P663
Evaluating Loss of STING/cGAS Expression Using a Novel Multiplex Immunohistochemistry Detection Approach
Apollina Goel1, Jeanette Rheinhardt1, Adam Markman1, Hideki Goda2, Etsuko Futaya2, Masaru Takahashi2, Hiroyuki Yokota2, Kenji Nishikawa2, Hisatake Okada2, Kenneth Bloom1, George Abe1, Joseph Krueger1
1Invicro, LLC, Boston, MA; 2Konica Minolta Inc. Bio-Healthcare unit, Hino-shi, JapanThe cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) pathway an important target in theexpanding landscape of next-generation immunotherapies (1-3). STING agonists can induce systemic anti-tumor
immunity and thus may be also synergistic with other immunotherapies (i.e. immune checkpoint blockade, cancer
vaccines, and CAR-T cell therapy). Absence of cGAS-STING pathway regulates response to oncolytic virotherapy(2). Advanced stages of solid cancers (i.e. melanoma, colon adenocarcinoma) frequently show loss of STING/cGAS
and would not be expected to respond to STING agonists (1, 3). There is an emerging need for predicting patient
response which can accurately classify patients according to STING/cGAS expression.

Quanticell Score (particles/cell ± SEM)

Immunoblotting*

STINGcGAS

Background

STINGcGAS

A375

39.0 ± 3.8

16.9 ± 0.5

7.9 ± 0.4

STINGlowcGAS-ve

SK-MEL-28

127.9 ± 6.0

164.0 ± 8.1

15.7 ± 0.3

STING-/+cGASlow

STING
AgonistIn this study, a novel immunohistochemistry (IHC)-based assay using fluorescent nanoparticles(Quanticell™) was tested for highly sensitive and quantitative analysis of STING and cGAS within thetumor contexture. The technique provides detection with high sensitivity (100-fold greater brightness
than quantum dots enabling the detection of analytes not detected by conventional IHC methods),
signal quantitation (300-fold greater linear dynamic range than IHC with no amplification steps toensure robustness) with contextual visualization (pathologist validation at cellular and sub-cellularresolution) to allow for quantitative evaluation of gene products in clinical tissues.

IHC

Quanticell™
C408300 sample with STING/cGAS PID scores of 108 ± 11
and 108 ± 13, respectively. H&E (A), chromogenic IHC (B)and representative images of one FOV (C, overlay of while& dual FL image) are shown. This sample is STING+/cGAS+
by Quanticell assay. IHC shows strong cGAS staining but
STING staining is difficult to visualize. In contrast,visualization and quantification of cGAS and STING is clear
with Quanticell.

The Quanticell™ assay provides STING/cGAS scores forcell lines above the background signal, andperformance was compared to cell lines with low
protein expression of STING/cGAS as determined byWestern blotting. *Adapted from (2)C408305 sample with STING and cGAS PID scores of 36 ± 1
and 17 ± 8, respectively. H&E (A), chromogenic IHC (B) andrepresentative images of one FOV (C, overlay of while & dual
FL image) are shown. This sample is STING+/cGAS+ byQuanticell assay. IHC shows weak cGAS staining and weak
STING staining. In contrast, visualization and quantificationof cGAS and STING is clear with Quanticell

STING/cGAS expression in control tissues for dual-color IHC or Quanticell™ assay▪ A corresponding IHC assay was utilized to gain information on robustness of preanalytical or analytical phase testing(5). Dual color chromogenic IHC was developed using a normal skin control.
▪ IHC analysis of STING/cGAS in melanoma shows infiltration of immune and lympho-vascular cells, both cell types are
cGAS+STING+ (1), as also shown by chromogenic IHC.
▪ In the Quanticell™ assay, staining performance and scores were calibrated against tumor adjacent skin, where cGAS
and STING expression are present in discrete layers of the dermis.

C408299 sample with STING/cGAS PID scores of 86 ± 14
and 96 ± 25, respectively. H&E (A), chromogenic IHC (B)and representative images of one FOV (C, overlay ofwhile& dual FL image) are shown. This sample isSTING+/cGAS+ by Quanticell assay. IHC shows cGAS andSTING staining but is difficult to distinguish. In contrast,visualization and quantification of cGAS and STING is clear
with Quanticell.

▪ Quanticell™ showed STING expression in all 4 layers of the epidermis (stratum corneum, granulosum, spinosum andbasale. However, cGAS expression is noted as localized to the stratum basale layer.

Study Aims
▪ Establish dual color Quanticell™ IHC assay by utilizing established melanoma cell lines, to measure STING/cGAS expression.
▪ Assess utility of Quanticell™ assay in biomarker evaluation of STING/cGAS in clinical melanoma samples.
▪ Compare Quanticell™ assay with chromogenic IHC to detect STING/cGAS expression in melanoma.

Clinical melanoma samples scored using the Quanticell™ assay for STING/cGAS expression▪

Quanticell™ Score: Tumor-adjacent Normal Skin

Background▪
N=24 (metastatic melanoma)▪ In melanoma, STING pathway is deregulated at various nodes and also with disease progression (1, 2).
▪ High expression of TMEM173 predicts favorable prognosis of metastatic melanoma patients treatedwith monoclonal antibody against CTLA-4 (1).
▪ An accurate measurement of STING/cGAS in tumor cells is paramount for STING agonist therapy(cGAS+STING+, cGAS-STING+) and oncolytic virotherapy (cGAS-STING-).
▪ The requirement for non-DAB detection in melanoma (due to melanin) poses challenges to IHC.

H&E

Validation of dual-color Quanticell™ assay using established melanoma cell lines

STING signaling via cGAS or agonists activate
innate immune response.

Schematic workflow of Quanticell™. The technique utilized regular IHC
primary antibody, biotinylated secondary antibody, and detection
using phosphor integrated fluorescent dots (PIDs). The assay offers afullylinear,quantitative
assay
that
extendedbeyonddetectable/saturated range of DAB. Adapted from (4).

Representative cGAS/STING Staining in Melanoma

Results

Introduction

38%

▪
▪

TMEM173 expression and OS (I-O therapy)

The median Quanticell™ score for STING and cGAS in tumor-adjacent normal skin was 37.2 and15.6, respectively.
The medianQuanticell™ score for STING and cGAS detection threshold for cell lines was 39.0 ± 3.8
Visualizationand 16.9 ± 0.5, respectively (A375 cell line).
72% samples showed tumor adjacent skin that could also be scored for STING and cGAS expression.
Based on the Quanticell™ score in internal control tissue, samples that showed altered expression for➢➢42%

STING: n=5; C408312, C408307, C408303, C408298, C408296
cGAS: n=3; C408313, C408299, C408298

C408313
C408312cGAS Quanticell score

Clinical samples: Archived clinical melanoma blocks (FFPE, n=20) from primary tumor site (stage III or IV) werepurchased (BioChain Institute Inc.). Sections (4 µm) were stained by chromogenic IHC and H&E (Invicro Boston) orQuanticell™ assay.
Chromogenic IHC: Dual-color IHC was performed sequentially using α-STING antibody (LSBio LS-C337801, 1:6,000dilution) and α-cGAS (Abcam ab224144, 1:300 dilution) with Vina Green kit (Biocare BRR807AH, HRP) or Leica Red kit(DS9390, AP), respectively using antigen retrieval (pH 9, 10 min) and staining (Bond RX, Leica Biosystems).
IHC-PID: We utilized Quanticell™ (Ver 1.1) that offer 2-color PID staining and image analysis. Sections were stainedwith α-STING or α-cGAS antibody (control, no primary antibody), biotinylated secondary antibody, followed by preassembled PIDs (green or red, respectively). Slides were counter stained by hematoxylin. For each clinical sample,
fluorescent images of both tumor region and internal “normal” skin were captured using DP80 camera (Olympus, 3-7
microscopic fields).
Image recognition algorithms: The number of PIDs on the digital image was calculated based on the light spot profile.
The number of PID particles/ unit area (100 μm2) was calculated for both fluorophores.
Data analysis: The IHC-PID data was visually compared to IHC-chromogenic data and Quanticell™ values provided.

cGAS Quanticell score

C408313

STING Quanticell score

C408307
STING Quanticell score

C408309

Methods

MedianC408312

C408310

Quanticell™ Score: Melanoma

C408305

C408308

C408303

C408307

C408302
C408300

C408306

C408299

C408305

C408298

C408303

C408297

C408302

C408296

C408301

C408295
C408294

C408300

0C408299

Nucleusrecognition

Bright spots
recognition

20406080100120140

C408298
C408297
C408296

Quanticell™ Score: Melanoma

C408295
C408294
020

4060

80

100

120

14011%
6%

Number or Area of nucleus Number of particles
Statistical Processing

Conclusions

Quanticell™ Score: Tumor-adjacent Normal Skin

Quanticell™ Score: Melanoma

12% 8%

C408298 sample with STING and cGAS PID scores of 80 ± 12
and 46 ± 9, respectively. H&E (A), chromogenic IHC (B) andrepresentative images of one FOV (C, overlay of while &dual FL image) are shown. This sample is STING+/cGAS+ byQuanticell assay. IHC shows strong cGAS staining but STING
staining is difficult to visualize. In contrast, visualization andquantification of cGAS and STING is clear with Quanticell.

STING+/cGAS+
44%

Quanticell™ assay results from tumor analysis show that:➢ 50% of patients have below average STING expression and may not be candidates for STING agonists.
➢ 17% patients have below average cGAS expression and may not be candidates for oncolytic virotherapy.

STING-/cGAS+
STING+/cGAS-

39%STING-/cGAS-(n=18)

160▪ STING/cGAS expression in the Grade III/IV melanoma samples analyzed varied widely. STING-IHC showed focal
nuclear and cytoplasmic positivity in tumor-associated skin and endogenous immune cells as expected, while cGASIHC showed dense heterogeneous, cytoplasmic and nuclear staining in melanoma.
▪ The Quanticell™ assay for STING showed downregulation to below average in 50% of the samples; it is not clear if
these patients will benefit from STING-agonist treatment compared to their counterparts.
▪ The Quanticell™ assay for cGAS showed downregulation to below average in 17% of the samples; it is not clear if
these patients will benefit from oncolytic viral therapy compared to their counterparts.
▪ In contrast to IHC, the Quanticell™ assay enabled clear, simultaneous, and sensitive visualization and quantitative
scoring of STING/cGAS expression, and may have value for predictive and prognostic application in STING agonistbased therapies and oncolytic viral therapy.

References
1. An X et al. An analysis of the expression and association with immune cell infiltration of the cGAS/STING pathway in pan-cancer. Mol Ther Nucleic Acids. 2019; 14:80-89.
2. Xia T et al. Recurrent loss of STING signaling in melanoma correlates with susceptibility to viral oncolysis. Cancer Research, 2016; 76:6747-6759.
3. Xia T et al. Deregulation of STING signaling in colorectal carcinoma constrains DNA damage responses and correlates with tumorigenesis. Cell Rep, 2016; 14:282-97
4. Gonda K et al. Quantitative diagnostic imaging of cancer tissues by using phosphor-integrated dots with ultra-high brightness. Sci Rep. 2017; 7:7509.
5. Torlakovic E E et al. Getting controls under control: the time is now for immunohistochemistry J Clin Pathol. 2015; 68(11): 879–882