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Novel Retinoid UAB30 Decreases Tumorigenicity and Cancer Stem Cell Maintenance in Human Neuroblastoma Patient-Derived Xenografts
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Scientific Poster Presentations: 2016 Clinical Congress
Short Bowel Syndrome Has Widespread Effects Beyond Altering Nutrient Absorption: RNA Sequencing a Zebrafish Short Bowel Syndrome Model Kathy Schall, MD, Matthew E Thornton, Kathleen A Holoyda, MD, Ching-Ling Lien, PhD, Tracy C Grikscheit, MD, FACS Children’s Hospital of Los Angeles, Los Angeles, CA INTRODUCTION: Most of the morbidities associated with short bowel syndrome (SBS) are attributed to effects of decreased enteral nutrition and administration of total parenteral nutrition (TPN). We hypothesized that acute SBS alone has significant systemic effects, and tested this in a zebrafish SBS model. METHODS: With IACUC approval, adult male wild-type zebrafish underwent SBS (laparotomy, proximal stoma, distal ligation, n¼3) or sham (laparotomy alone, n¼3) surgery. After 2 weeks, the proximal intestine was harvested, RNA isolated, and external RNA controls consortium (ERCC) controls spiked into each sample, sequenced, and aligned to reference genome with gene ontology (GO) enrichment analysis performed. CyclinD1, CyclinB1, SAA1, IFN-gamma, and CYP7A1 gene expression were confirmed by qPCR. RESULTS: RNA-seq analysis identified 1,346 up-regulated genes and 678 down-regulated genes in SBS zebrafish compared to sham. The up-regulated genes were involved in acute phase response signaling, complement system, coagulation, cell proliferation, cellular barrier, production of nitric oxide and reactive oxygen species, and bile acid biosynthesis. The down-regulated genes were involved in folate synthesis, gluconeogenesis, glycogenolysis, fattyacid oxidation and activation, and drug and steroid metabolism. CyclinD1 gene expression was 2-fold higher, CyclinB1 2.8-fold higher, SAA1 4.5-fold higher, IFN-gamma 2.1-fold higher, and CYP7A1 25-fold higher in SBS than sham by qPCR. CONCLUSIONS: The gene expression of SBS demonstrates complex and extensive alteration of multiple pathways, some previously implicated as effects of TPN. The systemic complications of SBS alone are significant and extend beyond the complications of therapy. Surgical Management of Benign Ovarian Neoplasms in Pediatric and Adolescent Females Dani O Gonzalez, MD, Jennifer N Cooper, PhD, Jennifer H Aldrink, MD, FACS, Geri D Hewitt, MD, Peter C Minneci, MD, FACS, Katherine J Deans, MD, FACS Nationwide Children’s Hospital, Columbus, OH, Icahn School of Medicine at Mount Sinai, New York, NY INTRODUCTION: Most ovarian neoplasms in pediatric and adolescent females are benign. Consensus is lacking amongst pediatric surgeons as to whether ovary-sparing surgical techniques are the gold standard of management. We aimed to determine the proportion of pediatric and adolescent patients with benign
J Am Coll Surg
ovarian neoplasms who are treated with ovary-sparing procedures across tertiary children’s hospitals, and to assess whether variability in management exists across hospitals and physician specialties. METHODS: Using the Pediatric Health Information System (PHIS), we studied patients aged 6-21 years who were treated in 2006-2014 for a benign ovarian neoplasm with either oophorectomy or ovary-sparing surgery. Inter-hospital variability and predictors of the type of surgery were evaluated using logistic mixed effects models with random hospital effects. RESULTS: A total of 1,164 patients with benign ovarian neoplasms were identified; 646 underwent oophorectomy and 518 underwent ovary-sparing surgery. Patients managed by pediatric surgeons (vs pediatric gynecologists) were less likely to undergo ovary-sparing procedures (OR: 0.27, 95% CI: 0.17-0.43, p<0.001), as were younger patients (OR: 0.94 per year of age, 95% CI: 0.90-0.98, p¼0.007) and those admitted through the emergency department (OR: 0.76, 95% CI: 0.58-0.99, p¼0.04). Across tertiary children’s hospitals, there was significant variability in the proportion of ovary-sparing procedures in these patients (range: 18% to 81%). This variability remained significant after adjusting for patient demographics, physician specialty, year of treatment, and several relevant hospital-level characteristics (p<0.001). CONCLUSIONS: Significant variability exists in the management of benign ovarian neoplasms across hospitals and specialties. Collaborative efforts between pediatric surgeons and gynecologists may improve implementation of evidence-based guidelines for ovary-sparing surgery. Novel Retinoid UAB30 Decreases Tumorigenicity and Cancer Stem Cell Maintenance in Human Neuroblastoma Patient-Derived Xenografts Laura L Stafman, MD, Anita Hjelmeland, PhD, Smitha Mruthyunjayappa, Evan F Garner, MD, Venkatram R Atigadda, PhD, Jerry Stewart, Clinton J Grubbs, PhD, Donald Muccio, PhD, Elizabeth A Beierle, MD, FACS University of Alabama Birmingham, Birmingham, AL INTRODUCTION: Thirteen-cis-retinoic acid (RA) is a differentiating agent and maintenance therapy for neuroblastoma, but use is limited by toxicities. A novel synthetic analogue of 9-cis-RA, UAB30, decreased tumorigenicity in immortalized neuroblastoma cells lines and has had no significant toxicities in adult trials. Tumor initiating cells are a subpopulation of cancer cells with stem cell properties that propagate tumors in immunocompromised mice, and can be characterized by cell surface markers and the ability to grow as neurospheres. We hypothesized that UAB30 would decrease tumorigenicity and cancer stem cell maintenance similar to RA in human neuroblastoma patient-derived xenografts (PDXs). METHODS: Cells from 2 human neuroblastoma PDXs were treated with either UAB30 or RA (50 uM for 72 hours). Viability and proliferation were assessed with alamarBlue and CellTiter 96
Vol. 223, No. 4S2, October 2016
Scientific Poster Presentations: 2016 Clinical Congress
assays, respectively. Migration was quantified using laminin-coated Transwell inserts. Expression of the stem cell marker CD133 was determined by flow cytometry. Sphere formation was assessed with in vitro limiting dilution analysis. Student’s t-test and chisquare statistics were used, with p < 0.05 significant. RESULTS: Treatment with either UAB30 or RA significantly decreased viability, proliferation, and migration in both neuroblastoma PDXs. UAB30 or RA treatment led to significantly decreased CD133 expression (18.5% vs. 7.98%) and sphere formation (Table). These effects were concentration-dependent. Table. PDX
Treatments compared
COA3
Control vs RA Control vs UAB30 UAB30 vs RA Control vs RA Control vs UAB30 UAB30 vs RA
COA6
c2 25.3 31.0 0.5 40.4 67 3.17
p Value
< 0.0001 < 0.0001 0.479 < 0.0001 < 0.0001 0.075
CONCLUSIONS: UAB30 decreased tumorigenicity and cancer stem cell maintenance in human neuroblastoma PDXs similar to RA. These data, along with the information that UAB30 has an improved side effect profile compared to RA, indicate that UAB30 warrants further exploration as a therapy for neuroblastoma. Vascular Endothelial Growth Factor Accelerates Compensatory Lung Growth by Increasing Alveolar Units Duy Dao, Prathima Nandivada, Amy Pan, Lorenzo Anez-Bustillos, MD, Meredith A Baker, MD, Gillian L Fell, MD, PhD, Thomas Martin, MD, Sara Vargas, MD, Mark Puder, MD, FACS Boston Children’s Hospital, Harvard Medical School, Boston, MA
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INTRODUCTION: Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis. We have shown that exogenous VEGF administration accelerates compensatory lung growth in mice after unilateral pneumonectomy. How VEGF alters pulmonary mechanics or architecture of regenerating lungs remains unknown. METHODS: Eight to 10-week-old male mice underwent left pneumonectomy, followed by daily intraperitoneal injection of saline or VEGF at 0.5 mg/kg/dose. Pulmonary mechanical properties were measured on postoperative day (POD) 4 or 10, immediately before euthanasia. Lung volume was determined by volume displacement method and morphometric studies were performed with pointcounting technique. Volume and surface area measurements were normalized against body weight. RESULTS: Lung volume to body weight ratio on POD 4 is significantly higher in the VEGF-treated mice compared to the saline group (0.0590.005 vs 0.0520.008 mL/g, p¼0.02). Area under the pressure-volume loop, which represents the number of alveolar units, was significantly increased by VEGF treatment on POD 4 (0.1240.028 vs 0.0970.033 cmH2O/mL/g, p¼0.04). Other pulmonary mechanical properties were similar between the 2 groups. On morphometric analyses, VEGF significantly increased parenchymal volume (0.0470.003 vs 0.0380.005 mL/g, p<0.01), alveolar volume (0.0300.001 vs 0.0230.003 mL/g, p<0.01), and septal surface area (19.01.4 vs 15.41.8 cm2/g, p<0.01) on POD 4. The alveolar to parenchymal volume ratio was similar in both groups, indicating preserved pulmonary architecture with VEGF treatment. CONCLUSIONS: VEGF accelerates compensatory lung growth by increasing alveolar units without altering pulmonary mechanics or architecture. These findings highlight the potential of VEGF as a novel therapy in the treatment of severe pulmonary hypoplasia associated with congenital diaphragmatic hernia.
Scientific Poster Presentations: 2016 Clinical Congress
Short Bowel Syndrome Has Widespread Effects Beyond Altering Nutrient Absorption: RNA Sequencing a Zebrafish Short Bowel Syndrome Model Kathy Schall, MD, Matthew E Thornton, Kathleen A Holoyda, MD, Ching-Ling Lien, PhD, Tracy C Grikscheit, MD, FACS Children’s Hospital of Los Angeles, Los Angeles, CA INTRODUCTION: Most of the morbidities associated with short bowel syndrome (SBS) are attributed to effects of decreased enteral nutrition and administration of total parenteral nutrition (TPN). We hypothesized that acute SBS alone has significant systemic effects, and tested this in a zebrafish SBS model. METHODS: With IACUC approval, adult male wild-type zebrafish underwent SBS (laparotomy, proximal stoma, distal ligation, n¼3) or sham (laparotomy alone, n¼3) surgery. After 2 weeks, the proximal intestine was harvested, RNA isolated, and external RNA controls consortium (ERCC) controls spiked into each sample, sequenced, and aligned to reference genome with gene ontology (GO) enrichment analysis performed. CyclinD1, CyclinB1, SAA1, IFN-gamma, and CYP7A1 gene expression were confirmed by qPCR. RESULTS: RNA-seq analysis identified 1,346 up-regulated genes and 678 down-regulated genes in SBS zebrafish compared to sham. The up-regulated genes were involved in acute phase response signaling, complement system, coagulation, cell proliferation, cellular barrier, production of nitric oxide and reactive oxygen species, and bile acid biosynthesis. The down-regulated genes were involved in folate synthesis, gluconeogenesis, glycogenolysis, fattyacid oxidation and activation, and drug and steroid metabolism. CyclinD1 gene expression was 2-fold higher, CyclinB1 2.8-fold higher, SAA1 4.5-fold higher, IFN-gamma 2.1-fold higher, and CYP7A1 25-fold higher in SBS than sham by qPCR. CONCLUSIONS: The gene expression of SBS demonstrates complex and extensive alteration of multiple pathways, some previously implicated as effects of TPN. The systemic complications of SBS alone are significant and extend beyond the complications of therapy. Surgical Management of Benign Ovarian Neoplasms in Pediatric and Adolescent Females Dani O Gonzalez, MD, Jennifer N Cooper, PhD, Jennifer H Aldrink, MD, FACS, Geri D Hewitt, MD, Peter C Minneci, MD, FACS, Katherine J Deans, MD, FACS Nationwide Children’s Hospital, Columbus, OH, Icahn School of Medicine at Mount Sinai, New York, NY INTRODUCTION: Most ovarian neoplasms in pediatric and adolescent females are benign. Consensus is lacking amongst pediatric surgeons as to whether ovary-sparing surgical techniques are the gold standard of management. We aimed to determine the proportion of pediatric and adolescent patients with benign
J Am Coll Surg
ovarian neoplasms who are treated with ovary-sparing procedures across tertiary children’s hospitals, and to assess whether variability in management exists across hospitals and physician specialties. METHODS: Using the Pediatric Health Information System (PHIS), we studied patients aged 6-21 years who were treated in 2006-2014 for a benign ovarian neoplasm with either oophorectomy or ovary-sparing surgery. Inter-hospital variability and predictors of the type of surgery were evaluated using logistic mixed effects models with random hospital effects. RESULTS: A total of 1,164 patients with benign ovarian neoplasms were identified; 646 underwent oophorectomy and 518 underwent ovary-sparing surgery. Patients managed by pediatric surgeons (vs pediatric gynecologists) were less likely to undergo ovary-sparing procedures (OR: 0.27, 95% CI: 0.17-0.43, p<0.001), as were younger patients (OR: 0.94 per year of age, 95% CI: 0.90-0.98, p¼0.007) and those admitted through the emergency department (OR: 0.76, 95% CI: 0.58-0.99, p¼0.04). Across tertiary children’s hospitals, there was significant variability in the proportion of ovary-sparing procedures in these patients (range: 18% to 81%). This variability remained significant after adjusting for patient demographics, physician specialty, year of treatment, and several relevant hospital-level characteristics (p<0.001). CONCLUSIONS: Significant variability exists in the management of benign ovarian neoplasms across hospitals and specialties. Collaborative efforts between pediatric surgeons and gynecologists may improve implementation of evidence-based guidelines for ovary-sparing surgery. Novel Retinoid UAB30 Decreases Tumorigenicity and Cancer Stem Cell Maintenance in Human Neuroblastoma Patient-Derived Xenografts Laura L Stafman, MD, Anita Hjelmeland, PhD, Smitha Mruthyunjayappa, Evan F Garner, MD, Venkatram R Atigadda, PhD, Jerry Stewart, Clinton J Grubbs, PhD, Donald Muccio, PhD, Elizabeth A Beierle, MD, FACS University of Alabama Birmingham, Birmingham, AL INTRODUCTION: Thirteen-cis-retinoic acid (RA) is a differentiating agent and maintenance therapy for neuroblastoma, but use is limited by toxicities. A novel synthetic analogue of 9-cis-RA, UAB30, decreased tumorigenicity in immortalized neuroblastoma cells lines and has had no significant toxicities in adult trials. Tumor initiating cells are a subpopulation of cancer cells with stem cell properties that propagate tumors in immunocompromised mice, and can be characterized by cell surface markers and the ability to grow as neurospheres. We hypothesized that UAB30 would decrease tumorigenicity and cancer stem cell maintenance similar to RA in human neuroblastoma patient-derived xenografts (PDXs). METHODS: Cells from 2 human neuroblastoma PDXs were treated with either UAB30 or RA (50 uM for 72 hours). Viability and proliferation were assessed with alamarBlue and CellTiter 96
Vol. 223, No. 4S2, October 2016
Scientific Poster Presentations: 2016 Clinical Congress
assays, respectively. Migration was quantified using laminin-coated Transwell inserts. Expression of the stem cell marker CD133 was determined by flow cytometry. Sphere formation was assessed with in vitro limiting dilution analysis. Student’s t-test and chisquare statistics were used, with p < 0.05 significant. RESULTS: Treatment with either UAB30 or RA significantly decreased viability, proliferation, and migration in both neuroblastoma PDXs. UAB30 or RA treatment led to significantly decreased CD133 expression (18.5% vs. 7.98%) and sphere formation (Table). These effects were concentration-dependent. Table. PDX
Treatments compared
COA3
Control vs RA Control vs UAB30 UAB30 vs RA Control vs RA Control vs UAB30 UAB30 vs RA
COA6
c2 25.3 31.0 0.5 40.4 67 3.17
p Value
< 0.0001 < 0.0001 0.479 < 0.0001 < 0.0001 0.075
CONCLUSIONS: UAB30 decreased tumorigenicity and cancer stem cell maintenance in human neuroblastoma PDXs similar to RA. These data, along with the information that UAB30 has an improved side effect profile compared to RA, indicate that UAB30 warrants further exploration as a therapy for neuroblastoma. Vascular Endothelial Growth Factor Accelerates Compensatory Lung Growth by Increasing Alveolar Units Duy Dao, Prathima Nandivada, Amy Pan, Lorenzo Anez-Bustillos, MD, Meredith A Baker, MD, Gillian L Fell, MD, PhD, Thomas Martin, MD, Sara Vargas, MD, Mark Puder, MD, FACS Boston Children’s Hospital, Harvard Medical School, Boston, MA
e161
INTRODUCTION: Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis. We have shown that exogenous VEGF administration accelerates compensatory lung growth in mice after unilateral pneumonectomy. How VEGF alters pulmonary mechanics or architecture of regenerating lungs remains unknown. METHODS: Eight to 10-week-old male mice underwent left pneumonectomy, followed by daily intraperitoneal injection of saline or VEGF at 0.5 mg/kg/dose. Pulmonary mechanical properties were measured on postoperative day (POD) 4 or 10, immediately before euthanasia. Lung volume was determined by volume displacement method and morphometric studies were performed with pointcounting technique. Volume and surface area measurements were normalized against body weight. RESULTS: Lung volume to body weight ratio on POD 4 is significantly higher in the VEGF-treated mice compared to the saline group (0.0590.005 vs 0.0520.008 mL/g, p¼0.02). Area under the pressure-volume loop, which represents the number of alveolar units, was significantly increased by VEGF treatment on POD 4 (0.1240.028 vs 0.0970.033 cmH2O/mL/g, p¼0.04). Other pulmonary mechanical properties were similar between the 2 groups. On morphometric analyses, VEGF significantly increased parenchymal volume (0.0470.003 vs 0.0380.005 mL/g, p<0.01), alveolar volume (0.0300.001 vs 0.0230.003 mL/g, p<0.01), and septal surface area (19.01.4 vs 15.41.8 cm2/g, p<0.01) on POD 4. The alveolar to parenchymal volume ratio was similar in both groups, indicating preserved pulmonary architecture with VEGF treatment. CONCLUSIONS: VEGF accelerates compensatory lung growth by increasing alveolar units without altering pulmonary mechanics or architecture. These findings highlight the potential of VEGF as a novel therapy in the treatment of severe pulmonary hypoplasia associated with congenital diaphragmatic hernia.