ERG/TFF3/HMWCK triple immunostain for detection of prostate cancer
This invention relates to a triple immunostaining assay (ERG/TFF3/HMWCK) for sensitive and specific detection of prostate cancer. Positive staining for at least one of ERG or TFF3 combined with negative staining of HMWCK in a sample such as prostate tissue sample is indicative of cancer.
1. A triple immunohistochemistry (IHC) assay for in situ detection of TFF3, ERG, and a high-molecular weight cytokeratin (HMWCK), comprising:
providing a prostate tissue sample comprising fixed cells;
contacting the sample with a first primary antibody directed to a first molecule selected from ERG, TFF3 or HMWCK, a second primary antibody directed to a second molecule selected from ERG, TFF3 or HMWCK, and a third primary antibody directed to a third molecule selected from ERG, TFF3 or HMWCK, to permit formation of antibody-antigen complexes in the sample which lead to the generation of detectable signals; and
identifying cells that display a detectable signal.
2. The triple IHC assay of claim 1 , further comprising contacting the sample with a first secondary binding reagent which binds to said first primary antibody and is labeled with a first reporter molecule, contacting the sample with a second secondary binding reagent which binds to said second primary antibody and is labeled with a second reporter molecule, and contacting the sample with a third secondary binding reagent which binds to said third primary antibody and is labeled with a third reporter molecule, wherein said first, second and third reporter molecules are capable of generating detectable signals.
3. The triple IHC assay of claim 2 , wherein said first, second and third reporter molecules generate three distinct signals.
4. The triple IHC assay of claim 2 , wherein said first, second and third reporter molecules each comprise an enzyme that acts on a chromogenic substrate to form a pigment.
5. The triple IHC assay of claim 4 , wherein said first, second and third reporter molecules form three distinct pigments.
6. The triple IHC assay of claim 5 , wherein said three distinct pigments are brown, red and blue in color.
7. The triple IHC assay of claim 2 , wherein the sample is contacted with said first primary antibody, followed by said first secondary binding reagent; then with said second primary antibody, followed by said second secondary binding reagent; and subsequently with said third primary antibody, followed by said third secondary binding reagent.
8. The triple IHC assay of claim 7 , wherein the first primary antibody is directed to ERG, the second primary antibody is directed to TFF3, and the third primary antibody is directed to a HMWCK.
9. The triple IHC assay of claim 8 , wherein the secondary binding reagents generate pigments of brown, red and blue colors for ERG, TFF3 and HMWCK, respectively.
10. The triple IHC assay of claim 1 , wherein cells displaying signals for at least one of ERG or TFF3 and lacking a signal for HMWCK are identified as cancerous cells.
11. The triple IHC assay of claim 1 , wherein said tissue sample is a needle biopsy sample.
12. A method of detecting prostate cancer in a subject, comprising
obtaining a prostate tissue sample comprising fixed cells from the subject;
contacting the sample with a first primary antibody directed to a first molecule selected from ERG, TFF3 or HMWCK, a second primary antibody directed to a second molecule selected from ERG, TFF3 or HMWCK, and a third primary antibody directed to a third molecule selected from ERG, TFF3 or HMWCK, to permit formation of antibody-antigen complexes in the sample which leads to the generation of detectable signals; and
identifying cells that display a detectable signal; and
determining cells as cancerous cells based on the presence of signals for at least one of ERG or TFF3 and the absence of signals for HMWCK.
13. A kit for performing a triple IHC assay, comprising antibodies directed to ERG, TFF3 and HMWCK, respectively.
14. The kit of claim 13 , further comprising three secondary binding agents which bind to the primary antibodies directed to ERG, TFF3 and HMWCK, respectively, and are labeled each with a reporter molecule capable of generating a detectable signal.
15. The kit of claim 14 , wherein the reporter molecules generate three distinct signals.
16. The kit of claim 15 , wherein the reporter molecules each comprise an enzyme that acts on a chromogenic substrate to form a pigment, and the reporter molecules form three distinct pigments.
17. The kit of claim 16 , wherein the three distinct pigments are brown, red and blue colors.