Made at the 4th Global Precision Medicine and Biomarkers Leaders Summit: Europe by Christopher Bunker. For more information visit http://www.global-engage.com/wp-content/uploads/2017/04/Global-Precision-Medicine-Biomarkers-Europe-2017.pdf. Detection of biomarkers within the morphological context of tissue samples allows to assess interactions between cell type as well as heterogeneity. ACD has developed a novel RNA ISH technology called RNAscope® to detect any RNA species in FFPE and fresh or fixed frozen tissue or cell samples. It can be multiplexed, performed manually or by automation, and in many cases combined with IHC. The unique probe design and signal amplification system allows the visualisation of each individual target RNA molecules as a single dot. The RNAscope assay has been applied in a broad range of research areas, including immuno-oncology, neuroscience, stem cells, inflammation and infectious disease and has been widely adopted in biotech and pharmaceutical R&D in various fields like drug safety, target and biomarker validation, the latter already in the process of the development of companion diagnostics.

1. Expanding Precision Medicine & Biomarker Strategy
Options with RNAscope®
A Clinically‐Validated, Quantitative RNA in situ Platform
Christopher Bunker, Ph.D | Advanced Cell Diagnostics | www.acdbio.com

2. Advanced Cell Diagnostics
RNAscope® ISH for Research and Partnered Diagnostics
900+ Peer‐reviewed publications: >1‐a‐day
>2,000 customers: All Major Pharma and Univ.
>14,000 Target Probes
GMP Manufacturing for IVD

3. >15,000 slides analyzed per year
>150 tissue tested: clinical, archival human, animal
>500 targets profiled for tissue expression
>14,000 catalog probes available
4 weeks TAT for typical projects
RNAscope Pharma Assay Services

4. RNAscope in Precision Medicine & Biomarkers
Single Target Specific | Single Molecule Sensitive | Single Cell Resolution
Biomarker Development
Patient Stratification & Selection
Target Validation & Tissue Profiling

5. PD‐L1 TIM3
CTLA4TIGIT
RNAscope: Precise and Specific Biomarker Detection

6. NSCLC Tumor Microenvironment Expression Patterns of:
PD‐L1 CTLA4 IDO1 IFNG
RNAscope: Precise and Specific Biomarker Detection

7. Ovarian Tumor Microenvironment Expression Patterns of:
TIM3 PD‐1 CD68 CXCL10
RNAscope: Precise and Specific Biomarker Detection

8. NSCLC Immuno‐Oncology Expression Landscape
PD‐L1 + PD1
PD‐L1 + PD‐L2
PD‐L1 + LAG3
PD‐L1 + CTLA4
…….
…….
…….
…….
…….
…….
Immune
CD45+PDL1
CD3+PDL1
CD8a+PDL1
CD44+PDL1
Checkpoint
CTLA4+PDL1
LAG3+FOXP3
HAVCR2+PDL1
ICOS+PDL1
IDO1+IFNg
LAG3+FOXP3
LAG3+PDL1
PD1+PDL1
PDL2+PDL1
TNFRSF18+PDL1
TNFRSF9+ICOS
CD80+PDL1
VTCN1+PDL1
Chemokine /
Cytokine
CSF1+PDL1
CCL2+CCR2
IDO1+IFNg
IL15+PDL1
TGFb1+PDL1
Other
CTNNB1+PDL1
EGFR+MET
KRT19+PDL1
Comprehensive, Precise and Specific Detection

9. RNAscope Universal ISH Platform: “ZZ” Probe Design
Lower region:
~ 18‐25‐base sequence
Upper region:
14‐base tail sequence
Target Probe Set:
Pool of 20 ZZ Pairs
RNA Target Region
1,000 bases
20 pairs of 2x25 bases
Preamplifer binding
site: 28‐base region
Sequence‐specific
binding site:
~ 50‐base region
Probe: ~ 20 ZZ oligo pool. 3 ZZ binding mRNA = Molecular Detection.

10. RNAscope Universal ISH Platform: “ZZ” Probe Design
Non‐specific
RNA
Specific
Target RNA
Probe: ~ 20 ZZ oligo pool. 3 ZZ binding mRNA = Molecular Detection.

11. Signal
Amplification
+ No Background
Single
Molecule Detection
in Single Cells
Universal:
Any Species,
Gene & Tissue
Proprietary
Probe Design
RNAscope Universal ISH Platform

12. Probes +
Detection
Reagents
Fully Automated
Visualize &
Quantify
FFPE
Tissues
Fully Automated for Leica & Ventana Instrumentation

13. RNAscope: Single Molecule Sensitivity
Wang, F. et al. RNAscope: A Novel In Situ RNA Analysis Platform for Formalin-Fixed Paraffin-Embedded Tissues. J of Mol
Diagnostics (2012). doi:10.1016/j.jmoldx.2011.08.002
Measuring HER2 mRNA in HeLa by RNAscope and in Extracted RNA

14. R = 0.84
RNAscope is Equivalent to qPCR in Sensitive
R = 0.91
FFPE Breast Cancer Tissue FFPE Cell Pellets

15. 0 1‐3 4‐9 10‐15 >15
Histogram of dots/cell
Expression H‐score from Cell‐by‐Cell Quantitative Analysis
Quantification of RNA Molecules Per Cell with HALO®

16. Cell‐by‐Cell Expression and Co‐Localization Analysis: PD‐L1 and LAG3
Cell‐Specific Quantification of Checkpoint Controls

17. Tumor vs Stromal Expression: PD‐L1 and LAG3
44.0361
20.2018
30.3217
20.7873
62.4328
44.6096
0
10
20
30
40
50
60
70
Stromal Region Tumor Region Entire CoreLAG3 PD‐L1 LAG3 PD‐L1 LAG3 PD‐L1
Positive Cells %
Entire
Core
Stromal
region
Tumor
region
T
S
Cell‐Specific Quantification of Checkpoint Controls

18. Cell‐Specific Quantification of Any Checkpoint Controls
NSCLC Immuno‐Oncology Analysis: % Cell Positive Heat‐Map

19. PD‐L1 CD8
PD‐L1 TGFB
PD‐L1 LAG3
PD‐L1 4‐1BB
Visual & Quantitative Analysis of Multiple I‐O Markers

20. IFNG expressed by
CD8‐negative cells in
tumor microenvironment
RNAscope Probes:
Hs‐CD8A (Green)
Hs‐IFNG (Red)
Rare Cell Detection: CD8‐, IFN Cells in NSCLC TME

21. Rare Cell Detection: CD3+, IFN TILs in NSCLC TME
IFNG expressing
infiltrating CD3+ T cell in
tumor microenvironment
RNAscope Probes:
Hs‐CD3D (Green)
Hs‐IFNG (Red)

22. Breast Cancer
(FFPE)
KRT19
CD8A
CD4
CD68
CD4
CD8
CD68
Multiplexed Cell Marker & Biomarker Detection

23. Liver Cancer
(FFPE)
KRT19
CD31
Ki67
CD45Ki67
CD31
CD45
Multiplexed Cell Marker & Biomarker Detection

24. RNAscope Universal ISH Platform: “ZZ” Probe Design
Non‐specific
RNA
Specific
Target RNA
Probe: ~ 20 ZZ oligo pool. 3 ZZ binding mRNA = Molecular Detection.

25. Positive Control, PPIB
RNAscope Data Quality Control: ~ 90% QC Pass
Target: PD‐L1Negative Control, dapB
Quality Controlled ISH Eliminates False Positives and False Negatives.

26. No Probe HPV‐16 HPV‐18 HPV‐45
Eff Tm = 81.5 + 16.6(log M [Na+]) + 0.41(%G+C) – 0.72(% formamide)
HPV E6/E7 Oncogene ~85%
Identical
No Cross-Hybridization @
85% Homology
RNAscope Specificity: Calculated and Predictable

27. RNAscope – RNAseq and Target Validation
Visual, cell‐specific assessment of disease‐associated tissue expression
RNAscope
Any/All Candidates
Probes in 2 weeks
Data in 1 day

28. Transcriptomics Discovery RNAscope Validation
“Validation of esophageal squamous cell carcinoma candidate genes from high‐
throughput transcriptomic studies.” Du et. Al (2013).
2990 genes > 2‐fold 
KRT17, EpCAM, CD44: >>2‐fold
Tumor cells Normal cells
Microarray NormalTumor
KRT17 CD44 EpCAM

29. RNAscope Collagen 11A1 Ovarian Cancer Biomarker
“A collagen‐remodeling gene signature regulated by TGF‐β signaling is associated
with metastasis and poor survival in ovarian cancer.” Cheon, et. al (2014).
COL11A is expressed
in inter/peri‐tumor
stromal cells, not
tumor cells
Better Dynamic Range and Lower Detection Limit Than IHC

30. PD‐L1: Greater Sensitivity of RNAscope versus IHC
RNAscope IHC: SP263 IHC: 28‐8
H Score: 0 H Score: 0H Score: 22
0.00
50.00
100.00
150.00
200.00
250.00
2C1 1G4 1E1 2C4 1D1 1F2 1B5 1A5 1G2 1C3 2C3 1D2 1F3 1F1 2G1 1C5 1C4 1E2 1C2 1B4 1D4 1E4 2B3 2A2 1E5 2E3 1A4 1C2 2G3 2F1 1B3 1A2 1B1 1F4 2D4 1D3 2F4 2E1 1B2 1D5 2F3 2F2 2G4 1G3 2A3 2D3 2B5 2D2 1A3 1E3 2D1 1C1 2A5 2E2 2G2 2B2

31. Cancer Immunity Cycle – RNAscope Assays for All
Chen & Mellman, Cell

32. Hs‐IDO1 in human esophagus cancer ACD045 4+ Hs‐IFNG in human vulvar cancer ACD056 3+
Hs‐ICAM1 in human lung cancer ACD072 2+ Hs‐ICOS in human stomach cancer ACD172 2+
Immuno‐Oncology Assays: ICAM1, ICOS, IDO1, IFNG

33. Hs‐CCL7 in human ovary cancer ACD318 2+ Hs‐CD3 in human lymphoma cancer ACD280 4+
Hs‐CD27 in human lymphoma cancer ACD280 4+ Hs‐CD70 in human lymphoma cancer ACD280 2+
Immuno‐Oncology Assays: CCL7, CD3, CD27, CD70

34. Immuno‐Oncology Assays: CX3CL1, CX3CR1, CXCL10, 13
Hs‐CX3CL1 in human lung cancer ACD219 4+ Hs‐CX3CR1 in human liver cancer ACD206 2+
Hs‐CXCL10 in human breast cancer 59083‐2 3+ Hs‐CXCL13 in human stomach cancer ACD226 4+

35. Hs‐CXCL9 in human stomach cancer ACD160 4+ Hs‐FOXP3 in human stomach cancer ACD172 3+
Hs‐GNLY in human lymphoma cancer ACD280 4+ Hs‐HAVCR2 in human testicular cancer ACD148 4+
Immuno‐Oncology Assays: CXCL9, FOXP3, GNLY, TIM3

36. Hs‐IL17A in human stomach cancer ACD213 3+ Hs‐IL18 in human esophagus cancer ACD045 2+
Hs‐IL20 in human bladder cancer ACD177 2+ Hs‐IL21 in human lymphoma cancer ACD279 3+
Immuno‐Oncology Assays: IL17A, IL18, IL20, IL21

37. Hs‐IL17A in human stomach cancer ACD213 3+ Hs‐IL18 in human esophagus cancer ACD045 2+
Hs‐IL20 in human bladder cancer ACD177 2+ Hs‐IL21 in human lymphoma cancer ACD279 3+
Immuno‐Oncology Assays: TNFA, 4‐1BB, OX40, OX40‐L

38. BaseScope: Advancements in RNAscope Technology
RNAscope 2.5
• Routine mRNA detection
• 3 ZZ oligo pairs = Visible
molecular detection
Amplification
Target‐specific
binding
Amplification
Target‐specific
binding
BaseScope Platform
• Isoforms and splice junctions
• Point mutations
• 1 ZZ oligo pair = Visible
molecular detection

39. MET 14
(H596)
MET WT
(A549)
BaseScope Assay for c‐MET Exon 14 Deletion
E13/15E13/15E12/13E12/13 E14/15E14/15 DapBDapBBaseScope Probes:

40. BaseScope Detection of KRAS Mutations in CRC
Cell Lines:
KRAS G12 (H29) and G12V (SW620) Cells
Archival Colorectal Cancer:
KRAS G12 and G12D

41. BaseScope Detection of KRAS Mutations in CRC

42. CAR Protein: Human
– Indistinguishable
from endogenous
RNAscope CAR‐T Cell Tumor Infiltration Analysis
CAR mRNA: Viral UTRs
– Unique tags for
RNAscope detection

43. RNAscope CAR‐T Cell Tumor Infiltration Analysis
RNAscope CAR Viral UTR Probe
Novartis/UPenn: O’Rourke et al., Sci. Transl. Med.,19 July 2017

44. Novartis/UPenn: O’Rourke et al., Sci. Transl. Med.,19 July 2017
RNAscope CAR‐T Detection Post‐Treatment in GBM
RNAscope CAR Viral UTR Probe

47. RNAscope Patient‐Selection in Phase 2 Clinical Trial
• Phase I: Demonstrated significant
correlation between RNAscope NRG1
and therapeutic benefit when treated
with MM‐121 + standard therapies
• RNAscope to identify patients with
Heregulin (NRG1) positive, locally
advanced or metastatic NSCLC.
• Enrolling patients with high NRG1
+ Control Probe - Control Probe
Identify
HRG+
patients
Merrimack Pharmaceuticals' MM‐121 anti‐ErbB3
using RNAscope Heregulin Biomarker Assay

48. Leica Biosystems and Merrimack Pharmaceuticals
to Collaborate on Development of a Heregulin
Companion Diagnostic for Seribantumab
NEWCASTLE UPON TYNE, England and CAMBRIDGE, Mass., April 14, 2016 /PRNewswire/ ‐‐ Leica Biosystems
and Merrimack Pharmaceuticals (NASDAQ: MACK) today announced a strategic partnership to develop
companion diagnostic assays to aid in identification of patients who might best respond to current and future
oncology therapies developed by Merrimack. The assays will be designed using Advanced Cell Diagnostics'
(ACD's) RNAscope technology and will be processed on Leica Biosystems' BOND clinical advanced staining
systems.

49. “FGFR1 RNA and Protein Expression, not Gene Copy Number,
Predict FGFR TKI Sensitivity Across All Lung Cancer Histologies”
Wynes (2014) Clin. Can. Res.
RNAscope Identifies FGFR TKI Responsive Patients

50. Bayer Develops Companion Diagnostic Test for
Cancer Patients Together with Leica Biosystems
Leverkusen,December 21,2016 - Bayer has entered into agreement with Leica Biosystems to
collaborate on the development of companion diagnostic tests based on tissue samples using
RNAscope® (a multiplex nucleic acid hybridization technology)...
Companion diagnostics network supports Bayer's personalized medicine approach… Companion
diagnostic tests aim to unlock molecular information from each patient to guide treatment decisions
with targeted medications for cancer.

51. RNAscope Platform: In Situ Expression Analysis
Any mRNA: specific exons, isoforms, mutations
Single‐Molecule Sensitivity & Quantitative
Multiplexed expression analysis
Tissue‐context and cell‐specificity
Specific Fast and Efficient