Method of using non-rare cells to detect rare cells

• US 10,613,089 B2
• Filed: 01/18/2019
• Issued: 04/07/2020
• Est. Priority Date: 01/30/2006
• Status: Active Grant

• Current Assignee: Epic Sciences, Inc., The Scripps Research Institute
• Original Assignee: The Scripps Research Institute
• Inventors: Kuhn, Peter, Kolatkar, Anand, Kunken, Joshua, Marrinucci, Dena, Yang, Xing, Stuelpnagel, John R.
• Primary Examiner(s): Yao, Lei
• Application Number:
  US16/252,472
• Publication Number:
  US 20190257834A1
• Time in Patent Office: 445 Days
• Field of Search: None
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: G01N 2800/52 Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• CPC Class Codes: G01N 2800/56 Staging of a disease; Further complications associated with the disease
• CPC Class Codes: G01N 33/5076 involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
• CPC Class Codes: G01N 33/5091 for testing the pathological state of an organism
• CPC Class Codes: G01N 33/574 for cancer
• CPC Class Codes: G01N 33/6875 Nucleoproteins

Abstract

The invention provides seminal computational approaches utilizing data from non-rare cells to detect rare cells, such as circulating tumor cells (CTCs). The invention is applicable at two distinct stages of CTC detection; the first being to make decisions about data collection parameters and the second being to make decisions during data reduction and analysis. Additionally, the invention utilizes both one and multi-dimensional parameterized data in a decision making process.

Claims

1. A method for detecting a circulating tumor cell (CTC) in a blood sample from a subject having metastatic cancer comprising:

a) plating a non-enriched blood sample comprising nucleated blood cells onto a slide, said non-enriched sample suspected of comprising circulating tumor cell (CTCs) and non-CTCs wherein said non-CTCs are present at a concentration that is at least 10 times that of the CTCs;

b) contacting the sample with a first immunuofluorescently detectable agent targeting a positive marker and a second immunuofluorescently detectable agent targeting a negative marker, wherein said positive marker is cytokeratin, wherein said negative marker is CD45, and wherein said detectable agent comprises an antibody;

c) scanning the slide with fluorescent scanning microscopy to generate a cell image of the nucleated cells in the sample; and

d) identifying the CTC in the sample based on assessment of said detectable agents and morphology in said cell image, wherein the CTC is cytokeratin positive, CD45 negative, has an intact nucleus, and is morphologically distinct from white blood cells.

2. The method of claim 1, wherein said positive marker is expressed on a specific cancer cell type or cancer tissue.

3. The method of claim 2, wherein said positive marker is expressed on a specific cancer cell type.

4. The method of claim 3, wherein said specific cell type is epithelial.

5. The method of claim 1, wherein said negative marker is expressed on non-CTCs.

6. The method of claim 5, wherein said non-CTCs are white blood cells.

7. The method of claim 1, further comprising a third immunofluorescently detectable agent.

8. The method of claim 7, wherein said third immunofluorescently detectable agent targets a content marker.

9. The method of claim 8, wherein said content marker is androgen receptor (AR).