Search

The phase 2 portion of this open-label phase 2/3 study assessed the efficacy and safety of blinatumomab as second salvage for aggressive relapsed or refractory (r/r) aggressive B-cell non-Hodgkin lymphoma (B-NHL) following platinum-based first salvage chemotherapy. Forty-one patients with aggressive disease (32% relapsed; 68% refractory) enrolled and received stepwise blinatumomab (9-28-112 μg/day) in a 70-day cycle 1 and an optional 28-day cycle 2; 19 (46%) completed cycle 1 and 3 (7%) completed cycle 2. The overall response rate after 12 weeks was 37%, including 9 (22%) complete metabolic responses. Eight (20%) patients (all responders) subsequently received stem cell transplants. Grade ≥3 adverse events were reported in 29 (71%) patients. Grade 3 cytokine release syndrome occurred in one patient. Grade 3 neurologic events occurred in 10 (24%) patients; all resolved. Blinatumomab monotherapy appears effective as second salvage therapy in patients with r/r aggressive B-NHL. Trial registration: NCT02910063. ispartof: LEUKEMIA & LYMPHOMA vol:61 issue:9 pages:2103-2112 ispartof: location:United States status: published

Introduction: In an open-label phase 2 study, blinatumomab demonstrated efficacy with a manageable safety profile as second salvage in patients with relapsed or refractory B-cell Non-Hodgkin's lymphoma (R/R B-NHL) following platinum-based salvage regimens (Coyle et al. Leukemia & Lymphoma. 2020: 1-10). Blinatumomab is a BiTE® (bispecific T-cell engager) immuno-oncology therapy that activates endogenous cytotoxic T cells to kill target B cells. Here, findings from the updated analysis are reported (NCT02910063). Methods: Patients aged >18 years with biopsy-confirmed B-NHL without prior complete response or complete metabolic response (CMR) following first-line treatment with anthracycline- based chemotherapy and anti-CD20 therapy, had progressive metabolic disease (PMD), no metabolic response (NMR), or partial metabolic response (PMR; Lugano Classification) after ≥2 cycles of platinum-based S1 therapy were eligible. Blinatumomab was given by continuous intravenous infusion for a single 70-day cycle 1 (9 µg/day for 7 days, 28 µg /day for 7 days, and 112 µg /day for 42 days, followed by a 14-day treatment free interval) and an optional 28-day second cycle (9 µg /day for 7 days, 28 µg /day for 7 days and 112 µg / day for 14 days) at the investigator's discretion. Primary endpoint was CMR by central PET. Additional endpoints included objective response rate (ORR; CMR plus PMR), overall survival (OS), progression- free survival (PFS), duration of response, post-response HSCT rate, and the incidence and severity of adverse events (AEs). Results: As of the data cut date (June 3, 2020) for this updated analysis, 41 patients were enrolled between 23 January 2017 and 15 January 2018; 28 (68%) patients were refractory and 13 (32%) relapsed to first-line therapy, 66% had progressive disease following first salvage (S1), and 9 (22%) had double or triple hit status at baseline (Figure 1). ORR was 37% (15/41; 95% CI, 22-53) after 12 weeks of treatment, including 9 (22%) patients who achieved CMR and 6 (15%) achieved PMR. Of the 41 patients enrolled, 17 (42%) were double refractory; of which, 3 (7%) achieved CMR, and 3 (7%) achieved PMR. Of the 41 patients who received blinatumomab, median OS (95% CI) was 11.2 (5.9-NE) months with median follow-up time of 27.9 months. Among the 9 patients who achieved CMR, median OS (95% CI) was NE (7.0, NE) and median PFS (95% CI) was 8.4 (4.9-11.6) months with median follow of time of 4.7 months; of which, 3 patients had disease progression and 0 died. Of the 13 patients who achieved HSCT, median OS (95% CI) was NE (13.1-NE) with 69% of patients alive at 30 months and median PFS (95% CI) was 8.4 (5.3-13.9) months with 21% of patients alive at 12 months (Figure 2 and 3). In total, 29 (71%) patients had grade ≥3 treatment-emergent AEs, including included infections (n=8; 20%), neutropenia (n=4; 10%), pulmonary embolism (n=1; 2%), and acute pancreatitis (n=1; 2%). Consistent with previous blinatumomab reports, neurologic events (NEs) were reported in 23 (56%) patients, including 10 (24%) with grade 3 NEs and 3 (7%) with NEs leading to treatment discontinuation. Grade 3 cytokine release syndrome was reported in only 1 patient. 7 (17%) patients discontinued treatment due to AEs and 7 (17%) had fatal AEs of which were related to disease progression. Conclusions: In conclusion, durable complete remissions can be achieved with a manageable safety profile using blinatumomab as second salvage in patients with aggressive R/R B-NHL following platinum based first salvage regimens Figure 1 Disclosures Coyle: Amgen: Other: Travel support. Morley:Janssen: Honoraria, Other: Fees; AbbVie: Honoraria, Other: Fees; Roche: Other: travel support; Amgen: Honoraria, Other: Fees, travel support. Rambaldi:Sanofi: Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Omeros: Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support from Gilead.; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company). Advisory board and speaker fees from Pfizer.; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company). Research grant from Amgen Inc.; BMS/Celgene: Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Astellas: Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Support of parent study and funding of editorial support. Received travel support., Research Funding; University of Milan: Current Employment. Furness:Amgen: Other: Travel Support. Desai:IQVIA: Current Employment. Mergen:Amgen: Current Employment, Current equity holder in publicly-traded company. OffLabel Disclosure: Durable complete remissions can be achieved with a manageable safety profile using blinatumomab as second salvage in patients with aggressive R/R B-NHL following platinum based first salvage regimens

Background: Tisagenlecleucel was approved in Europe for relapsed/refractory acute lymphoblastic leukemia (ALL) in young patients in 2018. In England, a national CAR T cell panel (NCCP ALL) ensures equity of access and assesses eligibility using inclusion and exclusion criteria based on the ELIANA study. All UK cases are discussed. Eligible cases are allocated to one of 9 JACIE-FACT IEC-approved centres based on age-appropriate service, capacity, distance and patient preference. We systematically reviewed all cases from panel inception providing an opportunity to analyse complete real-world ALL outcomes from this national access programme for tisagenlecleucel on an intention-to-treat (ITT) basis. Methods: We included all patients discussed in the NCCP ALL from Nov 2018 up to July 16th 2020. Clinical data were retrospectively reported in a standardised dataset to allow central analysis of disease and toxicity-related outcomes. Survival outcomes were assessed as for other CAR studies, using Kaplan-Meier estimates of survival from infusion. These included overall survival (interval to death from any cause) and event-free survival as defined in the ELIANA study (interval to death, disease relapse, or treatment failure defined in turn as failure to respond by day 30, with patients receiving further therapy being censored). In order to report outcomes on an ITT basis, survival outcomes were also assessed from time of treatment allocation for all patients considered eligible for tisagenlecleucel. Further, a more comprehensive event-free survival measure encompassing interval to molecular or frank relapse, further therapy, B cell recovery, death or treatment failure was analysed. Median follow up was calculated using a reverse Kaplan-Meier method. Results: Figure 1 demonstrates all patients: 66 patients were screened, 60 patients were deemed eligible for therapy (the ITT cohort), 57 patients were harvested and 49 infused. A total of 3 patients were not harvested and 2 not infused because of progressive disease (n=4) and 1 manufacturing failure. Patient and disease characteristics are summarised in Table 1. The cohort comprised patients with advanced ALL, having been treated with a median of 2.5 therapy lines not including HSCT (range 1-6), and with 47.5% of the cohort having had a prior HSCT. The median follow-up from infusion was 9.9 months and from treatment allocation for the ITT cohort was 11.9 months. The median lead time from allocation to infusion was 2 months. The CR/CRi rate was 95% in the first 90 days, 78.9% were MRD-. On an ITT basis, CR/CRi rate was 84.8%. Median OS and EFS as defined in the ELIANA study were not reached. Overall survival at 6 and 12 months for infused patients was 97.6% and 86.1%, and for the ITT cohort was 90.4% and 78.3%. EFS for infused patients at 6 and 12 months were 74.8% and 68.2%, and for the ITT cohort were 78.5% and 60.9%. Of 49 patients infused, 14 (28.5%) received further therapy including 6 (8.2%) who received allogeneic SCT for relapse or B cell recovery. For patients relapsing following CR (n=10 infused) there were 4/10 CD19- relapses. A composite EFS including molecular or frank relapse, further therapy, B cell recovery, death or treatment failure was analysed. Using this more stringent definition from infusion, the 6, 12 month EFS were 47.6%, and 33.6% and from treatment allocation for the ITT cohort were 63% and 34.3% Toxicity outcomes are summarised in Table 2. Severe (grade ≥3) CRS, neurotoxicity, infection or cytopenia after day 30 post infusion occurred in 20.4%, 10.2% 27.1% and 54.2% respectively. 71.4% of the cohort developed hypogammaglobulinaemia, (Figure 3A). The 6 and 12 month probabilities of B cell depletion were 67.1% and 51.3% respectively. Conclusions: Whilst registry data provide outcomes of large cohorts receiving Tisagenlecleucel, standardised data collection on complete populations are lacking. The framework of a national access scheme provides a unique opportunity to study outcomes on an ITT basis. The CR and MRD negative CR rates, as well as conventional EFS and OS and severe toxicities noted in our cohort compare favourably to published registry reports (Grupp et al., 2019). We found a greater proportion of CD19+ compared to CD19- relapses than noted in the ELIANA study. Consideration of OS and EFS on an ITT basis are informative for clinicians when screening patients for eligibility. If selected for presentation at ASH 2020, updated data will be presented Disclosures Ghorashian: Amgen: Honoraria; Novartis: Honoraria; UCLB: Patents & Royalties. O'Reilly:Gilead: Honoraria; Novartis: Honoraria, Other: Travel support. Roddie:Celgene: Honoraria; Gilead: Honoraria; Novartis: Honoraria. Neill:Novartis: Other: Funded attendance at academic conferences; Celgene: Other: Funded attendance at academic conferences. Pagliuca:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Patel:Novartis: Honoraria, Other: travel support. Hough:Novartis: Other: Travel support.

In recent years significant progress in cancer therapy has led to vastly improved survival rates in the adolescent and young adult diagnosed with cancer. Many patients receive treatment that results in the loss or compromise of future fertility. This has led to a greater focus on reproduction as an important aspect of survivorship. The simultaneous advances in assisted reproductive technology has meant that young people have a better opportunity for future fertility. Though national and international guidance recommend discussion and provision of fertility preservation where practicable there is considerable variation in clinical practice. Oncofertility centres set up in association with larger cancer centres has enabled a rapid access to specialist fertility services, and long term care of gonadal dysfunction in young cancer patients. It is hoped that development of oncofertility networks in the future will enable these resources to be accessed by all cancer centres and many more young people to avail of these services. In this chapter we provide a comprehensive understanding of the effect of cancer therapy on fertility and options of fertility preservation.

Outcomes for teenage and young adult (TYA) patients with acute lymphoblastic leukaemia (ALL) who relapse on contemporary risk-adapted paediatric protocols are largely unknown and there is no consensus on optimal salvage strategies. We assessed the treatment and outcome of TYA patients (aged 16-24 years) recruited to the UKALL2003 trial, who relapsed following attainment of complete morphological remission. Forty-two of 223 patients (18·8%) relapsed, the majority (n = 26, 62%) on treatment. Thirty-eight (90%) patients received salvage treatment, with 22 (58%) achieving second remission (CR2) and 21 patients receiving an allogeneic haematopoietic cell transplant (alloHSCT). Post-relapse outcomes were poor with a 5-year overall survival (OS) of 23% (95% confidence interval; 11-37%). Outcomes for patients relapsing on active treatment were inferior to those relapsing after completing treatment (5-year OS 9% vs. 52%, log-rank P = 0·001). No patient with B cell ALL relapsing on treatment was alive at the end of the study period. TYA patients with ALL who relapse on the UK paediatric protocol, UKALL2003, are largely unsalvageable with conventional approaches aimed at achieving CR2 followed by alloHSCT. Future efforts should be aimed at identifying those patients who are destined to relapse and exploring novel treatment approaches for this high-risk group and for those who do relapse.

Improving outcomes for teenagers and young adults (TYA) with cancer is a key element of the national cancer strategy in England. Recognition of the unique needs of this group has led to the development of recommendations for specific models of care and delivery of this care through the provision of dedicated clinical units in principal treatment centers (PTCs) across the United Kingdom. The aim of this study was to understand the current cancer patient experience for this patient group. We aimed to determine whether treatment experience is influenced by place of treatment and whether it has changed over time using patient-reported data from national cancer patient experience surveys. This study highlights that a prolonged pathway to diagnosis remains an issue for the TYA group and identifies areas on which quality improvement measures for TYA services should focus, including communication and involvement of the patient in treatment decisions. Positive experiences for the TYA group such as involvement in research were also highlighted. Treatment within a TYA PTC was associated with positive patient perception in a number of key areas highlighting the need for future studies to fully elucidate the impact of the full range of TYA services now available in the United Kingdom on both patient experience and outcome.

Background: Autologous hematopoietic stem cell transplantation (autoHSCT) following response to salvage chemotherapy is the standard of care for patients with relapsed/refractory (r/r) aggressive B-cell lymphoma after first-line chemotherapy. Patients without complete metabolic response (CMR) to first salvage (S1) therapy have limited options and poor outcomes based on historical data. Blinatumomab, a bispecific T-cell engager (BiTE®) antibody construct that directs cytotoxic T cells to lyse B cells expressing CD19, has demonstrated a survival benefit in B-cell acute lymphoblastic leukemia and has antitumor activity in patients with r/r aggressive B-cell non-Hodgkin lymphoma (B-NHL), including patients previously treated with autoHSCT. This open-label, multicenter, phase 2 portion of an adaptive phase 2/3 study (ClinicalTrials.gov, NCT02910063) assessed the efficacy and safety of blinatumomab as a second salvage (S2) therapy for patients with aggressive r/r NHL who have not achieved CMR following platinum-based S1 chemotherapy. Methods: Patients ≥18 years had biopsy-confirmed r/r aggressive B-NHL without a prior complete remission or CMR after first-line treatment with an anthracycline and anti-CD20 agent, and had either progressive metabolic disease (PMD), no metabolic response (NMR), or partial metabolic response (PMR; Lugano Classification) after ≥2 cycles of platinum-based S1 therapy. Patients with prior radiotherapy were PET+ ≥6 weeks after the last dose. Blinatumomab was given by continuous intravenous infusion for a single 70-day cycle 1 (9 μg/day for 7 days, 28 μg/day for 7 days, and 112 μg/day for 42 days, followed by a 14-day treatment-free interval) and an optional 28-day cycle 2 (9 μg/day for 7 days, 28 μg/day for 7 days, and 112 μg/day for 14 days). The primary endpoint was CMR by central PET. Additional endpoints were objective response rate (ORR [CMR + PMR]), post-response HSCT realization rates, and the incidence/severity of adverse events (AEs). Results: Of the 41 patients enrolled, most had PMD/progressive disease (66%) and had refractory (68%) or relapsed (32%) disease; 5 (12%) had NMR, and 9 (22%) had PMR (Table). All 41 patients received blinatumomab; 19 (46%) completed cycle 1 (Table). Twenty-two patients discontinued cycle 1 (disease progression, n=17; AE n=4; death, n=1). Among the 17 patients who discontinued cycle 1 due to disease progression, 8 (47%) completed at least 90% of planned treatment duration. Four patients started cycle 2; 3 (7%) completed cycle 2. One patient discontinued cycle 2 due to AEs. The ORR (within 12 weeks of starting blinatumomab) was 37% (15/41 patients; 95% CI, 22%, 53%); 9 (22%) patients achieved CMR (Table). Eight (20%) patients had HSCT in remission, 7 (17%) with autoHSCT (CMR, n=6; PMR, n=1), and 1 with allogeneic HSCT in PMR. Thirty-five patients did not have HSCT (n=32) or had delayed HSCT (n=3) due to PMD (n=17), lack of CMR (n=4), AE (n=4), patient preference (n=1), NMR or unknown (n=1), and other (n=8); 1 patient had missing information. Eight of 9 CMR patients (89%) were alive without relapse, with a median follow up time of 8.8 months. The Kaplan-Meier estimate at 9 months was 51%; median overall survival (OS) was not reached (Table). In total, 24 (59%) patients had grade ≥3 treatment-emergent AEs, and 10 (24%) had grade ≥4 treatment-emergent AEs. Seven (17%) patients discontinued treatment due to AEs. Consistent with previous blinatumomab reports, neurologic events (NEs) were reported in 23 (56%) patients, including 10 (24%) with grade 3 NEs and 3 (7%) with NEs leading to treatment discontinuation. Grade 3 cytokine release syndrome was reported in only 1 patient. Other grade ≥3 AEs included infections (n=8; 20%), bone marrow toxicity (n=7; 17%), thromboembolic events (n=3; 7%), hepatic disorders (n=2; 5%), and acute pancreatitis (n=1; 2%). Conclusions: In patients with r/r aggressive B-NHL and predominantly progressive disease following ≥2 cycles of platinum-based S1 chemotherapy, blinatumomab monotherapy as S2 therapy induced CMR/PMR in 37% of patients and led to HSCT in 20%. When considering that 66% of the patients enrolled had progressive disease and that 47% received the therapeutic dose, blinatumomab showed promising efficacy consistent with the efficacy and safety demonstrated in earlier blinatumomab B-NHL trials and potentially offers a treatment option for patients unresponsive to standard salvage regimens. Disclosures Coyle: Amgen Inc.: Other: non-financial relationship. Morley:Amgen Inc.: Honoraria, Other: non-financial; Roche: Honoraria, Other: non-financial, Research Funding. Rambaldi:Celgene: Consultancy; Roche: Consultancy; Omeros: Consultancy; Italfarmaco: Consultancy; Novartis: Consultancy; Amgen Inc.: Consultancy; Pfizer: Consultancy. Zhang:Amgen Inc.: Employment, Equity Ownership. Jung:Amgen Inc.: Employment, Equity Ownership. Franklin:Amgen Inc.: Employment, Equity Ownership.

Purpose. From a global perspective, the rates of allogeneic hematopoietic cell transplantation (alloHCT) are closely related to the economic status of a country. However, a potential association with outcome has not yet been documented. The goal of this study was to evaluate effects of health care expenditure (HCE), Human Development Index (HDI), team density, and center experience on nonrelapse mortality (NRM) after HLA-matched sibling alloHCT for adults with acute lymphoblastic leukemia (ALL). Patients and Methods. A total of 983 patients treated with myeloablative alloHCT between 2004 and 2008 in 24 European countries were included. Results. In a univariate analysis, the probability of day 100 NRM was increased for countries with lower current HCE (8% vs. 3%; p = .06), countries with lower HDI (8% vs. 3%; p = .02), and centers with less experience (8% vs. 5%; p = .04). In addition, the overall NRM was increased for countries with lower current HCE (21% vs. 17%; p = .09) and HDI (21% vs. 16%; p = .03) and for centers with lower activity (21% vs. 16%; p = .07). In a multivariate analysis, the strongest predictive model for day 100 NRM included current HCE greater than the median (hazard ratio [HR], 0.39; p = .002). The overall NRM was mostly predicted by HDI greater than the median (HR, 0.65; p = .01). Both lower current HCE and HDI were associated with decreased probability of overall survival. Conclusion. Both macroeconomic factors and the socioeconomic status of a country strongly influence NRM after alloHCT for adults with ALL. Our findings should be considered when clinical studies in the field of alloHCT are interpreted.

A single-cytosine-deletion in the herpes simplex virus gene encoding thymidine kinase (TK) was previously found in an acyclovir-resistant clinical isolate. A laboratory strain engineered to carry this mutation did not generate sufficient TK activity for detection by plaque autoradiography, which detected 0.25% wild-type activity. However, a drug sensitivity assay suggested that extremely low levels of TK are generated by this virus. The virus was estimated to express 0.09% of wild-type TK activity via a ribosomal frameshift 24 nucleotides upstream of the mutation. Remarkably, this appeared to be sufficient active TK to support a low level of reactivation from latently infected mouse trigeminal ganglia.

Introduction Treatment outcomes for teenage and young adult (TYA) patients with Acute Lymphoblastic Leukaemia (ALL) lag behind those of younger patients when treated on equivalent protocols, with higher rates of toxicity and relapse reported for those aged >10 years. Outcomes of relapsed ALL are poor so more accurate identification of those destined to relapse is required. Whilst minimal residual disease (MRD) is a powerful tool to predict those at low risk and in whom treatment can be de-escalated, improved strategies are required for early identification of high risk patients. This study aimed to determine if the morphological early response assessment could be used as a predictor of relapse in TYA patients with ALL. Methods All patients treated on the national prospective UKALL2003 trial aged 10 - 24 years who had not relapsed by day 35 (i.e. could potentially have treatment escalated) were included in the study. Early response in patients aged >10 years was defined as percentage of blasts by morphology at day 8: rapid early responders had ≤ 25% blasts and slow early responders >25% blasts. We analysed the following patient characteristics which were previously associated with differential outcomes: age at diagnosis, presenting white cell count, rapid or slow early response (RER or SER); MRD status at day 28; immunophenotype (B/T/other) and cytogenetic risk group (high risk, good risk, poor risk, T-ALL, other). Cox regression was used to assess the association of these factors with time to relapse and logistic regression (LR) was used to assess their association with relapse by the end of treatment (EOT: 2.5 years for girls and 3.5 years for boys). Using backwards selection (cut off for inclusion: p=0.1) a multivariable LR model was established and a receiver operating characteristic curve (ROC) was used to assess how well it could predict relapse before the EOT. Results 820 consecutive patients were included in the analysis of whom 597 were aged 10 - 15 years and 223 were aged 16 - 24 years. 332 patients were classified as MRD high risk and 184 as SER on the basis of morphology. RER was significantly associated with a reduced risk of relapse even after adjustment for other factors including MRD, age, WBC and risk status (Hazard Ratio (HR) 0.64 (95% CI 0.42 - 0.97, p 697 patients with an outcome available at the EOT could be included in the LR analysis. The final multivariable model included day 28 MRD, day 8 response, age, WBC and cytogenetic risk group. The predictor generated by this model gives a ROC area under the curve of 81.2% (95% CI: 76.8 - 85.6) but there were no cut points which resulted in acceptable detection (DR) and false positive rates (FPR). As the intervention for the poor prognosis group would potentially include an allogeneic transplant with associated high mortality and morbidity a low FPR would be key to clinical utility. In order to achieve a clinically useful DR of 79.5% we would have to accept a false positive rate of 30.5%. As only 11% pf patients relapsed by EOT but 36% of patients fell into our bad prognosis group this would mean that for every 100 patients, ~27 would be transplanted unnecessarily. In the subgroup analysis of those aged 16 - 24 early response was not significantly associated with either time to relapse (HR RER: 0.74 (0.36 - 1.51, p = 0.4) or relapse by the EOT (Odds ratio RER: 0.63 (0.26 - 1.49, p =0.3) Conclusions Slow early response is significantly associated with both time to relapse and relapse by EOT in this patient cohort aged 10 - 24 years and this effect is independent of other prognostic factors including MRD. However, even when over-fit the prognostic model generated (which included SER) resulted in FPRs which were too high to be clinically useful. Future work should focus on novel alternative methods of relapse risk prediction for the adolescent patient with ALL. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

The superior outcomes of teenage and young adult (TYA) patients with acute lymphoblastic leukemia (ALL) treated on contemporary pediatric minimal residual disease (MRD) directed chemotherapy regimens compared to adult ALL protocols are well established. However, there are limited data on the outcome of relapse following such regimens and consequently no consensus on optimal salvage strategies. UKALL 2003 was a prospective, randomised controlled trial investigating treatment modification according to MRD status at the end of induction in consecutively diagnosed children and young people with ALL recruited from the UK and Republic of Ireland between October 2003 and June 2011. The trial was initially open to children up to the age of 18 years but the upper age limit was increased to 20th birthday in 2006 and 25th birthday in 2007. Here we report the relapse outcomes of 16 - 24 year old TYA patients recruited to the trial. All TYA patients recruited to UKALL 2003 who subsequently relapsed were identified using the trial database, which also provided data regarding prognostic factors at presentation, MRD risk group, treatment allocation, site of and time to relapse and overall outcome. Individual centers were then contacted for follow up information including salvage chemotherapy, disease response, use of transplantation, subsequent outcome and cause of death. Of a total of 229 TYA patients recruited, 42 (18.3%) relapsed. The immunophenotype in those who subsequently relapsed was precursor B cell (B-ALL) in 30 patients and T cell (T-ALL) in 12. The median white cell count at diagnosis was 22x109/l and 1 patient had central nervous system (CNS) involvement. In B-ALL, 4 had good risk cytogenetics (all high hyperdiploid), 13 had intermediate risk, 6 had high risk (3 with MLL rearrangements), and 7 had unknown cytogenetics. End of induction MRD risk group was low risk in 2, intermediate risk in 11 and high risk in 29. The median time from diagnosis to relapse was 17 months (17.5 months for B-ALL and 14 months for T-ALL, p=0.1). The site of relapse was isolated bone marrow (BM) in 30 patients, combined BM and CNS in 4, combined BM and testicular in 1, and isolated CNS in 6 (T-ALL; 8 BM, 3 BM+CNS, 1 isolated CNS) (B-ALL; 23 BM, 1 BM+CNS, 1 BM + testicular, 5 isolated CNS). 36 of the 42 relapsed patients were given salvage chemotherapy, using a variety of regimens including R3 (n=9), Fludarabine/Cytarabine/Idarubicin based (n=16), and Clofarabine based (n=4). One patient received Blinatumamab and one entered the MARALL study (a Phase I/II study of the combination of Veltuzumab (anti-CD20) and Epratuzumab (anti-CD22) with intensive chemotherapy in patients with relapsed B-cell ALL). A second complete remission rate was achieved in 22 patients (61%) and a further 2 had an empty marrow without count recovery. 23 patients proceeded to an allogeneic hemopoietic stem cell transplant. The median overall survival (OS) from relapse was 213 days (actuarial 5 year OS of 18%). 9 patients remain alive; 8 in remission (1 unconfirmed) and 1 undergoing active treatment. The cause of death in the 33 patients who died was relapsed/refractory disease in 20, complications of re-induction treatment in 4, transplant related complications in 6 and unknown in 2. 1 further patient died of sepsis post transplant in the context of pancytopenia and suspected (though unproven) relapse. Those relapsing earlier than the median of 17 months had a shorter median survival and 5 year OS from relapse compared to those relapsing at >17 months (median 113.5 v 400 days, and OS 10% v 37%, p=0.0075). When this analysis was restricted to only the B-ALL cohort the effect was even more marked (median 151 v 595 days, OS 0% v 45%). No patient with B-ALL relapsing ≤24 months was salvageable. There were no differences in salvage approach between the early and late relapsing cohorts, nor differences in MRD risk. The late relapsing cohort did have more patients with good risk cytogenetics, and fewer with high risk cytogenetics. TYA patients with ALL who relapse despite intensive, contemporary MRD directed chemotherapy have an extremely poor outcome. Patients with B-ALL relapsing at 24 months from diagnosis or earlier are unsalvageable. Improving the outcomes of TYA ALL patients will depend on a) more accurate early identification of those destined to relapse and b) use of innovative therapies in those at high risk of relapse or with overt relapse. Disclosures No relevant conflicts of interest to declare.

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.

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 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.