Abstract 1821: FFPE preanalytical variables: Investigating the effect of delayed times to fixation on the proteome and phosphoproteome for FFPE kidney tumor samples and a comparison of tumor versus normal for matching FFPE and OCT frozen tissue
Authors: Fiona E. McAllister, Rachana Agarwal, Bich Nguyen, Yiyong Zhou, Sushmita Roy, Daniel Chelsky, Ping Guan, Mary Barcus, Hana Odeh, Lararsha Carithers, Helen Moore
Journal: Cancer Research (2015)

Abstract Variations in how human biospecimens are collected, processed and stored have been shown to significantly affect downstream molecular analyses. FFPE (formalin-fixed, paraffin-embedded) specimens are valuable resources for biospecimen based research, since many such samples are routinely collected and stored in biobanks. With an aim to evaluate the impact of delay to fixation on total protein and phosphoprotein profiles, we used a label free mass spectrometry based proteomic and phosphoproteomic analysis on FFPE renal cell carcinoma (RCC) tumor tissues from 20 patients at four “delay to fixation” time points (1h, 2h, 3h and 12h delay to fixation), collected under the National Cancer Institute's (NCI) Biospecimen Pre-analytical Variables (BPV) program. In addition, we compared tumor and adjacent normal kidney tissue at the four delay to fixation time points. This comparison was performed using both FFPE and frozen (OCT) tissue from case-matched tumor specimens to assess the relative impact of the two biospecimen preservation methods. We employed a label-free intensity based quantitation for the proteome profiling using high resolution Orbitrap mass spectrometry. A total of 3475 proteins and 1690 phosphoproteins were quantitated in the FFPE specimens and 3728 proteins and 1817 phosphoproteins in the OCT (frozen) tumor specimens. Very few significant changes were observed at the proteome level with different delay to fixation times. However, at the phosphoprotein level, significant numbers of phosphopeptides were observed to change and these changes were observed across the majority of patients. Approximately 8% of the phosphopeptides were significantly changed after a 12 h delay in time to fixation (vs. 1 h) compared to just 0.5% of the nonphosphopeptides. The observed changes to the phosphoproteome do not appear to be completely random. The phosphopeptides that are differentially expressed are enriched in proteins associated with renal cell death. In the tumor versus normal tissue comparison, large numbers of changes were observed at both the proteome and phosphoproteome level. These changes were consistent between the FFPE and OCTtumor specimens. In general, however, greater magnitude fold changes were observed in OCT versus the FFPE tumor specimens. Many of the differentially expressed proteins observed in this study have previously been identified in urine. These findings could potentially lead to the development of a urine-based biomarker assays for renal cell carcinoma that could aid in the early detection of this cancer. This work is funded by NCI Contract No. HHSN261200800001E. Citation Format: Fiona E. McAllister, Rachana Agarwal, Bich Nguyen, Yiyong Zhou, Sushmita Roy, Daniel Chelsky, Ping Guan, Mary Barcus, Hana Odeh, Lararsha Carithers, Helen Moore. FFPE preanalytical variables: Investigating the effect of delayed times to fixation on the proteome and phosphoproteome for FFPE kidney tumor samples and a comparison of tumor versus normal for matching FFPE and OCT frozen tissue. [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 1821. doi:10.1158/1538-7445.AM2015-1821