# US 11,079,382 B2 · Methods, kits, and systems for scoring the immune response to cancer

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- Number: US 11,079,382 B2
- Document ID: US11079382B2
- Status: Granted
- Kind: B2
- Application number: 15/246,409
- Filed: 2016-08-24
- Published: 2016-12-15
- Granted: 2021-08-03
- Earliest claimed: 2014-02-24
- Assignees: Ventana Medical Systems, Inc.
- Applicants: Ventana Medical Systems, Inc.
- Inventors: Noemi Sebastiao; William Day; Robert Ochs; Srinivas Chukka; Jim Martin; Michael Barnes; Joerg Bredno; Ting Chen; Alisa Tubbs; Yao Nie
- Web page: https://patentgrep.com/patent/US11079382B2

## CPC

- G01N 33/5751: of the skin, e.g. melanoma
- G01N 15/1433: using image recognition
- G01N 33/566: using specific carrier or receptor proteins as ligand binding reagents {where possible specific carrier or receptor proteins are classified with their target compounds}
- G01N 33/575: for cancer
- G01N 33/57515: of the breast
- G01N 33/6875: {Nucleoproteins}
- G01N 35/00029: {provided with flat sample substrates, e.g. slides (G01N35/028 takes precedence)}
- G16B 25/00: ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
- G16B 25/10: Gene or protein expression profiling; Expression-ratio estimation or normalisation
- G01N 2015/016: {White blood cells}
- G01N 2015/1006: {for cytology}
- G01N 2015/1486: {Counting the particles}
- G01N 2035/00138: {Slides}
- G01N 2333/4703: Regulators; Modulating activity
- G01N 2333/4739: Cyclin; Prad 1
- G01N 2333/4742: Keratin; Cytokeratin
- G01N 2333/70503: Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
- G01N 2333/7051: T-cell receptor (TcR)-CD3 complex
- G01N 2333/70517: CD8
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY

## Abstract

This disclosure describes methods, kits, and systems for scoring the immune response to cancer through examination of tissue infiltrating lymphocytes (TILs). Methods of scoring the immune response in cancer using tissue infiltrating lymphocytes include detecting CD3, CD8, CD20, and FoxP3 within the sample and scoring the detection manually or scoring the digital images of the staining with the aid of image analysis and algorithms.

## Claims

### Claim 1

A computer-implemented method for counting a number of types of cells in a single tissue specimen that has been stained with a multiplex assay, comprising:
imaging the single tissue specimen that has been stained with the multiplex assay that includes lymphocyte markers CD3, CD8, CD20, FoxP3, and tumor detection markers;
unmixing the image of single tissue specimen that has been stained with a multiplex assay into separate image channels for each marker of the multiplex assay;
identifying regions of interest in each image channel based on intensity information in each channel, wherein regions of low intensity in each channel are removed, and regions of high intensity represent cell signals;
generating a single surrogated image, wherein the surrogated image is a combination of the image channel information of all the lymphocyte markers;
applying a cell detection algorithm, wherein the cell detection algorithm is a membrane finding algorithm or a nucleus finding algorithm;
identifying features of the lymphocytes and combinations of lymphocytes in each image channel or image of combined channels, or a transformed image such as grayscale or absorbance image, or a surrogated image;
training a classification algorithm based on features of known lymphocytes and lymphocyte combinations;
applying the trained algorithm to features of the lymphocytes and combinations of lymphocytes in each image channel or image of combined channels, or a transformed image such as grayscale or absorbance image, or a surrogated image, that were identified to classify the detected cells as at least one of false positive cells, CD3 only T-cells, CD3 and CD8 T-cells, FP3 T-cells; and CD20 B-cells;
counting a number of each different type of cell classified;
generating a score the tissue specimen, wherein the score is based on the number each type of cell counted.

### Claim 2 (depends on 1)

The method of claim 1, wherein said multiplex assay comprises:
contacting a sample with CD3, CD8, CD20, and FoxP3 specific binding moieties,
removing excess specific binding moieties from the sample, and
detecting the specific binding moieties.

### Claim 3 (depends on 2)

The method of claim 2, wherein said multiplex assay further comprises contacting the sample with a specific binding moiety for a tumor marker.

### Claim 4 (depends on 3)

The method of claim 3, wherein the specific binding moiety for the tumor marker is selected from antibodies to cytokeratin for epithelial tumors (carcinomas) and antibodies to S100 for melanomas.

### Claim 5 (depends on 2)

The method of claim 2, wherein the specific binding moiety for CD3 is anti-CD3 rabbit or mouse monoclonal antibodies.

### Claim 6 (depends on 2)

The method of claim 2, wherein the specific binding moiety for CD8 is anti-CD8 rabbit or mouse monoclonal antibodies.

### Claim 7 (depends on 2)

The method of claim 2, wherein the specific binding moiety for CD20 is anti-CD20 rabbit or mouse monoclonal antibodies.

### Claim 8 (depends on 2)

The method of claim 2, wherein the specific binding moiety for FoxP3 is anti-FoxP3 rabbit or mouse monoclonal antibodies.

### Claim 9 (depends on 2)

The method of claim 2, wherein contacting comprises contacting the sample with CD3, CD8, CD20, and FoxP3 specific binding moieties which are selected from rabbit or mouse monoclonal antibodies.

### Claim 10 (depends on 9)

The method of claim 9, wherein detecting comprises contacting the sample with an anti-species antibody specific to the rabbit or mouse monoclonal antibodies.

### Claim 11 (depends on 10)

The method of claim 10, wherein the anti-species antibody is haptenated, and wherein detecting the haptenated antibody further comprises contacting the sample with an anti-hapten antibody conjugated to an enzyme.

### Claim 12 (depends on 11)

The method of claim 11, wherein detecting further comprises contacting the sample with a detection compound.

### Claim 13 (depends on 12)

The method of claim 12, wherein the detection compound is selected from a chromogen or a fluorophore.

### Claim 14 (depends on 2)

The method of claim 2, wherein the specific binding moieties are haptenated, and wherein detecting the specific binding moieties further comprises contacting the sample with an anti-hapten antibody conjugated to an enzyme.

### Claim 15 (depends on 14)

The method of claim 14, wherein detecting further comprises contacting the sample with a detection compound.

### Claim 16 (depends on 15)

The method of claim 15, wherein the detection compound is selected from a chromogen or a fluorophore.

### Claim 17 (depends on 1)

The method of claim 1, wherein the single tissue specimen is a tissue section.