Abstract 1994: Impact of laser capture microdissection on cancer signaling proteins and phosphoproteins

Abstract

Abstract Background: Tissues are heterogeneous in terms of cell composition and the percentage of tumor epithelium can vary tremendously in any clinical biopsy. Given the importance and focus on the cancer phosphoproteome due to the mechanism of action of nearly all targeted therapies, we and others have shown the importance of upfront cellular enrichment strategies such as laser capture microdissection (LCM). However, despite the improved data quality, retention of dynamic range, and correlations with IHC and genomic data shown with LCM based analysis; questions remained about the overall impact of LCM on protein and phosphoprotein measurements. The aim of the study was to investigate whether a real-world tissue processing workflow using infrared (IR) based LCM for the isolation of pure cell populations affect the overall expression and activation/deactivation levels of protein signaling molecules that are important in druggable target analysis and companion diagnosis. Materials and methods: Five Non Small Cell Lung Cancer samples were used for this study. Eight micron sections were cut and mounted on glass slides. Samples were microdissected or lysed within 30 minutes once stained. For each sample, using alternating adjacent slides, three different protein lysates were collected: the first lysate was generated after tumor epithelium enrichment was performed via LCM, the second one was obtained by lysing of the undissected whole tissue directly from the glass slide (TL) and the last was obtained by microdissecting the entire tissue slide (LCM-TL) without regard to the underpinning cell population. Lysates were then analyzed by Reverse Phase Protein Microarray to evaluate the expression/activation levels of 87 proteins. Unsupervised hierarchical clustering analysis was used to compare the expression/activation of the 87 analytes across the different collection methods. Results: Tumor percentage was estimated as 50% (n = 1), 60% (n = 1) and 70% (n = 3). Unsupervised hierarchical clustering analysis showed that for all five pairs (100%), the TL and matched LCM-TL samples clustered within the same arm of the dendrogram while only one of the LCM samples (with tumor percentage of 70%) clustered in the same arm of the dendrogram as the matched LCM-TL. On the other hand, two of the five LCM samples were contained in the same cluster. Conclusion: These results confirm the importance of using LCM for upfront cellular enrichment for the accurate measurement of the expression/activation levels of proteins for biomarker discovery, even when the tumor cells percentage in the specimen is high. Importantly, based on this pilot study we conclude, that the LCM process, using an IR based direct capture laser system, itself does not appear to have a significant impact on the integrity of proteome and phosphoproteome. Citation Format: Elisa Baldelli, Vienna Ludovini, Lucio Crinò, Lance Liotta, Emanuel Petricoin, Mariaelena Pierobon. Impact of laser capture microdissection on cancer signaling proteins and phosphoproteins. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1994. doi:10.1158/1538-7445.AM2015-1994