Quanticell™, a novel histopathology technology for visualization andquantitation of membrane PSMA demonstrated in cell lines and clinical tissue

Poster # 3192
Contact: Hemi Dimant, PhD
E-mail: hdimant@Invicro.com

Hemi Dimant1, Ankit Gandhi1, Michael Maker1, Adam Markman1, Jeanette Reinhardt1, Stefan Collins1, Jacob Hesterman1, Joseph Krueger1, Kenneth Bloom1, Julie Bailis2, Gloria Juan2

Background

1Invicro, A Konica Minolta Company, Boston, MA; 2Amgen, South San Francisco, CA; 3Amgen, Thousand Oaks, CA

68Ga-PSMAPET is a highly promising modality for staging
prostate cancer due to its higher detection rate compared toconventional imaging techniques. Both PET/CT and PET/MRI
combined with PSMA radiotracers are able to detect tumors
with very high levels of PSMA expression. However, PSMA-PET
lacks the sensitivity to distinguish low versus high expression.
Radioconjugates, such as Lutetium-177 (177Lu)-PSMA-617, andPSMA-BiTE® molecules (AMG 212, AMG 160) have been used todetect PSMA-positive disease. PSMA-PET and tissue-based
immunoassays have been used to evaluate PSMA expression inclinical tissues, however, standard immunohistochemistry andimmunofluorescence assays are not sensitive enough to detect
low PSMA expression in prostate cancer biopsies. Lack ofsensitivity may prevent proper response predictions and could
deny anti-PSMA treatment to qualified patients that harbor low
PSMA expression. Here, we present a new histopathology
assay, Quanticell, to enable the sensitive and specificdetection of PSMA in prostate tumors.

Specificity

Sensitivity

Comparison Between Quanticell Detection and DAB Detection Using Comparable Analysis Workflow

Specific Signal Only in PSMA-Expressing Cell LinesObjective
These studies were designed to evaluate Quanticell, a newassay based on novel phosphor-integrated dots (PID)
detection technology on PSMA-expressing cell lines andclinical tissue. The Quanticell technology was compared tostandard immunohistochemistry for:• Specificity• Sensitivity• Dynamic Range• Quantification

Methods

B

Novel Nanoparticle-based Technology

Quanticell Schematic. Quanticell consists of highly fluorescent, extremely uniform streptavidincoated nanoparticles. Quanticell technology involves a standard immunostaining workflow using
commercially available primary antibodies and biotinylated secondary antibodies.

QuantitativeGonda et al (2017). Science Reports, 8;7(1):7509

Quanticell Quantitation. The number of PID particles per cell based on fluorescence microcopy
and electron microscopy were plotted against the respective fluorescence intensity. A
dedicated analysis software was developed to quantitate the number of particles in microscopy
images.

Figure 1: The Quanticell protocol was developed on several celllines with different levels of PSMA expression. Specific PSMA staining
is demonstrated on high-expressing CHO-PSMA cells (A, lowerpanel), as compared to lack of signal in the non-expressing CHOcells (A, upper panel). VCAP prostate cancer cells, which express
low levels of PSMA were used to optimize the protocol (data notshown). (B) Quantitation of the Quanticell signal in CHO, VCAP andCHO-PSMA cell lines. Positive PSMA staining was determined in theVCAP cell line evident by signal that is higher than 3 standard
deviations above negative control.

Quantification and Dynamic Range
AB

BAA

Data Acquisition Process In Clinical Samples

Membrane Analysis Workflow: Input FOV Image

Figure 2: The sensitivity of the Quanticell assay was compared tochromogenic diaminobenzidine (DAB) for detection of PSMA. (A)Cells were stained by the Quanticell method (upper panel) andfor DAB (lower panel). We developed comparable analysis
workflows to quantitate the percent PSMA-positive cells
identified by each technique. In both detection methods, weused hematoxylin to identify and count the number of cells.
Fluorescent Quanticell signal or chromogenic DAB signal werethen overlayed on the hematoxylin image to determine % cells
that are PSMA-positive (B). The Quanticell assay identified PSMApositive VCAP cells with significant separation from the PSMAnegative CHO cells, demonstrating the improved sensitivity ofQuanticell in detecting low PSMA expression levels.

C PID Scores Provide Significant Class SeparationCD

Total PSMA Score

Correlation Between Total and Membrane PSMAMembrane PSMA Score

Summary
The Quanticell technology provides asensitive, specific method to quantitate target
expression levels and can be applied forevaluation of human tumor samples

Specificity➢ Anti-PSMA PID staining shows highspecificity of detection of PSMA
Sensitivity➢ An optimal signal to noise ratio wasachieved with a high PSMA-specific signal
Eand minimal background staining
Variance in Quanticell Signal Across Clinical Samples ➢ PID provides increased sensitivity indetecting low PSMA-expressing cells, ascompared to standard IHC
Quantification➢ The Quanticell assay is suitable forquantitation of PSMA levels per cell
Dynamic Range➢ The Quanticell assay has the potential toevaluate PSMA expression across a broad
range of expression levels

Figure 3: (A) From the whole slide image of stained clinical samples, high magnification fluorescence microscopy was used to acquire multiple fields of view (FOV) across the regions containing tumor cells. The image
acquisition was performed in three channels: PSMA staining by PID (red channel), sodium potassium ATPase to mark the cell membrane (Alexa Fluor 488), and hematoxylin staining to mark the cell nuclei (brightfield image). (B)To enable analysis of membrane-associated PSMA, the cell membrane was segmented and a mask of the membrane was applied to the Quanticell or PID channel. (C) Using an established imaging and image analysis
workflow, a proprietary algorithm for analysis was developed and conducted to score total PSMA expression (upper plot) and membrane-specific PSMA expression (lower plot). Analysis was conducted on biopsies from healthysubjects (n=5) and prostate cancer subjects (n=15). The PSMA signal (PID/cell) was determined per FOV, represented by each data point, with 5 FOVs analyzed per subject. (D) High correlation was observed between the total
PSMA score and the membrane-PSMA score. (E) From the total PSMA analysis, the highest FOV scores per subject across all 20 subjects were plotted to demonstrate the variance between subjects and the dynamic range of
Quanticell detection method.

Disclosures
This study was funded by Amgen, Inc.
JB is employed by and holds stock in Amgen Inc.
JG was employed by and held stock in Amgen Inc. at thetime the work was done.