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PURPOSE The US Food and Drug Administration–expanded access program (EAP) uses a single patient use (SPU) mechanism to provide patient access to investigational agents in situations where no satisfactory or comparable therapy is available. Genomic profiling of de novo and relapsed or refractory childhood cancer has led to increased identification of new drug targets in the last decade. The aim of this study is to examine the SPU experience for genomically targeted therapies in patients with pediatric cancer. PATIENTS AND METHODS All genomically targeted therapeutic SPUs obtained over a 5-year period were evaluated at four large pediatric cancer programs. Data were collected on the type of neoplasm, agents requested, corresponding molecularly informed targets, and clinical outcomes. RESULTS A total of 45 SPUs in 44 patients were identified. Requests were predominantly made for CNS and solid tumors (84.4%) compared with hematologic malignancies (15.6%). Lack of an available clinical trial was the main reason for SPU initiation (64.4%). The median time from US Food and Drug Administration submission to approval was 3 days (range, 0-12 days) and from Institutional Review Board submission to approval was 5 days (range, 0-50 days). Objective tumor response was seen in 39.5% (15 of 38) of all evaluable SPUs. Disease progression was the primary reason for discontinuation of drug (66.7%) followed by toxicity (13.3%). CONCLUSION SPU requests remain an important mechanism for pediatric access to genomically targeted agents given the limited availability of targeted clinical trials for children with high-risk neoplasms. Furthermore, this subset of SPUs resulted in a substantial number of objective tumor responses. The development of a multi-institutional data registry of SPUs may enable systematic review of toxicity and clinical outcomes and provide evidence-based access to new drugs in rare pediatric cancers.

PURPOSE Profiling of pediatric cancers through deep sequencing of large gene panels and whole exomes is rapidly being adopted in many clinical settings. However, the most impactful approach to genomic profiling of pediatric cancers remains to be defined. METHODS We conducted a prospective precision medicine trial, using whole-exome sequencing of tumor and germline tissue and whole-transcriptome sequencing (RNA Seq) of tumor tissue to characterize the mutational landscape of 127 tumors from 126 unique patients across the spectrum of pediatric brain tumors, hematologic malignancies, and extracranial solid tumors. RESULTS We identified somatic tumor alterations in 121/127 (95.3%) tumor samples and identified cancer predisposition syndromes on the basis of known pathogenic or likely pathogenic germline mutations in cancer predisposition genes in 9/126 patients (7.1%). Additionally, we developed a novel scoring system for measuring the impact of tumor and germline sequencing, encompassing therapeutically relevant genomic alterations, cancer-related germline findings, recommendations for treatment, and refinement of risk stratification or prognosis. At least one impactful finding from the genomic results was identified in 108/127 (85%) samples sequenced. A recommendation to consider a targeted agent was provided for 82/126 (65.1%) patients. Twenty patients ultimately received therapy with a molecularly targeted agent, representing 24% of those who received a targeted agent recommendation and 16% of the total cohort. CONCLUSION Paired tumor/normal whole-exome sequencing and tumor RNA Seq of de novo or relapsed/refractory tumors was feasible and clinically impactful in high-risk pediatric cancer patients.

Involvement of the pituitary gland by leukemic infiltration is exceedingly rare. Here, we describe a very late recurrence of B-cell acute lymphoblastic leukemia masquerading as a pituitary tumor and review the literature for previously reported cases. Our female patient presented 13 years after completion of therapy for B-ALL with headache, amenorrhea, galactorrhea and a pituitary mass. Subsequent studies revealed recurrence of her leukemia, and the pituitary lesion resolved after induction chemotherapy. Our case highlights the importance of considering leukemic infiltrate in the differential diagnosis of pituitary mass, particularly in a patient with a history of hematologic malignancy, sparing unnecessary surgical intervention and informing endocrine evaluation. In addition, the case also highlights difficulties with characterizing this recurrence as a very late relapse or clonal evolution of the original leukemia.

Relapse- and continuous complete remission (CCR)-associated pediatric acute myeloid leukemia (AML) patient bone marrows collected at the time of diagnosis (Dx), end of induction (EOI) and relapse were analyzed by single cell RNA sequencing. A novel AML blasts-associated 7-genes signature (CLEC11A, PRAME, AZU1, NREP, ARMH1, C1QBP, TRH) displayed a strong correlation with blast percentages and overall survival in the TARGET AML dataset (HR = 2.3; P-value = .007). Distinct clusters of AML-blasts at Dx were observed for relapse- and CCR-associated samples with differential expression of genes associated with survival. Relapse-associated samples demonstrated enrichment of exhausted T cells and M2 macrophages as opposed to inflammatory M1 macrophages in CCR-associated samples at Dx. EOI treatment resistant blast cells overexpressed fatty acid oxidation, tumor growth and stemness genes. Also, a relapse-associated EOI samples T cells subset showed downregulation of MHC Class I and regulatory genes. Altogether, this study deeply characterizes pediatric AML relapse-/CCR-associated tumor microenvironment transcriptome landscape.

This selection from the NCCN Guidelines for Adolescent and Young Adult (AYA) Oncology focuses on treatment and management considerations for AYA patients with cancer. Compared with older adults with cancer, AYA patients have unique needs regarding treatment, fertility counseling, psychosocial and behavioral issues, and supportive care services. The complete version of the NCCN Guidelines for AYA Oncology addresses additional aspects of caring for AYA patients, including risk factors, screening, diagnosis, and survivorship.

Ataxia-telangiectasia (A-T) is a rare autosomal recessive disorder characterized by progressive cerebellar degeneration that is typically diagnosed in early childhood. A-T is associated with a predisposition to malignancies, particularly lymphoid tumors in childhood and early adulthood. An adolescent girl with minimal neurologic symptoms was diagnosed with A-T 8 years after completing therapy for T-cell acute lymphoblastic leukemia, following a diagnosis of ATM-mutated breast cancer in her mother. We highlight the importance of recognizing ATM mutations in T-cell acute lymphoblastic leukemia, appreciating the phenotypic heterogeneity of A-T, and defining optimal cancer screening in A-T patients.

Background: Leukemia is the most common type of childhood cancer. Although the prognosis for many pediatric leukemias has improved, leukemias associated with the t(10;11) CALM-AF10 translocation remain difficult to treat. CALM-AF10 leukemias account for ~5-10% of childhood T-cell acute lymphoblastic leukemia (T-ALL)as well as a subset of acute myeloid leukemia (AML). CALM-AF10 leukemias exhibit increased expression of proleukemic HOXA genes, but relatively little is known about the cellular mechanisms that drive CALM-AF10 leukemogenesis. Our laboratory has demonstrated that the CALM protein contains a nuclear export signal (NES) that is critical for CALM-AF10-dependent leukemogenesis. The NES interacts with the CRM1/XPO1 nuclear export receptor, which shuttles proteins from the nucleus to the cytoplasm through the nuclear pore complex. We have shown that transcriptional activation of HOXA genes by CALM-AF10 is dependent on its interaction with CRM1. Importantly, CRM1 does not contain a recognized DNA binding domain, and it is not currently understood how the CALM-AF10/CRM1 complex interacts with regulatory regions of HOXA genes. To identify proteins that mediate the interaction between the CALM-AF10/CRM1 complex and DNA, we took advantage of a proximity-based labeling approach using BioID2, a second-generation biotin ligase. When fused to a protein of interest and in the presence of biotin, BioID2 biotinylates proteins in close proximity to the ligase. These biotinylated proteins can then be identified by mass spectrometry (MS). Methods: We prepared an expression plasmid in which BioID2 was cloned in-frame with CALM-AF10. Human Embryonic Kidney 293 (HEK293) cells were transiently transfected with BioID2-CALM-AF10 and grown in the presence or absence of biotin. MS was performed to identify candidate interacting proteins. We validated direct interactions of candidate proteins with CALM-AF10 using co-immunoprecipitation experiments in HEK293 cells transfected with a CALM-AF10 plasmid. We confirmed that candidate proteins are present in murine CALM-AF10 leukemia cells via Western blotting. In order to efficiently knockout (KO) candidate proteins, we have generated a human U937 cell line (which harbors a t(10;11) CALM-AF10 translocation) with a stable incorporated Cas9. To assess whether KO of EPS15, DVL2 or CTTN affects HOXA5 expression, we performed RT-qPCR in U937-Cas9 cells lines with confirmed KO. Results: We carried out three independent transfections/MS experiments, which identified 71, 95 and 61 proteins, respectively. Of the proteins identified, 12 candidates were common to all three experiments . Importantly, we identified Disruptor Of Telomeric silencing 1-Like (DOT1L), a protein known to interact with AF10, and Nuclear pore complex protein 214 (NUP214), a protein that interacts with CRM1 and that is involved in leukemogenic translocations. We chose EPS15, DVL2 and CTTN for further study, as each of these proteins plays a role in leukemogenesis. We performed initial validation of direct interactions via co-immunoprecipitation and found that all three proteins co-precipitate with CALM-AF10. Western blotting showed that all three proteins are expressed in a murine CALM-AF10 leukemia cell line. We effectively knocked out EPS15 protein expression in U937 cells, and showed that HOXA5 expression is reduced in the setting of EPS15 knockout. Conclusion: We used biotin ligase-dependent proximity-based labeling to identify candidate proteins that potentially interact with the CALM-AF10 fusion protein. Our identification of DOT1L validates the approach, since DOT1L is known to interact with CALM-AF10. We have started to investigate three candidate proteins - EPS15, DVL2 and CTTN - all of which are involved in leukemogenic transformation. We have shown that EPS15, DVL2 and CTTN are expressed in murine CALM-AF10 leukemia cells and directly interact with the CALM-AF10 fusion protein. Knockout of EPS15 in U937 cells results in decreased HOXA5 expression, suggesting the importance of EPS15 in CALM-AF10 leukemogenesis. Evaluation of the roles of these proteins in leukemogenesis may lead to identification of novel pathways involved in CALM-AF10 leukemogenesis. Disclosures No relevant conflicts of interest to declare.

Background : The CALM-AF10 translocation is found in 5-10% of T-cell acute lymphoblastic leukemias (T-ALL), and a subset of acute myeloid leukemias (AML). CALM-AF10 leukemias are characterized by elevated expression of proleukemic HOXA genes. Since HOXA genes are difficult to target, we hypothesized that identification of non-HOXA CALM-AF10 effector genes could potentially yield novel therapeutic targets. To discover novel CALM-AF10-regulated genes, we took advantage of our prior observation that the nuclear export factor CRM1/XPO1 tethers CALM-AF10 to HOXA genes by interacting with a nuclear export signal within CALM. Using microarrays, we identified a set of genes that showed decreased expression in response to the CRM1 inhibitor Leptomycin B (LMB), similar to Hoxa genes, in murine CALM-AF10 leukemia cells. Then using RNA-sequencing, we discovered a set of genes increased in murine hematopoietic stem cells transduced with CALM-AF10. There were 11 genes that were both decreased in response to LMB and increased in response to CALM-AF10, which included the Hoxa gene cluster, as well as Six1. We demonstrated that CALM-AF10 increases Six1 expression and localizes to the Six1 locus, as it does the Hoxa genes. SIX1, like the Hoxa genes, is a homeobox gene that is associated with embryogenesis and is quiescent post-embryologically. In addition, SIX1 and its cofactor EYA2 are overexpressed in numerous solid tumors, and an inhibitor of the SIX1/EYA2 complex (Compound 8430) has recently been described. While there is evidence of a role for SIX1 in solid tumors, its role in leukemias has not been explored. Objective : Evaluate the effect of a SIX1/EYA2 complex inhibitor on leukemia cell proliferation. Design/Methods : SIX1 gene and protein expression were assessed in CALM-AF10, Jurkat (T-ALL) and NOMO1 (AML) leukemia cell lines via Western Blot and RT-qPCR. CALM-AF10 leukemias were derived from murine models in our lab, Jurkat and NOMO1 cell lines were obtained from ATCC. The effect of compound 8430 - an inhibitor of the Six1/Eya2 interaction - on cell proliferation was evaluated using Cell-Titer-Glo Assays and liquid culture proliferation assays. In addition, we used the the CRM1 Nuclear Export Inhibitor KPT-330 alone and in combination with 8430 in these cell lines. SynergyFinder2 (https://synergyfinder.fimm.fi/) was used to assess synergy of 8430 and KPT-330. δ-score is a calculated value that indicates synergistic drug interaction, with a higher δ-score indicative of a synergistic effect of the drugs. Results : SIX1 gene and protein expression are increased in CALM-AF10 leukemia cell lines and Jurkat T-ALL cells, but not NOMO1 cells. Compound 8430 decreases cell proliferation in CALM-AF10 leukemias and Jurkat leukemia cell lines, however it did not affect the AML line NOMO1. Correspondingly, liquid cultures showed that 8430 alone slowed the proliferation of CALM-AF10 leukemia and the Jurkat cells, but not NOMO1 cells. The addition of KPT-330 to 8430 was synergistic in CALM-AF10 leukemia cells with a KPT-330 dose of 60 nM and multiple dose levels of 8430 (δ-scores 17-19) while in the Jurkat leukemia cells a dose of 30 nM of KPT-330 was synergistic at multiple dose levels of 8430 (δ-score 6-8) (Figure 1). Conclusions : The SIX1 homeobox gene is highly expressed during development, and its expression is silenced post-embryogenesis. Through an initial unbiased screen, we discovered that Six1 may play a role in CALM-AF10 leukemogenesis. We have determined that Six1 expression is upregulated in the presence of CALM-AF10. A role for Six1 in CALM-AF10 leukemogenesis is further supported by the ability of a SIX1/EYA2 inhibitor to slow the proliferation of CALM-AF10 leukemia cells. Importantly, based on our observation that 8430 slows proliferation of Jurkat cells, SIX1 inhibition may be relevant in other leukemias. Finally, our demonstration that 8430 synergizes with KPT-330, a Nuclear Export Inhibitor, suggests the possibility of a novel therapeutic approach for CALM-AF10 and other leukemias. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Purpose The majority of new diagnoses of pediatric cancer are made in resource-poor countries, where survival rates range from 5% to 25% compared with 80% in high-resource countries. Multiple factors, including diagnostic and treatment capacities and complex socioeconomic factors, contribute to this variation. This study evaluated the available resources and outcomes for pediatric patients with cancer at the first oncology treatment center in northern Tanzania. Methods Qualitative interviews were completed from July to August 2015 to determine available staff, hospital, diagnostic, treatment, and supportive care resources. A retrospective review of hospital admissions and clinic visits from January 2010 to August 2014 was completed. A total of 298 patients were identified, and data from 182 patient files were included in this review. Results Diagnostic, treatment, and supportive capacities are limited for pediatric cancer care. The most common diagnoses were Burkitt lymphoma (n = 32), other non-Hodgkin lymphoma (n = 26), and Wilms tumor (n = 25). A total of 40% of patients (n = 72) abandoned care. There was a 20% 2-year event-free survival rate, which was significantly affected by patient age, method of diagnosis, and year of diagnosis. Conclusion To our knowledge, this is the first review of pediatric cancer outcomes in northern Tanzania. The study identified areas for future development to improve pediatric cancer outcomes, which included strengthening of training and diagnostic capacities, development of registries and research databases, and the need for additional research to reduce treatment abandonment.

Introduction: While advances in front-line conventional chemotherapy have increased the likelihood of attaining remission in pediatric AML, relapse rates remain high (25-35%), and novel therapies are needed (Zhang, Savage et al. 2019). The clinical and molecular heterogeneity of AML makes it complex to study and creates challenges for the development of novel therapies (Bolouri, Farrar et al. 2018). It is important to identify cells and pathways underlying relapse to facilitate development of novel therapies. Single-cell RNA Sequencing (scRNA-Seq) allows in-depth analysis of the heterogeneous AML landscape to provide a detailed view of the tumor microenvironment, revealing populations of blasts and immune cells which may be relevant to relapse or complete remission. Methods: We analyzed ~36,500 cells from 14 pediatric AML bone marrow samples in our institutional biorepository, spanning different AML subtypes and 3 healthy children to generate a comprehensive scRNA-Seq landscape of immature AML-associated blasts and microenvironment cells. Samples collected at the time of diagnosis (Dx), end of induction (EOI), and relapse (Rel) were used to generate scRNA-Seq data using a droplet-based barcoding technique (Panigrahy, Gartung et al. 2019). After normalization of scRNA-Seq data, the cell clusters were identified using principal component analysis and Uniform Manifold Approximation and Projection (UMAP) approach (Becht et al, 2018). Differential expression, pathways and systems biology analysis between relapsed and remission patients reveal differences for specific cell clusters (Panigrahy, Gartung et al. 2019). To determine the clinical outcome association of our AML blast specific markers, survival analysis was performed on AML TARGET data (https://ocg.cancer.gov/programs/target) using cox proportional hazard survival approach. To characterize AML blast cells with high accuracy, we used support vector machine (SVM), an Artificial Intelligence based feature extraction and model development approach (Bhasin, Ndebele et al. 2016). Results:ScRNA-Seq analysis of paired Dx and EOI samples using UMAP identified three blast cell clusters with significant gene expression differences among different patients, indicating heterogeneity of AML blast cells (Fig 1a, b). Comparative analysis of the three Dx enriched blast cell clusters with other cells identified a "core blast cell signature" with overexpression of genes like AZU1, CLEC11A, FLT3, and NREP (Fig 1c). These core AML-blast genes were linked to significant activation of the Wnt/Ca2+, Phospholipase C, and integrin signaling pathways (Z score >2 and P-value The scRNA-Seq of AML specific blast cells from relapsed and remission samples exhibited a different clustering pattern indicating different transcriptome landscapes. Relapse-associated AML cell clusters expressed high levels of AZU1, S100A4, LGALS1, and GRK2 genes (Fig 2a). Analysis of the non-AML tumor microenvironment demonstrated enrichment of T/NK in relapsed samples, with differential expression of T cell regulatory/activation genes (Fig 2b, c). ScRNA-Seq showed enrichment of monocyte/macrophage cell clusters in remission samples with distinct relapse- and remission-specific clusters. Remission associated macrophage/monocyte clusters showed overexpression of S100A10, FTH1, CST3 and IFITM2 genes (Fig 2d). Similarly, enrichment of T cell and monocyte/macrophage clusters was observed in relapse and remission samples respectively during EOI. Conclusions: Using single cell transcriptomics we developed a novel potential gene signature to characterize heterogenous AML blast populations with high sensitivity. These genes and the pathways they regulate implicate potential therapeutic targets in pediatric AML. Single cell transcriptome analysis also enabled identification of cell clusters with modulated gene expression at both Dx and EOI that may be useful in predicting relapse/remission. Disclosures Bhasin: Canomiiks Inc: Current equity holder in private company, Other: Co-Founder.

Background : The CALM-AF10 translocation is found 5-10% of T-cell acute lymphoblastic leukemias (T-ALL), and a subset of acute myeloid leukemias (AML). CALM-AF10 leukemias are characterized by elevated expression of proleukemic HOXA genes. Since HOXA genes are difficult to target, we hypothesized that identification of non-HOXA CALM-AF10 effector genes could potentially yield novel therapeutic targets. To discover novel CALM-AF10-regulated genes, we took advantage of our prior observation that the nuclear export factor CRM1/XPO1 tethers CALM-AF10 to HOXA genes by interacting with a nuclear export signal within CALM. Using microarrays, we identified a set of genes that showed decreased expression in response to the CRM1 inhibitor, Leptomycin B (LMB), similar to Hoxa genes, in murine CALM-AF10 leukemia cells. Then using RNA-sequencing, we discovered a set of genes increased in murine hematopoietic stem cells transduced with CALM-AF10. There were 11 genes that were both decreased in response to LMB and increased in response to CALM-AF10, which included the Hoxa gene cluster, as well as Six1. Similar to HOXA genes, SIX1 is a homeobox gene that is associated with embryogenesis and is quiescent post-embryologically. Additionally, SIX1 and its cofactor EYA2 have been found to be overexpressed in numerous solid tumors, and inhibitor of the SIX1/EYA2 complex has recently been described. While there is evidence of a role for SIX1 in solid tumors, its role in leukemias has not been explored. Objective: To evaluate the role of SIX1 in CALM-AF10 leukemias. Design/Methods: RT-qPCR and Chromatin Immunoprecipitation (ChIP) were performed using bone marrow progenitors transduced with CALM-AF10 or an empty vector, with and without LMB. Methylcellulose colony assays assessed the ability of SIX1 to enhance self-renewal of hematopoietic progenitors. An inhibitor of the Six1/Eya2 interaction (compound 8430) was used to evaluate cell proliferation. Downstream targets of Six1 were evaluated using RT-qPCR in CALM-AF10 cells treated with Six1/Eya2 inhibitor (8430). Results: RT-qPCR confirmed overexpression of SIX1 in CALM-AF10 leukemia cells, and showed decreased SIX1 expression in the presence of LMB. Furthermore, ChIP revealed that CALM-AF10 binds to the SIX1 gene locus. Overexpression of SIX1 in fetal liver progenitors was sufficient to increase self-renewal potential. The 8430 Six1/Eya2 inhibitor slowed cell growth in CALM-AF10 cells compared to cells treated with DMSO alone. Finally, downstream targets such as Slc2a1, Cdk2, and Cyclina2 were decreased in 8430-treated CALM-AF10 leukemia cells. Conclusions: The SIX1 homeobox gene is highly expressed during embryogenesis, and its expression is silenced post-embryogenesis. Through an initial unbiased screen, we discovered that Six1 may play a role in CALM-AF10 leukemogenesis. We have determined that Six1 expression is upregulated in the presence of CALM-AF10. Further, we have shown a potential oncogenic role for Six1, as it was able to increase the self-renewal potential of hematopoietic progenitors. The role of Six1 in CALM-AF10 leukemia is further supported by the ability of a SIX1/EYA2 inhibitor to slow the growth of CALM-AF10 leukemia cells and decrease the expression of downstream targets of SIX1. These observations suggest that Six1 plays a pathogenic role in leukemogenesis, and may be a novel therapeutic target in CALM-AF10 leukemias. Disclosures No relevant conflicts of interest to declare.

Wilms tumor (WT) is the most common renal tumor of childhood and accounts for 5-7% of all childhood cancers. Remarkable progress in the treatment of WT has been made in the last several decades through collaborative clinical trials in the United States and Europe. With a multimodal approach including chemotherapy, surgery, and radiation therapy, overall survival for WT is greater than 90%. However, there remains a subgroup of patients for which cure remains a challenge, and late effects of therapy are significant. New insights into clinical and biologic prognostic markers are being used to improve risk stratification and to tailor therapy to individual patients. Current treatment strategies aim to maintain excellent survival while reducing late effects for low-risk patients and optimize therapy to improve cure rates for high-risk patients. This review contains 1 figures, 4 tables, and 45 references. Key Words: chemotherapy, COG, kidney neoplasms, nephroblastoma, NWTS, pediatric cancer, radiotherapy, treatment outcome, SIOP, Wilms tumor

Background: Leukemia is the most common type of childhood cancer. Although the prognosis for many pediatric leukemias has improved, leukemias associated with the t(10;11) CALM-AF10 translocation remain difficult to treat. CALM-AF10 leukemias account for ~5-10% of childhood T-cell acute lymphoid leukemia (T-ALL) as well as a subset of acute myeloid leukemia (AML). CALM-AF10 leukemias exhibit increased expression of proleukemic HOXA genes, but relatively little is known about the cellular mechanisms that drive CALM-AF10 leukemogenesis. Our laboratory has demonstrated that the CALM protein contains a nuclear export signal (NES) that is critical for CALM-AF10-dependent leukemogenesis. The NES interacts with the CRM1/XPO1 nuclear export receptor, which shuttles proteins from the nucleus to the cytoplasm through the nuclear pore complex. We have shown that transcriptional activation of HOXA genes by CALM-AF10 is critically dependent on its interaction with CRM1. Importantly, CRM1 does not contain a recognized DNA binding domain, and it is not currently understood how the CALM-AF10/CRM1 complex interacts with regulatory regions of HOXAgenes. In order to identify proteins that mediate the interaction between the CALM-AF10/CRM1 complex and DNA, we took advantage of a proximity-based labeling approach using BioID2, a second-generation biotin ligase. When fused to a protein of interest and in the presence of biotin, BioID2 biotinylates proteins in close proximity to the ligase. These biotinylated proteins can then be identified by mass spectrometry (MS). Methods: We prepared an expression plasmid in which BioID2 was cloned in-frame with CALM-AF10. We then transiently transfected Human Embryonic Kidney 293 (HEK293) cells with the BioID2-CALM-AF10 plasmid, grew them in the presence or absence of biotin, and performed streptavidin-pulldown followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to identify candidate interacting proteins. Proteins were considered candidates if they had a peptide spectrum match (PSM) score > 10 and at least a two-fold greater PSM score versus negative control. We validated direct interactions of candidate proteins with CALM-AF10 by performing co-immunoprecipitation experiments. Results: We first confirmed that the addition of BioID2 to CALM-AF10 does not affect the transcriptional activation of HOXA genes or CALM-AF10 mediated immortalization of hematopoietic stem cells. We carried out three independent transfections/LC-MS/MS experiments, which identified 71, 95 and 61 proteins, respectively. Of the proteins identified, 11 candidates were common to all three experiments.Of particular interest, we identified Disruptor Of Telomeric silencing 1-Like (DOT1L), a protein known to interact with AF10, and Nuclear pore complex protein 214 (NUP214), a protein that has been identified in leukemogenic translocations. The nine additional candidate proteins included: EPS15, DVL2, DVL3, and DDX3X -all known to play a role in leukemogenesis. We performed initial validation of direct interactions via co-immunoprecipitation and found that Epidermal Growth Factor Receptor Substrate 15(EPS15) co-precipitates with CALM-AF10. Conclusion: We used biotin ligase-dependent proximity-based labeling to identify candidate proteins that potentially interact with the CALM-AF10 fusion protein. Our identification of DOT1L validates the approach, since DOT1L is known to interact with CALM-AF10. We have started to investigate other candidate proteins, focusing on known translocation partners in various leukemias. Our screen identified EPS15, a protein involved in receptor-mediated endocytosis of epidermal growth factor and a known translocation partner for MLL/KMT2A. KMT2A-EPS15 translocations (t(1;11)(p32;q23)) have been identified in both AML and ALL, and KMT2A-EPS15 is among the eight most common KMT2A rearrangements. We have shown that EPS15 co-immunoprecipitates with CALM-AF10, suggesting that EPS15 may also play a role in CALM-AF10 leukemogenesis. Further evaluation of this interaction is underway, and may lead to identification of novel pathways involved in CALM-AF10 leukemogenesis. Disclosures No relevant conflicts of interest to declare.

Introduction: The Food and Drug Administration (FDA) expanded access program uses a single-patient use (SPU) mechanism to provide patient access to investigational agents prior to FDA approval and outside clinical trials, in situations where no satisfactory or comparable therapy is available. Genomic profiling of both newly diagnosed and relapsed/refractory childhood cancer has increased in the last decade, resulting in identification of new drug targets for pediatric malignancies. Recently, a review of SPU use in adult and pediatric patients at a single institution (Feit, JAMA Oncology 2019) showed that a markedly higher percentage of pediatric patients receive access through SPUs compared with adult patients, identifying this as an important means of pediatric drug access. However, little is known about the pediatric use and efficacy of SPUs in children. The aim of this study is to examine the SPU experience in pediatric and adolescent and young adult (AYA) cancer patients—specifically obtained for targeted therapies at three large pediatric cancer centers. Methods: All therapeutic SPUs obtained between January 1, 2014 and January 1, 2019 were evaluated at the Aflac Cancer & Blood Disorders Center (Atlanta, GA), Cincinnati Children’s Hospital (Cincinnati, OH), and the Memorial Sloan Kettering Cancer Center (New York, NY). Data were collected on the type of malignancy, agents requested, and corresponding molecularly informed targets, if applicable. Results: A total of 61 SPUs were approved in the five-year period, with 34 (55%) of them specific for access to agents based on somatic tumor mutations identified by genomic profiling. Among the disease groups, SPUs were most often requested for tumors affecting the central nervous system (CNS) (48%), followed by solid tumors (bone, liver, and kidney tumors) (26%), hematologic malignancies (leukemia/lymphoma) (21%), and other rare tumors (5%). Kinase inhibitors were the most frequently requested agents in the genomically defined category (n=34), specifically, FGFR (fibroblast growth factor receptor) inhibitors followed by drugs targeting NTRK 1/2/3 (tropomyosin receptor kinase (Trk) receptors). Most patients within this genomically targeted group (18/34) are currently receiving therapy with these agents. Conclusions: We found that SPUs represent an important means of access to therapeutic agents in the pediatric and AYA populations, with more than half of all SPUs based on rearrangements identified by genomic profiling. A broad range of agents were requested across CNS, solid tumor, and hematologic types. Furthermore, more than half of the patients remain on their respective SPU-approved drug. We are currently performing a more detailed analysis of clinical responses, as well as a time analysis from SPU initiation to approval for all patients in this cohort. Establishment of this cohort of patients across institutions will serve as the basis for a formal registry of pediatric SPUs, which will enable us to study their use and efficacy over time. Citation Format: Himalee Sabnis, Benjamin Mizukawa, Julia Glade-Bender, Jason Fangusaro, Stephen Roberts, Chanta Whitlow, Marilyn Winchester, Maureen O’Brien, Laura Agresta, Brian Turpin, Daniel Wechsler, Sharon Castellino, Neerav Shukla. Targeted therapies for children and young adults with cancer: Single-patient use (SPU) experience at three large pediatric cancer programs [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A50.

The NUP98 and NUP214 nucleoporins (NUPs) are recurrently fused to heterologous proteins in leukemia. The resulting chimeric oncoproteins retain the phenylalanine-glycine (FG) repeat motifs of the NUP moiety that mediate interaction with the nuclear export receptor Crm1. NUP fusion leukemias are characterized by HOXA gene upregulation; however, their molecular pathogenesis remains poorly understood. To investigate the role of Crm1 in mediating the leukemogenic properties of NUP chimeric proteins, we took advantage of the Sequestosome-1 (SQSTM1)-NUP214 fusion. SQSTM1-NUP214 retains only a short C-terminal portion of NUP214 which contains FG motifs that mediate interaction with Crm1. We introduced point mutations targeting these FG motifs and found that the ability of the resulting SQSTM1-NUP214FGmut protein to interact with Crm1 was reduced by more than 50% compared with SQSTM1-NUP214. Mutation of FG motifs affected transforming potential: while SQSTM1-NUP214 impaired myeloid maturation and conferred robust colony formation to transduced hematopoietic progenitors in a serial replating assay, the effect of SQSTM1-NUP214FGmut was considerably diminished. Moreover, SQSTM1-NUP214 caused myeloid leukemia in all transplanted mice, whereas none of the SQSTM1-NUP214FGmut reconstituted mice developed leukemia. These oncogenic effects coincided with the ability of SQSTM1-NUP214 and SQSTM1-NUP214FGmut to upregulate the expression of Hoxa and Meis1 genes in hematopoietic progenitors. Indeed, chromatin immunoprecipitation assays demonstrated that impaired SQSTM1-NUP214 interaction with Crm1 correlated with impaired binding of the fusion protein to Hoxa and Meis1 genes. These findings highlight the importance of Crm1 in mediating the leukemogenic properties of SQSTM1-NUP214, and suggest a conserved role of Crm1 in recruiting oncoproteins to their effector genes.

Endothelial cell apoptosis induced by hypoxia is implicated in the pathogenesis of vascular diseases. However, the underlying mechanism is not clearly elucidated. In this study, we found that hypoxia increased Mxi1-0 expression, and the Mxi1-0 siRNA could inhibit caspase-8 activation and apoptosis in HUVECs induced by hypoxia. In addition, hypoxia induced FOXO3 activation, while Mxi1-0 expression and apoptosis were inhibited by transfection with FOXO3 siRNA. Using ChIP assay, we confirmed that FOXO3a binds to the Mxi1-0 promoter region. Furthermore, hypoxia treatment leads to remarkable production of reactive oxygen species (ROS), while ROS scavenger N-acetyl-L-cysteine (NAC) inhibits hypoxia-induced ROS production, apoptosis and FOXO3a-mediated Mxi1-0 up-regulation. Finally, we found that the HIF-1α siRNA inhibited hypoxia-induced HIF-1α expression and ROS production, as well as FOXO3a/Mxi1-0 activation and apoptosis in HUVECs. Taken together, this study identifies a HIF-1α/FOXO3a/Mxi1-0/caspase-8 signaling pathway in hypoxia-induced endothelial cell apoptosis. These data also indicate that HIF-1α-dependent ROS production is required for FOXO3a-mediated Mxi1-0 up-regulation and apoptosis in hypoxic endothelial cells.

We present a case of Wilms Tumor in a patient with Alagille syndrome 10 months after liver transplant. We explore a suggested genetic connection between these 2 diseases. In children with Wilms Tumor, we propose a pathoembryologic explanation for not just the tumor, but also for the cause of associated benign ureteral and renal parenchymal aberrancies that are commonly seen in the Alagille population. We also discuss the diagnostic and therapeutic challenges that can arise in a liver transplant patient with Alagille syndrome who subsequently develops a renal mass.

Central nervous system (CNS) chloromas are an exceedingly rare presentation of CNS relapse in acute lymphoblastic leukemia (ALL). We report a relapsed ALL patient who presented with 2 separate chloromas and cerebrospinal fluid lymphoblastocytosis, and outline a treatment plan of systemic chemotherapy and CNS-directed radiation therapy. A review of the literature indicates that multiagent chemotherapy combined with CNS radiotherapy is effective, with hematopoietic stem cell transplantation used in half of reported cases. We conclude that intensive systemic multiagent chemotherapy with CNS-directed radiation therapy can be successfully used to treat relapsed pediatric ALL with CNS lymphoblastic chloroma.

Neuroblastoma (NB) is the third most common malignancy of childhood, and outcomes for children with advanced disease remain poor; amplification of the MYCN gene portends a particularly poor prognosis. Mxi1 antagonizes N-Myc by competing for binding to Max and E-boxes. Unlike N-Myc, Mxi1 mediates transcriptional repression and suppresses cell proliferation. Mxi1 and Mxi1-0 (an alternatively transcribed Mxi1 isoform) share identical Max and DNA binding domains but differ in amino-terminal sequences. Because of the conservation of these critical binding domains, we hypothesized that Mxi1-0 antagonizes N-Myc activity similar to Mxi1. SHEP NB cells and SHEP cells stably transfected with MYCN (SHEP/MYCN) were transiently transfected with vectors containing full-length Mxi1, full-length Mxi1-0, or the common Mxi domain encoded by exons 2 to 6 (ex2-6). After incubation in low serum, parental SHEP/MYCN cell numbers were reduced compared with SHEP cells. Activated caspase-3 staining and DNA fragmentation ELISA confirmed that SHEP/MYCN cells undergo apoptosis in low serum, while SHEP/MYCN cells transfected with Mxi1 or Mxi1-0 do not. However, SHEP/MYCN cells transfected with Mxi1 or Mxi1-0 and grown in normal serum showed proliferation rates similar to SHEP cells. Mxi ex2-6 did not affect cell number in low or normal serum, suggesting that amino terminal domains of Mxi1 and Mxi1-0 are critical for antagonism. In the absence of N-Myc, Mxi1 and Mxi1-0 induce apoptosis independently through the caspase-8–dependent extrinsic pathway, while N-Myc activates the caspase-9–dependent intrinsic pathway. Together, these data indicate that Mxi1 and Mxi1-0 antagonize N-Myc but also independently impact NB cell survival.

* Abbreviation: EBV — : Epstein-Barr virus Major advances have been made in the field of pediatric solid organ transplant, with significant improvements in long-term survival for children requiring this life-saving intervention.1 Developments in supportive care, organ allocation, HLA antigen matching, and immunosuppressive regimens have all contributed to this success.2–4 With more patients surviving longer, however, there has also been an increase in the long-term complications of transplantation.5 In addition to other organ toxicities, allograft loss, and impaired health-related quality of life,6,7 a dramatic increase in cancer incidence has been seen in organ transplant recipients.8,9 The overall increased incidence of malignancy in patients who have received solid organ transplants has been reported to range from a two- to six-fold increase above the general population,8,10 depending on the cohort and type of transplant. The cumulative incidence has been reported to be as high as 55% … Address correspondence to Daniel S. Wechsler, MD, PhD, Pediatric Hematology-Oncology, Duke University Medical Center, 397 Hanes House, DUMC Box 102382, Durham, NC 27710. E-mail: dan.wechsler{at}duke.edu

The leukemogenic CALM-AF10 fusion protein is found in patients with immature acute myeloid and T-lymphoid malignancies. CALM-AF10 leukemias display abnormal H3K79 methylation and increased HOXA cluster gene transcription. Elevated expression of HOXA genes is critical for leukemia maintenance and progression; however, the precise mechanism by which CALM-AF10 alters HOXA gene expression is unclear. We previously determined that CALM contains a CRM1-dependent nuclear export signal (NES), which is both necessary and sufficient for CALM-AF10-mediated leukemogenesis. Here, we find that interaction of CALM-AF10 with the nuclear export receptor CRM1 is necessary for activating HOXA gene expression. We show that CRM1 localizes to HOXA loci where it recruits CALM-AF10, leading to transcriptional and epigenetic activation of HOXA genes. Genetic and pharmacological inhibition of the CALM-CRM1 interaction prevents CALM-AF10 enrichment at HOXA chromatin, resulting in immediate loss of transcription. These results provide a comprehensive mechanism by which the CALM-AF10 translocation activates the critical HOXA cluster genes. Furthermore, this report identifies a novel function of CRM1: the ability to bind chromatin and recruit the NES-containing CALM-AF10 transcription factor.

Background: The NUP98 and NUP214 nucleoporins (NUPs) are recurrently fused to heterologous proteins in leukemia. The resulting chimeric oncoproteins retain the NUP phenylalanine-glycine (FG) repeat motifs that mediate interaction with the nuclear export receptor Crm1. NUP fusion leukemias are characterized by HOXA gene upregulation; however, their molecular pathogenesis remains poorly understood. To investigate the role of Crm1 in mediating the leukemogenic properties of NUP chimeric proteins, we studied the SQSTM1-NUP214 fusion. Methods: We synthesized a SQSTM1-NUP214 fusion protein which retains only a short C-terminal portion of NUP214 containing FG motifs that mediate interaction with Crm1, and then introduced point mutations targeting these FG motifs (SQSTM1-NUP214FGmut). We compared the activity of these two fusion proteins using co-immunoprecipitation with CRM1, methylcellulose colony assays, murine transplantation, RT-qPCR, and chromatin immunoprecipitation. Results: We found that the ability of the SQSTM1-NUP214FGmut protein to interact with Crm1 was reduced by more than 50% compared with SQSTM1-NUP214. Further, mutation of FG motifs affected transforming potential: while SQSTM1-NUP214 conferred robust colony formation to transduced hematopoietic progenitors in a serial replating assay, the effect of SQSTM1-NUP214FGmut was greatly diminished. Moreover, SQSTM1-NUP214 caused myeloid leukemia in all transplanted mice (6/6), whereas none of the SQSTM1-NUP214FGmut reconstituted mice developed leukemia (0/7). These oncogenic effects coincided with the ability of SQSTM1-NUP214 and SQSTM1-NUP214FGmut to upregulate the expression of Hoxa and Meis1 genes in hematopoietic progenitors. Indeed, chromatin immunoprecipitation assays demonstrated that impairing the interaction of SQSTM1-NUP214 with Crm1 reduced binding of the fusion protein to Hoxa and Meis1 loci. Conclusions: These findings highlight the importance of Crm1 in mediating the leukemogenic properties of SQSTM1-NUP214, and suggest a conserved role of Crm1 in recruiting oncoproteins to their effector genes. Disclosures No relevant conflicts of interest to declare.

Background: The CALM-AF10 translocation is detected in ~10% of T-cell acute lymphoblastic leukemias (T-ALLs), and in some acute myeloid leukemias (AMLs). CALM-AF10 leukemias are characterized by high expression of proleukemic HOXA genes, which serve a critical role in hematopoiesis. We hypothesized that identification of novel CALM-AF10 effector genes may yield new therapeutic targets in this difficult to treat leukemia. We took advantage of our prior observation that the nuclear export factor CRM1/XPO1 tethers CALM-AF10 to HOXA genes by interacting with a nuclear export signal (NES) in CALM. Using next generation sequencing, we determined that, SIX1, similar to HOXA genes, is increased in CALM-AF10 leukemias and decreased in response to CRM1 inhibition with Leptomycin B (LMB). Design/Methods: RT-qPCR and Chromatin Immunoprecipitation were performed using both bone marrow progenitors and murine embryonic fibroblasts (MEFs) transduced with CALM-AF10 or an empty vector, with and without LMB. The ability of SIX1 to enhance self-renewal of hematopoietic progenitors was examined by measuring the colony-forming ability of transduced fetal liver hematopoietic progenitor cells. CRISPR-Cas9 was used to silence SIX1 in Human Embryonic Kidney 293 (HEK293) cells. Results: RT-qPCR confirmed overexpression of SIX1 in both CALM-AF10 transduced MEFs and CALM-AF10 leukemias, with decreased SIX1 expression observed in the presence of LMB. ChIP analysis showed that CALM-AF10 binds to the SIX1 gene locus. Overexpression of SIX1 in fetal liver cells was sufficient to increase the self-renewal potential of colony-forming progenitors. SIX1 was successfully knocked out in HEK293 cells without a significant effect on HEK293 proliferation. Conclusions: The SIX1 homeobox gene is highly expressed during development and its expression is silenced post-embryogenesis. Increased SIX1 expression has been reported in numerous solid tumors. We have determined that SIX1 is upregulated in CALM-AF10 leukemias, and increases the self-renewal potential of hematopoietic progenitors. Using CRISPR-Cas9 to silence SIX1, we have demonstrated that SIX1 is not essential for cell survival, and that its inhibition may impair CALM-AF10 leukemia cell proliferation. Thus, SIX1 may play a pathogenic role in leukemogenesis and is a potential therapeutic target in CALM-AF10 leukemias. Disclosures No relevant conflicts of interest to declare.

Amplification of the MYCN proto-oncogene is associated with a poor prognosis in patients with metastatic neuroblastoma (NB). MYCN encodes the N-Myc protein, a transcriptional regulator that dimerizes with the Max transcription factor, binds to E-box DNA sequences, and regulates genes involved in cell growth and apoptosis. Overexpression of N-Myc leads to transcriptional activation and an increase in NB cell proliferation. Mxi1, a member of the Myc family of transcriptional regulators, also binds to Max. However, Mxi1 is a transcriptional repressor and inhibits proliferation of NB cells, suggesting that Mxi1 functions as an N-Myc antagonist. Our laboratory previously identified Mxi1-0, an alternatively transcribed Mxi1 isoform. Mxi1-0 has properties distinct from those of Mxi1; in contrast to Mxi1, Mxi1-0 is unable to suppress c-Myc-dependent transcription. We now show that Mxi1-0 expression increases in response to MYCN overexpression in NB cells, with a positive correlation between MYCN and MXI1-0 RNA levels. We also show that N-Myc expression differentially regulates the MXI1 and MXI1-0 promoters: Increased MYCN expression suppresses MXI1 promoter activity while enhancing transcription through the MXI1-0 promoter. Finally, induction of Mxi1-0 leads to increased proliferation, whereas expression of Mxi1 inhibits cell growth, indicating differential roles for these two proteins. These data suggest that N-Myc differentially regulates the expression of MXI1 and MXI1-0 and can alter the balance between the two transcription factors. Furthermore, MXI1-0 appears to be a downstream target of MYCN-dependent signaling pathways and may contribute to N-Myc-dependent cell growth and proliferation.

Iron is essential for normal cellular function. It participates in a wide variety of cellular processes, including cellular respiration, DNA synthesis, and macromolecule biosynthesis. Iron is required for cell growth and proliferation, and changes in intracellular iron availability can have significant effects on cell cycle regulation, cellular metabolism, and cell division. Perhaps not surprisingly then, neoplastic cells have been found to have higher iron requirements than normal, non-malignant cells. Iron depletion through chelation has been explored as a possible therapeutic intervention in a variety of cancers. Here, we will review iron homeostasis in non-malignant and malignant cells, the widespread effects of iron depletion on the cell, the various iron chelators that have been explored in the treatment of cancer, and the tumor types that have been most commonly studied in the context of iron chelation.

The t(10;11) chromosomal translocation gives rise to the CALM-AF10 fusion gene and is found in patients with aggressive and difficult-to-treat hematopoietic malignancies. CALM-AF10-driven leukemias are characterized by HOXA gene up-regulation and a global reduction in H3K79 methylation. DOT1L, the H3K79 methyltransferase, interacts with the octapeptide/leucine zipper domain of AF10, and this region has been shown to be necessary and sufficient for CALM-AF10-mediated transformation. However, the precise role of CALM in leukemogenesis remains unclear. Here, we show that CALM contains a nuclear export signal (NES) that mediates cytoplasmic localization of CALM-AF10 and is necessary for CALM-AF10-dependent transformation. Fusions of the CALM NES (NES(CALM)-AF10) or NES motifs from heterologous proteins (ABL1, Rev, PKIA, APC) in-frame with AF10 are sufficient to immortalize murine hematopoietic progenitors in vitro. The CALM NES is essential for CALM-AF10-dependent Hoxa gene up-regulation and aberrant H3K79 methylation, possibly by mislocalization of DOT1L. Finally, we observed that CALM-AF10 leukemia cells are selectively sensitive to inhibition of nuclear export by Leptomycin B. These findings uncover a novel mechanism of leukemogenesis mediated by the nuclear export pathway and support further investigation of the utility of nuclear export inhibitors as therapeutic agents for patients with CALM-AF10 leukemias.

VEGF expression induced by hypoxia plays a critical role in promoting tumor angiogenesis. However, the molecular mechanism that modulates VEGF expression under hypoxia is still poorly understood. In this study, we found that VEGF induction in hypoxic HepG2 cells is ROS-dependent. ROS mediates hypoxia-induced VEGF by upregulation of Mxi1-0. Furthermore, PI3K/AKT/HIF-1α signaling pathway is involved in ROS-mediated Mxi1-0 and VEGF expression in hypoxic HepG2 cells. Finally, Mxi1-0 could in turn regulate ROS generation in hypoxic HepG2 cells, creating a positive feedback loop. Taken together, this study demonstrate a positive regulatory feedback loop in which ROS mediates hypoxia-induced Mxi1-0 via activation of PI3K/AKT/HIF-1α pathway, events that in turn elevate ROS generation and promote hypoxia-induced VEGF expression. These findings could provide a rationale for designing new therapies based on inhibition of hepatocellular carcinoma (HCC) angiogenesis.

Cancer is the leading cause of death among the adolescent and young adult (AYA) population, excluding homicide, suicide, or unintentional injury. AYA patients should be managed by a multidisciplinary team of health care professionals who are well-versed in the specific developmental issues relevant to this patient population. The recommendations for age-appropriate care outlined in these NCCN Guidelines include psychosocial assessment, a discussion of infertility risks associated with treatment and options for fertility preservation, genetic and familial risk assessment for all patients after diagnosis, screening and monitoring of late effects in AYA cancer survivors after successful completion of therapy, and palliative care and end-of-life considerations for patients for whom curative therapy fails.

Lymphoblastic lymphoma is the second most common type of non-Hodgkin lymphoma seen in children. Approximately, 90% of lymphoblastic lymphomas arise from T cells, with the remaining 10% being B-cell-lineage derived. Although T-cell lymphoblastic lymphoma most frequently occurs in the anterior mediastinum (thymus), B-cell lymphoblastic lymphoma (B-LBL) predominates in extranodal sites such as skin and bone. Here, we describe a pediatric B-LBL patient who presented with extensive abdominal involvement and whose lymphoma cells displayed segmental duplication of the mixed lineage leukemia (MLL) gene. MLL duplication/amplification has been described primarily in acute myeloid leukemia and myelodysplastic syndrome with no published reports of discrete MLL duplication/amplification events in B-LBL. The MLL gene duplication noted in this case may represent a novel mechanism for tumorigenesis in B-LBL.

Parvovirus B19 is a small single-stranded DNA virus of the Parvoviridae family. Depending on host factors, it may produce a wide array of clinical disease states. Disease severity can range from self-limited to severe, requiring significant supportive care. Immunocompromised patients are generally affected more severely but rarely develop prolonged and persistent infections. Here, we describe a patient who was diagnosed with parvovirus during maintenance therapy for acute lymphoblastic leukemia and required therapy with intravenous immunoglobulin; the patient remained parvovirus positive according to a polymerase chain reaction testing but had no clinical symptoms for 27 months off chemotherapy.

Histiocytic sarcoma (HS) is a malignant tumor composed of proliferating cells of histiocytic origin. True HS is exceedingly rare, particularly in pediatric patients. These tumors are frequently aggressive, and outcome for patients with HS has traditionally been poor. There is currently no consensus on the optimal management of these tumors, with the literature consisting largely of case reports and small case series utilizing a wide variety of therapies. We describe a case of HS in an 8-year-old female who was successfully treated with an abbreviated leukemia chemotherapy regimen.

We describe a 4-year-old female patient with a persistent paraspinal mass following chemotherapy for Wilms tumor. A discordant response to chemotherapy prompted biopsy of the persistent mass, which revealed a ganglioneuroma. This report highlights the synchronous occurrence of different tumors in the same patient, and suggests that repeat biopsies should be considered when contiguous tumor masses do not respond as expected. Pediatr Blood Cancer. 2010;55:562–565. © 2010 Wiley-Liss, Inc.

We hypothesized that sirolimus, an mTOR inhibitor, may be effective in patients with autoimmune lymphoproliferative syndrome (ALPS) and treated patients who were intolerant to or failed other therapies. Four patients were treated for autoimmune cytopenias; all had a rapid complete or near complete response. Two patients were treated for autoimmune arthritis and colitis, demonstrating marked improvement. Three patients had complete resolution of lymphadenopathy and splenomegaly and all patients had a reduction in double negative T cells, a population hallmark of the disease. Based on these significant responses, we recommend that sirolimus be considered as second-line therapy for patients with steroid-refractory disease.