Introduction Cancers may escape immune surveillance and eradication through the expression of programmed death-

ligand 1 (PD-L1) on tumor cells and in the tumor microenvironment. PD-L1 expression has been reported in

various cell populations within the tumor, and its expression associated with prognosis for various tumors.

Further, clinical studies have shown that this pathway is an important target for immunotherapy and PD-L1

expression on tumor cells and in the tumor microenvironment has been associated with enhanced response.

Understanding the complex biological function of the PD-L1 pathway, not only on tumor cells but also within

the tumor microenvironment requires simultaneous interrogation of multiple biomarkers, ranging from cancer

immunology checkpoint markers, tumor infiltrating immune cell markers, and tumor specific markers, etc.

Multiplex immunohistochemistry (IHC) allows simultaneous detection of multiple markers to explore the

cellular composition of immune/stromal/cancer cells in the tumor microenvironment. Development of a

multiplex IHC assay remains challenging due to antibody species similarity and cross reactivity, stability of

fluorophores through multiple rounds of processing, balancing high and low signals and measurement of

weakly expressed markers. We present here the development of a fully automated multiplex IHC assay (PD-

L1, CD3, CD8, CD68 and FoxP3) using rabbit primary antibodies with a heat deactivation (HD) process

between each antigen staining cycles on the BenchMark ULTRA automated slide stainer.

Automated PD-L1 5Plex Fluorescence (FL) IHC Sequential Detection Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5

The staining was automated on BenchMark ULTRA automated slide stainer with total run time of ~10 hours for up to 30 slides.

HD is Effective to Prevent Cross-reactivity (tested on 3 NSCLC and 3 UBC specimens)

• Each Ab(+) ctrl slide was stained with the tested 1°Ab at 1X[ ], GaR-HRP and the TSA-fluor at the respective cycle, while

the other cycles used diluents to replace 1°Abs, GaR-HRP and TSA-fluors.

• Each HD slide was stained with the tested 1°Ab at 2X[ ], GaR-HRP, and TSA diluent at the respective cycle, followed with

1°Ab diluent, GaR-HRP and the TSA-fluor at each of subsequent cycles.

• Diluents were used in prior cycle(s) when evaluating HD on CD3, CD68, FoxP3 and PD-L1 Ab complex.

• HD on PD-L1 Ab complex (last cycle) was evaluated with FAM.

An Automated 5plex Fluorescent Immunohistochemistry Enabled Characterization of PD-L1 Expression and Tumor

Infiltrating Immune Cells in Lung and Bladder Cancer Specimens

www.roche.com www.ventana.com © 2016 Ventana Medical Systems, Inc., the BENCHMARK logo is the trademark of Roche. All other trademarks are the property of their respective owners.

CD8

Rb anti-CD8

(SP239) Spring Bio sci.

Cat # M5394)

HRP

TSA-R6G

CD3

Rb anti-CD3

(SP162) Spring Bio sci.

Cat # M4624)

FoxP3

Rb anti-FoxP3

(SP97) Spring Bio sci.

Cat # M3970)

CD68

Rb anti-CD68

(SP251) Spring Bio sci.

Cat # M5514)

PD-L1

Rb anti-PDL1

(SP142)

HD 1°/2°Ab complex HD 1°/2°Ab complex

Goat anti-

rabbit-HRP (Ventana Cat

# 253-4413

from #760-098)

Goat anti-

rabbit-HRP (same as the left)

HRP Goat anti-

rabbit-HRP (same as the left)

HRP Goat anti-

rabbit-HRP

(same as the left)

HRP Goat anti-

rabbit-HRP (same as the left)

HRP

TSA-DCC TSA-Red610 TSA-Cy5 TSA-FAM

1

PD-L1 5Plex FL IHC Assay is Reproducible

CD3

PD-L1 5Plex FL Staining (Decomposited Single-Channel Images)

Comparable to Single DAB Staining in a NSCLC Case

Conclusion Automated PD-L1 5Plex FL assay was developed using 5 rabbit 1°Ab for NSCLC and UBC specimens on

BenchMark Ultra instruments.

• The PD-L1 5Plex FL assay staining performance is reproducible, as no significant difference of staining

was observed across 3 instruments and 3 non-consecutive days.

• The PD-L1 5Plex FL assay staining has high agreement to that by single DAB staining.

• This automated PD-L1 5-Plex IHC assay could be utilized as a reliable and reproducible tool to further

characterize tumor and the tumor microenvironment. This could be used to gain a better understanding of

which patients may benefit most from immunotherapies.

PD-L1 5Plex FL IHC Agreement with Single DAB IHC

CD8(+) ctrl staining with R6G

HD CD8 Ab complex, detected with DCC

100 ms

25 ms lung lung

lung lung

lung

lung 100 ms

25 ms

100 ms

25 ms

CD3(+) ctrl staining with DCC

HD CD3 Ab complex, detected with Red610

CD68(+) ctrl staining with Red610

HD CD68 Ab complex, detected with Cy5

FoxP3(+) ctrl staining with Cy5

HD FoxP3 Ab complex, detected with FAM

PD-L1(+) ctrl staining with FAM

HD FoxP3 Ab complex, detected with FAM

HD 1°/2°Ab complex HD 1°/2°Ab complex

• Duplicate slides of 3 non-small cell lung cancer (NSCLC) and 3 urothelial bladder cancer (UBC) specimens were stained on 3 BenchMark Ultra instruments in 3 non-consecutive days.

• Each dot represents the average mode of intensity from 10 ROIs that were randomly selected from a region that is present across the sections of the same specimen.

• General linear model (GLM) analysis was used to perform analysis of variance upon the two factors – instruments and run (duplicate slides within an instrument).

• Intra-class correlation (ICC) was used to evaluate instrument reproducibility

GLM: instruments, p=0.407; run, p=0.502; ICC=0.45 GLM: instruments, p=0.139; run, p=0.419; ICC=0.40 GLM: instruments, p=0.420; run, p=0.275; ICC=0.87

GLM: instruments, p=0.153; run, p=0.158; ICC=0.58 GLM: instruments, p=0.293; run, p=0.325; ICC=0.85 GLM: instruments, p=0.166; run, p=0.341, ICC=0.73

DAPI PDL1 CD68

FoxP3 CD8 CD3

HE NSCLC

PD-L1 5Plex FL Staining of a NSCLC Case

CD3

CD8

CD68

FoxP3

PD-L1

1. Digital pathology tools: Number of positive cells (CD3, CD8, FoxP3) and % of positive area (PD-L1, CD68) from the same regions

of interest in tumor areas were compared between 5Plex FL and single DAB IHC staining by digital pathology tools.

Wenjun Zhang1, Antony Hubbard1, Nick Cummins1, John Hurley1, Mehrnoush Khojasteh1a, Joerg Bredno1a, Liping Zhang1, Ehab Elgabry1, Xiaoling Xia1, John Palting1, Dustin Harshman1, Frank Ventura1, Karl Garsha1, Zhenqiang James Lu1, Jorge Lozano1, Marcin Kowanetz2, Sanjeev

Mariathasan2, Dustin Smith2, J. Andrew Williams2, Lidija PesticDragovich1, Larry Morrison1, Lei Tang1 ; 1Ventana Medical Systems, Inc., Tucson, AZ; 1a Ventana Medical Systems, Inc., Mountain View, CA; 2Genentech, Inc., South San Francisco, CA

2. Manual pathological assessment : Overall % of PD-L1 (+) tumor cells (TC) and immune cells (IC) of each specimen were

compared between FL and DAB staining by pathologists’ evaluation of the scanned images.

𝑅2: Squared Pearson correlation measures

the linear correlation between two

methods

𝜌𝑐 : Concordance correlation coefficient

measures the agreement between two

methods

Composite 5-channel image

All images were taken under 20X