Alex's Lemonade Stand Foundation Research Articles

Multi-Cohort Meta-Analysis of Rnaseq Data to Determine Sex Bias in Pediatric Acute Myeloid Leukemia

Leukemia is the most common childhood cancer, and remains a leading cause of childhood cancer mortality. The most common leukemia in children, B-cell acute lymphoblastic leukemia, has a 92% cure rate (5-year overall survival). In contrast, pediatric acute myeloid leukemia (pAML) has a 68% cure rate, which has not changed significantly despite 40 years of trials. AML exhibits some of the most striking sex-specific disparities in childhood cancer, affecting disease risk, therapy response, and transplant outcomes. pAML cohorts are consistently about 45% female and 55% male, yielding a male excess risk of 22%. Some aggressive subtypes, such as NUP98::NSD1 fusions seen in many treatment failures, the male:female ratio rises to 2:1, and to 3:1 near puberty. Up to 50% of pAML patients show loss of X or Y, with loss of Y (LOY) prognostic for lifetime all-cause mortality in males. Even mutation-targeted therapies such as midostaurin show significant differences in efficacy by sex in Phase III trials. We sought to illuminate these differences via meta-analysis of bulk RNA sequencing (RNAseq) data from 2,000 TARGET (Therapeutically Applicable Research to Generate Effective Treatments) initiative patients and matched controls to identify sex-specific transcriptional differences. Subsequently, we interrogated these findings by performing a meta-analysis and cross-projection of publicly available single-cell RNA sequencing (scRNAseq) data from a variety of pediatric hematopoietic malignancies from eight published GEO datasets and two Alex's Lemonade Stand Foundation (ALSF) datasets, comprising 225 myeloid or myeloid-like pediatric leukemia patients and 23 non-leukemic specimens. Using Non-negative Matrix Factorization (NMF), we projected all scRNAseq data into the same latent space as the Chan Zuckerberg Initiative (CZI) human, matched-tissue scRNAseq. These NMF projections were then utilized to inform a comprehensive meta-analysis of both bulk and scRNAseq data from myeloid-like diseases. By leveraging both bulk and single-cell transcriptomic data, we aimed to elucidate the role of sex in the molecular underpinnings of pediatric hematopoietic malignancies, potentially unveiling novel therapeutic targets and contributing to personalized medicine approaches in these diseases. Combined with the development of new models for predisposition conditions such as RUNX1-FPDMM, our work begins to reveal the molecular underpinnings of sex bias in treatment response and immunogenicity for pAML patients, and creates a foundation to better understand how genetic lesions interact with age and sex to shape risk in this lethal malignancy.

Financial hardships and psychosocial outcomes among parents of children who die of cancer.

Caregivers experience financial hardship during a child's cancer treatment and after their child's death. These bereaved caregivers also experience negative psychosocial outcomes following the death of a child, but the relationship between financial hardship and negative psychosocial outcomes is poorly understood in this population. We surveyed self-selected bereaved caregivers as part of a publicly posted survey through Alex's Lemonade Stand Foundation in order to explore family experiences after losing a child to cancer. The survey contained questions regarding parent psychosocial and financial outcomes following their child's death. One-hundred seventy-six caregivers completed the survey a median of 7years after their child's death. The majority were female (91%), non-Hispanic White (97%), and married or living with a domestic partner (76%). Overall, 31% of caregivers reported that their child's death significantly impacted the financial well-being of their family, 23% experienced a decrease in income following their child's death, and 14% were still paying medical expenses. Financial hardship that the caregiver attributed to the child's death was associated with feeling lonely and isolated (adjusted relative risk [ARR]=1.7, 95% CI: 1.1-2.7) and living day to day (ARR = 1.8, 95% CI: 1.3-2.5), even after adjustment for household income and time since child's death. Caregivers experience multiple financial hardships following the death of a child to cancer, which endure for years after the child's death. These hardships are associated with negative psychosocial outcomes, demonstrating the need for both financial and psychosocial interventions for caregivers following the death of a child to cancer.

Rates of laboratory adverse events by course in paediatric leukaemia ascertained with automated electronic health record extraction: a retrospective cohort study from the Children's Oncology Group

Adverse events are often misreported in clinical trials, leading to an incomplete understanding of toxicities. We aimed to test automated laboratory adverse event ascertainment and grading (via the ExtractEHR automated package) to assess its scalability and define adverse event rates for children with acute myeloid leukaemia and acute lymphoblastic leukaemia. For this retrospective cohort study from the Children's Oncology Group (COG), we included patients aged 0-22 years treated for acute myeloid leukaemia or acute lymphoblastic leukaemia at Children's Healthcare of Atlanta (Atlanta, GA, USA) from Jan 1, 2010, to Nov 1, 2018, at the Children's Hospital of Philadelphia (Philadelphia, PA, USA) from Jan 1, 2011, to Dec 31, 2014, and at the Texas Children's Hospital (Houston, TX, USA) from Jan 1, 2011, to Dec 31, 2014. The ExtractEHR automated package acquired, cleaned, and graded laboratory data as per Common Terminology Criteria for Adverse Events (CTCAE) version 5 for 22 commonly evaluated grade 3-4 adverse events (fatal events were not evaluated) with numerically based CTCAE definitions. Descriptive statistics tabulated adverse event frequencies. Adverse events ascertained by ExtractEHR were compared to manually reported adverse events for patients enrolled in two COG trials (AAML1031, NCT01371981; AALL0932, NCT02883049). Analyses were restricted to protocol-defined chemotherapy courses (induction I, induction II, intensification I, intensification II, and intensification III for acute myeloid leukaemia; induction, consolidation, interim maintenance, delayed intensification, and maintenance for acute lymphoblastic leukaemia). Laboratory adverse event data from 1077 patients (583 from Children's Healthcare of Atlanta, 200 from the Children's Hospital of Philadelphia, and 294 from the Texas Children's Hospital) who underwent 4611 courses (549 for acute myeloid leukaemia and 4062 for acute lymphoblastic leukaemia) were extracted, processed, and graded. Of the 166 patients with acute myeloid leukaemia, 86 (52%) were female, 80 (48%) were male, 96 (58%) were White, and 132 (80%) were non-Hispanic. Of the 911 patients with acute lymphoblastic leukaemia, 406 (45%) were female, 505 (55%) were male, 596 (65%) were White, and 641 (70%) were non-Hispanic. Patients with acute myeloid leukaemia had the most adverse events during induction I and intensification II. Hypokalaemia (one [17%] of six to 75 [48%] of 156 courses) and alanine aminotransferase (ALT) increased (13 [10%] of 134 to 27 [17%] of 156 courses) were the most prevalent non-haematological adverse events in patients with acute myeloid leukaemia, as identified by ExtractEHR. Patients with acute lymphoblastic leukaemia had the greatest number of adverse events during induction and maintenance (eight adverse events with prevalence ≥10%; induction and maintenance: anaemia, platelet count decreased, white blood cell count decreased, neutrophil count decreased, lymphocyte count decreased, ALT increased, and hypocalcaemia; induction: hypokalaemia; maintenance: aspartate aminotransferase [AST] increased and blood bilirubin increased), as identified by ExtractEHR. 187 (85%) of 220 total comparisons in 22 adverse events in four AAML1031 and six AALL0923 courses were substantially higher with ExtractEHR than COG-reported adverse event rates for adverse events with a prevalence of at least 2%. ExtractEHR is scalable and accurately defines laboratory adverse event rates for paediatric acute leukaemia; moreover, ExtractEHR seems to detect higher rates of laboratory adverse events than those reported in COG trials. These rates can be used for comparisons between therapies and to counsel patients treated on or off trials about the risks of chemotherapy. ExtractEHR-based adverse event ascertainment can improve reporting of laboratory adverse events in clinical trials. US National Institutes of Health, St Baldrick's Foundation, and Alex's Lemonade Stand Foundation.

The role of complement-mediated signaling during antigen presentation

Abstract Local production of complement C3a and C5a at the immune synapse by activated dendritic cells (DCs) enhances T cell responses in a variety of experimental systems. We have shown that complement C3 plays a mechanistic role in driving synergistic anti-tumor effects of blocking indoleamine 2,3-dioxygenase (IDO) during radiation and chemotherapy in a murine brain tumor model. We hypothesize that, when anti-tumor responses are allowed to happen, locally produced complement C3 acts as a costimulation factor for activation of T cells. To study the role of complement C3 in promoting T cell responses against a nominal tumor antigen (gp10025–33 peptide expressed by B16F10 melanoma), we used T cells from syngeneic pmel-1 mice (TCR transgenic with CD8 T cells that recognize the cross-presented gp10025–33 peptide). We found florid deposition of complement C3 in B16F10 tumors when IDO-blockade was added to a pmel-1/gp10025–33 based vaccine. To model homeostatic conditions of the tumor microenvironment, we used an in vitro co-culture system with gp10025–33 peptide in stringent limited supply. Under these conditions, pmel-1 CD8 T cells stimulated by DCs from complement C3 knock-out (C3-KO) mice had decreased activation marker expression, proliferation, and cytokine effector function, relative to pmel-1 CD8 T cells stimulated by DCs from wild-type (WT) mice. These findings suggest that suppression of local complement production by DCs in the tumor microenvironment may inhibit anti-tumor immune responses. Supported by grants from Alex's Lemonade Stand Foundation, Press On Foundation, and Hyundai Hope On Wheels.

Results of the NLG2105 Phase I Trial Using the IDO Pathway Inhibitor Indoximod, in Combination with Radiation and Chemotherapy, for Children with Newly Diagnosed DIPG

Abstract Background We conducted a phase Ib dose-confirmation study of indoximod with radiation, followed by indoximod with cyclic temozolomide therapy, to evaluate safety and overall survival (OS) in children with newly diagnosed DIPG (diffuse intrinsic pontine glioma). Indoximod is a small-molecule inhibitor of the IDO pathway that reverses immune suppression imposed by tumor microenvironments. DIPG is a uniformly fatal orphan disease with no curative treatment options. Methods Children age 3 to 21 years, with newly diagnosed DIPG were eligible for treatment with oral indoximod (38.4 mg/kg/day divided BID, throughout) combined with fractionated conformal radiation therapy (54 Gy in 30 fractions), followed by cyclic oral chemo-immunotherapy using indoximod combined with temozolomide (200 mg/m2/day, days 1-5 of each 28-day cycle). The indoximod dose was the previously determined recommended phase-II dose (RP2D), and the study design called for phase I monitoring to confirm safety of this dose in DIPG patients. Results Thirteen children (median age 9 years, range 5 to 20 years) with newly diagnosed DIPG were treated using this indoximod-based radio-chemo-immunotherapy regimen. The 12-month OS was 62% (8/13) and estimated median OS was 14.5 months (follow-up range 4.8 to 22 months) with 4 patients remaining in follow-up. This compares favorably to the expected 12-month OS of approximately 45% and median OS of approximately 10.8 months from published historical controls (Kilburn, et al; 2017). Two patients experienced near-complete responses until showing relapse at 7.6 months and 13.3 months of study therapy. Patients were followed with quantitative volumetric analyses of MRIs and serial measurements of peripheral blood inflammatory monocytes, T cell activation, and pro-inflammatory cytokines. The most common adverse events attributed to indoximod were thrombocytopenia, diarrhea, nausea, vomiting, and fatigue. Conclusion Adding indoximod-based immunotherapy to conventional radiation and chemotherapy for up-front therapy of children with DIPG appears to be well tolerated with improved outcomes. Clinical trial identification NLG2105, NCT02502708. Legal entity responsible for the study NewLink Genetics Corporation. Funding NewLink Genetics Corporation, Alex's Lemonade Stand Foundation, Cannonball Kids cancer Foundation, Beloco Foundation, Eli’s Block Party Foundation, Hyundai Hope on Wheels Foundation, Northern Nevada Children’s Cancer Research Foundation, CAM Fund, Press On Foundation. Disclosure T.S. Johnson: Research grant / Funding (institution): NewLink Genetics Corporation. E.P. Kennedy: Shareholder / Stockholder / Stock options, Full / Part-time employment, Officer / Board of Directors: NewLink Genetics Corporation. N. Vahanian: Shareholder / Stockholder / Stock options, Full / Part-time employment, Officer / Board of Directors: NewLink Genetics Corporation. D.H. Munn: Advisory / Consultancy, Shareholder / Stockholder / Stock options, Licensing / Royalties: NewLink Genetics Corporation. All other authors have declared no conflicts of interest.

B7-H3-redirected chimeric antigen receptor T cells target glioblastoma and neurospheres

BackgroundThe dismal survival of glioblastoma (GBM) patients urgently calls for the development of new treatments. Chimeric antigen receptor T (CAR-T) cells are an attractive strategy, but preclinical and clinical studies in GBM have shown that heterogeneous expression of the antigens targeted so far causes tumor escape, highlighting the need for the identification of new targets. We explored if B7-H3 is a valuable target for CAR-T cells in GBM.MethodsWe compared mRNA expression of antigens in GBM using TCGA data, and validated B7-H3 expression by immunohistochemistry. We then tested the antitumor activity of B7-H3-redirected CAR-T cells against GBM cell lines and patient-derived GBM neurospheres in vitro and in xenograft murine models.FindingsB7-H3 mRNA and protein are overexpressed in GBM relative to normal brain in all GBM subtypes. Of the 46 specimens analyzed by immunohistochemistry, 76% showed high B7-H3 expression, 22% had detectable, but low B7-H3 expression and 2% were negative, as was normal brain. All 20 patient-derived neurospheres showed ubiquitous B7-H3 expression. B7-H3-redirected CAR-T cells effectively targeted GBM cell lines and neurospheres in vitro and in vivo. No significant differences were found between CD28 and 4-1BB co-stimulation, although CD28-co-stimulated CAR-T cells released more inflammatory cytokines.InterpretationWe demonstrated that B7-H3 is highly expressed in GBM specimens and neurospheres that contain putative cancer stem cells, and that B7-H3-redirected CAR-T cells can effectively control tumor growth. Therefore, B7-H3 represents a promising target in GBM.FundAlex's Lemonade Stand Foundation; Il Fondo di Gio Onlus; National Cancer Institute; Burroughs Wellcome Fund.

The fusion oncogene ASPSCR1‐TFE3 directs epigenetic‐induced autophagy in alveolar soft part sarcoma

Altered metabolism is considered to be one of the new hallmarks of cancer. Autophagy is one major avenue of altered cancer metabolism, enabling cell survival under metabolic stress and promoting chemoresistance. The nuclear localization of MiTF/TFE3 family transcription factors has associated with upregulated transcription of autophagy genes in pancreatic cancer. Alveolar soft part sarcoma (ASPS) is a rare but deadly soft-tissue sarcoma, with a predilection for adolescent and young adult victims. ASPS is noteworthy for its resistance to traditional cytotoxic chemotherapies. It consistently associates with a t(X;17) chromosomal translocation that produces the ASPSCR1-TFE3 target gene, bearing the DNA-binding domain from TFE3 and protein interaction domains from ASPSCR1. We have demonstrated that conditional expression of ASPSCR1-TFE3 is sufficient to drive alveolar soft part sarcomagenesis in the mouse with complete penetrance. Mouse tumors recapitulate human ASPS histology and transcriptomes. Our objective was to identify the direct targets of ASPSCR1-TFE3 and how these targets confer resistance to doxorubicin. The human cell lines ASPS-1 and FUUR-1, as well as mouse tumors driven by expression of ASPSCR1-TFE3 were subjected to nuclear fractionation and chromatin immunoprecipitation using antibodies against ASPSCR1 and RNAPol2. Cells and tumors were further characterized for their presence of auotphagic flux by detection of LC3-II and abundance of lysosomal proteins LAMP1 and CTSD. Furthermore, cells treated with doxorubicin were analyzed by gas-chromatography mass spectrometry (GC-MS) for metabolites involved in cellular respiration and glycolysis. Lastly, mice were treated with either control, monotherapy of chloroquine (15 mg/kg) or doxorubicin (10 mg/kg), or combination therapy for up to 5 months. Mice on combination therapy showed a stastical improvement in survival of 3 months over control and doxorubicin treatments. We report not only the first genome-wide localization of the ASPSCR1-TFE3 oncoprotein on chromatin from ASPS cell lines and mouse tumors, but also its association with actively transcribed genes. Among these are found many genes related to autophagy. We demonstrate high expression of autophagy-related proteins at baseline conditions in human tumors, cell lines and mouse tumors. We also demonstrate active autophagic flux even in the absence of stress conditions. Inhibition of autophagy strongly synergizes with chemotherapy to kill ASPS cells, suggesting a mechanism for resistance. Furthermore, mice treated with combination therapy of chloroquine and chemotherapy significantly extends life 3 months beyond control mice. We have therefore demonstrated the direct targets of ASPSCR1-TFE3 in ASPS, including a number of autophagy genes that are expressed in these tumors. Inhibition of autophagy in ASPS causes the tumor cells to be more susceptible to chemotherapeutic stress. Support or Funding Information Alex's Lemonade Stand Foundation Sarcoma Foundation of America Huntsman Cancer Foundation Chromatin immunoprecipitation of ASPSCR1-TFE3 peaks from the entire mouse genome. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Re-Induction Outcome for Pediatric Patients with Relapsed or Refractory B-Cell Precursor Acute Lymphoblastic Leukemia: A Retrospective Cohort Study of the Therapeutic Advances in Childhood Leukemia Consortium

IntroductionRemission induction rates after a second or greater relapse is a critical endpoint in phase II trials of childhood acute lymphoblastic leukemia (ALL). A robust benchmark is crucial for identification of novel multi-agent regimens worthy of further study. The Therapeutic Advances in Childhood Leukemia and Lymphoma (TACL) consortium previously reported the response rates of children with multiply relapsed and refractory (R/R) ALL treated between 1995 and 2004, which provided a benchmark for clinical trials. To define more recent treatment patterns and test the robustness of this benchmark, we performed a retrospective cohort review of children with R/R ALL who experienced second or greater treatment failure at TACL consortium sites between 2005 and 2013.Patients and MethodsEligible patients were identified at participating TACL institutions. This cohort was comprised of patients with medullary R/R B-cell precursor ALL who experienced at least 2 treatment failures or relapsed after hematopoietic stem cell transplant. Patient demographic data and details of the initial and R/R disease characteristics were abstracted from medical records and entered into a central database. This study was approved by the IRB of each participating institution.Treatment failure was defined by the presence or re-emergence of circulating blasts, M2/M3 BM, or extramedullary (EM) disease despite therapy. Complete remission (CR) was defined as M1 marrow, no EM disease and evidence of peripheral count recovery. For the purpose of statistical analysis, patients who met these criteria without platelet recovery (CRp) or normal blood count recovery (CRi) were included as CR. Univariate and multivariate logistic regression were utilized to evaluate the risk of re-induction failure. Predictors included in this preliminary analysis were NCI risk criteria at diagnosis, duration of the prior remission, the treatment attempt number, and the EM and BM status at the start of each therapy attempt.ResultsThis report includes 214 patients. Fifty-six percent were male. At initial diagnosis, 32% were at least 10 years old, 26% had initial white blood cell (WBC) counts over 50,000/µL, and 39% were classified as high risk by the NCI risk criteria (Table 1). Therapy involved various combinations of agents and ranged between 2 and 10 attempts. The CR rate was 42% for third treatment attempt and 24% for fourth and subsequent treatment attempts (Table 2). Treatment failures were significantly associated with increased number of treatment attempts (p < 0.001), shorter duration of previous CR (p < 0.001) and NCI risk category at diagnosis (p = 0.018).ConclusionThis preliminary analysis found similar CR rates in patients with third treatment failure compared to the 1st TACL retrospective study of the prior decade (42% vs. 44%, Ko et al, 2010) and an Austrian report with a small cohort of patients (Reismüller et al, 2013). Further analysis will be performed in comparison to the initial TACL retrospective study cohort once enrollment to this study has been completed (approximately 400 patients). A robust, contemporary historical control may serve as an alternative to a randomized control when outcome with past therapies in unacceptably poor.Table 1Patient Characteristics at Initial Diagnosis of Patients with ALL who received at least two treatment attempt (n = 214 patients)CharacteristicNo of patients%Age, years< 1 (infants)1881-91265910 and over7033WBC count/uL< 50K1286050K and over5626Unknown3014NCI risk criteria at diagnosisNon-infants, standard risk8238Non-infants, high risk8439Non-infants, unknown3014Infants188SexFemale9444Male12056CNS diseaseYes4220No14869Unknown2411Karyotype1Normal683011q23 (MLL gene) rearranged198Hypodiploidy73Hyperdiploidy2612iAMP2121t(12;21)63t(1;19)73t(9;22)157Other4621Unknown28121 Karyotype is available for 214 unique patients; 2 entries were reported for 7 patients, and 4 entries were reported for 1 patient.Table 2Achievement of CR/CRp/CRi After Treatment of R/R ALL by Preceding Remission Duration and Treatment AttemptThird treatment attemptFourth through tenth treatment attemptDuration of preceding CRResponseTotal%ResponseTotal%Not achieved (refractory)246338208623< 18 months duration28783611492218 to 36 months duration915603560≥ 36 months duration88100010All patients combined69164423414124 DisclosuresSun:Gateway for Cancer Researchy: Research Funding; Amgen: Research Funding. Wilkes:Healthcare Research and Quality: Research Funding; Alex's Lemonade Stand Foundation: Research Funding. Gaynon:Bristol Meyers Squibb: Membership on an entity's Board of Directors or advisory committees; Sigma Tau: Speakers Bureau; JAZZ: Speakers Bureau. Wayne:Medimmune: Honoraria, Other: travel support, Research Funding; NIH: Patents & Royalties; Kite Pharma: Honoraria, Other: travel support; Pfizer: Honoraria; Spectrum Pharmaceuticals: Honoraria, Other: travel support, Research Funding. Whitlock:Amgen: Honoraria.

Abstract 1613: β-lapachone: a novel targeted therapy for atypical teratoid rhabdoid tumors (ATRTs)

Abstract Atypical teratoid rhabdoid tumors (ATRTs) are aggressive malignant neoplasms that occur in the CNS, most commonly in children &amp;lt;3 years of age. Despite intensive chemo-radiotherapy and surgery, the outcomes for patients with this disease remain dismal, with ∼75% of patients dying from their disease at a median of 10 months after diagnosis. Thus, it is critical to identify and validate novel ATRT targets for new therapies. We found that the pathognomonic genetic lesion of ATRTs, loss of the SWI/SNF chromatin remodeling complex member INI1/hSNF5/SMARCB1, leads to high expression of NAD(P)H:quinone oxidoreductase 1 (NQO1), a two-electron oxidoreductase. Thus, we hypothesized that ATRTs will be vulnerable to drugs, such as ß-lapachone (ß-lap, ARQ761 in clinical form), that selectively kill NQO1-expressing cells. A panel of ATRT cell lines exhibited overall high NQO1 expression and low micromolar sensitivity to ß-lap (IC50 1.6-4 uM), with the exception of cell lines that were homozygous for the C609T NQO1 (*2) polymorphism, which exhibited no NQO1 activity and were resistant to ß-lap treatment (IC50 &amp;gt;15 uM). A two-hour pulse with ß-lap caused a burst of ROS-mediated DNA damage and PARP1 hyperactivation, resulting in NAD+ and ATP depletion and caspase-independent, mu-calpain-mediated apoptosis. Co-treatment with dicoumarol, a specific inhibitor of NQO1, prevented these effects by blocking the NQO1-dependent bioactivation of ß-lap. Low expression of NQO1 in normal brain and other pediatric tissues suggests a wide therapeutic window for ß-lap. These findings encourage the continued study of ß-lap and other NQO1 bioactivated therapeutics for the treatment of this recalcitrant disease. This work was supported by NIH/NCI R01 CA102972 to DAB and a REACH award from the Alex's Lemonade Stand Foundation to JFA and TWL. Citation Format: Zachary R. Moore, Sarai Stuart, Agnieszka Cholka, Ashwin Venkataraman, Jingying Xu, Anat Erdreich-Epstein, Dinesh Rakheja, David A. Boothman, James F. Amatruda, Theodore W. Laetsch. β-lapachone: a novel targeted therapy for atypical teratoid rhabdoid tumors (ATRTs). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1613. doi:10.1158/1538-7445.AM2015-1613

Alex's Lemonade Stand Foundation Infant and Childhood Primary Brain and Central Nervous System Tumors Diagnosed in the United States in 2007-2011.

The CBTRUS Statistical Report: Alex's Lemonade Stand Foundation Infant and Childhood Primary Brain and Central Nervous System Tumors Diagnosed in the United States in 2007–2011 comprehensively describes the current population-based incidence of primary malignant and non-malignant brain and CNS tumors in children ages 0–14 years, collected and reported by central cancer registries covering approximately 99.8% of the United States population (for 2011 only, data were available for 50 out of 51 registries). Overall, brain and CNS tumors are the most common solid tumor, the most common cancer, and the most common cause of cancer death in infants and children 0–14 years. This report aims to serve as a useful resource for researchers, clinicians, patients, and families.

Abstract 1295: The association between asthma and pediatric brain tumors in neurofibromatosis type 1

Abstract Background. Neurofibromatosis type 1 (NF1) is a common hereditary cancer syndrome, affecting an estimated 1/2,500 people worldwide. NF1 increases the risk of several tumor types, including pediatric brain tumors that affect an estimated 10-20% of individuals. While established risk factors for pediatric brain tumor development in this population have not been elucidated, consistent evidence from non-syndromic populations suggests a strong inverse association between asthma and brain tumors. As such, the objective of this study was to test the hypothesis that asthma is inversely associated with pediatric brain tumors in the NF1 population. Methods. In this analysis, we employed two data sources. The first dataset was assembled from the international NF1 Patient Registry Initiative (NPRI) (https://nf1registry.wustl.edu/) that enrolled individuals with NF1 over ∼2.5 years. Medical history data were collected through the online registry questionnaire. The second study used private health insurance claims data from 2006-2010 from Thompson Reuters MarketScan to assemble an NF1 group and their claims data on asthma- and brain tumor-related healthcare visits. Diagnoses were determined from ICD-9 codes. The NF1 group was defined by the presence of &amp;gt;2 NF1-related outpatient claims &amp;gt;30 days apart or one NF1-related inpatient claim. Within the NF1 group, individuals were classified as having asthma and/or brain tumors if they had &amp;gt;2 claims for these conditions &amp;gt;30 days apart. The study population for both data sources was limited to subjects who were &amp;lt;18 years old at the time of NPRI or insurance enrollment. Unconditional logistic regression was employed to calculate odds ratios (ORs) and 95% confidence intervals (CIs) for the association between pediatric brain tumors and asthma for both datasets. Results. A total of 409 individuals with NF1 were included in the analysis using NPRI data, resulting in 119 pediatric brain tumors. After adjusting for birth year, we observed an inverse association between pediatric brain tumors and asthma (OR=0.6; 95% CI 0.4-1.1). In the analysis using MarketScan data, the NF1 group included a total of 3,871 individuals &amp;lt;18 years at the time of insurance enrollment, of which 397 had &amp;gt;2 pediatric brain tumor-related claims. After controlling for number of months enrolled, birth year, and number of healthcare visits, subjects with brain tumor claims were significantly less likely to have asthma claims than those without brain tumor claims (OR=0.5; 95% CI 0.3-0.8). Conclusions. Similar to findings from non-syndromic populations, the results from this study provide strong epidemiologic evidence for an inverse association between asthma and pediatric brain tumors in the NF1 population. Acknowledgements. This work was supported by Alex's Lemonade Stand Foundation and NIH CTSA UL1 TR000448. Citation Format: Kimberly J. Johnson, Nancy Mueller, Evelyn Sharkey, Qian Liu, David H. Gutmann. The association between asthma and pediatric brain tumors in neurofibromatosis type 1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1295. doi:10.1158/1538-7445.AM2014-1295

Purged versus non-purged peripheral blood stem-cell transplantation for high-risk neuroblastoma (COG A3973): a randomised phase 3 trial

Myeloablative chemoradiotherapy and immunomagnetically purged autologous bone marrow transplantation has been shown to improve outcome for patients with high-risk neuroblastoma. Currently, peripheral blood stem cells (PBSC) are infused after myeloablative therapy, but the effect of purging is unknown. We did a randomised study of tumour-selective PBSC purging in stem-cell transplantation for patients with high-risk neuroblastoma. Between March 16, 2001, and Feb 24, 2006, children and young adults (<30 years) with high-risk neuroblastoma were randomly assigned at diagnosis by a web-based system (in a 1:1 ratio) to receive either non-purged or immunomagnetically purged PBSC. Randomisation was done in blocks stratified by International Neuroblastoma Staging System stage, age, MYCN status, and International Neuroblastoma Pathology classification. Patients and treating physicians were not masked to treatment assignment. All patients were treated with six cycles of induction chemotherapy, myeloablative consolidation, and radiation therapy to the primary tumour site plus meta-iodobenzylguanidine avid metastases present before myeloablative therapy, followed by oral isotretinoin. PBSC collection was done after two induction cycles. For purging, PBSC were mixed with carbonyl iron and phagocytic cells removed with samarium cobalt magnets. Remaining cells were mixed with immunomagnetic beads prepared with five monoclonal antibodies targeting neuroblastoma cell surface antigens and attached cells were removed using samarium cobalt magnets. Patients underwent autologous stem-cell transplantation with PBSC as randomly assigned after six cycles of induction therapy. The primary endpoint was event-free survival and was analysed by intention-to-treat. The trial is registered with ClinicalTrials.gov, number NCT00004188. 495 patients were enrolled, of whom 486 were randomly assigned to treatment: 243 patients to receive non-purged PBSC and 243 to received purged PBSC. PBSC were collected from 229 patients from the purged group and 236 patients from the non-purged group, and 180 patients from the purged group and 192 from the non-purged group received transplant. 5-year event-free survival was 40% (95% CI 33-46) in the purged group versus 36% (30-42) in the non-purged group (p=0·77); 5-year overall survival was 50% (95% CI 43-56) in the purged group compared with 51% (44-57) in the non-purged group (p=0·81). Toxic deaths occurred in 15 patients during induction (eight in the purged group and seven in the non-purged group) and 12 during consolidation (eight in the purged group and four in the non-purged group). The most common adverse event reported was grade 3 or worse stomatitis during both induction (87 of 242 patients in the purged group and 93 of 243 patients in the non-purged group) and consolidation (131 of 177 in the purged group vs 145 of 191 in the non-purged group). Serious adverse events during induction were grade 3 or higher decreased cardiac function (four of 242 in the purged group and five of 243 in the non-purged group) and elevated creatinine (five of 242 in the purged group and six of 243 non-purged group) and during consolidation were sinusoidal obstructive syndrome (12 of 177 in the purged group and 17 of 191 in the non-purged group), acute vascular leak (11 of 177 in the purged group and nine of 191 in the non-purged group), and decreased cardiac function (one of 177 in the purged group and four of 191 in the non-purged group). Immunomagnetic purging of PBSC for autologous stem-cell transplantation did not improve outcome, perhaps because of incomplete purging or residual tumour in patients. Non-purged PBSC are acceptable for support of myeloablative therapy of high-risk neuroblastoma.

Abstract 4847: Efficacy of different recruitment methods for a neurofibromatosis type 1 online registry.

Abstract Background. Neurofibromatosis Type 1 (NF1) is a hereditary cancer syndrome caused by a germline NF1 gene mutation. Affecting an estimated 70,000-100,000 Americans, cancer in individuals with NF1 is a leading cause of death. However, the factors that modify cancer risk in NF1 are largely unknown. To facilitate NF1 research aimed at defining epidemiologic and clinical cancer risk factors, we created an online patient registry (https://nf1registry.wustl.edu/). In the current study, we sought to evaluate different recruiting mechanisms and the characteristics of participants recruited by each mechanism. Methods. Potential participants were alerted to the registry through three main mechanisms: (1) paid online advertising (Facebook and Google ads), (2) posting on government, academic, and advocacy group websites, and (3) healthcare provider-based recruiting through established NF1 Clinical Programs. Referral patterns were tracked through the online questionnaire that included a question about where the participant first heard about the registry. We compared the total number and characteristics of participants recruited through each method for the period of 4/17/2011-10/22/2012. Statistical significance of differences in participant characteristics between recruiting mechanisms were evaluated using ANOVA and chi-square tests. Results. A total of 693 participants with self-reported NF1 participated in the registry during the study period. The most effective recruitment mechanism was paid online advertising that resulted in over half of the recruits (n=378), of which 75% responded to a Facebook ad. The second most effective method was postings on government, academic, and advocacy group websites (69 participants) followed by healthcare provider referrals (53 participants). The remainder of the participants reported hearing about the registry through a variety of other mechanisms. The mean age of participants was 30.7 years with the majority of participants reporting white race (72%) and female sex (62%). Age and sex were not significantly associated with the specific recruitment mechanism. However, there was a significant difference in referral patterns by race, with a higher percentage of Whites recruited by Facebook ads than other races and a higher percentage of other races reporting referral via a Google ad than Whites. Conclusions. We found that the internet, especially Facebook advertising, provides a highly effective means for rapidly assembling large numbers of patients with a rare disease to an online registry for clinical research studies. Although participants were similarly recruited by all three recruitment mechanisms with respect to age and sex, referral patterns appear to differ by race. Supported by: An American Cancer Society Institutional Research Grant, Alex's Lemonade Stand Foundation, and the St. Louis Children's Hospital Foundation Citation Format: Kimberly J. Johnson, Nancy L. Mueller, David H. Gutmann. Efficacy of different recruitment methods for a neurofibromatosis type 1 online registry. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4847. doi:10.1158/1538-7445.AM2013-4847

HbF-Associated Genetic Variation Marks an Erythroid Regulatory Element Essential for BCL11A Transcription and Subsequent Stage-Specific Globin Expression

Abstract 828Genome-wide association studies (GWAS) have identified common genetic variation within a 14-kb segment of intron-2 of the BCL11A gene that is associated with the level of fetal hemoglobin (HbF) and the clinical severity of the β-globin disorders sickle cell disease and β-thalassemia. Functional studies have validated BCL11A as a major repressor of HbF. Here, we explore the consequences of genetic variation on BCL11A expression. The HbF-associated variants overlap BCL11A sequences that possess an evolutionarily conserved enhancer chromatin signature, including the presence of H3K4me1 and H3K27ac and the absence of H3K4me3 histone modifications. Moreover, these sequences show a unique pattern of DNase I hypersensitivity in erythroid precursors compared to other BCL11A-expressing lineages including fetal brain and B-lymphocytes. Occupancy of the major erythroid transcription factors GATA1 and TAL1 is observed at these DNase I hypersensitive sites (DHS). Chromosome conformation capture studies demonstrate that these DHSs form a long-range interaction with the BCL11A promoter.To address the function of the putative regulatory element, we generated transgenic mice with the 12-kb intronic region attached to a minimal promoter and reporter gene. The element enhanced reporter gene expression in an erythroid-lineage restricted, definitive erythroid stage-specific manner. The minimal enhancer activity was mapped to the +58-kb DHS. Similar enhancer activity was observed in primary human erythroid precursors. To address the possible requirement of this element for BCL11A expression, we deleted the corresponding region in adult-stage murine erythroleukemia cells with TALEN-mediated gene targeting. Expression of BCL11A was dramatically reduced and embryonic globins Hbby and Hbb-bh1 were markedly derepressed with concomitant reduction of adult globin Hbb-b1. Analysis of heterozygous primary human erythroid precursors revealed that low-HbF haplotype alleles are preferentially occupied by GATA1 and TAL1 at sequences surrounding rs1427407. A composite half-E-box–GATA motif is disrupted at this SNP in the high-HbF allele. Furthermore, the low-HbF haplotype allele of BCL11A is expressed at a 1.7-fold higher level relative to the high-HbF allele. These data demonstrate that HbF-associated genetic variation in BCL11A modulates the function of a critical erythroid enhancer of BCL11A, and consequently expression of BCL11A itself. The quantitative level of BCL11A in turn regulates the relative expression of the stage-specific globin genes. The essential erythroid enhancer of the BCL11A gene might itself serve as a target for genetic manipulation as a novel approach to the human β-globin disorders. Disclosures:Porteus:Alex's Lemonade Stand Foundation: Research Funding.

Population Dynamics of Acute Myeloid Leukemia Clones by Barcode Tracking and High Throughput Sequencing

AbstractAbstract 1398 Introduction:Acute myeloid leukemia (AML) is a heterogeneous malignancy that continues to have a significant relapse rate in both adult and pediatric patients. This heterogeneity manifests itself between patients because different patients will have different gene mutations causing their AML (inter-patient heterogeneity). It also manifests as intra-patient heterogeneity as within a single patient there are different sub-clones that contribute differentially to primary disease and relapse. For example, relapse after chemotherapy can occur from a genetically distinct sub-clone that presumably was relatively resistant to the administered chemotherapy. The dynamics of clonal relapse and the contribution of intra-patient functional heterogeneity (of either genetic or epigenetic origin), however, remains poorly understood. We have developed an assay using a molecular barcode system and next generation sequencing to simultaneously track the fate of ∼14,000 different clones either in vitro or in vivo (Figure 1). Individual clones can therefore be tracked over time even in complex populations. We validated this assay on a human AML cell line, THP1, over multiple population doublings, describe how the heterogeneity can be monitored and quantified over time and describe that even in this cell line the population continues to undergo dynamic clonal changes. METHODS:A lentiviral barcode library consisting of ∼14,000 unique 20 basepair sequences (the molecular barcode) was transduced into THP1 cells at a multiplicity of infection of 0.05 (to assure that each cell only acquired a single barcode). Barcoded cells were purified by selection for GFP by flow cytometry. Cells were maintained in RPMI at a concentration of 2×106 cells/ml in triplicates and passaged every 2∼3 days. The percentage of GFP expression was assessed every 30 population doublings (PD). Cells were passaged to a maximum of 90 population doublings. Genomic DNA was extracted every 10 population doublings (PD), barcodes were amplified by PCR using primers bracketing the barcode region to produce a 250 bp product, and gel purified 250 bp products were submitted to Illumina™ high throughput sequencing to quantify the abundance of each barcode in the population. Barcodes were clustered allowing for a minimum quality score of 30 and a maximum of 1 mismatch. Clusters were ordered and plotted by frequency of occurrence from highest to lowest. The frequency of barcode clusters at specific PD was compared between the triplicates. RESULTS:The population doublings and percentage of GFP expression of barcoded THP1 cells was nearly identical in the triplicate cell passage of up to 90 population doublings (Figure 2. THP1 bc1 = barcoded THP1 tissue passage 1 of triplicate, THP1 bc2 = barcoded THP1 tissue passage 2 of triplicate, THP1 bc3 = barcoded THP1 tissue passage 3 of triplicate). A high fraction of these barcoded cells (> 90%) expressed GFP consistently throughout the 90 population doublings. On the other hand the heterogeneity, represented by clusters of barcodes in a single cell passage at PD 30, 60 and 90, varied between PD as shown in the stacked histogram (Figure 3). PD 30 was the reference PD and was sorted from highest cluster frequency to lowest; all other PDs were compared to PD 30. CONCLUSIONS:Subpopulations of cells are dynamic even in established cell lines, including the THP1 human AML cell line. Fluctuations in barcode frequencies were observed between PD within a sequential cell passage. A high degree of heterogeneity was therefore maintained and thus represents the highly dynamic nature of this AML cell line. This barcode assay can be a powerful tool to assess how population heterogeneity can be affected by environmental pressures such as chemotherapy. Our future effort is directed at challenging barcoded THP1 cells with cytarabine, a foundation of AML therapy, to determine how chemotherapy affects the functional heterogeneity of a dynamic heterogeneous population. In addition, we are also assessing how heterogeneity is influenced in vivo by first transplanting barcoded THP1 cells into NSG mice and then challenging the transplanted mice with chemotherapy. [Display omitted] [Display omitted] [Display omitted] Disclosures:Hsu:Child Health Research Institute and the Stanford CTSA (grant no. UL1 RR025744): Research Funding; Rosa Wann and Marjorie Shannon Fellowship: Research Funding. Porteus:Alex's Lemonade Stand Foundation: Research Funding.