Immunohistochemistry of colorectal cancer biomarker phosphorylation requires controlled tissue fixation

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.

Figure 1. Phosphoprotein IHC scoring.

H scores of staining intensity for all studied biomarkers, by treatment condition. “2+2” is the rapid fixation condition, “24 hr.” is the standard 24-hour room temperature condition without pre-fixation cold ischemia, and “Ischemia” denotes a 1-hour cold ischemic period prior to 24-hour room temperature fixation. The medians are represented by horizontal lines, the boxes in plots correspond to the 25^th and 75 percentiles of each distribution, the whiskers extend to the most extreme value that is within 1.5 times the distance between the first and third quartiles, and data beyond the whiskers are shown as dots.

Figure 2. Colon Cancer Phosphoprotein IHC.

Representative results from IHC analysis of two colonic carcinoma samples subjected to either the rapid fixation condition (“2+2”) or standard 24-hour room temperature condition without pre-fixation cold ischemia (“24 hr”). pERK, pMTOR, pMEK1/2 and pPRAS40 are shown for one case and pBAD and pAKT are shown from a second case. Two different cases are shown here because not all cases showed staining for all markers. All micrographs are taken at 200× with equivalent exposures.

Figure 3. Colon Cancer Phosphoprotein IHC.

Representative images of one colonic carcinoma case stained with antibodies to BRAF V600E, pEGFR, EGFR, and pMSK1, showing significant loss of pEGFR staining in the ischemia condition. All micrographs are taken at 200× with equivalent exposures.

Figure 4. Colon Cancer Phosphoprotein IHC.

Representative of a second colonic carcinoma case stained with antibodies to BRAF V600E, pEGFR, PTEN, and pMSK1, showing significant loss of pEGFR staining in the ischemia condition. All micrographs are taken at 200× with equivalent exposures.