compared to those with favorable ERM-D8 (n=45; 5-year CIR 62.8% vs 29.2%; p=1.01x10-3). ERM-D8 also improved risk stratification in patients with favorable cytogenetics. Favorable cytogenetics patients with unfavorable ERM-D8 (n=31) do significantly poorer than those with favorable ERM-D8 (n=51; 5-year CIR 36.4% vs 2.4%; p=1.04x10-4). CONCLUSION We describe a novel metric based on time-series GEP during first 8 days of remission-induction therapy that is independently predictive of risk of relapse even after adjusting for age, WBC, NCI risk, cytogenetics and MRD. | | Without Day 33 MRD | With Day 33 MRD | | --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | ------------------ | --------------- | --------- | | p-value | HR | 95% CI | p-value | HR | 95% CI | | ERM-D8 unfavorable vs favorable* | 7.9x10-4 | 3.19 | 1.62~6.29 | 8.5x10-3 | 2.63 | 1.28~5.42 | | Cytogenetics unfavorable vs favorable and others** | 0.021 | 2.45 | 1.14~5.26 | 0.23 | 1.69 | 0.72~3.98 | | Age, years 10 vs 1~10 | 0.37 | 1.39 | 0.67~2.89 | 0.43 | 1.35 | 0.64~2.84 | | WBC, x109/L over 50 vs below 50 | 0.05 | 1.99 | 1.00~2.96 | 0.25 | 1.69 | 0.69~4.13 | | Day 8 Response poor vs good | 0.96 | 1.02 | 0.40~2.59 | 0.65 | 0.81 | 0.32~2.06 | | Day 33 MRD >=1.0x10-2 vs

Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer with excellent treatment outcome where >80% are cured. However, relapse and therapy-related toxicities limit further improvements and greatly increase the cost of therapy. Vincristine (VCR) is cheap, well tolerated, and highly effective. Using VCR optimally will help improve the cost-benefit ratio favorably by allowing us to reduce toxicities like infections from myelosuppression and yet improving cure. The highly successful BFM-ALL treatment backbone starts with a single intrathecal methotrexate on Day 1 followed by 7 days of oral prednisolone (PRED). The persistence of absolute blasts count >1,000/µL at Day 8 (D8), known as PRED poor response, confers a significantly poorer treatment outcome. To avoid seeding the CNS with leukemia from traumatic taps, the new Ma-Spore ALL 2010 treatment protocol, omitted intrathecal methotrexate at Day 1 and replaced with VCR at Day 0. By June 2013, a total of 133 patients have been enrolled. We found that the number of poor PRED responders was halved from the historical 9.5% in the previous Ma-Spore ALL 2003 study (Yeoh et al. J Clin Oncol 2013) to only 4.7% of patients in the ALL 2010 study. In addition, the percentage of MRD standard risk patients (Day 33 blast count ≤1x10-4) increased from 38.9% in the Ma-Spore ALL 2003 to 51.8% in the Ma-Spore ALL 2010 study (P We investigated VCR and PRED combination in PRED and VCR-resistant (VCR-R) cell lines. Specifically, REH cell line is intrinsically resistant to PRED in vitro because of a mutation in its glucocorticoid receptor. We exposed the REH cell line to increasing concentrations of VCR over 6 months and generated a VCR resistant REH cell line (Fig. 2). This VCR-R REH cell line is resistant to both PRED or VCR when exposed individually in vitro. However when exposed to both PRED and VCR in combination, only 30% of the resistant cells survived (P We found that the drug efflux transporter multi-drug resistance protein 1 (MDR1) was preferentially highly expressed in our VCR-R cell line models. To determine if the highly expressed MDR1 is responsible for treatment resistance, we exposed the VCR-R cell lines to VCR, verapamil (an MDR1 inhibitor) and combination of both VCR and verapamil. The combination of VCR and verapamil increased the G2 cell cycle arrest by 3- folds compared to when VCR was used alone (Fig. 3), supporting the role of MDR1 in treatment resistance. Interestingly we also found that the combination of VCR and PRED led to a decrease in levels of MDR1 expression by western blot, suggesting that depletion of MDR1 may be a mechanism through which VCR and PRED combination therapy enhances leukemic cell killing. We also investigated microenvironment-mediated resistance to VCR and PRED using mesenchymal stromal cells (MSC) co-culture systems. It was found that after co-culture with MSC or in conditional medium containing soluble factors secreted by MSC, leukemic cells were resistant to VCR and PRED mono-treatment, but the resistance was abrogated after combinatorial therapy. In conclusion, VCR in combination with PRED improves D8 peripheral blood treatment response during early induction in our Ma-Spore 2010 trial. This synergistic combination results from its ability to reverse resistance from intrinsic overexpression of MDR1 in resistant leukemia cells and decrease microenvironment-contributed resistance by mesenchymal cells. Disclosures: No relevant conflicts of interest to declare.

Contemporary acute lymphoblastic leukemia (ALL) therapy involves the use of powerful anti-mitotic agents such as vincristine (VCR). VCR targets the microtubule cytoskeleton and disrupts important cellular processes, leading to cell death. Nonetheless, around 20% of patients relapse and become resistant to most drugs. At present, mechanisms of resistance toward VCR remain elusive despite its extensive clinical use, warranting a more in-depth study with respect to ALL. Five VCR-resistant (VCR-R) subclones were established by gradually exposing five ALL VCR-sensitive (VCR-S) cell lines (Jurkat, REH, SEM, RS4;11, 697) to incremental doses of VCR. Comparing resistant to sensitive cells, the IC50 at 48h increased 3-6 logs. Both VCR-S and VCR-R cell lines were subjected to cell cycle analysis by flow cytometry using propidium iodide staining. After a 48h exposure to VCR, cell death for VCR-S cells was the result of a typical G2/M phase arrest. In contrast, a G2/M phase arrest did not precede the VCR-induced cell death of VCR-R cells. VCR-R subclones were also observed to be more resistant to vinblastine, an alternative tubulin binding agent. In addition, there was increased resistance towards other chemotherapeutic drugs such as prednisolone, dexamethasone, daunorubicin and doxorubicin, indicating that resistance to VCR may contribute to cross-resistance after a prolonged incubation. The PI3K/Akt/mTOR signaling pathway is known to play important roles in cancer cell proliferation and survival. The combination of inhibitors with other chemotherapeutic agents also has the potential to enhance leukemic cell death. To further investigate if the PI3K/Akt/mTOR signaling pathway is involved in VCR resistance, we screened a panel of twelve kinase inhibitors (Merck) which selectively targets this pathway. Interestingly, PDK1/Akt/Flt Dual Pathway Inhibitor KP372-1, was highly potent toward all cell lines with IC50 less than 100nM. Our results also showed that the administration of PI3Kα Inhibitor VIII, Akt Inhibitor IV and PI-103, as well as pan-selective PI3K inhibitors Wortmannin and LY294002 in combination with VCR was able to promote increased levels of cell death. Cell cycle analysis also revealed that the combination treatment elicits different methods of cell death between VCR-S and VCR-R cells. This is especially evident upon the co-treatment with pan-selective inhibitors. Optimising the sensitivity of ALL cells and/or reversing resistance to VCR promise to maximise the therapeutic value of this important chemotherapy. This study provides evidence that VCR resistance potentially contributes to cross-resistance. Our data also shows that the PI3K-Akt pathway may be involved in VCR resistance in ALL, providing a valid rationale for further studies and the future application of inhibitors in clinical settings. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 822. doi:1538-7445.AM2012-822

Introduction: With the improvement of treatment outcome in pediatric acute lymphoblastic leukemia (ALL), about 80% of the patients can be cured. However, around 10% of patients have poor outcome due to drug resistance which calls for novel drugs such as BH3-mimetics. We have previously demonstrated that GX15-070 (GX), a BH3-mimetic, could effectively induce cell death. In this study, we investigate the mechanisms of GX induced cell death in ALL cells. Method: Seven ALL cell lines were used in this study. Cell viability was determined by MTS assay (Promega). Western blot was used to detect protein expression level. Cholesterol (CHO) level was measured by Amplex® Red Cholesterol Assay Kit (Promega). RT-PCR was done by Roche Light Cycler using SYBR Green. Results: There was a dose-dependent cell death induced in all 7 ALL cell lines after treatment with GX. Interestingly, genes that are involved in the CHO synthesis pathway such as LDLR, HMGCR, HMGSC1 and SQLE were upregulated after GX treatment. Furthermore, GX mediated reduction in cellular CHO levels was observed. To verify that CHO depletion could induce cell death in ALL cells, Lovastatin was applied. Our results showed that Lovastatin not only induced a dose-dependent cell death in all 7 ALL cell lines but also upregulated the genes mentioned above. Co-treatment of 0.1µM GX and 25µM Lovastatin could enhance the cell death by further decreasing CHO level. In addition, GX-induced cell death could also be enhanced by 10mM 2-Deoxy-D-glucose (2-DG) co-treatment. 2-DG is an inhibitor of glycolysis which provides metabolites for CHO metabolism. Next, to mimic a culture condition whereby CHO is depleted, we incubated the cells in HBSS without Fetal bovine serum (FBS) for 24h. This CHO depleted condition decreased the cellular CHO level to 46% compared to the cells that were cultured in 10% FBS and RPMI medium. After incubation in HBSS for 24h, 0.1µM GX could eliminate more cells (80%) compared to the same dose of GX in serum supplemented RPMI medium (20%). Both HBSS incubation and GX treatment caused a decrease in anti-apoptotic protein Mcl-1 expression levels, while pro-apoptotic protein BIM and cleaved-PARP protein levels were upregulated. The two treatments did not alter the protein levels of other Bcl-2 family members. These results indicate that Mcl-1 expression is tightly controlled by cellular metabolism and CHO depletion can result in cell death. The mechanism of GX induced cell death may entail the suppression of Mcl-1 expression, hence sensitizing ALL cells to apoptosis. Conclusion: GX can regulate CHO metabolism to induce cell death in ALL cells which are normally dependent on aerobic glycolysis. This highlights the possibility of manipulating CHO metabolism to provide a specific way to treat leukemic cells. The administration of GX in the clinic, alone or in combination, may improve the treatment outcome of leukemia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3227. doi:1538-7445.AM2012-3227

beta-Thalassaemia major patients have chronic anaemia and since 3-4 per cent of Singaporeans carry the beta-gene, prenatal diagnosis is essential. We evaluated the amplification refractory mutation system (ARMS) technique as a routine test for prenatal diagnosis of beta-major. Six mutations along the beta-gene were studied--41-42 (-TCTT), IVSII #654 (C-T), 17 beta (A-T), -28 TATA (A-G), IVSI #5 (G-C), and IVSI #1 (G-T). Our results indicate that prenatal diagnosis using these mutations can be offered to 90 per cent (35/39) of our Chinese couples and 54.6 per cent (12/22) of our Malay couples at risk. Confirmation of ARMS results was carried out using allele-specific oligonucleotide hybridization. Prenatal diagnosis using ARMS was successfully carried out in nine cases which included a set of triplets and twins. The triplets were diagnosed with the beta-trait carrying the 41-42 mutation. The couple with twins possessed the #654 mutation and one twin was diagnosed with the beta-trait and the other with #654 homozygosity. Genomic sequencing of the undefined mutations in the Chinese couples revealed rarer mutations at -29 and an ATG-AGG base substitution at the initiation codon for translation. In the Malay couples, genomic sequencing detected mutations at codon 15 (TGG-TAG) and codon 26 (GAG-AAG). We conclude that ARMS with its direct detection of amplified products by gel electrophoresis provides an accurate, rapid, and simpler method for our beta-thalassaemia prenatal diagnosis programme in Singapore.

Abstract 567FN2 The successful incorporation of risk-associated factors in contemporary treatment regimens has remarkably improved the treatment outcome of pediatric ALL. Currently, the most commonly used prognostic factors such as the age and white blood cell (WBC) count which are tightly correlated with certain cytogenetic/molecular subtypes, are mainly related to leukemic factors. Beyond that, host pharmacogenetic factors, i.e., how the host genetic makeup influences the metabolism of chemotherapeutic agents, are expected to have additional but smaller impact on treatment outcome. Modern, successful multi-agent chemotherapy incorporates more than 8 different chemotherapeutic agents to treat pediatric ALL. Variability in individual patients in handling these drugs arises from their pharmacogenetic factors. However, such pharmacogenetic variants are rarely evaluated in existing prognostication schema. We studied both tumor- and host pharmacogenetic factors to determine the impact of germline variations on the treatment outcome in the Malaysia-Singapore (Ma-Spore) ALL 2003 study essentially guided by the MRD-risk stratification. A total of 463 patients were included, with a median follow-up of 3.8 years (0-8.2). Genotyping was carried out for 20 germline polymorphisms in 11 genes (i.e., GSTM1, GSTT1, GSTP1, NQO1, MTHFR, MTHFD1, SLC19A1, ABCB1, TYMS, CCR5, and IL15). The genotypic influence on the event-free survival (EFS) probability was estimated by Cox regression, adjusted for patients' characteristics (including the ethnicity, sex, age, lineage, and initial WBC count) and cytogenetic subtypes. An event was defined as any of: induction failure (N=20), relapse (N=32, any site) or death (N=25, any cause). Only cytogenetic subtypes showed significant impact on the EFS (P=0.015). After adjusting for host-related and leukemic factors, ABCB1 3435C>T (rs1045642), CCR5 246A>G (rs1799987), and IL15 67276493G>C (rs17015014) were also found to significantly and independently affect the survival probabilities. Specifically, ABCB1 3435T/T and CCR5 246G/A predicted lower EFS probabilities compared to 3435C/C and 246G/G, respectively (P=0.012, HR=2.496, 95%CI=1.223-5.092, Fig.1a and P=0.033, HR=1.820, 95%CI=1.051-3.153, Fig.1b), while IL15 67276493G/C was associated with a higher survival probability compared to 67276493G/G (P=0.030, HR=0.545, 95%CI=0.315-0.942, Fig.1c). In addition, the risk increased with increasing number of risk genotypes (i.e., ABCB1 3435T/T, CCR5 246G/A, and IL15 67276493G/G, which were associated with the worst survival respectively; Fig.1d), implicating a cumulative adverse effect (P Further analyses revealed that compared to the reference genotypes respectively, ABCB1 3435T/T particularly correlated with the highest risk of relapse (P=0.011); CCR5 246G/A was associated with a higher risk of failure of induction therapy (including induction failure and death during induction; P=0.022); and IL15 67276493G/C conferred a protection from septic death (P=0.041). We subsequently investigated whether these polymorphisms could still influence the outcome in cytogenetic groups with known prognosis. ABCB1 3435C/T and T/T in combination were predictive of a poorer survival in 184 patients with favorable subtypes including t(12;21), t(1;19), and hyperdiploidy (P=0.027, HR=5.271, 95%CI=1.211-22.944); CCR5 246G/A appeared to retain its significance in 29 patients with unfavorable subtypes including t(11q23)/MLL rearrangements and t(9;22) (P=0.052, HR=7.708, 95%CI=0.983-60.471); and in 229 patients without common chromosomal abnormalities, IL15 67276493C/C was associated with the best survival (P=0.027, HR=0.291, 95%CI=0.098–0.868). Our results suggest that risk assignment for contemporary ALL treatment can be refined by incorporating the host pharmacogenetic profile into existing strategy of risk stratification that uses mainly leukemic factors. Disclosures: No relevant conflicts of interest to declare.

Background: Acute lymphoblastic leukemia (ALL) is the most common form of cancer in children. Although considered highly curable, about 20% of children with ALL relapse and majority of them will die of the resistant disease. Sensitivity to the glucocorticoid, prednisolone (PRED), is critical for cure and the development of combination therapy with PRED may improve treatment outcome. Triptolide, a diterpene triepoxide extracted from the Chinese plant Tripterygium wilfordii has been reported to be immunosuppressive, anti-inflammatory, and anti-proliferative in a broad spectrum of diseases. In this study, we investigate the effect of triptolide to induce apoptosis in ALL cells in combination with prednisolone. Methods: Three clinical important leukemia cells lines 697, Sup-B15 and RS4;11 that represent TCF3-PBX1, BCR-ABL1 and MLL-AF4 respectively were used. They were exposed to triptolide as well as in combination with PRED. Cell viability was determined by MTT assay (Promega). Cell Death Detection ELISA (Roche) was used for measuring apoptosis levels. Western blot was used to detect protein expression levels. Caspase-3 and -9 activities were measured using Caspase-Glo Assay kits (Promega). Cell cycle analysis was performed by Propidium Iodide (PI) staining using flow cytometry. Results: Triptolide can induce cell death at ED50 of 15nM for 697, 13nM for Sup-B15, and 13nM for RS4;11 at 24h time point. The ED50 further decreased to 3nM for 697, 4nM for Sup-B15 and 5nM for RS4;11 at 48h time point. Apoptosis induction and cell cycle arrest was observed upon triptolide treatment in these three cell lines. The levels of caspase-3 and caspase-9 were significantly increased with triptolide treatment, suggesting that triptolide induced apoptosis was caspase-dependent. Although PRED treatment as a single agent can also induce apoptosis, cell cycle arrest and caspase activation in these three cell lines, the co-treatment of PRED and triptolide significantly enhanced these effects compared to single treatment (p Conclusion: Triptolide can induce apoptosis in ALL cell lines. Combined therapy with PRED and triptolide is a novel promising therapy for this hematological malignancy that warrants further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2563. doi:10.1158/1538-7445.AM2011-2563

Glucocorticoids such as prednisolone (PRED) are widely used in the treatment of pediatric acute lymphoblastic leukemia. In PRED-induced apoptosis, Bcl-2 family members play important regulatory roles. However, the exact members involved remain unknown. In this study, the roles of Bcl-2 family members in PRED-induced apoptosis and their prognostic value to day 8 PRED response are evaluated.Four clinically important acute lymphoblastic leukemia cell lines, three PRED-sensitive (697, Sup-B15, and RS4;11) and one PRED-resistant (REH) were studied. Thirty paired patient bone marrow samples were obtained at diagnosis (day 0) and after 7 days (day 8) of PRED monotherapy. Twenty-five patients had PRED good response and five PRED poor response. Differential expressions of Bcl-2 members were observed in those samples and BIM was further investigated using gene silencing technology in representative cell line Sup-B15.The proapoptotic BH3-only Bcl-2 family member BIM was upregulated only in PRED-sensitive cells. Receiver operating characteristic curve analysis showed that BIM expression was highly predictive of PRED response (area under the curve = 0.81; p = 0.032) in paired patient bone marrow samples and is, most excitingly, independent of molecular subtype. Patients whose BIM protein expression levels fail to upregulate at day 8 compared to day 0 (D8/D0 ratio0.93) have significantly poorer event-free survival (60%) than those patients whose BIM protein expression levels did upregulate (92%). By silencing BIM in PRED-sensitive cells, PRED-induced apoptosis was inhibited.Upregulation of BIM by PRED in acute lymphoblastic leukemia cells regardless of molecular subtype is significantly prognostic of outcomes, confirming BIM's essential regulatory role in the PRED-induced apoptosis.

Abstract 1850 Introduction: Resistance to treatment remains the most important cause of relapse in contemporary acute lymphoblastic leukemia (ALL) therapy which calls for novel drugs to improve treatment outcome. We have shown previously that single agent treatment of BH3-mimetics like ABT-737 and obatoclax (GX15-070) resulted in a dose dependent apoptotic cell death and synergistic with prednisolone. However, little is known about the mechanisms and genomic responses underlying these BH3-mimetics. Since ABT-737 is a Bad-like mimetic while obatoclax is a Bim-like mimetic, we hypothesized that combination of these two different BH3-mimetics will increase the efficacy of cell death and enable reduced doses. Methods: Seven ALL cell lines and a chronic myeloid leukemia cell line (K562) were used in this study. The 8 cells lines were exposed to ABT-737 or obatoclax as well as a combination of both and then subjected to whole genome gene expression analysis using Affymetrix HGU133 Plus 2.0 microarrays. The sets of differentially expressed genes were subsequently used for pathway analyses to identify the associated network functions using Ingenuity software. Cell viability was determined by MTS assay (Promega) and synergism was calculated using CalcuSyn software version 2.1. Western blot was used to detect protein expression level changes of the BCL-2 family members and cleaved PARP. Caspase-3,-8 and -9 activities were measured using Caspase-Glo™ Assay kits (Promega). Results: Treatment of both ABT-737 and obatoclax resulted in a dose dependent cell death in all 8 cell lines at 24h time point. All 7 ALL cell lines were sensitive to ABT-737 treatment with IC50 ranging between 0.05μM and 1.6μM, while K562 was less sensitive, with an IC50 of 31μM. All the 8 cell lines were sensitive to obatoclax treatment with similar IC50 ranging between 0.6μM and 5.7μM. Simultaneous in vitro exposure of ABT-737 and obatoclax to all cell lines in a 1:10 ratio resulted in synergistic levels of cell death with combination index (CI) values that are distinctly less than one. The levels of cleaved PARP and caspases -3, -8 and -9 activities increased significantly after combination treatment compared to individual treatment of each chemical. Interestingly, we did not observe any change in protein levels of Bcl-2 family members (including Bid, Bim, Bax, PUMA, Bcl-2, Mcl-1 and Bcl-w) after individual or combination treatment, indicating that the mechanism of synergism may be independent of the regulation of Bcl-2 family members. Groups of differentially regulated probe sets (p Conclusion: In this study, we reported that synergism of ABT-737 and obatoclax could be achieved in all 8 leukemia cell lines. Gene microarray analysis suggests that leukemia cells differ in their genomic response to the two drugs, and combination gains over single treatment. Although ABT-737 exhibits higher potency, its sensitivity seems to be cell-type dependent. The administration of BH3-mimetics in the clinic, alone or in combination, may overcome the limitations of single agent treatment and improve the treatment outcome of leukemia. Disclosures: No relevant conflicts of interest to declare.

Chemotherapeutic regimens for pediatric acute lymphoblastic leukemia (ALL) treatment entail the use of glucocorticoids such as prednisolone (PRED). Around 5% of patients fail to attain remission due to drug resistance. We have previously shown that the pro-apoptotic BH3-only BCL-2 family member BIM was up-regulated in only in PRED-sensitive cells. After the specific silencing of BIM by siRNA, inhibition of apoptosis was observed and validated by levels of cleaved PARP and caspase activity, providing a clear indication of BIM's significance in PRED induced apoptosis. In this study, protein expression levels of BIM were further evaluated using patient bone marrow. We also examined the ability of two BH3 mimetics, GX15-070 (obatoclax) and ABT-737, to induce apoptosis in ALL cell lines and the effect of drug combinations. Patient bone marrow samples were obtained at diagnosis (Day 0) and after 7 days (Day 8) of PRED monotherapy. Response to PRED was defined as Good (PGR, peripheral blast count < 1000 /µl), and Poor (PPR, peripheral blast count ≥1000 /µl). Twenty-five PGR and five PPR patients were included in this study. Using western immunoblotting, the protein expression of BIM was reproducibly observed in patient samples. The value of ODBim/ODActin at Day 8 was significantly higher than that on Day 0 (p-Value=0.0006) for PGR patients. In contrast, this value did not differ greatly in PPR patients. At Day 0, BIM expression levels of the entire patient cohort was comparable, but levels were significantly different at Day 8 (p-Value=0.01). These results indicate that the upregulation of BIM was induced by PRED in ALL patients and further confirms the essential role of BIM in this apoptotic pathway. Treatment with GX15-070 and ABT-737 as single agents yielded an increase in cell death in a dose dependent manner both in PRED-sensitive and -resistant cell lines. Apoptosis induced by both GX15-070 and ABT-737 could be inhibited by caspase-9 inhibitor (Z-LEHD-FMK) and caspase-3 inhibitor (Z-DEVD-FMK). Simultaneous exposure of ALL cells to PRED and GX15-070 or ABT-737 resulted in synergistic levels of cell death in SUP-B15 and RS4;11 cell lines with combination index (CI) values that are significantly less than one (as calculated by CalcuSyn software version 2.1). This highlights the possibility of enhancing cell death in ALL cells even with a reduction in drug dosage. In conclusion, BH3-only BCL-2 family member BIM, is vital for PRED-induced apoptosis in ALL. BH3 mimetics GX15-070 and ABT-737 can induce apoptosis in ALL cells regardless of PRED response. The synergism between PRED and BH3-mimetics is likely to minimize severe side effects, and more importantly, provide alternative therapies especially for patients that are resistant to conventional chemotherapeutic drugs in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1033.

The loss of mitochondrial membrane permeability is central to apoptosis. The mitochondrial permeability transition pore (PTP) complex, comprising of at least voltage-dependent anion channel 1 (VDAC1), adenine nucleotide transporter (ANT) and cyclophilin D (CyD), mediates the permeability of the mitochondrial membranes. We investigated the involvement of VDAC1, ANT and CyD in prednisolone (PRED)-induced apoptosis in 2 ALL cell lines: Sup-B15 (sensitive) and REH (resistant). After 24h exposure to PRED (10.0µg/ml), VDAC1 protein was consistently upregulated only in PRED-sensitive cell line, but remained unchanged in the resistant one. Co-administration of non-specific VDAC1 inhibitor, 4, 4′-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) with PRED, blocked PRED-induced apoptosis and caspase-3 activity only in sensitive cell line. Surprisingly, complete VDAC1 silencing using siRNA (Dharmacon) failed to abrogate PRED-induced apoptosis, suggesting that VDAC1 is not critical. We next investigated the other members of the PTP complex i.e., ANT and CyD. Co-administration of PRED and bongkrekic acid (BA) a specific inhibitor of ANT, also resulted in a dose-dependent inhibition of apoptosis and the caspase-3 activity. The combination of DIDS and BA, even at reduced doses by 60% and 96% respectively, is highly synergistic in inhibiting PRED-induced apoptosis and surprisingly reduced VDAC1 upregulation in PRED-induced apoptosis, unlike each agent alone. PRED did not alter the level of CyD protein and administration of specific CyD inhibitor, cyclosporine A (CsA), did not affect PRED-induced apoptosis. We conclude that ANT protein is central in the mitochondrial PTP mediated apoptosis during PRED treatment of ALL; VDAC1 and CyD are dispensable. ANT is upstream to VDAC1 and it upregulates VDAC1 during PTP activation in PRED-induced apoptosis. Our investigation provides the basis for looking at the role of mitochondrial PTP complex proteins especially ANT in PRED treatment in childhood ALL. This may provide new avenues to reverse resistance to therapy in patients with relapsed ALL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1197.

Abstract 2624 Poster Board II-600 Although childhood ALL is the prototype for a drug-responsive malignancy, some patients relapse or develop severe toxicity due to significant inter-individual variability. Leukemic factors like the molecular subtype account for the majority of the variation in prognosis; some studies have suggested that polymorphisms in key genes involved in drug transport, uptake, metabolism or targeting are responsible for smaller magnitude of variance in terms of therapeutic efficacy. Thus far, genetic characteristics are not widely applied in modern risk-stratifying system for ALL patients. And due to the significant differences of ethnic genotype/allele frequencies, the variance is probably much larger than leukemic factors alone. We integrated molecular and pharmacogenetic profiles to assess the contribution of germline variations in early treatment response using minimal residual disease MRD status in the Malaysia-Singapore ALL 2003 study. Three hundred and seventy children with ALL (200 Chinese and 170 Malays) were included and tested for t(12;21), t(9;22), t(1;19) and t(11q23)/MLL rearrangements. Genotypes of 20 polymorphisms in 11 different candidate genes were collected (Phase II enzymes: GSTM1, GSTT1, GSTP1, NQO1; folate metabolism: MTHFR, MTHFD1, TYMS, SLC19A1; transporter: ABCB1; candidates reported in other studies: CCR5, IL15). MRD levels were measured by real-time quantitative PCR at Week-5 (TP1, end-of-induction) and Week-12 (TP2) after the start of therapy. Chinese and Malays were analyzed separately to minimize confounding effect as most studied polymorphisms had significantly different genotype/allele frequencies between the two ethnic groups. All genotypes, together with patient characteristics of sex, leukemia lineage, molecular subtype and NCI risk group, were included as variables to build a prediction model using Classification and Regression Tree (CART) analysis. Patients with ≥0.1% TP1 MRD and ≥0.01% TP2 MRD had significantly shorter event-free survival (EFS) compared to those with undetectable MRD level ( Disclosures: No relevant conflicts of interest to declare.

Background: Several large cohort studies–like from BFM and St Jude groups-have shown that minimal residual disease (MRD) is the single most important predictor of treatment outcome in childhood acute lymphoblastic leukemia. However, lingering concerns about clonal evolution of IgH/TCR rearrangements may result in false-negative MRD. Hence, the AIEOP-BFM ALL 2000 study utilized at least 2 sensitive MRD markers with at least 10−4 sensitivity for risk-stratification. This stringent minimum of 2 sensitive MRD markers criteria limits the applicability of MRD stratification to Methods: A total of 362 patients (B-lineage=333, T-lineage=29) were enrolled in Ma-Spore ALL 2003 from July 2002 to August 2008, with a median age of 5.61 years (range 0.14 to 15.29). The treatment protocol is based on the modified BFM ALL 2000 backbone without randomization. Risk-stratification is based primarily on MRD levels at end of induction (Day-33) and week 12, Day-8 PR, and presence of BCR-ABL or MLL. In patients whom we are unable to quantify MRD, are assigned IR risk group. Screening for IgH/TCR rearrangements is carried out using multiplex BIOMED-2 primers (JJM van Dongen et al 2003) with standardization with the European MRD Study Group, which we are an active member. Selection of MRD markers is based on frequency and stability of IgH/TCR rearrangements. MRD quantifications were carried out using Real-time PCR (LightCycler 1.0, Roche Diagnostics). We define three distinct treatment subgroups: Standard-Risk (SR) who had MRD 1×10−3; remaining patients formed Intermediate-Risk (IR). The SR patients received decelerated therapy while HR group was treated under intensified therapy or bone marrow transplant. Results: Of 362 patients enrolled, Ma-Spore risk stratification is possible in 360 patients (n=2 delayed treatment), out of which 239 have completed 2 years therapy, 15 had induction failures, and 22 had relapses (isolated BM=17, isolated CNS=4, isolated testis=1.) Patients stratified into SR=29% (n=106/360); IR=50% (n=180/360), and HR=21% (n=74/360). At the end of the study, 4-years overall survival (0S) was SR=94.2±2.5%; IR=95.3±1.6%; HR=66.2±6.5%; event-free survival (EFS) was SR=91.8±3%; IR=86.2±3.4%; HR=52.9±6.4%, and leukemia-free survival (LFS) was SR=94.8±2.6%; IR=90.5±3.2%; HR=62.7±6.6%. Within the HR subgroup, using multivariate analysis, high MRD levels at week 12 (>10−3) is significantly associated with adverse treatment outcome (OS=27.5±15.8%, EFS=17.9±14.4%; LFS=23.1±17.8%), whilst poor prednisolone responders alone in the absence of high risk MRD or high risk BCR-AL or MLL-AF4 did well (OS=84.1±7.4%, EFS=84.1±7.4%, LFS= no event). Of the 362 patients, at least 91% (n=309/339) of patients had at least 1 sensitive MRD marker; the remaining patients have no diagnostic samples (n=9), died before time-point 1(n=10), and default before time-point 1(n=4). For risk-stratification using only MRD levels, 45.7% of the patients (n=128/280) were low-risk with MRD10−3 at week 12; and the remaining 51.1% (n=143) were stratified as intermediate-risk MRD. Overall survival in MRD low-risk group was 93.2±2.5%, MRD intermediate-risk was 93.6±2.4% and MRD high-risk MRD was 45±18.8%. Conclusion: We have demonstrated that a simplified MRD methodology using a single PCR-based marker (IgH/TCR) can be successfully implemented to tailor therapy for childhood ALL without adversely affecting outcome. It is cost-effective and can be applied to a larger group of patients (91%). This is particularly useful for countries with limited resources to pursue risk stratification incorporating MRD. Deceleration of therapy in the SR group would also help to reduce long-term side effects and better quality life in these children.