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10123 - PD-L1, Melanoma, TME

INTRODUCTION
Programmed death-ligand 1 (PD-L1) is a transmembrane protein that binds to the inhibitory receptor programmed death 1 receptor (PD-1), causing a down regulation of immune responses. PD-L1 is typically expressed on normal cells but has been observed in immune cells and tumor cells[1] while PD-1 is typically expressed on cytotoxic T-cells and other immune cells. Tumor cells can upregulate PD-L1 expression and avoid being attacked by the body’s immune system, making an interruption of the PD-1/PD-L1 interaction an attractive method for assisting the immune system in destroying tumor cells[2].

This APP can be used to determine the proportion of PD-L1 positive cells. It quantifies all cells and classifies PD-L1 positive cells based on brown/DAB positive membrane staining.

KEYWORDS
PD-L1, PD-1, T-cell, cancer, oncology, IHC, membrane, skin, melanoma, digital pathology, image analysis, B7-H1, hematoxylin

METHODS
A hematoxylin input band is filtered with a median filter and combined with image intensity to smoothen out and mitigate granularity of nuclei. Nuclei are then detected using two different size scales of polynomial blob filters in combination with polynomial local linear filters that delimit the cells. Membranes are detected using two different scales of polynomial local linear filters on a DAB input band. Postprocessing steps grow the nuclei blobs and combine/separate adjacent nuclei appropriately to match the hematoxylin band. The classified PD-L1 positive membrane staining is verified by size, shape and intensity constraints and then applied to the adjacent cells to quantify the number of PD-L1 positive cells.

QUANTITATIVE OUTPUT VARIABLES
The output variables obtained from this protocol are:

  • Negative Cells: Number of negative cells
  • PD-L1 Positive Cells: Number of PD-L1 positive cells
  • Total Cells: Number of total cells
  • PD-L1 Positive Percentage: Percentage of PD-L1 positive cells relative to total number of cells

AUXILIARY APPS
APP: 01 Tumor Detect
The Tumor Detection APP is used for initial tumor region detection and is included with the APP.

WORKFLOW
Step 1: Use the Tumor Detect APP and/or outline tumor regions manually if necessary.
Step 2: Run the PD-L1 APP. Click the save button to transfer the results to the database.

STAINING PROTOCOL
Staining protocols have been developed by Dako and Ventana for the following three assays which have been found to produce comparable results[3] and are available from their website(s):

  • Dako 22C3
  • Dako 28-8
  • Ventana SP263

ADDITIONAL INFORMATION
The APP utilizes the EngineTM and Viewer software modules, where EngineTM offers an execution platform to expand processing capability and speed of image analysis. Viewer gives a fast review together with several types of image adjustment properties ex. outlining of regions, annotations and direct measures of distance, curve length, radius, etc. By adding the AuthorTM module the APP can be customized to fit other purposes. AuthorTM offers a comprehensive and dedicated set of tools for creating new fit-for-purpose analysis APPs, and no programming experience is required.

REFERENCES
USERS
This APP was developed in collaboration with NordiQC.

LITERATURE

  1. Zou, Weiping, and Lieping Chen. "Inhibitory B7-family molecules in the tumour microenvironment." Nature Reviews Immunology 8.6 (2008): 467-477.
  2. Pardoll, Drew M. "The blockade of immune checkpoints in cancer immunotherapy." Nature Reviews Cancer 12.4 (2012): 252-264.
  3. Hirsch, Fred R., et al. "PD-L1 immunohistochemistry assays for lung cancer: results from phase 1 of the blueprint PD-L1 IHC assay comparison project." Journal of Thoracic Oncology 12.2 (2017): 208-222.
RUO
FIGURE 1
FIGURE 1
Overview of a single PD-L1 stained TMA core with melanoma tissue.
FIGURE 2
FIGURE 2
The same core as in Figure 1. The core has been automatically outlined, followed by manual editing to exclude stromal and necrotic tissue.
FIGURE 3
FIGURE 3
Region of melanoma tissue after PD-L1 analysis. Red labels show PD-L1positive cells and blue labels show PD-L1 negative cells.
FIGURE 4
FIGURE 4
Original image region of a medium-positive core with melanoma tissue
FIGURE 5
FIGURE 5
Same region as shown in Figure 4 after PD-L1 analysis with labels showing positive (red) and negative (blue) cells.