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10095 - PHH3+CK5, Neck Cancer

INTRODUCTION
Phosphohistone H3 (PHH3) is a mitotic marker and has prognostic capabilities when applied to breast cancer tissue. The mitotic index can be obtained by PHH3-immunohistochemical staining, which can be used as a supplement for diagnosis.

To identify the PHH3 positive cells within the tumor regions of an image, these regions must be outlined manually, which can be a tedious and time-consuming task. However, tumor detection in neck tissue sections can be automated by performing a Physical Double Staining (PDS) with cytokeratin 5 (CK5). CK5 is overexpressed in tumors located in the oral cavity, oropharyngeal, hypopharyngeal and laryngeal areas. CK5 can be used as a tumor marker, and a nucleus can thus be classified as belonging to a tumor based on the staining of the surrounding tissue.

This protocol automatically detects and quantifies PHH3 positive cells within tumor regions. No manual outlining of the tumor is needed, since the tumor region is automatically identified based on the PHH3+CK5 PDS. This APP has been developed to quantify the number of PHH3 positive cells and their area. Furthermore, the tumor area is quantified with and without these positive cells.

KEYWORDS
PHH3, CK5, physical double staining, neck cancer, image analysis

METHODS
The first image processing step involves an automated detection and outlining of the tumor tissue, i.e. the region of interest (ROI) [See Example: Figure 2]. The APP then detects the PHH3 positive nuclei within the tumor ROI. The detection of the PHH3 positive nuclei is based on the calculation of a color-deconvolution band. This band is used as the input parameter to a cell classification protocol that identifies the positively stained nuclei. Nuclei are managed with a postprocessing protocol to reduce the number of false positives by merging closely connected nuclei and removing very small nuclei.

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

- POS (N): The number of positive nuclei

- CLEAR (A): The area of tumor without positive nuclei

- POS (A): The area of positive nuclei

- TOTAL (A): The total tumor area area (CLEAR (A) + POS (A))

AUXILIARY APPs (included)
APP: "01 ROI Detect"
The APP "01 ROI Detect" automatically detects the CK5 stained tumor tissue and limits the subsequent analysis to be within this region.

WORKFLOW
Step 1: Load and run the APP “01 ROI detect” for tumor identification.
Step 2: Load and run the APP “02 Analyze” for the quantification of PHH3 positive nuclei within the tumor ROI.

STAINING PROTOCOL
There is no stainig protocol available.

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
There are no references available.

RUO
FIGURE 1
FIGURE 1
Raw image of tissue physically double stained with PHH3 and CK5.
FIGURE 2
FIGURE 2
Result of analysis with the auxiliary APP: The CK5 stained tumor tissue has automatically been identified and outlined with a blue ROI.
FIGURE 3
FIGURE 3
Result of analysis with the APP: Detected PHH3 positive nuclei are marked with a green label. Notice how only PHH3 positive nuclei within the blue tumor ROI have been identified by the APP.