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Silvia Calabuig-Fariñas
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MA04 - Models and Biomarkers (ID 122)
- Event: WCLC 2019
- Type: Mini Oral Session
- Track: Biology
- Presentations: 2
- Now Available
- Moderators:Montse Sanchez-Cespedes, Kwok-kin Wong
- Coordinates: 9/08/2019, 13:30 - 15:00, Interlaken (1988)
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MA04.03 - Lung Tumorspheres Characterization Reveals Cancer Stem-Like Cells Potential Targets and Prognostic Markers in Non-Small Cell Lung Cancer (Now Available) (ID 2269)
13:30 - 15:00 | Author(s): Silvia Calabuig-Fariñas
- Abstract
- Presentation
Background
Non-small cell lung cancer (NSCLC) is the leading cause of death cancer-related worldwide due to late diagnosis and high resistance against treatments. This resistance has been associated to cancer stem-like cells (CSCs), a highly tumorigenic subpopulation for which the identification of targets and biomarkers is still under development.
Method
Tissue samples from 8 NSCLC patients were successfully established and cultured using a sphere-forming assay for CSCs enrichment. Adherent counterparts were used as differentiated control cells. Proliferation, chemorresistance, invasion and differentiation capacities were tested in vitro, whereas tumor initiation capacity was determined in vivo. The expression of 44 CSCs-related genes was assessed by qPCR and protein expression of the best contributors to distinguish adherent cells from tumorspheres was determined by immunoblot and immunofluorescence. The prognostic role of these genes was evaluated in a cohort of 661 resected NSCLC patients from TCGA and validated in an independent cohort of 114 resected lung adenocarcinoma patients.
Result
Patient-derived tumorspheres showed unlimited exponential growth, high resistance against chemotherapy, great invasion and differentiation capacities in vitro in addition to a higher tumorigenic potential than adherent cells in vivo. The expression of 17 genes was significantly overexpressed in lung tumorspheres, being NANOG, NOTCH3, CD44, CDKN1A, SNAI1, and ITGA6 the best contributors. Proteins encoded by these genes were consistently increased in tumorspheres from adenocarcinoma patients and showed differential localization and expression patterns. The expression of CDKN1A, SNAI1 and ITGA6 was associated to prognosis based on Cox regression analysis (Z-score > 1.5), so their absolute regression coefficients from a multivariate model were used to calculate a gene expression score. Kaplan-Meier survival analysis showed that patients with high score have shorter OS in the entire cohort [37.7 vs. 60.4 mo., p = 0.001] and the adenocarcinoma subcohort [36.6 vs. 53.5 mo., p = 0.003], but not in squamous cell carcinoma one. Multivariate analysis indicated that this gene expression score was an independent biomarker of prognosis for OS in both, the entire cohort [HR: 1.498; 95% CI, 1.167-1.922; p = 0.001] and the adenocarcinoma subcohort [HR: 1.869; 95% CI, 1.275-2.738; p = 0.001]. The prognostic value of this score was confirmed in an independent cohort of 114 lung adenocarcinoma patients (42.90 vs. NR mo, p = 0.020).
Conclusion
Proteins encoded by NANOG, NOTCH3, CD44, CDKN1A, SNAI1, and ITGA6 are potential targets against lung CSCs. Elevated gene expression levels of CDKN1A, SNAI1 and ITGA6 are associated with worse prognosis.
Funded by CB16/12/00350 from CIBEROnc, PI12-02838, and PI15-00753 from ISCIII and Fundacion Arnal Planelles.
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MA04.09 - Study of Exosomes in NSCLC for Biomarkers Searching (Now Available) (ID 2417)
13:30 - 15:00 | Author(s): Silvia Calabuig-Fariñas
- Abstract
- Presentation
Background
Exosomes are small membranous vesicles secreted by a most type of cells (especially in tumoral processes), around 40-130 nm of size, that carry relevant information to distant tissues and being able to modulate its physiology. Exosomes have been detected in different clinical samples and may play a key role in NSCLC, participating in several processes such as horizontal transfer of RNA from tumor to microenvironmental cells, angiogenesis, pre-metastatic niche formation, immunosuppression; and could also be key elements in stem cell differentiation (from different origins).
The principal objective of this study was to analyze the exosomes cargo from NSCLC cell lines and primary cultures with diverse characteristics under different growth conditions: suspension cultures with cancer stem cells (CSCs) features and monolayer cultures.
Method
Primary cultures from resected NSCLC patients and NSCLC cell lines were successfully established. Differentiated tumor cells were cultured under adherent conditions (2D) whereas CSCs were established in suspension cultures (3D tumorspheres). Exosomes isolation was performed by ultracentrifugation. Exosomes characterization was carried out through nanovesicles tracking analysis (NTA) and electron microscopy; and the determination of surface markers through immunoblot and flow cytometry. Exosomal DNA was extracted in order to determine the mutational status of the EGFR and RAS genes by BEAMing Digital PCR (Sysmex). Transcriptomic analysis has been carried out from exosomal RNA through whole genome gene expression microarrays, (Affymetrix). The data was normalized by Robust Multi-Array Average (RMA) and analyzed using Transcriptome Analysis Console (TAC), MultiExperiment Viewer (MeV) software and Partek Genomics Suite. Statistical significance was established at (p ≤ 0.01).
Result
In reference to the characterization, NTA and electron microscopy showed that exosomes were obtained free of cellular debris and their size ranges from 108-125 nm, according to the size of tumor-derived microvesicles. Exosomal surface markers analyzed by immunoblot and flow cytometry were detected in samples, confirming proper isolation. Mutational analysis of EGFR and RAS genes performed on exosomal DNA shown the same pattern displayed by the origin cells. Transcriptomic analysis of the exosomal content showed that the expression of mRNAs, miRNAs and precursors were significantly different between 3D and 2D-derived exosomes. Finally, a pathway enrichment analysis was carried out to know in which biological processes (cancer-related) are involved.
Significant differential expressions were also found between mRNAs, miRNAs and pre-miRs present in exosomes from adenocarcinoma (ADC) vs. squamous cell carcinoma (SCC). Interestingly, 7 miRNAs differentially expressed in exosomes (miR-200c; miR-29a; miR-339; miR-224; miR-31; miR-21; miR-33a) had already been identified as overexpressed in tumor tissue from NSCLC patients by our group. Moreover, miR-339 y miR-21 were related to prognosis (p < 0.05) in ADC group.
Conclusion
Differences in exosomal mRNA, miRNAs and pre-miRs expression have been observed between: i) lung-tumorspheres vs. more differentiated tumor cells and ii) ADC vs. SCC cultures. In addition, the same mutational pattern was detected in exosomes as compared with their parental cultures. Therefore, exosomes can be a useful source for biomarkers analysis in NSCLC.
Supported by grant GV/2018/026, PI18/00266, & Asociación Española Contra el Cáncer (AECC Valencia).
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P2.03 - Biology (ID 162)
- Event: WCLC 2019
- Type: Poster Viewing in the Exhibit Hall
- Track: Biology
- Presentations: 2
- Moderators:
- Coordinates: 9/09/2019, 10:15 - 18:15, Exhibit Hall
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P2.03-08 - Analysis of Immunosuppressive Factors Produced by CSCs Revealed Galectin-3 as Immune Modulator with Prognostic Value in NSCLC Adenocarcinoma (ID 1855)
10:15 - 18:15 | Author(s): Silvia Calabuig-Fariñas
- Abstract
Background
The study of the tumor microenvironment is leading to a better understanding of the tumor escape from immunosurveillance and immunotherapy response. Cancer stem cells (CSCs) are targets poorly recognized by the immune surveillance system given that they favor an immunosuppressive microenvironment. The aim of this work is to study the interactions between CSCs and the immune microenvironment in NSCLC.
Method
Tumor cells from 8 resected NSCLC patients and 12 cell lines were cultured using a sphere forming assay for CSCs enrichment. Adherent cultures were established as differentiated controls. The gene expression of IL-4, IL-10, IL6, IL8 LGALS-3 was analyzed by RTqPCR. Gene expression results were validated at a protein level by a sensitivity bead-based multiplex immunoassay using the Millipore kit for Luminex 100/200. The prognostic value of these factors in silico was determined in a cohort of 661 patients from The Cancer Genome Atlas (TCGA) cohort. Prognostic value was assessed by Cox regression and Kaplan‐Meier curves ﴾long rank‐test﴿, considering significant when p<0.05.
Result
Patients’ median age was 67 years [57-74], 62% were male, and 62.5% were adenocarcinomas (ADC). Gene expression analysis revealed that lungspheres had significantly higher expression of LGALS3 compared to differentiated adherent cells. On the contrary, adherent cells had significantly higher expression of IL6. In concordance with gene expression levels, we observed significant differences in the secretion of these two soluble factors (IL-6 and Galectin-3) between adherent cells and tumorspheres. Neither expression nor secretion levels of IL-10 and IL-4 were detectable. We had not observed significant results for IL-8. Survival analysis showed that ADC patients with higher LGALS3 expression had significantly decreased overall survival (OS, 40.49 vs. 87.90 months, p= 0.005) and relapse-free survival (25.28 vs. 41.25 months, p=0.003) than the group with LGALS3 expression below the median. The multivariate analysis showed that LGALS3 expression can be established as an independent prognostic biomarker for OS [HR=1.75; 95% CI, 1.19-2.59; p=0.004] and for PFS in lung adenocarcinoma [HR=1.68; 95% CI, 1.22-2.32; p= 0.001].
Conclusion
Our results suggest that Galectin-3 highly secreted by lung CSCs can be involved in the modulation of the immune microenvironment. Moreover, Galectin-3 is an independent prognostic biomarker for overall survival and relapse-free survival in lung adenocarcinomas.
Supported by grants CB16/12/00350, PI18/00266 and PI15-00753 from ISCIII and ACIF/2018/275 from GVA and FSE
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P2.03-16 - Agreement Between Different Methodologies for Non-Invasive p.T790M and EGFR Sensitizing Mutation Testing (ID 1965)
10:15 - 18:15 | Author(s): Silvia Calabuig-Fariñas
- Abstract
Background
Tyrosine kinase inhibitors (TKIs) are the current standard of care for patients with advanced EGFR-mutant non-small cell lung cancer (NSCLC). However, most patients progressed within 1 to 2 years. The EGFR p.T790M mutation is the most common resistance mechanism to first and second generation EGFR TKIs. The identification of p.T790M mutation is of considerable clinical relevance as osimertinib has demonstrated clinical efficacy in this setting. Guidelines recommend testing for the p.T790M mutation in blood at relapse to TKIs, and re-biopsy only in case of a negative result. Several blood based methodologies for detection of EGFR mutations have been developed in the recent years. However, the number of comparison studies between platforms is very limited.
Method
This is a multicenter, cross-sectional study (ClinicalTrials.gov Identifier: NCT03363139) performed by the Spanish Lung Cancer Group. Samples from 75 consecutive EGFR mutant NSCLC patients were collected at disease progression to first line TKI treatment. The presence of EGFR mutations in the cfDNA was evaluated in 39 samples by 7 methodologies, namely: Cobas® EGFR Mutation Test v2 (Roche Diagnostics), Therascreen EGFR Plasma RGQ PCR Kit (Qiagen), QuantStudio® 3D Digital PCR System (Thermofisher), a 5′-nuclease real-time PCR (TaqMan®) assay in presence of PNA, OncoBEAM EGFR (Sysmex Inostics), NGS with two different gene panels: Oncomine® (Thermofisher) and Lung Cancer Panel (Qiagen). The agreement between methodologies was assessed using the kappa coefficient (K) and its corresponding 95% confidence intervals (95% CI). For quantitative variables the concordance correlation coefficient (ccc) was used.
Result
Complete results are available for 39 patients. Overall, the agreement between all methodologies for the detection of p.T790M mutation as well as the original EGFR sensitizing mutation was good (K=0.669; 95CI: 0.504-0.835 and K=0.750 95CI: 0.599-0.899 respectively). Remarkably, the agreement between FDA-approved methodologies for p.T790M detection was almost perfect (K=0.926; 95CI: 0.712-1) and good for the EGFR sensitizing mutations (K=0.657; 95CI: 0.417-0.902). Similarly, the agreement between NGS-based methodologies for the detection of p.T790M and the EGFR activating mutations was very high (K=0.843; 95CI: 0.567-1 and K=0.872 95CI: 0.595-1 respectively). Moreover, concordance between both technologies for p.T790M and EGFR sensitizing mutation mutant allele frequency was excellent (ccc=0.956; 95CI: 0.906-1 and ccc=0.980 95CI: 0.950-1 respectively). The proportion of samples that were positive for p.T790M detection varied from 28% (PCR based technologies) to 37% depending on the methodology.
Conclusion
NGS and PCR-based methodologies show a good to excellent agreement for the detection of EGFR mutations, including the p.T790M. Our results support the use of liquid biopsies for non-invasive testing of clinically relevant mutations (Data from the whole cohort will be presented at the meeting).
