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E. Conde
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P1.02 - Poster Session with Presenters Present (ID 454)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 12/05/2016, 14:30 - 15:45, Hall B (Poster Area)
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P1.02-043 - Multiplexed FISH (ALK/ROS1, RET, NTRK1) in Lung Adenocarcinomas: Novel Dual ALK/ROS1 Probe and Automated Scanning System (ID 5743)
14:30 - 14:30 | Author(s): E. Conde
- Abstract
Background:
Although the use of NGS (next-generation sequencing) is indeed feasible for the study of druggable rearrangements, the sensitivity and specificity of targeted NGS has been challenged on several grounds: use of fixed tissue, poor quality/insufficient sample, RNA-contamination risk or long turn-around time. Our relatively high rate of failures (7.6%) with the RNA workflow of targeted NGS (data not shown), prompted us to investigate possible alternatives. The objective of this study is to demonstrate an efficient solution based on multiplexed fluorescence in situ hybridization (FISH) for the comprehensive characterization of fusions (ALK, ROS1, RET and NTRK1) in lung adenocarcinomas (ACs). We have implemented for the first time in the literature a novel four-colour ALK/ROS1 probe and used an automated scanning system for scoring these four translocations.
Methods:
A total of 64 patients with early stage lung ACs who underwent surgery at HM Sanchinarro University Hospital were considered. All slides were carefully reviewed to identify the different histological patterns. Afterwards we performed FISH to study ALK, ROS1, RET and NTRK1 in each pattern. We used both commercially (RET and NTRK1) and non-commercially (ALK/ROS1 dual) available Vysis break-apart probes (Abbott Molecular, USA). Slides were captured and scored with the BioView (Rehovot, Israel) automated imaging and analysis system, using dedicated applications for each probe. Positive cases were all confirmed with targeted NGS (Oncomine Focus Assay[TM], Life Technologies, USA).
Results:
Seven out of 420 slides did not hybridize (1.6%). We found two ALK (2.8%) and two ROS1 (2.8%) positive tumors, while no translocations were identified in RET or NTRK1. The rearrangements were present in all the different histological patterns within a given tumour, with a similar proportion of positive cells. The two major patterns of positivity were represented. The mean percentage of positive cells was 65% (range 46 to 84%). The NGS results confirmed the FISH findings.
Conclusion:
Simultaneous FISH testing for ALK and ROS1 is feasible on a single slide. The strategy reported herein provided reduced overall scoring time and ensured sensitive counting. This model might be easier to reconcile (lower cost, shorter turn-around time, and less failure rate) with subsequent comprehensive genotyping. Therefore, it could be used prospectively in advanced lung ACs to align the use of several technologies. Acknowledgements This study was partially funded by Abbott Molecular and Instituto de Salud Carlos III (ISCIII), Fondo de Investigaciones Sanitarias (FIS), Fondos FEDER-Plan Estatal de I+D+I 2013-2016 (PI14-01176).
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P2.01 - Poster Session with Presenters Present (ID 461)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 12/06/2016, 14:30 - 15:45, Hall B (Poster Area)
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P2.01-064 - Molecular Context of Immune Microenvironment in Early-Stage Lung Squamous Cell Carcinoma (ID 5914)
14:30 - 14:30 | Author(s): E. Conde
- Abstract
Background:
Although it has been proposed that the number of somatic mutations, together with high PD-L1 and CD8 expression, defines a type of tumour microenvironment predictive of response to immune checkpoint inhibitors, this data has been challenged because the methodologies are difficult to reproduce (e.g. tissue microarrays, whole exome sequencing or complicated scoring approaches). This situation prompted us to investigate possible alternatives that are easier to reconcile with daily practice.
Methods:
A total of 40 consecutive patients with early stage lung squamous cell carcinoma who underwent surgery at HM Sanchinarro University Hospital were considered. Automated immunohistochemistry (IHC) for PD-L1 expression was performed on whole tissue sections (Benchmark ULTRA, OptiView, Ventana Medical Systems, USA) with three different antibodies: SP142 (Ventana), SP263 (Ventana), and E1L3N (Cell Signaling). PD-L1 IHC was considered positive according to the criteria used in the corresponding clinical trials. P53 aberrant IHC expression was used as a surrogate marker for TP53 mutations. Whole tissue sections were also automatically scored for CD8+ tumour-infiltrating lymphocytes (TILs) density (off-label algorithm on the iScan Coreo). Afterwards, we performed targeted next-generation sequencing (NGS) in 52 genes (Oncomine Focus Assay[TM], Life Technologies, USA). The prognostic impact of all these variables was also evaluated.
Results:
Correlation between E1L3N and SP142 or SP263 was similar when scoring tumour cells (TCs) (0.94). There was a significant association between the intraepithelial and peritumour stromal CD8+ TILs density and overall survival when using the image analysis software (p=0.032). The presence of >247 CD8+ cells/mm[2] had a 94% specificity and a 67% sensitivity for identifying patients with at least 5% SP142 PD-L1+ TCs. The highest percentage of PD-L1+ TCs were found in samples with CDK6 (2.6%) or MYC (2.6%) amplifications. Cases with FGFR1 amplification (7.9%) were negative for all PD-L1 antibodies. P53 aberrant expression and PD-L1 expression in TCs also seem to be related.
Conclusion:
The methodology presented herein could help align the use of targeted NGS and the immune microenvironment assessment to increase the clinical value of immune checkpoint inhibitors. Acknowledgements This study was partially funded by Instituto de Salud Carlos III (ISCIII), Fondo de Investigaciones Sanitarias (FIS), Fondos FEDER-Plan Estatal de I+D+I 2013-2016 (PI14-01176).
