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Characterization of lung tumourspheres reveals cancer stem-like cells potential targets and prognostic markers in non-small cell lung cancer
abstracts
Annals of Oncology
1443P
Characterization of lung tumourspheres reveals cancer stem-like cells potential targets and prognostic markers in non-small cell lung cancer
Background: Non-small cell lung cancer (NSCLC) is the first cause of death cancerrelated worldwide mainly due to high therapeutic resistance. This resistance is related to cancer stem-like cells (CSCs), for which the identification of targets and markers is still ongoing. Methods: Primary cultures from 8 NSCLC patients were established as tumorspheres and as monolayers. CSCs properties were tested for both conditions in vitro and in vivo. The expression of 50 CSCs-related genes was assessed by RTqPCR and proteins of significantly overexpressed genes were examined by immunoblot and immunofluorescence. The prognostic role of these genes was analyzed in a cohort of 661 NSCLC patients from TCGA and validated in an independent cohort of 114 lung adenocarcinoma (ADC) patients. Results: Tumorspheres exhibited self-renewal, unlimited exponential growth, drug resistance, great invasion and differentiation capacities in vitro and higher tumorigenic potential than monolayers in vivo. 17 genes were significantly overexpressed in tumorspheres, being NANOG, NOTCH3, CD44, CDKN1A, SNAI1, and ITGA6 the major contributors to distinguish them from adherent cells. Proteins encoded by these genes showed differential localization and expression patterns in ADC tumorspheres. The expression of CDKN1A, SNAI1 and ITGA6 was associated to prognosis, so a score was built based on their regression coefficients from a multivariate model. TCGA patients with high CSCs score show shorter OS in the entire cohort [37.7 vs. 60.4 mo., p ¼ 0.001] and the ADC subcohort [36.6 vs. 53.5 mo., p ¼ 0.003]. Multivariate analysis indicated that this score is an independent biomarker of prognosis for OS in the entire cohort from TCGA [HR: 1.498; 95% CI, 1.167-1.922; p ¼ 0.001] and the ADC subcohort [HR: 1.869; 95% CI, 1.275-2.738; p ¼ 0.001]. The prognostic value is confirmed in an independent cohort of 114 lung adenocarcinoma patients OS (42.90 vs. NR mo, p ¼ 0.020). Conclusions: Lung tumorspheres are a useful method for CSCs enrichment. Elevated expression of CDKN1A, SNAI1 and ITGA6 genes is associated with worse prognosis in NSCLC. Funded by CB16/12/00350 from CIBEROnc, PI12-02838, and PI15-00753 from ISCIII, Fundacion Arnal Planelles and Domingo Martınez. Legal entity responsible for the study: Fundaci on de Investigaci on Hospital General de Valencia. Funding: CIBEROnc, Instituto de Salud Carlos III; Fundacion Arnal Planelles and Domingo Martınez. Disclosure: All authors have declared no conflicts of interest.
1444P
Pulmonary tumour-draining vein exosomal lincRNA-p21 levels impacts non-small cell lung cancer prognosis
nolas4, J. Moises3, J.J. Castellano1, A. Navarro1, L. Molins2, J. Canals1, R. Marrades3, N. Vi~ noz1, M. Casadevall1, Y. Li1, B. Han1, D. Martinez4, J. Martin1, A. Garisoain1, C. Mu~ J. Ramirez5, M. Monzo1 1 Human Anatomy, Univeristy of Barcelona, School of Medicine, Barcelona, Spain, 2 Thoracic Surgery, Hospital Clinic of Barcelona, Barcelona, Spain, 3Pneumology, Hospital Clinic of Barcelona, Barcelona, Spain, 4Dept. Medical Oncology, Hospital Clinic y Provincial de Barcelona, Barcelona, Spain, 5Pathology, Hospital Clinic y Provincial de Barcelona, Barcelona, Spain Background: In resected non-small cell lung cancer (NSCLC) considerable recurrence rates has been reported (30%). Tumor secretes small extracellular vesicles known as exosomes, which plays a role in the metastasis process. Pulmonary tumor-draining vein blood has proved to be better source than peripheral vein to analyze the prognosis
Volume 30 | Supplement 5 | October 2019
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Immunological impact of surgery in NSCLC patients
A. Yanagihara1, H. Kagamu2, H. Sakaguchi1, H. Ishida1, H. Nitanda1, R. Taguchi1, R. Yoshimura1, O. Yamaguchi2, K. Hashimoto2, K. Kodaira2 1 Department of General Thoracic Surgery, Saitama Medical University International Medical Center, Hidaka, Japan, 2Department of Respiratory Medicine, Saitama Medical University International Medical Center, Hidaka, Japan Background: Immune checkpoint inhibitors (ICI) improve survival in patients with advanced non-small cell lung cancer (NSCLC) and have become one of standard therapy. It was demonstrated that nivolumab therapy for resectable NSCLC patients prior to surgery resulted in major pathological responses in 40% of the patients. On the other hand, adjuvant nivolumab therapy for melanoma resulted in longer recurrence-free survivals. Thus, it is still uncertain which host immunological status is appropriate for ICI therapy, before or after surgery. In this study, we examined the peripheral blood, and tumor tissues to elucidate immunological impact of surgery in NSCLC patients. Methods: The present study comprised of 20 surgically resectable early (stage I, II, or IIIA) NSCLC patients in Saitama Medical University International Medical Center. Peripheral blood samples were collected before and after surgical resection of NSCLC. PBMCs were analyzed with LSR FortessaTM. Results: Interestingly, more effector memory (EM) CCR7-CD45RA- CD8þ T cells and CD62Llow CD4þ T cells were observed in early stage NSCLC patients, compared with stage IV NSCLC patients. Significant decrease of the percentages of EM CD8þ T cells (P ¼ 0.0045) and CD62Llow CD4þ T cells (P < 0.0001) were detected after surgical removal of cancer. In contrast, central memory (CM) CCR7þCD45RA- CD8þ T cells significantly increased (P ¼ 0.0037). It is likely that the decrease of EM CD8þ T cells and CD62Llow CD4þ T cells reflected conversion of EM T cells to CM T cells due to disappearance of cancer antigens, because the decrease was not observed in the patients of incomplete resection. PD-1 expression on both CD8þ and CD4þ T cells was up-regulated after surgery. The percentages of myeloid derived suppressor cells (MDSC) (P < 0.0001) increased and natural killer cells (NK) decreased (P ¼ 0.0001). Conclusions: After surgical removal of lung cancer, EM type T cells were converted to CM type T cells. It seems that surgery may have negative influence for innate immunity. Because PD-1 blockade therapy only activates primed effector T cells, the immunological status before surgery seems more appropriate for ICI therapy. Legal entity responsible for the study: Saitama Medical University International Medical Center. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.
1447P
The prognostic value of selected immunological panel in predicting the prognosis of early-stage resectable non-small cell lung cancer
S. Zhao1, Y. He1, X. Zhang1, C. Zhou2 Oncology, Shanghai Pulmonary Hospital, Tongji University, Shanghai, China, 2 Shanghai Pulmonary Hospital, Tongji University, Shanghai, China
1
Background: Lung cancer is one of the most fatal cancer types globally. Currently, the immune status in the tumor microenvironment (TME) is highly valued. The immune
doi:10.1093/annonc/mdz258 | v587
Downloaded from https://academic.oup.com/annonc/article-abstract/30/Supplement_5/mdz258.004/5577078 by guest on 09 October 2019
nas3, J.D. de-Maya-Girones4, A. Herreros Pomares1, E. Jantus-Lewintre2, S. Calabuig-Fari~ R. Lucas5, A. Blasco6, R. Guijarro7, M. Martorell8, E. Escorihuela1, S. Alonso9, M.D. Chiara10, E. Dure´ndez1, C. Gandia4, R. Sirera11, R. Farras4, C. Camps12 1 Molecular Oncology Laboratory-CIBERONC, Fundaci on de Investigaci on Hospital General Universitario de Valencia, Valencia, Spain, 2Department of Biotechnology, Universidad Polite´cnica de Valencia, Molecular Oncology Laboratory-CIBERONC, Fundacion de Investigacion Hospital General Universitario de Valencia, Valencia, Spain, 3 Department of Pathology, Universitat de Vale`ncia, Molecular Oncology LaboratoryCIBERONC, Fundacion de Investigaci on Hospital General Universitario de Valencia, on Prıncipe Valencia, Spain, 4Oncogenic Signalling Laboratory, Centro de Investigaci 5 Felipe, Valencia, Spain, Department of History of Science and Documentation, 6 Universitat de Vale`ncia, Valencia, Spain, Department of Medical Oncology-CIBERONC, Hospital General Universitario de Valencia, Valencia, Spain, 7Department of Thoracic Surgery-CIBERONC, Hospital General Universitario de Valencia, Valencia, Spain, 8 Department of Pathology, Hospital General Universitario de Valencia, Valencia, Spain, 9 Program of Predictive and Personalized Medicine of Cancer, Institut de Reserca Germans Trias i Pujol (PMPPC-IGTP), Badalona, Spain, 10Institute of Sanitary Research of Asturias-CIBERONC, Hospital Central de Asturias, Oviedo, Spain, 11Department of Biotechnology-CIBERONC, Universidad Polite´cnica de Valencia, Valencia, Spain, 12 Department of Medical Oncology, Hospital General Universitario de Valencia; Department of Medicine, Hospital General Universitario de Valencia, Molecular Oncology Laboratory-CIBERONC, Fundaci on de Investigaci on Hospital General Universitario de Valencia, Valencia, Spain
impact of exosomes (Navarro A, et al. Cancers 2019). LincRNA-p21 is a long non-coding RNA which upregulation in tumor tissue induced by hypoxia was related to poor prognosis in NSCLC (Castellano JJ. et al. JTO 2016). We aimed to evaluate the presence of lincRNA-p21 in exosomes and its potential as prognosis biomarker in resected NSCLC patients. Methods: H23 and H1299 NSCLC cell lines were cultured in exosome-free medium 48h under normoxic/hypoxic conditions. LincRNA-p21 was inhibited using a siRNA and quantified using a TaqMan assay. Exosomes were purified by ultracentrifugation and characterized by electron microscopy, western blot and Nanosight. LincRNA-p21 was analyzed in exosomes from pulmonary tumor-draining vein, obtained during surgery, from 54 NSCLC patients. Results: LincRNA-p21 was present in exosomes showing 18 times more representation than in their origin cells. Under hypoxic conditions exosomal lincRNA-p21 was upregulated in both cell lines (p < 0.05). Cellular lincRNA-p21 silencing resulted in a reduction in exosomal lincRNA-p21 (p ¼ 0.04) indicating a relation between cellular and exosomal levels. The analysis in patient samples showed that lincRNA-p21 was present in patient exosomes obtained from tumor-draining vein. The survival analysis showed that high exosomal lincRNA-p21 levels were associated with shorter time to relapse (TTR) (37 vs 56 months; p ¼ 0.03) and shorter overall survival (OS) (46 vs 66 months; p ¼ 0.04). In the multivariate analysis exosomal lincRNA-p21 emerged as an independent prognostic marker for TTR (HR: 3.1; 95%CI: 1.02-9.4; p ¼ 0.04) and OS (HR: 5.3; 95%CI: 1.6-17.2; p ¼ 0.006) together with disease stage. Conclusions: Tumor exosomes carry LincRNA-p21, which increases under hypoxia, and its analysis in tumor-draining vein arises as a promising prognosis marker. Legal entity responsible for the study: The authors. Funding: SAF2017-88606-P (AEI/FEDER, UE). Disclosure: All authors have declared no conflicts of interest.
Annals of Oncology
1443P
Characterization of lung tumourspheres reveals cancer stem-like cells potential targets and prognostic markers in non-small cell lung cancer
Background: Non-small cell lung cancer (NSCLC) is the first cause of death cancerrelated worldwide mainly due to high therapeutic resistance. This resistance is related to cancer stem-like cells (CSCs), for which the identification of targets and markers is still ongoing. Methods: Primary cultures from 8 NSCLC patients were established as tumorspheres and as monolayers. CSCs properties were tested for both conditions in vitro and in vivo. The expression of 50 CSCs-related genes was assessed by RTqPCR and proteins of significantly overexpressed genes were examined by immunoblot and immunofluorescence. The prognostic role of these genes was analyzed in a cohort of 661 NSCLC patients from TCGA and validated in an independent cohort of 114 lung adenocarcinoma (ADC) patients. Results: Tumorspheres exhibited self-renewal, unlimited exponential growth, drug resistance, great invasion and differentiation capacities in vitro and higher tumorigenic potential than monolayers in vivo. 17 genes were significantly overexpressed in tumorspheres, being NANOG, NOTCH3, CD44, CDKN1A, SNAI1, and ITGA6 the major contributors to distinguish them from adherent cells. Proteins encoded by these genes showed differential localization and expression patterns in ADC tumorspheres. The expression of CDKN1A, SNAI1 and ITGA6 was associated to prognosis, so a score was built based on their regression coefficients from a multivariate model. TCGA patients with high CSCs score show shorter OS in the entire cohort [37.7 vs. 60.4 mo., p ¼ 0.001] and the ADC subcohort [36.6 vs. 53.5 mo., p ¼ 0.003]. Multivariate analysis indicated that this score is an independent biomarker of prognosis for OS in the entire cohort from TCGA [HR: 1.498; 95% CI, 1.167-1.922; p ¼ 0.001] and the ADC subcohort [HR: 1.869; 95% CI, 1.275-2.738; p ¼ 0.001]. The prognostic value is confirmed in an independent cohort of 114 lung adenocarcinoma patients OS (42.90 vs. NR mo, p ¼ 0.020). Conclusions: Lung tumorspheres are a useful method for CSCs enrichment. Elevated expression of CDKN1A, SNAI1 and ITGA6 genes is associated with worse prognosis in NSCLC. Funded by CB16/12/00350 from CIBEROnc, PI12-02838, and PI15-00753 from ISCIII, Fundacion Arnal Planelles and Domingo Martınez. Legal entity responsible for the study: Fundaci on de Investigaci on Hospital General de Valencia. Funding: CIBEROnc, Instituto de Salud Carlos III; Fundacion Arnal Planelles and Domingo Martınez. Disclosure: All authors have declared no conflicts of interest.
1444P
Pulmonary tumour-draining vein exosomal lincRNA-p21 levels impacts non-small cell lung cancer prognosis
nolas4, J. Moises3, J.J. Castellano1, A. Navarro1, L. Molins2, J. Canals1, R. Marrades3, N. Vi~ noz1, M. Casadevall1, Y. Li1, B. Han1, D. Martinez4, J. Martin1, A. Garisoain1, C. Mu~ J. Ramirez5, M. Monzo1 1 Human Anatomy, Univeristy of Barcelona, School of Medicine, Barcelona, Spain, 2 Thoracic Surgery, Hospital Clinic of Barcelona, Barcelona, Spain, 3Pneumology, Hospital Clinic of Barcelona, Barcelona, Spain, 4Dept. Medical Oncology, Hospital Clinic y Provincial de Barcelona, Barcelona, Spain, 5Pathology, Hospital Clinic y Provincial de Barcelona, Barcelona, Spain Background: In resected non-small cell lung cancer (NSCLC) considerable recurrence rates has been reported (30%). Tumor secretes small extracellular vesicles known as exosomes, which plays a role in the metastasis process. Pulmonary tumor-draining vein blood has proved to be better source than peripheral vein to analyze the prognosis
Volume 30 | Supplement 5 | October 2019
1446P
Immunological impact of surgery in NSCLC patients
A. Yanagihara1, H. Kagamu2, H. Sakaguchi1, H. Ishida1, H. Nitanda1, R. Taguchi1, R. Yoshimura1, O. Yamaguchi2, K. Hashimoto2, K. Kodaira2 1 Department of General Thoracic Surgery, Saitama Medical University International Medical Center, Hidaka, Japan, 2Department of Respiratory Medicine, Saitama Medical University International Medical Center, Hidaka, Japan Background: Immune checkpoint inhibitors (ICI) improve survival in patients with advanced non-small cell lung cancer (NSCLC) and have become one of standard therapy. It was demonstrated that nivolumab therapy for resectable NSCLC patients prior to surgery resulted in major pathological responses in 40% of the patients. On the other hand, adjuvant nivolumab therapy for melanoma resulted in longer recurrence-free survivals. Thus, it is still uncertain which host immunological status is appropriate for ICI therapy, before or after surgery. In this study, we examined the peripheral blood, and tumor tissues to elucidate immunological impact of surgery in NSCLC patients. Methods: The present study comprised of 20 surgically resectable early (stage I, II, or IIIA) NSCLC patients in Saitama Medical University International Medical Center. Peripheral blood samples were collected before and after surgical resection of NSCLC. PBMCs were analyzed with LSR FortessaTM. Results: Interestingly, more effector memory (EM) CCR7-CD45RA- CD8þ T cells and CD62Llow CD4þ T cells were observed in early stage NSCLC patients, compared with stage IV NSCLC patients. Significant decrease of the percentages of EM CD8þ T cells (P ¼ 0.0045) and CD62Llow CD4þ T cells (P < 0.0001) were detected after surgical removal of cancer. In contrast, central memory (CM) CCR7þCD45RA- CD8þ T cells significantly increased (P ¼ 0.0037). It is likely that the decrease of EM CD8þ T cells and CD62Llow CD4þ T cells reflected conversion of EM T cells to CM T cells due to disappearance of cancer antigens, because the decrease was not observed in the patients of incomplete resection. PD-1 expression on both CD8þ and CD4þ T cells was up-regulated after surgery. The percentages of myeloid derived suppressor cells (MDSC) (P < 0.0001) increased and natural killer cells (NK) decreased (P ¼ 0.0001). Conclusions: After surgical removal of lung cancer, EM type T cells were converted to CM type T cells. It seems that surgery may have negative influence for innate immunity. Because PD-1 blockade therapy only activates primed effector T cells, the immunological status before surgery seems more appropriate for ICI therapy. Legal entity responsible for the study: Saitama Medical University International Medical Center. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.
1447P
The prognostic value of selected immunological panel in predicting the prognosis of early-stage resectable non-small cell lung cancer
S. Zhao1, Y. He1, X. Zhang1, C. Zhou2 Oncology, Shanghai Pulmonary Hospital, Tongji University, Shanghai, China, 2 Shanghai Pulmonary Hospital, Tongji University, Shanghai, China
1
Background: Lung cancer is one of the most fatal cancer types globally. Currently, the immune status in the tumor microenvironment (TME) is highly valued. The immune
doi:10.1093/annonc/mdz258 | v587
Downloaded from https://academic.oup.com/annonc/article-abstract/30/Supplement_5/mdz258.004/5577078 by guest on 09 October 2019
nas3, J.D. de-Maya-Girones4, A. Herreros Pomares1, E. Jantus-Lewintre2, S. Calabuig-Fari~ R. Lucas5, A. Blasco6, R. Guijarro7, M. Martorell8, E. Escorihuela1, S. Alonso9, M.D. Chiara10, E. Dure´ndez1, C. Gandia4, R. Sirera11, R. Farras4, C. Camps12 1 Molecular Oncology Laboratory-CIBERONC, Fundaci on de Investigaci on Hospital General Universitario de Valencia, Valencia, Spain, 2Department of Biotechnology, Universidad Polite´cnica de Valencia, Molecular Oncology Laboratory-CIBERONC, Fundacion de Investigacion Hospital General Universitario de Valencia, Valencia, Spain, 3 Department of Pathology, Universitat de Vale`ncia, Molecular Oncology LaboratoryCIBERONC, Fundacion de Investigaci on Hospital General Universitario de Valencia, on Prıncipe Valencia, Spain, 4Oncogenic Signalling Laboratory, Centro de Investigaci 5 Felipe, Valencia, Spain, Department of History of Science and Documentation, 6 Universitat de Vale`ncia, Valencia, Spain, Department of Medical Oncology-CIBERONC, Hospital General Universitario de Valencia, Valencia, Spain, 7Department of Thoracic Surgery-CIBERONC, Hospital General Universitario de Valencia, Valencia, Spain, 8 Department of Pathology, Hospital General Universitario de Valencia, Valencia, Spain, 9 Program of Predictive and Personalized Medicine of Cancer, Institut de Reserca Germans Trias i Pujol (PMPPC-IGTP), Badalona, Spain, 10Institute of Sanitary Research of Asturias-CIBERONC, Hospital Central de Asturias, Oviedo, Spain, 11Department of Biotechnology-CIBERONC, Universidad Polite´cnica de Valencia, Valencia, Spain, 12 Department of Medical Oncology, Hospital General Universitario de Valencia; Department of Medicine, Hospital General Universitario de Valencia, Molecular Oncology Laboratory-CIBERONC, Fundaci on de Investigaci on Hospital General Universitario de Valencia, Valencia, Spain
impact of exosomes (Navarro A, et al. Cancers 2019). LincRNA-p21 is a long non-coding RNA which upregulation in tumor tissue induced by hypoxia was related to poor prognosis in NSCLC (Castellano JJ. et al. JTO 2016). We aimed to evaluate the presence of lincRNA-p21 in exosomes and its potential as prognosis biomarker in resected NSCLC patients. Methods: H23 and H1299 NSCLC cell lines were cultured in exosome-free medium 48h under normoxic/hypoxic conditions. LincRNA-p21 was inhibited using a siRNA and quantified using a TaqMan assay. Exosomes were purified by ultracentrifugation and characterized by electron microscopy, western blot and Nanosight. LincRNA-p21 was analyzed in exosomes from pulmonary tumor-draining vein, obtained during surgery, from 54 NSCLC patients. Results: LincRNA-p21 was present in exosomes showing 18 times more representation than in their origin cells. Under hypoxic conditions exosomal lincRNA-p21 was upregulated in both cell lines (p < 0.05). Cellular lincRNA-p21 silencing resulted in a reduction in exosomal lincRNA-p21 (p ¼ 0.04) indicating a relation between cellular and exosomal levels. The analysis in patient samples showed that lincRNA-p21 was present in patient exosomes obtained from tumor-draining vein. The survival analysis showed that high exosomal lincRNA-p21 levels were associated with shorter time to relapse (TTR) (37 vs 56 months; p ¼ 0.03) and shorter overall survival (OS) (46 vs 66 months; p ¼ 0.04). In the multivariate analysis exosomal lincRNA-p21 emerged as an independent prognostic marker for TTR (HR: 3.1; 95%CI: 1.02-9.4; p ¼ 0.04) and OS (HR: 5.3; 95%CI: 1.6-17.2; p ¼ 0.006) together with disease stage. Conclusions: Tumor exosomes carry LincRNA-p21, which increases under hypoxia, and its analysis in tumor-draining vein arises as a promising prognosis marker. Legal entity responsible for the study: The authors. Funding: SAF2017-88606-P (AEI/FEDER, UE). Disclosure: All authors have declared no conflicts of interest.