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Virus-specific T-cells (VSTs) from third-party donors mediate short- and long-term antiviral effects in allogeneic hematopoietic stem cell transplant (HSCT) recipients with relapsed or refractory viral infections. We investigated early administration of third-party VSTs, together with antiviral therapy in patients requiring treatment for first cytomegalovirus (CMV) or Epstein-Barr virus (EBV) infection. Thirty HSCT patients were treated with 1 to 4 VST infusions (2 × 107 cells/m2; CMV n=27, EBV n=3) at a median of 4 days after initiation of antiviral treatment. The overall viral response rate was 100%, with a complete response (CR) rate of 94%. Of the 28 patients who achieved a CR, 23 remained virus PCR negative (n=9) or below quantitation limit (n=14) for the duration of follow-up. Four patients had brief episodes of quantifiable reactivation not requiring additional therapy, and one required a second infusion after initial CR, remaining PCR negative thereafter. All 3 patients treated for EBV post-transplant lymphoproliferative disorder achieved sustained CR. Rates of aGVHD and cGVHD after infusion were 13% and 23%, respectively. There were no serious infusion-related adverse events. VST infusion was associated with rapid recovery of CD8+CD45RA−CD62L− and a slower recovery of CD4+CD45RA−CD62L− effector memory T-cells; CMV-specific T-cells comprised up to 13% of CD8+ cells. At 1 year post-transplant, non-relapse mortality was 10%, cumulative incidence of relapse was 7%, overall survival was 88% and 25 of 27 patients had ECOG status of 0 or 1. Early administration of third-party VSTs in conjunction with antiviral treatment appears safe and leads to excellent viral control and clinical outcomes. Registered on Australian New Zealand Clinical Trials Registry as #ACTRN12618000343202.

Cytomegalovirus (CMV) is a human herpes virus that causes significant morbidity and mortality in immunosuppressed children. CMV primary infection causes a clinically mild disease in healthy children, usually in early childhood; the virus then utilises several mechanisms to establish host latency, which allows for periodic reactivation, particularly when the host is immunocompromised. It is this reactivation that is responsible for the significant morbidity and mortality in immunocompromised children. We review CMV infection in the primary immunodeficient host, including early identification of these infants by newborn screening to allow for CMV infection prevention strategies. Furthermore, clinical CMV is discussed in the context of children treated with secondary immunodeficiency, particularly paediatric cancer patients and children undergoing haematopoietic stem cell transplant (HSCT). Treatments for CMV are highlighted and include CMV immunotherapy.

Phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling is commonly dysregulated in acute lymphoblastic leukemia (ALL). The TACL2014-001 phase I trial of the mTOR inhibitor temsirolimus in combination with cyclophosphamide and etoposide was performed in children and adolescents with relapsed/refractory ALL. Temsirolimus was administered intravenously (IV) on days 1 and 8 with cyclophosphamide 440 mg/m2 and etoposide 100 mg/m2 IV daily on days 1-5. The starting dose of temsirolimus was 7.5 mg/m2 (DL1) with escalation to 10 mg/m2 (DL2), 15 mg/m2 (DL3), and 25 mg/m2 (DL4). PI3K/mTOR pathway inhibition was measured by phosphoflow cytometry analysis of peripheral blood specimens from treated patients. Sixteen heavily-pretreated patients were enrolled with 15 evaluable for toxicity. One dose-limiting toxicity of grade 4 pleural and pericardial effusions occurred in a patient treated at DL3. Additional dose-limiting toxicities were not seen in the DL3 expansion or DL4 cohort. Grade 3/4 non-hematologic toxicities occurring in three or more patients included febrile neutropenia, elevated alanine aminotransferase, hypokalemia, mucositis, and tumor lysis syndrome and occurred across all doses. Response and complete were observed at all dose levels with a 47% overall response rate and 27% complete response rate. Pharmacodynamic correlative studies demonstrated dose-dependent inhibition of PI3K/mTOR pathway phosphoproteins in all studied patients. Temsirolimus at doses up to 25 mg/m2 with cyclophosphamide and etoposide had an acceptable safety profile in children with relapsed/refractory ALL. Pharmacodynamic mTOR target inhibition was achieved and appeared to correlate with temsirolimus dose. Future testing of next-generation PI3K/mTOR pathway inhibitors with chemotherapy may be warranted to increase response rates in children with relapsed/refractory ALL.

7039 Background: Disease relapse and infection cause significant morbidity and mortality after allogeneic HSCT for acute myeloid leukemia (AML) and myelodysplasia (MDS). Wilms’ tumour 1 (WT1) and preferentially expressed antigen in melanoma (PRAME) are both commonly overexpressed in these conditions. We assessed the safety of a novel combination of tumour associated antigen (TAA) and multipathogen (MP) specific T cells administered prophylactically after HSCT in a phase 1 trial. Methods: Patients with overexpression of WT1 or PRAME by ddPCR on diagnostic tumour samples were eligible. TAA and MP specific T cells were ex vivo expanded from stem cell donors by stimulating apheresis derived mononuclear cells with tumour, viral or fungal peptides. T cells specific for CMV, EBV, Adenovirus (AdV) and Aspergillus (Asp) antigens were produced separately and pooled in equal parts into a MP product. Patients received 1 infusion of MP specific and up to 4 infusions of TAA specific T cells at 4-weekly intervals from 28 days post HSCT (cell dose 2x107/m2 per infusion). Results: Ten HSCT recipients have received a total of 38 infusions. Median age was 48 years (17-67), disease AML (n = 6) or high risk MDS (n = 4), DRI intermediate (n = 4) or high (n = 6), conditioning myeloablative (n = 8) or reduced intensity (n = 2), donor source sibling (n = 7) or matched unrelated (n = 3). Median expression of WT1 on diagnostic bone marrow was 1442 copies/104 copies ABL (0-3870), PRAME 131 copies/104 copies ABL (4-12300). Patients received WT1 (n = 4), PRAME (n = 5) or both WT1 and PRAME specific T cells (n = 1). Mean tumour antigen specificity in the TAA product was 1.4% and 13.3% of CD3+ cells for WT1 and PRAME respectively. Mean total pathogen specificity in the MP product was approximately 44% (CMV = 14.0%, EBV = 14.8% and AdV = 11.6% of CD3+ cells, Asp = 14.8% of CD4+ cells). All patients received MP specific T cells. No immediate infusion related adverse events were reported. At a median 540 days post transplant (80-1265), 8 of 10 patients remain alive and in complete disease remission. Two patients did not proceed after completing 3 of 5 infusions due to the development of graft versus host disease (GVHD); both are in remission. There were 2 deaths; one patient with progressive disease who had persistent high risk MDS pre and post HSCT, and one with multiorgan failure who had multiple post transplant complications (venoocclusive disease, sepsis, GVHD), both prior to infusion. Low level viral reactivations occurred (CMV n = 5, EBV n = 7, BKV n = 3, HHV6 n = 4, AdV n = 1), however none required treatment and there were no cases of viral tissue disease or EBV PTLD. There were no invasive fungal infections. Conclusions: Prophylactic infusions of donor derived WT1/PRAME and multipathogen specific T cells post HSCT are well tolerated and associated with low rates of infection and relapse. Clinical trial information: NCT02895412.

We report on the Australian experience of blinatumomab for treatment of 24 children with relapsed/refractory precursor B-cell acute lymphoblastic leukaemia (B-ALL) and high-risk genetics, resulting in a minimal residual disease (MRD) response rate of 58%, 2-year progression-free survival (PFS) of 39% and 2-year overall survival of 63%. In total, 83% (n = 20/24) proceeded to haematopoietic stem cell transplant, directly after blinatumomab (n = 12) or following additional salvage therapy (n = 8). Four patients successfully received CD19-directed chimeric antigen receptor T-cell therapy despite prior blinatumomab exposure. Inferior 2-year PFS was associated with MRD positivity (20%, n = 15) and in KMT2A-rearranged infants (15%, n = 9). Our findings highlight that not all children with relapsed/refractory B-ALL respond to blinatumomab and factors such as blast genotype may affect prognosis.

Introduction: Reactivation of viruses such as cytomegalovirus (CMV) or Epstein Barr virus (EBV) after allogeneic hemopoietic stem cell transplant (aHSCT) is associated with increased non relapse mortality and a requirement for antivirals with mainly hemopoietic and renal toxicities that can further compromise transplant outcomes and increase health care costs. The use of 3 rd party virus specific T cells (VSTs) has been effective in treating recurrent and refractory viral reactivation after transplant and leads to rapid restoration of viral immunity. We investigated whether early administration of 3 rd party VSTs together with antiviral therapy could safely enhance immune recovery and improve viral control in patients requiring treatment for their initial CMV and EBV infection after aHSCT. Methods: We performed a single arm phase 1 clinical trial in which aHSCT patients requiring treatment for their first CMV or EBV reactivation (or EBV driven malignancy) received infusions of partially HLA matched 3 rd party VSTs within 7 days of commencing standard antiviral treatment. Patients were required to have a viral copy number of at least 1,000 copies/mL for CMV, 10,000 copies/mL of blood for EBV, or proven tissue infection irrespective of copy number for treatment initiation. T cell products were expanded from G-CSF stimulated aphereses from normal donors following peptide stimulation and CD137 magnetic bead selection. T cells were cultured for up to 12 days before specificity testing and cryopreservation. Patients were eligible to receive up to 4 doses of VSTs at 4 week intervals with products selected with a minimum of 1 of 6 HLA matches at HLA-A, -B and -DRB1 with antiviral activity demonstrated through the shared HLA molecule. The primary endpoint of the study was infusion safety. Results: Thirty aHSCT patients were treated with 1-4 VST infusions (27 CMV, 3 EBV) commencing at a median of 4 days after initiation of antiviral treatment. 27 patients were transplanted for hematological malignancies, 3 for immune deficiencies. Conditioning was myeloablative in 12 patients and the majority of patients (22/30) received in vivo T cell depletion. 7/27 CMV seropositive recipients were transplanted from CMV seronegative donors. A total of 41 infusions were given, most frequently targeting antigens presented through shared HLA molecules A2 and/or B7. All infusions were administered at a cell dose of 2 x 10 7/m 2. VST products were CD3 + (median 97.9%, range 96.2 - 99.4%), with median percentage of CD3 +CD8 + 85.8% (range 23.2 - 95.5%). There was one infusion related adverse event consisting of fever that resolved rapidly after admission and antibiotics. Overall viral response rate was 100% with a complete response rate of 94% (Figure 1). Of the 28 patients who achieved a CR (after either 1 or 2 infusions), 22 remained virus PCR negative (n = 8) or below the limit of quantitation (n = 14) for the duration of follow up. 3 patients had brief episodes of quantifiable reactivation not requiring additional therapy and 3 patients required a second infusion following initial CR. All remained PCR negative after their 2 nd CR. All 3 patients treated for EBV PTLD achieved sustained CR. Overall rates of acute and chronic GVHD post-infusion were 33% (10/30) and 20% (6/30) respectively (grade IIII/IV aGVHD 10%, severe cGVHD 7%). VST infusion was associated with a reduction in activation and inhibitory marker expression on CD4 + and CD8 + lymphocytes within the first 30 days and recovery of CD8 + (and more slowly CD4 +) CD45RA -CD62L - effector memory cells. Within the first 100 days after infusion there was an increase in interferon-γ responsiveness of blood lymphocytes. CMV and EBV specific tetramer positive T cells were detected comprising up to 13% of CD8 + cells for up to 6 months post infusion. At 1 year post transplant, non relapse mortality was 10%, cumulative incidence of relapse was 7% and overall survival was 87%. Conclusion: The combination of traditional antiviral treatment and early administration of 3 rd party VSTs is safe and achieves high rates of viral control without evidence of increased acute or chronic GVHD and with evidence of enhanced immune responses to viral antigens. 1 year overall survival was high with low rates of non relapse mortality and relapse. These encouraging results require confirmation in a prospective randomized study comparing best available therapy with best available therapy combined with early VST administration. Figure 1 Figure 1. Disclosures Ritchie: Novartis: Honoraria; BMS: Research Funding; Takeda: Research Funding; CRISPR Therapeutics: Research Funding; Amgen Inc: Honoraria, Research Funding; CSL: Honoraria.

Background The Philadelphia chromosome t(9;22), a reciprocal translocation between chromosomes 9 and 22, results in the gene fusion BCR-ABL1, and occurs in 2-3% of childhood acute lymphoblastic leukemia (ALL). It is detected using cytogenetic and molecular techniques: karyotype, fluorescence in-situ hybridization (FISH) for t(9;22) and reverse transcription polymerase chain reaction (RT-PCR) for BCR-ABL1. Detection has implications for treatment, with the addition of tyrosine kinase inhibitors to chemotherapy regimens improving outcome. Low level BCR-ABL1 transcripts have been reported in blood of healthy individuals. We have observed this finding in bone marrow in newly diagnosed ALL in the absence of the t(9;22) by karyotype or FISH. The significance of low level positivity at diagnosis has not been determined in the setting of childhood Philadelphia chromosome negative (Ph-) ALL. Here we report, for the first time, the molecular evolutionary characteristics of children and adolescents with low level BCR-ABL1 positivity found at diagnosis to relapse. Methods We reviewed 327 patients aged 0-17 years diagnosed with ALL or Acute Leukemia of Ambiguous lineage (ALAL) at The Children's Hospital at Westmead, Sydney, Australia from 1 January 2010 to 30 June 2020. Those positive for the BCR-ABL1 gene fusion by RT-PCR, and negative for t(9;22) by karyotype or FISH were included. Demographics, cytogenetics at diagnosis and relapse, and outcome data were extracted from the medical record. Qualitative BCR-ABL1 analysis was performed using multiplex RT-PCR, followed by nested PCR, on RNA extracted from diagnostic bone marrow (sensitivity 5x10-6). If positive, quantitation was performed using real-time RT-PCR with results expressed as the ratio of BCR-ABL1 over ABL1 (sensitivity 1x10-5). Each PCR included positive and negative controls. Results Of 313 (96%) evaluable patients diagnosed with ALL or ALAL at our institution in the study period, 54 (17%) were positive by RT-PCR for BCR-ABL1 in diagnostic bone marrow. Seven patients were excluded as they had Ph+ ALL-specific treatment after the detection of t(9;22) by karyotype, FISH or other methods. Forty-seven (15%) children with Ph- ALL had low level BCR-ABL1 detected by qualitative PCR. Demographic and cytogenetic characteristics for these patients are summarized in Table 1. All were diagnosed with ALL, the majority (77%) of precursor B-cell lineage including 2 with infant ALL. The e1a2 transcript was identified in 43 (91%) patients, with other transcript types as follows: e4a2 in 1 (2%), e13a2 in 1 (2%), and splicing variants in 2 (4%). BCR-ABL1 quantitation was performed in 43 (91%) and was quantifiable only in 12 (28%) patients, with a median of 0.0008% (range 0.0003 - 0.095%). Forty-five (96%) patients were treated with Berlin-Frankfurt-Munster ALL chemotherapy regimens. The two infant ALL patients were treated on the Interfant06 trial. One received a bone marrow transplant (BMT) in first remission then died after relapse; the other relapsed and died before BMT. Seven (15%) of 47 relapsed, occurring at a median of 21 months (range 2 - 41 months) after diagnosis. Characteristics of these patients are presented in Table 2. Four patients were tested for BCR-ABL1 by RT-PCR in relapse marrow samples; all were negative. No patient with low level BCR-ABL1 positivity at initial diagnosis was diagnosed with Ph+ ALL at relapse. There was no difference in 5-year relapse-free (80% vs 83%, P = .451) or overall survival (86% vs 91%, P = .368) between children with low level BCR-ABL1 positivity (n=47) and those without (n=259). Conclusion BCR-ABL1 low level positivity detected by RT-PCR in the bone marrow of children with newly diagnosed ALL is a relatively common finding, and did not adversely affect outcome for patients treated for Ph- ALL using a contemporary risk-adapted approach. Importantly, this finding did not influence the molecular evolutionary characteristics at the time of relapse in our patient group. Disclosures No relevant conflicts of interest to declare.

10512 Background: PI3K/mTOR signaling, a critical pathway in cell proliferation, metabolism, and apoptosis, is often dysregulated in acute lymphoblastic leukemia (ALL). A phase 1 trial of the mTOR inhibitor temsirolimus combined with etoposide and cyclophosphamide was performed in children with relapsed/refractory (r/r) ALL. Methods: Temsirolimus was administered intravenously (IV) on days 1 and 8 with cyclophosphamide 440 mg/m2 and etoposide 100 mg/m2 IV daily days 1-5. The starting dose level (DL) of temsirolimus was 7.5 mg/m2 (DL1) with escalation to 10 mg/m2 (DL2), 15 mg/m2 (DL3), and 25 mg/m2 (DL4). MRD was performed centrally. PI3K pathway inhibition was measured by phosphoflow cytometry (PFC) analysis of peripheral blood (PB) from treated patients (pts). Results: Sixteen heavily pretreated r/r ALL pts ages 2-19 years with marrow blasts > 25% were enrolled; 15 were evaluable [10 B-ALL/5 T-ALL]. One dose-limiting toxicity (DLT) of grade (Gr) 4 pleural and pericardial effusions with pneumonitis/lung infection leading to Gr 5 cardiorespiratory arrest occurred in a pt treated at DL3. No further DLTs were seen in the DL3 expansion and DL4 cohorts. Gr 3/4 non-hematologic toxicities occurring in ≥ 3 pts included febrile neutropenia, elevated ALT, hypokalemia, mucositis, and tumor lysis syndrome and were independent of dose. Of 15 evaluable pts, 4 (27%; 2 B-ALL/2 T-ALL) had a complete response (CR) after cycle 1, comprised of 1 pt at each DL. Three had MRD < 0.01%. Three pts (20%; 2 B-ALL/1 T-ALL) had partial response (PR). Overall response rate (CR+PR = ORR) was 47%. Pharmacodynamic PFC studies compared phosphoprotein levels pre (day 0) and post treatment (days 3-5) in 9 consenting pts with available PB. All tested pts showed basal activation of PI3K pathway signaling. Dose-dependent inhibition of mTOR targets phosphorylated (p) S6 and/or p4EBP1 was observed in 9/9 and 6/9 pts, respectively, following temsirolimus and chemotherapy treatment. Various patterns of compensatory upregulation of pPI3K, pmTOR, pAkt, and/or pERK was observed. Conclusions: Temsirolimus at 25 mg/m2 combined with salvage etoposide and cyclophosphamide has an acceptable safety profile in high-risk pediatric patients with r/r ALL. Responses were observed at all DLs. mTOR target inhibition was achieved and appeared to correlate with dose level. Future testing of other PI3K/mTOR pathway inhibitors in combination with chemotherapy may be warranted with a goal of further increasing response in r/r ALL. Clinical trial information: NCT01614197.

Studies on twins with concordant acute lymphoblastic leukemia (ALL) have revealed that ETV6-RUNX1 gene fusion is a common, prenatal genetic event with other driver aberrations occurring subclonally and probably postnatally. The fetal cell type that is transformed by ETV6-RUNX1 is not identified by such studies or by the analysis of early B-cell lineage phenotype of derived progeny. Ongoing, clonal immunoglobulin (IG) and cross-lineage T-cell receptor (TCR) gene rearrangements are features of B-cell precursor leukemia and commence at the pro-B-cell stage of normal B-cell lineage development. We reasoned that shared clonal rearrangements of IG or TCR genes by concordant ALL in twins would be informative about the fetal cell type in which clonal advantage is elicited by ETV6-RUNX1. Five pairs of twins were analyzed for all varieties of IG and TCR gene rearrangements. All pairs showed identical incomplete or complete variable-diversity-joining junctions coupled with substantial, subclonal and divergent rearrangements. This pattern was endorsed by single-cell genetic scrutiny in one twin pair. Our data suggest that the pre-leukemic initiating function of ETV6-RUNX1 fusion is associated with clonal expansion early in the fetal B-cell lineage.

Background Administration of partially HLA-matched third party virus-specific T cells (VST) from a cryopreserved cell bank is safe and effective after failure of standard antiviral therapy to resolve viral infection occurring after allogeneic stem cell transplantation (HSCT). Aim In this phase I trial, we assessed the safety and efficacy of administering partially HLA-matched third party VST at the time of initial antiviral therapy following HSCT rather than waiting for failure of at least two weeks of standard antiviral treatment. Methods A cryopreserved cell bank of VST directed at cytomegalovirus (CMV), Epstein Barr virus (EBV) or adenovirus (Adv) was established using G-CSF mobilised peripheral blood from healthy stem cell donors. After stimulation with peptide mixes, VST were selected by expression of CD137+ cells and cultured with cytokines. HSCT recipients were treated with up to 4 doses of 2x107 of VST/m2, the first commencing within 7 days of initial antiviral treatment for viral reactivation. Results A total of 188 doses of VST were manufactured from 7 donors with 12 product manufacturing runs (CMV n=3, EBV n=4 and Adv n=5). Median virus specificity was 75% for CMV, 83% for EBV and 37% for Adv. Thirty HSCT recipients were treated with VST a median of 55 days post-transplant. Data from 25 patients treated for initial viral reactivation were available for analysis (CMV n=22, EBV n=2, Adv n=1). Median age was 58 years (0-71). Patients underwent transplant for myeloid malignancies (n=16), lymphoid malignancies (n=5) and non-malignant conditions (n=4). Patients with malignant disease were transplanted in CR1 (n=8), CR2 (n=3), >CR2 (n=3) or with active disease (n=7). Conditioning was myeloablative in 11 patients and reduced intensity in 14 patients. Donors were matched unrelated (n=20), haploidentical (n=4) or siblings (n=1). 21 patients received some form of T cell depletion (most commonly pre-transplant thymoglobuline in vivo). All patients received VST within 7 days of commencing initial antiviral therapy. 18 patients received a single VST infusion, 6 received 2 and 1 received 4 VST infusions. There were 3 mild infusion related adverse events (vomiting, hypertension, fever). 3 patients had aGVHD pre-infusion (2 grade 1 skin, 1 grade 3 GI). Two patients died of acute GVHD (1 patient with resolved grade 3 GI GVHD pre-VST infusion developed grade 4 GI GVHD 89 days post- infusion as immunosuppression was weaned; the other patient developed de novo liver and GI GVHD 30 days post infusion in the context of a rapid wean in immunosuppression for severe BK virus haemorrhagic cystitis). 2 patients developed de novo grade 1 GVHD post-infusion. 2 patients developed mild limited cGVHD and 1 patient developed extensive cGVHD after VST infusion. 23/25 patients (92%) had complete viral clearance of the infection for which VST were given, 2 had a partial viral response. Median time to best viral response was 20 days. There were 5 deaths (refractory aGVHD in 2 patients, pulmonary VOD/CMV pneumonitis, disease relapse, and sepsis/aspiration pneumonia). 4 of 25 patients died within 12 months of transplant for a 1 year NRM of 12%. At a median follow up of 431 days (112-1391) post-transplant, 20 of 25 patients (80%) remain alive (Figure 1). Conclusion Infusion of third party partially HLA-matched donor-derived VST at the time of first antiviral treatment for CMV, EBV and Adv post HSCT is associated with minimal infusion toxicity, a low rate of moderate to severe GVHD and complete viral clearance in 92% of recipients. Overall survival in this group of high-risk patients requiring treatment for viral reactivation after HSCT is high. A randomised trial will be performed to determine whether administration of third-party VST in addition to standard anti-viral treatment improves transplant outcomes. Figure 1 Disclosures Gottlieb: Haemalogix P/L: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy; Gilead: Consultancy; AbbVie: Consultancy; University of Sydney: Employment; Merck: Consultancy. Ritchie:Amgen: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy; BMS: Research Funding; Takeda: Research Funding; Beigene: Research Funding; Imago: Research Funding; Novartis: Honoraria; Sanofi: Honoraria.

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.

In a small fraction ( approximately 2%) of cases of childhood acute lymphoblastic leukemia (ALL) clinical presentation of leukemia is preceded, some 2-9 months earlier, by a transient, remitting phase of nonclassical aplastic anemia, usually in connection with infection. The potential "preleukemic" nature of this prodromal phase has not been fully explored. We have retrospectively analyzed the blood and bone marrow of a child who presented with aplastic anemia 9 months before the development of ETV6-RUNX1 fusion gene positive ALL. High resolution SNP genotyping arrays identified 11 regions of loss of heterozygosity, with and without concurrent copy number changes, at the presentation of ALL. In all cases of copy number change, the deletion or gain identified by single nucleotide polymorphism (SNP) analysis was confirmed in the ALL blasts by FISH. Retrospective analysis of aplastic phase bone marrow showed that the ETV6-RUNX1 fusion was present along with all of the additional genetic changes assessed, albeit subclonal to ETV6-RUNX1. These data identify for the first time the leukemic genotype of an aplasia preceding clinical ALL and indicate that multiple secondary genetic abnormalities can contribute to a dominant subclone several months before a diagnosis of ALL. These data have implications for the biology of ALL and for management of similar patients.

Identical twins have provided unique insights on timing or sequence of genetic events in acute lymphoblastic leukaemia (ALL). To date, this has mainly focused on ALL with MLL or ETV6-RUNX1 fusions, with hyperdiploid ALL remaining less well characterised. We examined three pairs of monozygotic twins, two concordant and one discordant for hyperdiploid ALL, for single-nucleotide polymorphism (SNP)-defined copy number alterations (CNAs), IGH/L plus TCR gene rearrangements and mutations in NRAS, KRAS, FLT3 and PTPN11 genes. We performed whole exome sequencing in one concordant twin pair. Potential 'driver' CNAs were low, 0-3 per case, and all were different within a pair. One patient had an NRAS mutation that was lacking from leukaemic cells of the twin sibling. By exome sequencing, there were 12 nonsynonymous mutations found in one twin and 5 in the other, one of which in SCL44A2 was shared or identical. Concordant pairs had some identical IGH/L and TCR rearrangements. In the twin pair with discordant hyperdiploid ALL, the healthy co-twin had persistent low level hyperdiploid CD19+ cells that lacked a CNA present in the ALL cells of her sibling. From these data, we propose that hyperdiploid ALL arises in a pre-B cell in utero and mutational changes necessary for clinical ALL accumulate subclonally and postnatally.

Chimeric fusion genes are highly prevalent in childhood acute lymphoblastic leukemia (ALL) and are mostly prenatal, early genetic events in the evolutionary trajectory of this cancer. ETV6-RUNX1–positive ALL also has multiple (∼ 6 per case) copy number alterations (CNAs) as revealed by genome-wide single-nucleotide polymorphism arrays. Recurrent CNAs are probably “driver” events contributing critically to clonal diversification and selection, but at diagnosis, their developmental timing is “buried” in the leukemia's covert natural history. This conundrum can be resolved with twin pairs. We identified and compared CNAs in 5 pairs of monozygotic twins with concordant ETV6-RUNX1–positive ALL and 1 pair discordant for ETV6-RUNX1 positive ALL. We compared, within each pair, CNAs classified as potential “driver” or “passenger” mutations based upon recurrency and, where known, gene function. An average of 5.1 (range 3-11) CNAs (excluding immunoglobulin/T-cell receptor alterations) were identified per case. All “driver” CNAs (total of 32) were distinct within each of the 5 twin pairs with concordant ALL. “Driver” CNAs in another twin with ALL were all absent in the shared ETV6-RUNX1–positive preleukemic clone of her healthy co-twin. These data place all “driver” CNAs secondary to the prenatal gene fusion event and most probably postnatal in the sequential, molecular pathogenesis of ALL.

Abstract 28 Identical twins have provided substantial insight into the natural history, developmental timing and sequence of genetic events in ALL. Much of these data are focussed on ALL with ETV6-RUNX1 fusions. More limited data exists for the most prevalent subset of childhood ALL with hyperdiploidy though retrospective screening from cord blood (Maia et al. Genes Chromosomes and Cancer, 2004) provide evidence that hyperdiploidy itself can be an early, possibly initiating and pre-natal event. We have now explored the genetic complexity of hyperdiploid ALL using four pairs of monozygotic twins, three concordant and one discordant for ALL. In parallel with our prior studies on ETV6-RUNX1 ALL (Bateman et al. Blood, 2010), we hypothesized that hyperdiploidy itself would be a consistent pre-natal event and all other potential ‘driver’ mutations would be secondary and probably post-natal. If correct, this twin pair should share identical hyperdiploid karyotypes but have different copy number alterations (CNA) or other secondary mutations (e.g. in FLT3 or RAS). We used Affymetrix SNP 6.0 mapping arrays to identify CNA using the Genome Orientated Laboratory File (GOLF) v3.2 software package and dCHIP. The SNP array was performed using leukaemic DNA compared to matched remission DNA for all cases. The mutation screen included the activating D835 FLT3, NRAS exon 2 (codons 12 and 13) exon 3 (codon 61), KRAS exon 2 (codons 12 and 13) and exon 3 (codon 61), PTPN11 (exon 3 and exon 13). In the discordant twin pair, Fluorescence In-Situ Hybridisation (FISH) was used to interrogate the pre-leukaemic cells of the unaffected twin for the CNA found in the corresponding affected twin. In the identical twin pairs concordant for hyperdiploid ALL, the whole chromosome gains were identical within each twin pair apart from an additional but sub-clonal gain of chromosome 10 in one twin of twin pair set 2. The median number of whole chromosome gains was 56 (Range 55 to 59). The total number of CNA found was 6 (Range 0 to 4 per case) (excluding T cell receptor and immunoglobulin rearrangements). These included genes previously found to be recurrently altered in hyperdiploid ALL such as PAX5, CDKN2A/2B and ADD3. All the CNA abnormalities detected were different within each twin pair. The mutation screen revealed an NRAS exon 2 codon 13, GGT to GAT (glycine to aspartic acid) mutation in one twin of twin pair set 1, which was absent in leukaemic DNA of the co-twin. In the twin set discordant for ALL, the CNA at 19p13.3 (incl TCF3 gene) found in the leukaemic DNA of twin 1 were absent from the hyperdiploid pre-leukaemic cells of the unaffected twin 2. These pre-leukaemic cells were found at a consistent frequency between 0.0012% and 0.002% (over a period of 13 months). These data accord with the view that hyperdiploidy is a pre-natal and probably initiating event in childhood B cell precursor ALL and that the set of additional ‘driver’ genetic changes including CNA and sequence based mutations are all secondary. As all of the latter are single twin restricted, it is likely, though not formally proven, that they occur post-natally and perhaps more proximal to disease diagnosis. These data extend our understanding of the history of B cell precursor ALL. Whole genome sequencing is required to complete the picture of essential genetic events and their timing. 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.

Monozygotic twin pairs with concordant ALL have provided unique insights into the molecular pathogenesis and natural history of childhood leukaemia. Data from twin pair studies and neonatal blood spot screening indicate that ETV6-RUNX1 usually arises as an early or initiating pre-natal event. Its consequence appears to be the generation of a clinically silent or covert but persistent pre-leukaemic clone. Conversion to overt, clinical ALL then requires the acquisition of one or more additional genetic lesions that functionally complement ETV6-RUNX1, often including deletions of the non-rearranged ETV6 allele. Recent genome wide single nucleotide polymorphism (SNP) array based studies have revealed considerably more genetic complexity than previously suspected, with ETV6-RUNX1 cases having an average of 6 (range 1–21) genomic losses or gains (Mullighan et al., Nature2007, 446: 758). It is however unclear from these descriptive screens or audits when these multiple changes arise in relation to the presumed initiating gene fusion and what functional contribution they make. We have used a series of identical twin pairs with ETV6-RUNX1 positive B precursor ALL to test the proposition that, as we reported previously for ETV6 deletion (Maia et al., Blood2001, 98: 478), all presumed functional or ‘driver’ genomic changes are post-natal in origin and therefore secondary to ETV6-RUNX1 fusion. If this were to be correct then we anticipated that genomic deletions and gains should be different or distinct within each twin pair. We used 250K Sty and 250K Nsp Affymetrix SNP mapping arrays on 5 pairs of identical twins concordant for ETV6-RUNX1 gene fusion positive ALL. We identified copy number variation using the “in-house” Genome Orientated Laboratory File v2.2.9 software package. The SNP array was performed using leukaemic DNA compared to matched remission DNA for 4 out of 5 cases. The fifth case was compared to a pool of remission DNA. The total number of genetic aberrations found was 51 (excluding T cell receptor and immunoglobulin rearrangements): 36 of these lesions were deletions (mean = 7.2) and 15 amplifications. The commonest aberration, found in 8 out of the 10 children, was a deletion on 12p13.2 involving the ETV6 gene. This was discordant in all cases, consistent with our previous reports using microsatellite markers. Other aberrations included deletions of PAX5, CDKN1B, CDKN2A and CD200/BTLA. The status of these, and other, presumed ‘drivers’ of leukaemogenesis were always different when diagnostic DNA of twins, within a pair, were compared i.e. either the genetic change was absent in one but present in the other, or the alteration was present in both but had distinct genomic boundaries. However in 2 of 5 twin pairs concordant, identical lesions were detected. These were idiosyncratic or very rare genomic changes in ALL and were either in gene sparse regions or involved loci with no known or likely contribution to B cell regulation or leukaemogenesis (e.g. CRYGD). We consider the most likely explanation for these shared genetic events in twin cases is that they arise simultaneously with (or immediately prior to) ETV6-RUNX1 fusion, and in the same incipient pre-ALL stem cell, as collateral damage or ‘passenger’ mutations. These data indicate that the common and presumed ‘driver’ genetic changes that accompany ETV6-RUNX1 in ALL are all secondary to gene fusion and most probably post-natal in origin. It remains to be established whether they contribute at all to the sustained pre-leukaemic state and whether they arise independently of each other and sequentially or as a timed suite or bolus perhaps proximate to diagnosis.

Children with Down syndrome (DS) have a 30 fold increased risk of developing leukaemia compared to their non-DS counterparts. While it is now known that cooperating mutations in the haemopoietic transcription factor GATA1 occur in all cases of acute megakaryoblastic leukaemia in Down syndrome patients, the additional genetic events which confer an increased risk of acute lymphoblastic leukaemia in Down syndrome (ALL-DS) are unknown. We initiated a search for mutations in the coding region of candidate genes including RAS, B-RAF, FLT3, KIT and JAK2 in a series of ALL-DS cases. No mutations were identified. We then carried out high resolution (Affymetrix 250K Nsp and 250K Sty) SNP array analysis of leukaemic blast cell DNA from 9 cases of ALL-DS, the majority of which had matched remission DNA. Overall, the whole and partial chromosome gains and losses were in agreement with the karyotype, but in all cases these were updated and refined. There were between 1 and 12 additional copy number alterations per case, with small focal deletions comprising 1 or 2 genes being more frequent than gains. The most common of these was a focal deletion of the CDKN2A gene (4 cases), all of which had either a partial deletion or copy number neutral LOH of the whole of 9p. The other most common focal deletions were of 12p13.3 (ETV6 gene) and 9p13.2 (PAX5), found in 2 cases each. Other regions affected included 3q13.2 (BTLA), 5q33.3 (EBF1), 13q14.2 (RB1) and 20p12.2 (including c20orf94), identified in 1 case each. These results indicate that the secondary genetic events in ALL-DS are similar to those for ALL overall (Mullighan et al., Nature446: 758–764, 2007). The number and pattern of submicroscopic genetic abnormalities more closely resembles that of ETV6-RUNX1 positive ALL than high hyperdiploid ALL (which includes acquired trisomy 21).

Adenovirus (AdV) infection is an important cause of morbidity and mortality post BMT in both children and adults. The incidence of AdV in retrospective or single-centre prospective studies is reported between 4.9% and 41%. There are no published multi-institutional prospective clinical studies. We conducted a prospective cohort study from January 2001 to December 2004 in Australia and New Zealand for allogeneic and autologous BMT in children and adults, to find the incidence of AdV and disease post BMT and confirm risk factors for developing AdV. The 37 centres involved in the study completed a brief questionnaire every month to capture all recognized cases of AdV, and were periodically contacted to collect detailed data on patients who had AdV post transplant. The centres did not change their current practice of surveillance for AdV for this study. No centres were prospectively monitoring for adenoviraemia by polymerase chain reaction (PCR) in blood. We found an overall incidence of AdV disease or infection of 52/3468 (1.5%; 95% Confidence Interval [CI] 1.1 to 2.0), with 46/596, (7.7%; CI 5.7 to 10.2) in the paediatric population and 6/2872 (0.21%; CI 0.1 to 0.45) in the adult population. Although only 596/3468 (17%) of transplants were performed in children, 48% (CI 39 to 69) of all patients with AdV infection were 5 years of age or under. Using the established criteria1 of 52 patients with AdV infection, 14 had definite adenoviral disease and 27 probable adenoviral disease. Four (7.6%) patients died of adenovirus, two of multi-organ failure, one of liver failure and one of pneumonitis. There was a greater risk of developing AdV with an allogeneic transplant than an autologous transplant (RR 9.48, CI 4.2 to 20.9 p Children who had an allogeneic transplant with a T cell depleted graft were at greater risk of developing AdV than those with a T cell replete graft (RR 2.2, CI 1.20 to 4.1, p=0.02) as were those who had an unrelated compared to a related donor (RR 1.99, CI 1.02 to 3.85, p=0.05). Unrelated cord BMT compared to other unrelated transplants was not a statistically significant risk factor for developing AdV in this study (RR 1.1 CI 0.55 to 2.14 p = 0.98). The median day of detection of an AdV isolate for all patients was day 38 post transplant. However, there was a biphasic pattern, with 23.4% of all patients having the first AdV isolate identified greater than Day 100. 38.5% (CI 25.3– 53.0) of patients had isolation of AdV in more than one site. The most common site of an isolate was the gastrointestinal tract in 71% of patients, followed by positive blood PCR in 25%. 31 out of the 52 patients with AdV had another virus isolated apart from AdV; in 15 out of these 31 patients this virus was CMV, either reactivation or CMV disease. This prospective population-based study confirms the importance of AdV in BMT, particularly in paediatric T cell depleted unrelated donor BMT and highlights the occurrence of AdV infection greater than 100 days after transplant.

Introduction: Fludarabine phosphate is commonly used as part of conditioning regimens in children undergoing allogeneic haematopoietic stem cell transplantation (HSCT) for both malignant (n = 47) and non-malignant (n = 39) diseases, but its use has been associated with engraftment failure or mixed chimerism1. The aim of this study was to examine whether suboptimal engraftment is associated with fludarabine pharmacokinetics. Material (or patients) and methods: The clinical pharmacokinetics of fludarabine phosphate was evaluated by studying the free concentrations of the active metabolite, (9B-D-arabinosyl-2 fludarabine, F-Ara-A). Eighty-six HSCTs were studied in 84 children 0.1 to 19 years. Glomerular filtration rate (GFR) was determined by measuring the plasma clearance of diethylenetriaminepentacetic acid (DTPA). The children in this study were treated according to specific HSCT conditioning protocols which varied with respect to the total administered fludarabine dose and the number of days over which it was administered (range: 3 to 6). A weight-based dose of 1 to 2 mg/kg was used in 13 transplants where the patient weighed Results: Mixed chimerism or graft rejection occurred following 10 (of 86) transplants and, of these, 8 were being treated for malignant diseases. First dose free F-Ara-A AUC was significantly lower for these transplants compared with the remainder: median 3.0 (2.4 - 3.8) mg/L.h versus 4.1 (3.3 - 5.2) mg/L.h, p = 0.038, but the difference in clearance was not significant: 6.3 (5.2 - 11.0) L/h versus 4.6 (2.7 - 8.1) L/h, p = 0.06. The recovery of neutrophils also correlated significantly with first dose free AUC (r = -0.22, p= 0.044) but not with clearance (r=0.193, p = 0.075). Recovery of platelets was not significantly associated with any estimate of free F-Ara-A exposure. Within the fludarabine dose range of 25 to 40 mg/m2, there was a linear association with median free F-Ara-A AUC (r 2= 0.977), which was represented by the equation median F-Ara-A AUC (mg/l.h) = 0.1306 x fludarabine dose (mg/m2). There was a significant negative correlation between clearance and GFR (r = -0.31, p < 0.005) and GFR was significantly higher in patients with suboptimal engraftment: median 126 (116 - 152) versus and 111 (92 - 133) ml/min/1.73 m2 (p = 0.015). Suboptimal engraftment was significantly associated with poorer overall survival using Kaplan Meier survival analysis (p = 0.002 using log rank test). Conclusions: The data suggests that patients with good renal function are at greater risk of suboptimal engraftment since they have lower exposure to free F-Ara-A. These children may therefore require higher doses of fludarabine. We found that mg/m2 fludarabine dose was linear with free F-Ara-A AUC between the 25 and 40 mg/m2 dose range. 1.Lee JH, Joo YD, Kim H, Ryoo HM, Kim MK, Lee GW, et al. Randomized trial of myeloablative conditioning regimens: busulfan plus cyclophosphamide versus busulfan plus fludarabine. J Clin Oncol 2013 Feb 20; 31(6): 701-709. Disclosures No relevant conflicts of interest to declare.