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EP-4740016-A1 - BRIGHTFIELD TRIPLEX IMMUNOHISTOCHEMISTRY ASSAY FOR EVALUATING THE COLOCALIZATION OF THE ER, PR, AND KI-67 BIOMARKERS IN CELLS

EP4740016A1EP 4740016 A1EP4740016 A1EP 4740016A1EP-4740016-A1

Abstract

The present disclosure is directed to a triplex immunohistochemical assay for detecting the colocalization of the ER, PR, and Ki-67 biomarkers in cells or cell nuclei. It is believed that the brightfield triplex immunohistochemical assay of the present disclosure may serve as a prognostic assay for ER-positive breast cancer, facilitating the identification of disease-free survivors among ER-positive breast cancer patients treated with hormone therapy.

Inventors

  • HORII, RIE
  • NITTA, HIRO
  • UENO, TAKAYUKI

Assignees

  • Ventana Medical Systems, Inc.
  • Japanese Foundation For Cancer Research

Dates

Publication Date
20260513
Application Date
20240705

Claims (20)

  1. 1. An affinity histochemical or affinity cytochemical method for assessing expression of the PR, ER, and Ki-67 biomarkers in a sample, the method comprising: (a) contacting the sample with a human PR biomarker-specific reagent under conditions that permit specific binding of the PR biomarker-specific reagent to the sample; (b) contacting the sample with a first set of detection reagents that interact with the human PR biomarker-specific reagent to facilitate deposition of a first brightfield detectable moiety on the sample; (c) contacting the sample with a human ER biomarker-specific reagent under conditions that permit specific binding of the ER biomarker-specific reagent to the sample; (d) contacting the sample with a second set of detection reagents that interact with the human ER biomarker-specific reagent to facilitate deposition of a second brightfield detectable moiety on the sample; (e) contacting the sample with a human Ki-67 biomarker-specific reagent under conditions that permit specific binding of the Ki-67 biomarker-specific reagent to the sample; (f) contacting the sample with a third set of detection reagents that interact with the human Ki-67 biomarker-specific reagent to facilitate deposition of a third brightfield detectable moiety on the sample; and (g) identifying cell nuclei within the sample expressing each of the PR, ER, and Ki-67 biomarkers based on a colocalization of signals from each of the deposited first, second, and third brightfield detectable moieties.
  2. 2. The method of claim 1, wherein the human PR biomarker-specific reagent is an anti-PR monoclonal antibody.
  3. 3. The method of any one of the preceding claims, wherein the human ER biomarker-specific reagent is an anti -ER monoclonal antibody.
  4. 4. The method of any one of the preceding claims, wherein the human Ki-67 biomarkerspecific reagent is an anti-Ki-67 monoclonal antibody.
  5. 5. The method of any of the preceding claims, wherein the first set of detection reagents include: (i) a first secondary antibody specific to the human PR biomarker-specific reagent; and (ii) a conjugate including the first brightfield detectable moiety.
  6. 6. The method of claim 5, wherein the first secondary antibody specific to the human PR biomarker-specific reagent includes a first enzyme.
  7. 7. The method of any of the preceding claims, wherein the second set of detection reagents include: (i) a second secondary antibody specific to the human ER biomarker-specific reagent; and (ii) a conjugate including the second brightfield detectable moiety.
  8. 8. The method of claim 7, wherein the second secondary antibody specific to the human ER biomarker-specific reagent includes a first enzyme.
  9. 9. The method of any of the preceding claims, wherein the third set of detection reagents include: (i) a third secondary antibody specific to the human Ki-67 biomarker-specific reagent; and (ii) a conjugate including the third brightfield detectable moiety.
  10. 10. The method of claim 9, wherein the third secondary antibody specific to the human Ki-67 biomarker-specific reagent includes a first enzyme.
  11. 11. The method of any of the preceding claims, wherein the first, second, and third brightfield detectable moieties are selected from the group consisting of TAMRA, Dabsyl, Dabcyl, Cy3, CyB, Cy3.5, Cy5, Cy5.5, Cy7, rhodamine 800 and fluorescein.
  12. 12. The method of claim 1, wherein the sample is a breast tissue sample.
  13. 13. The method of claim 12, wherein the breast tissue sample is from a subject diagnosed with breast cancer.
  14. 14. The method of 13, wherein the breast cancer is Luminal A breast cancer.
  15. 15. The method of any of the preceding claims, wherein an inactivation composition is applied to the sample prior to the contacting the sample with the human ER biomarker-specific reagent.
  16. 16. The method of any of the preceding claims, wherein an inactivation composition is applied to the sample prior to the contacting the sample with the human Ki-67 biomarker-specific reagent.
  17. 17. A method of selecting a patient with a breast tumor to receive a hormone therapy, the method comprising: (a) affinity histochemically staining a sample derived from the breast tumor with a human PR biomarker-specific reagent under conditions that permit specific binding of the PR biomarker-specific reagent to the sample; (b) affinity histochemically staining the sample derived from the breast tumor with a human ER biomarker-specific reagent under conditions that permit specific binding of the ER biomarker-specific reagent to the sample; (c) affinity histochemically staining the sample derived from the breast tumor with a human Ki-67 biomarker-specific reagent under conditions that permit specific binding of the Ki-67 biomarker-specific reagent to the sample; and (d) determining a number of proliferating tumor nuclei that are both ER+ and PR+ within the histochemically stained sample; wherein the patient is selected to receive the hormone therapy if a ratio of the determined number of proliferating tumor nuclei that are both ER+ and PR+ to a total number of ER+ proliferating tumor nuclei is greater or equal to a predetermined cutoff value.
  18. 18. The method of claim 17, wherein the predetermined cutoff value is between about 0.4 to about 0.6.
  19. 19. The method of claim 17, wherein the predetermined cutoff value is between about 0.45 to about 0.55.
  20. 20. The method of claim 17, wherein the predetermined cutoff value is about 0.5.

Description

BRIGHTFIELD TRIPLEX IMMUNOHISTOCHEMISTRY ASSAY FOR EVALUATING THE COLOCALIZATION OF THE ER, PR, AND KI-67 BIOMARKERS IN CELLS CROSS REFERENCE TO RELATED APPLICATIONS [0001] The present disclosure claims the benefit of the filing date of United States Provisional Patent Application No. 63/525,406 filed on July 7, 2023, the disclosure of which is hereby incorporated by reference herein in its entirety. BACKGROUND OF THE DISCLOSURE [0002] Breast cancer accounts for about 23% of all cancers worldwide and is responsible for hundreds of thousands of deaths each year. Breast cancers vary in their response to different treatments, and it is important to select an appropriate treatment regimen for each patient. Receptor status is a common classification system that is used to select treatments for a patient with breast cancer. Breast tumors may have (be positive for) or lack (be negative for) estrogen receptor (ER) protein, HER2 (also known as ErbB2) protein, and/or progesterone receptor (PR) protein. Breast tumors are also routinely screened for HER2 gene amplification, as another measure of whether the tumor is HER2 positive or negative. Some breast tumors are negative for all three markers (ER, PR, and HER2) and are referred to as "triple negative" tumors. [0003] Estrogen receptor and/or progesterone receptor positive tumors are typically treated with hormone-blocking therapy (such as tamoxifen); while HER2 positive tumors are treated with HER2-targeting therapeutics such as trastuzumab or lapatinib. [0004] Another biomarker, Ki-67, is often combined with the biomarkers ER, PR, and HER2 in an immunohistochemical score "IHC4" to evaluate risk of recurrence (for example see Cuzick et al., J. Clin. Oncol. 29:4273-8, 2011, and Barton et al., Br. J. Cancer 1-6, April 24, 2012). The IHC4 score, however, lacks reproducibility. [0005] There remains a need to improve current molecular screening methods to accurately assess the risk for recurrence and to select appropriate therapies in the clinic. BRIEF SUMMARY OF THE DISCLOSURE [0006] The present disclosure is directed to a brightfield triplex immunohistochemical (IHC) assay for detecting the colocalization of the ER, PR, and Ki-67 biomarkers in cells or cell nuclei. It is believed that the brightfield triplex immunohistochemical assay of the present disclosure may serve as a prognostic assay for ER-positive breast cancer, facilitating the identification of disease-free survivors among ER-positive breast cancer patients treated with hormone therapy. It is also believed that the brightfield triplex immunohistochemical assay of the present disclosure may serve as a companion assay to identify ER-positive breast cancer patients who should be treated with a CDK4/6 inhibitor because of their poor prognosis. It is further believed that the disclosed brightfield triplex immunohistochemical assay could serve as a replacement for costly gene expression tests (e.g., Oncotype DX, MammaPrint, Prosigna, Breast Cancer Index). [0007] A first aspect of the present disclosure is an affinity histochemical or affinity cytochemical (e.g., immunohistochemical method) for assessing expression of the PR, ER, and Ki-67 biomarkers in a sample, the method comprising: contacting the sample with a human PR biomarker-specific reagent under conditions that permit specific binding of the PR biomarkerspecific reagent to the sample; contacting the sample with a first set of detection reagents that interact with the human PR biomarker-specific reagent to facilitate deposition of a first brightfield detectable moiety on the sample; contacting the sample with a human ER biomarker-specific reagent under conditions that permit specific binding of the ER biomarker-specific reagent to the sample; contacting the sample with a second set of detection reagents that interact with the human ER biomarker-specific reagent to facilitate deposition of a second brightfield detectable moiety on the sample; contacting the sample with a human Ki-67 biomarker-specific reagent under conditions that permit specific binding of the Ki-67 biomarker-specific reagent to the sample; contacting the sample with a third set of detection reagents that interact with the human Ki-67 biomarker-specific reagent to facilitate deposition of a third brightfield detectable moiety on the sample; and identifying cell nuclei within the sample expressing each of the PR, ER, and Ki-67 biomarkers based on a colocalization of signals from each of the deposited first, second, and third brightfield detectable moieties. In some embodiments, the first, second, and third brightfield detectable moieties are each different. [0008] In some embodiments, the human PR biomarker-specific reagent is an anti-PR monoclonal antibody. In some embodiments, the human PR biomarker-specific reagent is Clone 1E2. [0009] In some embodiments, the human ER biomarker-specific reagent is an anti-ER monoclonal antibody. In some embodiments, the human ER biomarker-specific reag