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Background A study within a trial (SWAT) is a self-contained research study embedded within one or more host trials to evaluate or explore alternative ways of delivering or organising a particular trial process. There is limited evidence of SWATs evaluating trial processes other than recruitment and retention. Purpose Embedding a SWAT into a host trial provides a potential method of evaluating an aspect of intervention implementation, such as engagement or compliance with the intervention. Research Design This paper presents two case studies of SWATs which aim to test the use of video animations to improve intervention implementation, with particular focus on enhancing understanding, engagement and compliance. These are important aspects of intervention implementation as they are directly linked to intervention effectiveness and therefore, important to study. Results In this paper, we present the potential benefits of conducting SWATs of intervention implementation processes as well as discussing the methodological considerations for embedding a SWAT of this nature within a host trial. Benefits include the opportunity to test minor refinements to intervention implementation within trials through robust randomised SWATs, and the possibility of increasing trial efficiency by maximising the quality or quantity of intervention implementation. Methodological considerations surrounding the design and conduct of the SWAT as well as statistical and health economics considerations are discussed in this paper. Conclusions This paper presents a novel application of SWAT methodology in investigating intervention implementation processes within trial conduct.

Background For older trauma patients who sustain trauma in rural areas, the risk of adverse outcomes associated with advancing age, is compounded by the challenges encountered in rural healthcare such as geographic isolation, lack of resources, and accessibility. Little is known of the experience and challenges faced by rural clinicians who manage trauma in older adults. An understanding of stakeholders’ views is paramount to the effective development and implementation of a trauma system inclusive of rural communities. The aim of this descriptive qualitative study was to explore the perspectives of clinicians who provide care to older trauma patients in rural settings. Method We conducted semi-structured interviews of health professionals (medical doctors, nurses, paramedics, and allied health professionals) who provide care to older trauma patients in rural Queensland, Australia. A thematic analysis consisting of both inductive and deductive coding approaches, was used to identify and develop themes from interviews. Results Fifteen participants took part in the interviews. Three key themes were identified: enablers of trauma care, barriers, and changes to improve trauma care of older people. The resilience of rural residents, and breadth of experience of rural clinicians were strengths identified by participants. The perceived systemic lack of resources, both material and in the workforce, and fragmentation of the health system across the state were barriers to the provision of trauma care to older rural patients. Some changes proposed by participants included tailored education programs that would be taught in rural centres, a dedicated case coordinator for older trauma patients from rural areas, and a centralised system designed to streamline the management of older trauma patients coming from rural regions. Conclusions Rural clinicians are important stakeholders who should be included in discussions on adapting trauma guidelines in the rural setting. In this study, participants formulated pertinent and concrete recommendations that could improve trauma care in rural settings.

Patient reported outcomes (PROs) are a pillar of modern-day patient-centered care and clinical trials. PROs complement clinical information with the patient’s own report about their experiences of health, without influence or interpretation by other people. However, choosing an appropriate PRO measure from the many available remains challenging for clinicians and researchers. One of the common pitfalls in instrument selection is that the instrument is often developed with a different patient population than the group being cared for or researched. This difference can result in salient items of importance to the patients, being under-reported or missed altogether. We highlight, through the reporting of some of our own data, that PRO instrument development does not stop with a validation study and we provide suggestions for future research for further improvement in this space.

In successive UK clinical trials (UKALL 2003, UKALL 2011) for paediatric acute lymphoblastic leukaemia (ALL), polyethylene glycol-conjugated E. coli L-asparaginase (PEG-EcASNase) 1000 iu/m

A cancer diagnosis can have significant financial impacts for patients, often resulting from unexpected out-of-pocket expenses and a reduced capacity to work. These financial implications have been well characterised quantitatively in common cancers. However, less is known about the lived experience of financial stress, particularly outside the United States and in rarer cancers. This study aimed to explore the perceived financial impact of acute myeloid leukaemia (AML)-a rare haematological malignancy where patients may be particularly vulnerable to financial stress due to the lengthy, specialised and centralised care. The findings provide insight into the patients' lived experience of the personal financial impact of the disease. This Australian qualitative study was undertaken with 11 adults in remission from AML and recruited from their treating hospital. Semi-structured interviews were transcribed, and data were managed using NVivo. Themes were identified through inductive and deductive analysis using open, axial and thematic coding. Four themes were identified: burden of AML-attributable costs (e.g. out-of-pocket parking and medication expenses); accommodating the AML-impact on paid work (e.g. early retirement and modifying job tasks); the consequence of financial strain from AML (e.g. using savings and accessing Government welfare) and concerns about the future and future familial financial burden (e.g. securing finances and worry about depleting financial resources). A reduction in or stopping work was perceived as the most burdensome to their current and future finances. The findings demonstrate people with AML experience financial difficulty even within a publically funded healthcare system. Opportunities exist for health services to alleviate some financial burden through reducing or abolishing parking fees for oncology patients and ensuring adequate access to social workers to facilitate access to Government welfare. Improving patients' financial difficulties contributes to improved quality of life, which is congruent to cancer survivorship.

Background The majority of older people (> 65 years) in hospital have frailty and are at increased risk of readmission or death following discharge home. In the UK, following acute hospitalisation, around one third of older people with frailty are referred on for rehabilitation, termed ‘intermediate care’ services. Although this rehabilitation can reduce early readmission to hospital ( Methods A multi-centre individually randomised controlled trial, to evaluate the clinical and cost-effectiveness of the HOPE programme. This individualised, graded and progressive 24-week exercise programme is delivered by NHS physiotherapy teams to people aged 65 and older with frailty, identified using the Clinical Frailty Scale, following discharge from acute hospitalisation and linked intermediate care rehabilitation pathways. The primary outcome is physical health-related quality of life, measured using the physical component summary score of the modified Short Form 36- item health questionnaire (SF36) at 12 months. Secondary outcomes include self-reported physical and mental health, functional independence, death, hospitalisations, care home admissions. Plans include health economic analyses and an embedded process evaluation. Discussion This trial seeks to determine if extended rehabilitation, via the HOPE programme, can improve physical health-related quality of life for older people with frailty following acute hospitalisation. Results will improve awareness of the rehabilitation needs of older people with frailty, and provide evidence on the clinical and cost-effectiveness of the targeted exercise intervention. There is potential for considerable benefit for health and social care services through widespread implementation of trial findings if clinical and cost-effectiveness is demonstrated. Trial registration ISRCTN 13927531. Registered on April 19, 2017.

Patient reported outcomes (PROs) are a pillar of modern-day patient-centered care and clinical trials. PROs complement clinical information with the patient's own report about their experiences of health, without influence or interpretation by other people. However, choosing an appropriate PRO measure from the many available remains challenging for clinicians and researchers. One of the common pitfalls in instrument selection is that the instrument is often developed with a different patient population than the group being cared for or researched. This difference can result in salient items of importance to the patients, being under-reported or missed altogether. We highlight, through the reporting of some of our own data, that PRO instrument development does not stop with a validation study and we provide suggestions for future research for further improvement in this space.

Aim: Outcomes of children with high-risk (HR) relapsed acute lymphoblastic leukaemia (ALL) (N = 393), recruited to ALLR3 and ALL-REZ BFM 2002 trials, were analysed. Minimal residual disease (MRD) was assessed after induction and at predetermined time points until haematopoietic stem cell transplantation (SCT). Methods: Genetic analyses included karyotype, copy-number alterations and mutation analyses. Ten-year survivals were analysed using Kaplan-Meier and Cox models for multivariable analyses. Results: Outcomes of patients were comparable in ALLR3 and ALL-REZ BFM 2002. The event-free survival of B-cell precursor (BCP) and T-cell ALL (T-ALL) was 22.6% and 26.2% (P = 0.94), respectively, and the overall survival (OS) was 32.6% and 28.2% (P = 0.11), respectively. Induction failures (38%) were associated with deletions of NR3C1 (P = 0.002) and BTG1 (P = 0.03) in BCP-ALL. The disease-free survival (DFS) and OS in patients with good vs poor MRD responses were 57.4% vs 22.6% (P < 0.0001) and 57.8% vs 32.0% (P = 0.0004), respectively. For BCP- and T-ALL, the post-SCT DFS and OS were 42.1% and 56.8% (P = 0.26) and 51.6% and 55.4% (P = 0.67), respectively. The cumulative incidences of post-SCT relapse for BCP- and T-ALL were 36.9% and 17.8% (P = 0.012) and of death were 10.7% and 25.5% (P = 0.013), respectively. Determinants of outcomes after SCT were acute graft versus host disease, pre-SCT MRD (≥10 −3), HR cytogenetics and TP53 alterations in BCP-ALL. Conclusion: Improvements in outcomes for HR ALL relapses require novel compounds in induction therapy to improve remission rates and immune targeted therapy after induction to maintain remission after SCT. Trial registration: ALLR3: NCT00967057; ALL REZ-BFM 2002: NCT00114348

PURPOSE: Acute myeloid leukemia (AML) is a rare hematologic malignancy accounting for 0.8% of new cancer diagnoses in Australia. High mortality and morbidity affect work productivity through workforce dropout and premature death. This study sought to estimate the productivity loss attributable to AML in the Australian population over 10 years and to estimate the costs of this productivity loss. Productivity was measured using productivity-adjusted life years (PALYs), a similar concept to quality-adjusted life years, but adjusts for the productivity loss attributable to disease, rather than impaired health. MATERIALS AND METHODS: Dynamic life tables modeled the Australian working population (age 15-65 years) between 2020 and 2029. The model population had two cohorts: those with and without AML. Differences in life years, PALYs, and costs represented the health and productivity impact of AML. Secondary analyses evaluated the impact of different scenarios. RESULTS: Over the next 10 years, there will be 7,600 years of life lost and 7,337 PALYs lost because of AML, amounting to Australian dollars (AU$) 1.43 billion in lost gross domestic product ($971 million in US dollars). Secondary analyses highlight potential savings of approximately AU$52 million if survival rates were improved by 20% and almost AU$118 million in savings if the return-to-work rates increased by 20% on the current estimates. CONCLUSION: Our study demonstrates that even in low-incidence cancer, high mortality and morbidity translate to profound impacts on years of life, productivity, and the broader economy. Better treatment strategies are likely to result in significant economic gains. This highlights the value of investing in research for improved therapies.

BackgroundThe statistics of healthcare worker (HCW) COVID-19 infections do not convey the lived experience of HCWs during the pandemic. This study explores the working conditions and issues faced by Australian HCWs.MethodsQualitative analysis of free-text responses from Australian HCWs from 3 August to 5 August 2020 from an open letter calling for better respiratory protection for HCWs, transparent reporting of HCW COVID-19 infections and diversity in national infection control policy development. The open letter was sent to an email list of 23,000 HCWs from a previous campaign and promoted on social media.ResultsAmong 2,733 HCWs who signed the open letter during the study period, 407 free-text responses were analysed. Doctors and nurses accounted for 58% and 35% of respondents, respectively. Most respondents came from Victoria (48%); New South Wales (18%); Queensland (12%) or Western Australia (12%). Dominant themes included concerns about: work health and safety standards; guidelines on respiratory protection including the omission of fit-testing of P2/N95 respirators; deficiencies in the availability, quality, appropriateness and training of personal protective equipment; a top-down workplace culture that enabled bullying in response to concerns about safety that culminated a loss of trust in leadership, self-reported COVID-19 infections in some respondents and moral injury.ConclusionOccupational moral injury in HCWs is the consequence of lapses in leadership at policy-making and organisational levels that have violated the normative expectations of HCWs. The challenge for healthcare leaders is to address workplace culture, consultation and engagement with HCWs in order to prevent this hidden pandemic from spreading throughout the health system.

Background Frailty is characterised by increased vulnerability to falls, disability, hospitalisation and care home admission. However, it is relatively reversible in the early stages. Older people living with frailty often have multiple health and social issues which are difficult to address but could benefit from proactive, person-centred care. Personalised care planning aims to improve outcomes through better self-management, care coordination and access to community resources. Methods This feasibility cluster randomised controlled trial aims to recruit 400 participants from 11 general practice clusters across Bradford and Leeds in the north of England. Eligible patients will be aged over 65 with an electronic frailty index score of 0.21 (identified via their electronic health record), living in their own homes, without severe cognitive impairment and not in receipt of end of life care. After screening for eligible patients, a restricted 1:1 cluster-level randomisation will be used to allocate practices to the PROSPER intervention, which will be delivered over 12 weeks by a personal independence co-ordinator worker, or usual care. Following initial consent, participants will complete a baseline questionnaire in their own home including measures of health-related quality of life, activities of daily living, depression and health and social care resource use. Follow-up will be at six and 12 months. Feasibility outcomes relate to progression criteria based around recruitment, intervention delivery, retention and follow-up. An embedded process evaluation will contribute to iterative intervention optimisation and logic model development by examining staff training, intervention implementation and contextual factors influencing delivery and uptake of the intervention. Discussion Whilst personalised care planning can improve outcomes in long-term conditions, implementation in routine settings is poor. We will evaluate the feasibility of conducting a cluster randomised controlled trial of personalised care planning in a community population based on frailty status. Key objectives will be to test fidelity of trial design, gather data to refine sample size calculation for the planned definitive trial, optimise data collection processes and optimise the intervention including training and delivery. Trial registration ISRCTN12363970 – 08/11/18.

ObjectiveThe aim of this review was to systematically identify, appraise and synthesise evidence on work-related outcomes experienced by younger to middle-aged adults (aged 16–50 years) with arthritis.MethodsEligible studies were identified in Medline, PsycINFO, Embase and CINAHL in January 2020. Quantitative and qualitative studies containing self-reported data on work-related outcomes on younger/middle-aged adults with arthritis were included. Quality assessment was undertaken using validated quality appraisal tools from the Joanna Briggs Institute.ResultsThirty-four studies were identified for inclusion. Work outcomes were organised around five themes: (1) arthritis-related work productivity outcomes, (2) arthritis-related work participation outcomes, (3) other arthritis-related workplace outcomes, (4) barriers to work participation associated with arthritis and (5) enablers to work participation associated with arthritis. Arthritis was associated with work limitations on the Workplace Activity Limitations Scale (average scores ranging from 5.9 (indicating moderate workplace difficulty) to 9.8 (considerable workplace difficulty)), and higher work disability prevalence rates (range: 6%–80%) relative to healthy populations. Arthritis was not associated with decreased absenteeism on the Work Productivity and Activity Impairment Questionnaire (mean (SD) 7.9% (14.0%)), indicating low levels of absenteeism, similar to healthy populations. As work outcomes were commonly binary, person-centred (qualitative) perspectives on barriers and enablers augmented the quantitative findings.ConclusionArthritis is commonly associated with poorer work outcomes for younger/middle-aged adults relative to healthy populations. Additional research focusing solely on the workplace needs of younger/middle-aged population groups is required to inform tailored interventions and workplace support initiatives to maximise productive working years.

It is increasingly appreciated that some patients with cancer will experience financial burden due to their disease but little is known specifically about patients with haematological malignancies. Therefore, this study aimed to measure financial toxicity experienced by patients with haematological malignancies in the context of a publicly funded health care system.All current patients diagnosed with leukaemia, lymphoma or multiple myeloma, from two major metropolitan health services in Melbourne, Australia were invited to complete a survey capturing; patient demographics, employment status, income sources, financial coping and insurances, OOP expenses and self-reported financial toxicity using a validated measure.Of the 240 people approached, 113 (47 %) participated and most had leukaemia (62 %). Forty-seven (42 %) participants experienced some degree of financial toxicity using the Comprehensive Score for financial toxicity (COST) instrument. On multivariate linear regression, older age (65 years, p = 0.007), higher monthly income ($8000, p = 0.008), not having and being forced into unemployment or early retirement (p 0.001) remained significantly associated with less financial toxicity.Financial toxicity is present in Australian haematology patients and those at higher risk may be patients of working age, those without private health insurance and patients that have been forced to retire early or have become unemployed due to their diagnosis.

Background: A low proportion of adults with cancer are recruited to clinical trials. Cancer Council Victoria provides funding to clinical trial sites through its statewide Cancer Trials Management Scheme (CTMS). Historically, there appeared to be a relationship between budget-allocated funding and the number of patients recruited. A randomized controlled trial was conducted to test whether additional funding in 2013 would increase trial recruitment. Methods: A total of 18 trial centers ("sites") received usual CTMS funds, whereas 16 intervention sites received usual funds plus additional funds, proportional to recruitment in 2011; additional payments to sites in the intervention group ranged from $6,750 to $234,000 AUD (≈$6,750-$234,000 USD at the time). This represented an average 11.8% (interquartile range [IQR], 8.0%, 12.3%) increase in sites' budgets. Sites were required to use the funds with the aim of increasing recruitment. The study end point was the number of new participants recruited to trials in 2013. An online survey assessed strategies used to increase recruitment. Results: The median number of new trial recruits per site in 2013 was 21 (IQR, 5-39) in the control arm and 12.5 (IQR, 3.5-44.5) in the intervention arm. The ratio of new trial recruitment numbers at the intervention sites compared with control sites in 2013, adjusting for respective 2012 numbers and institution type, was 0.99 (95% CI, 0.69, 1.43; P=.96). The survey revealed most intervention sites used funding to increase staffing. Conclusions: Additional funding at a site level did not lead to a contemporaneous increase in trial recruitment.

Introduction The use of dexamethasone in acute lymphoblastic leukaemia therapy contributes to short- and long-term toxicities. The UKALL 2011 randomised trial investigated whether a more intense dexamethasone dose (10 mg/m2/d x 14d, short vs 6 mg/m2/d x 28d, standard) would lead to a more rapid cytoreduction and reduced adverse effects associated with longer durations of steroids in induction. The impact of dose and duration on dexamethasone pharmacokinetics was investigated. Methods Blood samples were obtained on one of the first three and last three days of induction dexamethasone dosing at time points up to 8 h after oral administration. Plasma dexamethasone levels were quantified in 1084 plasma samples obtained from 174 children and a population pharmacokinetic model developed. Results Drug exposure varied significantly between patients, with a >12-fold variation in AUC0–12h values and a marked overlap in dexamethasone exposures between dose levels. Intuitively, AUC0–12h was significantly higher with short dosing (10 mg/m2/d), but cumulative exposure was significantly higher with standard dosing over 28 days, after a higher cumulative dose. Concomitant rasburicase administration was associated with a 60% higher dexamethasone clearance. Day 8 bone marrow response was comparable between dosing arms, but those with 5%. No statistical differences were observed between arms in terms of steroid-related toxicity or minimal residual disease at the end of induction. Conclusion The potential significance of dexamethasone AUC0–12h on early response and higher cumulative exposure on the standard arm suggest that duration of therapy and exposure may be more important factors than absolute dose from a clinical pharmacology perspective.

Instagram has more than one billion monthly users, which presents a unique research opportunity particularly in rare diseases or hard to reach populations. This study focuses on acute myeloid leukaemia, a rare haematological malignancy and aims to characterise who posts acute myeloid leukaemia-related content and the type of content created. The findings can provide information and a method for future studies, particularly those focused on online or social media based interventions. Acute myeloid leukaemia-related Instagram posts were identified by searching specific and relevant hashtags (#). A content analysis systematically classified themes in the data. A convenience sample of 100 posts (138 photos) were manually extracted and coded. Data are described using descriptive statistics and demonstrated by qualitative examples. The most frequent users in our sample were patients (66%), patient support networks (24%) and professional organisations (10%). Patients who were communicating their health update (31%) were the most frequently posted content and 25% of these posts described a symptom experience. Our findings demonstrate that patients and their support networks are frequenting Instagram and therefore may be able to receive and benefit from tailored intervention, however there is an identified gap in health-organisations participating in this virtual online community.

// Anindita Dutta 1, 2, * , David N. Potier 1, * , Michael J. Walker 1 , Oliver J. Gray 3 , Catriona Parker 4 , Mark Holland 4 , Andrew J.K. Williamson 1 , Andrew Pierce 1 , Richard D. Unwin 1, 5 , Shekhar Krishnan 2 , Vaskar Saha 2, 4, * , Anthony D. Whetton 1, 3, * 1 Stem Cell and Leukaemia Proteomics Laboratory, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK 2 Tata Translational Cancer Research Centre, Kolkata, India 3 Stoller Biomarker Discovery Centre, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK 4 Children’s Cancer Group, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK 5 Current address: Centre for Advanced Discovery and Experimental Therapeutics, Central Manchester University Hospitals NHS Foundation Trust and Institute of Human Development, University of Manchester, Manchester, UK * These authors have contributed equally to this work Correspondence to: Anthony D. Whetton, email: tony.whetton@manchester.ac.uk Keywords: legumain, AEP, biomarker, SRM, protease Received: May 16, 2016 Accepted: September 15, 2016 Published: September 23, 2016 ABSTRACT Cancer Biomarkers have the capability to improve patient outcomes. They have potential applications in diagnosis, prognosis, monitoring of disease progression and measuring response to treatment. This type of information is particularly useful in the individualisation of treatment regimens. Biomarkers may take many forms but considerable effort has been made to identify and quantify proteins in biological fluids. However, a major challenge in measuring protein in biological fluids, such as plasma, is the sensitivity of the assay and the complex matrix of proteins present. Furthermore, determining the effect of proteases in disease requires measurement of their activity in biological fluids as quantification of the protein itself may not provide sufficient information. To date little progress has been made towards monitoring activity of proteases in plasma. The protease asparaginyl endopeptidase has been implicated in diseases such as breast cancer, leukaemia and dementia. Here we describe a new approach to sensitively and in a targeted fashion quantify asparaginyl endopeptidase activity in plasma using a synthetic substrate peptide protected from nonspecific hydrolysis using D-amino acids within the structure. Our selected reaction monitoring approach enabled asparaginyl endopeptidase activity to be measured in human plasma with both a high dynamic range and sensitivity. This manuscript describes a paradigm for future development of assays to measure protease activities in biological fluids as biomarkers of disease.

The EBF1-PDGFRB gene fusion accounts for 0.5%) at week 14. Two UKALL 2011 patients, positive for EBF1-PDGFRB, received imatinib; 1 died 6 months after a matched unrelated bone marrow transplant as a result of undefined encephalopathy, and the other remained in remission 10 months after diagnosis.

Background:On a global scale, it is estimated that adults in their peak income-earning years are disproportionately impacted by arthritis (1). Younger adults with arthritis are less likely to be employed and are more likely to face productivity challenges at work when compared to healthy similar-aged peers (2). The work-related impacts of arthritis on younger adults remain largely unexplored and are rarely considered in routine clinical care for arthritis.Objectives:To systematically identify, appraise and synthesise the available evidence on work impacts experienced by individuals aged 16-50 years with arthritis.Methods:Eligible studies from 2000 - 2020 were identified in OVID Medline, PsycINFO, Embase, and CINAHL databases using a comprehensive search strategy. Quantitative and qualitative studies containing self-reported data on the work impacts of arthritis on younger people were included. Quality assessment was undertaken using validated quality appraisal tools (3).Results:From a yield of 300 studies, 35 were included in the review. After quality assessment and exclusion of the lowest-ranked studies, 28 studies (17 quantitative, 11 qualitative) were analysed. Work outcomes data were organised into five themes (1-3 for quantitative outcomes, 4-5 for qualitative outcomes): (1) the impacts of arthritis on work productivity; (2) the impacts of arthritis on work participation; (3) other arthritis attributable workplace challenges; (4) barriers to work participation associated with arthritis, and (5) enablers to work participation associated with arthritis. For quantitative themes, arthritis was strongly associated with other workplace challenges: scores on the Workplace Activity Limitations Scale ranged from 5.9 (moderate workplace difficulty) to 9.8 (considerable workplace difficulty); and work disability relative to the healthy population (prevalence ranging from 6% - 80%). For qualitative themes, barriers to work participation included lack of workplace support; enablers included workplace support and intrinsic motivation to work.Conclusion:Arthritis is associated with poorer work outcomes for younger people relative to healthy peers. The available evidence was heterogeneous across studies. Additional research focusing solely on the unique workplace needs of younger population groups is required. This would inform the development of tailored intervention or workplace support strategies to maximise productive working years.References:[1]European League Against Rheumatism. Horizon 2020 Framework Programme: EULAR’s positions and recommendations. EULAR 2011.[2]Jetha A. (2015). The impact of arthritis on the early employment experiences of young adults: A literature review. Disabil Health J. 8(3) 317-324.[3]Joanna Briggs Institute. Critical Appraisal Tools. The University of Adelaide 2019.Disclosure of Interests:None declared

Carbonic anhydrase IX (CA IX) is a hypoxia-regulated enzyme, overexpressed in many types of human cancer. CA IX is involved in pH homeostasis, contributing to extracellular acidification and tumourigenesis. Acidification of the extracellular milieu can impact upon cellular uptake of chemotherapeutic drugs by favouring weak acids (e.g. melphalan), but limiting access of weak bases (e.g. doxorubicin). We investigated whether alterations of CA IX activity affected anti-cancer drug uptake and toxicity. CA inhibitor acetazolamide (AZM) enhanced doxorubicin toxicity but reduced melphalan toxicity in cell lines that highly expressed CA IX under anoxic conditions (HT29 and MDA435 CA9/18). The toxicity changes reflected modification of passive drug uptake. AZM did not alter toxicity or uptake in cells with low CA IX activity (HCT116 and MDA435 EV1). AZM lowered intracellular pH in HT29 and MDA435 CA9/18 cells under anoxic conditions. CA IX activity has chemomodulatory properties and is an attractive target for anti-cancer therapy.

Contains fulltext : 172831.pdf (Publisher’s version ) (Closed access) Somatic genetic abnormalities are initiators and drivers of disease and have proven clinical utility at initial diagnosis. However, the genetic landscape and its clinical utility at relapse are less well understood and have not been studied comprehensively. We analyzed cytogenetic data from 427 children with relapsed B-cell precursor ALL treated on the international trial, ALLR3. Also we screened 238 patients with a marrow relapse for selected copy number alterations (CNAs) and mutations. Cytogenetic risk groups were predictive of outcome postrelapse and survival rates at 5 years for patients with good, intermediate-, and high-risk cytogenetics were 68%, 47%, and 26%, respectively (P < .001). TP53 alterations and NR3C1/BTG1 deletions were associated with a higher risk of progression: hazard ratio 2.36 (95% confidence interval, 1.51-3.70, P < .001) and 2.15 (1.32-3.48, P = .002). NRAS mutations were associated with an increased risk of progression among standard-risk patients with high hyperdiploidy: 3.17 (1.15-8.71, P = .026). Patients classified clinically as standard and high risk had distinct genetic profiles. The outcome of clinical standard-risk patients with high-risk cytogenetics was equivalent to clinical high-risk patients. Screening patients at relapse for key genetic abnormalities will enable the integration of genetic and clinical risk factors to improve patient stratification and outcome. This study is registered at www.clinicaltrials.org as #ISCRTN45724312.

Despite the success of contemporary treatment protocols in childhood acute lymphoblastic leukaemia (ALL), relapse within the central nervous system (CNS) remains a challenge. To better understand this phenomenon, we have analysed the changes in incidence and pattern of CNS relapses in 5564 children enrolled in four successive Medical Research Council-ALL trials between 1985 and 2001. Changes in the incidence and pattern of CNS relapses were examined and the relationship with patient characteristics was assessed. The factors affecting outcome after relapse were determined. Overall, relapses declined by 49%. Decreases occurred primarily in non-CNS and combined relapses with a progressive shift towards later (> or =30 months from diagnosis) relapses (P

Despite the high cure rates in childhood acute lymphoblastic leukemia (ALL), relapsed ALL remains a significant clinical problem. Genetic heterogeneity does not adequately explain variations in response to therapy. The chemoprotective tumor microenvironment may additionally contribute to disease recurrence. This study identifies metabolic reprogramming of leukemic cells by bone marrow stromal cells (BMSC) as a putative mechanism of drug resistance. In a BMSC-extracellular matrix culture model, BMSC produced chemoprotective soluble factors and facilitated the emergence of a reversible multidrug resistant phenotype in ALL cells. BMSC environment induced a mitochondrial calcium influx leading to increased reactive oxygen species (ROS) levels in ALL cells. In response to this oxidative stress, drug resistant cells underwent a redox adaptation process, characterized by a decrease in ROS levels and mitochondrial membrane potential with an upregulation of antioxidant production and MCL-1 expression. Similar expanded subpopulations of low ROS expressing and drug resistant cells were identified in pre-treatment bone marrow samples from ALL patients with slower response to therapy. This suggests that the bone marrow microenvironment induces a redox adaptation in ALL subclones that protects against cytotoxic stress and potentially gives rise to minimal residual disease. Targeting metabolic remodeling by inhibiting antioxidant production and antiapoptosis was able to overcome drug resistance. Thus metabolic plasticity in leukemic cell response to environmental factors contributes to chemoresistance and disease recurrence. Adjunctive strategies targeting such processes have the potential to overcome therapeutic failure in ALL.

The resistance of hypoxic cells to conventional chemotherapy is well documented. Using both adenovirus-mediated gene delivery and small molecules targeting hypoxia-inducible factor-1 (HIF-1), we evaluated the impact of HIF-1 inhibition on the sensitivity of hypoxic tumor cells to etoposide. The genetic therapy exploited a truncated HIF-1alpha protein that acts as a dominant-negative HIF-1alpha (HIF-1alpha-no-TAD). Its functionality was validated in six human tumor cell lines using HIF-1 reporter assays. An EGFP-fused protein demonstrated that the dominant-negative HIF-1alpha was nucleus-localized and constitutively expressed irrespective of oxygen tension. The small molecules studied were quinocarmycin monocitrate (KW2152), its analog 7-cyanoquinocarcinol (DX-52-1), and topotecan. DX-52-1 and topotecan have been previously established as HIF-1 inhibitors. HT1080 and HCT116 cells were treated with either AdHIF-1alpha-no-TAD or nontoxic concentrations (0.1 microM

Extracellular vesicles have been described in non-paracrine cellular interactions in cancer. We report a similar phenomenon in B-cell precursor (BCP) acute lymphoblastic leukaemia (ALL). Using advanced microscopy and high throughput screening, we further characterise a subset of large vesicles (LEVs) identified in cell lines, murine models of human BCP-ALL and clinical samples. Primary ALL blasts and cell lines released heterogeneous anucleate vesicles

Contains fulltext : 138929.pdf (Publisher’s version ) (Open Access) The outcomes of Central Nervous System (CNS) relapses in children with acute lymphoblastic leukaemia (ALL) treated in the ALL R3 trial, between January 2003 and March 2011 were analysed. Patients were risk stratified, to receive a matched donor allogeneic transplant or fractionated cranial irradiation with continued treatment for two years. A randomisation of Idarubicin with Mitoxantrone closed in December 2007 in favour of Mitoxantrone. The estimated 3-year progression free survival for combined and isolated CNS disease were 40.6% (25.1, 55.6) and 38.0% (26.2, 49.7) respectively. Univariate analysis showed a significantly better survival for age

Though modern chemotherapy regimens cure over 80% of children with acute lymphoblastic leukaemia (ALL), the outcome of relapsed disease remains suboptimal. Current curative strategies for relapsed patients are based on high dose cytotoxic therapy often followed by an allogeneic stem cell transplant. Drug-related toxicities limits the intensification of therapy and can result in treatment delays as well as deaths in remission. The drug L-Asparaginase (ASNase) is not associated with a dose-limiting toxicity and has been widely used not only in strategies for relapsed ALL1 but also in early phase clinical trials.2 ASNase is a bacterial protein and the main disadvantage of its repeated use is sensitization leading to clinical hypersensitivity reported in up to 60% or silent inactivation of the drug in 30% of patients receiving native Escherichia Coli–ASNase.3 As most patients receive intensive ASNase during frontline therapy, there is then the question of its continued efficacy on subsequent use in relapsed patients. One option is the use of the noncross reacting Erwinia product, but this has a shorter half life and is prohibitively expensive. The polyethylene-conjugated ASNase (PEG-ASNase) is associated with less antigenic reactions, as a lower dose is required and it can be given less frequently. It is thus increasingly being used in both frontline and relapsed-therapeutic protocols. The optimal dose remains unclear and the drug is administered ranging from 2500 IU m−2 (refs. 2, 4) (weekly) to 1000 IU m−2(ref. 1) (fortnightly). Before 2003, all de novo childhood ALL patients in UK received native ASNase. Since then patients have received PEG-ASNase, 1000 IU m−2 given intramuscularly, twice in induction and once in delayed intensification with high-risk patients receiving up to an additional nine doses.5 Relapsed patients in the international ALLR3 trial (2003–2013, ISCRTN45724312) were given the same dose of PEG-ASNase, two doses in induction and once during consolidation1 (Supplementary Figure 1). To assess the effect of PEG-ASNase given during initial therapy on its subsequent use in relapse patients, we recruited after obtaining ethical approval and written consent, patients on the ALLR3 clinical trial who had previously been treated on the UKALL 2003 protocol. Thirty-three patients were analysed between Jan 2009—May 2011. Trough ASNase activity was measured in plasma samples 7–14 days after each PEG-ASNase dose. ASNase activity was measured using a modified indoxine method.6 A trough level of ⩾100 IU/l was taken to represent adequate therapeutic activity.7 Antibodies against PEG-ASNase and native E. coli–ASNase (Asparaginase Medac) were measured by indirect enzyme-linked immunosorbent assays developed and validated at Medac GmbH (Hamburg, Germany) from samples taken for activity analysis and in a sample obtained once ASNase therapy was completed but before transplantation (Supplementary Figure 2). Of the 33 patients recruited (Table 1), a total of 21 patients had ASNase activity measured at least at one time point (maximum of three). Only one patient did not show adequate activity with the rest demonstrating activity ⩾200 IU l−1 (Supplementary Figure 2). We were able to serially monitor nine patients. With the exception of the previously described patient with inadequate levels, the other eight were found to have adequate activity at more than one time point. Antibody assays were performed in 19 of the 21 patients with ASNase activity results and an additional 14 patients in whom we did not have activity data. Only one of the 33 patients was detected to have antibody against both PEG-ASNase and native ASNase. This was the same patient who did not have adequate ASNase activity levels. Thus 1000 IU m−2 of PEG-ASNase given intramuscularly provides adequate therapeutic levels in patients who relapse of a PEG-ASNase frontline protocol. For patients treated initially with PEG-ASNase on frontline protocols who subsequently relapse, silent inactivation does not appear to pose a significant clinical problem. Table 1 Distribution of patients in the study At the time these assays were performed, 390 patients, recruited from participating centres in UK, Netherlands, Australia and New Zealand, had completed the first three blocks of therapy containing ASNase (Supplementary Figure 1).1 Patients recruited to ALLR3 with hypersensitivity to E Coli-derived ASNase (either native or PEG) received the Erwinia Chrysanthemi derivative Erwinase. We identified 354 patients who received PEG-ASNase, 20 who received Erwinase and 16 patients who received no ASNase in the ALLR3 trial (Table 1). For a period of time, Erwinase was not available and patients with hypersensitivity (n=14) did not receive any ASNase. Similarly, patients who developed pancreatitis (n=2) during frontline therapy did not receive any ASNase in ALLR3. We examined the impact of not receiving ASNase on the outcome of these relapsed patients. Progression-free and overall survival were analysed using the Kaplan–Meier plot (unstratified) and log rank test. The 3-year progression-free survival and overall survival of the 16 patients who did not receive ASNase were 42.9 (95% CI 30.3,55.5) and 49.2% (95% CI 36.5,61.9). This was not significantly different from the progression-free survival of 46.4% (95% CI 35.4,57.1) (P=0.377) and overall survival of 53.5% (95% CI 41.8,63.9) (P=0.365) of the 354 patients who received PEG-ASNase (The 20 patients who received Erwinase when it became available at a later date have not been analysed here). The outcome of relapsed patients is related to the duration of first remission, site of relapse and immunophenotype,8 with late isolated extramedullary and progenitor-B-cell phenotype having the best outcomes. In this latter category, there were no patients who had not received ASNase. To eliminate bias, a matched case-control analysis with a 3:1 ratio including all non-PEG patients and the maximum number of PEG patients randomly selected to obtain balance in both groups (same percentages) was performed. The progression-free survival and overall survival in the PEG and non-PEG groups were not significantly different (Figure 1). Unlike that previously reported for patients treated on frontline protocols,9 survival was similar for patients with relapsed ALL irrespective of whether they received ASNase or not. A recent report from the frontline ALL2003 trial has also failed to show a difference in outcome in patients with ASNase-induced pancreatitis who received no further ASNase.10 Figure 1 Kaplan–Meier estimates of (a) progression-free and (b) overall survival in relapsed ALL patients who received no asparaginase (n=16) and a 3:1 risk-stratified matched cohort of those who received PEG-Asparaginase (ASNase) (n=48). Of the 354 patients who received PEG-ASNase as part of their relapse therapy, 241 and 113 patients, respectively had received native or PEG-ASNase during frontline treatment. Twenty-two (6%) patients, 12/241(5%) who had received native ASNase and 10/113 (9%) who had received PEG-ASNase in frontline therapy reported grade 3-4 toxicities associated with ASNase. Ten developed pancreatitis, seven had thrombosis, four had encephalopathy and one had hypersensitivity. The toxicity data for patients in ALLR3 who received E. Coli–ASNase or PEG-ASNase in the frontline protocol are comparable, though the slightly lower incidence with previous native ASNase may reflect the expected higher incidence of silent antibodies. PEG-ASNase at this dose can be administered at relapse, with no excess toxicity in patients who have previously received E. Coli–ASNase in frontline therapy. The numbers analysed here are small, a reflection of the rarity of the disease, and thus interpretations of this data must be viewed cautiously. However we feel that our data will inform consortiums in the rational design of therapeutic strategies incorporating ASNase for those with recurrent ALL. This includes optimising both the dose and timing of ASNase. For patients relapsing of a frontline protocol using PEG-ASNase exclusively, the drug may be used again safely and a dose of 1000 IU m−2 appears to be adequate for most patients. Furthermore, as in ALLR3, the timing of PEG-ASNase administration can be optimised to potentially maximise the effect of dexamethasone,11 as well as high dose methotrexate and cytarabine blocks in a Capizzi design.12 All patients in this study analysed for ASNase activity relapsed of the UKALL 2003 trial and received 3-12 doses (based on risk stratification) of PEG-ASNase at a dose of 1000 IU m−2.5 Thus we do not have comparable activity data from patients who initially received E. Coli–ASNase in earlier frontline protocols, though we found no differences in toxicity. The use of PEG-ASNase after E. Coli–ASNase has been previously reported to be associated with a high incidence of hypersensitivity and drug inactivation owing to the presence of antibodies.13 However if antibody titres are low, PEG-ASNase may still provide adequate activity levels.14 A dose of 2500 IU m−2 given weekly has been shown to provide therapeutic levels in relapsed ALL patients who have previously received E. Coli–ASNase.15 The monitoring of ASNase activity/antibody and increasing the dose of PEG-ASNase in those who have inadequate activity/silent antibodies could optimise drug administration and lead to a better outcome. This merits investigation in future trials for relapsed ALL.

BACKGROUND: The determinants of outcome in children with rALL are the duration of first remission (CR1), site of relapse and immunophenotype. High risk (HR) relapses are defined as those occurring with a CR1 of METHODS: Clofarabine was added to the UKALLR3 consolidation block of cyclophosphamide, etoposide (CCE) and used as induction therapy, with dexamethasone and PEG-Asparaginase for HR patients. The previous induction block with mitoxantrone (M) was given as consolidation and all patients were eligible for stem cell transplantation (SCT) with any donor after a third intensification block. The outcomes assessed were improvements in CR2, MRD and progression-free survival (PFS) when compared to historical controls of patients receiving idarubicin (I) or M induction in UKALLR3. A Fleming-style design, based on observed response and toxicity, was incorporated to allow an increase in the dose of cyclophosphamide from 300 mg/m2 to 440 mg/m2. RESULTS: 61, 39 at lower and 22 at the higher dose of cyclophosphamide, CCE patients were compared to 30 I and 69 M patients with HR rALL. Patients in the CCE group had a lower median age at presentation, but other prognostic variables were comparable. CR2 rates of 73%, 83%, 71% and low MRD (≤10-4) was seen in 32%, 0%, 25% of CCE, I and M groups. The higher cyclophosphamide dose was associated with improved CR rates, lower MRD but also increased toxicity levels in CCE compared to M group patients. The proportions of patients reaching transplantation were 43%, 60% and 55% of CCE, I and M patients respectively. 73/82 eligible patients received a SCT, 48 (66%) with matched and 25 (34%) with mismatched donors. The 2-year PFS with CCE, M and I regimens were 17% (11,23), 27% (19,34) and 30% (25,36) respectively (p=0.08). Outcomes of matched sibling, matched unrelated and mismatched SCT were comparable (p=0.9). Seventeen patients with a post induction MRD CONCLUSIONS: We define two groups of HR rALL patients based on MRD levels attained post induction, independent of the induction regimen. Approximately a quarter of HR patients continue to have chemosensitive disease as evidenced by rapid MRD clearance ( Disclosures Off Label Use: Clofarabine 1st relapse childhood ALL.

Abstract 2573 Introduction: Polyethylene glycol conjugated L-asparaginase (PEG-ASNase) is used in varying doses between 1000–2500 U/m2 in the therapy of ALL and associated with a wide variation in pharmacokinetics. UKALL 2003 used intramuscular PEG-ASNase at 1000 U/m2. Patients & Methods: Patients enrolled in the UKALL 2003 trial (ISRCTN: 07355119), were consented for trough asparaginase activity analysis during therapy. National Cancer Institute (NCI) standard risk (SR) patients with rapid early response and non-high risk (HR) cytogenetics (Lancet Oncol. 2010;11:429) received a 3 drug induction (Dexamethasone, Vincristine & PEG-ASNase). All other patients received additional Daunorubicin. PEG-ASNase was given on days 4 and 18 of induction, and at least once post induction. Trough plasma asparaginase activity was measured by the indooxine method (Anal. Biochem. 2002;309:117). The lower limit of assay detection was 34 U/L. Adequate asparaginase activity was defined as a trough level of > 100 U/L. IgG and IgM antibodies to PEG-ASNase and native asparaginase were measured by indirect ELISA. Asparaginase activity was correlated with defined risk factors, minimal residual disease (MRD) at day 28 of induction and development of anti-asparaginase antibodies using the chi-squared test. Results: Between July 2008 to July 2011, 482 patients aged 1–25 years from 27 centres were recruited. Numbers of samples assayed in induction were 335 & 371 after first and second doses respectively. Overall, 86% (n=606/706) of samples had adequate activity during induction time points. There was > 10 fold variation in activity levels (Figure 1). Three hundred and nine out of 706 samples had activity > 3 times the therapeutic threshold, while 51/100 samples with inadequate activity had no detectable drug levels (median: below detection limit, range: < 34–99 U/L). Thus, increasing the dose of PEG-ASNase in induction is unlikely to benefit patients with inadequate activity. Compared to SR patients, NCI HR patients had a higher incidence of inadequate asparaginase activity in induction (p=0.002). Inadequate asparaginase activity correlated with high MRD (≥ 10−4) in SR patients (p=0.045), especially those with good risk cytogenetics (p=0.012), and in particular the high hyperdiploid subgroup (p=0.03). Inadequate asparaginase activity during induction did not correlate with MRD in HR patients (p=0.699), possibly because these patients received in addition Daunorubicin (Table 1). Results of serial asparaginase activity (at least one time point each in induction and post induction), measured in 282 patients are summarised in Table 2. Antibodies were detected in 18 of 81 patients tested. All had anti-PEG and 7 in addition also had anti-asparaginase antibodies. While all antibody positive patients had inadequate asparaginase activity at one time point, 17 had adequate activity prior to antibody detection, suggesting immune-mediated drug inactivation at re-exposure. Antibodies were not detected in 14/15 patients who had inadequate activity at first exposure, so the mechanism here remains unclear. The reported incidence of asparaginase toxicity in this study was 6.6% (n=32/482). This included hypersensitivity (n=17/482) that was almost exclusively seen in HR patients (n=16/17), thrombosis (n=10/482) and pancreatitis (n=5/482). Conclusions: Intramuscular PEG-ASNase given fortnightly at 1000 U/m2 during induction provides adequate asparaginase activity in 86% of patients. Monitoring asparaginase activity may benefit patients who receive 3 drug induction and improve the resolution of the current prognostic classification. Disclosures: Off Label Use: PEG-asparaginase. Essink:medac: Employment. Kuehnel:medac: Employment.

6514 Background: The proportion of adults with cancer recruited to clinical trials is low. Cancer Council Victoria awards funding to clinical trial sites through its Clinical Trials Management Scheme (CTMS). With this state-wide scheme, there appeared to be a temporal relationship between growth or stability in funding and the number of patients recruited. We undertook a RCT to evaluate the hypothesis that additional funding would improve trial recruitment. Methods: Participating sites (n = 34) were stratified into four strata based on 2011 recruitment numbers. Control sites (n = 18) received usual CTMS funds only while intervention sites (n = 16) received usual funds plus additional funds, proportional to the number of patients recruited in 2011. Additional funding was a median increase of 300% (IQR: 112.5%, 459%) relative to usual CTMS funds and was an average 11.8% (IQR: 8%, 12.3%) increase in a site’s budget. Additional funds were provided in early 2013. Sites were to use funds with the aim of increas...

Hypoxia is a physiological abnormality that has been detected in all solid tumours analysed to date. Studies using polarographic needle electrodes have shown an unequivocal link between the extent of tumour hypoxia and poor treatment outcome. The practical limitations of polarographic needle electrodes have warranted investigation into alternative strategies enabling routine assessment of tumour hypoxia in the clinical setting. This review focuses on the clinical evaluation of exogenous and endogenous markers of tumour hypoxia that may fulfil this role.

Acute lymphoblastic leukaemia (ALL) is a heterogeneous disease at the genetic level. Chromosomal abnormalities, gene deletions, mutations and amplifications relevant for diagnosis, prognosis or treatment allocation have been identified. This knowledge is mostly derived from presentation samples and focussed on predicting for relapse after frontline therapy. Studies genetically characterising a large cohort of relapsed patients treated uniformly on a single protocol have rarely been reported. In this study, we assessed the outcome of B-cell precursor (BCP) ALL patients treated on the international relapse trial, ALLR3, according to the presence of established chromosomal abnormalities and the most prevalent gene deletions. Among 416 childhood BCP-ALL patients, a representative cohort of 372 (89%) patients were classified into three risk groups based on cytogenetic analysis at presentation and/or relapse: (1) good risk (GR, n=181, 49%) - ETV6-RUNX1, high hyperdiploidy; (2) poor risk (PR, n=69, 19%) - BCR-ABL1, MLL translocation, near haploidy, low hypodiploidy, intrachromosomal amplification of chromosome 21 (iAMP21), t(17;19)(q23;p13) and, in the absence of GR abnormalities, deletions of 13q and abnormalities of 17p; and (3) intermediate risk (IR, n=122, 33%) – all other cases with abnormal or normal cytogenetics. Relapses were categorised as very early ( Patients at first relapse with bone marrow involvement (minimum 50% blasts in the marrow) were screened by multiplex ligation-dependent probe amplification (P335 kit, MRC Holland) for the most prevalent micro-deletions seen in paediatric ALL. Among 285 eligible patients, a representative cohort of 187 (66%) patients was analysed. Over two-thirds (126, 67%) of patients harboured at least one micro-deletion at relapse: CDKN2A/B (77, 41%), IKZF1 (44, 24%), PAX5 (37, 20%), ETV6 (31, 17%), PAR1 (P2RY8-CRLF2) (18, 10%), EBF1 (7, 4%), BTG1 (6, 3%) and RB1 (6, 3%). IKZF1 and PAX5 deletions were significantly less prevalent among GR patients compared to IR and PR patients: IKZF1 8/81 (10%) v 22/53 (42%) v 12/38 (32%) (p A subset of 87 patients with a marrow relapse was screened successfully by MLPA at both presentation and relapse to estimate the degree of clonal evolution. The majority of patients (47/87, 54%) displayed evidence of evolution defined as the gain or loss of at least one micro-deletion. The acquisition of a micro-deletion was more common than loss (54 v 22). There was a trend towards late relapses being more likely to show evidence of clonal evolution compared to very early and early relapses: 34/54 (63%) v 12/28 (43%) v 1/5 (20%), p=0.065, respectively. However, there was no evidence of an association between clonal evolution and cytogenetic risk group or outcome. In conclusion, cytogenetic risk groups strongly correlated with both the duration of first remission and outcome after first relapse. However, the most prevalent eight micro-deletions, which were frequently gained or lost between presentation and relapse, did not influence outcome after first relapse. Disclosures: No relevant conflicts of interest to declare.

Clinical and laboratory studies in childhood acute lymphoblastic leukemia (ALL) suggest that the bone marrow microenvironment protects blast cells, thus influencing disease relapse. This study was designed to identify the mechanisms by which bone marrow stromal cells could mediate chemoresistance in ALL cells. An in vitro organotypic extracellular matrix-bone marrow stromal cells (ECM-BMSC) culture system, that sustained primary cells without the addition of exogenous factors, was created. ECM-BMSC derived conditioned medium (CM) conferred broad-spectrum chemoprotection to ALL and other cancer cells. Multi drug resistant sub-clones (MDRS), derived by exposing ALL cells in CM to Mitoxantrone, retained chemoresistance even beyond 50 passages in normal medium. Chemoprotective effect was retained in the Proteinase K, RNase and heat resistant, < 3kDa fraction of CM; transmission electron microscopy demonstrated the presence of stromal cell derived exosomes in this fraction. Confocal microscopy confirmed the uptake of ultra-centrifuged fluorescently labelled (PKH 67) stromal exosomes by ALL cells which then developed a resistant phenotype. Stromal derived exosomes and MDRS were enriched in miRNA expression (TaqMan Human miRNA array V3), compared to parent cells, with 92% concordance. Ingenuity™ pathway-based analysis of the unique miRNA pattern predicted dysregulation of mitochondrial function and oxidative phosphorylation. SILAC based phosphoproteomics and U133A V2 gene expression array analysis revealed aberrant PI3K/AKT and oxidative phosphorylation activity. A kinase inhibitor screen showed that PI3K/AKT inhibitors were most potent in reversing CM mediated chemoresistance. Exposure to CM initially (24 hours), pAKT activity and ROS level decreased to lower than the basal level, with upregulation of CDKN1B and MCL-1, and a decreased mitochondrial membrane potential. shRNA knockdown of CDKN1B restored normal growth rate but not chemosensitivity. MDRS that were resistant to exogenous H2O2 induced apoptosis, regained sensitivity after MCL-1 knockdown. Upregulation of ROS and downregulation of MCL-1 using PEITC lead to cell death in chemo sensitive and resistant cells. Our study identifies that the microenvironment produces soluble factors that induces a redox adaptation in ALL cells leading to chemoresistance. This can be overcome by the simultaneous inhibition of the antioxidant system (to increase ROS) and suppressing antiapoptic proteins (to eliminate the mitochondrial membrane safeguard). Tumour protection by the microenvironment is likely to be common to other cancers. Future therapeutic strategies need to consider targeting the host-tumor interactions in adjunct to conventional chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1506. doi:1538-7445.AM2012-1506

Abstract 3303 Introduction: Despite improvement in frontline therapy in childhood acute lymphoblastic leukemia (ALL), central nervous system (CNS) relapse remains a significant clinical problem. The ALLR3 trial (ISCRTN 45724312) was designed specifically to address this issue with the use of drugs known to penetrate the CNS. The trial incorporated a randomization between Mitoxantrone and Idarubicin during induction. Mitoxantrone showed an early benefit in all patients resulting in closure of the randomization in December 2007 (ASH Annual Meeting Abstracts, Nov 2009; 114:3390). Subsequently all patients now receive Mitoxantrone. Here we report on the outcome of patients with isolated CNS relapse (iCNSr) or combined CNS relapse (involvement of CNS and bone marrow, cCNSr). Methods: CNS involvement was defined as ≥5 WBC/μl with morphological evidence of blasts in the cerebrospinal fluid (CSF). Combined relapse (cCNSr) was defined as CNS disease with ≥ 5% blasts in the bone marrow. Time to relapse was classified as, Very Early: within 18 months of first diagnosis; Early: after 18 months of first diagnosis but within 6 months of stopping therapy and Late: more than 6 months after stopping therapy. All patients received 3 blocks of chemotherapy. Subsequently, allogenic stem cell transplant (allo-SCT) was offered to all very early relapses (iCNSr & cCNSr), early iCNSr (irrespective of immunophenotype), all T-cell cCNSr (irrespective of time to relapse) and early or late pre-B cCNSr that had a minimal residual disease level of ≥ 104 at the end of induction. All other patients were offered chemotherapy and cranial radiotherapy. Results: Of a total of 330 relapsed patients, 102 (31%) had CNS involvement. Of these 63 (62%) had iCNSr and 39 (38%) had cCNSr. The incidence of CNS disease was higher in males (M:F, CNS relapses 2.5:1 vs all relapses 1.5:1). CNS relapses had a higher proportion of T-cell disease (pre B:T CNS relapses 3.6:1 vs all relapses 7.8:1]. The number of patients presenting in very early, early and late phases were 19 (19%), 55 (54%) and 28 (27%) respectively. All late iCNSr patients were males. Almost all late relapses (iCNSr and cCNSr) (27/28) were of a pre B phenotype. At the end of induction phase, 91/102 (89%) achieved complete remission (CR) and 82/102 (80%) remained in CR after 3 blocks of chemotherapy. The estimated 3-year overall survival (OS) and progression free survival (PFS) for all patients with CNS disease was 45.5% (95%CI 32.9, 58.0) & 43.4% (95%CI 32.0, 54.7) respectively. There were no significant differences in survival with respect to site of the disease (combined vs isolated), gender or immunophenotype (pre B vs T). As shown in Table 1, CNS relapse patients who received Mitoxantrone had a significantly improved outcome when compared to those who received Idarubicin. This was most evident in those who had i) iCNSr, ii) pre-B phenotype and iii) allo-SCT, when analyzed on an intention to treat basis. This represents a considerable improvement in outcome compared to the results obtained in these sub-groups of patients in the previous UK ALLR2 study (Roy A et.al. Br. J. Haem. 2005;130:67-75). Conclusion: Mitoxantrone is highly effective in children with relapsed pre B ALL who have CNS involvement. As there were no other differences between patients treated on Mitoxantrone or Idarubicin, effective systemic therapy is as important as CNS directed therapy, if not more, in treating patients with CNS relapse. Disclosures: Off Label Use: Most drugs used in this protocol are off label as the majority of drugs used in childhood ALL are not liscensed for use in children.

Abstract 3390 Poster Board III-278 Introduction: While the outcome of children with acute lymphoblastic leukemia (ALL) has steadily improved, the prognosis for those who relapse (rALL) remains poor. Partly this has been due to the lack of a standardised approach to children with rALL. The international collaborative trial ALLR3 stratified patients into standard (SR), intermediate (IR) and high risk (HR) groups based on the time from first diagnosis to relapse, site of relapse and immunophenotype. All patients received 3 blocks of therapy, with a minimal residual disease (MRD) assessment at the end of blocks 1 and 3. Allogeneic stem cell transplantation (allo-SCT) was offered to all HR and those IR who had a MRD of ≥10-4 at week 5. In children in whom MRD was unavailable, allo-SCT was offered to those who relapsed within 24 months of stopping therapy. All other patients continued on chemotherapy for 24 months. A day 1 and 2 randomisation was performed between mitoxantrone and idarubicin. Results: 239 patients were enlisted, of whom 216 were randomised and 109 idarubicin and 103 mitoxantrone patients are analysable. There were non-significant differences between the groups with regards to time to relapse, site of relapse and cytogenetic subtypes.108 (51%) were in CR2 at a median follow up of 36 months (range 1-70) and Progression Free Survival (PFS) at 3 years was 50.3% (95% CI 42.9%, 57.3%). Forty four percent of idarubicin and 40% of mitoxantrone patients had disease levels of ≥10-4 at week 5 (p=0.90) at the end of block 1. The PFS at 3 years for those who received idarubicin was 35.9% (25.9%, 45.9%) and mitoxantrone 64.6% (54.2%, 73.2%) (p=0.0004). Adjusted for differences in risk group, country, age, gender and cytogenetic subtype, this difference continues to be significant (p=0.003). Overall mitoxantrone was better tolerated and less toxic than idarubicin. A competing risks model showed that the difference between the two drugs is primarily related to disease control (p=0.02), rather than toxicity of treatment (p=0.09). In the patients who were transplanted, 35% and 5% of patients who received idarubicin (n = 48) or mitoxantrone (n = 44) respectively have relapsed. Based on the intention to treat analyses, there were non significant differences in patients treated without an allo-SCT in both arms. IR patients who were transplanted did worse (p = 0.01) in the idarubicin group but had comparable results in the mitoxantrone group to those who received chemotherapy. As a result the randomisation in ALLR3 has now been closed, though the study continues to accrue to better answer the MRD stratification question. Conclusions: Mitoxantrone is a well tolerated and highly effective drug for the treatment of children with rALL. While MRD at week 5 failed to predict the differences in outcome for the two randomised groups, it identified that children in the IR group with Disclosures: Off Label Use: Although Idarubicin and Mitoxantrone have been shown to be effective in ALL, they are not licensed for use in children in the UK. .

Summary Background Although survival of children with acute lymphoblastic leukaemia has improved greatly in the past two decades, the outcome of those who relapse has remained static. We investigated the outcome of children with acute lymphoblastic leukaemia who relapsed on present therapeutic regimens. Methods This open-label randomised trial was undertaken in 22 centres in the UK and Ireland and nine in Australia and New Zealand. Patients aged 1–18 years with first relapse of acute lymphoblastic leukaemia were stratified into high-risk, intermediate-risk, and standard-risk groups on the basis of duration of first complete remission, site of relapse, and immunophenotype. All patients were allocated to receive either idarubicin or mitoxantrone in induction by stratified concealed randomisation. Neither patients nor those giving interventions were masked. After three blocks of therapy, all high-risk group patients and those from the intermediate group with postinduction high minimal residual disease (≥10 −4 cells) received an allogenic stem-cell transplant. Standard-risk and intermediate-risk patients with postinduction low minimal residual disease ( −4 cells) continued chemotherapy. The primary outcome was progression-free survival and the method of analysis was intention-to-treat. Randomisation was stopped in December, 2007 because of differences in progression-free and overall survival between the two groups. This trial is registered, reference number ISCRTN45724312. Findings Of 239 registered patients, 216 were randomly assigned to either idarubicin (109 analysed) or mitoxantrone (103 analysed). Estimated 3-year progression-free survival was 35·9% (95% CI 25·9–45·9) in the idarubicin group versus 64·6% (54·2–73·2) in the mitoxantrone group (p=0·0004), and 3-year overall survival was 45·2% (34·5–55·3) versus 69·0% (58·5–77·3; p=0·004). Differences in progression-free survival between groups were mainly related to a decrease in disease events (progression, second relapse, disease-related deaths; HR 0·56, 0·34–0·92, p=0·007) rather than an increase in adverse treatment effects (treatment death, second malignancy; HR 0·52, 0·24–1·11, p=0·11). Interpretation As compared with idarubicin, mitoxantrone conferred a significant benefit in progression-free and overall survival in children with relapsed acute lymphobastic leukaemia, a potentially useful clinical finding that warrants further investigation. Funding Cancer Research UK, Leukaemia and Lymphoma Research, Cancer Council NSW, and Sporting Chance Cancer Foundation.