Alterations of molecular pattern in clinical biospecimen by pre-analytical conditions.

Abstract

e22263 Background: Biomarker analysis and patient stratification in the clinical situation is highly affected by the quality of biospecimen, which strongly depends on the pre-analytical conditions in which they were acquired. Warm and cold ischemia time that tissues are exposed to is 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 molecular effects of warm and cold ischemia and consequences for clinical decision making. Methods: Normal and colorectal cancer (CRC) tissues from patients (n = 50) were analyzed by comparing endoscopy samples with samples fixed within 10, 20 and 45 minutes post surgery (cold ischemia). In addition, normal liver tissue (n = 43) before and after 10 minutes of warm ischemia were collected and compared to normal and metastatic liver tissue samples. Gene expression was analyzed using Affymetrix GeneChip technologies and protein expression/phosphorylation was examined by IHC, MSD and NanoPro1000 technology. Results: The gene expression profiling showed significant changes in individual patients in up to 690 genes due to warm ischemia in normal liver tissue and in up to 4,116 genes in primary CRC tissue due to cold ischemia. Strongly regulated genes as well as stable genes were identified and further validated. Clinically highly relevant was the finding that, the phosphorylation of EGFR-pathway proteins was affected by pre-analytical conditions. This was validated using the NanoPro1000 technology, to also information on the level of distinct isoform phosphorylation. Conclusions: Pre-analytical conditions had a significant impact on gene expression and phosphorylation of proteins. An understanding of pre-analytical influences is mandatory when testing surgical specimens in the clinical, while drug development, or identification of predictive biomarkers. To obtain reliable data, tissue processing for research and diagnostic purposes needs to be highly standardized. Molecular alterations identified in this study have to be analyzed with caution in clinical decision making, research and development programs.