Abstract
Phosphorylated signaling molecules are biomarkers of cancer pathophysiology and resistance to therapy, but because phosphoprotein analytes are often labile, poorly controlled clinical laboratory practices could prevent translation of research findings in this area from the bench to the bedside. We therefore compared multiple biomarker and phosphoprotein immunohistochemistry (IHC) results in 23 clinical colorectal carcinoma samples after either a novel, rapid tissue fixation protocol or a standard tissue fixation protocol employed by clinical laboratories, and we also investigated the effect of a defined post-operative “cold” ischemia period on these IHC results. We found that a one-hour cold ischemia interval, allowed by ASCO/CAP guidelines for certain cancer biomarker assays, is highly deleterious to certain phosphoprotein analytes, specifically the phosphorylated epidermal growth factor receptor (pEGFR), but shorter ischemic intervals (less than 17 minutes) facilitate preservation of phosphoproteins. Second, we found that a rapid 4-hour, two temperature, formalin fixation yielded superior staining in several cases with select markers (pEGFR, pBAD, pAKT) compared to a standard overnight room temperature fixation protocol, despite taking less time. These findings indicate that the future research and clinical utilities of phosphoprotein IHC for assessing colorectal carcinoma pathophysiology absolutely depend upon attention to preanalytical factors and rigorously controlled tissue fixation protocols.
Highlights
In targeted therapy of colorectal cancer, there has been recent attention placed on the importance of identifying the activation states of cell signaling molecules to the prediction of responses to therapy
Using 23 colorectal carcinoma surgical samples collected with defined cold ischemic intervals and formalin fixation protocols, we studied the phosphorylation state of numerous PI3 Kinase pathway targets, as well as the BRAF V600E mutation and other markers using immunohistochemistry [12,13]
The findings of this study clearly demonstrate that a substantial post-surgical, pre-fixation cold ischemic interval confounds the attempt to identify the phosphorylation states of a key PI3 Kinase pathway element in formalin-fixed tissue
Summary
In targeted therapy of colorectal cancer, there has been recent attention placed on the importance of identifying the activation states of cell signaling molecules to the prediction of responses to therapy. A case in point is the EGFR-mediated reactivation of MAPK signaling, which contributes to the insensitivity of BRAFmutant colorectal carcinomas to RAF inhibition with Vemurafenib [1] This pEGFR reactivation event is subject to modulation by EGFR inhibitors, and the combined polytherapy BRAF and EGFR inhibitors overcome this resistance, with beneficial response in vitro and in animal models in vivo. The extension of these prior laboratory findings to the clinic, and our ability to affect alternative treatments, is entirely dependent on our ability to accurately preserve and detect the activation states in question. This is problematic, because phosphorylation states have been known for some time to be highly sensitive to preanalytical conditions [7,8,9], including warm ischemic times [10] (which are not investigated here)
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