One-Step Preservation of Phosphoproteins and Tissue Morphology at Room Temperature for Diagnostic and Research Specimens

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.

Reverse Phase Protein Arrays: Mapping the Path Towards Personalized Medicine

“…Following procurement, the tissue is immediately subjected to induced stress and hypoxia and surges of wound repair related signal pathway proteins and transcription factors [67, 68]. Excised tissue is still considered a living cellular entity, and as such, it reacts to ex vivo trauma beginning with oxidative, hypoxic and metabolic stress, nutrient deprivation, wounding, finally resulting in cell death [41, 69, 70]. Consequently, phosphorylation activity of certain kinase substrates may increase due to the persistence of functional signaling, or activation by other stress-response signals [41, 69, 71].…”

“…These new tissue fixatives are being incorporated and evaluated in large national biobanking efforts and specifically provide the molecular preservation equivalent to snap-frozen tissue, while retaining formalin-like histomorphologic details [41, 70]. There is a broad range of clinical assay variability, including pre-analytical and post-analytical events, which could potentially influence the final data output.…”

“…There is a broad range of clinical assay variability, including pre-analytical and post-analytical events, which could potentially influence the final data output. The promise of tissue protein biomarkers to provide revolutionary diagnostic and therapeutic information will only be possible with the specific and rapid preservation of phosphoproteins at the time of excision, providing true information representative of the in vivo state of the signaling network within the tissue [70]. …”

Novel neoadjuvant therapy paradigms for bladder cancer: Results from the National Cancer Center Institute Forum

“…The workshop participants reached the consensus that the analysis of any proposed biomarker should be feasible using routine clinical materials (i.e., formalin- fixed, paraffin-embedded tissues; FFPE) with attention to new strategies for specimen collection, preservation, processing and analysis that minimize preanalytic confounders inherent to the processing of FFPE tissue [5–7]. These recommendations reflected several practical considerations including: 1) pre-therapy tumor is often sampled by transurethral resection (TUR) and tumor material may be limited and/or fully submitted for pathology diagnosis; 2) the volume of tumor remaining post-therapy may also be limited, especially if downstaging from prior treatment has occurred; and, 3) accurate biomarker comparisons require material to be available from patients both pre- and post-therapy which most commonly involves the use of FFPE material.…”

Combined Effect of Tissue Stabilization and Protein Extraction Methods on Phosphoprotein Analysis

“…9,10 Recently, a novel fixation method has been proposed for both histomorphology and phosphoprotein analysis. 11 If the tissue is not fixed chemically, it can be cryo-stabilized. Snapfreezing works quickly, but the cryogenic equipment needed to process and store the samples is expensive and not available in many clinical environments.…”