Bright field triple immunohistochemical assay for assessing co-localization of ER, PR and KI-67 biomarkers in cells

Abstract

The present disclosure relates to a triple immunohistochemical assay for detecting co-localization of ER, PR and Ki-67 biomarkers in a cell or nucleus. It is believed that the bright field triple immunohistochemistry assay of the present disclosure can be used as a prognostic assay for ER-positive breast cancer, facilitating the identification of disease-free survivors in ER-positive breast cancer patients treated with hormone therapy.

Description

[0001] Cross-references to related applications

[0002] This disclosure claims the benefit of U.S. Provisional Patent Application No. 63 / 525,406, filed July 7, 2023, the disclosure of which is incorporated herein by reference in its entirety. Background Technology

[0003] Breast cancer accounts for approximately 23% of all cancers worldwide and causes hundreds of thousands of deaths each year. Breast cancer varies in its response to different treatments, making it crucial to choose the appropriate treatment plan for each patient. Receptor status is a common classification system used to select treatment options for patients with breast cancer. Breast tumors may have (positive) or lack (negative) estrogen receptor (ER) protein, HER2 (also known as ErbB2) protein, and / or progesterone receptor (PR) protein. Routine screening for HER2 gene amplification is also performed on breast tumors as another measure of whether a 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.

[0004] Estrogen receptor and / or progesterone receptor positive tumors are usually treated with hormone blocking therapy (such as tamoxifen); while HER2 positive tumors are treated with HER2 targeted therapy (such as trastuzumab or lapatinib).

[0005] Another biomarker, Ki-67, is often combined with the biomarkers ER, PR, and HER2 in the immunohistochemical score “IHC4” to assess the risk of recurrence (see, for example, Cuzick et al., J. Clin. Oncol. 29:4273-8, 2011, and Barton et al., Br. J. Cancer 1-6, April 24, 2012). However, the IHC4 score lacks reproducibility.

[0006] There is still a need to improve current molecular screening methods to accurately assess the risk of relapse and select appropriate therapies in clinical practice. Summary of the Invention

[0007] The present disclosure relates to brightfield triplex immunohistochemistry (IHC) assays for detecting co-localization of ER, PR, and Ki-67 biomarkers in cells or nuclei. It is believed that the brightfield triplex immunohistochemistry assays of the present disclosure can be used as a prognostic assay for ER-positive breast cancer, facilitating the identification of disease-free survivors in ER-positive breast cancer patients treated with hormone therapy. It is also believed that the brightfield triplex immunohistochemistry assays of the present disclosure can be used as a companion assay to identify ER-positive breast cancer patients who should be treated with CDK4 / 6 inhibitors because of their poor prognosis. It is further believed that the disclosed brightfield triplex immunohistochemistry assays can be used as a replacement for expensive gene expression tests (e.g., Oncotype DX, MammaPrint, Prosigna, Breast Cancer Index).

[0008] A first aspect of the present disclosure is an affinity histochemical or affinity cytochemical method (e.g., an immunohistochemical method) for assessing expression of 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 allow specific binding of the PR biomarker-specific 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 allow 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 allow 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 nuclei within the sample that express each of the PR, ER, and Ki-67 biomarkers based on co-localization 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.

[0009] 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.

[0010] In some embodiments, the human ER biomarker-specific reagent is an anti-ER monoclonal antibody. In some embodiments, the human ER biomarker-specific reagent is SP1.

[0011] In some embodiments, the human Ki-67 biomarker-specific reagent is an anti-Ki-67 monoclonal antibody. In some embodiments, the human Ki-67 biomarker-specific reagent is clone 30-9.

[0012] In some embodiments, the first set of detection reagents comprises: (i) a first secondary antibody specific for a human PR biomarker-specific reagent; and (ii) a conjugate comprising a first brightfield detectable moiety. In some embodiments, the first secondary antibody specific for a human PR biomarker-specific reagent comprises a first enzyme.

[0013] In some embodiments, the second set of detection reagents comprises: (i) a second secondary antibody specific for a human ER biomarker-specific reagent; and (ii) a conjugate comprising a second brightfield detectable moiety. In some embodiments, the second secondary antibody specific for a human ER biomarker-specific reagent comprises a first enzyme.

[0014] In some embodiments, the third set of detection reagents comprises: (i) a third secondary antibody specific for a human Ki-67 biomarker-specific reagent; and (ii) a conjugate comprising a third brightfield detectable moiety. In some embodiments, the third secondary antibody specific for a human Ki-67 biomarker-specific reagent comprises a first enzyme.

[0015] In some embodiments, 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.

[0016] In some embodiments, the sample is a breast tissue sample. In some embodiments, the breast tissue sample is from a subject diagnosed with breast cancer. In some embodiments, the breast cancer is luminal A breast cancer.

[0017] In some embodiments, the inactivation composition is applied to the sample prior to contacting the sample with the human ER biomarker-specific reagent. In some embodiments, the inactivation composition is applied to the sample prior to contacting the sample with the human Ki-67 biomarker-specific reagent.

[0018] A second aspect of this disclosure is a method for selecting a patient with breast cancer to receive hormone therapy, the method comprising: performing affinity histochemical staining on a sample derived from the breast cancer with a human PR biomarker-specific reagent under conditions allowing the PR biomarker-specific reagent to bind specifically to the sample; performing affinity histochemical staining on the sample derived from the breast cancer with a human ER biomarker-specific reagent under conditions allowing the ER biomarker-specific reagent to bind specifically to the sample; performing affinity histochemical staining on the sample derived from the breast cancer with a human Ki-67 biomarker-specific reagent under conditions allowing the Ki-67 biomarker-specific reagent to bind specifically to the sample; determining the number of proliferating tumor nuclei that are both ER+ and PR+ (i.e., those nuclei that are Ki-67+) in the histochemically stained sample; wherein if the ratio of the determined number of proliferating tumor nuclei that are both ER+ and PR+ to the total number of ER+ proliferating tumor nuclei is greater than or equal to a predetermined cutoff value, then the patient is selected to receive the hormone therapy. In some embodiments, histochemical staining of PR, ER, and Ki-67 biomarkers can be performed in any order. In other embodiments, histochemical staining of PR, ER, and Ki-67 biomarkers is performed in the order described above.

[0019] In some embodiments, the predetermined cutoff value is between about 0.3 and about 0.7. In some embodiments, the predetermined cutoff value is between about 0.4 and about 0.6. In some embodiments, the predetermined cutoff value is between about 0.45 and about 0.55. In some embodiments, the predetermined cutoff value is about 0.48. In some embodiments, the predetermined cutoff value is about 0.49. In some embodiments, the predetermined cutoff value is about 0.5. In some embodiments, the predetermined cutoff value is about 0.51. In some embodiments, the predetermined cutoff value is about 0.52.

[0020] In some embodiments, the hormone therapy is a selective estrogen receptor modulator. In some embodiments, the hormone therapy is a selective estrogen receptor degrader. In some embodiments, the hormone therapy is an aromatase inhibitor. In some embodiments, the patient has previously been diagnosed with luminal type A breast cancer. In some embodiments, the patient has previously received endocrine therapy. In some embodiments, the patient has previously received adjuvant chemotherapy.

[0021] In some embodiments, affinity histochemical staining of a sample with a human PR biomarker-specific reagent includes: (i) contacting the sample with the human PR biomarker-specific reagent under conditions that allow the PR biomarker-specific reagent to bind specifically to the sample; and (ii) contacting the sample with a first set of detection reagents, the first set of detection reagents interacting with the human PR biomarker-specific reagent to promote the deposition of a first brightfield detectable portion on the sample. In some embodiments, affinity histochemical staining of a sample with a human ER biomarker-specific reagent includes: (i) contacting the sample with a human ER biomarker-specific reagent under conditions that allow the ER biomarker-specific reagent to bind specifically to the sample; and (ii) contacting the sample with a second set of detection reagents, the second set of detection reagents interacting with the human ER biomarker-specific reagent to promote the deposition of a second brightfield detectable portion on the sample. In some embodiments, affinity histochemical staining of the sample with a human Ki-67 biomarker-specific reagent includes: (i) contacting the sample with the human Ki-67 biomarker-specific reagent under conditions that allow the Ki-67 biomarker-specific reagent to specifically bind to the sample; and (ii) contacting the sample with a third set of detection reagents that interact with the human Ki-67 biomarker-specific reagent to promote the deposition of a third brightfield detectable portion on the sample.

[0022] In some embodiments, determining the number of proliferating tumor nuclei expressing both ER+ and PR+ within an affinity-histochemically stained sample includes identifying nuclei expressing each of the PR, ER, and Ki-67 biomarkers within the sample based on co-localization of signals from each of the deposited first, second, and third bright-field detectable portions. In some embodiments, the first, second, and third bright-field detectable portions are selected from the group consisting of TAMRA, Dabsyl, Dabcyl, Cy3, CyB, Cy3.5, Cy5, Cy5.5, Cy7, Rhodamine 800, and fluorescein. In some embodiments, the first, second, and third bright-field detectable portions are each distinct.

[0023] A third aspect of this disclosure is a method for selecting a patient with breast cancer to receive hormone therapy, the method comprising: performing affinity histochemical staining on a sample derived from breast cancer with a human PR biomarker-specific reagent under conditions allowing the PR biomarker-specific reagent to bind specifically to the sample; performing affinity histochemical staining on the sample derived from breast cancer with a human ER biomarker-specific reagent under conditions allowing the ER biomarker-specific reagent to bind specifically to the sample; performing affinity histochemical staining on the sample derived from breast cancer with a human Ki-67 biomarker-specific reagent under conditions allowing the Ki-67 biomarker-specific reagent to bind specifically to the sample; calculating the ratio of the number of cells or nuclei that are positive for staining all three of ER, Ki-67, and PR to the total number of cells that are (i) ER+, Ki-67+, and PR+ and (ii) ER+, Ki-67+, and PR-; wherein if the calculated ratio is greater than or equal to a predetermined cutoff value, the patient is selected to receive the hormone therapy. In some embodiments, histochemical staining of PR, ER, and Ki-67 biomarkers can be performed in any order. In other embodiments, histochemical staining of PR, ER, and Ki-67 biomarkers is performed in the order described above.

[0024] In some embodiments, the predetermined cutoff value is between about 0.4 and about 0.6. In some embodiments, the predetermined cutoff value is between about 0.45 and about 0.55. In some embodiments, the predetermined cutoff value is about 0.5. In some embodiments, the ratio is calculated using the following formula:

[0025] [ER + Ki-67 + PR + ] / ([ER + Ki-67 + PR + ] + [ER + Ki-67 + PR - ]).

[0026] In some embodiments, affinity histochemical staining of a sample with a human PR biomarker-specific reagent includes: (i) contacting the sample with the human PR biomarker-specific reagent under conditions that allow the PR biomarker-specific reagent to bind specifically to the sample; and (ii) contacting the sample with a first set of detection reagents, the first set of detection reagents interacting with the human PR biomarker-specific reagent to promote the deposition of a first brightfield detectable portion on the sample. In some embodiments, affinity histochemical staining of a sample with a human ER biomarker-specific reagent includes: (i) contacting the sample with a human ER biomarker-specific reagent under conditions that allow the ER biomarker-specific reagent to bind specifically to the sample; and (ii) contacting the sample with a second set of detection reagents, the second set of detection reagents interacting with the human ER biomarker-specific reagent to promote the deposition of a second brightfield detectable portion on the sample. In some embodiments, affinity histochemical staining of the sample with a human Ki-67 biomarker-specific reagent comprises: (i) contacting the sample with the human Ki-67 biomarker-specific reagent under conditions that allow the Ki-67 biomarker-specific reagent to specifically bind to the sample; and (ii) contacting the sample with a third group of detection reagents that interact with the human Ki-67 biomarker-specific reagent to promote the deposition of a third brightfield detectable portion on the sample. In some embodiments, the first brightfield detectable portion, the second brightfield detectable portion, and the third brightfield detectable portion are selected from the group consisting of TAMRA, Dabsyl, Dabcyl, Cy3, CyB, Cy3.5, Cy5, Cy5.5, Cy7, Rhodamine 800, and fluorescein.

[0027] A fourth aspect of the invention is a method for selecting a patient with breast tumor to receive treatment with cyclin-dependent kinase 4 and 6 inhibitors, the method comprising: performing affinity histochemical staining on a sample derived from a breast tumor using a human PR biomarker-specific reagent under conditions allowing the PR biomarker-specific reagent to bind specifically to the sample; performing affinity histochemical staining on the sample derived from a breast tumor using a human ER biomarker-specific reagent under conditions allowing the ER biomarker-specific reagent to bind specifically to the sample; performing affinity histochemical staining on the sample derived from a breast tumor using a human Ki-67 biomarker-specific reagent under conditions allowing the Ki-67 biomarker-specific reagent to bind specifically to the sample; calculating the number of cells or nuclei that are positive for staining all three of ER, Ki-67, and PR, and the number of cells that are (i) ER+, Ki-67+, and PR+ and (ii) ER+, Ki-67+, and PR- The ratio of the total number of cells; wherein if the calculated ratio is less than a predetermined cutoff value, the patient is selected to receive hormone therapy. In some embodiments, histochemical staining for PR, ER, and Ki-67 biomarkers can be performed in any order. In other embodiments, histochemical staining for PR, ER, and Ki-67 biomarkers is performed in the order described above.

[0028] In some embodiments, the predetermined cutoff value is between about 0.4 and about 0.6. In some embodiments, the predetermined cutoff value is between about 0.45 and about 0.55. In some embodiments, the predetermined cutoff value is about 0.5. In some embodiments, the ratio is calculated using the following formula:

[0029] [ER + Ki-67 + PR + ] / ([ER + Ki-67 + PR + ] + [ER + Ki-67 + PR - ]).

[0030] In some embodiments, affinity histochemical staining of a sample with a human PR biomarker-specific reagent includes: (i) contacting the sample with the human PR biomarker-specific reagent under conditions that allow the PR biomarker-specific reagent to bind specifically to the sample; and (ii) contacting the sample with a first set of detection reagents, the first set of detection reagents interacting with the human PR biomarker-specific reagent to promote the deposition of a first brightfield detectable portion on the sample. In some embodiments, affinity histochemical staining of a sample with a human ER biomarker-specific reagent includes: (i) contacting the sample with a human ER biomarker-specific reagent under conditions that allow the ER biomarker-specific reagent to bind specifically to the sample; and (ii) contacting the sample with a second set of detection reagents, the second set of detection reagents interacting with the human ER biomarker-specific reagent to promote the deposition of a second brightfield detectable portion on the sample. In some embodiments, affinity histochemical staining of the sample with a human Ki-67 biomarker-specific reagent comprises: (i) contacting the sample with the human Ki-67 biomarker-specific reagent under conditions that allow the Ki-67 biomarker-specific reagent to specifically bind to the sample; and (ii) contacting the sample with a third group of detection reagents that interact with the human Ki-67 biomarker-specific reagent to promote the deposition of a third brightfield detectable portion on the sample. In some embodiments, the first brightfield detectable portion, the second brightfield detectable portion, and the third brightfield detectable portion are selected from the group consisting of TAMRA, Dabsyl, Dabcyl, Cy3, CyB, Cy3.5, Cy5, Cy5.5, Cy7, Rhodamine 800, and fluorescein.

[0031] The fifth aspect of this disclosure is a method for classifying a patient with breast cancer as ER+, Ki-67+, PR+ dominant or ER+, Ki-67+ dominant, the method comprising: performing affinity histochemical staining on a sample derived from the patient with a human PR biomarker-specific reagent under conditions allowing the PR biomarker-specific reagent to bind specifically to the sample; performing affinity histochemical staining on the sample derived from the patient with a human ER biomarker-specific reagent under conditions allowing the ER biomarker-specific reagent to bind specifically to the sample; performing affinity histochemical staining on the sample derived from the patient with a human Ki-67 biomarker-specific reagent under conditions allowing the Ki-67 biomarker-specific reagent to bind specifically to the sample; calculating the number of cells or nuclei that are positive for staining for all three of ER, Ki-67, and PR, and the number of cells that are (i) ER+, Ki-67+, and PR+ and (ii) ER+, Ki-67+ The ratio of the total number of cells with PR- to the total number of cells with PR-; wherein if the calculated ratio is greater than or equal to a predetermined threshold, the patient is classified as ER+, Ki-67+, PR+ dominant; or wherein if the calculated ratio is less than a predetermined threshold, the patient is classified as ER+, Ki-67+ dominant. In some embodiments, affinity histochemical staining for PR, ER, and Ki-67 biomarkers can be performed in any order. In other embodiments, histochemical staining for PR, ER, and Ki-67 biomarkers is performed in the order described above.

[0032] In some embodiments, the predetermined cutoff value is between about 0.4 and about 0.6. In some embodiments, the predetermined cutoff value is between about 0.45 and about 0.55. In some embodiments, the predetermined cutoff value is about 0.5.

[0033] In some embodiments, if a patient is classified as ER+, Ki-67+, PR+ dominant, that patient is selected to receive hormone therapy. In some embodiments, if a patient is classified as ER+, Ki-67+ dominant, that patient is selected to receive cyclin-dependent kinase 4 and 6 inhibitors.

[0034] In some embodiments, affinity histochemical staining of a sample with a human PR biomarker-specific reagent includes: (i) contacting the sample with the human PR biomarker-specific reagent under conditions that allow the PR biomarker-specific reagent to bind specifically to the sample; and (ii) contacting the sample with a first set of detection reagents, the first set of detection reagents interacting with the human PR biomarker-specific reagent to promote the deposition of a first brightfield detectable portion on the sample. In some embodiments, affinity histochemical staining of a sample with a human ER biomarker-specific reagent includes: (i) contacting the sample with a human ER biomarker-specific reagent under conditions that allow the ER biomarker-specific reagent to bind specifically to the sample; and (ii) contacting the sample with a second set of detection reagents, the second set of detection reagents interacting with the human ER biomarker-specific reagent to promote the deposition of a second brightfield detectable portion on the sample. In some embodiments, affinity histochemical staining of the sample with a human Ki-67 biomarker-specific reagent comprises: (i) contacting the sample with the human Ki-67 biomarker-specific reagent under conditions that allow the Ki-67 biomarker-specific reagent to specifically bind to the sample; and (ii) contacting the sample with a third group of detection reagents that interact with the human Ki-67 biomarker-specific reagent to promote the deposition of a third brightfield detectable portion on the sample. In some embodiments, the first brightfield detectable portion, the second brightfield detectable portion, and the third brightfield detectable portion are selected from the group consisting of TAMRA, Dabsyl, Dabcyl, Cy3, CyB, Cy3.5, Cy5, Cy5.5, Cy7, Rhodamine 800, and fluorescein.

[0035] The sixth aspect of this disclosure is a method for classifying patients with breast cancer as potentially responders or potentially non-responders to hormone therapy, the method comprising: performing affinity histochemical staining on a sample derived from the patient with a human PR biomarker-specific reagent under conditions allowing the PR biomarker-specific reagent to bind specifically to the sample; performing affinity histochemical staining on the sample derived from the patient with a human ER biomarker-specific reagent under conditions allowing the ER biomarker-specific reagent to bind specifically to the sample; performing affinity histochemical staining on the sample derived from the patient with a human Ki-67 biomarker-specific reagent under conditions allowing the Ki-67 biomarker-specific reagent to bind specifically to the sample; calculating the number of cells or nuclei that are positive for staining for all three of ER, Ki-67, and PR, and the number of cells that are (i) ER+, Ki-67+, and PR+ and (ii) ER+, Ki-67+ The ratio of the total number of cells with PR- to the total number of cells with PR-; wherein if the calculated ratio is greater than or equal to a predetermined threshold, the patient is classified as a likely responder; or wherein if the calculated ratio is less than a predetermined threshold, the patient is classified as a likely non-responder. In some embodiments, histochemical staining for PR, ER, and Ki-67 biomarkers can be performed in any order. In other embodiments, histochemical staining for PR, ER, and Ki-67 biomarkers is performed in the order described above. Attached Figure Description

[0036] This patent or application document contains at least one color drawing. Upon request and payment of the necessary fees, a copy of the published patent or application with color drawings will be provided to the Patent Office.

[0037] Figure 1 illustrates a method for assessing ER, Ki-67 and PR biomarkers in a sample using triple immunohistochemistry according to an embodiment of the present disclosure.

[0038] Figure 2 illustrates a method for assessing ER, Ki-67 and PR biomarkers in a sample using triple immunohistochemistry according to an embodiment of the present disclosure.

[0039] Figure 3 illustrates a method for assessing ER, Ki-67 and PR biomarkers in a sample using triple immunohistochemistry according to an embodiment of the present disclosure, and further illustrates the selection of different brightfield dyes for staining each of the different biomarkers.

[0040] Figures 4A to 4C illustrate single immunohistochemical assays in which different biomarkers are stained with different brightfield dyes. Figure 4A shows cells stained purple; Figure 4B shows cells stained blue / cyan; and Figure 4C shows cells stained yellow.

[0041] Figures 5A to 5C illustrate the colocalization of three different signals from three different brightfield dyes in cells or cell nuclei, where “PR” is purple, “ER” is blue / cyan, and “Ki-67” is yellow.

[0042] Figure 6A illustrates the colocalization of three different signals in a sample stained with three different brightfield dyes, such as colocalization signals from purple, blue / cyan, and yellow staining.

[0043] Figure 6B illustrates the colocalization of two different signals in samples stained with three different brightfield dyes.

[0044] Figure 7 depicts the disease-free survival of ER+, Ki-67+, PR+ symptomatic cases and ER+, Ki-67+ symptomatic cases.

[0045] Figure 8 shows the breast cancer-specific survival of ER+, Ki-67+, PR+ dominant cases and ER+, Ki-67+ dominant cases.

[0046] Figure 9 illustrates the reclassification of luminal type A breast cancer into ER+, Ki-67+, PR+ dominant subtypes and ER+, Ki-67+ dominant subtypes.

[0047] Figure 10 illustrates a method for treating a subject with breast cancer by classifying a sample derived from a subject as Ki-67+, Ki-67+, PR+ dominant, or Ki-67+, PR+ dominant according to an embodiment of the present disclosure. Detailed Implementation

[0048] It should also be understood that, unless expressly indicated otherwise, in any method claimed herein that includes more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order of the steps or actions of the method.

[0049] As used herein, unless the context clearly indicates otherwise, the singular forms “a / an” and “the / that” include plural referents. Similarly, unless the context clearly indicates otherwise, the word “or” is intended to include “and”. The term “including” is defined as inclusive, such as “including A or B” meaning including A, B, or A and B.

[0050] As used herein in the specification and claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when items are listed separately, “or” or “and / or” should be interpreted as inclusive, meaning it includes multiple elements or at least one element from a list of elements, but also includes more than one element, and optionally includes additional unlisted items. Only terms indicating the opposite, such as “only one” or “exactly one,” or “consisting of…” as used in the claims, will refer to the inclusion of several elements or exactly one element from a list of elements. Generally, the term “or” as used herein should only be interpreted as indicating an exclusive alternative (i.e., “one or the other, but not two”) when preceded by exclusive terms such as “or,” “one of,” “only one,” or “exactly one.” “Constitutes substantially of…” as used in the claims should have the ordinary meaning used in the field of patent law.

[0051] The terms “comprising,” “including,” and “having” are used interchangeably and have the same meaning. Similarly, the terms “comprising,” “including,” and “having” are used interchangeably and have the same meaning. Specifically, the definition of each term is consistent with the definition of “comprising” under ordinary U.S. patent law, and therefore each term can be understood as an open-ended term meaning “at least the following,” and can also be interpreted as not excluding additional features, limitations, aspects, etc. Thus, for example, “an apparatus having components a, b, and c” means that the apparatus includes at least components a, b, and c. Similarly, the phrase “a method relating to steps a, b, and c” means that the method includes at least steps a, b, and c. Furthermore, although the steps and processes may be described herein in a specific order, those skilled in the art will recognize that the order of steps and processes may vary.

[0052] As used herein in the specification and claims, with respect to a list of one or more elements, the phrase "at least one" should be understood as at least one element selected from any one or more elements in the list, but does not necessarily include at least one of each element specifically listed in the list, nor exclude any combination of elements in the list. In addition to the elements specifically identified in the list of elements referred to by the phrase "at least one," this definition also allows for the optional presence of other elements, whether or not they are related to the specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B," or equivalently, "at least one of A and / or B") in one embodiment may refer to at least one, optionally including more than one A, with no B (and optionally including elements other than B); in another embodiment, it refers to at least one, optionally including more than one B, with no A (and optionally including elements other than A); in yet another embodiment, it refers to at least one, optionally including more than one A, and at least one, optionally including more than one B (and optionally including other elements); and so on.

[0053] As used herein, the term "administration" means the physical introduction of a composition comprising a therapeutic agent into a subject using any of the various methods and delivery systems known to those skilled in the art. Routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration routes, such as by injection or infusion. As used herein, the phrase "parenteral administration" means other methods of administration besides enteral and local administration (typically administered by injection), and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrasheath, intralymphatic, intralesional, intracapsular, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. In some embodiments, the formulation is administered via a non-gastrointestinal route (orally in some embodiments). Other non-gastrointestinal routes include local, epidermal, or mucosal administration routes, such as intranasal, vaginal, rectal, sublingual, or local administration. Administration may also be performed, for example, once, multiple times, and / or over one or more extended time periods.

[0054] As used herein, the term “antibody” refers to and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired antigen-binding activity.

[0055] As used herein, the term "antibody fragment" refers to a molecule other than a complete antibody that comprises a portion of the complete antibody and binds to the antigen bound by that complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; bisomatic antibodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0056] As used herein, the terms “binding,” “specific binding,” “specifically binding to,” or “specific to” refer to a measurable and reproducible interaction, such as the binding between a target and a specific binder, which determines the presence of a target in the presence of a heterogeneous population of molecules, including biological molecules. For example, a binding entity that specifically binds to a target is an antibody that binds to that target with greater affinity, strength, ease, and / or longer duration compared to binding to other targets. In one embodiment, the degree of binding of an antibody to an irrelevant target is less than about 10% of the antibody's binding to the target, for example, as measured by radioimmunoassay (RIA). In some embodiments, the dissociation constant (Kd) of an antibody that specifically binds to a target is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM. In some embodiments, the antibody specifically binds to an epitope on a protein that is conserved across proteins of different species. In another embodiment, specific binding may include, but is not required to be, exclusive binding.

[0057] As used herein, the term "biomarker" refers to any molecule or group of molecules found in a biological sample that can be used to characterize that biological sample or the subject from which the biological sample was obtained. For example, a biomarker can be a molecule or group of molecules whose presence, absence, or relative abundance is: a characteristic of a particular cell or tissue type or state; or a characteristic of a particular pathological condition or state; or an indication of the severity of a pathological condition, the likelihood of progression or resolution of the pathological condition, and / or the likelihood that the pathological condition will respond to a particular treatment. As another example, a biomarker can be a cell type or microorganism (such as bacteria, mycobacteria, fungi, viruses, etc.), or a substituent molecule or a group of molecules thereof.

[0058] As used in this article, the phrase "biomarker-specific reagent" refers to a specific detection reagent, such as a primary antibody, that can bind directly and specifically to one or more biomarkers in a cell sample.

[0059] As used herein, the term “cancer” refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth lead to the formation of malignant tumors that invade adjacent tissues and may also metastasize to distant sites of the body via the lymphatic system or bloodstream. The term “cancer” is generally used interchangeably with “tumor” (unless a tumor is specifically referred to as a “benign” tumor, i.e., an abnormal cluster of cells lacking the ability to invade adjacent tissues or metastasize) and encompasses malignant solid tumors (e.g., carcinoma, sarcoma) and malignant growth in which there may not be a detectable cluster of solid tumors (e.g., certain hematologic malignancies). Non-limiting examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of this type of cancer include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal carcinoma), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, hepatocellular carcinoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, superficial diffuse melanoma, malignant lentigines melanoma, acral lentigines melanoma, nodular melanoma, multiple myeloma, and B-cell lymphoma (including low-grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL)). NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-lytic cell NHL; giant mass NHL; mantle cell lymphoma; AIDS-related lymphoma and Waldenström macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloid leukemia; and post-transplant lymphoproliferative disorders (PTLD), as well as abnormal angiogenesis associated with nevus hamartomas, edema (such as that associated with brain tumors), Megs syndrome, brain and head and neck cancers and related metastases. In some embodiments, cancers suitable for treatment with the antibodies of the present invention include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-Hodgkin lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid tumors, head and neck cancer, ovarian cancer, mesothelioma, and multiple myeloma. In some embodiments, the cancers are selected from: small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast cancer, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma.In some embodiments, the cancer is selected from non-small cell lung cancer, colorectal cancer, glioblastoma, and breast cancer, including metastatic forms of those cancers. In specific embodiments, the cancer is melanoma or lung cancer, appropriately metastatic melanoma or metastatic lung cancer.

[0060] As used herein, the terms “chromogen,” “chromogenic compound,” “mostly detectable,” or “dye,” etc., refer to substances that can be converted into colored compounds under specific conditions (e.g., when acted upon by an enzyme) or under specific chemical / reaction conditions. Examples of enzyme-substrate combinations include: (i) horseradish peroxidase (HRP) with catalase as a substrate, wherein catalase oxidizes a dye precursor [e.g., o-phenylenediamine (OPD) or 3,3',5,5'-tetramethylbenzidine hydrochloride (TMB)]; (ii) alkaline phosphatase (AP) with p-nitrophenyl phosphate as a chromogenic substrate; and (iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g., p-nitrophenyl-β-D-galactosidase) or a fluorescent substrate (e.g., 4-methylumbelliferone-β-D-galactosidase). Many other enzyme-substrate combinations can be utilized by those skilled in the art. For a general overview of these contents, see U.S. Patent Nos. 4,275,149 and 4,318,980.

[0061] As used in this article, the term "colocalize" refers to staining that occurs in the same or substantially the same place, such as staining that occurs in the same or substantially the same place or is located in the same or substantially the same place.

[0062] As used herein, the term "detection reagent" means any reagent used to stain a cellular sample by depositing near a biomarker-specific reagent that causes the detectable portion to bind to the biomarker. Non-limiting examples include secondary detection reagents (such as secondary antibodies capable of binding to primary antibodies, any reagent that specifically binds to biotin or avidin), tertiary detection reagents (such as tertiary antibodies capable of binding to secondary antibodies), enzymes directly or indirectly associated with specific binders, chemicals that react with such enzymes to affect the deposition of fluorescent or chromogenic staining agents, washing reagents used between staining steps, etc.

[0063] As used herein, the term "estrogen receptor" or "ER" refers to a nuclear hormone family member of the intracellular receptor activated by 17β-estradiol. The estrogen receptor is overexpressed in approximately 70% of breast cancer cases, termed "ER-positive" (ER+). The ESR1 gene encodes a transcription factor that activates the estrogen receptor and its ligands. A typical protein contains an N-terminal ligand-independent transactivation domain, a central DNA-binding domain, a hinge domain, and a C-terminal ligand-dependent transactivation domain. The protein localizes to the nucleus, where it can form a homodimer or heterodimer with estrogen receptor 2. The protein encoded by this gene regulates the transcription of many estrogen-inducible genes that play roles in growth, metabolism, sexual development, pregnancy, and other reproductive functions and are expressed in many non-reproductive tissues. The receptor encoded by the ESR1 gene plays a crucial role in breast cancer, endometrial cancer, and osteoporosis.

[0064] As used herein, the term “formalin-fixed paraffin-embedded (FFPE) tissue section” refers to a piece of tissue fixed in formaldehyde (e.g., phosphate-buffered saline with 3% to 5% formaldehyde) or Bouin solution, embedded in paraffin, cut into thin sections, and then mounted on a flat surface (e.g., a microscope slide), such as a biopsy tissue obtained from a subject.

[0065] As used herein, the term "monoclonal antibody" ("mAb") refers to a non-naturally occurring formulation of an antibody molecule consisting of a single molecule, i.e., an antibody molecule whose primary sequence is substantially identical and which exhibits single binding specificity and affinity for a specific epitope. An mAb is an example of an isolated antibody. MAbs can be produced by hybridoma, recombinant, transgenic, or other techniques known to those skilled in the art.

[0066] As used herein, the term "immunohistochemistry" refers to a method for determining the presence or distribution of an antigen in a sample by detecting the interaction between the antigen and a specific binding agent (such as an antibody) within the sample. The sample is contacted with the antibody under conditions that allow antibody-antigen binding. Antibody-antigen binding can be detected using a detectable tag conjugated to the antibody (direct detection) or a detectable tag conjugated to a second antibody that specifically binds to a first antibody (indirect detection).

[0067] As used herein, the term "Ki-67" refers to a nuclear protein associated with cell proliferation and ribosomal RNA transcription. Inactivation of the antigen Ki-67 leads to inhibition of ribosomal RNA synthesis. For example, Ki-67 is used as a marker of proliferation. The MKI67 gene promotes protein C-terminal binding activity and is involved in the regulation of chromosome segregation and mitotic nuclear division.

[0068] As used herein, the terms “primary antibody” and “secondary antibody” refer to different antibodies, where a primary antibody is a polyclonal or monoclonal antibody derived from a single species (rabbit, mouse, goat, donkey, etc.) that specifically recognizes an antigen (e.g., a biomarker) in the sample under study (e.g., a human biological sample), and a secondary antibody is an antibody (usually a polyclonal antibody) derived from a different species that specifically recognizes the primary antibody, for example, in its Fc region.

[0069] As used herein, the term "progesterone receptor" or "PR" refers to the intracellular steroid receptor that specifically binds to progesterone. The progesterone receptor is overexpressed in some breast cancer cases, termed "PR positive" (PR+). The PGR gene encodes a member of the steroid receptor superfamily. The encoded protein mediates the physiological effects of progesterone, which plays a central role in reproductive events associated with the establishment and maintenance of pregnancy. This gene uses two distinct promoters and translation initiation sites in its first exon to produce several transcriptional variants, including both protein-coding and non-protein-coding variants. Apart from an additional 165 amino acids found at the N-terminus of isoform B, the two isoforms (A and B) are identical and mediate their own response genes and physiological effects with little overlap.

[0070] As used herein, the term “sample” should refer to any material obtained from a subject who can be tested for the presence or absence of a biomarker, such as a tissue sample or a cytological sample.

[0071] As used herein, the term “slide” refers to any substrate of suitable size on which a biological sample is placed for analysis (e.g., a substrate made wholly or partially of glass, quartz, plastic, silicon, etc.), and such as “microscope slide”, such as a standard 3-inch x 1-inch microscope slide or a standard 75 mm x 25 mm microscope slide.

[0072] When used as a noun, the term "stain" refers to any substance that can be used to visualize specific molecules or structures in cell samples for microscopic analysis, including bright-field microscopy, fluorescence microscopy, electron microscopy, etc. When used as a verb, the term "stain" refers to any process that causes a stain to deposit on a cell sample.

[0073] As used herein, the terms "subject" or "individual" refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0074] As used herein, the term "tumor" refers to all proliferative cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. In some embodiments, a tumor is a malignant cancerous tumor (i.e., cancer). In some embodiments, a tumor is a solid tumor or a non-solid tumor or a soft tissue tumor. Examples of soft tissue tumors include leukemias (e.g., chronic myeloid leukemia, acute myeloid leukemia, adult acute lymphoblastic leukemia, acute myeloid leukemia, mature B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, multi-lymphocytic leukemia, or hairy cell leukemia) or lymphomas (e.g., non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, or Hodgkin's disease). Solid tumors include cancers of any human tissue other than the blood, bone marrow, or lymphatic system. Solid tumors can be further classified into epithelial cell-derived solid tumors and non-epithelial cell-derived solid tumors. Examples of epithelial cell solid tumors include gastrointestinal tumors, colon tumors, colorectal tumors (e.g., basal colorectal cancer), breast tumors, prostate tumors, lung tumors, kidney tumors, liver tumors, pancreatic tumors, ovarian tumors (e.g., endometrioid ovarian cancer), head and neck tumors, oral cavity tumors, stomach tumors, duodenal tumors, small intestine tumors, large intestine tumors, anal tumors, gallbladder tumors, labial tumors, nasopharyngeal tumors, skin tumors, uterine tumors, male reproductive organ tumors, urinary organ tumors (e.g., urothelial carcinoma, dysplastic urothelial carcinoma, transitional cell carcinoma), bladder tumors, and skin tumors. Non-epithelial cell-derived solid tumors include sarcomas, brain tumors, and bone tumors.

[0075] SUMMARY

[0076] This disclosure relates to a multiplex immunohistochemical assay for assessing the expression of ER, PR, and Ki-67 biomarkers in a sample, such as by detecting the co-localization of ER, PR, and Ki-67 biomarkers in cells or cell nuclei. It is believed that the multiplex immunohistochemical assay of this disclosure can be used as a prognostic assay for ER-positive breast cancer, facilitating the identification of disease-free survivors in ER-positive breast cancer patients treated with hormone therapy. In fact, the applicant has surprisingly discovered that the ER and PR status of proliferating cancer cells (i.e., Ki-67-positive cancer cells) is a potent prognostic factor for patients with ER-positive breast cancer.

[0077] Methods of assessing expression of ER, Ki-67, and PR biomarkers

[0078] This disclosure provides a method for assessing the expression of ER, Ki-67, and PR biomarkers (such as the co-localization expression of each of the ER, Ki-67, and PR biomarkers in the cell nucleus).

[0079] In some embodiments, the method employs affinity histochemistry or affinity cytochemistry assays to stain each of the ER, Ki-67, and PR biomarkers, such as with different detectable portions. Affinity histochemistry and cytochemistry staining techniques typically involve contacting a sample deposited on a slide or other solid support with a biomarker-specific reagent under conditions sufficient to allow specific binding between the biomarker-specific reagent and the biomarker of interest. The binding of the biomarker-specific reagent to the biomarker promotes the deposition of the detectable portion on the sample near the site containing the biomarker. The detectable portion can be used to locate and / or quantify the biomarker targeted by the biomarker-specific reagent. Thus, the presence and / or relative amount of the target in the sample can be detected by detecting the signal generated by the detectable portion.

[0080] In some embodiments, the method employs multiple affinity histochemistry or affinity cytochemistry assays (e.g., immunohistochemistry) in which a single tissue sample is stained for the presence of ER, Ki-67, and PR biomarkers. In some embodiments, the multiple affinity histochemistry or affinity cytochemistry assays (e.g., immunohistochemistry) of this disclosure employ different bright-field detectable portions, such that each of the ER, Ki-67, and PR biomarkers is stained with a different bright-field detectable portion. After staining the tissue sample in multiple affinity histochemistry or affinity cytochemistry assays (e.g., immunohistochemistry), the stained sample can be evaluated manually or using one or more automated digital pathology techniques. The evaluated stained sample can then be used for risk assessment and / or selected treatment regimens. These and other embodiments are further described herein, for example.

[0081] Affinity histochemical or affinity cytochemical assays

[0082] In some embodiments, techniques such as immunohistochemistry (IHC) are used to assess the expression of ER, Ki-67, and / or PR biomarkers. In some embodiments, the samples are tissue sections (including, but not limited to, formalin-fixed paraffin-embedded (FFPE) tissue sections and freshly frozen tissue sections).

[0083] IHC assays involve contacting a sample with a biomarker-specific reagent under conditions that promote specific binding between the biomarker (e.g., ER, PR, and / or Ki-67) and the biomarker-specific reagent (also referred to herein as a “specific binding entity” or “specific binder”), and removing unbound biomarker-specific reagent from the sample (e.g., by washing with a washing buffer). If the biomarker-specific reagent is directly conjugated to a detectable portion (referred to as a “direct detection IHC assay”), the sample can be analyzed directly. For example, a biomarker-specific reagent can be directly conjugated to a bright-field detectable portion and analyzed directly.

[0084] Alternatively, the sample can be contacted with a set of detection reagents that interact with biomarker-specific reagents to promote the deposition of a detectable moiety on or near the biomarker, thereby generating a detectable signal localized to the biomarker. For example, the detection reagents may contain a secondary antibody specific to the biomarker-specific reagent, wherein the secondary antibody is conjugated to an enzyme. The detection reagents may further contain a conjugate comprising a bright-field detectable moiety, wherein an enzyme conjugated to the secondary antibody acts on the conjugate to deposit the bright-field detectable moiety on or near the biomarker.

[0085] Typically, one or more inactivation and / or washing steps are performed between the application of different reagents to avoid non-specific staining of the tissue. Biomarker-labeled sections may optionally be additionally labeled with contrast agents (such as hematoxylin staining) to visualize macromolecular structures within the cellular sample. One or more other biomarkers of the sample may also be tested.

[0086] sample

[0087] Any type of sample compatible with IHC assays can be used.

[0088] In some embodiments, the sample may be a tissue section, or, for example, a tissue section that has been processed to remove at least a portion of the tissue section for further genetic analysis. In some embodiments, the sample may be a cytological sample, deposited or printed on a microscope slide. In some embodiments, the cytological sample may be in the form of a block of sliced ​​cells, such as a block of cells formed from paraffin or other matrices (such as gels, aerogels, polymers, proteins) that hold the cells in a 3D structure for slicing. In some embodiments, the cytological sample may be in the form of a cell clump, for example, formed by precipitation or centrifugation, such as a clump of sliceable material for placement on a microscope slide.

[0089] In other embodiments, the sample is in the form of a fine-needle aspirate or a core extracted from a tissue sample, whether in vivo or ex vivo. In still other embodiments, the sample may be a representative sample such as that described in U.S. Patent Publication No. 2020 / 0049599, the disclosure of which is incorporated herein by reference in its entirety.

[0090] Samples used in the methods disclosed herein can be prepared using any method known in the art. Samples can be obtained from subjects undergoing routine screening or from subjects suspected of having a condition or suspected of having a condition such as cancer. In some embodiments, samples are derived from subjects diagnosed with cancer (e.g., subjects previously diagnosed with breast cancer) or subjects suspected of having cancer (e.g., subjects suspected of having breast cancer).

[0091] In some embodiments, the sample originates from a tumor in a subject (such as a subject diagnosed with or suspected of having breast cancer). In some embodiments, the sample originates from a metastatic breast tumor in a subject. In some embodiments, the sample originates from a patient previously diagnosed with luminal type A breast cancer. In some embodiments, the sample originates from a patient previously diagnosed with luminal type B breast cancer. In some embodiments, the sample originates from a subject who has undergone prior treatment for cancer (e.g., breast cancer or luminal type A breast cancer); or a subject currently undergoing treatment for cancer (e.g., breast cancer or luminal type A breast cancer).

[0092] In other embodiments, the sample may be free of any abnormalities, diseases, symptoms, etc., and is referred to as a "normal" sample. Such normal samples can be used, in addition to many other applications, as controls for comparison with other samples. For example, it can be useful to test for cancer in a patient (i.e., a human subject) by collecting tissue samples from multiple sites, and these samples can be used as controls and compared with later samples to determine whether a particular cancer has spread beyond its primary site.

[0093] In some embodiments, the sample has been previously screened in one or more genetic tests. In some embodiments, the sample has been previously identified as ER positive. In some embodiments, the previously identified sample includes proliferating cells, such as by assessing the expression of one or more proliferation biomarkers.

[0094] In some embodiments, the sample is a fixed sample. Fixed samples maintain cellular and tissue components in a state as close to that of life as possible and allow them to undergo preparative procedures without significant alteration. This prevents autolysis and bacterial degradation processes that begin after cell death and stabilizes the cellular and tissue components of the sample, making them resistant to subsequent tissue processing stages. Fixatives can be classified as cross-linking agents (such as aldehydes, e.g., formaldehyde, polyoxymethylene, and glutaraldehyde, as well as non-aldehyde cross-linking agents), oxidizing agents (e.g., metal ions and complexes, such as osmium tetroxide and chromic acid), protein denaturing agents (e.g., acetic acid, methanol, and ethanol), fixatives of unknown mechanism (e.g., mercuric chloride, acetone, and picric acid), combination reagents (e.g., Carnoy's fixative, methanol-Carnoy's fixative, Bouaner's solution, B5 fixative, Rothmann's solution, and Randle's solution), microwave fixatives, and various fixatives (e.g., exclusion volume fixation and vapor fixation). Fixatives may also include additives such as buffers, detergents, tannins, phenols, metal salts (such as zinc chloride, zinc sulfate, and lithium salts), and lanthanum. The most commonly used fixative in sample preparation is formaldehyde, typically in the form of a formalin solution (an aqueous solution of formaldehyde (and usually a buffered aqueous solution of formaldehyde)). In some embodiments, the samples used in the methods of this disclosure are fixed by means of fixation in a formalin-based fixative. In one example, the fixative is 10% neutral buffered formalin. Despite these examples, tissues can be fixed using any fixation medium compatible with the specific reagents and assays used for the biomarkers.

[0095] In some embodiments, the fixed sample is embedded in an embedding medium. The embedding medium is an inert material in which tissues and / or cells are embedded to help preserve them for future analysis. Embedding also allows cell samples to be sliced ​​into thin sections. Embedding media include paraffin, collodion, OCT™ compounds, agar, plastics, or acrylic resins. In some embodiments, the sample is fixed in a formalin-based fixative and embedded in paraffin to form a formalin-fixed paraffin-embedded (FFPE) block.

[0096] In some embodiments, if the sample is embedded in paraffin, it can be dewaxed using a suitable dewaxing process.

[0097] In some embodiments, biological samples are pretreated with an enzyme inactivation composition to substantially or completely inactivate endogenous peroxidase activity. For example, some cells or tissues contain endogenous peroxidases. The use of HRP-conjugated antibodies may result in high, nonspecific background staining. This nonspecific background can be reduced by pretreating the sample with the enzyme inactivation composition disclosed herein. In some embodiments, the sample is pretreated with hydrogen peroxide (about 1% to about 3% by weight of a suitable pretreatment solution) to reduce endogenous peroxidase activity. Once the endogenous peroxidase activity is reduced or inactivated, an assay kit is added, and the enzyme present in the assay kit is then inactivated (as described above). The disclosed enzyme inactivation compositions and methods can also be used as methods for inactivating the activity of endogenous enzyme peroxidase. Additional inactivation compositions are described in U.S. Patent Publication No. 2018 / 0120202, the disclosure of which is incorporated herein by reference in its entirety.

[0098] Automated dyeing system

[0099] The IHC assays described herein can be performed on an automated staining apparatus, manually, or as a combination of automated and manual steps. In some embodiments, the automated staining apparatus includes: one or more reservoirs (such as for storing various reagents used in the labeling protocol), one or more reagent dispensing units in fluid communication with the one or more reservoirs for dispensing reagents onto a sample, a waste removal system for removing used reagents and other waste from the sample, and a control system coordinating the operation of the one or more reagent dispensing units and the waste removal system. In addition to performing the labeling step, the automated staining apparatus may also be configured to perform labeling-assisted steps (or be compatible with a separate system for performing such auxiliary steps), including but not limited to: slide baking (for adhering the sample to the slide), dewaxing (also known as deparaffinizing), antigen retrieval, counterstaining, dehydration and cleaning, and covering with a coverslip.

[0100] The entire contents of this paper are incorporated herein by reference in Prichard, Overview of Automated Immunohistochemistry, Arch Pathol Lab Med., Vol. 138, pp. 1578–1582 (2014), which describes several specific examples of automated staining devices and their various features, including the intelliPATH (Biocare Medical), WAVE (Celerus Diagnostics), DAKO OMNIS and DAKO LINK 48 automated stainer (Agilent Technologies), BENCHMARK (Ventana Medical Systems, Inc.), Leica BOND and LAB VISION automated stainer (Thermo Scientific) automated AHC labeling systems. Additionally, Ventana Medical Systems, Inc. is the assignee of several U.S. patents disclosing systems and methods for performing automated analyses, including U.S. Patent Nos. 5,650,327, 5,654,200, 6,296,809, 6,352,861, 6,827,901, and 6,943,029, and U.S. Patent Application Publication Nos. 20030211630 and 20040052685, each of which is incorporated herein by reference in its entirety.

[0101] Automated staining equipment typically operates on one of the following principles: (1) open single-slide labeling, in which the slide is placed horizontally and reagent is dispensed as a pool onto the surface of the slide containing the tissue sample (such as performed on the DAKO Automated Stainer Link 48 (Agilent Technologies) and INTELLIPATH (Biocare Medical) labelers); (2) liquid-covering techniques, in which reagent is covered by or dispensed through an inert fluid layer deposited on the sample (such as performed on the BENCHMARK and DISCOVERY labelers); (3) capillary gap labeling, in which the slide surface is placed close to another surface (which may be another slide or cover plate) to form a narrow gap through which capillary forces draw liquid reagent and bring the liquid reagent into contact with the sample (such as the labeling principle used by the DAKOTECHMATE, Leica BOND, and DAKO OMNIS labelers).

[0102] Some iterations of capillary gap marking do not mix the fluid in the gap (e.g., on DAKO TECHMATE and Leica BOND). In a variant of capillary gap marking called dynamic gap marking, the sample is applied to a slide using capillary force, and then parallel surfaces are translated relative to each other to agitate the reagents during incubation to achieve reagent mixing (e.g., the marking principle performed on the DAKO OMNIS slide marker (Agilent)). In translational gap marking, a translational head is located on the slide. The lower surface of this head is spaced from the slide by a first gap small enough to allow the liquid on the slide to form a liquid meniscus during slide translation. A mixing extension, with a lateral dimension smaller than the width of the slide, extends from the lower surface of the translational head to define a second gap smaller than the first gap between the mixing extension and the slide. During head translation, the lateral dimensions of the mixing extension are sufficient to generate lateral movement in the liquid on the slide in a direction substantially extending from the second gap to the first gap (see WO 2011 / 139978A1, the disclosure of which is hereby incorporated herein by reference in its entirety). It is also suggested to use inkjet technology to deposit reagents on the slide (see WO 2016 / 170008A1, the disclosure of which is hereby incorporated herein by reference in its entirety). This list of labeling techniques is not intended to be exhaustive, and any fully automated or semi-automated systems or manual methods used to perform biomarker labeling may be incorporated into this method.

[0103] Specific binding entity

[0104] This disclosure employs at least three different specific binding entities, namely binding entities that are specific to the ER, Ki-67 and PR biomarkers.

[0105] As used in this article, the phrases “specific binder,” “specific binding entity,” and “biomarker-specific reagent” refer to any substance that can specifically bind to a target chemical structure (such as a biomarker expressed by the sample or a biomarker-specific reagent that binds to the sample) associated with a cell sample. Examples include antibodies and their antigen-binding fragments; and engineered specific binding structures, including ADNECTIN (a scaffold based on fibronectin 10 FN3; Bristol-Myers-Squibb Co.), AFFIBODY (a scaffold based on the Z domain of protein A from Staphylococcus aureus; Affibody AB, Solna, Sweden), AVIMER (a scaffold based on domain A / LDL receptor; Amgen, Thousand Oaks, California), dAb (a scaffold based on VH or VL antibody domains; GlaxoSmithKline PLC, Cambridge, UK), DARPin (a scaffold based on ankyrin repeat protein; Molecular Partners AG, Zurich, Switzerland), ANTICALIN (a scaffold based on lipid transport protein; Pieris AG, Freising, Germany), and NANOBODY (a scaffold based on VHH (Camelidae Ig); Ablynx). N / V (Ghent, Belgium), TRANS-BODY (transferrin-based scaffold; Pfizer Inc., NY, New York), SMIP (Emergent Biosolutions, Inc., Rockville, Maryland), and TETRANECTIN (C-type lectin domain-based scaffold (CTLD), tetralinkin; Borean Pharma A / S, Aarhus, Denmark). A review of these engineered specific binding structures is presented in Wurch et al., Development of Novel Protein Scaffolds as Alternatives to Whole Antibodies for Imaging and Therapy: Status on DISCOVERY Research and Clinical Validation, Current Pharmaceutical Biotechnology, Vol. 9, pp. 502-509 (2008), which is incorporated herein by reference.

[0106] In some embodiments, the biomarker-specific reagent that is specific to the ER, Ki-67, and PR biomarkers is an antibody, such as a monoclonal antibody (such as a mouse monoclonal antibody or a rabbit monoclonal antibody).

[0107] Non-limiting examples of human Ki-67 biomarker-specific antibodies include antibodies belonging to the MIB@ family, such as MIB-1, MIB-2, MIB-5, MIB-7, MIB-21, and MIB-24. Examples of human Ki-67 biomarker-specific antibodies include the anti-Ki-67(30-9) monoclonal antibody available from Ventana Medical Systems, Inc. (Tucson, Arizona). Other examples of human Ki-67 biomarker-specific antibodies include clone 30-9 (Roche) and clone Ki-67 (BioLegend).

[0108] Non-limiting examples of human ER biomarker-specific antibodies include an anti-ER (SP1) monoclonal antibody available from Ventana Medical Systems, Inc. (Tucson, Arizona). Another example of a human ER biomarker-specific antibody includes the anti-ER monoclonal antibody 1D5 available from Invitrogen. High-affinity monoclonal antibodies for recognizing the ER receptor are described in U.S. Patent No. 7,569,675, the disclosure of which is incorporated herein by reference in its entirety.

[0109] Non-limiting examples of human ER biomarker-specific antibodies include an anti-PR (1E2) monoclonal antibody available from Ventana Medical Systems, Inc. (Tucson, Arizona). A high-affinity monoclonal antibody for recognizing the PR receptor is described in U.S. Patent No. 7,569,675, the disclosure of which is incorporated herein by reference in its entirety.

[0110] Test reagents and detectable components

[0111] Detection of biomarkers in a sample is achieved by depositing a detectable portion adjacent to a biomarker-specific reagent that binds to the sample. In some embodiments, the detectable portion is directly or indirectly conjugated to a biomarker-specific reagent (e.g., a monoclonal antibody, including any of those described herein), and thus deposited on the sample when the biomarker-specific reagent binds to its target (generally referred to as a direct labeling method). In other embodiments, deposition of the detectable portion is achieved by applying a set of detection reagents to the sample after the application of the biomarker-specific reagent, wherein the detection reagents bind to or otherwise react with the biomarker-specific reagent in a manner that affects the deposition of the detectable portion (generally referred to as an indirect labeling method).

[0112] For example, and in other embodiments, the biomarker-specific reagents (e.g., human ER biomarker-specific antibodies, human Ki-67 biomarker-specific antibodies, and human PR biomarker-specific antibodies) do not include a detectable portion. In these embodiments, the sample is then contacted with a set of detection reagents that interact with a specific binder to promote the deposition of the detectable portion (e.g., a chromogen) on or adjacent to the biomarker, thereby generating a detectable signal localized to the biomarker.

[0113] In some embodiments where an indirect method is used, the detectable moiety is deposited via an enzymatic reaction targeting a biomarker-specific reagent (e.g., a monoclonal antibody). Suitable enzymes for such reactions are well known and include, but are not limited to, oxidoreductases, hydrolases, and peroxidases. Specific enzymes explicitly included are horseradish peroxidase (HRP), alkaline phosphatase (AP), acid phosphatase, glucose oxidase, β-galactosidase, β-glucuronidase, and β-lactamase. The enzyme may be directly conjugated to the biomarker-specific reagent (e.g., a monoclonal antibody) or indirectly associated with the biomarker-specific reagent via a labeled conjugate. As used herein, a “labeled conjugate” includes:

[0114] (a) Specific detection reagents; and

[0115] (b) An enzyme conjugated to a specific detection reagent, wherein the enzyme can react with a chromogenic substrate, a signal transduction conjugate and / or an enzyme-reactive dye under appropriate reaction conditions to achieve in situ generation of the dye and / or deposition of the dye on a tissue sample.

[0116] In non-limiting examples, the specific detection reagent for the labeled conjugate can be a secondary detection reagent (such as a species-specific secondary antibody bound to a primary antibody, an anti-hapten antibody bound to a primary antibody conjugated to a hapten, or a biotin-binding protein bound to a biotinylated primary antibody), a tertiary detection reagent (such as a species-specific tertiary antibody bound to a secondary antibody, an anti-hapten antibody bound to a secondary antibody conjugated to a hapten, or a biotin-binding protein bound to a biotinylated secondary antibody), or other such arrangements. An enzyme thus positioned to bind a biomarker-specific reagent (e.g., a monoclonal antibody) to a sample can then be used in various protocols to deposit a detectable portion.

[0117] In non-limiting examples, the specific detection reagent for the labeled conjugate can be a secondary detection reagent (such as a species-specific secondary antibody bound to a primary antibody, an anti-hapten antibody bound to a primary antibody conjugated to a hapten, or a biotin-binding protein bound to a biotinylated primary antibody), a tertiary detection reagent (such as a species-specific tertiary antibody bound to a secondary antibody, an anti-hapten antibody bound to a secondary antibody conjugated to a hapten, or a biotin-binding protein bound to a biotinylated secondary antibody), or other such arrangements. An enzyme thus positioned to bind a biomarker-specific reagent to a sample can then be used in various protocols to deposit a detectable moiety. In some cases, the enzyme reacts with a chromogenic compound / substrate.

[0118] In other embodiments, the detectable portion is deposited via a signal conjugate comprising a potentially reactive portion configured to react with an enzyme to form a reactive species that can bind to the sample or other detection components. These reactive species are capable of reacting with the sample in the vicinity of their formation (i.e., near the enzyme) but rapidly convert to non-reactive species, preventing the signal conjugate from depositing at sites distant from the enzyme deposition site. Examples of potentially reactive portions include quinone methyl (QM) analogs, such as those described in WO2015124703A1, and tyrosine conjugates, such as those described in WO2012003476A2, the full text of which is incorporated herein by reference. In some instances, the potentially reactive moiety is directly conjugated to a dye, such as N,N'-biscarboxypentyl-5,5'-disulfonyl-indole-dicarbonylcyanine (Cy5), 4-(dimethylamino)azobenzene-4'-sulfonamide (DABSYL), tetramethylrhodamine (DISCO Violet), and rhodamine 110 (rhodamine). In other instances, the potentially reactive moiety is conjugated to one member of a specific binding pair, and the dye is linked to the other member of the specific binding pair. In still other instances, the potentially reactive moiety is linked to one member of a specific binding pair, and an enzyme is linked to the other member of the specific binding pair, wherein the enzyme (a) reacts with a chromogenic substrate to affect dye production, or (b) reacts with the dye to affect the deposition of a dye (such as DAB). Examples of specific binding pairs include: (1) a biotin or biotin derivative (such as desulfobiotin) linked to a potential reactive moiety, and a biotin-binding entity (such as avidin, streptavidin, desacchariavidin (such as NEUTRAVIDIN) or a biotin-binding protein (such as CAPTAVIDIN) having a nitrated tyrosine residue at its biotin binding site linked to a dye or an enzyme (such as a peroxidase linked to a biotin-binding protein when the dye is DAB); and (2) a hapten linked to a potential reactive moiety, and a hapten linked to a dye or an enzyme (such as a peroxidase linked to a biotin-binding protein when the dye is DAB).

[0119] Any detection reagent or detectable moiety compatible with multiplex affinity histochemistry or affinity cytochemistry (e.g., immunohistochemistry) may be used in the methods of this disclosure. In some embodiments, the detectable moiety is a molecule detectable via bright-field microscopy. Non-limiting examples of bright-field detectable moieties compatible with IHC, including multiplex IHC, and methods for using bright-field detectable moieties are disclosed in US 10,041,950, the disclosure of which is incorporated herein by reference in its entirety. Specific examples of brightfield detectable components (also known as chromogens) include, but are not limited to, diaminobenzidine (DAB), 4-(dimethylamino)azobenzene-4'-sulfonamide (DABSYL), tetramethylrhodamine, N,N'-biscarboxypentyl-5,5'-disulfonyl-indole-dicarbonylcyanine (Cy5) and rhodamine 110 (rhodamine), 4-nitrophenyl phosphate (pNPP), Solid Red, bromochloroindole phosphate (BCIP), nitroblue tetrazolium (NBT), BCIP / NBT, Solid Red, AP Orange, AP Blue, tetramethylbenzidine (TMB), and 2,2'-azido-di-[3-ethylbenzothiazoline sulfonate]. (ABTS), 4-chloronaphthol (4-CN), nitrophenyl-β-D-galactopyranoside (ONPG), o-phenylenediamine (OPD), 5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-Gal), methylumbelliferyl-β-D-galactopyranoside (MU-Gal), p-nitrophenyl-α-D-galactopyranoside (PNP), 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc), 3-amino-9-ethylcarbazole (AEC), magenta, iodonitrotetrazol (INT), tetrazolium blue, or tetrazolium violet. In some embodiments, the brightfield detectable portion is translucent. Given the translucency of the brightfield detectable portion, different color combinations may occur if two or more brightfield detectable portions are deposited at the same or approximately the same location. Against the backdrop of staining specific biomarkers within cells or cell nuclei, the translucency of the bright-field detectable portion facilitates the detection of different expression patterns of two or more biomarkers due to the appearance of specific colors produced by combinations of different bright-field detectable portions.

[0120] In some embodiments, the bright-field detectable portion is selected from TAMRA, Dabsyl, Dabcyl, Cy3, CyB, Cy3.5, Cy5, Cy5.5, Cy7, Rhodamine 800, and fluorescein. In some embodiments, the bright-field detectable portion is a conjugate comprising at least two chromogens, such as at least two chromogens selected from TAMRA, Dabsyl, Dabcyl, Cy3, CyB, Cy3.5, Cy5, Cy5.5, Cy7, Rhodamine 800, and fluorescein. In some embodiments, the bright-field detectable portion is selected from those described in U.S. Patent Publication No. 2021 / 0055285, the disclosure of which is incorporated herein by reference in its entirety.

[0121] Non-limiting examples of commercially available assay reagents or kits containing assay reagents suitable for the methods of the present invention include: the VENTANA ULTRAVIEW assay system (with an enzyme-conjugated secondary antibody, said enzyme comprising HRP and AP); the VENTANA IVIEW assay system (with a biotinylated anti-species secondary antibody and a streptavidin-conjugated enzyme); the VENTANA OptiView assay system (with an anti-species secondary antibody conjugated to a hapten and an anti-hapten tertiary antibody conjugated to an enzyme polymer); VENTANA amplification kits (with unconjugated secondary antibodies that can be used with any of the aforementioned VENTANA assay systems to amplify the amount of enzyme deposited at the primary antibody binding site); and VENTANA OptiView. Amplification systems (secondary anti-happen antibodies conjugated to haptens, tertiary anti-happen antibodies conjugated to enzyme polymers, and tyrosine conjugated to the same hapten. In use, the secondary antibody is contacted with the sample to achieve binding with the primary antibody. The sample is then incubated with the anti-hapten antibody to achieve association between the enzyme and the secondary antibody. The sample is then incubated with tyrosine to achieve deposition of additional hapten molecules. The sample is then incubated again with the anti-hapten antibody to achieve deposition of additional enzyme molecules. The sample is then incubated with the detectable portion to achieve dye deposition); VENTANA DISCOVERY, DISCOVERY OMNIMAP, DISCOVERY ULTRAMAP anti-hapten antibody, secondary antibody, chromogen, fluorophore, and dye kits, each of which is available from Ventana Medical Systems, Inc. (Tucson, Arizona); POWERVISION and POWERVISION+ IHC detection systems (directly connected to HRP or AP Secondary antibodies polymerized into compact polymers carrying both enzymes and antibodies at a high ratio; the DAKOENVISION™+ system (enzyme-labeled polymers conjugated with secondary antibodies); and the ULTRAPLEX multiplex colorimetric IHC technology from CELL IDx (hapten-labeled primary antibodies combined with enzyme-labeled or fluorescently labeled anti-hapten secondary antibodies).

[0122] Commercially available bright-field detectable components include Discovery Red, Discovery Yellow, Discovery Blue, Discovery Violet, Discovery Silver, Discovery Blue-Green, and Discovery Green, each of which is available from Roche Diagnostics.

[0123] Other suitable detectable conjugates, including different detectable moieties, are disclosed in PCT Publication No. WO / 2022 / 043491, the contents of which are incorporated herein by reference in their entirety. For example, PCT Publication No. WO / 2022 / 043491 discloses detectable moieties with different “core” structures, such as a coumarin core, a phenoloxazinone core, a 4-hydroxy-3-phenoloxazinone core, a 7-amino-4-hydroxy-3-phenoloxazinone core, a thiononium core, a phenoloxazine core, a phenoloxathia-3-one core, or a xanthine core. Any of these detectable moieties may be suitable for labeling ER, Ki-67, and / or PR biomarkers.

[0124] Other detection reagents, detectable components, and detection strategies are described in U.S. Patent Nos. 11,249,085, 11,249,085, and 10,168,336; and in U.S. Patent Publication No. 2012 / 0171668, the disclosure of which is incorporated herein by reference in its entirety.

[0125] Typically, washing steps are performed between the application of different reagents to prevent unwanted nonspecific labeling of tissues. For example, washing can be performed after each of these pretreatment steps by applying a washing buffer once or multiple times. Washing buffers are typically neutral buffered saline solutions, which may also contain small amounts of detergent. Non-limiting examples of washing buffers include, for instance, phosphate-buffered saline (PBS), PBS-Tween 20, tris(hydroxymethyl)aminomethane buffered saline (TBS), TBS-Tween 20 (polysorbate 20), tris(hydroxymethyl)aminomethane-HCl, tris(hydroxymethyl)aminomethane-HC-Tween 20, phosphate buffer (PB), AP buffer, etc.

[0126] Counterstaining and morphological staining

[0127] In some embodiments, samples may be counterstained to aid in the manual or automated identification of morphologically relevant regions. Examples of counterstains include chromogenic nuclear counterstains such as hematoxylin (staining from blue to purple), methylene blue (staining blue), toluidine blue (staining cell nuclei dark blue and polysaccharides pink to red), nuclear solid red (also known as Kernechtrot dye, staining red), and methyl green (staining green); and non-nuclear chromogenic stains such as eosin (staining pink). In some embodiments, the counterstain is a nonnuclear chromogenic staining agent, such as eosin (stains pink); a fluorescent nuclear staining agent, including 4',6-diamino-2-phenylindole (DAPI, stains blue), propidium iodide (stains red), Hoechst staining agent (stains blue), nuclear green DCS1 (stains green), riboflavin (Hoechst S769121, stains yellow at neutral pH and blue at acidic pH), DRAQ5 (stains red), and DRAQ7 (stains red); a fluorescent nonnuclear staining agent, such as fluorophore-labeled phalloidin (stains filamentous actin, the color depending on the conjugated fluorophore).

[0128] In some embodiments, sequential sections of biomarker-labeled slides may be morphologically stained to identify specific regions of interest within them for evaluation of the biomarker-stained sample. Many morphological staining agents are known, including but not limited to hematoxylin and eosin (H&E) staining agents and Listerine staining agents (methylene blue and basic fuchsin). In specific embodiments, at least one sequential section of each biomarker-labeled slide is H&E stained. Any method of applying H&E staining can be used, including manual and automated methods. In one embodiment, at least one section of the sample is an H&E-stained sample stained on an automated staining system. Automated systems for performing H&E staining typically operate on one of two staining principles: batch staining (also known as “immersion”) or individual slide staining. Batch stainers typically use a bucket or tank in which many slides are simultaneously immersed. On the other hand, individual slide stainers apply reagent directly to each slide, and no two slides share the same aliquot of reagent. Commercially available examples of H&E stainers include the VENTANA HE 600 series H&E stainer (independent slide stainer) from Roche; the DAKO COVERSTAINER (batch stainer) from Agilent Technologies; and the LEICA ST4020 small linear stainer, LEICA ST5020 multi-stage stainer, and LEICAST5010 automated stainer XL series H&E stainers (batch stainers) from Leica Biosystems Nussloch GmbH.

[0129] Multiple affinity histochemistry or affinity cytochemistry methods

[0130] In some embodiments, the affinity histochemical or affinity cytochemical assays of this disclosure, such as IHC assays, are provided in multiple formats.

[0131] Multiplex IHC formats involve affinity staining of multiple biomarkers in a single sample, where at least some of the biomarkers are differentially labeled. For example, a dual IHC assay targeting two different biomarkers in the same sample, where each of the two biomarkers is stained with a different bright-field detectable portion (e.g., ER and PR; ER and Ki-67; Ki-67 and PR) would be considered a “multiplex IHC assay.” Similarly, a triple IHC assay targeting three biomarkers, where each of the three biomarkers is stained with a different bright-field detectable portion, would also be considered a “multiplex IHC assay.” The assay reagents (including any detectable portions used in such methods) should be compatible with multiplex immunohistochemistry.

[0132] Multiplex IHC assays can be performed to stain samples (in any or a predetermined order) for the expression of ER, Ki-67, and PR biomarkers. In some embodiments, a triplet approach is provided in which human ER, Ki-67, and PR biomarkers are differentially stained in the same sample, such as with three different bright-field detectable portions (e.g., three different bright-field detectable portions with different detectable signals and / or different distinguishable signals).

[0133] Referring to Figure 1, in some embodiments, the triple IHC assay of this disclosure includes staining the tissue sample for the presence of the PR biomarker (step 10), the ER biomarker (step 20), and the Ki-67 biomarker (step 30). Although Figure 1 depicts the sequential staining of the PR, ER, and Ki-67 biomarkers, the biomarkers can be stained in any order. For example, the ER biomarker can be stained first, followed by the PR biomarker, and then the Ki-67 biomarker. Similarly, the ER biomarker can be stained first, followed by the Ki-67 biomarker, and then the ER biomarker. Likewise, the PR biomarker can be stained first, followed by the Ki-67 biomarker, and then the ER biomarker. Alternatively, the Ki-67 biomarker can be stained first, followed by the ER biomarker, and then the PR biomarker. Alternatively, the Ki-67 biomarker can be stained first, followed by the PR biomarker, and then the ER biomarker.

[0134] In some embodiments, the method includes the steps of: (i) contacting a biological sample with a first human biomarker-specific agent against one of ER, Ki-67, or PR; and (ii) contacting the biological sample with a first detection reagent to label the first human biomarker-specific agent with a first bright-field detectable portion. In some embodiments, the first human biomarker-specific agent is a first primary monoclonal antibody specific to one of ER, Ki-67, or PR. In some embodiments, the first detection reagent includes a secondary antibody specific to the first primary monoclonal antibody, wherein the secondary antibody is conjugated to a first enzyme (e.g., horseradish peroxidase or alkaline phosphatase). In some embodiments, the first detection reagent includes a conjugate containing a first bright-field detectable portion such that when the first enzyme acts on the conjugate containing the first bright-field detectable portion, the first bright-field detectable portion is deposited on or adjacent to one of the ER, Ki-67, or PR biomarkers. In some embodiments, the first enzyme is inactivated before introducing further detection reagents.

[0135] In some embodiments, the method further includes the steps of: (iii) contacting a biological sample with a second human biomarker-specific agent targeting another of ER, Ki-67, or PR; and (iv) contacting the biological sample with a second detection reagent to label the second human biomarker-specific agent with a second bright-field detectable portion. In some embodiments, the second human biomarker-specific agent is a second primary monoclonal antibody specific to another of ER, Ki-67, or PR. In some embodiments, the second detection reagent comprises a secondary antibody specific to the second primary monoclonal antibody, wherein the secondary antibody is conjugated to a second enzyme (e.g., horseradish peroxidase or alkaline phosphatase). In some embodiments, the second detection reagent comprises a conjugate containing a second bright-field detectable portion such that when the second enzyme acts on the conjugate containing the second bright-field detectable portion, the second bright-field detectable portion is deposited on or adjacent to another of the ER, Ki-67, or PR biomarkers. In some embodiments, the second bright-field detectable portion is different from the first bright-field detectable portion. In some embodiments, the second enzyme is inactivated before the introduction of further detection reagents.

[0136] In some embodiments, the method includes the steps of: (v) contacting a biological sample with a third-person biomarker-specific agent targeting a third of ER, Ki-67, or PR; and (vi) contacting the biological sample with a third detection reagent to label the third-person biomarker-specific agent with a third bright-field detectable portion. In some embodiments, the third-person biomarker-specific agent is a third-level monoclonal antibody specific to a third of ER, Ki-67, or PR. In some embodiments, the third detection reagent includes a secondary antibody specific to the third-level monoclonal antibody, wherein the secondary antibody is conjugated to a third enzyme (e.g., horseradish peroxidase or alkaline phosphatase). In some embodiments, the third detection reagent includes a conjugate containing a third bright-field detectable portion such that when the third enzyme acts on the conjugate containing the third bright-field detectable portion, the third bright-field detectable portion is deposited on or adjacent to the third of the ER, Ki-67, or PR biomarkers. In some embodiments, the third bright-field detectable portion is different from the first and second bright-field detectable portions.

[0137] In the context of multiplex assays that sequentially detect multiple bright-field detectable fractions, it is desirable to inactivate any reagents or endogenous enzymes between successive bright-field detectable fraction detection steps. Therefore, it is believed that enzymes present in any bright-field detectable fraction detection step will not interfere with those in subsequent bright-field detectable fraction detection steps. This, in turn, is considered to improve the visualization and detection of the different bright-field detectable fractions used in multiplex assays. Any enzyme inactivation composition known in the art can be used for this purpose. In some embodiments, an enzyme inactivation composition is applied after each detection step to inactivate the reagent or endogenous enzyme. Exemplary enzyme inactivation compositions are disclosed in U.S. Patent No. 11,162,877, the disclosure of which is incorporated herein by reference in its entirety.

[0138] Referring to Figure 2, in some embodiments, the method includes the steps of: (i) contacting a biological sample with a human PR biomarker specific agent (step 11); and (ii) contacting the biological sample with a first detection reagent to label the human PR biomarker specific agent with a first brightfield detectable portion (step 12), thereby staining the biological sample for the presence of the PR biomarker (step 10). In some embodiments, the first detection reagent includes a secondary antibody specific to the human PR biomarker specific agent, and wherein the secondary antibody is conjugated to a first enzyme (e.g., horseradish peroxidase or alkaline phosphatase). In some embodiments, the first detection reagent includes a conjugate containing the first brightfield detectable portion, such that when the first enzyme acts on the conjugate containing the first brightfield detectable portion, the first brightfield detectable portion is deposited on or adjacent to the PR biomarker. In some embodiments, the first bright-field detectable portion is selected from TAMRA, Dabsyl, Dabcyl, Cy3, CyB, Cy3.5, Cy5, Cy5.5, Cy7, Rhodamine 800, and fluorescein. In some embodiments, the first bright-field detectable portion is a conjugate comprising at least two chromogens (such as at least two chromogens selected from TAMRA, Dabsyl, Dabcyl, Cy3, CyB, Cy3.5, Cy5, Cy5.5, Cy7, Rhodamine 800, and fluorescein). In other embodiments, the first bright-field detectable portion is selected from Discovery Violet, Discovery Blue-Green, or Discovery Yellow. In some embodiments, the enzyme inactivation composition is introduced into the biological sample prior to staining for the presence of a second biomarker.

[0139] In some embodiments, the method includes the steps of: (iii) contacting a biological sample with a human ER biomarker-specific agent (step 21); and (iv) contacting the biological sample with a second detection reagent to label the human ER biomarker-specific agent with a second bright-field detectable portion (step 22), thereby staining the biological sample for the presence of the ER biomarker (step 20). In some embodiments, the second detection reagent includes a secondary antibody specific to the human ER biomarker-specific agent, and wherein the secondary antibody is conjugated to a second enzyme (e.g., horseradish peroxidase or alkaline phosphatase). In some embodiments, the second detection reagent includes a conjugate containing a second bright-field detectable portion, such that when the second enzyme acts on the conjugate containing the second bright-field detectable portion, the second bright-field detectable portion is deposited on or adjacent to the ER biomarker. In some embodiments, the second bright-field detectable portion is selected from TAMRA, Dabsyl, Dabcyl, Cy3, CyB, Cy3.5, Cy5, Cy5.5, Cy7, Rhodamine 800, and fluorescein. In some embodiments, the second bright-field detectable portion is a conjugate comprising at least two chromogens, such as at least two chromogens selected from TAMRA, Dabsyl, Dabcyl, Cy3, CyB, Cy3.5, Cy5, Cy5.5, Cy7, Rhodamine 800, and fluorescein. In other embodiments, the second bright-field detectable portion is selected from Discovery Violet, Discovery Blue-Green, or Discovery Yellow. In some embodiments, the enzyme inactivation composition is introduced into the biological sample prior to staining for the presence of a third biomarker.

[0140] In some embodiments, the method includes the steps of: (v) contacting a biological sample with a human Ki-67 biomarker specific agent (step 31); and (vi) contacting the biological sample with a third detection reagent to label the human Ki-67 biomarker specific agent with a third brightfield detectable portion (step 32), thereby staining the biological sample for the presence of the Ki-67 biomarker (step 30). In some embodiments, the third detection reagent includes a secondary antibody specific to the human Ki-67 biomarker specific agent, wherein the secondary antibody is conjugated to a third enzyme (e.g., horseradish peroxidase or alkaline phosphatase). In some embodiments, the third detection reagent includes a conjugate containing a third brightfield detectable portion such that when the third enzyme acts on the conjugate containing the third brightfield detectable portion, the third brightfield detectable portion is deposited on or adjacent to the Ki-67 biomarker. In some embodiments, the third bright-field detectable portion is selected from TAMRA, Dabsyl, Dabcyl, Cy3, CyB, Cy3.5, Cy5, Cy5.5, Cy7, Rhodamine 800, and fluorescein. In some embodiments, the third bright-field detectable portion is a conjugate comprising at least two chromophores (such as at least two chromophores selected from TAMRA, Dabsyl, Dabcyl, Cy3, CyB, Cy3.5, Cy5, Cy5.5, Cy7, Rhodamine 800, and fluorescein). In other embodiments, the third bright-field detectable portion is selected from Discovery Violet, Discovery Blue-Green, or Discovery Yellow. As described above, steps 10, 20, and 30 can be performed in any order.

[0141] An exemplary method for staining tissue samples in the presence of PR, ER, and Ki-67 is depicted in Figure 3.

[0142] Staining assessment / score

[0143] After staining, the stained samples can be evaluated for the expression of ER, Ki-67, and PR biomarkers. In some embodiments, the stained samples are evaluated for the colocalization of the expression of ER, Ki-67, and PR biomarkers in cells or cell nuclei. In some embodiments, staining assessment / scoring is performed manually by a pathologist. In other embodiments, the stained samples are scored using a digital pathology system, such as automatic scoring of stained samples.

[0144] In some embodiments, this disclosure includes contacting a sample with a first detection reagent, a second detection reagent, and a third detection reagent, the first detection reagent comprising a component labeled with a first brightfield detectable portion of ER, Ki-67, or PR, the second detection reagent comprising a component labeled with a second brightfield detectable portion of ER, Ki-67, or PR, and the third detection reagent comprising a component labeled with a third brightfield detectable portion of ER, Ki-67, or PR, wherein each of the first, second, and third brightfield detectable portions provides a different first detectable signal, a different second detectable signal, and a different third detectable signal; and detecting the colocalization of the different first, second, and third detectable signals (given the translucency of the brightfield detectable portion) to identify cells or nuclei that are positive for each of ER, Ki-67, and PR.

[0145] Figures 4A through 4C illustrate individual tissue samples partially stained with different bright fields for the presence of one of the ER, PR, or Ki-67 biomarkers in a single-field IHC assay. Figures 4A through 4C illustrate that different signals (e.g., different colors) can be observed for each differentially stained sample.

[0146] When tissue samples are stained for the presence of the ER, PR, and Ki-67 biomarkers in a triple assay as described herein, the cells or nuclei expressing each of the three biomarkers comprise a combination of each of the individual signals (e.g., each of different colors) used to stain a single biomarker. These different signals or colors co-localized to a single cell or the nucleus of a single cell may result in a mixture of individual signals or colors. These co-localized signals or colors can be interpreted during manual or automated scoring as ER+, PR+, and Ki-67 positive cells. For example, co-localization of yellow and blue signals may result in a green signal, which can be visually observed by a pathologist (given the translucency of the brightfield detectable portion used). Similarly, co-localization of yellow, blue, and purple signals may produce another different color signal, which can then be interpreted by a pathologist.

[0147] For example, this is illustrated in Figure 5A, which shows cells or nuclei stained with a first color (purple), cells or nuclei stained with a second color (blue-green), cells or nuclei stained with a third color (yellow), and cells or nuclei stained with a combination of the first, second, and third colors. Cells or nuclei stained with a combination of the first, second, and third colors represent cells or nuclei expressing each of the three biomarkers. This is in Figure 5BFurther examples are illustrated in Figures 5C, 6A, and 6B, which illustrate cells having: (i) a single stain; (ii) two co-localization stains; and (iii) three co-localization stains. In some embodiments, cells containing three co-localization stains are scored and / or cells containing three co-localization stains are used for downstream analysis.

[0148] In some embodiments, the pathologist will identify one or more regions of interest in a tissue sample that are differentially stained due to the presence of the ER, PR, and Ki-67 biomarkers, and manually determine the number of cells or nuclei colocalized with the three biomarkers (such as manual assessment based on colocalization of the three different signals or colors).

[0149] In other embodiments, a digital pathology system can be used to evaluate biological samples that are differentially stained in a triple assay. A digital pathology system comprises two basic components: (1) a scanning or image acquisition system for generating digital images of the stained sample; and (2) an image analysis system for identifying and quantifying specific features within the generated digital images.

[0150] Image acquisition systems may include scanning platforms, such as slide scanners capable of scanning stained slides at 20x, 40x, or other magnifications to produce high-resolution digital images of the entire slide, including, for example, slide scanners. In some embodiments, a slide scanner includes at least: (1) a microscope with lens objectives, (2) a light source (such as halogen, light-emitting diode, white light, and / or multispectral light sources, depending on the dye), (3) an automated device for moving the slide (or moving optics around the slide), (4) one or more digital cameras for image capture, and (5) a computer and associated software for controlling the automated device and manipulating, managing, and viewing the digital slides. In some embodiments, digital data at multiple different XY locations (and in some cases, at multiple Z planes) on the slide are captured by the charge-coupled device (CCD) of the camera, and the images are combined to form a composite image of the entire scanned surface. Common methods for achieving this include: (1) tiling-based scanning, in which the slide stage or optics moves in very small increments to capture square image frames that slightly overlap with adjacent squares. In some embodiments, the captured squares are then automatically matched to form a composite image; and (2) line-based scanning, in which the slide stage moves along a single axis during acquisition to capture several composite image “strips.” In some embodiments, the image strips can then be matched to form a larger composite image.

[0151] A detailed overview of the various scanners (both fluorescence and bright field) can be found in Farahani et al., Whole slideimaging in pathology: advantages, limitations, and emerging perspectives, Pathology and Laboratory Medicine Int'l, Vol. 7, pp. 23–33 (June 2015), the contents of which are incorporated herein by reference in their entirety. Examples of commercially available slide scanners include: 3D Histech PANNORAMIC SCAN II; DigiPath PATHSCOPE; Hamamatsu NANOZOOMER RS, HT, and XR; Huron TISSUESCOPE 4000, 4000XT, and HS; Leica SCANSCOPE AT, AT2, CS, FL, and SCN400; Mikroscan D2; Olympus VS120-SL; Omnyx VL4 and VL120; PerkinElmer LAMINA; Philips ULTRA-FAST SCANNER; Sakura Finetek VISIONTEK; Unic PRECICE 500 and PRECICE 600x; and Zeiss AXIO SCAN.Z1. In some embodiments, the scanning device is a digital pathology device, such as that disclosed in any of U.S. Patent No. 9,575,301; U.S. Patent Application Publication No. 2014 / 0178169; U.S. Patent No. 9,575,301; U.S. Patent Application Publication No. 2014 / 0178169; U.S. Patent Publication No. 2021 / 0092308; and / or U.S. Patent Application Publication No. 2021 / 0088769, the contents of each of which are incorporated herein by reference in their entirety.

[0152] Exemplary commercial image analysis software packages include: the VENTANA VIRTUOSO software suite (Ventana Medical Systems, Inc.); the TISSUE STUDIO, DEVELOPER XD, and IMAGE MINER software suite (Definiens); the BIOTOPIX, ONCOTOPIX, and STEREOTOPIX software suite (Visiopharm); and the HALO platform (Indica Labs, Inc.).

[0153] According to this disclosure, samples stained in triple IHC can be imaged on a scanner system to generate high-quality digital images of the stained samples. The digital images are then analyzed by an image analysis system to identify and classify one or more relevant objects in the samples.

[0154] In some embodiments, the triplet staining images are first demixed to provide images with different color channels, each color channel image representing the contribution of one of the three bright-field detectable portions used in the triplet assay. Demixing, also known as deconvolution, essentially separates an image with a mixture of staining into contributions from a single stain, thereby allowing individual evaluation of the single stain. By applying deconvolution methods, individual cells and / or regions of a sample can be classified based on multiple biomarkers. Thus, for example, in an ER, Ki-67, and PR triplet assay, each tumor nucleus can be classified based on contributions from ER, Ki-67, and PR-related stains. Exemplary bright-field deconvolution methods are disclosed, for example, in PCT / EP2015 / 061226, PCT / EP2015 / 067384, PCT / EP2016 / 081329, and PCT / EP2018 / 070956.

[0155] In some embodiments, image analysis may identify all cells in one or more tumor regions of interest, and then classify cells as expressing one, two, or all three of the ER, PR, and / or Ki-67 biomarkers, such as by identifying the contribution of different bright-field detectable fractional signals in each cell or nucleus. In some embodiments, the contribution of each of the different bright-field detectable fractional signals in the identified cells or nuclei may be compared to a predetermined threshold signal amount (e.g., intensity amount). In some embodiments, if the contribution of any of the different bright-field detectable fractional signals exceeds the predetermined threshold signal amount, the cell or nucleus may be classified as positive for a specific biomarker.

[0156] After staining samples to assess the expression of ER, Ki-67 and PR biomarkers, the samples can be classified as: (i) ER+, Ki-67+, PR+ dominant cases; or (ii) ER+, Ki-67+ dominant cases.

[0157] In some embodiments, the ratio of the number of cells or nuclei that are positive for all three of ER, Ki-67, and PR to the total number of cells that are (i) ER+, Ki-67+, and PR+ and (ii) ER+, Ki-67+, and PR- is calculated, as described below:

[0158] [ER + Ki-67+ PR + ] / ([ER + Ki-67 + PR + ] + [ER + Ki-67 + PR - ])

[0159] The calculated ratio is then compared to a predetermined threshold. If the calculated ratio is greater than or equal to the predetermined threshold, the stained sample is classified as an ER+, Ki-67+, PR+ dominant case. Conversely, if the calculated ratio is less than the predetermined threshold, the stained sample is classified as an ER+, Ki-67+ dominant case.

[0160] In some embodiments, the predetermined threshold is 35%. In some embodiments, the predetermined threshold is 40%. In some embodiments, the predetermined threshold is 41%. In some embodiments, the predetermined threshold is 42%. In some embodiments, the predetermined threshold is 43%. In some embodiments, the predetermined threshold is 44%. In some embodiments, the predetermined threshold is 45%. In some embodiments, the predetermined threshold is 46%. In some embodiments, the predetermined threshold is 47%. In some embodiments, the predetermined threshold is 48%. In some embodiments, the predetermined threshold is 49%. In some embodiments, the predetermined threshold is 50%. In some embodiments, the predetermined threshold is 51%. In some embodiments, the predetermined threshold is 52%. In some embodiments, the predetermined threshold is 53%. In some embodiments, the predetermined threshold is 54%. In some embodiments, the predetermined threshold is 55%. In some embodiments, the predetermined threshold is 55%. In some embodiments, the predetermined threshold is 60%. In some embodiments, the predetermined threshold is 61%. In some embodiments, the predetermined threshold is 62%. In some embodiments, the predetermined threshold is 63%. In some embodiments, the predetermined threshold is 64%. In some embodiments, the predetermined threshold is 65%.

[0161] Examples of dominant ER+, Ki-67+, and PR+ cases are illustrated in Figure 6A. Examples of dominant ER+ and Ki-67+ cases are illustrated in Figure 6B. Notably, Figure 6A shows the colocalization of three different signals or colors in cells or nuclei. On the other hand, Figure 6B shows the colocalization of two different signals or colors in cells (where colocalized cells appear green due to the colocalization of blue staining (ER+) and yellow staining (Ki-67+)).

[0162] Therapeutic methods based on assessment of ER, KI-67, and PR

[0163] The assessment of ER, Ki-67, and PR biomarkers is useful for identifying tumors that will benefit from treatment with one or more therapeutic agents, such as one or more hormone therapy drugs (e.g., selective estrogen receptor modulators (SERMs), selective estrogen receptor degraders (SERDs), and aromatase inhibitors (AIs)). In fact, the applicant surprisingly found that the ER and PR status of proliferative cancer cells (i.e., Ki-67-positive cancer cells) is a potent prognostic factor for patients with ER-positive breast cancer (see the example in this paper, which shows that in a population of ER-positive and HER2-negative breast cancer patients using hormone therapy, ER+, Ki-67+, PR+ dominant patients had significantly better clinical outcomes than ER+, Ki-67+ dominant patients).

[0164] In some embodiments, this disclosure identifies tumors that would benefit from administration of one or more therapeutic agents based on cells or nuclei that positively express each of ER, Ki-67, and PR. In some embodiments, if a sample is classified as an ER+, Ki-67+, PR+ dominant case, the sample may be further classified as likely to respond to a therapy, such as hormone therapy. In other embodiments, if a sample is classified as an ER+, Ki-67+ dominant case, the sample may be further classified as unlikely to respond to a therapy, such as hormone therapy. One or more appropriate therapeutic agents may then be administered to patients identified as likely to respond to the therapy. For example, specific hormone therapy may be administered to patients identified as likely to respond to the therapy according to the manufacturer's instructions and recommended course of treatment. Patients identified as unlikely to respond are switched to an alternative course of treatment.

[0165] In some embodiments, the assays and methods described herein can be used as screening tests to identify patients eligible for treatment with SERM, SERD, or AI. In some embodiments, the assays and methods disclosed herein can be used to predict or aid in predicting response to therapy with one or more of SERM, SERD, or AI; or the results of any assay or method disclosed herein can be used to facilitate treatment with one or more of SERM, SERD, or AI. Similarly, the assays and methods described herein can be used to stratify subjects into two or more categories based on the likelihood of a subject responding to treatment with SERM, SERD, or AI. For example, based on the assessment of the expression of ER, Ki-67, and PR biomarker components, subjects requiring treatment can be stratified into a first category, including those who are likely to respond to treatment with one or more of SERM, SERD, or AI; or a second category, including those who are likely not to respond to treatment with one or more of SERM, SERD, or AI.

[0166] In yet another embodiment, if a sample is classified as an ER+, Ki-67+ dominant case, it may be further classified as potentially responsive to therapy using cyclin-dependent kinase 4 and 6 (CDK4 / 6) inhibitors (see, for example, Figure 10).

[0167] In some embodiments, the assays and methods described herein can be used as screening tests to identify patients eligible for CDK4 / 6 inhibitor therapy. In some embodiments, the assays and methods disclosed herein can be used to predict or aid in predicting response to therapy with a CDK4 / 6 inhibitor; or the results of any assay or method disclosed herein can be used to facilitate treatment with a CDK4 / 6 inhibitor. Similarly, the assays and methods described herein can be used to stratify subjects into two or more categories based on the likelihood of a subject responding to treatment with a CDK4 / 6 inhibitor. For example, based on assessments of the expression of ER, Ki-67, and PR biomarker components, subjects requiring treatment can be stratified into a first category, including those who are likely to respond to CDK4 / 6 inhibitor therapy; or a second category, including those who are likely not to respond to CDK4 / 6 inhibitor therapy.

[0168] This disclosure also relates to methods for selecting or identifying subjects who are suitable candidates for treatment with a therapy for cancer (e.g., with one or more hormone therapies). Such individuals include subjects who are expected to respond to the therapy (e.g., with one or more hormone therapies) and therefore have an increased likelihood of benefiting from administering the therapy compared to other patients with different characteristics (e.g., unresponsiveness to the therapy). In some embodiments, a suitable candidate is one who is reasonably likely to benefit from the treatment or at least sufficiently likely to benefit from the treatment to justify administering the treatment in terms of its risks and side effects. This disclosure also covers methods for selecting or identifying subjects who are not suitable candidates for treatment with a therapy for cancer (e.g., with one or more hormone therapies). Such subjects include cancer patients who are predicted to be unresponsive or weakly responsive to the therapy and therefore have a reduced likelihood of benefiting from administering the therapy compared to other patients with different characteristics (e.g., responsiveness to the therapy), or a low or substantially no likelihood of benefiting from such treatment, making different or additional treatments potentially necessary. In some embodiments, a patient’s suitability for use of one or more hormone therapies is determined based on an assessment of the expression of ER, Ki-67 and PR in samples derived from the patient, as described herein.

[0169] In some embodiments, the assays and methods disclosed herein can be used to treat cancer (e.g., breast cancer). For example, for subjects whose tumor samples are assessed as ER+, Ki-67+, PR+ dominant cases, a therapeutically effective amount of one or more hormone therapy drugs may be administered. For example, one or more of a selective estrogen receptor modulator, a selective estrogen receptor degrader, or an aromatase inhibitor may be administered to subjects requiring such treatment.

[0170] In other embodiments, for subjects whose tumor samples are assessed as ER+, Ki-67+ dominant cases, a therapeutically effective amount of one or more CDK4 / 6 inhibitors (e.g., abemaciclib) may be administered.

[0171] In other embodiments, this disclosure relates to a method of treating a subject with cancer, such as a human patient, the method comprising: (a) selecting a subject as a suitable candidate for treatment with one or more hormonal therapy agents; and (b) administering a therapeutically effective amount of one or more hormonal therapy agents to the selected subject based on the expression of ER, Ki-67, and PR as described herein. In some embodiments, selecting a subject for treatment with one or more hormonal therapy agents comprises (i) obtaining a biological sample from the subject with cancer; (ii) assessing the expression of ER, Ki-67, and PR biomarkers as described herein in the obtained biological sample; and (iii) selecting the subject candidate for treatment with one or more hormonal therapy agents if the obtained biological sample is determined to be an ER+, Ki-67+, PR+ dominant case.

[0172] Methods of stratifying and / or classifying patients

[0173] This disclosure also provides methods for stratifying patients or patient populations into those likely to relapse and those unlikely to relapse. In fact, the applicant has discovered that the triple IHC assay of this disclosure can be used to predict recurrence in ER-positive breast cancer. The applicant has also discovered that the triple IHC assay of this disclosure can be used to predict recurrence in ER-positive breast cancer without performing any breast cancer gene expression testing. As described in the examples herein, in ER-positive and HER2-negative breast cancer populations, with hormone therapy, the clinical outcomes of ER+, Ki-67+, PR+ dominant patients are significantly better than those of ER+, Ki-67+ dominant patients.

[0174] Furthermore, the applicant has found that patients with luminal surface A subtype can be reclassified into ER+, Ki-67+, PR+ subtypes and ER+, Ki-67+ subtypes. The applicant has demonstrated that the clinical outcomes of patients with dominant ER+, Ki-67+, PR+ are significantly better than those with dominant ER+, Ki-67+ (see Figure 9).

[0175] Kits

[0176] This disclosure also provides kits comprising antibodies and detection reagents suitable for staining samples in multiplex or singlex immunoenzyme assays. In some embodiments, the kit comprises (i) a set of primary antibodies and (ii) detection reagents optionally for performing the immunoenzyme assay, wherein the set of primary antibodies comprises anti-ER antibody, anti-Ki-67 antibody, and anti-PR antibody. In some embodiments, the anti-ER, anti-Ki-67, and anti-PR antibodies are monoclonal antibodies. In some embodiments, the anti-ER, anti-Ki-67, and anti-PR antibodies are mouse monoclonal antibodies. In some embodiments, the anti-ER, anti-Ki-67, and anti-PR antibodies are rabbit monoclonal antibodies. In some embodiments, the human PR biomarker-specific reagent is clone 1E2. In some embodiments, the human ER biomarker-specific reagent is SP1. In some embodiments, the human Ki-67 biomarker-specific reagent is clone 30-9. In some embodiments, the set of primary antibodies comprises clone 1E2, SP1 clone, and clone 30-9. In other embodiments, the primary antibody group consists essentially of clone 1E2, clone SP1, and clone 30-9. In some embodiments, the primary antibody group consists of clone 1E2, clone SP1, and clone 30-9.

[0177] In some embodiments, the kit further includes secondary antibodies specific to anti-ER, anti-Ki-67, and anti-PR antibodies. In some embodiments, the secondary antibody is conjugated to peroxidase or alkaline phosphatase.

[0178] In some embodiments, the kit further includes a first brightfield detectable portion, a second brightfield detectable portion, and a third brightfield detectable portion. In some embodiments, the first brightfield detectable portion, the second brightfield detectable portion, and the third brightfield detectable portion are selected from TAMRA, Dabsyl, Dabcyl, Cy3, CyB, Cy3.5, Cy5, Cy5.5, Cy7, Rhodamine 800, and fluorescein. In some embodiments, the first brightfield detectable portion, the second brightfield detectable portion, and the third brightfield detectable portion comprise conjugates containing at least two chromogens. In some embodiments, the brightfield detectable portion is selected from Discovery Yellow, Discovery Blue-Green, and Discovery Violet, each available from Ventana Medical Systems, Inc. (Tucson, Arizona).

[0179] Examples - Co-expression of estrogen receptor (ER), progesterone receptor (PR), and KI-67 in single breast cancer cells indicates a good prognosis in ER-positive breast cancer Additional embodiments

[0180] background

[0181] ER-positive breast cancer is biologically and clinically classified into two subtypes: luminal A and luminal B. This classification is primarily based on the state of cell proliferation. In ER-positive breast cancer, at least two distinct pathways drive cell proliferation. One is the classical pathway, in which ER binds to estrogen-response elements (EREs), leading to the expression of downstream molecules, including progesterone (PR). The other is a non-classical pathway, in which a complex of ER and related factors binds to sites different from EREs. Growth factor signaling has been shown to enhance the non-classical pathway. It is hypothesized that examining the PR status in ER-positive proliferating cells can determine which pathway is more dominant in ER-positive breast cancer.

[0182] method

[0183] To test this hypothesis, a newly developed triple immunohistochemical (IHC) assay was used, which simultaneously detects three molecules under a bright-field microscope. This study included postmenopausal patients treated with neoadjuvant endocrine therapy using aromatase inhibitors at Saitama Prefectural Cancer Center from January 2007 to September 2016. ER, PR, and Ki-67 expression were assessed in a single slide using a triple IHC assay with anti-ER antibody (clone SP1), anti-PR antibody (clone 1E2), and anti-Ki-67 antibody (clone 30-9). ER, PR, and Ki-67 expression were assessed in single nuclei of cancer cells (567 to 4871 cells) from multiple regions of each case. ER-positive proliferating cells were defined as ER-positive and Ki-67-positive cells. The PR status of ER-positive proliferating cells was assessed. Tumors were classified as PR-positive when more than 50% of ER-positive proliferating cells in a clinical case expressed PR. Lumen A and lumbar B breast cancers were defined based on the pre-treatment Ki-67 marker index using a 14% cutoff value. Statistical analyses included the Mann-Whitney test, log-rank test, and Cox proportional hazards model.

[0184] result

[0185] Pretreatment tissues from 55 patients were evaluated (see table below). The median age was 62 years (range: 54–80). Patients were assigned to either a PR-positive or PR-negative group. There were no differences between the two groups in age or pretreatment T and N stages. The median pretreatment Ki-67 marker index was 5.9% in the PR-positive group and 9.9% in the PR-negative group, which was statistically significant (P = 0.01). Clinical responses to neoadjuvant endocrine therapy were compared, and no differences were observed. The median posttreatment Ki-67 marker index was 3.6% in the PR-positive group and 13.1% in the PR-negative group, which was statistically significant (P = 0.035). Survival was compared between the two groups. The PR-positive group showed significantly better disease-free survival (DFS) than the PR-negative group (P = 0.0079). To adjust for background differences, multivariate analysis showed that the PR-positive group had significantly better DFS than the PR-negative group, regardless of clinical stage, Ki-67 marker index, and PR status (P = 0.042) (see, e.g., Figure 7). After adjusting for clinical stage, Ki-67 marker index, and PR status, breast cancer-specific survival (BCSS) was also better in the PR-positive group than in the PR-negative group (P = 0.043) (see, e.g., Figure 8). Interestingly, in patients with luminal type A tumors, the PR-positive group showed better DFS than the PR-negative group (P = 0.022) (see, e.g., Figure 9).

[0186]

[0187] in conclusion

[0188] PR status in ER-positive proliferating cells is an independent prognostic factor for DFS and BCSS, and patients with luminal type A tumors are further divided into two prognostic groups.

[0189] SUMMARY Methods of assessing expression of ER, Ki-67, and PR biomarkers Affinity histochemical or affinity cytochemical assays Staining assessment / score Figure 5B Therapeutic methods based on assessment of ER, KI-67, and PR Methods of stratifying and / or classifying patients Kits Examples - Co-expression of estrogen receptor (ER), progesterone receptor (PR), and KI-67 in single breast cancer cells indicates a good prognosis in ER-positive breast cancer Additional embodiments

[0190] Another aspect of this disclosure is an immunohistochemical method for assessing the expression of PR, ER, and Ki-67 biomarkers in a sample, the method comprising: contacting the sample with a human PR biomarker-specific reagent under conditions allowing the PR biomarker-specific reagent to bind specifically to the sample; contacting the sample with a first set of detection reagents, the first set of detection reagents interacting with the human PR biomarker-specific reagent to promote the deposition of a first brightfield detectable portion on the sample; contacting the sample with a human ER biomarker-specific reagent under conditions allowing the ER biomarker-specific reagent to bind specifically to the sample; contacting the sample with a second set of detection reagents, the second set of detection reagents interacting with the human ER biomarker-specific reagent to promote the deposition of a second brightfield detectable portion on the sample; contacting the sample with a human Ki-67 biomarker-specific reagent under conditions allowing the Ki-67 biomarker-specific reagent to bind specifically to the sample; and contacting the sample with a third set of detection reagents, the third set of detection reagents interacting with the human Ki-67 ... to promote the deposition of a second brightfield detectable portion on the sample; contacting the sample with a human Ki-67 biomarker-specific reagent to promote the deposition of a second brightfield detectable portion on the sample; contacting the sample with a human Ki-67 biomarker-specific reagent to promote the deposition of a second brightfield detectable portion on the sample; contacting the sample with a human Ki-67 biomarker-specific reagent to promote the deposition of a second bright Biomarker-specific reagent interactions facilitate the deposition of a third brightfield detectable fraction on the sample; and the nuclei expressing each of the PR, ER, and Ki-67 biomarkers within the sample are identified based on the co-expression of signals from each of the deposited first, second, and third brightfield detectable fractions.

[0191] Another aspect of this disclosure is an immunohistochemical method for assessing the expression of PR and ER biomarkers and cell proliferation biomarkers (e.g., Ki-67) in a sample, the method comprising: contacting the sample with a human PR biomarker-specific reagent under conditions allowing the PR biomarker-specific reagent to bind specifically to the sample; contacting the sample with a first set of detection reagents, the first set interacting with the human PR biomarker-specific reagent to promote the deposition of a first brightfield detectable portion on the sample; contacting the sample with a human ER biomarker-specific reagent under conditions allowing the ER biomarker-specific reagent to bind specifically to the sample; and contacting the sample with a second set of detection reagents, the second set of detection reagents interacting with the human ER ... to promote the deposition of a first brightfield detectable portion on the sample; contacting the sample with a second set of detection reagents, the second set interacting with the human ER biomarker-specific reagent to promote the deposition of a first brightfield detectable portion on the sample; contacting the sample with a second set of detection reagents, the second set interacting with the human ER biomarker-specific reagent to promote the deposition of a first brightfield detectable portion on the sample; contacting the sample with a second set of detection reagents, the second set interacting with the second set of detection reagents to promote the deposition of a first brightfield detectable portion on the sample; contacting the sample with a second set of detection reagents, the second set interacting with the second set of detection reagents to promote the deposition of a first brightfield detectable portion on the sample; contacting the sample with a second The biomarker-specific reagents interact to promote the deposition of a second brightfield detectable portion on the sample; the sample is contacted with a human cell proliferation biomarker-specific reagent under conditions that allow the cell proliferation biomarker-specific reagent to bind specifically to the sample; the sample is contacted with a third set of detection reagents, which interact with the human cell proliferation biomarker-specific reagent to promote the deposition of a third brightfield detectable portion on the sample; and the nuclei expressing each of PR, ER, and cell proliferation biomarkers within the sample are identified based on the co-expression or co-localization of signals from each of the deposited first, second, and third brightfield detectable portions.

[0192] Another aspect of this disclosure includes: staining a first of the ER, BR, and Ki-67 biomarkers in the sample with a yellow or yellowish-brown stain; staining a second of the ER, PR, and Ki-67 biomarkers in the sample with a blue-green or blue-greenish-brown stain; and staining a third of the ER, PR, and Ki-67 biomarkers in the sample with a purple or purpleish-purple stain. In some embodiments, the sample is evaluated to determine the colocalization of signals from the yellow (or yellowish-brown) stain, the blue-green (or blue-greenish-brown) stain, and the purple (or purpleish-purple) stain. In some embodiments, the sample is a breast tumor sample. In some embodiments, the sample is a sample previously diagnosed as luminal type A breast cancer.

[0193] Another aspect of this disclosure is an affinity histochemical or affinity cytochemical method for assessing the expression of PR, ER, and Ki-67 biomarkers in a sample, the method comprising: (a) contacting the sample with a human ER biomarker-specific reagent under conditions allowing the ER biomarker-specific reagent to bind specifically to the sample; (b) contacting the sample with a first set of detection reagents, the first set of detection reagents interacting with the human ER biomarker-specific reagent to promote the deposition of a first brightfield detectable portion on the sample; (c) contacting the sample with a human PR biomarker-specific reagent under conditions allowing the PR biomarker-specific reagent to bind specifically to the sample; (d) contacting the sample with a second set of detection reagents, the second set of detection reagents interacting with the human PR biomarker-specific reagent to promote the deposition of a second brightfield detectable portion on the sample; (e) contacting the sample with a human Ki-67 biomarker-specific reagent under conditions allowing the Ki-67 biomarker-specific reagent to bind specifically to the sample; (f) The sample is brought into contact with a third set of detection reagents, which interact with the human Ki-67 biomarker-specific reagent to promote the deposition of a third brightfield detectable portion on the sample; and (g) the cell nuclei expressing each of the PR, ER, and Ki-67 biomarkers within the sample are identified based on the colocalization of signals from each of the deposited first, second, and third brightfield dyes.

[0194] Another aspect of this disclosure is an affinity histochemical or affinity cytochemical method for assessing the expression of PR, ER, and Ki-67 biomarkers in a sample, the method comprising: (a) contacting the sample with a human ER biomarker-specific reagent under conditions allowing the ER biomarker-specific reagent to bind specifically to the sample; (b) contacting the sample with a first set of detection reagents, the first set of detection reagents interacting with the human ER biomarker-specific reagent to promote the deposition of a first brightfield detectable portion on the sample; (c) contacting the sample with a human Ki-67 biomarker-specific reagent under conditions allowing the Ki-67 biomarker-specific reagent to bind specifically to the sample; (d) contacting the sample with a second set of detection reagents, the second set of detection reagents interacting with the human Ki-67 biomarker-specific reagent to promote the deposition of a second brightfield detectable portion on the sample; (e) contacting the sample with a human PR biomarker-specific reagent under conditions allowing the PR biomarker-specific reagent to bind specifically to the sample; (f) The sample is brought into contact with a third set of detection reagents, which interact with the human PR biomarker-specific reagent to promote the deposition of a third brightfield detectable portion on the sample; and (g) the cell nuclei expressing each of the PR, ER, and Ki-67 biomarkers within the sample are identified based on the colocalization of signals from each of the deposited first, second, and third brightfield dyes.

[0195] Another aspect of this disclosure is an affinity histochemical or affinity cytochemical method for assessing the expression of PR, ER, and Ki-67 biomarkers in a sample, the method comprising: (a) contacting the sample with a human Ki-67 biomarker-specific reagent under conditions allowing the Ki-67 biomarker-specific reagent to bind specifically to the sample; (b) contacting the sample with a first set of detection reagents, the first set of detection reagents interacting with the human Ki-67 biomarker-specific reagent to promote the deposition of a first brightfield detectable portion on the sample; (c) contacting the sample with a human ER biomarker-specific reagent under conditions allowing the ER biomarker-specific reagent to bind specifically to the sample; (d) contacting the sample with a second set of detection reagents, the second set of detection reagents interacting with the human ER biomarker-specific reagent to promote the deposition of a second brightfield detectable portion on the sample; (e) contacting the sample with a human PR biomarker-specific reagent under conditions allowing the PR biomarker-specific reagent to bind specifically to the sample; (f) The sample is brought into contact with a third set of detection reagents, which interact with the human PR biomarker-specific reagent to promote the deposition of a third brightfield detectable portion on the sample; and (g) the cell nuclei expressing each of the PR, ER, and Ki-67 biomarkers within the sample are identified based on the colocalization of signals from each of the deposited first, second, and third brightfield dyes.

[0196] Another aspect of this disclosure is an affinity histochemical or affinity cytochemical method for assessing the expression of PR, ER, and Ki-67 biomarkers in a sample, the method comprising: contacting a sample with a human Ki-67 biomarker-specific reagent, a human PR biomarker-specific reagent, and a human ER biomarker-specific reagent under conditions allowing the human Ki-67 biomarker-specific reagent, the human PR biomarker-specific reagent, and the human ER biomarker-specific reagent to specifically bind to the sample; sequentially contacting the sample with a first detection reagent, a second detection reagent, and a third detection reagent, the first detection reagent, the second detection reagent, and the third detection reagent interacting with the human Ki-67 biomarker-specific reagent, the human PR biomarker-specific reagent, and the human ER biomarker-specific reagent to promote the deposition of a first brightfield detectable portion, a second brightfield detectable portion, and a third brightfield detectable portion on the sample; and identifying the expression of Ki-67 and PR within the sample based on the colocalization of signals from each of the deposited first detectable portion, second detectable portion, and third detectable portion. The cell nucleus of each of the ER biomarkers. In some embodiments, contact of the sample with the human Ki-67 biomarker-specific reagent, the human PR biomarker-specific reagent, and the human ER biomarker-specific reagent can be performed in any sequential order. In some embodiments, contact of the sample with the human Ki-67 biomarker-specific reagent, the human PR biomarker-specific reagent, and the human ER biomarker-specific reagent can be performed simultaneously.

[0197] Another aspect of this disclosure is a kit comprising: (i) a human Ki-67 biomarker-specific reagent, (ii) a human PR biomarker-specific reagent, and (iii) a human ER biomarker-specific reagent. In some embodiments, the kit further comprises a first detection reagent, a second detection reagent, and a third detection reagent. In some embodiments, the first detection reagent, the second detection reagent, and the third detection reagent each comprise a different detectable portion. In some embodiments, the different detectable portion is a chromogen. In some embodiments, the different detectable portion is selected from the group consisting of TAMRA, Dabsyl, Dabcyl, Cy3, CyB, Cy3.5, Cy5, Cy5.5, Cy7, Rhodamine 800, and fluorescein. In some embodiments, the human Ki-67 biomarker-specific reagent is a monoclonal antibody. In some embodiments, the monoclonal antibody is selected from the group consisting of MIB-1, MIB-2, MIB-5, MIB-7, MIB-21, MIB-24, and clone 30-9. In some embodiments, the human ER biomarker-specific reagent is a monoclonal antibody. In some embodiments, the monoclonal antibody is 1D5. In some embodiments, the human PR biomarker-specific reagent is a monoclonal antibody. In some embodiments, the monoclonal antibody is an 1E2 clone.

[0198] Another aspect of this disclosure is a method for selecting a patient with breast cancer to receive hormone therapy, the method comprising: (a) contacting a sample with a human PR biomarker-specific reagent under conditions allowing the PR biomarker-specific reagent to bind specifically to the sample; (b) contacting the sample with a first set of detection reagents, the first set of detection reagents interacting with the human PR biomarker-specific reagent to promote the deposition of a first brightfield detectable portion on the sample; (c) contacting the sample with a human ER biomarker-specific reagent under conditions allowing the ER biomarker-specific reagent to bind specifically to the sample; (d) contacting the sample with a second set of detection reagents, the second set of detection reagents interacting with the human ER biomarker-specific reagent to promote the deposition of a second brightfield detectable portion on the sample; (e) contacting the sample with a human Ki-67 biomarker-specific reagent under conditions allowing the Ki-67 biomarker-specific reagent to bind specifically to the sample; and (f) contacting the sample with a third set of detection reagents, the third set of detection reagents interacting with the human Ki-67 biomarker-specific reagent to promote the deposition of a second brightfield detectable portion on the sample; (g) Biomarker-specific reagent interactions to promote deposition of a third brightfield detectable fraction on the sample; (h) Identifying cell nuclei expressing each of the PR, ER, and Ki-67 biomarkers within the sample based on the colocalization of signals from each of the deposited first, second, and third brightfield detectable fractions; and (h) Determining the number of proliferating tumor nuclei that are both ER+ and PR+ within the sample after histochemical staining; wherein the patient is selected to receive the hormone therapy if the ratio of the determined number of proliferating tumor nuclei that are both ER+ and PR+ to the total number of ER+ proliferating tumor nuclei is greater than or equal to a predetermined cutoff value; wherein the predetermined cutoff value is between about 0.4 and about 0.6.

[0199] Another aspect of this disclosure is a method for selecting a patient with breast cancer to receive hormone therapy, the method comprising: (a) contacting a sample with a human PR biomarker-specific reagent under conditions allowing the PR biomarker-specific reagent to bind specifically to the sample; (b) contacting the sample with a first set of detection reagents, the first set of detection reagents interacting with the human PR biomarker-specific reagent to promote the deposition of a first bright-field detectable portion on the sample; (c) contacting the sample with a human ER biomarker-specific reagent under conditions allowing the ER biomarker-specific reagent to bind specifically to the sample; (d) contacting the sample with a second set of detection reagents, the second set of detection reagents interacting with the human ER biomarker-specific reagent to promote the deposition of a second bright-field detectable portion on the sample; (e) contacting the sample with a human Ki-67 biomarker-specific reagent under conditions allowing the Ki-67 biomarker-specific reagent to bind specifically to the sample; and (f) contacting the sample with a third set of detection reagents, the third set of detection reagents interacting with the human Ki-67 biomarker-specific reagent to promote the deposition of a second bright-field detectable portion on the sample; (g) Biomarker-specific reagents interact to promote deposition of a third brightfield detectable fraction on the sample; (h) Identify cell nuclei expressing each of the PR, ER, and Ki-67 biomarkers within the sample based on the colocalization of signals from each of the deposited first, second, and third brightfield detectable fractions; and (h) Calculate the ratio of the number of cells or nuclei that are positive for staining all three of ER, Ki-67, and PR to the total number of cells that are (i) ER+, Ki-67+, and PR+ and (ii) ER+, Ki-67+, and PR-; wherein if the calculated ratio is greater than or equal to a predetermined cutoff value, the patient is selected to receive the hormone therapy; wherein the predetermined cutoff value is between about 0.4 and about 0.6.

[0200] Another aspect of this disclosure is a method for selecting a patient with breast cancer to receive hormone therapy, the method comprising: (a) contacting a sample with a human PR biomarker-specific reagent under conditions allowing the PR biomarker-specific reagent to bind specifically to the sample; (b) contacting the sample with a first set of detection reagents, the first set of detection reagents interacting with the human PR biomarker-specific reagent to promote the deposition of a first bright-field detectable portion on the sample; (c) contacting the sample with a human ER biomarker-specific reagent under conditions allowing the ER biomarker-specific reagent to bind specifically to the sample; (d) contacting the sample with a second set of detection reagents, the second set of detection reagents interacting with the human ER biomarker-specific reagent to promote the deposition of a second bright-field detectable portion on the sample; (e) contacting the sample with a human Ki-67 biomarker-specific reagent under conditions allowing the Ki-67 biomarker-specific reagent to bind specifically to the sample; and (f) contacting the sample with a third set of detection reagents, the third set of detection reagents interacting with the human Ki-67 biomarker-specific reagent to promote the deposition of a second bright-field detectable portion on the sample; (g) Biomarker-specific reagents interact to promote deposition of a third brightfield detectable fraction on the sample; (h) Identify cell nuclei expressing each of the PR, ER, and Ki-67 biomarkers within the sample based on the colocalization of signals from each of the deposited first, second, and third brightfield detectable fractions; and (h) Calculate the ratio of the number of cells or cell nuclei that are positive for staining all three of ER, Ki-67, and PR to the total number of cells that are (i) ER+, Ki-67+, and PR+ and (ii) ER+, Ki-67+, and PR-; wherein if the calculated ratio is less than a predetermined cutoff value, the patient is selected to receive the hormone therapy; wherein the predetermined cutoff value is between about 0.4 and about 0.6.

[0201] Another aspect of this disclosure is a method for classifying patients with breast cancer as ER+, Ki-67+, PR+ dominant or ER+, Ki-67+ dominant, the method comprising: (a) contacting a sample with a human PR biomarker-specific reagent under conditions allowing the PR biomarker-specific reagent to bind specifically to the sample; (b) contacting the sample with a first set of detection reagents, the first set of detection reagents interacting with the human PR biomarker-specific reagent to promote the deposition of a first brightfield detectable portion on the sample; (c) contacting the sample with a human ER biomarker-specific reagent under conditions allowing the ER biomarker-specific reagent to bind specifically to the sample; (d) contacting the sample with a second set of detection reagents, the second set of detection reagents interacting with the human ER biomarker-specific reagent to promote the deposition of a second brightfield detectable portion on the sample; (e) contacting the sample with a human Ki-67 biomarker-specific reagent under conditions allowing the Ki-67 biomarker-specific reagent to bind specifically to the sample; (f) The sample is contacted with a third set of assay reagents, which interact with a human Ki-67 biomarker-specific reagent to promote the deposition of a third brightfield detectable portion on the sample; (g) cell nuclei expressing PR, ER, and Ki-67 biomarkers within the sample are identified based on the colocalization of signals from each of the deposited first, second, and third brightfield detectable portions; and the number of cells or cell nuclei that are positive for staining for all three of ER, Ki-67, and PR is calculated as a ratio to the total number of cells that are (i) ER+, Ki-67+, and PR+ and (ii) ER+, Ki-67+, and PR-; wherein if the calculated ratio is greater than or equal to a predetermined threshold, the patient is classified as ER+, Ki-67+, PR+ dominant; or if the calculated ratio is less than a predetermined threshold, the patient is classified as ER+, Ki-67+ dominant.

[0202] Another aspect of this disclosure is a method for classifying patients with breast cancer as potentially responders or potentially non-responders to hormone therapy, the method comprising: (a) contacting a sample with a human PR biomarker-specific reagent under conditions allowing the PR biomarker-specific reagent to bind specifically to the sample; (b) contacting the sample with a first set of detection reagents, the first set of detection reagents interacting with the human PR biomarker-specific reagent to promote the deposition of a first brightfield detectable portion on the sample; (c) contacting the sample with a human ER biomarker-specific reagent under conditions allowing the ER biomarker-specific reagent to bind specifically to the sample; (d) contacting the sample with a second set of detection reagents, the second set of detection reagents interacting with the human ER biomarker-specific reagent to promote the deposition of a second brightfield detectable portion on the sample; (e) contacting the sample with a human Ki-67 biomarker-specific reagent under conditions allowing the Ki-67 biomarker-specific reagent to bind specifically to the sample; and (f) contacting the sample with a third set of detection reagents, the third set of detection reagents interacting with the human Ki-67 biomarker-specific reagent to promote the deposition of a second brightfield detectable portion on the sample; (g) Biomarker-specific reagents interact to facilitate the deposition of a third brightfield detectable portion on the sample; (g) Identify cell nuclei expressing each of the PR, ER, and Ki-67 biomarkers within the sample based on the colocalization of signals from each of the deposited first, second, and third brightfield detectable portions; and calculate the ratio of the number of cells or cell nuclei that are positive for staining all three of ER, Ki-67, and PR to the total number of cells that are (i) ER+, Ki-67+, and PR+ and (ii) ER+, Ki-67+, and PR-; wherein if the calculated ratio is greater than or equal to a predetermined threshold, the patient is classified as a likely responder; or if the calculated ratio is less than a predetermined threshold, the patient is classified as a likely non-responder.

[0203] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in and / or listed in the application data sheets are incorporated herein by reference in their entirety. Modifications may be made to various aspects of the embodiments as necessary to provide further embodiments employing the concepts of various patents, applications, and publications.

[0204] Although this disclosure has been described with reference to numerous illustrative embodiments, it should be understood that many other modifications and embodiments can be devised by those skilled in the art within the spirit and scope of the principles of this disclosure. More specifically, reasonable variations and modifications can be made to the components and / or arrangements of the subject matter combination within the scope of the foregoing disclosure, the drawings, and the appended claims without departing from the spirit of this disclosure. In addition to variations and modifications in the components and / or arrangements, alternative uses will also be apparent to those skilled in the art.

Claims

1. An affinity histochemical or affinity cytochemical method for assessing the expression of PR, ER, and Ki-67 biomarkers in a sample, the method comprising: (a) Contact the sample with a human PR biomarker-specific reagent under conditions that allow the PR biomarker-specific reagent to specifically bind to the sample; (b) Contact the sample with a first set of detection reagents, the first set of detection reagents interacting with the human PR biomarker-specific reagent to promote the deposition of a first brightfield detectable portion on the sample; (c) Contact the sample with a human ER biomarker-specific reagent under conditions that allow the ER biomarker-specific reagent to specifically bind to the sample; (d) Contact the sample with a second set of detection reagents, the second set of detection reagents interacting with the human ER biomarker-specific reagents to promote the deposition of a second brightfield detectable portion on the sample; (e) Contact the sample with a human Ki-67 biomarker-specific reagent under conditions that allow the Ki-67 biomarker-specific reagent to specifically bind to the sample; (f) Contact the sample with a third set of detection reagents, which interact with the human Ki-67 biomarker-specific reagents to promote the deposition of a third brightfield detectable portion on the sample; as well as (g) Identify cell nuclei expressing each of the PR, ER, and Ki-67 biomarkers within the sample based on the colocalization of signals from each of the deposited first, second, and third bright-field detectable portions.

2. The method according to claim 1, wherein the human PR biomarker specific reagent is an anti-PR monoclonal antibody.

3. The method according to any one of the preceding claims, wherein the human ER biomarker specific reagent is an anti-ER monoclonal antibody.

4. The method according to any one of the preceding claims, wherein the human Ki-67 biomarker specific reagent is an anti-Ki-67 monoclonal antibody.

5. The method according to any one of the preceding claims, wherein the first set of detection reagents comprises: (i) A primary or secondary antibody that is specific to the human PR biomarker-specific reagent; (ii) includes the conjugate of the first bright field detectable portion.

6. The method of claim 5, wherein the first secondary antibody, which is specific to the human PR biomarker specific reagent, comprises a first enzyme.

7. The method according to any one of the preceding claims, wherein the second set of detection reagents comprises: (i) A second secondary antibody that is specific to the human ER biomarker-specific reagent; (ii) includes the conjugate of the second bright-field detectable portion.

8. The method of claim 7, wherein the second secondary antibody, which is specific to the human ER biomarker specific reagent, comprises a first enzyme.

9. The method according to any one of the preceding claims, wherein the third group of detection reagents comprises: (i) A third secondary antibody that is specific to the human Ki-67 biomarker-specific reagent; (ii) includes the conjugate of the third bright field detectable portion.

10. The method of claim 9, wherein the third secondary antibody, which is specific to the human Ki-67 biomarker specific reagent, comprises a first enzyme.

11. The method according to any one of the preceding claims, wherein the first bright-field detectable portion, the second bright-field detectable portion and the third bright-field detectable portion are selected from the group consisting of TAMRA, Dabsyl, Dabcyl, Cy3, CyB, Cy3.5, Cy5, Cy5.5, Cy7, Rhodamine 800 and fluorescein.

12. The method according to claim 1, wherein the sample is a breast tissue sample.

13. The method of claim 12, wherein the breast tissue sample is derived from a subject diagnosed with breast cancer.

14. The method according to 13, wherein the breast cancer is luminal type A breast cancer.

15. The method according to any one of the preceding claims, wherein the inactivating composition is applied to the sample before the sample is contacted with the human ER biomarker-specific reagent.

16. The method according to any one of the preceding claims, wherein the inactivating composition is applied to the sample before the sample is contacted with the human Ki-67 biomarker-specific reagent.

17. A method for selecting patients with breast tumors to receive hormone therapy, the method comprising: (a) Affinity histochemical staining of the sample derived from the breast tumor using a human PR biomarker-specific reagent, under conditions that allow the PR biomarker-specific reagent to bind specifically to the sample; (b) Affinity histochemical staining of the sample derived from the breast tumor using a human ER biomarker-specific reagent, provided that the ER biomarker-specific reagent is allowed to bind specifically to the sample; (c) Affinity histochemical staining of the sample derived from the breast tumor using a human Ki-67 biomarker-specific reagent, under conditions allowing the Ki-67 biomarker-specific reagent to specifically bind to the sample; and (d) Determine the number of proliferating tumor nuclei that are both ER+ and PR+ in the histochemically stained sample; If the ratio of the number of proliferating tumor nuclei of both ER+ and PR+ to the total number of ER+ proliferating tumor nuclei is greater than or equal to a predetermined cutoff value, then the patient is selected to receive the hormone therapy.

18. The method of claim 17, wherein the predetermined cutoff value is between about 0.4 and about 0.

6.

19. The method of claim 17, wherein the predetermined cutoff value is between about 0.45 and about 0.

55.

20. The method of claim 17, wherein the predetermined cutoff value is about 0.

5.

21. The method according to any one of claims 17 to 20, wherein the hormone therapy is a selective estrogen receptor modulator.

22. The method according to any one of claims 17 to 20, wherein the hormone therapy is a selective estrogen receptor degrader.

23. The method according to any one of claims 17 to 20, wherein the hormone therapy is an aromatase inhibitor.

24. The method according to any one of claims 17 to 23, wherein the patient has previously been diagnosed with luminal type A breast cancer.

25. The method according to any one of claims 17 to 24, wherein the patient has previously been treated with endocrine therapy.

26. The method according to any one of claims 17 to 24, wherein the patient has previously been treated with adjuvant chemotherapy.

27. The method according to any one of claims 17 to 26, wherein affinity histochemical staining of the sample with the human PR biomarker-specific reagent comprises: (i) Contact the sample with the human PR biomarker-specific reagent under conditions that allow the PR biomarker-specific reagent to specifically bind to the sample; (ii) Contacting the sample with a first set of detection reagents, the first set of detection reagents interacting with the human PR biomarker-specific reagent to promote the deposition of a first bright-field detectable portion on the sample.

28. The method according to any one of claims 17 to 27, wherein affinity histochemical staining of the sample with the human ER biomarker-specific reagent comprises: (i) Contact the sample with the human ER biomarker-specific reagent under conditions that allow the ER biomarker-specific reagent to specifically bind to the sample; (ii) Contacting the sample with a second set of detection reagents, the second set of detection reagents interacting with the human ER biomarker-specific reagents to promote the deposition of a second bright-field detectable portion on the sample.

29. The method according to any one of claims 17 to 28, wherein affinity histochemical staining of the sample with the human Ki-67 biomarker-specific reagent comprises: (i) contacting the sample with the human Ki-67 biomarker-specific reagent under conditions that allow the Ki-67 biomarker-specific reagent to specifically bind to the sample; and (ii) contacting the sample with a third set of detection reagents that interact with the human Ki-67 biomarker-specific reagent to promote the deposition of a third brightfield detectable portion on the sample.

30. The method according to any one of claims 17 to 29, wherein determining the number of proliferating tumor nuclei expressing both ER+ and PR+ within an affinity-histochemically stained sample comprises identifying nuclei expressing each of the PR, ER, and Ki-67 biomarkers within the sample based on the colocalization of signals from each of the deposited first, second, and third bright-field detectable portions.

31. The method according to any one of claims 27 to 30, wherein the first bright-field detectable portion, the second bright-field detectable portion and the third bright-field detectable portion are selected from the group consisting of TAMRA, Dabsyl, Dabcyl, Cy3, CyB, Cy3.5, Cy5, Cy5.5, Cy7, Rhodamine 800 and fluorescein.

32. A method for selecting patients with breast tumors to receive hormone therapy, the method comprising: (a) Affinity histochemical staining of the sample derived from the breast tumor using a human PR biomarker-specific reagent, under conditions that allow the PR biomarker-specific reagent to bind specifically to the sample; (b) Affinity histochemical staining of the sample derived from the breast tumor using a human ER biomarker-specific reagent, provided that the ER biomarker-specific reagent is allowed to bind specifically to the sample; (c) Affinity histochemical staining of the sample derived from the breast tumor using a human Ki-67 biomarker-specific reagent, provided that the Ki-67 biomarker-specific reagent is allowed to bind specifically to the sample; (d) Calculate the ratio of the number of cells or nuclei that are positive for all three of ER, Ki-67 and PR to the total number of cells that are positive for (i) ER+, Ki-67+ and PR+ and (ii) both ER+, Ki-67+ and PR-. If the calculated ratio is greater than or equal to a predetermined cutoff value, then the patient is selected to receive the hormone therapy.

33. The method of claim 32, wherein the predetermined cutoff value is between about 0.4 and about 0.

6.

34. The method of claim 32, wherein the predetermined cutoff value is between about 0.45 and about 0.

55.

35. The method of claim 32, wherein the predetermined cutoff value is about 0.

5.

36. The method of claim 32, wherein the ratio is calculated using the following formula: [ER + , Ki-67 + , PR + ] / ([ER + , Ki-67 + , PR + ] + [ER + , Ki-67 + , PR - ])。 37. The method according to any one of claims 32 to 36, wherein affinity histochemical staining of the sample with the human PR biomarker-specific reagent comprises: (i) Contact the sample with the human PR biomarker-specific reagent under conditions that allow the PR biomarker-specific reagent to specifically bind to the sample; (ii) Contacting the sample with a first set of detection reagents, the first set of detection reagents interacting with the human PR biomarker-specific reagent to promote the deposition of a first bright-field detectable portion on the sample.

38. The method according to any one of claims 32 to 37, wherein affinity histochemical staining of the sample with the human ER biomarker-specific reagent comprises: (i) Contact the sample with the human ER biomarker-specific reagent under conditions that allow the ER biomarker-specific reagent to specifically bind to the sample; (ii) Contacting the sample with a second set of detection reagents, the second set of detection reagents interacting with the human ER biomarker-specific reagents to promote the deposition of a second bright-field detectable portion on the sample.

39. The method according to any one of claims 32 to 38, wherein affinity histochemical staining of the sample with the human Ki-67 biomarker-specific reagent comprises: (i) contacting the sample with the human Ki-67 biomarker-specific reagent under conditions that allow the Ki-67 biomarker-specific reagent to specifically bind to the sample; and (ii) contacting the sample with a third set of detection reagents that interact with the human Ki-67 biomarker-specific reagent to promote the deposition of a third brightfield detectable portion on the sample.

40. The method according to any one of claims 37 to 39, wherein the first bright-field detectable portion, the second bright-field detectable portion, and the third bright-field detectable portion are selected from the group consisting of TAMRA, Dabsyl, Dabcyl, Cy3, CyB, Cy3.5, Cy5, Cy5.5, Cy7, Rhodamine 800, and fluorescein.

41. A method for selecting a patient with breast cancer to receive treatment with cyclin-dependent kinase 4 and 6 inhibitors, the method comprising: (a) Affinity histochemical staining of a sample derived from the breast tumor using a human PR biomarker-specific reagent, provided that the PR biomarker-specific reagent is allowed to bind specifically to the sample; (b) Affinity histochemical staining of the sample derived from the breast tumor using a human ER biomarker-specific reagent, provided that the ER biomarker-specific reagent is allowed to bind specifically to the sample; (c) Affinity histochemical staining of the sample derived from the breast tumor using a human Ki-67 biomarker-specific reagent, provided that the Ki-67 biomarker-specific reagent is allowed to bind specifically to the sample; (d) Calculate the ratio of the number of cells or nuclei that are positive for all three of ER, Ki-67 and PR to the total number of cells that are (i) ER+, Ki-67+ and PR+ and (ii) ER+, Ki-67+ and PR-. If the calculated ratio is less than a predetermined cutoff value, then the patient is selected to receive hormone therapy.

42. The method of claim 41, wherein the predetermined cutoff value is between about 0.4 and about 0.

6.

43. The method of claim 41, wherein the predetermined cutoff value is between about 0.45 and about 0.

55.

44. The method of claim 41, wherein the predetermined cutoff value is about 0.

5.

45. The method of claim 41, wherein the ratio is calculated using the following formula: [ER + , Ki-67 + , PR + ] / ([ER + , Ki-67 + , PR + ] + [ER + , Ki-67 + , PR - ])。 46. ​​The method according to any one of claims 41 to 45, wherein affinity histochemical staining of the sample with the human PR biomarker-specific reagent comprises: (i) Contact the sample with the human PR biomarker-specific reagent under conditions that allow the PR biomarker-specific reagent to specifically bind to the sample; (ii) Contacting the sample with a first set of detection reagents, the first set of detection reagents interacting with the human PR biomarker-specific reagent to promote the deposition of a first bright-field detectable portion on the sample.

47. The method according to any one of claims 41 to 46, wherein affinity histochemical staining of the sample with the human ER biomarker-specific reagent comprises: (i) Contact the sample with the human ER biomarker-specific reagent under conditions that allow the ER biomarker-specific reagent to specifically bind to the sample; (ii) Contacting the sample with a second set of detection reagents, the second set of detection reagents interacting with the human ER biomarker-specific reagents to promote the deposition of a second bright-field detectable portion on the sample.

48. The method according to any one of claims 41 to 47, wherein affinity histochemical staining of the sample with the human Ki-67 biomarker-specific reagent comprises: (i) contacting the sample with the human Ki-67 biomarker-specific reagent under conditions that allow the Ki-67 biomarker-specific reagent to specifically bind to the sample; and (ii) contacting the sample with a third set of detection reagents that interact with the human Ki-67 biomarker-specific reagent to promote the deposition of a third brightfield detectable portion on the sample.

49. The method according to any one of claims 46 to 48, wherein the first bright-field detectable portion, the second bright-field detectable portion, and the third bright-field detectable portion are selected from the group consisting of TAMRA, Dabsyl, Dabcyl, Cy3, CyB, Cy3.5, Cy5, Cy5.5, Cy7, Rhodamine 800, and fluorescein.

50. A method for classifying patients with breast cancer as ER+, Ki-67+, PR+ dominant or ER+, Ki-67+ dominant, the method comprising: (a) Affinity histochemical staining of the sample derived from the patient using a human PR biomarker-specific reagent, provided that the PR biomarker-specific reagent is allowed to bind specifically to the sample; (b) Affinity histochemical staining of the sample derived from the patient using a human ER biomarker-specific reagent, provided that the ER biomarker-specific reagent is allowed to bind specifically to the sample; (c) Affinity histochemical staining of the sample derived from the patient using a human Ki-67 biomarker-specific reagent, provided that the Ki-67 biomarker-specific reagent is allowed to bind specifically to the sample; (d) Calculate the ratio of the number of cells or nuclei that are positive for all three of ER, Ki-67 and PR to the total number of cells that are (i) ER+, Ki-67+ and PR+ and (ii) ER+, Ki-67+ and PR-. in If the calculated ratio is greater than or equal to a predetermined threshold, the patient is classified as ER+, Ki-67+, PR+ dominant; or If the calculated ratio is less than the predetermined threshold, the patient is classified as ER+, Ki-67+ dominant.

51. The method of claim 50, wherein the predetermined cutoff value is between about 0.4 and about 0.

6.

52. The method of claim 50, wherein the predetermined cutoff value is between about 0.45 and about 0.

55.

53. The method of claim 50, wherein the predetermined cutoff value is about 0.

5.

54. The method according to any one of claims 50 to 53, wherein if the patient is classified as ER+, Ki-67+, PR+ dominant, then the patient is selected to receive hormone therapy.

55. The method according to any one of claims 50 to 53, wherein if the patient is classified as ER+, Ki-67+ dominant, then the patient is selected to receive cyclin-dependent kinase 4 and 6 inhibitors.

56. The method according to any one of claims 50 to 55, wherein affinity histochemical staining of the sample with the human PR biomarker-specific reagent comprises: (i) Contact the sample with the human PR biomarker-specific reagent under conditions that allow the PR biomarker-specific reagent to specifically bind to the sample; (ii) Contacting the sample with a first set of detection reagents, the first set of detection reagents interacting with the human PR biomarker-specific reagent to promote the deposition of a first bright-field detectable portion on the sample.

57. The method according to any one of claims 50 to 56, wherein affinity histochemical staining of the sample with the human ER biomarker-specific reagent comprises: (i) Contact the sample with the human ER biomarker-specific reagent under conditions that allow the ER biomarker-specific reagent to specifically bind to the sample; (ii) Contacting the sample with a second set of detection reagents, the second set of detection reagents interacting with the human ER biomarker-specific reagents to promote the deposition of a second bright-field detectable portion on the sample.

58. The method according to any one of claims 50 to 57, wherein affinity histochemical staining of the sample with the human Ki-67 biomarker-specific reagent comprises: (i) contacting the sample with the human Ki-67 biomarker-specific reagent under conditions that allow the Ki-67 biomarker-specific reagent to specifically bind to the sample; and (ii) contacting the sample with a third set of detection reagents that interact with the human Ki-67 biomarker-specific reagent to promote the deposition of a third brightfield detectable portion on the sample.

59. The method according to any one of claims 56 to 58, wherein the first bright-field detectable portion, the second bright-field detectable portion, and the third bright-field detectable portion are selected from the group consisting of TAMRA, Dabsyl, Dabcyl, Cy3, CyB, Cy3.5, Cy5, Cy5.5, Cy7, Rhodamine 800, and fluorescein.

60. A method for classifying patients with breast cancer into those who may respond to hormone therapy or those who may not, the method comprising: (a) Affinity histochemical staining of the sample derived from the patient using a human PR biomarker-specific reagent, provided that the PR biomarker-specific reagent is allowed to bind specifically to the sample; (b) Affinity histochemical staining of the sample derived from the patient using a human ER biomarker-specific reagent, provided that the ER biomarker-specific reagent is allowed to bind specifically to the sample; (c) Affinity histochemical staining of the sample derived from the patient using a human Ki-67 biomarker-specific reagent, provided that the Ki-67 biomarker-specific reagent is allowed to bind specifically to the sample; (d) Calculate the ratio of the number of cells or nuclei that are positive for all three of ER, Ki-67 and PR to the total number of cells that are (i) ER+, Ki-67+ and PR+ and (ii) ER+, Ki-67+ and PR-. in If the calculated ratio is greater than or equal to a predetermined threshold, the patient is classified as a likely responder; or If the calculated ratio is less than the predetermined threshold, the patient is classified as a possible non-responder.

61. An affinity histochemical or affinity cytochemical method for assessing the expression of PR, ER, and Ki-67 biomarkers in a sample, the method comprising: (a) Contact the sample with a human ER biomarker-specific reagent under conditions that allow the ER biomarker-specific reagent to specifically bind to the sample; (b) Contact the sample with a first set of detection reagents, the first set of detection reagents interacting with the human ER biomarker-specific reagents to promote the deposition of a first bright-field detectable portion on the sample; (c) Contact the sample with a human PR biomarker-specific reagent under conditions that allow the PR biomarker-specific reagent to specifically bind to the sample; (d) Contact the sample with a second set of detection reagents, the second set of detection reagents interacting with the human PR biomarker-specific reagents to promote the deposition of a second brightfield detectable portion on the sample; (e) Contact the sample with a human Ki-67 biomarker-specific reagent under conditions that allow the Ki-67 biomarker-specific reagent to specifically bind to the sample; (f) Contact the sample with a third set of detection reagents, which interact with the human Ki-67 biomarker-specific reagents to promote the deposition of a third brightfield detectable portion on the sample; as well as (g) Identify cell nuclei expressing each of the PR, ER, and Ki-67 biomarkers within the sample based on the colocalization of signals from each of the deposited first, second, and third bright-field detectable portions.

62. An affinity histochemical or affinity cytochemical method for assessing the expression of PR, ER, and Ki-67 biomarkers in a sample, the method comprising: (a) Contact the sample with a human ER biomarker-specific reagent under conditions that allow the ER biomarker-specific reagent to specifically bind to the sample; (b) Contact the sample with a first set of detection reagents, the first set of detection reagents interacting with the human ER biomarker-specific reagents to promote the deposition of a first bright-field detectable portion on the sample; (c) Contact the sample with a human Ki-67 biomarker-specific reagent under conditions that allow the Ki-67 biomarker-specific reagent to specifically bind to the sample; (d) Contact the sample with a second set of detection reagents, the second set of detection reagents interacting with the human Ki-67 biomarker-specific reagent to promote the deposition of a second brightfield detectable portion on the sample; (e) Contact the sample with a human PR biomarker-specific reagent under conditions that allow the PR biomarker-specific reagent to specifically bind to the sample; (f) Contact the sample with a third set of detection reagents, which interact with the human PR biomarker-specific reagents to promote the deposition of a third brightfield detectable portion on the sample; as well as (g) Identify cell nuclei expressing each of the PR, ER, and Ki-67 biomarkers within the sample based on the colocalization of signals from each of the deposited first, second, and third bright-field detectable portions.

63. An affinity histochemical or affinity cytochemical method for assessing the expression of PR, ER, and Ki-67 biomarkers in a sample, the method comprising: (a) Contact the sample with a human Ki-67 biomarker-specific reagent under conditions that allow the Ki-67 biomarker-specific reagent to specifically bind to the sample; (b) Contact the sample with a first set of detection reagents, the first set of detection reagents interacting with the human Ki-67 biomarker-specific reagent to promote the deposition of a first bright-field detectable portion on the sample; (c) Contact the sample with a human ER biomarker-specific reagent under conditions that allow the ER biomarker-specific reagent to specifically bind to the sample; (d) Contact the sample with a second set of detection reagents, the second set of detection reagents interacting with the human ER biomarker-specific reagents to promote the deposition of a second brightfield detectable portion on the sample; (e) Contact the sample with a human PR biomarker-specific reagent under conditions that allow the PR biomarker-specific reagent to specifically bind to the sample; (f) Contact the sample with a third set of detection reagents, which interact with the human PR biomarker-specific reagents to promote the deposition of a third brightfield detectable portion on the sample; as well as (g) Identify cell nuclei expressing each of the PR, ER, and Ki-67 biomarkers within the sample based on the colocalization of signals from each of the deposited first, second, and third bright-field detectable portions.

64. An affinity histochemical or affinity cytochemical method for assessing the expression of PR, ER, and Ki-67 biomarkers in a sample, the method comprising: (a) Contact the sample with a human Ki-67 biomarker-specific reagent under conditions that allow the Ki-67 biomarker-specific reagent to specifically bind to the sample; (b) Contact the sample with a first set of detection reagents, the first set of detection reagents interacting with the human Ki-67 biomarker-specific reagent to promote the deposition of a first bright-field detectable portion on the sample; (c) Contact the sample with a human PR biomarker-specific reagent under conditions that allow the PR biomarker-specific reagent to specifically bind to the sample; (d) Contact the sample with a second set of detection reagents, the second set of detection reagents interacting with the human PR biomarker-specific reagents to promote the deposition of a second brightfield detectable portion on the sample; (e) Contact the sample with a human ER biomarker-specific reagent under conditions that allow the ER biomarker-specific reagent to specifically bind to the sample; (f) Contact the sample with a third set of detection reagents, which interact with the human ER biomarker-specific reagents to promote the deposition of a third brightfield detectable portion on the sample; as well as (g) Identify cell nuclei expressing each of the PR, ER, and Ki-67 biomarkers within the sample based on the colocalization of signals from each of the deposited first, second, and third bright-field detectable portions.

65. An affinity histochemical or affinity cytochemical method for assessing the expression of PR, ER, and Ki-67 biomarkers in a sample, the method comprising: (a) The sample is brought into contact with human Ki-67 biomarker-specific reagent, human PR biomarker-specific reagent and human ER biomarker-specific reagent under conditions that allow the human Ki-67 biomarker-specific reagent, the human PR biomarker-specific reagent and the human ER biomarker-specific reagent to specifically bind to the sample; (b) The sample is sequentially contacted with a first detection reagent, a second detection reagent, and a third detection reagent, wherein the first detection reagent, the second detection reagent, and the third detection reagent interact with the bound human Ki-67 biomarker-specific reagent, the bound human PR biomarker-specific reagent, and the bound human ER biomarker-specific reagent to promote the deposition of a first bright-field detectable portion, a second bright-field detectable portion, and a third bright-field detectable portion on the sample; as well as (c) Identify the cell nuclei expressing each of the Ki-67, PR and ER biomarkers within the sample based on the colocalization of signals from each of the deposited first, second and third detectable portions.

66. The method of claim 65, wherein the contact of the sample with the human Ki-67 biomarker-specific reagent, the human PR biomarker-specific reagent, and the human ER biomarker-specific reagent can be performed in any order.

67. The method of claim 65, wherein the contact between the sample and the human Ki-67 biomarker-specific reagent, the human PR biomarker-specific reagent, and the human ER biomarker-specific reagent can be performed simultaneously.

68. A reagent kit comprising: (i) Human Ki-67 biomarker-specific reagent, (ii) Human PR biomarker-specific reagent, and (iii) Human ER biomarker-specific reagent.

69. The kit according to claim 68, further comprising a first detection reagent, a second detection reagent, and a third detection reagent.

70. The kit of claim 69, wherein the first detection reagent, the second detection reagent, and the third detection reagent each comprise different detectable portions.

71. The kit of claim 70, wherein each different detectable portion is a chromogen.

72. The kit according to claim 70, wherein the different detectable portions are selected from the group consisting of TAMRA, Dabsyl, Dabcyl, Cy3, CyB, Cy3.5, Cy5, Cy5.5, Cy7, Rhodamine 800, and fluorescein.

73. The kit according to any one of claims 68 to 72, wherein the human Ki-67 biomarker specific reagent is a monoclonal antibody.

74. The kit according to claim 73, wherein the monoclonal antibody is selected from the group consisting of MIB-1, MIB-2, MIB-5, MIB-7, MIB-21, MIB-24 and clone 30-9.

75. The kit according to any one of claims 68 to 72, wherein the human ER biomarker specific reagent is a monoclonal antibody.

76. The kit according to claim 75, wherein the monoclonal antibody is 1D5.

77. The kit according to any one of claims 68 to 72, wherein the human PR biomarker specific reagent is a monoclonal antibody.

78. The kit according to claim 77, wherein the monoclonal antibody is an 1E2 clone.

79. A method for selecting patients with breast tumors to receive hormone therapy, the method comprising: (a) Contact the sample with a human PR biomarker-specific reagent under conditions that allow the PR biomarker-specific reagent to specifically bind to the sample; (b) Contact the sample with a first set of detection reagents, the first set of detection reagents interacting with the human PR biomarker-specific reagent to promote the deposition of a first brightfield detectable portion on the sample; (c) Contact the sample with a human ER biomarker-specific reagent under conditions that allow the ER biomarker-specific reagent to specifically bind to the sample; (d) Contact the sample with a second set of detection reagents, the second set of detection reagents interacting with the human ER biomarker-specific reagents to promote the deposition of a second brightfield detectable portion on the sample; (e) Contact the sample with a human Ki-67 biomarker-specific reagent under conditions that allow the Ki-67 biomarker-specific reagent to specifically bind to the sample; (f) Contact the sample with a third set of detection reagents, which interact with the human Ki-67 biomarker-specific reagents to promote the deposition of a third brightfield detectable portion on the sample; (g) Identifying cell nuclei expressing each of the PR, ER, and Ki-67 biomarkers within the sample based on the colocalization of signals from each of the deposited first, second, and third bright-field detectable regions; and (h) Determine the number of proliferating tumor nuclei that are both ER+ and PR+ in the histochemically stained sample; If the ratio of the number of proliferating tumor nuclei of both ER+ and PR+ to the total number of ER+ proliferating tumor nuclei is greater than or equal to a predetermined cutoff value, then the patient is selected to receive the hormone therapy; wherein the predetermined cutoff value is between about 0.4 and about 0.

6.

80. A method for selecting patients with breast tumors to receive hormone therapy, the method comprising: (a) Contact the sample with a human PR biomarker-specific reagent under conditions that allow the PR biomarker-specific reagent to specifically bind to the sample; (b) Contact the sample with a first set of detection reagents, the first set of detection reagents interacting with the human PR biomarker-specific reagent to promote the deposition of a first brightfield detectable portion on the sample; (c) Contact the sample with a human ER biomarker-specific reagent under conditions that allow the ER biomarker-specific reagent to specifically bind to the sample; (d) Contact the sample with a second set of detection reagents, the second set of detection reagents interacting with the human ER biomarker-specific reagents to promote the deposition of a second brightfield detectable portion on the sample; (e) Contact the sample with a human Ki-67 biomarker-specific reagent under conditions that allow the Ki-67 biomarker-specific reagent to specifically bind to the sample; (f) Contact the sample with a third set of detection reagents, which interact with the human Ki-67 biomarker-specific reagents to promote the deposition of a third brightfield detectable portion on the sample; (g) Identifying cell nuclei expressing each of the PR, ER, and Ki-67 biomarkers within the sample based on the colocalization of signals from each of the deposited first, second, and third bright-field detectable regions; and (h) Calculate the ratio of the number of cells or nuclei that are positive for all three of ER, Ki-67 and PR to the total number of cells that are positive for both (i) ER+, Ki-67+ and PR+ and (ii) ER+, Ki-67+ and PR-. If the calculated ratio is greater than or equal to a predetermined cutoff value, the patient is selected to receive the hormone therapy; wherein the predetermined cutoff value is between about 0.4 and about 0.

6.

81. A method for classifying patients with breast cancer as ER+, Ki-67+, PR+ dominant or ER+, Ki-67+ dominant, the method comprising: (a) Contact the sample with a human PR biomarker-specific reagent under conditions that allow the PR biomarker-specific reagent to specifically bind to the sample; (b) Contact the sample with a first set of detection reagents, the first set of detection reagents interacting with the human PR biomarker-specific reagent to promote the deposition of a first brightfield detectable portion on the sample; (c) Contact the sample with a human ER biomarker-specific reagent under conditions that allow the ER biomarker-specific reagent to specifically bind to the sample; (d) Contact the sample with a second set of detection reagents, the second set of detection reagents interacting with the human ER biomarker-specific reagents to promote the deposition of a second brightfield detectable portion on the sample; (e) Contact the sample with a human Ki-67 biomarker-specific reagent under conditions that allow the Ki-67 biomarker-specific reagent to specifically bind to the sample; (f) Contact the sample with a third set of detection reagents, which interact with the human Ki-67 biomarker-specific reagents to promote the deposition of a third brightfield detectable portion on the sample; (g) Identifying cell nuclei expressing each of the PR, ER, and Ki-67 biomarkers within the sample based on the colocalization of signals from each of the deposited first, second, and third bright-field detectable regions; and (h) Calculate the ratio of the number of cells or nuclei that are positive for all three of ER, Ki-67 and PR to the total number of cells that are (i) ER+, Ki-67+ and PR+ and (ii) ER+, Ki-67+ and PR-. in If the calculated ratio is greater than or equal to a predetermined threshold, the patient is classified as ER+, Ki-67+, PR+ dominant; or If the calculated ratio is less than the predetermined threshold, the patient is classified as ER+, Ki-67+ dominant.

82. A method for classifying patients with breast cancer into those who may respond to hormone therapy or those who may not, the method comprising: (a) Contact the sample with a human PR biomarker-specific reagent under conditions that allow the PR biomarker-specific reagent to specifically bind to the sample; (b) Contact the sample with a first set of detection reagents, the first set of detection reagents interacting with the human PR biomarker-specific reagent to promote the deposition of a first brightfield detectable portion on the sample; (c) Contact the sample with a human ER biomarker-specific reagent under conditions that allow the ER biomarker-specific reagent to specifically bind to the sample; (d) Contact the sample with a second set of detection reagents, the second set of detection reagents interacting with the human ER biomarker-specific reagents to promote the deposition of a second brightfield detectable portion on the sample; (e) Contact the sample with a human Ki-67 biomarker-specific reagent under conditions that allow the Ki-67 biomarker-specific reagent to specifically bind to the sample; (f) Contact the sample with a third set of detection reagents, which interact with the human Ki-67 biomarker-specific reagents to promote the deposition of a third brightfield detectable portion on the sample; (g) Identifying cell nuclei expressing each of the PR, ER, and Ki-67 biomarkers within the sample based on the colocalization of signals from each of the deposited first, second, and third bright-field detectable regions; and (h) Calculate the ratio of the number of cells or nuclei that are positive for all three of ER, Ki-67 and PR to the total number of cells that are (i) ER+, Ki-67+ and PR+ and (ii) ER+, Ki-67+ and PR-. in If the calculated ratio is greater than or equal to a predetermined threshold, the patient is classified as a likely responder; or If the calculated ratio is less than the predetermined threshold, the patient is classified as a possible non-responder.

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