- Email: [email protected]
Development and validation of an RNA-seq based prognostic signature in neuroblastoma
abstracts
Annals of Oncology
1924P
Exosomes in NSCLC as a source of biomarkers
nas2, C. Suarez3, M. Mosqueda4, A. Moreno-Manuel4, E. Dure´ndez1, S. Calabuig-Fari~ S. Torres Martinez4, A. Herreros Pomares1, S. Gallach1, E. Escorihuela1, E. de la Cueva5, A. Martinez-Romero6, E. Serna7, J.M. Paramio3, E. Jantus-Lewintre8, C.J. Camps Herrero9 1 Molecular Oncology Laboratory-CIBERONC, Fundaci on de Investigaci on Hospital General Universitario de Valencia, Valencia, Spain, 2Department of Pathology, Universitat de Vale`ncia, Fundaci on de Investigaci on Hospital General Universitario de Valencia, Molecular Oncology Laboratory-CIBERONC, Valencia, Spain, 3Centro de Investigaciones Energe´ticas, Medioambientales y Tecnol ogicas (CIEMAT), Molecular Oncology Unit, Instituto de Investigaci on Hospital 12 de Octubre (imas12), Madrid, 4 on de Investigaci on Hospital General Spain, Molecular Oncology Laboratory, Fundaci on Universitario de Valencia, Valencia, Spain, 5Core Facilities Unit, Centro de Investigaci 6 on Prıncipe Prıncipe Felipe, Valencia, Spain, Cytomics Laboratory, Centro de Investigaci 7 Felipe, Valencia, Spain, Physiology Department, Universitat de Valencia, Valencia, Spain, 8Department of Biotechnology, Universidad Polite´cnica de Valencia, Molecular Oncology Laboratory-CIBERONC, Fundaci on de Investigaci on Hospital General Universitario de Valencia, Valencia, Spain, 9Medicine Department, Universitat de Valencia, Medical Oncology, Hospital General de Valencia- CIBERONC, Valencia, Spain Background: Exosomes are small membranous vesicles (around 40-130 nm), that have been detected in different biological samples, that play a key role in NSCLC and being relevant in stem cell differentiation as well. The main objective of this study was to analyze the exosomes cargo from NSCLC cell cultures growth in monolayer (2D) and suspension conditions (3D, lung tumorespheres). Methods: Cultures were established from NSCLC resected patients and cell lines. Exosomes isolation was performed by ultracentrifugation. Characterization was carried out by NTA, electron microscopy, immunoblot and flow cytometry. Mutational status of EGFR and RAS genes was analyzed by BEAMing dPCR. Transcriptomic analysis has been carried out from exosomal RNA with microarrays, (p 0.01). Consequently, XAGE1B (significantly expressed gene in exosomes) was analyzed by RTqPCR in 189 paired fresh-frozen tumor and normal tissue samples of resected NSCLC. Prognostic value was assessed by Kaplan-Meier curves (log rank-test), considering significant p < 0.05. Results: Exosomal characterization through NTA and electron microscopy showed an exosomes size from 108-125 nm. Specific markers were detected by IB and FC. Mutational analysis of EGFR and RAS genes in exosomes shown the same pattern displayed by the origin cells. Transcriptomic analysis showed that the expression of mRNAs, miRNAs and precursors were significantly different between 3D and 2Dderived exosomes. A pathway enrichment was carried out to know in which processes (cancer-related) are involved. Significant differential expression was also found between ADC vs SCC–derived exosomes. Concretely, XAGE1B is overexpressed in ADC-derived exosomes (p ¼ 0.00003). This overexpression in ADC was validated in NSCLC cohort (p ¼ 0.002). Furthermore, it has revealed a significant association with patient prognosis for overall survival in the ADC group (n ¼ 74)(OS 49.8 vs. NR months, p ¼ 0.043). Conclusions: Differences in exosomal cargo have been observed between: i) 3D vs. 2D cultures and ii) ADC vs. SCC. In addition, the same mutational pattern was detected in exosomes as compared with parental cells. Therefore, exosomes can be a useful source of biomarkers in NSCLC analysis. Supported by grant GV/2018/026, PI18/00266, & AECC Valencia. Legal entity responsible for the study: Fundaci on de Investigaci on del Hospital General Universitario de Valencia.
Volume 30 | Supplement 5 | October 2019
Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.
1925P
The role of Pim-1 in the development and progression of papillary thyroid carcinoma
X. Zhu1, K. Yang1, Q. Wen2, F. Xiang3, J. Shuai3 Zhejiang Cancer Institution, Zhejiang Cancer Hospital, Hangzhou, China, 2Head and Neck Surgery, Zhejiang Cancer Hospital, Hangzhou, China, 3Stomatology College, Zhejiang Chinese Medical University, Hangzhou, China
1
Background: Pim-1 is an oncogene and has been proved to play pivotal role in proliferation, apoptosis and angiogenesis. Thyroid cancer represents the most common malignancy in the endocrine system and displays a marked increase in the incidence in recent years. Papillary thyroid cancer (PTC) is among the most frequent thyroid malignancies and accounts for more than 85%. Therefore, it is worthwhile to discuss the function of Pim-1 in the development and progression of PTC. Methods: Gene set enrichment analysis (GSEA) of Pim-1 in the PTC was performed on the TCGA databases. 177 PTC paraffin blocks were selected to make the tissue microarrays and the levels of Pim-1 protein was investigated by immunohistochemistry. Meanwhile, one of the Pim-1 kinase inhibitor, SGI-1776, was used to evaluate the function of Pim-1 on the PTC cell BCPAP and TPC-1. CCK-8 and colony formation assay was carried out to measure the cell proliferation. Apoptosis rate and migration capacity were determined by flow cytometry and wound-healing methods respectively. Results: GSEA results revealed that Pim-1 expression was significantly associated with the immune system of PTC. IHC result showed that Pim-1 was overexpressed in the PTC tissues compared with normal adjacent tissues. Meanwhile, Pim-1 had a significant relationship with the T-stage, lymph node involvement, capsule invasion and gender. Female patients and patients with higher T-stage, positive lymph node involvement, positive capsule invasion have a higher Pim-1 level. After SGI-1776 treatment, Pim-1 expressions were obviously downregulated in both BCPAP and TPC-1. Decreased Pim-1 led to the proliferation depression, apoptosis rate elevation and the migration capacity reduction in both cell lines. Conclusions: Taken together, our current data showed the important role of Pim-1 in the tumorigenesis of PTC. High Pim-1 level linked to immune status and aggressive malignant behavior of PTC. It was also suggested that PIM-1 kinase might be a novel molecular biomarker of PTC. Legal entity responsible for the study: The authors. Funding: Zhejiang Province Natural Science Foundation of China. Disclosure: All authors have declared no conflicts of interest.
1926P
Development and validation of an RNA-seq based prognostic signature in neuroblastoma
J-G. Zhou, H. Ma Department of Oncology, Zunyi Medical University Affiliated Hospital, Zunyi, China Background: Credible prognostic stratification remains a challenge for neuroblastoma (NBL) with variable clinical manifestations. RNA expression signatures might predict the outcomes; notwithstanding, independent cross-platform validation is still rare. Methods: RNA expression data were obtained from NBL patients and then analyzed. In TARGET-NBL data, an RNA-based prognostic signature was developed and validated. Survival prediction was assessed using a time-dependent receiver operating characteristic (ROC) curve. Functional enrichment analysis of the RNAs was conducted using bioinformatics methods. Results: A total of 1,119 differentially expressed RNAs and 149 prognosis-related RNAs were identified sequentially. Then, in the training cohort, 12 RNAs were identified as significantly associated with overall survival (OS) and were combined to develop a model that stratified NBL patients into low- and high-risk groups. Twelve RNA signature high-risk patients had poorer OS in the training cohort (n ¼ 105, Hazard Ratios (HR)¼ 0.10 (0.05-0.20), P < 0.001) and in the validation cohort (n ¼ 44, HR ¼ 0.25 (0.09-0.69), P ¼ 0.008). ROC curve analysis also showed that both the training and validation cohorts performed well in predicting OS (12-month AUC values of 0.852 and 0.438, 36-month AUC values of 0.824 and 0.737, and 60-month AUC values of 0.802 and 0.702, respectively). Moreover, these 12 RNAs may be involved in certain events that are known to be associated with NBL through functional enrichment analysis. Conclusions: This study identified and validated a novel 12-RNA prognostic signature to reliably distinguish NBL patients at low and high risk of death. Further larger, multicenter prospective studies are desired to validate this model. Legal entity responsible for the study: The authors. Funding: National Natural Science Foundation of China (Grant No. 81660512). Disclosure: All authors have declared no conflicts of interest.
doi:10.1093/annonc/mdz268 | v777
Downloaded from https://academic.oup.com/annonc/article-abstract/30/Supplement_5/mdz268.053/5576701 by guest on 24 October 2019
Methods: 78 patients with advanced NSCLC were enrolled in the study at HGUV and CHUS. EpCAM positive CTCs from these patients were isolated and analysed using both CellSearch technology and a qRT-PCR based approach at different times: at baseline and before the 2nd and 5th cycle treatment. A panel of genes with a relevant role for NSCLC aggressiveness and the resistance to platinum-based treatments were analysed. Results: From all patients included in the study 46% were positive for CTCs using CellSearch system at baseline, showing only 12% of patients 5 CTCs. Additionally, patients with 5 CTCs showed poor PFS (4.66 vs 7.12 months, p ¼ 0.040) and OS (3,9 vs 13.43 months, p < 0.001) rates compared with those patients with <5CTCs. In addition, patients with high CTCs levels during treatment also had a more aggressive disease evolution in terms of PFS and OS (p ¼ 0.007 and p ¼ 0.001, respectively). From the analyzed gene panel, patients with lower CHOKa expression at baseline had increased PFS (7.03 vs. 3.9 months, p ¼ 0.009) and OS (11.4 vs. 4.07 months, p ¼ 0.001). Furthermore, we found low CHOK a levels as a powerful marker to discriminate patients with a good response to chemotherapy from those that progressed during treatment administration (14.35 vs 5.07 months, respectively, p ¼ 0.011). Conclusions: We demonstrated that quantification and gene expression characterization of CTCs represents an adequate strategy to identify prognostic biomarkers in NSCLC patients Supported by RTC-2014-1532-1 from MINECO and CB16/12/00350CB16/12/00328 from CIBERONC. Legal entity responsible for the study: FIHGUV. Funding: Supported from RTC-2014-1532-1 (Spanish Ministry of Economy, Industry and Competitiveness) and CB16/12/00350 and CB16/12/00328 from CIBERONC. Disclosure: All authors have declared no conflicts of interest.
Annals of Oncology
1924P
Exosomes in NSCLC as a source of biomarkers
nas2, C. Suarez3, M. Mosqueda4, A. Moreno-Manuel4, E. Dure´ndez1, S. Calabuig-Fari~ S. Torres Martinez4, A. Herreros Pomares1, S. Gallach1, E. Escorihuela1, E. de la Cueva5, A. Martinez-Romero6, E. Serna7, J.M. Paramio3, E. Jantus-Lewintre8, C.J. Camps Herrero9 1 Molecular Oncology Laboratory-CIBERONC, Fundaci on de Investigaci on Hospital General Universitario de Valencia, Valencia, Spain, 2Department of Pathology, Universitat de Vale`ncia, Fundaci on de Investigaci on Hospital General Universitario de Valencia, Molecular Oncology Laboratory-CIBERONC, Valencia, Spain, 3Centro de Investigaciones Energe´ticas, Medioambientales y Tecnol ogicas (CIEMAT), Molecular Oncology Unit, Instituto de Investigaci on Hospital 12 de Octubre (imas12), Madrid, 4 on de Investigaci on Hospital General Spain, Molecular Oncology Laboratory, Fundaci on Universitario de Valencia, Valencia, Spain, 5Core Facilities Unit, Centro de Investigaci 6 on Prıncipe Prıncipe Felipe, Valencia, Spain, Cytomics Laboratory, Centro de Investigaci 7 Felipe, Valencia, Spain, Physiology Department, Universitat de Valencia, Valencia, Spain, 8Department of Biotechnology, Universidad Polite´cnica de Valencia, Molecular Oncology Laboratory-CIBERONC, Fundaci on de Investigaci on Hospital General Universitario de Valencia, Valencia, Spain, 9Medicine Department, Universitat de Valencia, Medical Oncology, Hospital General de Valencia- CIBERONC, Valencia, Spain Background: Exosomes are small membranous vesicles (around 40-130 nm), that have been detected in different biological samples, that play a key role in NSCLC and being relevant in stem cell differentiation as well. The main objective of this study was to analyze the exosomes cargo from NSCLC cell cultures growth in monolayer (2D) and suspension conditions (3D, lung tumorespheres). Methods: Cultures were established from NSCLC resected patients and cell lines. Exosomes isolation was performed by ultracentrifugation. Characterization was carried out by NTA, electron microscopy, immunoblot and flow cytometry. Mutational status of EGFR and RAS genes was analyzed by BEAMing dPCR. Transcriptomic analysis has been carried out from exosomal RNA with microarrays, (p 0.01). Consequently, XAGE1B (significantly expressed gene in exosomes) was analyzed by RTqPCR in 189 paired fresh-frozen tumor and normal tissue samples of resected NSCLC. Prognostic value was assessed by Kaplan-Meier curves (log rank-test), considering significant p < 0.05. Results: Exosomal characterization through NTA and electron microscopy showed an exosomes size from 108-125 nm. Specific markers were detected by IB and FC. Mutational analysis of EGFR and RAS genes in exosomes shown the same pattern displayed by the origin cells. Transcriptomic analysis showed that the expression of mRNAs, miRNAs and precursors were significantly different between 3D and 2Dderived exosomes. A pathway enrichment was carried out to know in which processes (cancer-related) are involved. Significant differential expression was also found between ADC vs SCC–derived exosomes. Concretely, XAGE1B is overexpressed in ADC-derived exosomes (p ¼ 0.00003). This overexpression in ADC was validated in NSCLC cohort (p ¼ 0.002). Furthermore, it has revealed a significant association with patient prognosis for overall survival in the ADC group (n ¼ 74)(OS 49.8 vs. NR months, p ¼ 0.043). Conclusions: Differences in exosomal cargo have been observed between: i) 3D vs. 2D cultures and ii) ADC vs. SCC. In addition, the same mutational pattern was detected in exosomes as compared with parental cells. Therefore, exosomes can be a useful source of biomarkers in NSCLC analysis. Supported by grant GV/2018/026, PI18/00266, & AECC Valencia. Legal entity responsible for the study: Fundaci on de Investigaci on del Hospital General Universitario de Valencia.
Volume 30 | Supplement 5 | October 2019
Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.
1925P
The role of Pim-1 in the development and progression of papillary thyroid carcinoma
X. Zhu1, K. Yang1, Q. Wen2, F. Xiang3, J. Shuai3 Zhejiang Cancer Institution, Zhejiang Cancer Hospital, Hangzhou, China, 2Head and Neck Surgery, Zhejiang Cancer Hospital, Hangzhou, China, 3Stomatology College, Zhejiang Chinese Medical University, Hangzhou, China
1
Background: Pim-1 is an oncogene and has been proved to play pivotal role in proliferation, apoptosis and angiogenesis. Thyroid cancer represents the most common malignancy in the endocrine system and displays a marked increase in the incidence in recent years. Papillary thyroid cancer (PTC) is among the most frequent thyroid malignancies and accounts for more than 85%. Therefore, it is worthwhile to discuss the function of Pim-1 in the development and progression of PTC. Methods: Gene set enrichment analysis (GSEA) of Pim-1 in the PTC was performed on the TCGA databases. 177 PTC paraffin blocks were selected to make the tissue microarrays and the levels of Pim-1 protein was investigated by immunohistochemistry. Meanwhile, one of the Pim-1 kinase inhibitor, SGI-1776, was used to evaluate the function of Pim-1 on the PTC cell BCPAP and TPC-1. CCK-8 and colony formation assay was carried out to measure the cell proliferation. Apoptosis rate and migration capacity were determined by flow cytometry and wound-healing methods respectively. Results: GSEA results revealed that Pim-1 expression was significantly associated with the immune system of PTC. IHC result showed that Pim-1 was overexpressed in the PTC tissues compared with normal adjacent tissues. Meanwhile, Pim-1 had a significant relationship with the T-stage, lymph node involvement, capsule invasion and gender. Female patients and patients with higher T-stage, positive lymph node involvement, positive capsule invasion have a higher Pim-1 level. After SGI-1776 treatment, Pim-1 expressions were obviously downregulated in both BCPAP and TPC-1. Decreased Pim-1 led to the proliferation depression, apoptosis rate elevation and the migration capacity reduction in both cell lines. Conclusions: Taken together, our current data showed the important role of Pim-1 in the tumorigenesis of PTC. High Pim-1 level linked to immune status and aggressive malignant behavior of PTC. It was also suggested that PIM-1 kinase might be a novel molecular biomarker of PTC. Legal entity responsible for the study: The authors. Funding: Zhejiang Province Natural Science Foundation of China. Disclosure: All authors have declared no conflicts of interest.
1926P
Development and validation of an RNA-seq based prognostic signature in neuroblastoma
J-G. Zhou, H. Ma Department of Oncology, Zunyi Medical University Affiliated Hospital, Zunyi, China Background: Credible prognostic stratification remains a challenge for neuroblastoma (NBL) with variable clinical manifestations. RNA expression signatures might predict the outcomes; notwithstanding, independent cross-platform validation is still rare. Methods: RNA expression data were obtained from NBL patients and then analyzed. In TARGET-NBL data, an RNA-based prognostic signature was developed and validated. Survival prediction was assessed using a time-dependent receiver operating characteristic (ROC) curve. Functional enrichment analysis of the RNAs was conducted using bioinformatics methods. Results: A total of 1,119 differentially expressed RNAs and 149 prognosis-related RNAs were identified sequentially. Then, in the training cohort, 12 RNAs were identified as significantly associated with overall survival (OS) and were combined to develop a model that stratified NBL patients into low- and high-risk groups. Twelve RNA signature high-risk patients had poorer OS in the training cohort (n ¼ 105, Hazard Ratios (HR)¼ 0.10 (0.05-0.20), P < 0.001) and in the validation cohort (n ¼ 44, HR ¼ 0.25 (0.09-0.69), P ¼ 0.008). ROC curve analysis also showed that both the training and validation cohorts performed well in predicting OS (12-month AUC values of 0.852 and 0.438, 36-month AUC values of 0.824 and 0.737, and 60-month AUC values of 0.802 and 0.702, respectively). Moreover, these 12 RNAs may be involved in certain events that are known to be associated with NBL through functional enrichment analysis. Conclusions: This study identified and validated a novel 12-RNA prognostic signature to reliably distinguish NBL patients at low and high risk of death. Further larger, multicenter prospective studies are desired to validate this model. Legal entity responsible for the study: The authors. Funding: National Natural Science Foundation of China (Grant No. 81660512). Disclosure: All authors have declared no conflicts of interest.
doi:10.1093/annonc/mdz268 | v777
Downloaded from https://academic.oup.com/annonc/article-abstract/30/Supplement_5/mdz268.053/5576701 by guest on 24 October 2019
Methods: 78 patients with advanced NSCLC were enrolled in the study at HGUV and CHUS. EpCAM positive CTCs from these patients were isolated and analysed using both CellSearch technology and a qRT-PCR based approach at different times: at baseline and before the 2nd and 5th cycle treatment. A panel of genes with a relevant role for NSCLC aggressiveness and the resistance to platinum-based treatments were analysed. Results: From all patients included in the study 46% were positive for CTCs using CellSearch system at baseline, showing only 12% of patients 5 CTCs. Additionally, patients with 5 CTCs showed poor PFS (4.66 vs 7.12 months, p ¼ 0.040) and OS (3,9 vs 13.43 months, p < 0.001) rates compared with those patients with <5CTCs. In addition, patients with high CTCs levels during treatment also had a more aggressive disease evolution in terms of PFS and OS (p ¼ 0.007 and p ¼ 0.001, respectively). From the analyzed gene panel, patients with lower CHOKa expression at baseline had increased PFS (7.03 vs. 3.9 months, p ¼ 0.009) and OS (11.4 vs. 4.07 months, p ¼ 0.001). Furthermore, we found low CHOK a levels as a powerful marker to discriminate patients with a good response to chemotherapy from those that progressed during treatment administration (14.35 vs 5.07 months, respectively, p ¼ 0.011). Conclusions: We demonstrated that quantification and gene expression characterization of CTCs represents an adequate strategy to identify prognostic biomarkers in NSCLC patients Supported by RTC-2014-1532-1 from MINECO and CB16/12/00350CB16/12/00328 from CIBERONC. Legal entity responsible for the study: FIHGUV. Funding: Supported from RTC-2014-1532-1 (Spanish Ministry of Economy, Industry and Competitiveness) and CB16/12/00350 and CB16/12/00328 from CIBERONC. Disclosure: All authors have declared no conflicts of interest.