Abstract
BackgroundThere is an urgent need to measure phosphorylated cell signaling proteins in cancer tissue for the individualization of molecular targeted kinase inhibitor therapy. However, phosphoproteins fluctuate rapidly following tissue procurement. Snap-freezing preserves phosphoproteins, but is unavailable in most clinics and compromises diagnostic morphology. Formalin fixation preserves tissue histomorphology, but penetrates tissue slowly, and is unsuitable for stabilizing phosphoproteins. We originated and evaluated a novel one-step biomarker and histology preservative (BHP) chemistry that stabilizes signaling protein phosphorylation and retains formalin-like tissue histomorphology with equivalent immunohistochemistry in a single paraffin block.ResultsTotal protein yield extracted from BHP-fixed, routine paraffin-embedded mouse liver was 100% compared to snap-frozen tissue. The abundance of 14 phosphorylated proteins was found to be stable over extended fixation times in BHP fixed paraffin embedded human colon mucosa. Compared to matched snap-frozen tissue, 8 phosphoproteins were equally preserved in mouse liver, while AMPKβ1 Ser108 was slightly elevated after BHP fixation. More than 25 tissues from mouse, cat and human specimens were evaluated for preservation of histomorphology. Selected tissues were evaluated in a multi-site, independent pathology review. Tissue fixed with BHP showed equivalent preservation of cytoplasmic and membrane cytomorphology, with significantly better nuclear chromatin preservation by BHP compared to formalin. Immunohistochemical staining of 13 non-phosphorylated proteins, including estrogen receptor alpha, progesterone receptor, Ki-67 and Her2, was equal to or stronger in BHP compared to formalin. BHP demonstrated significantly improved immunohistochemical detection of phosphorylated proteins ERK Thr202/Tyr204, GSK3-α/β Ser21/Ser9, p38-MAPK Thr180/Tyr182, eIF4G Ser1108 and Acetyl-CoA Carboxylase Ser79.ConclusionIn a single paraffin block BHP preserved the phosphorylation state of several signaling proteins at a level comparable to snap-freezing, while maintaining the full diagnostic immunohistochemical and histomorphologic detail of formalin fixation. This new tissue fixative has the potential to greatly facilitate personalized medicine, biobanking, and phospho-proteomic research.
Highlights
Protein kinase inhibitors constitute a large percentage of current lead compounds for molecular targeted cancer therapy [1]
We evaluated the state of protein and post-translationally modified proteins from mouse, cat, and human tissues fixed in our biomarker and histology preservative (BHP) for (i) retention of phosphorylated levels over extended fixation time and in comparison to snap-frozen material, (iii) overall yield of extractable biomolecules, (iv) preservation of tissue morphology in multiple organs and tissues, and (v) preservation of key diagnostic immunohistochemistry antigens
Protein yield from BHP fixed paraffin embedded tissue The amount of protein that can be extracted from small tissue samples is a key limiting factor for many proteomic analysis technologies
Summary
Protein kinase inhibitors constitute a large percentage of current lead compounds for molecular targeted cancer therapy [1]. Sub-populations of patients that may respond to such targeted kinase inhibitors need to be identified for individualization of therapy. Accurate quantitative measurement of the state of phosphoprotein cellular signaling pathways directly in human diagnostic tissue samples will be a critical driver for the future of molecular diagnostics [2]. There is an urgent need to measure phosphorylated cell signaling proteins in cancer tissue for the individualization of molecular targeted kinase inhibitor therapy. We originated and evaluated a novel one-step biomarker and histology preservative (BHP) chemistry that stabilizes signaling protein phosphorylation and retains formalin-like tissue histomorphology with equivalent immunohistochemistry in a single paraffin block
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