After After
Before Before

10096 - Ki-67 + CK5 VDS, Neck Cancer

INTRODUCTION
The Ki-67 protein is associated with cellular proliferation, and the protein is present in the nucleus of all cells that are in the active phase of the cell cycle, but absent in resting cells [see REFFERENCES: 1]. The cell proliferation rate can be assessed by Ki-67-immunohistochemical (IHC) staining, and this can be correlated to the tumor grade and the clinical course [see REFFERENCES: 2].

This protocol can be used to assess tumors by determining the Ki-67 positivity.
Two serial sections stained for Ki-67 and CK5, respectively, must be used in this APP. Tumor regions are identified automatically on the CK5 stained slides using the VirtualDoubleStaining™ technique, and the outlined tumor regions are overlaid on the Ki-67 stained slide, thus automatically identifying tumor regions.

KEYWORDS
Ki-67, CK5, proliferation,VirtualDoubleStaining™, VDS™,  neck, throat, immunohistochemistry, quantitative, digital pathology, image analysis.

METHODS
The Ki-67 and CK5 serial sections are linked together and aligned via the TissuealignTM workflow that allows for an easy user-assisted computational alignment.

Tumor areas are automatically detected from the CK5 slide and outlined as regions of interest (ROIs). The ROIs are superimposed onto the aligned Ki-67 tissue slide to outline the tumor region on the Ki-67 slide. The subsequent analysis is now limited to the inside of the tumor regions only.

Ki-67 negative and positive nuclei are detected and delineated from their surroundings to accurately quantify nuclei in highly concentrated areas.

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

- Neg Nuclei (N): The number of negative nuclei profiles

- Pos Nuclei (N): The number of positive nuclei profiles

- Total Nuclei (N): The total number of nuclei profiles

- Proliferation Index (N) % = Pos Nuclei (N) / Total Nuclei (N): The ratio of number of positive nuclei profiles

AUXILIARY APPs (included)
APP: “01 Tumor Detection”
The auxiliary APP '01 Tumor Detection" is used for automatic tumor detection.

WORKFLOW
Step 1: Load the auxiliary APP for tumor region identification: “01 Tumor Detection”.

Step 2: Load the APP for identification and scoring of Ki-67 stained cells: “02 Analyze”.

STAINING PROTOCOL
There is no staining 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. In addition the APP also utilizes the Tissuealign™ incl. VirtualDoubleStaining™ module, that facilitates a user-assisted computational alignment of serial sections and automatic tumor detection.
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
1. The Ki-67 protein: from the known and the unknown, T. Scholzen et al., J. Cell Physiol. 2000, 182 (3): 311-22

2. www.nordiqc.org/epitope.php?id=1

RUO
Figure 1
Figure 1
CK5 stained slide.
Figure 2
Figure 2
The serial Ki-67 stained slide.
Figure 3
Figure 3
Illustration of the tissue alignment workflow linking the two differently stained serial sections together. This workflow requires the module Tissuealign™ with VirtualDoubleStaining™.
Figure 4
Figure 4
Tumor regions are automatically identified on the CK5 slide, and outlined with a blue ROI.
Figure 5
Figure 5
The tumor region is superimposed onto the serial Ki-67 stained slide.
Figure 6
Figure 6
Analysis identifying the Ki-67 negative (blue label) and positive (red label) nuclei. Notice how the Ki-67 analysis is restricted to the inside of the automatically identified tumor region.