Validation of a novel one-step tissue fixation chemistry that preserves phosphoproteins and histomorphologyVirginia A. Espina1, Claudius Mueller1, Svetlana Rassulova2, Holly S. Gallimore2, Elisa Baldelli1,3, Svetlana Senina1,

Joel Pachter4, Kirsten H. Edmiston2, Lance A. Liotta1

1 George Mason University, Manassas, VA, 2Inova Fairfax Hospital, Department of Surgery, Falls Church, VA, 3S.Maria della Misericordia Hospital, Perugia, Italy, 4University 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 aliveand reacting to ex vivo stress [1]. During this “cold ischemia time” cells within the tissue reactand adapt to the absence of vascular perfusion, ischemia, hypoxia, acidosis, andaccumulation of cellular waste. Challenged by the realization that phosphoprotein signalingpathways were reactive and fluctuating immediately following procurement, we developed anon-formalin fixative chemistry for the preservation of biomarker molecules andhistomorphology in one step, in epithelial and calcified tissues, using standard clinicalpathology processing protocols [2] (US Patent #8,460,859).Technology and potential advantages: A novel, non-formalin, one-step phosphoproteinpreservation chemistry was created, characterized in a wide variety of human and animaltissues, independently validated by pathologists, published, patented, licensed, and sampleswere distributed to the scientific community. Our preservative stabilizes phosphoproteins,immunohistochemical antigens, glycoproteins, nucleic acids, decalcifies bone, and rendersexquisite diagnostic cellular histomorphology superior, or equivalent, to formalin. This fixativereduces biospecimen pre-analytical variability in research or clinical molecular profiling. Ournovel preservative makes it possible to ask, and answer, research questions, and conductclinical trial studies that were never before possible. For example, we can now assessmolecular targets in calcified tissue, and reliably monitor phosphorylated signal transductionproteins - the substrates of kinase drug targets.Progress to date: Completed independent performance validation of the fixative in a widevariety of tissues and labile cancer related antigens. The histomorphology of our fixative wasequal or superior to formalin under the following histomorphology rankings: overall color andfidelity, cell size, preservation of nuclear membrane, preservation of nucleoli and nuclearchromatin, preservation of overall cell structure, and nuclear:cytoplasmic ratio maintained.RNA quality was equal to frozen brain tissue prior to paraffin embedding, and is compatiblewith laser capture microdissection and qRT-PCR. In the case of calcified tissues, our fixativeaided a simplified processing of bony tissues (18.5 hour reduction in processing time) whilesupporting immunohistology, histomorphology, and FISH, superior to or equivalent toformalin.

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/decalcificationsolution that is compatible with immunohistochemistry.TheraLin preserves nuclear volume, does not requireadditional decalcification, preserves phosphoprotein epitopes,and is compatible with laser capture microdissection, and withvarious Hematoxylin formulations. Top right panel shows bonesamples 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 inTheraLin or NBF for 3 or 24 hours prior to freezing. RNA was extracted using the Qiagen AllPrep DNA/RNA/ProteinMini kit (frozen and TheraLin fixed samples) or the Qiagen FFPE miRNeasy kit (NBF samples). 200 ng of RNA werereverse transcribed to cDNA and 0.5uL cDNA was used to amplify three different sized amplicons of Beta-ActinmRNA. B) LCM/qRT-PCR. Microdissected (Arcturus PixCell IIe) mouse brain was fixed or frozen. RNA was directlyreverse transcribed without isolation. TheraLin fixed tissue showed lower Ct values compared to frozen tissueindicating 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 bp438 bp651 bp

Frozen BHP 3h BHP 24hNBF 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

1A4AE1/AE3AFPBCCBcl-2BrachyuryCA125CalretininCD 10CD 20CD 21CD 23CD 3CD 31CD 34CD 38CD 45CD 5CD 56CD 68CDX2CK 20CK 7CK 8/18CK HMWCrAE-CadherinEGFREMAER αHEPAHer2HMB45HMB-45Ki-67MDM2OsterixPankeratinPax 8P-GlycoproteinPhospho-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)PRRCCS100SMASox9ThyroglobulinTTF1VimentinWT1

8A 8B

Figure 4. Histomorphology of murine tissue andwhole embryos. 31 murine tissues, including wholemouse embryos, show adequate preservation inTheraLin 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 samples27 tissue types

Preservation of clonal heterogeneity in tumor tissue. TheraLin fixed tissue has beenused to conduct Immuno-Laser Capture Microdissection for full genome sequencing ofspecific cell populations.Simultaneous preservation and decalcification of human bone metastasis formolecular analysis. TheraLin obviates the need for a separate decalcification step andpreserves histomophology and IHC superior to formalin for bone, as judged by independentpathologist analysis. TheraLin preserves and decalcifies entire mouse embryos in one step.TheraLin fixation replaces specimen freezing and eliminates the requirement for dryice shipping. Comparison of signal pathway phosphoprotein networks in vivo, in corebiopsies 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 braintissue. TheraLin preserves nuclear chromatin detail, without chromatin smudge artifacts,that is superior to formalin.Carbohydrate chemistry in tissue: Preservation of mucin and glycoproteins. SuperiorPAS staining of mucin, enabling visualization of glycocalyx gradients.Biodefense research: Inactivation of viral biohazard select agents by TheraLin intissue specimens. Rapid inactivation of select agents in tissue by TheraLin, whilepreserving biomarkers, improves safety of tissue handling and eliminates the one-monthquarantine 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 andquantity in cells fixed in TheraLin was verified to be maintained at high quality (RIN 8.2) for72 hours. This time exceeds the recommended maximum length of time of fixation, prior toparaffin embedding, for Anatomical Pathology Clinical Practice Guidelines. TheraLin hasmade 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 demonstratesviral inactivation during tissue fixation.Liver samples from animals infected with theBSL2 strain of Rift Valley Fever Virus showedtotal inactivation of RVFV after 7 days fixationin 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 andglycoproteins. A) Human colon stained with PeriodicAcid Schiff (PAS) stain. B) Thyroid tissue stained withMasson’s Trichrome stain demonstrating porositygradients within the colloid.

5A 5B