Abstract 2062: Pre-analytical conditions strongly influence molecular pattern in clinical biospecimen
Authors: Unger Florian T. , Lange Nicole , Uhlig Philipp , Juhl Hartmut , David Kerstin A. 
Published: August 2015

Research in personalized medicine in oncology and its implementation in the clinical situation is strongly affected by the quality of collected biospecimen, which strongly depends on the pre-analytical conditions in which they were acquired. Therefore, warm and cold ischemia times that tissues are exposed to during collection are of critical importance. Insufficient quality of such tissues may lead to spurious results and data misinterpretation resulting in biased stratification of patients. The present study was conducted to gain a better understanding of the effects of warm and cold ischemia on the molecular composition of tissue specimens and especially on critical clinical biomarkers whose expression and activity level inform targeted therapy evaluation in cancer.

Normal and cancer tissue from patients undergoing CRC (n = 50) surgery were analyzed comparing samples taken by endoscopy with postsurgical samples fixed within 10, 20 and 45 minutes postsurgery. In addition, we collected normal liver tissue (n = 43) before and after 10 minutes of ischemia and compared those samples with normal and metastatic cancer tissue fixed at the same timepoints. Tissue samples were analyzed using a whole genome gene expression array as well as different technologies for protein expression and protein phosphorylation with an emphasis on the epidermal growth factor receptor (EGFR)-pathway.

This study showed that surgery, vascular clamping and subsequent processing had a significant impact on the expression of genes and the phosphorylation status of proteins. The gene expression profiling showed a high variability between patients. Significant changes of expression in individual patients occurred in up to 690 genes in normal tissue and in up to 4,116 genes in primary cancer tissue comparing presurgery and ischemia time points postsurgery. Clinically highly relevant was the finding that the phosphorylation of EGFR-pathway proteins is affected by surgery and postsurgical tissue processing. For example, the expression level of EGFR changed more then 2-fold in cancer tissue of 30% of the patients. Furthermore, signaling protein phosphorylation in the corresponding downstream pathways was also affected. These observed changes were validated using the NanoPro1000 technology, in order to gain information on the level of distinct isoform phosphorylation.

An understanding of tissue data variability in relation to processing techniques during and postsurgery is mandatory when testing surgical specimens in the context of clinical diagnostics, drug development, or identification of predictive biomarkers. To obtain reliable expression data, tissue processing for research and diagnostic purposes needs to be highly standardized. Alterations in molecular patterns due to ischemia, identified in this study have to be analyzed with caution in research and development programs.

Citation Format: Florian T. Unger, Nicole Lange, Philipp Uhlig, Hartmut Juhl, Kerstin A. David. Pre-analytical conditions strongly influence molecular pattern in clinical biospecimen. [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 2062. doi:10.1158/1538-7445.AM2015-2062