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T-cell acute lymphoblastic leukemia (T-ALL) is frequently characterized by glucocorticoid (GC) resistance, which is associated with inferior outcomes, thus highlighting the need for novel therapeutic approaches for GC-resistant T-ALL. The pre-T-cell receptor (pTCR)/TCR signaling pathways play a critical role in cell fate decisions during physiological thymocyte development, with an interplay between TCR and glucocorticoid receptor (GR) signaling determining the T-lymphocyte selection process. We performed an shRNA screen in vitro and in vivo in T-ALL cell lines and patient-derived xenograft (PDX) samples to identify vulnerabilities in the pTCR/TCR pathway and identified a critical role for the lymphocyte cell-specific kinase (LCK) in cell proliferation. LCK knockdown or inhibition with dasatinib (DAS) caused cell cycle arrest. Combination of DAS with dexamethasone (DEX) resulted in significant drug synergy leading to cell death. The efficacy of this drug combination was underscored in a randomized phase II-like murine trial, recapitulating an early phase human clinical trial. T-ALL expansion in immunocompromised mice was significantly impaired using this drug combination, compared to mice receiving control vehicle or single drug treatment, highlighting the immediate clinical relevance of this drug combination for high-risk T-ALL patients. Our results thus provide a strategy to improve the efficacy of current chemotherapy platforms and circumvent GC resistance.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with inferior outcome compared with that of B cell ALL. Here, we show that Runt-related transcription factor 2 (RUNX2) was upregulated in high-risk T-ALL with KMT2A rearrangements (KMT2A-R) or an immature immunophenotype. In KMT2A-R cells, we identified RUNX2 as a direct target of the KMT2A chimeras, where it reciprocally bound the KMT2A promoter, establishing a regulatory feed-forward mechanism. Notably, RUNX2 was required for survival of immature and KMT2A-R T-ALL cells in vitro and in vivo. We report direct transcriptional regulation of CXCR4 signaling by RUNX2, thereby promoting chemotaxis, adhesion, and homing to medullary and extramedullary sites. RUNX2 enabled these energy-demanding processes by increasing metabolic activity in T-ALL cells through positive regulation of both glycolysis and oxidative phosphorylation. Concurrently, RUNX2 upregulation increased mitochondrial dynamics and biogenesis in T-ALL cells. Finally, as a proof of concept, we demonstrate that immature and KMT2A-R T-ALL cells were vulnerable to pharmacological targeting of the interaction between RUNX2 and its cofactor CBF beta. In conclusion, we show that RUNX2 acts as a dependency factor in high-risk subtypes of human T-ALL through concomitant regulation of tumor metabolism and leukemic cell migration.

Patients with an ABL-class fusion have a high risk of relapse on standard chemotherapy but are sensitive to tyrosine kinase inhibitors (TKI). In UKALL2011, we screened patients with post-induction MRD ≥1% and positive patients (12%) received adjuvant TKI. As the intervention started during UKALL2011, not all eligible patients were screened prospectively. Retrospective screening of eligible patients allowed the outcome of equivalent ABL-class patients who did and did not receive a TKI in first remission to be compared. ABL-class patients who received a TKI in first remission had a reduced risk of relapse/refractory disease: 0% vs. 63% at four years (P = 0·009).

Outcomes for adult patients with acute lymphoblastic leukemia (ALL) are dismal and have not kept pace with their pediatric counterparts, with five-year survival rates of less than 45%. TP53 mutations are infrequent in ALL, but activity of the oncoprotein MDM2 may otherwise phenotypically disrupt and circumvent normal p53 function, positing the p53-MDM2 signaling axis as a potential therapeutic target for the engagement of intrinsic cell death programs. Given the clinical safety and responses to p53-MDM2 antagonist idasanutlin (RG7388) in other hematological cancers and solid tumors, we aimed to evaluate the therapeutic potential of idasanutlin in ALL. Single-agent activity of idasanutlin was investigated in 17 high-risk ALL patient and patient-derived xenograft samples (aged 4 to 51 years), both B-ALL (n=15) and T-ALL (n=2), including KMT2A-rearranged, TCF3-rearranged, and Philadelphia-positive ALL. An ex vivo coculture of ALL blasts and hTERT-immortalized mesenchymal stem cells (MSC) was employed to support growth of the ALL blasts during short-term culture, complemented by a fluorescent image-based microscopy platform which identifies and discriminates the two cellular compartments using random forest machine learning algorithms based on cellular nuclear staining. Idasanutlin demonstrated sub-micromolar, dose-dependent anti-leukemic activity against 15 of 17 samples tested, with half maximal effective concentrations (EC50) in the range of 10 to 220 nM (mean EC50 = 45.1nM); the two exceptions were later determined to harbor homozygous inactivating TP53 mutations; p.Y220C and p.S241P within the p53 DNA binding domain. The idasanutlin EC50 concentrations determined are clinically achievable, well below the peak plasma concentrations reported in patients for other disease indications. Furthermore, idasanutlin concentrations below 10µM had no impact upon MSC survival. Conforming to p53-MDM2 auto-regulatory feedback mechanisms, we demonstrated that idasanutlin efficiently stabilized and activated p53 at the protein level within 6 hours when treated with their respective idasanutlin EC50, to a level greater than 4-fold increased relative to their respective vehicle-only controls (p=0.001, n=7). Further, p53-regulated transcriptional target gene products, MDM2 and p21, were increased by 5-fold and 2-fold respectively, validating engagement of the p53 pathway by idasanutlin (p=0.036 and 0.125, respectively). By contrast, idasanutlin did not elicit increased expression of either p53-regulated transcriptional target gene product in the identified TP53-mutant patient samples (n=2). On-target specificity of idasanutlin was further confirmed in a NALM6 isogenic cell line model, whereby the TP53 wildtype line was sensitive to idasanutlin (EC50 = 74nM) and effectively activated p53 signaling whereas the TP53 homozygous null line was highly resistant (EC50 = ~10µM). To determine whether the decreased cell numbers and engagement of p53 signaling observed were accompanied by cell death, the capacity of idasanutlin to induce apoptosis in the ALL samples was next investigated. Patient-derived ALL samples (n=6) were treated with vehicle or idasanutlin at their respective EC50s for 24 and 48 hours, and then analyzed by flow cytometry. There was an increase in annexin-V positive cells within 24 hours compared to the vehicle-only treated cells (mean±SD 14.3±6.6% vs 27.0±21.2% respectively (p=0.125). By 48 hours significant apoptosis was attained, with a mean±SD of 59.0±23.8% annexin-V positive cells compared to the mean of vehicle-treated cells at 29.1±11.6% (p=0.004). Cleaved poly(ADP-ribose) polymerase (PARP) levels were also increased greater than 3-fold compared to vehicle-only control cells as assessed by immunoblotting (p=0.045, n=3), corroborating these findings. These data emphasize the potential of pharmacologically targeting the p53-MDM2 axis in ALL, demonstrating potent, on-target, cytotoxic activity in a range of high-risk ALL cytogenetic subgroups. Taken together, these findings support further preclinical investigations into idasanutlin and other p53-MDM2 antagonists and potential combinations to improve the treatment of adult ALL. Disclosures Irving: F. Hoffmann-La Roche: Research Funding.

No abstract available.

There is a growing interest in fertility preservation as emerging research is highlighting the prevalence of infertility among young cancer survivors and its negative impact on quality of life. Previous qualitative research has identified barriers of fertility preservation care among professionals. The aim of this study was to assess the prevalence of these barriers among pediatric and adolescent oncology healthcare professionals and evaluate factors that influence them.Based on previously identified barriers and experts' input, a questionnaire was developed and sent to 88 professionals drawn from the multidisciplinary pediatric and adolescent oncology team of a large Principal Treatment Centre. Multivariate analysis was performed to evaluate which factors influence professional adherence to fertility preservation care.In total, 48 (55%) professionals responded and were included in the analysis. All pediatric and adolescent oncology healthcare professionals reported at least one barrier to fertility preservation care. Even though some interdisciplinary differences were observed, the most frequently endorsed barriers were focusing on patients' characteristics (age, health status, urgency of cancer treatment, and lack of interest in fertility issues). The least frequently endorsed barriers were related to organizational aspects (availability of fertility specialists, time constrains, and ability to raise fertility issues). Nurses and allied healthcare professionals endorsed knowledge or policy gaps as barriers to a greater degree than medical doctors.Results suggest that educational support provision, especially for nurses and allied healthcare professionals, and strengthening interdisciplinary collaborations could help overcome observed barriers and facilitate fertility discussions with pediatric and young cancer patients.

Purpose: Methotrexate administration is associated with\ud frequent adverse neurological events during treatment for\ud childhood acute lymphoblastic leukemia. Here, we present\ud evidence to support the role of common drug interactions\ud and low vitamin B12 levels in potentiating methotrexate\ud neurotoxicity.\ud Methods: We review the published evidence and highlight\ud key potential drug interactions as well as present clinical\ud evidence of severe methotrexate neurotoxicity in conjunction\ud with nitrous oxide anesthesia and measurements of\ud vitamin B12 levels among pediatric leukemia patients during\ud therapy.\ud Results: We describe a very plausible mechanism for\ud methotrexate neurotoxicity in pediatric leukemia patients\ud involving reduction in methionine and consequential disruption\ud of myelin production. We provide evidence that a\ud number of commonly prescribed drugs in pediatric leukemia\ud management interact with the same folate biosynthetic\ud pathways and/or reduce functional vitamin B12 levels and\ud hence are likely to increase the toxicity of methotrexate in\ud these patients. We also present a brief case study supporting\ud out hypothesis that nitrous oxide contributes to methotrexate\ud neurotoxicity and a nutritional study, showing that \ud patients.\ud Conclusions: Use of nitrous oxide in pediatric leukemia\ud patients at the same time as methotrexate use should be\ud avoided especially as many suitable alternative anesthetic\ud agents exist. Clinicians should consider monitoring levels\ud of vitamin B12 in patients suspected of having methotrexate-\ud induced neurotoxic effects.

The timing and developmental sequence of events for BCR-ABL1+ acute lymphoblastic leukemia (ALL), usually associated with IKAROS (IKZF1) deletions, are unknown. We assessed the status of BCR-ABL1 and IKZF1 genes in 2 pairs of monozygotic twins, one pair concordant, the other discordant for Philadelphia chromosome positive (Ph+) ALL. The twin pair concordant for ALL shared identical BCR-ABL1 genomic sequence indicative of monoclonal, in utero origin. One twin had IKZF1 deletion and died after transplantation. The other twin had hyperdiploidy, no IKZF1 deletion, and is still in remission 8 years after transplantation. In the twin pair discordant for ALL, neonatal blood spots from both twins harbored the same clonotypic BCR-ABL1 sequence. Low level BCR-ABL1+ cells were present in the healthy co-twin but lacked the IKZF1 deletion present in the other twin's leukemic cells. The twin with ALL relapsed and died after transplantation. The co-twin remains healthy and leukemia free. These data show that in childhood Ph+ ALL, BCR-ABL1 gene fusion can be a prenatal and possibly initiating genetic event. In the absence of additional, secondary changes, the leukemic clone remains clinically silent. IKZF1 is a secondary and probable postnatal mutation in these cases, and as a recurrent but alternative copy number change is associated with poor prognosis.

B-cell precursor childhood acute lymphoblastic leukemia with ETV6-RUNX1 (TEL-AML1) fusion has an overall good prognosis, but relapses occur, usually after cessation of treatment and occasionally many years later. We have investigated the clonal origins of relapse by comparing the profiles of genomewide copy number alterations at presentation in 21 patients with those in matched relapse (12-119 months). We identified, in total, 159 copy number alterations at presentation and 231 at relapse (excluding Ig/TCR). Deletions of CDKN2A/B or CCNC (6q16.2-3) or both increased from 38% at presentation to 76% in relapse, suggesting that cell-cycle deregulation contributed to emergence of relapse. A novel observation was recurrent gain of chromosome 16 (2 patients at presentation, 4 at relapse) and deletion of plasmocytoma variant translocation 1 in 3 patients. The data indicate that, irrespective of time to relapse, the relapse clone was derived from either a major or minor clone at presentation. Backtracking analysis by FISH identified a minor subclone at diagnosis whose genotype matched that observed in relapse ∼ 10 years later. These data indicate subclonal diversity at diagnosis, providing a variable basis for intraclonal origins of relapse and extended periods (years) of dormancy, possibly by quiescence, for stem cells in ETV6-RUNX1+ acute lymphoblastic leukemia.

Rare cases of possible materno-fetal transmission of cancer have been recorded over the past 100 years but evidence for a shared cancer clone has been very limited. We provide genetic evidence for mother to offspring transmission, in utero, of a leukemic cell clone. Maternal and infant cancer clones shared the same unique BCR-ABL1 genomic fusion sequence, indicating a shared, single-cell origin. Microsatellite markers in the infant cancer were all of maternal origin. Additionally, the infant, maternally-derived cancer cells had a major deletion on one copy of chromosome 6p that included deletion of HLA alleles that were not inherited by the infant (i.e., foreign to the infant), suggesting a possible mechanism for immune evasion.

In-depth molecular investigation of familial leukemia has been limited by the rarity of recognized cases. This study examines the genetic events initiating leukemia and details the clinical progression of disease across multiple families harboring germ-line CEBPA mutations. Clinical data were collected from 10 CEBPA-mutated families, representing 24 members with acute myeloid leukemia (AML). Whole-exome (WES) and deep sequencing were performed to genetically profile tumors and define patterns of clonal evolution. Germline CEBPA mutations clustered within the N-terminal and were highly penetrant, with AML presenting at a median age of 24.5 years (range, 1.75-46 years). In all diagnostic tumors tested (n = 18), double CEBPA mutations (CEBPAdm) were detected, with acquired (somatic) mutations preferentially targeting the C-terminal. Somatic CEBPA mutations were unstable throughout the disease course, with different mutations identified at recurrence. Deep sequencing of diagnostic and relapse paired samples confirmed that relapse-associated CEBPA mutations were absent at diagnosis, suggesting recurrence was triggered by novel, independent clones. Integrated WES and deep sequencing subsequently revealed an entirely new complement of mutations at relapse, verifying the presentation of a de novo leukemic episode. The cumulative incidence of relapse in familial AML was 56% at 10 years (n = 11), and 3 patients experienced ≥3 disease episodes over a period of 17 to 20 years. Durable responses to secondary therapies were observed, with prolonged median survival after relapse (8 years) and long-term overall survival (10-year overall survival, 67%). Our data reveal that familial CEBPA-mutated AML exhibits a unique model of disease progression, associated with favorable long-term outcomes.

The ETV6-RUNX1 fusion gene, found in 25% of childhood acute lymphoblastic leukemia (ALL) cases, is acquired in utero but requires additional somatic mutations for overt leukemia. We used exome and low-coverage whole-genome sequencing to characterize secondary events associated with leukemic transformation. RAG-mediated deletions emerge as the dominant mutational process, characterized by recombination signal sequence motifs near breakpoints, incorporation of non-templated sequence at junctions, ∼30-fold enrichment at promoters and enhancers of genes actively transcribed in B cell development and an unexpectedly high ratio of recurrent to non-recurrent structural variants. Single-cell tracking shows that this mechanism is active throughout leukemic evolution, with evidence of localized clustering and reiterated deletions. Integration of data on point mutations and rearrangements identifies ATF7IP and MGA as two new tumor-suppressor genes in ALL. Thus, a remarkably parsimonious mutational process transforms ETV6-RUNX1-positive lymphoblasts, targeting the promoters, enhancers and first exons of genes that normally regulate B cell differentiation. © 2014 Nature America, Inc

The t(12;21) translocation that generates the ETV6-RUNX1 (TEL-AML1) fusion gene, is the most common chromosomal rearrangement in childhood cancer and is exclusively associated with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The translocation arises in utero and is necessary but insufficient for the development of leukemia. Single-nucleotide polymorphism array analysis of ETV6-RUNX1 patient samples has identified multiple additional genetic alterations; however, the role of these lesions in leukemogenesis remains undetermined. Moreover, murine models of ETV6-RUNX1 ALL that faithfully recapitulate the human disease are lacking. To identify novel genes that cooperate with ETV6-RUNX1 in leukemogenesis, we generated a mouse model that uses the endogenous Etv6 locus to coexpress the Etv6-RUNX1 fusion and Sleeping Beauty transposase. An insertional mutagenesis screen was performed by intercrossing these mice with those carrying a Sleeping Beauty transposon array. In contrast to previous models, a substantial proportion (20%) of the offspring developed BCP-ALL. Isolation of the transposon insertion sites identified genes known to be associated with BCP-ALL, including Ebf1 and Epor, in addition to other novel candidates. This is the first mouse model of ETV6-RUNX1 to develop BCP-ALL and provides important insight into the cooperating genetic alterations in ETV6-RUNX1 leukemia.

l-Asparaginase is a key therapeutic agent for treatment of childhood acute lymphoblastic leukemia (ALL). There is wide individual variation in pharmacokinetics, and little is known about its metabolism. The mechanisms of therapeutic failure with l-asparaginase remain speculative. Here, we now report that 2 lysosomal cysteine proteases present in lymphoblasts are able to degrade l-asparaginase. Cathepsin B (CTSB), which is produced constitutively by normal and leukemic cells, degraded asparaginase produced by Escherichia coli (ASNase) and Erwinia chrysanthemi. Asparaginyl endopeptidase (AEP), which is overexpressed predominantly in high-risk subsets of ALL, specifically degraded ASNase. AEP thereby destroys ASNase activity and may also potentiate antigen processing, leading to allergic reactions. Using AEP-mediated cleavage sequences, we modeled the effects of the protease on ASNase and created a number of recombinant ASNase products. The N24 residue on the flexible active loop was identified as the primary AEP cleavage site. Sole modification at this site rendered ASNase resistant to AEP cleavage and suggested a key role for the flexible active loop in determining ASNase activity. We therefore propose what we believe to be a novel mechanism of drug resistance to ASNase. Our results may help to identify alternative therapeutic strategies with the potential of further improving outcome in childhood ALL.

Glioblastoma (GBM) is a lethal malignancy whose clinical intransigence has been linked to extensive intraclonal genetic and phenotypic diversity and the common emergence of therapeutic resistance. This interpretation embodies the implicit assumption that cancer stem cells or tumor-propagating cells are themselves genetically and functionally diverse. To test this, we screened primary GBM tumors by SNP array to identify copy number alterations (a minimum of three) that could be visualized in single cells by multicolor fluorescence in situ hybridization. Interrogation of neurosphere-derived cells (from four patients) and cells derived from secondary transplants of these same cells in NOD-SCID mice allowed us to infer the clonal and phylogenetic architectures. Whole-exome sequencing and single-cell genetic analysis in one case revealed a more complex clonal structure.This proof-of-principle experiment revealed that subclones in each GBM had variable regenerative or stem cell activity, and highlighted genetic alterations associated with more competitive propagating activity in vivo. Citation Format: Sara Grazia Maria Piccirillo, Sue Colman, Nicola E. Potter, Frederik W. van Delft, Suzanne Lillis, Maria-Jose Carnicer, Lyndal Kearney, Colin Watts, Mel Greaves. Genetic and functional diversity of propagating cells in glioblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2318. doi:10.1158/1538-7445.AM2015-2318

We have prospectively analysed and correlated the gene expression profiles of children presenting with acute leukaemia to the Royal London and Great Ormond Street Hospitals with morphological diagnosis, immunophenotype and karyotype. Total RNA extracted from freshly sorted blast cells was obtained from 84 lymphoblastic [acute lymphoblastic leukaemia (ALL)], 20 myeloid [acute myeloid leukaemia (AML)] and three unclassified acute leukaemias and hybridised to the high density Affymetrix U133A oligonucleotide array. Analysis of variance and significance analysis of microarrays was used to identify discriminatory genes. A novel 50-gene set accurately identified all patients with ALL and AML and predicted for a diagnosis of AML in three patients with unclassified acute leukaemia. A unique gene set was derived for each of eight subtypes of acute leukaemia within our data set. A common profile for children with ALL with an ETV6-RUNX1 fusion, amplification or deletion of ETV6, amplification of RUNX1 or hyperdiploidy with an additional chromosome 21 was identified. This suggests that these rearrangements share a commonality in biological pathways that maintains the leukaemic state. The gene TERF2 was most highly expressed in this group of patients. Our analyses demonstrate that not only is microarray analysis the single most effective tool for the diagnosis of acute leukaemias of childhood but it has the ability to identify unique biological pathways. To further evaluate its prognostic value it needs to be incorporated into the routine diagnostic analysis for large-scale clinical trials in childhood acute leukaemias.

Introduction The STIL-TAL1 fusion is found in 16% cases of paediatric and adolescent T-ALL, making it one of the most common T-ALL subgroups. Our study considers this leukaemia subtype in the context of a complex ecosystem that is diverse, evolving and subject to selective pressures. We used single cell methods to understand the order of co-operating mutational events and the clonal evolution of mutations in genes that are re-iteratively targeted, such as PTEN. Methods Diagnostic DNA from five STIL-TAL1 positive T-ALL cases was exome sequenced using Agilent SureSelect Human all Exon kit plus Illumina paired end sequencing. Driver copy number alterations and NOTCH1/PTEN exon 7 mutation status had been identified in a previous study and candidate driver mutations for inclusion in single cell experiments were validated by sequencing or Q-PCR using custom assays. Where more than one mutation was present within the same exon of a candidate driver gene, cloning experiments were carried out to verify the independent mutation sequences. Material from xenograft transplants was available in three of the five cases to assess their clonal heterogeneity in the leukaemia initiating cell compartment. Single cell multiplex Q-PCR was used to examine the single cell genetics of the pre-defined mutation events. Briefly, single cells were sorted and lysed prior to multiplex specific (DNA) target amplification and Q-PCR using the 96.96 dynamic microfluidic array and the BioMark HD (Fluidigm, UK). Copy number assays for the 1p33 deletion and custom assays for the patient specific STIL-TAL1 fusion breakpoints were used to confirm that the 1p33 deletion leading to this gene fusion was a clonal event. Results The only aberrant events common to all five samples were CKDN2A copy number loss and the 1p33 deletion that results in the STIL-TAL1 fusion. Exome sequencing revealed further mutations in known T-ALL drivers including NOTCH1, PTEN and PHF6 as well as candidate driver mutations in FREM2, PIK3CD, RPL14, BMPR1A and CDH18. Both NOTCH1 and PTEN demonstrated re-iterative inactivation and this was investigated in detail for PTEN. Case 1 had multiple PTEN exon 7 mutations and sub-clonal copy number loss. Case 2 had parallel frameshift mutations in PTEN exons 5 and 7. Case 3 contained an exon 8 mutation and multiple PTEN exon 7 mutations. In this case the three most frequent PTEN exon 7 indels were validated and tracked in a single cell multiplex Q-PCR experiment. This revealed a branching sub-clonal genetic architecture (see figure 1) in which all malignant cells at the proposed apex of the branching architecture harboured the STIL-TAL1 fusion and CDKN2A deletion with copy number losses of 4p, 6q and FREM2 and PTEN mutations occurring as sub-clonal events. PTEN indels 2 and 3 were found co-localised in the same sub-clone. Preliminary analysis of the paired mouse xenograft bone marrow did not detect PTEN exon 7 indels 1 – 3 in 84 single cells. However, bulk Sanger Sequencing analysis did identify the PTEN exon 8 mutation in the mouse. Ongoing work is in progress to determine whether single cells of the xenograft carry alternative PTEN exon 7 mutations detected in the diagnostic sample exome data and to characterise in which diagnostic sub-clone the PTEN exon 8 mutation resides. Conclusions This study demonstrates how exome sequencing and single cell multiplex Q-PCR can be used as complementary tools to understand the sub-clonal complexity of STIL-TAL1 T-ALL. PTEN inactivation is sub-clonal by single cell analysis, demonstrating the parallel evolution of multiple independent PTEN inactivated sub-clones, highlighting PTEN inactivation as a key event in this T-ALL subgroup. In a wider cohort of 20 patients collected by our group at least 50% had PTEN inactivation as assessed by sequencing of exon 7 and copy number data alone. Results indicate a strong evolutionary pressure selecting for mutational events that result in inactivation of the PTEN-PI3Kinase pathway. These events occur via multiple mechanisms, including copy number loss and truncating mutations, which are not limited to the known T-ALL hotspot in exon 7. Current work is focussing on using a similar approach to examine the clonal evolution of NOTCH1 mutations in STIL-TAL1 T-ALL samples in diagnostic and xenograft samples of cases 4 and 5. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Cerebral venous thrombosis (CVT) is a significant complication of therapy in children and young adults with acute lymphoblastic leukaemia (ALL). Previous reports indicate a considerable risk of mortality and morbidity in relation to CVT. In addition, there is potential for suboptimal treatment of ALL due to avoidance of Asparaginase and the need for reversal of anticoagulant therapy for invasive procedures such as intrathecal chemotherapy. Anticoagulation may be complicated by haemorrhage. This retrospective study aimed to identify the clinical features, therapy and outcome of CVT occurring during treatment of ALL in individuals, aged 1 to 24 years, who participated in the Medical Research Council UK ALL 2003 (MRC UKALL 2003) randomised controlled trial. Cases of CVT were identified from adverse event reporting. All reports were screened for central nervous system thrombosis and grade 4 serious adverse events in the category of coagulation/thrombosis. Reporting centres were asked for additional clinical information. 3126 children and young adults were enrolled in the study between 2003 and 2011, with median age 5 years, including 1776 males (56.8%). The 3 initial treatment regimens: A, B and C: corresponded to standard-risk, intermediate-risk and high-risk, respectively. 55% were treated with regimen A, 32% regimen B and 13% regimen C. 48 individuals (1.5%) developed CVT and data were returned for 42. Median age was 10 (range: 1 to 18) years and 25 cases were male (59.5%). 71.4% of cases (30/42) occurred during induction therapy. 18 cases were in individuals treated with regimen A, 22 regimen B and 2 regimen C. Median time between the most recent Asparaginase dose and CVT was 12.5 days (range: 1-111 days). 29 cases (68.4%) had received 2 doses of Asparaginase prior to thrombosis, 5 cases >2 doses and 8 cases 1 dose. Presenting symptoms were: seizures, 22 cases (52.3%); neurological impairment, 20 (47.6%); headache, 19 (45.2%); reduced conscious level, 8 (19.0%); nausea/vomiting, 8 (19.0%). Identified thrombotic risk factors included: hospitalisation, 22 (52.3%); active infection, 5; recent immobility, 5; dehydration, 6; combined oral contraceptive pill, 2. None were obese, had a high white cell count at time of CVT diagnosis or a history of recent major surgery. 13 (31.0%) had >1 thrombotic risk factor and 16 (38.1%) had no additional clinical risk factor identified. 10 had a thrombophilia screen performed, one with a repeatedly low protein S level (male, 1 year of age). 7 cases (16.7%) had cerebral infarction on imaging and 11 (26.2%) were complicated by intracranial haemorrhage (ICH). 38 cases (90.5%) received anticoagulant therapy, 37 of which received low molecular weight heparin (LMWH) and one unfractionated heparin. One case had catheter-directed thrombolysis. 4 were not anticoagulated, 3 due to ICH. 2 received Antithrombin replacement therapy. Anticoagulation therapy was continued for a median of 3 months. 65.0% of cases due more Asparaginase were re-exposed (26/40), 19 of which received thromboprophylaxis during re-exposure. 2 remain on long-term therapeutic anticoagulation. In 23 cases (54.8%) CVT resulted in a delay or change to planned treatment. Neurological morbidity was reported in 4 cases (9.5%): seizure disorder, 2; hemiplegia, 1; residual hemiplegia and hemianopia with severe developmental delay, 1. 36 cases (85.7%) remain in first remission. Survival in those with CVT was 90.1%, comparable to actuarial 5-year survival of the entire cohort (91.5%). There were 4 deaths, three due to leukaemia (two in first relapse) and one due to pneumonia as a complication of stem cell transplant. There were no deaths due to CVT or bleeding on anticoagulant therapy. This study highlights the clinical features of CVT in children and young adults treated for ALL in the MRC UKALL 2003 trial, in particular the timing in relation to Asparaginase treatment, presenting features and neurological outcome. These data support LMWH as safe treatment for CVT in this clinical setting. We have demonstrated the tendency of clinicians to adjust treatment regimen and/or avoid further Asparaginase exposure in response to CVT. However, overall survival at 5 years did not differ for the cohort with CVT. Future studies should aim to further evaluate clinical predictors, alongside potential coagulation and genetic markers, in order to identify a high-risk group that may benefit from measures to reduce CVT during ALL therapy. Disclosures No relevant conflicts of interest to declare.

Background Germline mutations in the N-terminal of CCAAT/enhancer binding protein α (CEBPA) are a feature of autosomal dominant AML. Despite the strong penetrance of these mutations, the age of disease onset varies considerably and is usually precipitated by acquiring a C-terminal mutation. Although biallelic CEBPA-mutations in sporadic AML are associated with favorable clinical outcomes, little is known about long-term survival or the secondary molecular events linked with familial cases. Aims We sought to establish the long term clinical outcomes in familial CEBPA-mutated AML and to examine the patterns of secondary mutations associated with leukemic transformation. Methods and Results Disease specific and follow up information was collected in 16 affected patients, from 7 pedigrees, published between 2004 and 2011. In 94% of patients (n=15), at least 3 years follow up was achieved. All pedigrees had a germline N-terminal CEBPA mutation and 17 of 18 documented disease episodes had an acquired C-terminal mutation. The age at AML diagnosis varied from 2-39 years (median 24.5 yrs) with a single asymptomatic carrier detected (now 23 yrs). With the exception of 1 patient diagnosed in 1963, all cases received combination chemotherapy at diagnosis. Additional consolidation comprised autologous stem cell transplantation (SCT, n=3) and allogeneic SCT in 1 patient failing to achieve CR post induction therapy. Ten patients had at least 1 further disease episode, the first recurrence presenting after a median of 2.1 years (range 0.5-14 yrs), 5 continued in CR and 1 patient was lost to follow up. In 3 out of 4 patients, where CEBPA was screened at recurrence, the acquired C-terminal mutations differed from diagnosis, signifying new episodes of AML. The median overall survival (OS) for the entire cohort was 20 years (1.1-46 yrs, n=16) and 17.3 years for patients with multiple disease episodes, reflecting durable responses to second line therapies. To identify potential co-operating mutations in CEBPA pedigrees, whole exome sequencing (WES) was performed in 7 tumor samples from 5 patients across 3 pedigrees, all with the germline mutation p.P23fs (Figure 1). All tumor DNA samples were sequenced with matched remission or skin DNA as a germline control. The number of acquired mutations in familial tumors was similar to sporadic disease, with 10-22 (median=14) non-synonymous tier 1 mutations per sample. In addition to the acquired C-terminal CEBPA mutation, these included established AML loci such as EZH2, TET2, WT1, GATA2, NRAS, CSF3R and the recently identified cohesin complex gene, SMC3. Of note, FLT3-ITD, NPM1 and DNMT3A mutations were absent in all tumors. A minimum of 2 established mutations were identified in each tumor and, at present, we can only speculate on which additional mutations are ‘driver' or ‘passenger' events. Reflecting findings in sporadic AML, biallelic CEBPA and GATA2 mutations co-occurred in both siblings from Pedigree 1 and were subsequently identified by Sanger sequencing in the child III.2 (Figure 1). All 3 patients continue in long term remission following chemotherapy. We were able to trace the clonal evolution in patient I.2 (Pedigree 3) by WES of 3 consecutive tumor samples which arose over a 17 year period. At diagnosis (Dx) the patient received induction and consolidation chemotherapy and remained disease free for 14 years. The second disease episode (R1) was treated with chemotherapy followed by autologous SCT and the third presentation (R2) was chemo-refractory. Tumor DNA from R2 was clonally related to Dx, sharing 7 identical mutations, including the original C-terminal CEBPA deletion. In contrast, R1 appeared molecularly distinct from Dx and R2, most likely representing a new clone which was subsequently eradicated with treatment. Conclusion This is the first report of long term clinical outcomes in familial CEBPA-mutated AML. Although many patients experienced disease recurrence, our extended follow up showed that OS remained favorable despite multiple episodes of disease. Assessment of C-terminal CEBPA mutations provided a unique insight into the recurrence of AML, with some patients appearing to develop completely new leukemic episodes. Although the penetrance of germline mutations is high, healthy carriers and late onset disease are noted, emphasizing the need for clinical vigilance and screening of all related potential SCT donors. Disclosures: No relevant conflicts of interest to declare.

Acute Lymphoblastic Leukaemia (ALL) in children is a disease strongly categorised by age at diagnosis of overt disease, leukaemic subtype and recurrent genetic alterations. ALL in infants is a rare subset often associated with MLL rearrangements, B cell precursor lineage lacking CD10 (pro-B ALL) and a prenatal origin in utero. Infant T-cell ALL (T-ALL) is a very rare and poorly defined entity with a worse outcome in general. The availability to us of a unique series of 13 infant T-ALL cases (≤12 months) along with 12 remission cases allowed us to perform a genome-wide study of the clonotypic alterations present in these leukaemias and to elucidate more definitively the critical genomic hits required to drive the emergence of overt disease. The main clinical and demographic characteristics observed in our series were a median age of 9 months, no predominance of gender and a high WBC count (≥ 50,000) in 12 out of 13 cases at diagnosis. The infants were treated according to Brazilian GBTLI protocol (n=3), BFM protocol (n=2), EORTC (n=3) and INTERFANT (n=5). All samples were analysed at diagnosis for the main T-ALL associated abnormalities and our results showed 4 cases mutated for NOTCH1. One case also presented with an FBXW7 mutation concomitant with wild type NOTCH1. Two cases showed PTEN mutations and all cases were germ line for IL7R. Furthermore, we did not observe any case with SIL-TAL1 or TLX3 rearrangements. TCRG & D were both rearranged in 7 cases while 1 case presented with only TCRG rearrangement and 1 case with only TCRD rearranged. Seven cases also showed rearrangement of TCRB and 1 case showed no receptor gene rearrangement at all. This latter case also had a classical ETP profile. MLL rearrangements were detected by FISH in 4 out of 13 cases. All diagnostic samples were analysed by SNP-array to identify genomic losses and gains. As a summary of the SNP data, a recurrent somatic chromosome 3 deletion was observed in 3 cases. The losses, similar in size, result in the complete deletion of MLF1 and have not previously been described in T-ALL. MLF1 plays a key role in AML and myelodysplastic syndrome leukaemogenesis. Q-PCR and FISH were used to confirm the copy number of MLF1 in those cases analysed. Akin to paediatric T-ALL we observed a PTEN deletion in 1 patient and 2 further cases with an 11p13 deletion that could lead to activation of LMO2. One case harboured a large 11q14.1-11q23.2 deletion that includes the important ‘driver’ gene ATM. The deletion of 11q including ATM has been reported in 30% of patients with CLL and at a low rate in ALL. One patient also harboured a TCRB-MYB translocation known to be associated with a young age of onset of T-ALL and an expression signature of proliferation/mitosis. Five out of 13 cases that were negative for MLL gene rearrangement, did show deletions of CDKN2A. These results were verified by Q-PCR copy number analysis and FISH. Overall, the current data suggest that infant T-ALL presents both different and infrequent genetic abnormalities in general when compared to T-ALL in older children and adults. Disclosures: No relevant conflicts of interest to declare.

Abstract 876 Immunophenotypic instability during early phase of ALL treatment is a frequent observation during flow cytometric minimal residual disease (FC MRD) monitoring. Antigens typically involved include CD10, CD20, CD34 and CD45 and these changes do not usually revoke initial disease classification and do not hamper FC MRD detection. We previously described a subtype of B-cell precursor (BCP) ALL with striking immunophenotypic instability towards monocytoid lineage within the first month of therapy (switching ALL-swALL). Blasts expressing both B and monocytoid markers emerged at early time points on days 8 and 15 of treatment while later only those blasts with pure monocytoid phenotype were present. Incidence of swALL in childhood was unexpectedly high (3-4% of all pediatric BCP ALL) as confirmed in two national reference labs. This phenomenon was associated with aberrant expression of CD2 (LFA-2) on diagnostic blasts (Mejstrikova et al, ASH 2010). The leukemic origin of monocytoid blasts was proven by the detection of clone-specific immunoreceptor gene rearrangements (Ig-TCR). No common genetics aberration was found in a cohort of swALL (n=17), MLL gene was always in germline configuration. We found an increased rate of alteration in IKZF1 gene compared to control BCP ALL cases (p=0.012). An expression analysis of the key hematopoietic regulators showed a difference in CEBPα expression, which is an important transcription factor in transdifferentiation of B cells into macrophages (Xie et al., Cell 2004). Expression of CEBPα is increased in swALL compared to other BCP ALL cases (p=0.01) already at diagnosis prior switching, however, the expression is lower compared to AML (p=0.002). We analyzed CEBPα the methylation status of the promoter region of this gene. Demethylation of CEBPα promoter region analyzed by bisulfite sequencing (295bp-594bp of promoter region) was found in 10/12 swALL cases, while it was seen in only 6/28 control BCP ALLs (Fisher test, p=0.0004). The only cases having demetylation in CEBPα were 5/5 BCR-ABLpos and 1/4 ETV6-RUNX1pos. Whole genome ERRBS method (Enhanced Reduced Representation Bisulfite Sequencing) confirmed this methylation pattern of CEBPα in 7 patients (4 swALL,3 BCP ALL). In order to establish an in vivo model to study the underlying molecular mechanisms, we transplanted ALL cells from 7 swALL patients intrafemorally into NOD-SCIDIL2Rgammanull (NSG) mice. Successfulstable engraftment was achieved only in 2 out of 7 swALL cases (28%) (Fisher test, p=0.049).Interestingly in these two cases, the 200 bp promoter region of CEBPα was methylated to some extent at diagnosis and completely methylated after engraftment into mice, suggesting the possibility of a selective advantage in this context. We treated engrafted animals with prednisolone and in both cases we observed demethylation of CEBPα promoter. Because the rate of engraftment of ALL in NSG is usually very high, these observations may indicate that the biology of this particular subset of patient is distinct. Conclusion: We described a novel subtype of BCP-ALL with the demethylation of CEBPα promoter region, increased CEBPα expression and immunophenotypic shift towards monocytic lineage during first weeks of the therapy. We identified CEBPα as the potential regulator of this lineage plasticity. Disclosures: No relevant conflicts of interest to declare.

Abstract 2521 Purpose: The outcome for Teenagers and Young Adults (TYA) with T-cell Acute Lymphoblastic Leukaemia (T-ALL) has improved significantly using paediatric based chemotherapy protocols. Event free survival figures from the recently closed UKALL2003 highlight this observation. It remains poorly understood however why their outlook remains inferior to children aged 1 – 15 years whilst receiving identical treatment. Possible explanations for this phenomenon are distinct tumour biology, host factors and treatment adherence. The primary aim of this study is to characterise the tumour associated genetic events leading to TYA T-ALL and establish whether T-ALL in Teenagers and Young Adults is a distinct disease entity. The secondary aims are to identify new prognostic markers and potential targets to optimise therapy. Methods: DNA and RNA were extracted from 60 TYA T-ALL (15 – 25 years) historic samples. The DNA was derived from blasts at diagnosis or relapse, and matched with germ line DNA where available. The DNA was examined at high resolution for Copy Number Alterations (CNAs) and Loss of Heterozygosity (LOH) using the Affymetrix SNP6.0 platform and analysed by CNAG, dCHIP and Partek. The most frequent CNAs were confirmed by MLPA (P-383-T-ALL). In addition the DNA was screened for mutations in NOTCH1/FBXW7/PTEN using conventional Sanger sequencing and for mutations in NRAS, KRAS, CBL, FLT3 and SHP2 using denaturing high performance liquid chromatography. In cases without CDKN2A gene deletion we initially performed CDKN2A gene promoter methylation by SABiosciences EpiTect Methyl qPCR Arrays, followed up by Methylation Specific PCR (MSP). Where available, RNA was extracted from viable cells and the quality confirmed by Bioanalyser. Next the RNA was reverse transcribed to cDNA and subjected to real-time PCR gene expression analysis for CDKN2A and T-ALL related oncogenes. Results: The most frequent CNA was deletion of CDKN2A in 72.7% of patient samples (91.6% homozygous deletions). Other frequent CNAs were loss of MLLT3 (31%), STIL (27%), PTEN (19%), LEF1 (15%) and gain of MYB (12%). These CNAs were confirmed by MLPA. The STIL-TAL fusion occurred in 27% of patients. The only recurrent region of Copy Number Neutral LOH encompassed 9p24.3-p13.3, including the gene CDKN2A (27%). NOTCH1, FBXW7 and PTEN mutations occurred at expected frequencies. Rare mutations were identified in NRAS and CBL, and these mutations represented known variants. Bisulphite modified DNA was subjected to CDKN2A gene promoter methylation analysis. All samples examined were negative by Epitect Methyl Array, including the positive control cell line Raji. Promoter methylation was positive by MSP for the Raji cell line and some TYA T-ALL cases without CDKN2A gene deletion. Conclusions: This data identifies the frequency of recurrent mutations in T-ALL of Teenagers and Young Adults but reveals no striking differences with those observed in T-ALL in younger children. We are in the process of analysing additional cases to complete a total of 75 patients, including whole exome sequencing of a subset of cases. This sample size will allow us to detect genomic aberrations with a 5% incidence within this cohort (>90% probability) and to determine any prognostic significance. The most frequently observed aberration is CDKN2A gene deletion. In some cases without CDKN2A deletion we identified CDKN2A gene promoter methylation, offering an alternative mechanism of CDKN2A inactivation. To be able to appreciate potential cooperation between genetic abnormalities, we are correlating aberrant expression of the oncogenes TAL1, LYL1, LMO1, LMO2, TLX1, TLX3, NKX2, ERG and MEF2C with the genomic abnormalities identified. Disclosures: Gribben: Celgene: Honoraria; Roche: Honoraria; Merck: Honoraria; Mundipharma: Honoraria; Pharmacyclics: Honoraria.

Abstract 1348 Philadelphia-positive (Ph+) acute lymphoblastic leukaemia (ALL), characterised by the BCR-ABL1 fusion gene, occurs in approximately 30% of adult and 5% of childhood ALL and is associated with a poor prognosis. It is considered a single clinical entity with identifiable and recurrent copy number alterations (CNA); notably deletions of the lymphoid transcriptional regulator IKAROS (encoded by IKZF1), PAX5, and CDKN2A/B that are presumed to cooperate with BCR–ABL 1 in lymphoid leukaemogenesis. In particular, IKZF1 deletions are present in 80% of BCR-ABL1 positive ALL cases, and have been implicated as an independent indicator of poor prognosis in childhood ALL. Our previous studies of twin pairs either concordant or discordant for BCR-ABL1+ ALL indicate that the fusion gene is a first hit that occurs prenatally. However, the order and sequence of acquisition of CNA is unknown. We recently reported a complex sub-clonal genetic architecture for leukaemic blasts and leukaemia-propagating (‘stem’) cells in childhood ETV6-RUNX1-positive ALL (Anderson et al., Nature 469: 356–361, 2011). In the present study, we aimed to determine whether similar sub-clonal genetic diversity occurs in BCR-ABL1+ ALL. We carried out five colour FISH to diagnostic blast cells from eight BCR-ABL1 positive cases with differentially-labelled probes for BCR, ABL1, IKZF1, CDKN2A and PAX5. In a subset of cases we also performed Affymetrix single nucleotide polymorphism (SNP 6.0) arrays to determine the specific boundaries of deletions. Four out of the eight cases screened had concurrent IKZF1, PAX5 and CDKN2A deletions. In one case the order of acquisition of these deletions was uninformative, with 97% of cells exhibiting a single FISH pattern (BCR-ABL1+ with monoallelic deletions of all three genes). In the second case, a linear clonal progression was observed with IKZF1 deleted first, PAX5 second and CDKN2A third. In the two remaining cases a branching sub-clonal pattern was observed. In one of these monoallelic IKZF1, CDKN2A and PAX5 deletions all arose independently in different sub-clones; i.e. IKZF1 was deleted first in one subclone, CDKN2A first in another and PAX5 first in a third sub-clone. In the final case we also studied matched diagnosis and relapse samples. Here, SNP array analysis revealed different deletions in all three genes at diagnosis and relapse. Genomic fusion breakpoint analysis revealed an identical BCR-ABL1 genomic sequence at diagnosis and relapse, confirming the same clonal origin of leukaemia. The different deletion boundaries in IKZF1, PAX5 and CDKN2A permitted us to design specific FISH probes to distinguish between ‘diagnostic’ and ‘relapse’ deletions and to track their evolution. The predominant clone at relapse was not a direct evolutionary product of any of the major clones found at diagnosis. The dominant sub-clone at diagnosis was BCR-ABL1+, with a large 9p deletion (encompassing PAX5 and CDKN2A) and a focal CDKN2A deletion, all sub-clonal to a focal IKZF1 deletion. At relapse, the dominant sub-clone had acquired a different IKZF1 deletion, which was sub-clonal to two different (focal, biallelic) deletions of CDKN2A and a different monoallelic PAX5 deletion. The large 9p deletion was not present at relapse. These results indicate the existence of a pre-leukaemic BCR-ABL1 fusion gene positive clone that has given rise to at least two sub-clones, each with different IKZF1, PAX5 and CDKN2A deletions, that have evolved independently. These data indicate that the sub-clonal architecture in this poor prognosis subtype of ALL is genetically diverse, and that key ‘driver’ CNA can arise independently and in no preferential order. Disclosures: No relevant conflicts of interest to declare.

Abstract 1708 Introduction: Anecdotic observations of B precursor (BCP) ALL patients with aberrant CD2 expression at diagnosis and a significant shift of phenotype towards monocytes during induction treatment lead us to investigate the nature, incidence and characteristics of monocytic transdifferentiation. Patients and Design: Patients with a significant shift of blast phenotype (decrease of CD19 associated with increase of CD14 and CD33, Fig) during the induction phase of treatment were labeled as “swALL”. All Czech patients diagnosed with BCP ALL (n=698) during the course of study served as a control group. A Swiss patient with swALL was confirmed in the Prague laboratory and added to the presented cohort. Results: In total, 13 patients with swALL have been identified between 09/96 and 05/10. Monocytic nature was confirmed by cytometry (FC) and/or microscopy in all cases. Comprehensive polychromatic FC investigation not only identified typical CD14pos CD19neg monocytic cells coexisting with unequivocal residual leukemic blasts but also cells in intermediate stages that shared B lymphoid and monocytic characteristics (Fig). Although at diagnosis CD14bright monocytic cells were present in low numbers in bone marrow (BM) (mean±SD; 1.7±2.02%, others: 0.57±0.65%, p=0.006) in all 12 analyzed swALL cases, their frequency increased at day 8 BM (17±21%, others: 1.2±1.2%, p We next asked whether the observed transdifferentiation could be recapitulated in vitro. The diagnostic BM cells from swALL (n=3) or other BCP ALL (n=9) were cultured for 8 days with or without prednisolone (0-0.5-5-10-100μg/ml). Between days 3 and 8 of culture, the number of CD14posCD19dim cells was significantly greater in swALL cells than in other BCP ALL cells (each day, p The incidence of swALL ranged from 0.99% (5 of 507) (1996-2007, before we started to use 8-color FC screening panel evaluating simultaneously monocytic and B cell lineage between diagnosis and day 33) to 4.3% (7 of 163) in patients diagnosed between 2007-10 and prospectively screened. Patients with swALL were treated according to the standard protocols for ALL. Eight of 13 swALL had MRD levels at day 33 >5×10-4, 4 of 13 were prednison poor responders. One patient developed an AML with monocytic phenotype 8 months after the diagnosis. Discussion: Examples of phenotype plasticity are known from both experimental studies and malignant diseases. None of the previously defined factors (monosomy 7, MLL rearrangements) was detected in our cohort. Despite the striking association with CD2 we have not identified a causal relationship between CD2 and plasticity yet. Conclusion: SwALL occurs in 1–4% of B precursor ALL. Although the monocytic cells in these patients have little obvious leukemic features both in microscopy and in standard FC, they are derived from the leukemic clone. Prognosis differs, ranging from standard risk to secondary AML with monocytic phenotype. Optimal treatment appears to be the standard ALL treatment but larger cohorts may indicate special approaches to these patients. SwALL presents with a homogeneous molecular genetics, lack of changes previously shown to be associated with immunophenotype instability, and a strong association with initial CD2 expression. In half of the patients IKZF1 deletions are present. Supported by: P301/10/1877, NS10480-3, NPV2B06064, MSM0021620813, NS/1000-4, GAUK15710, NS/10472-3, NS/10473-3. Disclosures: No relevant conflicts of interest to declare.

Abstract 980 Poster Board I-2 Introduction: Materno-fetal transmission of malignant clone is one of the rare causes of leukemia in infants. It has been recorded some 20 times over the past 100 years but definitive, genetic evidence for a maternal origin has been lacking. Here we present a detailed genetic analysis of materno-fetal transmission in a case with p190 type BCR-ABL leukemia. Patients course: A mother was found to have p190 type BCR-ABL positive acute lymphoblastic leukemia (ALL) one month after delivery of a baby, and died during induction therapy by uncontrolled bacillus cereus encephalitis. Her baby developed p190 type BCR-ABL positive B-precursor lymphoblastic lymphoma (B-LBL stage3 jaw tumor and pleural effusion) at 11-month old. Non-Hodgkin's lymphoma type chemotherapy according to Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) ALB-NHL03, followed by Hyper-CVAD imatinib therapy without stem cell transplantation were given to her, leading to complete remission for more than 31 months. Material and methods: We first cloned the BCR-ABL genomic breakpoint region from the infant's pleural effusion (PE). The breakpoint was designated as a fusion between BCR intron 1 and ABL intron1. Then, We conducted Q-PCR by using BCR-ABL genomic breakpoint specific primer and sequencing. Fifteen polymorphic STR markers were amplified in the paternal blood DNA and Patient's PE and PB DNA samples using the Powerplex-16 system. HLA serotyping was by microdroplet lymphocyte cytotoxity. Genotyping was carried out using a reversed SSO HLA DNA typing method. The infant jaw tumor was further analyzed by Affymetrix high resolusion (250K) SNP arrays. Results: Short tandem repeat profiles indicated that the infant jaw tumor was, unambiguously, of maternal origin. Amplification of BCR-ABL genomic breakpoints on maternal leukemic cells and infant lymphoma cells revealed that these leukemia clones shared the same unique BCR-ABL genomic sequence, indicating a shared, single cell origin. Positivity of BCR-ABL specific PCR on Guthrie card blood spot also indicated that the leukemic clone was present in the blood at birth. Additionally the infant, maternal-derived leukemic cells had uniparental disomy of chromosome 6p with a deletion of non-inherited maternal MHC allele (NIMA), suggesting a mechanism for immune evasion in the baby. Conclusions: This is the first case with definite evidence of in utero materno-fetal transmission of mother's leukemic cells. This case also suggests that materno-feto transfer of leukemic cells is more common than as reflected in the frequency of clinically diagnosed cases, where the immuno-surveillance, predominantly directed against major HLA locus antigens, may be the principal constraint. Disclosures: No relevant conflicts of interest to declare.

Abstract 1583 Poster Board I-609 Approximately a quarter of B cell precursor childhood acute lymphoblastic leukemia (ALL) is characterized by an ETV6-RUNX1 (TEL-AML1) fusion gene and has an overall good prognosis. The majority of these children will be treated on the standard risk arm of the United Kingdom ALL treatment protocols. Relapse usually occurs after cessation of treatment but remarkably can present many years later. The incidence of ETV6-RUNX1 at relapse has been reported to be less than or similar to de novo ALL. Molecular studies on neonatal bloodspots and on twins with concordant ALL have demonstrated the prenatal origin of major subtypes of childhood ALL, including most ETV6-RUNX1 fusion gene positive cases. In addition these investigations have suggested the existence of a preleukaemic stem cell requiring additional mutations or ‘hits’ in order to develop frank leukemia. To understand the genetic basis and clonal origin of late relapses we have compared the profiles of genome-wide copy number alterations (CNA) at relapse versus presentation in samples matched with remission DNA from 24 patients. The selected samples had tumor cell purity >75% before DNA extraction. DNA copy number alteration data was generated using the Affymetrix 500K SNP arrays. LOH analysis was performed using CNAG 3.0 and dCHIP 2008. Overall we identified 168 CNA at presentation and 252 at relapse (excluding deletions at IgH and TCR loci), equating to 6.96 and 10.3 CNA at presentation and relapse respectively. Although the number of CNA increased at relapse, no single gene or pathway was uniquely targeted in relapse. The most frequent alterations involved loss of 12p3.2 (ETV6), 9p21.3 (CDKN2A/B), 6q16.2-3 and gain of 21q22.1-22.12. A novel observation was gain of part or whole of chromosome 16 (2 patients at presentation, 5 at relapse) and deletion of the oncogene Plasmocytoma Variant Translocation 1 (PVT1) in 3 patients. Pathway analysis demonstrated frequent involvement at presentation and relapse of genes implicated in both B cell development (44 versus 46%) and cell cycle control (46 versus 71%). In order to study the clonal origin of relapse, we devised a classification describing the change in CNA between presentation and relapse in each individual patient. The clonal relationship between the presentation and relapse clone was established by the persistence of both the ETV6-RUNX1 fusion and at least 1 Ig and/or TCR rearrangement. We used a classification focussed on ‘driver’ CNA, defined as CNA that target genes functionally involved in leukemogenesis or CNA that are recurrently targeted as described in the literature. The four categories of relapse were type 1 (the dominant clone at presentation presented unchanged at relapse), type 2 (the relapse clone was derived from the major subclone at presentation with additional CNA), type 3 (the relapse clone was derived from a minor clone at presentation with gains and losses of CNA) and type 4 (the relapse clone is derived from an ancestral or preleukemic clone at initial presentation with all CNA gained). Twenty-one of the 24 patients were classifiable in this way (Figure 1). Although comparative relapse / presentation CNA profiles cannot identify precise clonal origins of relapse, the data indicate that irrespective of time to relapse (5 years) derived from pre-leukemic cells lacking CNA. This data indicate diverse clonal origins of relapse and extended periods of dormancy, possibly via quiescence, for stem cells in ETV6-RUNX1+ ALL. Relapse type Remission duration (years) < 2 2 - 5 > 5 1 • • 2 • ••••••• •• 3 •• •• ••• 4 •• Figure 1. Each patient is represented by a black dot. Each patient is classified on the basis of the relapse type and remission duration. Disclosures No relevant conflicts of interest to declare.

Abstract LBA-5 Intra-clonal, mutational complexity is a hallmark feature of cancer and provides the substrate for sub-clonal selection, progression and therapeutic resistance. There is limited insight however into detailed sub-clonal, genetic architecture in cancers and their propagating 'stem' cells. Childhood acute lymphoblastic leukaemia (ALL) genotypes are characterized by chimeric fusion genes (or hyperdiploidy) coupled with recurrent, copy number alterations (CNA), primarily in genes regulating cell cycle or differentiation. For ETV6-RUNX1-positive cases of B precursor ALL, the temporal sequence of events is known with the fusion gene usually arising as a pre-natal, initiating event. Recurrent CNA, presumed to be functional or 'driver' mutations, are secondary to gene fusion and probably post-natal in origin. We have interrogated clonal architecture by multiplexed FISH analysis of single cells using probes labelled with three or four distinct fluorochromes. We pre-selected diagnostic samples from 30 ETV6-RUNX1-positive cases that we found to have deletion of ETV6 and CDKN2A or PAX5 or combinations of these. The application of multiple FISH probes allowed us to score all cells (200 per patient sample) for up to six genetic lesions: ETV6-RUNX1 fusion (and multiple copies of the fusion), deletion of the unrearranged ETV6 allele, extra copies of chromosome 21q (via RUNX1 signals), and one or two copy deletions of PAX5 and CDKN2A. The genetic classification of individual cells using this method allowed a designation of sub-clones and the assembly of putative ancestral, or evolutionary, trees. Although sub-clones were previously known to exist at diagnosis of ALL, the extent of sub-clonal diversity revealed by this study was unanticipated and very marked. Sub-clones, of varying sizes (1–90% of total cells), numbered 3 to 14 per case. This must be an under-estimate of genetic complexity as only selected mutations were included in the screen. The common CNA (ETV6, CDKN2A and PAX5 deletions, chromosome 21q gains) arose independently and recurrently in sub-clones and in no preferential order. Matched relapse and diagnosis pairs were available on 5 patients. In all of these, the clones at relapse could be matched to individual sub-clones at diagnosis, with relapse originating from a major or minor subclone at diagnosis. Importantly, the relapse clone often diversified further, reiterating the evolution pathway of the primary clones. These data indicate that sub-clonal diversification does not arise via linear, clonal succession but rather has a complex, branching architecture reminiscent of Charles Darwin's 1837 evolutionary speciation model. The ancestral trees so revealed in ALL are single time point snapshots and therefore disguise temporal or sequential dynamics. We show that sub-clonal architecture changes in the months leading up to a diagnosis of ALL and is re-ordered by treatment and subsequent relapse. An important prediction derived from this pattern of sub-clonal architecture is that leukaemia propagating or 'stem' cells in ALL (and other cancers) should themselves be genetically diverse. Preliminary transplantation experiments in NOD/SCID/IL2Rgamma(null) mice show that this is the case, since multiple, genetically distinct sub-clones regenerated in vivo (Figure1). The complexity of genetic architecture in ALL has substantial implications for the cancer stem cell concept and for the efficacy of generic or targeted treatments. Disclosures: No relevant conflicts of interest to declare. Download : Download high-res image (120KB) Download : Download full-size image Figure 1 . Sub-clones present in diagnostic sample before intratibial injection into NOD/SCID/IL2Rgamma(null) mice, assayed by 3 colour FISH for the ETV6-RUNX1 gene fusion, ETV6 deletion, RUNX1 gain and CDKN2A deletion. All four leukaemic subclones were present in the mice after leukaemic engraftment at 10 -12 weeks, but with the CDKN2A deleted sub-clones predominant.