Wendy Allen-Rhoades

Cross-species identification of a plasma microRNA signature for detection, therapeutic monitoring, and prognosis in osteosarcoma.

Osteosarcoma (OS) is the primary bone tumor in children and young adults. Currently, there are no reliable, noninvasive biologic markers to detect the presence or progression of disease, assess therapy response, or provide upfront prognostic insights. MicroRNAs (miRNAs) are evolutionarily conserved, stable, small noncoding RNA molecules that are key posttranscriptional regulators and are ideal candidates for circulating biomarker development due to their stability in plasma, ease of isolation, and the unique expressions associated with specific disease states. Using a qPCR‐based platform that analyzes more than 750 miRNAs, we analyzed control and diseased‐associated plasma from a genetically engineered mouse model of OS to identify a profile of four plasma miRNAs. Subsequent analysis of 40 human patient samples corroborated these results. We also identified disease‐specific endogenous reference plasma miRNAs for mouse and human studies. Specifically, we observed plasma miR‐205‐5p was decreased 2.68‐fold in mice with OS compared to control mice, whereas, miR‐214, and miR‐335‐5p were increased 2.37‐ and 2.69‐fold, respectively. In human samples, the same profile was seen with miR‐205‐5p decreased 1.75‐fold in patients with OS, whereas miR‐574‐3p, miR‐214, and miR‐335‐5p were increased 3.16‐, 8.31‐ and 2.52‐fold, respectively, compared to healthy controls. Furthermore, low plasma levels of miR‐214 in metastatic patients at time of diagnosis conveyed a significantly better overall survival. This is the first study to identify plasma miRNAs that could be used to prospectively identify disease, potentially monitor therapeutic efficacy and have prognostic implications for OS patients.

Biomarker significance of plasma and tumor miR-21, miR-221, and miR-106a in osteosarcoma

Osteosarcoma is the most common malignant bone tumor in children and young adults. Despite the use of surgery and multi-agent chemotherapy, osteosarcoma patients who have a poor response to chemotherapy or develop relapses have a dismal outcome. Identification of biomarkers for active disease may help to monitor tumor burden, detect early relapses, and predict prognosis in these patients. In this study, we examined whether circulating miRNAs can be used as biomarkers in osteosarcoma patients. We performed genome-wide miRNA profiling on a discovery cohort of osteosarcoma and control plasma samples. A total of 56 miRNAs were upregulated and 164 miRNAs were downregulated in osteosarcoma samples when compared to control plasma samples. miR-21, miR-221 and miR-106a were selected for further validation based on their known biological importance. We showed that all three circulating miRNAs were expressed significantly higher in osteosarcoma samples than normal samples in an independent cohort obtained from the Children’s Oncology Group. Furthermore, we demonstrated that miR-21 was expressed significantly higher in osteosarcoma tumors compared with normal bone controls. More importantly, lower expressions of miR-21 and miR-221, but not miR-106a, significantly correlated with a poor outcome. In conclusion, our results indicate that miR-21, miR-221 and miR-106a were elevated in the circulation of osteosarcoma patients, whereas tumor expressions of miR-21 and miR-221 are prognostically significant. Further investigation of these miRNAs may lead to a better prognostic method and potential miRNA therapeutics for osteosarcoma.Osteosarcoma is the most common malignant bone tumor in children and young adults. Despite the use of surgery and multi-agent chemotherapy, osteosarcoma patients who have a poor response to chemotherapy or develop relapses have a dismal outcome. Identification of biomarkers for active disease may help to monitor tumor burden, detect early relapses, and predict prognosis in these patients. In this study, we examined whether circulating miRNAs can be used as biomarkers in osteosarcoma patients. We performed genome-wide miRNA profiling on a discovery cohort of osteosarcoma and control plasma samples. A total of 56 miRNAs were upregulated and 164 miRNAs were downregulated in osteosarcoma samples when compared to control plasma samples. miR-21, miR-221 and miR-106a were selected for further validation based on their known biological importance. We showed that all three circulating miRNAs were expressed significantly higher in osteosarcoma samples than normal samples in an independent cohort obtained from the Childrens Oncology Group. Furthermore, we demonstrated that miR-21 was expressed significantly higher in osteosarcoma tumors compared with normal bone controls. More importantly, lower expressions of miR-21 and miR-221, but not miR-106a, significantly correlated with a poor outcome. In conclusion, our results indicate that miR-21, miR-221 and miR-106a were elevated in the circulation of osteosarcoma patients, whereas tumor expressions of miR-21 and miR-221 are prognostically significant. Further investigation of these miRNAs may lead to a better prognostic method and potential miRNA therapeutics for osteosarcoma.

Coamplification of Myc/Pvt1 and homozygous deletion of Nlrp1 locus are frequent genetics changes in mouse osteosarcoma.

Osteosarcomas (OSs) are characterized by high levels of genomic instability (GI). To gain insights into the GI and its contribution toward understanding the genetic basis of OS, we characterized 19 primary and 13 metastatic mouse tumors in a genetically engineered novel mouse model of OS by a combination of genomic techniques. Through the bone‐specific deletion of the wild‐type Trp53 locus or activation of a metastatic‐promoting missense R172Hp53 allele, C57BL/6 mice developed either localized or metastatic OS. Subsequent tumors were isolated and primary cultures created from primary bone and/or distal metastatic lesions, for example, lung and liver. These tumors exhibited high levels of GI with complex chromosomal rearrangements, amplifications, and deletions comparable to human OS. The combined genomic approaches identified frequent amplification of chromosome 15D1 and loss of 11B4 by CGH and/or SKY. Both 15D1 and 11B4 have homology with frequently altered chromosomal bands 8q24 and 17p13 in human OS, respectively. Subsequent array CGH, FISH, and qRT‐PCR analysis identified coamplification and overexpression of Myc/Pvt1 transcripts from the 15D1 amplicon and loss and decreased expression of the Nlrp1b from 11B4. The Nlrp1 gene is the key mediator of apoptosis and interacts strongly with caspase 2.

Local therapy to distant metastatic sites in stage IV rhabdomyosarcoma

To determine the impact of surgery and/or radiation therapy on distant metastatic sites (DMS) in children with stage IV rhabdomyosarcoma (RMS).

miR-130b directly targets Arhgap1 to drive activation of a metastatic CDC42-PAK1-AP1 positive feedback loop in Ewing sarcoma

Ewing Sarcoma (ES) is a highly aggressive bone tumor with peak incidence in the adolescent population. It has a high propensity to metastasize, which is associated with dismal survival rates of approximately 25%. To further understand mechanisms of metastasis we investigated microRNA regulatory networks in ES. Our studies focused on miR‐130b due to our analysis that enhanced expression of this microRNA has clinical relevance in multiple sarcomas, including ES. Our studies provide insights into a novel positive feedback network involving the direct regulation of miR‐130b and activation of downstream signaling events contributing toward sarcoma metastasis. Specifically, we demonstrated miR‐130b induces proliferation, invasion, and migration in vitro and increased metastatic potential in vivo. Using microarray analysis of ES cells with differential miR‐130b expression we identified alterations in downstream signaling cascades including activation of the CDC42 pathway. We identified ARHGAP1, which is a negative regulator of CDC42, as a novel, direct target of miR‐130b. In turn, downstream activation of PAK1 activated the JNK and AP‐1 cascades and downstream transcriptional targets including IL‐8, MMP1 and CCND1. Furthermore, chromatin immunoprecipitation of endogenous AP‐1 in ES cells demonstrated direct binding to an upstream consensus binding site within the miR‐130b promoter. Finally, small molecule inhibition of PAK1 blocked miR‐130b activation of JNK and downstream AP‐1 target genes, including primary miR‐130b transcripts, and miR‐130b oncogenic properties, thus identifying PAK1 as a novel therapeutic target for ES. Taken together, our findings identify and characterize a novel, targetable miR‐130b regulatory network that promotes ES metastasis.

Pediatric Solid Tumors of Infancy: An Overview

1. Wendy Allen-Rhoades, MD, FAAP* 2. Sarah B. Whittle, MD, MS, FAAP* 3. Nino Rainusso, MD, FAAP* 1. *Department of Pediatrics, Section of Hematology-Oncology, Baylor College of Medicine and Texas Childrens Hospital Cancer and Hematology Centers, Houston, TX * Abbreviations: AFP: : α-fetoprotein ASCT: : autologous stem cell transplant CT: : computed tomography FDA: : Food and Drug Administration MRI: : magnetic resonance imaging OMS: : opsoclonus myoclonus syndrome VIP: : vasoactive intestinal peptide 123I-MIBG: : 123I-meta-iodo-benzylguanidine Pediatricians should recognize the role of age, genetic factors, and syndromes that predispose to the development of certain pediatric solid tumors. Many symptoms of common childhood illnesses that progress or do not resolve in a timely manner should require a detailed evaluation and prompt referral to a cancer specialist. After completing this article, readers should be able to: 1. Recognize the presenting signs and symptoms of pediatric solid tumors (eg, abdominal mass, constipation, shortness of breath, back pain, bone pain, fever, and hypertension). 2. Identify the signs and symptoms of retinoblastoma, neuroblastoma, hepatoblastoma, and Wilms tumor. 3. Recommend genetic evaluation and close disease surveillance for patients with certain solid tumors or particular predisposing conditions. 4. Recognize general aspects of the multidisciplinary treatment approach in children with retinoblastoma, neuroblastoma, hepatoblastoma, and Wilms tumor. Pediatric solid tumors are a group of nonhematologic, extracranial cancers that occur during childhood. This heterogeneous group of tumors represents approximately 40% of all pediatric cancers (Fig 1). Many pediatric solid tumors are referred to as embryonal or developmental cancers because they arise in young children or adolescents as a result of alterations in the processes of organogenesis or normal growth. In this review, we address common symptoms developed in children diagnosed as having malignant solid tumors and offer a general description of the most common pediatric solid tumors in infants and young children. Common malignant solid tumors in adolescents will be addressed in a separate article. Figure 1. Percentage distribution of pediatric cancers and solid tumors in children …

Pediatric Solid Tumors in Children and Adolescents: An Overview

1. Wendy Allen-Rhoades, MD* 2. Sarah B. Whittle, MD, MS* 3. Nino Rainusso, MD* 1. *Department of Pediatrics, Section of Hematology-Oncology, Baylor College of Medicine, Texas Childrens Cancer and Hematology Centers, Houston, TX * Abbreviations: 131I: : iodine 131 AFP: : α-fetoprotein β-HCG: : β-human chorionic gonadotropin CT: : computed tomography EWS: : Ewing sarcoma GCT: : germ cell tumor MRI: : magnetic resonance imaging OS: : osteosarcoma PTC: : papillary thyroid cancer SNL: : sentinel lymph node XP: : xeroderma pigmentosum Pediatricians should recognize the role of age, genetic conditions, and environmental exposures in the development of malignant solid tumors in children and adolescents. After completing this article, readers should be able to: 1. Identify the signs and symptoms of extracranial germ cell tumors, osteosarcoma, Ewing sarcoma, thyroid cancer, and melanoma in pediatric patients. 2. Identify the genetic conditions and environmental exposures associated with different cancer types in adolescents. 3. Recognize general aspects of the multidisciplinary treatment approach in patients with extracranial germ cell tumors, osteosarcoma, Ewing sarcoma, thyroid cancer, and melanoma. Although hematologic and central nervous system malignancies continue to be the most common cancers in adolescents, extracranial malignant solid tumors represent, as a group, 52% of cancers in patients in the 15- to 19-year-old age group (Fig 1). The tumor distribution of malignant pediatric solid tumors in adolescents is different compared with that of younger children, in whom embryonal or developmental cancers, such as retinoblastoma, neuroblastoma, or hepatoblastoma, are more prevalent. The most common malignant solid tumors in adolescents are extracranial germ cell tumors (GCTs), bone and soft tissue sarcomas, melanoma, and thyroid cancer. The diagnosis and treatment of adolescents with cancer also have particular challenges related to patient age, such as adherence to therapy, need for psychological support, concerns about body image, and fertility preservation. In this review, we offer a general description of the clinical presentation and treatment of the most common malignant pediatric solid tumors in adolescents. Figure 1. Distribution of pediatric cancers in adolescents aged …

Detection of Plasma MicroRNA Signature in Osteosarcoma Patients

Circulating microRNAs are increasingly being used as noninvasive prognostic and predictive biomarkers for various types of cancer. We describe a method that uses real-time quantitative PCR (qPCR) for establishing a signature plasma microRNA profile that can distinguish patients with osteosarcoma from healthy control samples.

Abstract A70: Targeting pediatric bone sarcoma stem cell with metabolic inhibitors

Our project tests whether metabolic inhibitors are able to target putative sarcoma stem cells in osteosarcoma and Ewing sarcoma. The cure rate for the two most common bone sarcomas affecting children and adolescents, osteosarcoma and Ewing sarcoma, has plateaued around 70% over the past twenty years, and the cure rate for patients with metastatic bone sarcomas has not significantly improved over 20% in several decades. The subpopulation of cells in these tumors referred to as sarcoma stem cells are thought to be responsible for resistance to current therapies, relapses and metastases. To date therapies targeting the sarcoma stem cell population have not been identified. We studied the ability of metabolic inhibitors to eliminate the sarcoma stem cell population. A panel of established and verified osteosarcoma (HOS, 143B, MNNG, SAOS2) and Ewing sarcoma (TC71, TC32, MHH, A4573, CHLA9, CHLA10) cell lines was grown in 2-dimensional culture and treated with metabolic inhibitors, including 2-deoxyglucose, dichloroacetate, oxamate, lonidamine, 3-Bromopyruvate, and metformin for 72hrs establishing dose response curves for each individual agent. Most agents showed IC50s in the millimolar range. Combinations of two metabolic inhibitors at their IC50 were then tested and evaluated for synergy and if the combination would allow for dosing at physiologically attainable concentrations. The combination of 2-deoxyglucose and metformin proved toxic to the osteosarcoma and Ewing sarcoma cell lines (p The cells were then grown in 3-dimensional cultures using Magnetic 3D Bioprinting kit(n3D Biosciences, Houston, TX) and exposed to physiologically attainable concentrations of 2-deoxyglucose (500uM) and metformin (5uM), based on Phase I studies of these individual agents in humans. The resulting 3-dimentional cell cultures were incubated for 10 days with 2-deoxyglucose and metformin and there was a significant reduction in cell proliferation as measured by trypan blue dye exclusion and reduced aldehyde dehydrogenase expression. Additionally, the functional assay of cancer stem cells, sphere-forming efficiency, was impeded in all cell lines treated with the combination of 2-deoxyglucose and metformin. Finally, the ability to establish orthotopic tumors in immune deficient mice was significantly reduced for intratibialy-injected cells pretreated with 2-deoxyglucose and metformin. In vivo testing of the combination of 2-deoxyglucose and metformin to treat established xenograft osteosarcoma and Ewing sarcomas is currently underway, and resulting tumors will be harvested and assessed for aldehyde dehydrogenase expression, sphere-forming efficiency, and further characterization of the stem cell properties and metabolic alterations. Our study will provide the preclinical basis for testing metabolic inhibitors, in particular the combination of 2-deoxyglucose and metformin, in clinical trials for pediatric bone sarcomas. Citation Format: Matteo Trucco, Nino Rainsusso, Piti Techavichit, Ronald Bernardi, Ryan Shuck, Laura Satterfield, Wendy Allen-Rhoades, Larry Donehower, David Loeb, Jason Yustein. Targeting pediatric bone sarcoma stem cell with metabolic inhibitors. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A70.

Abstract B24: Patient-derived tumor xenograft to study cancer stem cells is pediatric sarcomas

Tumor heterogeneity is recognized as an important hallmark in cancer. This characteristic allows cancer cells to evolve in time, to survive radiation and cyotoxic therapies, and to thrive in different organ microenvironments. Therefore, the presence of distinct tumor subpopulations, as mainstay feature of tumor heterogeneity, may play a role in tumor progression and treatment resistance. Patient-derived xenografts (PDXs) have been used to discover the molecular signaling pathways involved in tumorigenesis, metastasis formation and radiation/chemotherapy resistance in different epithelial-adult cancers. Our research interest in pediatric sarcomas is to understand the processes that drive tumor heterogeneity, to isolate and characterize cancer stem cells (CSCs), and to elucidate the mechanisms of chemoresistance using a PDX model. Pediatric patients with suspected or established diagnosis of sarcoma evaluated at Texas Children9s Hospital signed an informed consent to provide tumor samples for subcutaneous implantation of tumor explants in the flank of nonobese diabetic severe combined immunodeficient gamma (NSG) mice. Tumor explants were implanted on the same day of collection when was possible. First and subsequent PDX generations were harvested for tumor propagation, tumor histology, tumor cell karyotyping , RNA/DNA extraction for genetic studies and fluorescent activated cell sorting (FACS) analysis for ALDH, CD133, EphA2 and CD47. Forty-six tumor samples have been collected since June 2013. Half of tumor samples (n=21) corresponded to osteosarcoma patients and 59% of all tumor specimens were obtained at the time of the initial biopsy. Approximately 66% of biopsies have been performed by Interventional Radiology in our institution. All PDXs maintained the same histological characteristics that original tumors, and also presented specific genetic translocations associated with the different sarcoma types. Chemotherapy resistant and very aggressive tumors (metastatic) had short tumor latency and generate tumors in all transplanted mice. We did not observe the formation of metastases (lungs or liver) in any of the transplanted PDX mice. PDXs significantly increased the amount of tumor material necessary to perform cell culture under stem-like cell conditions and to analyze the expression of CSC markers by FACS. These studies would be very difficult to perform directly from patient tumor biopsies. All tumors harvested (n=15) contained 2-9% of ALDH Hi or stem-like cells. None of the PDXs analyzed expressed CD133 but synovial sarcoma. We found that >90% of sarcoma cells expressed CD47 and EphA2 at different intensity levels. Whole genome sequencing of different tumor cell subpopulations and limiting dilution experiments to validate the presence of CSCs are ongoing. Core needle biopsies would represent the most common source to obtain tumor specimens before treatment in pediatric sarcomas. The development of PDXs allows the propagation of tumor cell subpopulations, including CSCs, without the biological changes impose by cell culture. PDXs resemble the original tumor and constitute a powerful tool to study tumor heterogeneity. We plan to perform orthotopic transplantation and tail vein injection of PDX cells in an attempt to develop a PDX model of metastatic disease in pediatric sarcomas. Citation Format: Nino Rainusso, Jose Hernandez, Rex Marco, Sanjeev Vasudevan, Norma Quintanilla, John Hicks, Wendy Allen-Rhoades, Matteo Trucco, Jason Yustein. Patient-derived tumor xenograft to study cancer stem cells is pediatric sarcomas. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B24.