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Validation of a novel one-step tissue fixation chemistry that preserves phosphoproteins and histomorphology Virginia A. Espina 1 , Claudius Mueller 1 , Svetlana Rassulova 2 , Holly S. Gallimore 2 , Elisa Baldelli 1,3 , Svetlana Senina 1 , Joel Pachter 4 , Kirsten H. Edmiston 2 , Lance A. Liotta 1 1 George Mason University, Manassas, VA, 2 Inova Fairfax Hospital, Department of Surgery, Falls Church, VA, 3 S.Maria della Misericordia Hospital, Perugia, Italy, 4 University of Connecticut, Farmington, CT Abstract Performance Features of TheraLin Fixative Conclusions One-step Preservation and Decalcification Unmet need: A significant and underappreciated issue is the fact that excised tissue is alive and reacting to ex vivo stress [1]. During this “cold ischemia time” cells within the tissue react and adapt to the absence of vascular perfusion, ischemia, hypoxia, acidosis, and accumulation of cellular waste. Challenged by the realization that phosphoprotein signaling pathways were reactive and fluctuating immediately following procurement, we developed a non-formalin fixative chemistry for the preservation of biomarker molecules and histomorphology in one step, in epithelial and calcified tissues, using standard clinical pathology processing protocols [2] (US Patent #8,460,859). Technology and potential advantages: A novel, non-formalin, one-step phosphoprotein preservation chemistry was created, characterized in a wide variety of human and animal tissues, independently validated by pathologists, published, patented, licensed, and samples were distributed to the scientific community. Our preservative stabilizes phosphoproteins, immunohistochemical antigens, glycoproteins, nucleic acids, decalcifies bone, and renders exquisite diagnostic cellular histomorphology superior, or equivalent, to formalin. This fixative reduces biospecimen pre-analytical variability in research or clinical molecular profiling. Our novel preservative makes it possible to ask, and answer, research questions, and conduct clinical trial studies that were never before possible. For example, we can now assess molecular targets in calcified tissue, and reliably monitor phosphorylated signal transduction proteins - the substrates of kinase drug targets. Progress to date: Completed independent performance validation of the fixative in a wide variety of tissues and labile cancer related antigens. The histomorphology of our fixative was equal or superior to formalin under the following histomorphology rankings: overall color and fidelity, cell size, preservation of nuclear membrane, preservation of nucleoli and nuclear chromatin, preservation of overall cell structure, and nuclear:cytoplasmic ratio maintained. RNA quality was equal to frozen brain tissue prior to paraffin embedding, and is compatible with laser capture microdissection and qRT-PCR. In the case of calcified tissues, our fixative aided a simplified processing of bony tissues (18.5 hour reduction in processing time) while supporting immunohistology, histomorphology, and FISH, superior to or equivalent to formalin. 1. This fixative formulation is now commercially available as TheraLin™ from Grace Bio-Labs (www.gracebio.com). 2. TheraLin retains cellular morphology and antigenicity equivalent to formalin fixed tissue and is compatible with immunohistochemistry, PCR, qRT-PCR, reverse phase protein microarrays, and laser capture microdissection. 3. TheraLin has successfully undergone clinical pathology validation at multiple international sites. This work was funded by IMAT R21CA125698-01A1 and R33CA157403-01 to L. Liotta and V. Espina Figure 6. TheraLin is a one-step fixation/decalcification solution that is compatible with immunohistochemistry. TheraLin preserves nuclear volume, does not require additional decalcification, preserves phosphoprotein epitopes, and is compatible with laser capture microdissection, and with various Hematoxylin formulations. Top right panel shows bone samples without additional decalcification. TheraLin fixative components Precipitating fixative – stabilize proteins Permeation enhancer – rapid penetration Phosphatase & Kinase inhibitors – inhibit reactive cell signaling post excision Reversible cross-linkers – stabilize proteins, facilitate extraction of biomolecules post fixation Carboxylic acid – maintain nuclear morphology RNA is Preserved for Downstream PCR and qRT-PCR Figure 8. RNA is preserved during fixation with TheraLin. A) Human colon mucosa was snap frozen or fixed in TheraLin or NBF for 3 or 24 hours prior to freezing. RNA was extracted using the Qiagen AllPrep DNA/RNA/Protein Mini kit (frozen and TheraLin fixed samples) or the Qiagen FFPE miRNeasy kit (NBF samples). 200 ng of RNA were reverse transcribed to cDNA and 0.5uL cDNA was used to amplify three different sized amplicons of Beta-Actin mRNA. B) LCM/qRT-PCR. Microdissected (Arcturus PixCell IIe) mouse brain was fixed or frozen. RNA was directly reverse transcribed without isolation. TheraLin fixed tissue showed lower Ct values compared to frozen tissue indicating preservation of RNA. (BHP=TheraLin, NBF=neutral buffered formalin) References: 1. Espina V. et al, A portrait of tissue phosphoprotein stability in the clinical tissue procurement process. Molecular & Cellular Proteomics (2008), 7: p1998- 2018; DOI 10.1074/mcp.M700596-MCP200. 2. Mueller C. et al, One-step preservation of phosphoproteins and tissue morphology at room temperature for diagnostic and research specimens. PLoS One, (2011) 6(8): p. e23780. Methods 170 bp 438 bp 651 bp Frozen BHP 3h BHP 24h NBF 3h NBF 24h Seven Pathology Groups • Centro di Riferimento Oncologico, Aviano, Italy • University of Brescia, Italy • Istituto Nazionale Tumori, Milano, Italy • Istituto Regina Elena, Rome, Italy • Istituto Oncologico del Mediterraneo, Catania, Italy • Istituto Ortopedico Rizzoli, Bologna, Italy • St. James’ Hospital, Dublin, Ireland • Bone Cancer (50) • Breast Cancer (20) • Lung Cancer (19) • Ovarian Cancer (11) • Prostate Cancer (15) • Renal Cancer (10) • Uterus/Ovary (13) • Pancreas (2) • Other Tissues (10) • Gall bladder (5) • Liver (10) • Nerve (2) • Testis (3) • Thyroid (18) • Tonsil (5) • Skin (4) • Spleen (2) • Stomach (7) IHC Stains Better or Equal to FFPE 1A4 AE1/AE3 AFP BCC Bcl-2 Brachyury CA125 Calretinin CD 10 CD 20 CD 21 CD 23 CD 3 CD 31 CD 34 CD 38 CD 45 CD 5 CD 56 CD 68 CDX2 CK 20 CK 7 CK 8/18 CK HMW CrA E-Cadherin EGFR EMA ER α HEPA Her2 HMB45 HMB-45 Ki-67 MDM2 Osterix Pankeratin Pax 8 P-Glycoprotein Phospho-Acetyl- CoA Carboxylase (Ser79) Phospho-Akt (Ser473) Phospho-Bcl-2 (Ser70) Phospho-eIF4G (Ser1108) Phospho-ERK (Thr202/Tyr204) Phospho-GSK3 α/β (Ser21/Ser9) Phospho-p38 MAPK (Thr180/Tyr182) PR RCC S100 SMA Sox9 Thyroglobulin TTF1 Vimentin WT1 8A 8B Figure 4. Histomorphology of murine tissue and whole embryos. 31 murine tissues, including whole mouse embryos, show adequate preservation in TheraLin with paraffin embedding. 18.5 day mouse embryo Figure 3. Ki-67 antigenicity is preserved on cut tissue section slides. Formalin and TheraLin matched tissue sections were cut and stored on slides then stained side-by-side. Robust Ki-67 reactivity by IHC is evident up to 131 days after tissue sections were cut onto slides. 206 samples 27 tissue types Preservation of clonal heterogeneity in tumor tissue. TheraLin fixed tissue has been used to conduct Immuno-Laser Capture Microdissection for full genome sequencing of specific cell populations. Simultaneous preservation and decalcification of human bone metastasis for molecular analysis. TheraLin obviates the need for a separate decalcification step and preserves histomophology and IHC superior to formalin for bone, as judged by independent pathologist analysis. TheraLin preserves and decalcifies entire mouse embryos in one step. TheraLin fixation replaces specimen freezing and eliminates the requirement for dry ice shipping. Comparison of signal pathway phosphoprotein networks in vivo, in core biopsies taken before/after administration of molecular targeted therapy, is now possible. TheraLin has been validated in clinical research trials. Nuclear Chromatin: Improved nuclear histomorphology of eye, testes, and brain tissue. TheraLin preserves nuclear chromatin detail, without chromatin smudge artifacts, that is superior to formalin. Carbohydrate chemistry in tissue: Preservation of mucin and glycoproteins. Superior PAS staining of mucin, enabling visualization of glycocalyx gradients. Biodefense research: Inactivation of viral biohazard select agents by TheraLin in tissue specimens. Rapid inactivation of select agents in tissue by TheraLin, while preserving biomarkers, improves safety of tissue handling and eliminates the one-month quarantine time for formalin fixed tissue. RNA Preservation. A modified protocol based on the PAXgene Tissue miRNA Kit (PreAnalytix) was optimal for RNA extraction from TheraLin fixed cells. RNA quality and quantity in cells fixed in TheraLin was verified to be maintained at high quality (RIN 8.2) for 72 hours. This time exceeds the recommended maximum length of time of fixation, prior to paraffin embedding, for Anatomical Pathology Clinical Practice Guidelines. TheraLin has made possible the RNA analysis of microdissected brain vessels. Under evaluation Figure 1. Immuno-LCM for PCNA positive cells in prostate tissue. TheraLin fixed tissue is compatible with laser capture microdissection and immuno-LCM, coupled with downstream next gen sequencing (Ion Torrent). Figure 7. Viral plaque assay demonstrates viral inactivation during tissue fixation. Liver samples from animals infected with the BSL2 strain of Rift Valley Fever Virus showed total inactivation of RVFV after 7 days fixation in TheraLin. Figure 2. Bayesian unsupervised clustering of signal transduction proteins for microdissected matched FFPE, TheraLin fixed, or frozen lung tissues. The matched frozen and TheraLin samples clustered together, indicating similar molecular characteristics. None of the FFPE samples clustered with the matched frozen or TheraLin-fixed samples. (BHP=TheraLin fixed tissue, FFPE=formalin fixed paraffin embedded) Figure 5. TheraLin preserves mucins and glycoproteins. A) Human colon stained with Periodic Acid Schiff (PAS) stain. B) Thyroid tissue stained with Masson’s Trichrome stain demonstrating porosity gradients within the colloid. 5A 5B
