Determining tissue expression profiles of therapeutic target candidates is essential for safety assessment and the potential for adverse events. Immunohistochemistry is often not possible due to lack of reagents with the required specificity and sensitivity coupled with extensive time in attempts to validate IHC. RNAscope® in situ hybridization (ISH) technology is a universal assay able to detect and characterize tissue distribution of any target mRNA. RNAscope ISH has molecular sensitivity, high specificity and robust performance in FFPE tissue. Fully automated RNAscope ISH is performed on Ventana Discovery and Leica Biosystem Bond RX instrumentation; detection of marker RNAs is achieved in 8 hours and visualized via bright field microscopy. Marker RNAs can be quantified with cellular resolution using digital image analysis software. We present RNAscope methods for routine detection of very low copy RNAs, typical of most targets in normal tissues, as well as applications for detection of cell‐type, proliferation and apoptosis markers in animal tissues used in safety assessment and toxicity studies. We demonstrate robust performance in 25 tissues from rat, dog and cynomolgus monkey using probes to low‐, medium‐ and high‐expression housekeeping gene RNAs (POLR2A, PPIB and UBC). Specific signals and well‐maintained morphology was achieved efficiently for all 25 tissues. Based on optimized epitope retrieval conditions, tissues were grouped into two multi‐tissue microarrays for high‐throughput marker analysis. We examined and present tissue‐specific expression patterns of endothelial marker CD31, macrophage marker CD68, proliferation markers Ki‐67 and Cyclin E1, and apoptosis molecules, Puma, Fas/CD95 and DR5. The presentation demonstrates RNAscope as a method that can be replicated in any lab and suitable for efficient and reliable detection of any target candidate or safety/toxicity biomarker mRNA from any species for interrogation in any tissue.

Robust assays to evaluate biomarkers in tissue are needed for preclinical safety assessment and toxicity studies. Here we present the application of the fully automated RNAscope® 2.5 LS Assay on Leica BOND RX for RNA in situ hybridization in formalin-fixed paraffin-embedded (FFPE) tissues from three commonly used animal models (rat, cynomolgus monkey, and dog). We demonstrate robust assay performance with high signal-tonoise ratio and well-maintained morphology in 25 different tissues from each species. Based on these tests, we provide recommendations for proper control gene for sample qualification of each tissue type, as well as optimal pretreatment protocol selection. For specific target RNA markers, we successfully detected cell type specific markers such as CD31 (PECAM1) and CD68, proliferation marker Ki-67 (MKI67), and cell cycle marker Cyclin E1 (CCNE1), as well as apoptosis-related molecules Puma (BBC3), Fas (CD95), and DR5 (TNFRSF10B). This study demonstrates that the RNAscope® 2.5 LS Assay can be an attractive platform for biomarker analysis in tissues for preclinical safety assessment and general animal studies. Introduction Preclinical drug safety assessment in animal models has been well established as a routine laboratory practice to evaluate the pathological alterations induced by novel therapeutic agents1. This preliminary evaluation serves a major role in the development of new treatments prior to trials in humans. Histopathological techniques have been traditionally applied for general morphological evaluation by hematoxylin and eosin (H&E) stain, and also for evaluation of specific biomarkers by immunohistochemistry (IHC). IHC assay has been commonly applied to assess therapeutic targets and toxicity-related biomarkers. However, consistent and systematic application of IHC techniques has been hindered by inconsistent performance of various antibody clones, time-consuming antibody development and validation, and general lack of reagents for some animal models. RNA in situ hybridization (ISH) technology presents an attractive alternative method for pathological evaluation of biomarkers in tissues from various preclinical animal models, because nucleic acid-based probes specific for any biomarkers associated with drug toxicity or mechanism can be developed and validated rapidly. The RNAscope® technology, an advanced platform for in situ RNA detection, enables detection of almost any RNA biomarker with single-molecule detection sensitivity and high specificity in formalin-fixed paraffin-embedded (FFPE) tissues2. It provides a universal solution to characterize tissue distribution of drug targets and biomarkers in a highly specific and sensitive manner, without the need to wait for antibody development and validation. The RNAscope® assay can be performed in fully automated staining systems, including Ventana Discovery XT, Ventana Discovery ULTRA, and Leica BOND RX instruments. The assay allows visualization of each individual RNA molecule as a punctate dot under a standard bright field microscope. The RNA dots can be quantified by counting the number of signal dots in individual cells, either manually or by image analysis tools, including HALOTM (Indica Labs) and SpotStudioTM (ACD) software. In this study, we demonstrate the feasibility Preclinical drug safety assessment in animal models is used to evaluate the pathological effects induced by novel therapeutic agents. Here we present the use of the RNAscope® 2.5 LS Assay for the evaluation of biomarkers in tissues from three preclinical animal models. In this study we: • Identify optimal pretreatment conditions for different tissues in different species • Provide recommendations for control gene selection for tissue qualification • Detect specific RNA markers in various FFPE tissues from multiple species 1 1Advanced Cell Diagnostics, Inc 3960 Point Eden Way Hayward, CA, USA 94545 2Drug Safety Research and Development, Pfizer Global Research and Development, Groton, CT 06340, USA 2 Application Note of evaluating RNA biomarkers in 25 types of tissues from three commonly used animal models using the RNAscope® 2.5 LS Reagent Kit-BROWN on the Leica BOND RX instrument. Robust RNA detection was achieved following a standard protocol in almost all of the tissues tested, with minor alterations to the prestaining conditions for a few tissues. Here, we identified the threshold of pretreatment needed for different tissue types. We also provide recommendations for control probes to be applied for tissue qualification, and present the evaluation of RNA biomarkers including cell type specific markers (CD68 and endothelial marker PECAM1), proliferation marker Ki-67 (MKI67), and cell cycle marker Cyclin E1 (CCNE1). Overall, our study shows that the fully automated RNAscope® 2.5 LS Assay is capable of detecting a broad range of RNA targets in all major tissue types with little to no optimization needed, and thus well suited for the histopathological evaluation of biomarkers in the assessment of drug-derived toxicity in various tissues and animal models. Materials and Methods FFPE tissues Multiple tissues from three commonly used animals (rat, dog, and cynomolgus monkey) were harvested using a standard protocol at the drug safety research and development laboratory of PfizerGroton (Table 1). Tissues were cut into 3 mm thickness then fixed in 10% neutral-buffered formalin (NBF) for 24-48 hours. Fixed tissues were dehydrated in a graded series of ethanol and xylene, followed by infiltration of melted paraffin at 56°C in an automated processor. Tissue microarrays (TMAs) were constructed, sectioned at a thickness of 5 μm and mounted on the SuperFrost® Plus slides (Fisherbrand Cat # 12-550-15). Automated RNAscope® 2.5 LS assay Ready-to-use reagents from RNAscope® 2.5 LS Reagent Kit-BROWN were loaded onto the Leica BOND RX instrument according to the user manual (Doc. No. 322100-USM). FFPE tissue sections were baked and deparaffinized on the instrument, followed by epitope retrieval (using Leica Epitope Retrieval Buffer 2 at 95°C or at 88°C for 15 min) and protease treatment (15 min at 40°C). Probe hybridization, signal amplification, colorimetric detection, and counterstaining were subsequently performed. A schematic of the RNAscope® 2.5 LS Assay workflow on Leica BOND RX is presented in Figure 1A. RNAscope® probes Control probes of low-, medium-, and high-expressing housekeeping genes (POLR2A, PPIB, and UBC, respectively) were designed and tested for tissues from each species (Table 2). Because the sequences of the human probes for housekeeping genes are over 95% homologous to the respective target mRNA sequences of cynomolgus monkey, human probes were used to test samples of cynomolgus monkey. The bacterial probe DapB was used as a negative control. Probes for the cell type biomarkers, proliferation markers, and apoptosis-related molecules used in this study were designed for each species. As summarized in Table 3, speciesspecific target probes were tested for all RNA targets except two genes, CD68 and KI67, for which human probes were used to detect cynomolgus monkey genes, due to 90-95% homology between the probe sequence and target mRNA sequence. Image acquisition and data analysis Images were acquired using a Leica Biosystems Aperio AT2 Digital Pathology Scanner. RNA markers were analyzed based on the average RNA dot number per cell. RNA quantity was scored based on manual counting described as follows. Staining results were categorized into five grades according to the number of dots visualized under a bright-field microscope. 0: No staining or less than 1 dot to every 10 cells (40X magnification); 1+: 1-3 dots/cell (visible at 20-40X magnification); 2+: 4-10 dots/cell, very few dot clusters (visible at 20-40X magnification); 3+: >10 dots/cell, and less than 10% positive cells have dot clusters (visible at 20X magnification); and 4+: >10 dots/cell, and more than 10% positive cells have dot clusters (visible at 20X magnification). Results Optimal pretreatment condition for different tissues in different species The standard protocol of RNAscope® 2.5 LS Reagent Kit is designed to work for the majority of FFPE tissues. In this study, to achieve optimal detection of RNA molecules in each tissue type, we compared two different pretreatment conditions, standard and mild, with a modification in the epitope retrieval step (Figure 1A). The standard Animal Models: Rat, Dog, and Cynomolgus Hematopoietic system Thymus, Lymph Node, Spleen, Tonsil GI tract Esophagus, Stomach, Duodenum, Jejunum, Colon Urinary tract Kidney, Urinary bladder Reproductive system Epididymis, Prostate, Testis, Ovary Skin/soft tissues Skin, Skeletal muscle Endocrine glands/ exocrine glands Liver, Pancreas, Adrenal gland Respiratory system Lung, Bronchus Nervous system Spinal cord, Retina Cardiovascular system Heart TABLE 1. Tissue types from three commonly used animal models.

Abstract Heat induced target retrieval (HITR) is a widely adopted approach for Immunohistochemistry (IHC) and in situ hybridization (ISH) applications on formalin fixed and paraffin embedded (FFPE) tissues to facilitate target access and increase detection sensitivity. A variety of factors, such as fixative type, fixation time, tissue type, epitope abundance, antibody affinity, and probe length greatly affect the efficiency of HITR. Given such variability, over the decades, multiple HITR buffer formulations and protocols were invented to fit the needs in different experimental scenarios. We have developed a novel HITR solution that allows highly efficient target retrieval for RNA ISH application based on the RNAscope technology. When tested on 15 different tumor types, the new HITR solution resulted in significantly better sensitivity and/or tissue morphology comparing to using traditional HITR buffers, including Citrate, EDTA, and Tris based buffers with various pH. Furthermore, this new HITR solution is highly tolerant to sample variability. When used on multiple human tumor tissue microarrays (TMAs), including breast, colon, lung, and gastric cancers, the new HITR solution improved overall signal-to-noise ratio and reduced core disqualification rate, compared to traditional citrate based HITR buffer for ISH. Moreover, when tested on multiple mouse tissues fixed with different types of fixatives for varying periods of time (6 - 72 hours), a single experimental condition with the new HITR solution showed comparable results in both detection sensitivity and morphological integrity in all the samples. It also allowed detection of a target (Tbp) with extremely low abundance (<5 copies/cell). Given that all the RNAscope probes are synthetic oligonucleotide probes with length shorter than 60 bases, the new HITR solution, in combination with the RNAscope technology, will allow universal detection of any RNA targets with single molecule detection sensitivity in FFPE tissues with minimal requirement for analytical condition optimization. Citation Format: Li-chong Wang, Liuliu Pan, Kuang-Jung Chang, Daniel Kim, Xingyong Wu, Hongwei Wang, Casey Kernag, Bingqing Zhang, Mingxiao He, Nan Su, Xiao-Jun Ma, Yuling Luo. Developing a universal target retrieval solution and protocol for RNA in situ hybridization based on RNAscope technology. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4904. doi:10.1158/1538-7445.AM2015-4904

Introduction: Endobronchial ultrasound (EBUS)-guided sampling of the mediastinum has traditionally been performed by fine needle aspiration (FNA) for cytologic evaluation. The ProCore fine needle biopsy, used primarily to obtain intra-abdominal tissue core biopsies, has not been widely used for EBUS. The aim of this study was to evaluate our experience with EBUS-Procore of mediastinal nodes, and to compare its diagnostic utilization to that of conventional EBUS-FNA. Material and Methods: A retrospective review of all EBUS cases of mediastinal masses using a ProCore needle from December 2013 to March 2014 was performed. Patient demographics, anatomic site, number of procedure passes, on-site immediate evaluation, final pathology reports, and ancillary test results were recorded. All specimen slides and cell blocks were evaluated scoring lesion and contamination cellularity (0 Z acellular, 1 Z scant, 2 Z moderate, and 3 Z abundant). Results: There were 18 EBUS cases of mediastinal lymph nodes using Procore in patients (12 female, 6 male) of average age 57 years (range, 3380 years). FNA was followed by Procore biopsy in all cases, except one where only Procore was performed. Cases included benign (granulomas), atypical, and malignant (carcinoma) diagnoses. More FNAs were satisfactory (with 2 unsatisfactory cases) than Procores (with 7 unsatisfactory cases). The 2 non-diagnostic FNA cases remained unsatisfactory with subsequent Procore. More passes were performed with FNA (4/case) than Procore (2.6/case). On average, FNA provided greater cellularity of lesional material (smear score Z 2; cell block score Z 1.4) than Procore (smear score Z 1.4; cell block score Z 1.4). However, FNAs contained more bronchial contamination (smear score Z 1.6; cell block score Z 1) than Procore (smear score Z 1; cell block score Z 0.4). Both sample types contained similar bronchial and blood contamination. Ancillary studies (special stains, immunostains, FISH, molecular) were equally successful using cell blocks from both specimen types. Conclusions: Our preliminary experience shows that EBUS-guided Procore sampling of mediastinal lymph nodes is feasible. Although FNA samples appear to provide more diagnostic cellular material in this pilot series, fewer subsequent Procore passes were required to achieve satisfactory specimens. Procore specimens also contained less obscuring bronchial contamination. Evaluation of more cases and more experience with this new technique is necessary to determine if the diagnostic yield using Procore during EBUS can be improved.

Introduction: Endobronchial ultrasound (EBUS)-guided sampling of the mediastinum has traditionally been performed by fine needle aspiration (FNA) for cytologic evaluation. The ProCore fine needle biopsy, used primarily to obtain intra-abdominal tissue core biopsies, has not been widely used for EBUS. The aim of this study was to evaluate our experience with EBUS-Procore of mediastinal nodes, and to compare its diagnostic utilization to that of conventional EBUS-FNA. Material and Methods: A retrospective review of all EBUS cases of mediastinal masses using a ProCore needle from December 2013 to March 2014 was performed. Patient demographics, anatomic site, number of procedure passes, on-site immediate evaluation, final pathology reports, and ancillary test results were recorded. All specimen slides and cell blocks were evaluated scoring lesion and contamination cellularity (0 Z acellular, 1 Z scant, 2 Z moderate, and 3 Z abundant). Results: There were 18 EBUS cases of mediastinal lymph nodes using Procore in patients (12 female, 6 male) of average age 57 years (range, 3380 years). FNA was followed by Procore biopsy in all cases, except one where only Procore was performed. Cases included benign (granulomas), atypical, and malignant (carcinoma) diagnoses. More FNAs were satisfactory (with 2 unsatisfactory cases) than Procores (with 7 unsatisfactory cases). The 2 non-diagnostic FNA cases remained unsatisfactory with subsequent Procore. More passes were performed with FNA (4/case) than Procore (2.6/case). On average, FNA provided greater cellularity of lesional material (smear score Z 2; cell block score Z 1.4) than Procore (smear score Z 1.4; cell block score Z 1.4). However, FNAs contained more bronchial contamination (smear score Z 1.6; cell block score Z 1) than Procore (smear score Z 1; cell block score Z 0.4). Both sample types contained similar bronchial and blood contamination. Ancillary studies (special stains, immunostains, FISH, molecular) were equally successful using cell blocks from both specimen types. Conclusions: Our preliminary experience shows that EBUS-guided Procore sampling of mediastinal lymph nodes is feasible. Although FNA samples appear to provide more diagnostic cellular material in this pilot series, fewer subsequent Procore passes were required to achieve satisfactory specimens. Procore specimens also contained less obscuring bronchial contamination. Evaluation of more cases and more experience with this new technique is necessary to determine if the diagnostic yield using Procore during EBUS can be improved.