WO2016174185A1

WO2016174185A1 - Multiplex immunohistochemical panel for the identification of metastatic carcinoma in cytology fluid specimens

Info

Publication number: WO2016174185A1
Application number: PCT/EP2016/059580
Authority: WO
Inventors: Esteban A. ROBERTS; Lauren BEHMAN; Charalambos SOLOMIDES
Original assignee: Ventana Medical Systems, Inc.; F. Hoffmann-La Roche Ag; Thomas Jefferson University
Priority date: 2015-04-30
Filing date: 2016-04-29
Publication date: 2016-11-03

Abstract

Disclosed are methods of detecting metastatic carcinoma in a human subject by analyzing a single small, tissue sample for the presence or absence of the EP-Cam, calretinin, and CD163 markers, wherein the patient is diagnosed with metastatic carcinoma if the EP-Cam, calretinin, and CD163 markers are detected.

Description

Multiplex Immunohistochemical Panel for the Identification of Metastatic Carcinoma in

Cytology Fluid Specimens

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of the filing date of U.S. Provisional Patent

Application No. 62/287,573 filed January 27, 2016, and claims the benefit of the filing date of U.S. Provisional Patent Application No. 62/155,342 filed April 30, 2015, the disclosures of which are hereby incorporated herein by reference.

BACKGROUND OF THE DISCLOSURE

[0002] Immunohistochemistry, or IHC, refers to the process of detecting, localizing, and quantifying antigens (e.g. a protein), in a biological sample (e.g. a tissue sample), and using specific binding moieties, such as antibodies specific to the particular antigens. This detection technique has the advantage of being able to show exactly where a given protein or marker is located within the tissue sample. It is also an effective way to examine the tissues themselves. IHC and can be performed on various biological samples, such as tissue (e.g., fresh frozen, formalin fixed paraffin embedded) and cytological samples. Recognition of the targets may be detected through the use of various labels (e.g., chromogenic, fluorescent, luminescent, radiometric).

[0003] The cytologic differential diagnosis of mesothelial cells and adenocarcinoma in fluid specimens can be challenging. Difficulties in differential diagnosis result from the need to use multiple specimens to interrogate a single biomarker at a time which results in the loss of contextual information within a sample/ across samples.

BRIEF SUMMARY OF THE DISCLOSURE

[0004] In one aspect of the present disclosure is a method of detecting metastatic carcinoma in a patient by analyzing a single tissue sample for the presence or absence of malignant cells by measuring the presence or absence of an epithelial marker target of non-mesothelial origin, a marker of mesothelial cells, and a marker of macrophages, wherein the patient is diagnosed with metastatic carcinoma if the malignant cells are detected. [0005] In another aspect of the present disclosure is a method of detecting metastatic carcinoma in a patient by analyzing a single tissue sample for the presence or absence of malignant cells by measuring the presence or absence of the EP-Cam, calretinin, and CD 163 markers, wherein the patient is diagnosed with metastatic carcinoma if the malignant cells are detected. In some embodiments, the method comprises the steps of applying a first primary antibody specific for one of the EP-Cam, calretinin, or CD 163 markers; applying first detection reagents to detect the first primary antibody; applying a second primary antibody specific for another one of the EP-Cam, calretinin, or CD 163 markers; applying second detection reagents to detect the second primary antibody; applying a third primary antibody specific for a third one of the EP-Cam, calretinin, or CD 163 markers; and applying third detection reagents to detect the third primary antibody. In some embodiments, each of the first, second, and third detection reagents comprise (i) a secondary antibody specific to each of the primary antibodies, wherein the secondary antibody is conjugated to an enzyme such that the secondary antibody labels the marker with an enzyme; and (ii) a chromogenic substrate; wherein each of the first, second, and third detection reagents comprise different components (e.g. enzymes, secondary antibodies, chromogenic moieties).

[0006] In some embodiments, a first chromogenic substrate comprises one of Dabsyl, Cy5, or TAMRA; a second chromogenic substrate comprises another one of Dabsyl, Cy5, or TAMRA; and a third chromogenic substrate comprises a third one of Dabsyl, Cy5, or TAMRA. In some embodiments, the first primary antibody is specific for EP-Cam; the second primary antibody is specific for calretinin; and the third primary antibody is specific for CD 163. In some embodiments, the first chromogenic reagent comprises TAMRA; the second chromogenic reagent comprises Cy5; and the third chromogenic reagent comprises Dabsyl. In some embodiments, the chromogenic substrates are selected such that a peak wavelength for each is separated by at least about 50nm. In some embodiments, the antibodies are detected simultaneously. In some embodiments, the tissue sample is a serous effusion or a pleural effusion. In some embodiments, a size of the tissue sample ranges from about 3 microns to about 6 microns. In some embodiments, the metastatic carcinoma is selected from the group consisting of adenocarcinomas, basal cell carcinomas, squamous cell carcinomas, and transitional cell carcinomas.

[0007] In another aspect of the present disclosure is a method for the diagnosis of metastatic cancer in a human subject, wherein the metastatic cancer is characterized by the presence or absence of EP-Cam, calretinin, and CD 163 markers comprising: (a) applying a first primary antibody specific for one of the EP-Cam, calretinin, or CD 163 marker to a tissue sample, wherein a presence of one of the markers creates a first antibody-marker complex; (b) applying first detection reagents that detect the first antibody-marker complex; (c) applying a second primary antibody specific for another of the EP-Cam, calretinin, or CD 163 markers to the tissue sample, wherein a presence of another one of the markers creates a second antibody-marker complex; (d) applying second detection reagents that detect the second antibody-marker complex; (e) applying a third primary antibody specific for another of the EP-Cam, calretinin, or CD 163 markers to the tissue sample, wherein a presence of a third one of the markers creates a third antibody-marker complex; (f) applying third detection reagents that detect the third antibody-marker complex; and (g) diagnosing metastatic cancer where the first, second, and third detection reagents are detected.

[0008] In some embodiments, each of the first, second, and third detection reagents comprise

(i) a secondary antibody specific to each of the primary antibodies, wherein the secondary antibody is conjugated to an enzyme such that the secondary antibody labels the marker with an enzyme; and (ii) a chromogenic substrate; and wherein each of the first, second, and third detection reagents comprise different components. In some embodiments, a first chromogenic substrate comprises one of Dabsyl, Cy5, or TAMRA; a second chromogenic substrate comprises another one of Dabsyl, Cy5, or TAMRA; and a third chromogenic substrate comprises a third one of Dabsyl, Cy5, or TAMRA. In some embodiments, the first primary antibody is specific for EP-Cam; the second primary antibody is specific for calretinin; and the third primary antibody is specific for CD 163. In some embodiments, the first chromogenic reagent comprises TAMRA; the second chromogenic reagent comprises Cy5; and the third chromogenic reagent comprises Dabsyl. In some embodiments, the chromogenic substrates are selected such that a peak wavelength for each is separated by at least about 70nm. In some embodiments, the antibodies are detected simultaneously. In some embodiments, the tissue sample is a serous effusion. In some embodiments, a size of the tissue sample ranges from about 3 microns to about 6 microns. In some embodiments, the metastatic carcinoma is selected from the group consisting of adenocarcinomas, basal cell carcinomas, squamous cell carcinomas, and transitional cell carcinomas. The host species of the primary antibodies or any modification of that primary antibody include any biological or chemical labeling of the primary antibody.

[0009] In another aspect of the present disclosure is a method for diagnosis metastatic cancer in a human subject, wherein the metastatic cancer is characterized by the presence or absence of EP- Cam, calretinin, and CD 163 markers comprising, in any order, at least the steps of: (a) applying a first primary antibody specific for EP-Cam to a tissue sample, wherein a presence of EP-Cam creates an antibody-EP-Cam marker complex; (b) applying first detection reagents that detect the antibody- Ber-EP4 complex; (c) applying a second primary antibody specific for calretinin to the tissue sample, wherein a presence of calretinin creates an antibody-calretinin marker complex; (d) applying second detection reagents that detect the antibody-calretinin marker complex; (e) applying a third primary antibody specific for CD 163 to the tissue sample, wherein a presence of CD 163 creates an antibody- CD 163 marker complex; (f) applying third detection reagents that detect the antibody-CD 163 marker complex; (g) diagnosing metastatic cancer where the first, second, and third detection reagents are detected.

[0010] In some embodiments, each of the first, second, and third detection reagents comprise

(i) a secondary antibody specific to each of the primary antibodies, wherein the secondary antibody is conjugated to an enzyme such that the secondary antibody labels the marker with an enzyme; and (ii) a chromogenic substrate; and wherein each of the first, second, and third detection reagents comprise different components. In some embodiments, a first chromogenic substrate comprises one of Dabsyl, Cy5, or TAMRA; a second chromogenic substrate comprises another one of Dabsyl, Cy5, or TAMRA; and a third chromogenic substrate comprises a third one of Dabsyl, Cy5, or TAMRA. In some embodiments, the first chromogenic reagent comprises TAMRA; the second chromogenic reagent comprises Cy5; and the third chromogenic reagent comprises Dabsyl.

[0011] In another aspect of the present disclosure is a method of detecting metastatic carcinoma in a patient by analyzing a single tissue sample for the presence or absence of malignant cells, comprising the steps of: (a) contacting a biological sample with a first detection probe, the first detection probe comprising a first primary antibody selected from one of an antibody specific for an epithelial marker target of non-mesothelial origin, an antibody specific for mesothelial cells, or an antibody specific for macrophages; (b) contacting the biological sample with first chromogenic detection reagents comprising a first enzyme; (c) inactivating the first enzyme; (d) contacting a biological sample with a second detection probe, the second detection probe comprising a second primary antibody selected from another of an antibody specific for an epithelial marker target of non- mesothelial origin, an antibody specific for mesothelial cells, or an antibody specific for macrophages; (e) contacting the biological sample with second chromogenic detection reagents comprising a second enzyme; (f) inactivating the second enzyme; (g) contacting a biological sample with a third detection probe, the third detection probe comprising a third primary antibody selected from a third one of an antibody specific for an epithelial marker target of non-mesothelial origin, an antibody specific for mesothelial cells, or an antibody specific for macrophages; (h) contacting the biological sample with third chromogenic detection reagents comprising a third enzyme; and (i) detecting signals from the first, second, and third detection reagents, wherein each of the first, second, and third detection reagents comprise a different chromogenic moiety, and wherein the patient is diagnosed with metastatic carcinoma if the malignant cells are detected.

[0012] In some embodiments, the epithelial marker target of non-mesothelial origin is EP-

Cam. In some embodiments, the marker for mesothelial cells is calretinin. In some embodiments, the marker for macrophages is CD 163. In some embodiments, each of the first, second, and third detection reagents further comprise a chromogenic substrate; and wherein each of the first, second, and third detection reagents comprise different components. In some embodiments, a first chromogenic substrate comprises one of Dabsyl, Cy5, or TAMRA; a second chromogenic substrate comprises another one of Dabsyl, Cy5, or TAMRA; and a third chromogenic substrate comprises a third one of Dabsyl, Cy5, or TAMRA. In some embodiments, the first primary antibody is specific for EP-Cam; the second primary antibody is specific for calretinin; and the third primary antibody is specific for CD 163. In some embodiments, the first chromogenic reagent comprises TAMRA; the second chromogenic reagent comprises Cy5; and the third chromogenic reagent comprises Dabsyl. In some embodiments, the chromogenic substrates are selected such that a peak wavelength for each is separated by at least about 70nm. In some embodiments, the markers are detected simultaneously. In some embodiments, the markers are detected sequentially. In some embodiments, the tissue sample is a serous effusion. In some embodiments, a size of the tissue sample ranges from about 3 microns to about 6 microns. In some embodiments, the metastatic carcinoma is selected from the group consisting of adenocarcinomas, basal cell carcinomas, squamous cell carcinomas, and transitional cell carcinomas.

[0013] In another aspect of the present disclosure is a method of detecting metastatic carcinoma in a patient by analyzing a single tissue sample for the presence or absence of malignant cells, comprising the steps of: (a) contacting a biological sample with a first detection probe, the first detection probe comprising a first antibody selected from one of an antibody specific for an epithelial marker target of non-mesothelial origin, an antibody specific for mesothelial cells, or an antibody specific for macrophages; (b) contacting the biological sample with a first labeling conjugate that specifically binds to the first detection probe, wherein the first labeling conjugate comprises a first enzyme; (c) contacting the biological sample with a first signaling conjugate comprising a first latent reactive moiety and a first chromogenic moiety; (d) inactivating the first enzyme; (e) contacting a biological sample with a second detection probe, the second detection probe comprising a second antibody selected from another one of an antibody specific for an epithelial marker target of non- mesothelial origin, an antibody specific for mesothelial cells, or an antibody specific for macrophages; (f) contacting the biological sample with a second labeling conjugate that specifically binds to the second detection probe, wherein the second labeling conjugate comprises a second enzyme; (g) contacting the biological sample with a second signaling conjugate comprising a second latent reactive moiety and a second chromogenic moiety; (h) inactivating the second enzyme; (i) contacting a biological sample with a third detection probe, the third detection probe comprising a third antibody selected from a third one of an antibody specific for an epithelial marker target of non- mesothelial origin, an antibody specific for mesothelial cells, or an antibody specific for macrophages; (i) contacting the biological sample with a third labeling conjugate that specifically binds to the third detection probe, wherein the third labeling conjugate comprises a third enzyme; (j) contacting the biological sample with a third signaling conjugate comprising a third latent reactive moiety and a third chromogenic moiety; (k) optionally inactivating the third enzyme; and (j) detecting signals from the first, second, and third chromogenic moieties, wherein each of the first, second, and third chromogenic moieties are each different, and wherein the patient is diagnosed with metastatic carcinoma if the malignant cells are detected.

[0014] In some embodiments, the first chromogenic moiety comprises one of Dabsyl, Cy5, or TAMRA; a second chromogenic moiety comprises another one of Dabsyl, Cy5, or TAMRA; and a third chromogenic moiety comprises a third one of Dabsyl, Cy5, or TAMRA. In some embodiments, the first primary antibody is specific for EP-Cam; the second primary antibody is specific for calretinin; and the third primary antibody is specific for CD 163. In some embodiments, the first chromogenic reagent comprises TAMRA; the second chromogenic reagent comprises Cy5; and the third chromogenic reagent comprises Dabsyl.

[0015] In another aspect of the present disclosure is a method of detecting metastatic carcinoma in a patient by analyzing a single tissue sample for the presence or absence of EP-Cam, calretinin, and CD 163, wherein the patient is diagnosed with metastatic carcinoma if EP-Cam, calretinin, and CD 163 are detected.

[0016] In another aspect of the present disclosure is a kit for detecting metastatic carcinoma comprising a first primary antibody specific to EP-Cam; a second primary antibody specific to calretinin; and a third primary antibody specific to CD 163. In some embodiments, the kit further comprises a first labeling conjugate specific to the first primary antibody; a second labeling conjugate specific to the second primary antibody; and a third labeling conjugate specific to the third primary antibody. In some embodiments, the kit further comprises first, second, and third chromogenic substrates. In some embodiments, the kit further comprises one or more enzyme inactivation compositions.

[0017] Cytological examination of effusions is of paramount importance because the finding of malignant cells denotes advanced cancer. In this regard, Applicants have discovered a method for the multiplexed immunohistochemical detection of a panel of markers indicative of metastatic carcinoma. Applicants have found that the claimed methods offer the capability to unequivocally diagnose metastatic carcinoma from a small section of tissue, e.g. ranging from about 3 to 6 microns in size, thus allowing for the conservation of tissue for use in additional molecular testing routines, if needed. Applicants have found that this is possible even when the tumor content within the tissue sample is very low. Applicants have determined that the specific order of antigen detection and chromogen deposition is necessary to generating a three-color signal amenable to diagnostic interpretation (see Figures 11A and 11B).

BRIEF DESCRIPTION OF THE DRAWING FIGURES

[0018] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided to the Office upon request and the payment of the necessary fee.

[0019] Figure 1 provides a flowchart providing a method of multiplex detection;

[0020] Figure 2 provides a flowchart providing a method of multiplex detection;

[0021] Figure 3 illustrates a method of multiplex detection utilizing three different primary antibodies and various chromogenic detection reagents;

[0022] Figures 4A provides a DAB staining example with an antibody specific for EP-Cam in an appendix specimen;

[0023] Figures 4B provides a DAB staining example with an antibody specific for Calretinin in an appendix specimen;

[0024] Figures 4C provides a DAB staining example with an antibody specific for CD 163 in an appendix specimen;

[0025] Figures 4D provides a DAB staining example with an antibody specific for EP-Cam in an effusion specimen;

[0026] Figures 4E provides a DAB staining example with an antibody specific for Calretinin in an effusion specimen; [0027] Figures 4F provides a DAB staining example with an antibody specific for CD 163 in an effusion specimen;

[0028] Figure 5A provides an example of chromogenic multiplex detection of metastatic carcinoma, utilizing a panel comprising EP-CAM-Cy5 (stained blue); Calretinin-Dabsyl (stained yellow); and CD163 -TAMRA (stained magenta) in an appendix specimen;

[0029] Figure 5B provides an annotated version of a portion of the image of Figure 5A, where EP-CAM-Cy5 is labeled "a;" Calretinin-Dabsyl is labeled "c;" and CD163 -TAMRA is labeled "b" in an appendix specimen;

[0030] Figure 6A provides an example of chromogenic multiplex detection of metastatic carcinoma, utilizing a panel comprising EP-CAM-Cy5 (stained blue); Calretinin-Dabsyl (stained yellow); and CD163 -TAMRA (stained magenta) in an effusion specimen;

[0031] Figure 6B provides an annotated version of a portion of the image of Figure 6A, where EP-CAM-Cy5 is labeled "a," Calretinin-Dabsyl is labeled "c," and CD163 -TAMRA is labeled "b" in an effusion specimen;

[0032] Figure 7A provides an example of an effusion specimen demonstrating that the order of antibody addition is critical to successful chromogenic multiplex of metastatic carcinoma panel, where EP-Cam is stained blue; Calretinin is stained magenta; and CD-I 63 is stained yellow;

[0033] Figure 7B provides an annotated version of a portion of the image of Figure 7A, where EP-Cam is labeled "Α;" Calretinin is labeled "B" or stained magenta; and CD-I 63 is labeled "C;" (and "X" indicates a chromogenic overlap of stains);

[0034] Figure 8A provides an example of chromogenic multiplex detection of metastatic carcinoma in an appendix specimen, utilizing a panel comprising Ep-CAM-TAMRA (stained magenta), Calretinin-Dabsyl (stained yellow), and CD163-Cy5 (stained blue);

[0035] Figure 8B provides an annotated version of a portion of the image of Figure 8A, where Ep-CAM-TAMRA is labeled "B," Calretinin-Dabsyl is labeled "C," and CD163-Cy5 is labeled "Α;"

[0036] Figure 9A provides an example of chromogenic multiplex detection of metastatic carcinoma in an effusion specimen, utilizing a panel comprising Ep-CAM-TAMRA (stained magenta), Calretinin-Dabsyl (stained yellow), and CD163-Cy5 (stained blue);

[0037] Figures 9B provides an annotated version of a portion of the image of Figure 9A, where Ep-CAM-TAMRA is labeled "B," Calretinin-Dabsyl is labeled "C," and CD163-Cy5 is labeled "Α;" [0038] Figures 10A and IOC provide cohort effusion specimen examples of chromogenic multiplex detection of metastatic carcinoma utilizing a panel comprising Ep-CAM-TAMRA (stained magenta), Calretinin-Cy5 (stained blue), and CD163- Dabsyl (stained yellow);

[0039] Figures 10B and 10D provide annotated versions of a portion of the images of

Figures 10A and IOC, where Ep-CAM-TAMRA is labeled "B," Calretinin-Cy5 is labeled "A," and CD163- Dabsyl is labeled "C;"

[0040] Figure 11A provides a cohort effusion specimen example of chromogenic multiplex detection of metastatic carcinoma utilizing a panel comprising Ep-CAM-TAMRA (stained magenta), Calretinin-Cy5 (stained blue) and CD 163 -Dabsyl (stained yellow), demonstrating CD 163 yellow cytoplasmic/membrane staining and blue cytoplasmic/nuclear stain for Calretinin; and

[0041] Figures 11B provides an annotated version of a portion of the image of Figure 11 A, where Ep-CAM-TAMRA is labeled "B," Calretinin-Cy5 is labeled "A," and CD 163 -Dabsyl is labeled "C," again demonstrating CD 163 yellow cytoplasmic/membrane staining and blue cytoplasmic/nuclear stain for Calretinin.

DETAILED DESCRIPTION

[0042] In general, the present disclosure is directed to a method, including an automated method, of detecting metastatic carcinoma in a patient by analyzing a single tissue sample for the presence or absence of certain markers indicative of metastatic carcinoma, including epithelial marker targets of non-mesothelial origin, markers for mesothelial cells, and markers for macrophages. The present disclosure is also directed to detecting metastatic cancer in a single small tissue sample, e.g. a tissue sample having a size ranging from about 3 microns to about 6 microns, using a unique sequence for the application of primary antibodies and a unique combination of chromogenic substrates.

[0043] As used herein, the singular terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise.

[0044] The terms "comprising," "including," "having," and the like are used interchangeably and have the same meaning. Similarly, "comprises," "includes," "has," and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of "comprising" and is therefore interpreted to be an open term meaning "at least the following," and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, "a device having components a, b, and c" means that the device includes at least components a, b and c. Similarly, the phrase: "a method involving steps a, b, and c" means that the method includes at least steps a, b, and c. Moreover, while the steps and processes may be outlined herein in a particular order, the skilled artisan will recognize that the ordering steps and processes may vary.

[0045] Alkaline phosphatase (AP) is an enzyme that can be conjugated to a labeled molecule. AP removes (by hydrolysis) and transfers phosphate group organic esters by breaking the phosphate -oxygen bond, and temporarily forming an intermediate enzyme-substrate bond.

[0046] As used herein, the term "antibody," refers to immunoglobulins or immunoglobulin- like molecules, including by way of example and without limitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, (e.g., in mammals such as humans, goats, rabbits and mice) and antibody fragments that specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of binding to other molecules. Antibody further refers to a polypeptide ligand comprising at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of an antigen. Antibodies may be composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region. Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody. The term antibody also includes intact immunoglobulins and the variants and portions of them well known in the art.

[0047] A "biological sample" can be any solid or fluid sample obtained from, excreted by or secreted by any living organism, including without limitation, single celled organisms, such as bacteria, yeast, protozoans, and amoebas among others, multicellular organisms (such as plants or animals, including samples from a healthy or apparently healthy human subject or a human patient affected by a condition or disease to be diagnosed or investigated, such as cancer). For example, a biological sample can be a biological fluid obtained from, for example, blood, plasma, serum, urine, bile, ascites, saliva, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion, a transudate, an exudate (for example, fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (for example, a normal joint or a joint affected by disease). A biological sample can also be a sample obtained from any organ or tissue (including a biopsy or autopsy specimen, such as a tumor biopsy) or can include a cell (whether a primary cell or cultured cell) or medium conditioned by any cell, tissue or organ. [0048] "Cy5" refers to N,N'-biscarboxypentyl-5,5'-disulfonato-indo-dicarbocyanine.

[0049] "DABSYL" refers to 4-(dimethylamino) azobenzene-4'-sulfonamide.

[0050] "DAB" refers to (3,3'-diaminobenzidine) which is oxidized in the presence of peroxidase and hydrogen peroxide resulting in the deposition of a brown, alcohol-insoluble precipitate at the site of enzymatic activity. DAB (3, 3 '-diaminobenzidine) produces a dark brown reaction product and can be used for immunohistochemical applications.

[0051] "Diagnosed," as used herein, refers to the recognition of a disease by its signs and symptoms or genetic analysis, pathological analysis, histological analysis, and the like.

[0052] Horseradish peroxidase (HRP) is an enzyme that can be conjugated to a labeled molecule. It produces a colored, fluorimetric, or luminescent derivative of the labeled molecule when incubated with a proper substrate, allowing it to be detected and quantified. HRP acts in the presence of an electron donor to first form an enzyme substrate complex and then subsequently acts to oxidize an electronic donor.

[0053] "Multiplex," "multiplexed," or "multiplexing" refers to detecting multiple targets in a sample concurrently, substantially simultaneously, or sequentially.

[0054] The term "primary antibody" refers to an antibody which binds specifically to a target protein antigen in a tissue sample. A primary antibody is generally the first antibody used in an immunohistochemical procedure.

[0055] The term "secondary antibody" herein refers to an antibody which binds specifically to a primary antibody, thereby forming a bridge between the primary antibody and a subsequent reagent (e.g. a label, an enzyme, etc.), if any. The secondary antibody is generally the second antibody used in an immunohistochemical procedure.

[0056] "TAMRA" refers to carboxytetramethylrhodamine.

[0057] Detection Probes

[0058] The detection probes of the present disclosure include primary antibodies specific for a target (e.g. proteins or markers present in a tissue sample). In some embodiments, the detection probes are indirect detection probes, i.e. the detection probes are not configured for the purpose of direct visualization of the target. In these embodiments, a secondary antibody will bind to the primary antibody to effectuate labeling and detection.

[0059] In some embodiments, the detection probes utilized are those that are specific to epithelial marker targets that are of non-mesothelial origin, such as Epithelial Cell Adhesion Molecule ("EP-Cam"). EP-Cam is a transmembrane glycoprotein mediating Ca2+-independent homotypic cell-cell adhesion in epithelia. More specifically, Ep-CAM consists of two glycoproteins, 34 and 39 kDa, and sometimes designated epithelial antigen, epithelial specific antigen, and epithelial glycoprotein. EpCAM is also believed to be involved in cell signaling, migration, proliferation, and differentiation. The glycoproteins are located on the cell membrane surface and in the cytoplasm of virtually all epithelial cells with the exception of most squamous epithelia, hepatocytes, renal proximal tubular cells, gastric parietal cells and myoepithelial cells.

[0060] In paraffin sections, the protein is detected with monoclonal antibodies, including

Ber-EP4 and MOC-31. Ber-EP4 is a newly characterized monoclonal antibody that reliably labels epithelial tissues but does not react with mesothelial cells. The Ep-CAM primary antibody is available from Ventana Medical Systems, Inc. (Tucson, Arizona) (760-4383).

[0061] In some embodiments, the detection probes utilized are those that are specific to mesothelial cell targets, such as calretinin. The anti-Calretinin primary antibody is available from Ventana Medical Systems, Inc. (Tucson, Arizona) (790-4467). The anti-Calretinin (SP65) Primary Antibody (CONFIRM anti-Calretinin (SP65)) is directed against the calcium binding protein, calretinin, expressed by normal and reactive mesothelium, eccrine glands of skin, Sertoli cells of the testis, ovarian stromal cells and adrenal cortical cells. The anti-Calretinin antibody exhibits a nuclear and cytoplasmic staining pattern and may be used to aid in the identification of mesothelioma, and in distinguishing mesothelioma from adenocarcinoma. The antibody is intended for qualitative staining in sections of formalin-fixed, paraffin-embedded tissue.

[0062] In some embodiments, the detection probes utilized are those that are specific macrophages, such as CD 163. The anti-CD 163 primary antibody is available from Ventana Medical Systems, Inc. (Tucson, Arizona) (760-4437). CD163 was recently identified as an acute phase- regulated transmembrane protein whose function is to mediate the endocytosis of haptoglobin- hemoglobin complexes. This receptor is expressed on the surface of monocytes (low expression) and tissue macrophages (high expression). It is a member of the cysteine-rich scavenger receptor superfamily, encoded by a gene localized on human chromosome 12pl3.3. Solubilized in plasma, CD 163 functions as an anti- inflammatory signal and has many roles in disease processes that range from autoimmune conditions such as rheumatoid arthritis to atherosclerosis. Previous work has shown that the CD 163 gene can be regulated by glucocorticoids, IL-10, and other inflammatory modulators, and is highly expressed in inflamed tissues, consistent with its role in the resolution of inflammation.

[0063] Detection Reagents [0064] In some embodiments, the primary antibodies are detected indirectly, and specific reagents are utilized to enable detection of the primary antibodies and hence the target. The multiplex detection methods described herein utilize chromogenic detection reagents or chromogenic detection kits. Chromogenic detection reagents comprise an enzyme and a chromogenic substrate for the enzyme. The enzyme acts on the chromogenic substrate to produce a colored, detectable signal. In some embodiments, the detection reagents include a secondary antibody specific for the primary antibody, wherein the secondary antibody is conjugated to an enzyme, such that the target may be labeled with the enzyme (hereinafter, "labeling conjugate" or "labeling conjugates"). In some embodiments, the detection reagents include labeling conjugates and signaling conjugates.

[0065] Labeling Conjugates

[0066] As defined above, the labeling conjugate comprises a secondary antibody conjugated to an enzyme. Each of the secondary antibodies employed herein are specific to a single primary antibody. As such, the labeling conjugate, through the primary antibody, is able to label the target or marker with an enzyme. One approach to configuring a labeling conjugate has been to directly couple an enzyme to the anti-species antibody. Conjugates of this kind, which may or may not include various linkers, are also described in U.S. Pat. No. 7,695,929, the disclosure of which is incorporated herein by reference in its entirety.

[0067] The labeling conjugates may employ any kind of enzyme. In some embodiments, suitable enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, β-galactosidase, β-glucuronidase or β-lactamase. In other embodiments, enzymes include oxidoreductases or peroxidases (e.g. HRP, AP). The enzyme of the labeling conjugate catalyzes conversion of the chromogenic substrate to a reactive moiety which covalently binds to the biological sample proximally to or directly on the target.

[0068] Specific examples of labeling conjugates suitable for use with with present methods include OmniMap xMs HRP, UltraMap xRb AP, and UltraMap xMs AP, each of which are available from Ventana Medical Systems, Inc. (Tucson, Arizona), each of which are secondary antibodies, namely anti-species antibodies specific to the primary antibodies employed herein.

[0069] Chromogenic Substrates

[0070] The processes of the present disclosure utilize chromogenic detection reagents, where chromogenic moieties are ultimately used to detect the presence or absence of a target (or marker) within the sample. In general, chromogenic detection reagents comprise an enzyme and a chromogenic substrate for the enzyme. The enzyme acts on the chromogenic substrate to produce a colored, detectable signal (chromogenic moiety). Examples of enzymes and chromogenic substrates are disclosed within US Patent Publication No. 2013/0034853, the disclosure of which is hereby incorporated by reference herein in its entirety.

[0071] In some embodiments, the chromogenic substrates are signaling conjugates which comprise a latent reactive moiety and a chromogenic moiety. In some embodiments, the latent reactive moiety of the signaling conjugate is configured to undergo catalytic activation to form a reactive species that can covalently bond with the sample or to other detection components. The catalytic activation is driven by one or more enzymes (e.g., oxidoreductase enzymes and peroxidase enzymes, like horseradish peroxidase) and results in the formation of a reactive species. These reactive species are capable of reacting with the chromogenic moiety proximal to their generation, i.e. near the enzyme. Specific examples of signaling conjugates are disclosed in US Patent Publication No. 2013/0260379, the disclosure of which is hereby incorporated by reference herein in its entirety.

[0072] In some embodiments, the chromogenic substrates or signaling conjugates are selected such that peak detectable wavelengths of any chromogenic moiety do not overlap with each other and are readily detectable by a pathologist or an optical detector (e.g. a scanner). In some embodiments, the chromogenic moieties are selected such that the peak wavelengths of the different chromogenic moieties are separated by at least about 50nm. In other embodiments, the chromogenic moieties are selected such that the peak wavelengths of the different chromogenic moieties are separated by at least about 70nm. In yet other embodiments, the chromogenic moieties are selected such that the peak wavelengths of the different chromogenic moieties are separated by at least about lOOnm.

[0073] In yet further embodiments, the chromogenic moieties are selected such that the chromogenic moieties, when introduced to the tissue specimen, provide for different colors (e.g. yellow, blue, magenta). In some embodiments, the chromogenic moieties are selected such that they provide a good contrast between each other, e.g. a separation of colors that are optically recognizable. In some embodiments, the chromogenic moieties are selected such that when placed in close proximity of each other provide for a signal or color that is different than the signals or colors of either of the chromogenic moieties when observed alone.

[0074] In some embodiments, the ordering of chromogenic substrates is important. For example, a particular chromogenic substrate may not be as sensitive when used to detect one particular target as compared with another. Moreover, proper ordering may prevent masking of signals in close proximity to each other.

[0075] In some embodiments, the chromogenic substrates comprise Dabsyl, Cy5, or

TAMRA. In some embodiments, the chromogenic substrates are selected from QM- -Dabsyl, QM- - Cy5, and TYR-TAMRA. In other embodiments, the chromogenic substrates are selected from Rhodamine 110, Rhodamine 6G, and Texas Red® (sulforhodamine 101 acid chloride dye is a bright red-fluorescent dye).

[0076] Methods of Multiplex Detection

[0077] The present disclosure is directed to the detection of a panel of three primary antibodies in a multiplex assay, e.g. a first primary antibody specific to an epithelial marker of non- mesothelial origin (e.g. Ber-EP4), a second primary antibody specific to macrophages (e.g. CD163); and a third primary antibody specific to mesothelial cells (e.g. calretinin). Without wishing to be bound by any particular theory, it is believed that the detection of these three types of markers provides for the detection of malignant cells (e.g. adenocarcinomas, basal cell carcinomas, squamous cell carcinomas, and transitional cell carcinomas) within a tissue sample (e.g. a serous effusion, pleural effusion, or peritoneal effusion) and allows for the diagnosis or differential diagnosis of metastatic carcinoma, and allows for diagnosis in small tissue samples (e.g. one that is about four microns in size).

[0078] Samples for use in the methods disclosed herein can be prepared using any method known in the art. The samples can be obtained from a subject for routine screening or from a subject that is suspected of having a disorder or suspected as having a disorder, such as cancer. In some embodiments, the samples are analyzed for the diagnosis or differential diagnosis of metastatic cancer by detecting targets, including biomarkers (e.g. proteins), within the tissue sample. The described embodiments of the disclosed method can also be applied to samples that do not have abnormalities, diseases, disorders, etc., referred to as "normal" samples. Such normal samples are useful, among other things, as controls for comparison to other samples. For example, it may be useful to test a patient, i.e. a human subject, for cancer by taking tissue samples from multiple locations, and these samples may be used as controls and compared to later samples to determine whether a particular cancer has spread beyond its primary origin.

[0079] In some embodiments, the tissue samples are effusion specimens. In some embodiments, the tissue samples are serous effusions. In other embodiments, the tissue samples are pleural effusions. In yet other embodiments, the tissue samples are peritoneal effusions. In some embodiments, the tissue samples used for analysis range in size from about 2 microns to about 8 microns in any dimension. In other embodiments, the tissue samples used for analysis range in size from about 3 microns to about 6 microns in any dimension. In yet other embodiments, the tissue samples range in size from about 3 microns to about 5 microns in any dimension. In yet other embodiments, the tissue samples range in size from about 3.5 microns to about 4.5 microns in any dimension. In yet further embodiments, the tissue samples are about 4 microns in size in any dimension. The skilled artisan will be able to select an appropriate tissue size depending on the type of tissue, how much tissue is available for testing and for further assays (e.g. other IHC assays, other in situ hybridization assays, other polymerase chain reaction assays, etc.), and any existing diagnosis (e.g. more or less tissue may be needed when a patient has already been diagnosed with metastatic carcinoma).

[0080] In some embodiments, a method of detecting targets (e.g. EP-cam, calretinin, and/or

CD163) in a biological sample includes contacting the biological sample with detection probes (e.g. primary antibodies specific for EP-Cam, calretinin, and/or CD163), contacting the biological sample with labeling conjugates, and contacting the biological sample with chromogenic substrates or signaling conjugates. FIGs. 1 and 2 provide flowcharts delineating the steps of certain embodiments of the methods of the present disclosure. In particular, the method sets forth a sequential multiplex detection scheme where at step 1 the sample is contacted with a detection probe or primary antibody. When the detection probe is introduced into the sample, it will form a detection probe-target complex (e.g. an antibody-target complex or an antibody-Ber-EP4 complex).

[0081] A subsequent step 2 includes contacting the sample with detection reagents. The detection reagents may include labeling conjugates and chromogenic substrates or signaling conjugates as illustrated in steps 3a and 3b of FIG. 2. A further subsequent step 4 comprises contacting the sample with an enzyme inhibition composition. A dashed line indicates that the process of steps 1 through 4 may be repeated one or more times to provide for the sequential multiplex detection of targets within the tissue sample. The method also comprises a step 5 of illuminating sample with light and detecting the targets at step 6. While FIGs. 1 and 2 illustrate that all of the targets are detected simultaneously, the targets may be detected at any time during the multiplex method disclosed herein.

[0082] FIG. 3 further illustrates a multiplex detection assay according to the methods of the claimed disclosure. In this particular embodiment, the marker EP-Cam is detected using a chromogenic substrate that, one enacted upon by the enzyme (H P), provides a magenta color. Similarly, the marker calretinin is detected using a chromogenic substrate that, once enacted upon by the enzyme (AP), produces a blue color. Likewise, the marker CD 163 is detected using a chromogenic substrate that, once enacted upon by the enzyme (AP), produces a yellow color. In an exemplary embodiment, a primary antibody is first added to detect EP-Cam, followed by the introduction of chromogenic reagents (e.g. OmniMap xMs HRP and TYR-TAMRA) to provide the magenta color. Next, a primary antibody to detect calretinin is added second, followed by the introduction of chromogenic reagents (e.g. UltraMap xRb AP and QM-AP-Cy5) to provide the blue color. Finally, a primary antibody to detect CD 163 is added third, followed by the introduction of chromogenic reagents (UltraMap xMs AP and QM-AP-Dabsyl) to provide the yellow color. [0083] In some embodiments, the method comprises the steps of (i) contacting a biological sample with a first detection probe (e.g. one of an antibody specific for EP-Cam, calretinin, or CD 163) to form a first antibody-marker complex; (ii) contacting the biological sample with a first labeling conjugate wherein the first labeling conjugate comprises a first enzyme (where the first labeling conjugate is an anti-species antibody that specifically binds to the first detection probe and is configured to label the target with an enzyme); (iii) contacting the biological sample with a first signaling conjugate comprising a first latent reactive moiety and a first chromogenic moiety; (iv) inactivating the first enzyme, such as by contacting the sample with a first enzyme inactivation composition to substantially inactivate or completely inactivate the first enzyme contained in the biological sample.

[0084] After the first enzyme is inactivated, the method further comprises the steps of (v) contacting a biological sample with a second detection probe (e.g. another of an antibody specific for EP-Cam, calretinin, or CD 163) to form a second antibody-marker complex; (vi) contacting the biological sample with a second labeling conjugate wherein the second labeling conjugate comprises a second enzyme (where the second labeling conjugate is an anti-species antibody that specifically binds to the second detection probe and is configured to label the target with an enzyme); (vii) contacting the biological sample with a second signaling conjugate comprising a second latent reactive moiety and a second chromogenic moiety; (viii) inactivating the second second enzyme, such as by contacting the sample with a first enzyme inactivation composition to substantially inactivate or completely inactivate the first enzyme contained in the biological sample.

[0085] After the second enzyme is inactivated, the method further comprises the steps of (ix) contacting a biological sample with a third detection probe (e.g. another of an antibody specific for Ber-EP4, calretinin, or CD163) to form a third antibody-marker complex; (x) contacting the biological sample with a third labeling conjugate wherein the third labeling conjugate comprises a third enzyme (where the third labeling conjugate is an anti-species antibody that specifically binds to the third detection probe and is configured to label the target with an enzyme); (xi) contacting the biological sample with a third signaling conjugate comprising a third latent reactive moiety and a third chromogenic moiety; and (xii) detecting signals from the first, second, and third chromogenic moieties, wherein each of the first, second, and third chromogenic moieties are each different, and wherein the patient is diagnosed or differentially diagnosed with metastatic carcinoma if malignant cells comprising the Ber-EP4, calretinin, and CD163 markers are detected. In some embodiments, the third enzyme is optionally inactivated. [0086] The multiplex detection assays of the present disclosure may be simultaneous of sequential. For example, each of the detection probes may be added simultaneously or sequentially, but before any labeling conjugate is added. As another example, three detection probes may be sequentially applied at step 1, prior to introduction of any detection reagents.

[0087] In general, detection reagents utilized in the methods described herein are different in at least two of their components. First, each of the secondary antibodies employed are different and specific to each of the primary antibodies used. Second, each of the detection reagents utilized have different chromogenic moieties. The enzymes utilized may be the same or different. In some embodiments, the first, second, and third enzymes are the same or different. For example, the first and second enzymes may be the same while the third enzyme is different. In some embodiments, the first, second, and third chromogenic moieties are different. In some embodiments, the first, second, and third chromogenic moieties each comprise a peak wavelength for detection, and wherein each of the peak wavelengths for the first, second, and third chromogenic moieties are different. In some embodiments, the different peak wavelengths differ by at least about 70nm.

[0088] Conditions suitable for introducing the signaling conjugates or chromogenic substrates with the biological sample are used, and typically include providing a reaction buffer or solution that comprises a peroxide (e.g., hydrogen peroxide), and that has a salt concentration and pH suitable for allowing or facilitating the enzyme to perform its desired function. In general, this step of the method is performed at temperatures ranging from about 35° C. to about 40° C, although the skilled artisan will be able to select appropriate temperature ranges appropriate for the enzymes and signalizing conjugates selected. For example, it is believed that these conditions allow the enzyme and peroxide to react and promote radical formation on the latent reactive moiety of the signaling conjugate. The latent reactive moiety, and therefore the signaling conjugate as a whole, will deposit covalently on the biological sample, particularly at one or more tyrosine residues proximal to the immobilized enzyme conjugate, tyrosine residues of the enzyme portion of the enzyme conjugate, and/or tyrosine residues of the antibody portion of the enzyme conjugate. The biological sample is then illuminated with light and the target may be detected through absorbance of the light produced by the chromogenic moiety of the signaling conjugate.

[0089] In the context of a multiplex assay where multiple chromogenic reagents are detected sequentially, it is desirable to inactivate any reagent or endogenous enzymes between successive chromogenic detection steps. As a result, it is believed that enzymes present in any one chromogenic detection step will not interfere with those in a later chromogenic detections step. This in turn is believed to improve upon the visualization and detection of the different chromogens used in the multiplex assay. Any enzyme inactivation composition known in the art may be used for this purpose. In some embodiments, an enzyme inactivation composition is applied to inactivate the reagent or endogenous enzymes after each detection step. Exemplary enzyme inactivation compositions are disclosed in co-pending application US 62/159,297, the disclosure of which is incorporated by reference herein in its entirety.

[0090] In some embodiments, a denaturation step prevents the enzyme used in a first set of chromogenic detection reagents from acting on a second chromogenic substrate. In some embodiments, the denaturant is a substance that denatures the enzyme in the first chromogenic detection reagent set. In some embodiments, the denaturant is, for example, formamide, an alkyl- substituted amide, urea or a urea-based denaturant, thiourea, guanidine hydrochloride, or derivatives thereof. Examples of alky 1- substituted amides include, but are not limited to, N-propylformamide, N- butylformamide, N-isobutylformamide, and Ν,Ν-dipropylaformamide. In some embodiments, the denaturant is provided in a buffer. For example, formamide may be provided in a hybridization buffer comprising 20 mM dextran sulfate (50-57% % formamide (UltraPure formamide stock), 2 SSC (20xSSC stock containing 0.3 M citrate and 3M NaCl), 2.5mM EDTA (0.5M EDTA stock), 5 mM Tris, pH 7.4 (1 mM Tris, pH 7.4 stock), 0.05% Brij-35 (10% stock containing polyoxyethylene (23) lauryl ether), pH 7.4. In some embodiments, the sample is treated with the denaturant for a period of time and under conditions sufficient to denature the first target probe detection enzyme, for example alkaline phosphatase. In some embodiments, the sample is treated with the denaturant for about 15 to about 30 minutes, preferably about 20 to 24 minutes at about 37° C. In some embodiments, the sample is treated with the denaturant for a period of time and under conditions sufficient to denature the target enzyme while preserving hybridization of the second nucleic acid probe to the target.

[0091] The specimen processing apparatus can be an automated apparatus, such as the

BENCHMARK XT instrument and SYMPHONY instrument sold by Ventana Medical Systems, Inc. Ventana Medical Systems, Inc. is the assignee of a number of United States patents disclosing systems and methods for performing automated analyses, including U.S. Pat. Nos. 5,650,327, 5,654,200, 6,296,809, 6,352,861, 6,827,901 and 6,943,029, and U.S. Published Patent Application Nos. 20030211630 and 20040052685, each of which is incorporated herein by reference in its entirety. Alternatively, specimens can be manually processed. [0092] In some embodiments if the specimen is a sample embedded in paraffin, the sample can be deparaffinized using appropriate deparaffinizing fluid(s). After a waste remover removes the deparaffinizing fluid(s), any number of substances can be successively applied to the specimen. The substances can be for pretreatment (e.g., protein-crosslinking, expose nucleic acids, etc.), denaturation, hybridization, washing (e.g., stringency wash), detection (e.g., link a visual or marker molecule to a probe), amplifying (e.g., amplifying proteins, genes, etc.), counterstaining, coverslipping, or the like.

[0093] After the specimens are processed, a user can transport specimen-bearing slides to the imaging apparatus. The imaging apparatus used here is a brightfield imager slide scanner. One brightfield imager is the iScan Coreo™ brightfield scanner sold by Ventana Medical Systems, Inc. In automated embodiments, the imaging apparatus is a digital pathology device as disclosed in International Patent Application No.: PCT/US2010/002772 (Patent Publication No.: WO/2011/049608) entitled IMAGING SYSTEM AND TECHNIQUES or disclosed in U.S. Patent Application Publication No. 2014/0178169, filed on February 3, 2014, entitled IMAGING SYSTEMS, CASSETTES, AND METHODS OF USING THE SAME. International Patent Application No. PCT/US2010/002772 and U.S. Patent Application Publication No. 2014/0178169 are incorporated by reference in their entities. In other embodiments, the imaging apparatus includes a digital camera coupled to a microscope.

[0094] Detection Kits

[0095] Also disclosed are kits for detecting metastatic carcinoma in a tissue sample comprising a first primary antibody specific to an epithelial marker of non-mesothelial origin (e.g. Ber-EP4); a second primary antibody specific to mesothelial cells (e.g. calretinin); and a third primary antibody specific to macrophages (e.g. CD163). In some embodiments, the kit further comprises a first labeling conjugate specific to the first primary antibody; a second labeling conjugate specific to the second primary antibody; and a third labeling conjugate specific to the third primary antibody. In some embodiments, the kit further comprises first, second, and third chromogenic substrates or signaling conjugates, wherein each of the first, second, and third signaling conjugates are different. In some embodiments, the kit further comprises one or more enzyme inactivation compositions specific to the enzymes of the labeling conjugates. In some embodiments, the kits include instructions for the multiplex detection of markers indicating of metastatic carcinoma. In some embodiments, the kits include instructions for the differential diagnosis of metastatic carcinoma. In other embodiments, the kits include instructions for the administration of appropriate active pharmaceutical ingredients based on the diagnosis reached. The kits may include other reagents, e.g. buffers, primary stains, blocking agents, microscope coverslips, etc.

[0096] Example 1— Tissue Models and Clinical Specimens

[0097] Appendix specimens, shown in the literature to contain cell types expressing all three targets in Table 1, were provided by the Tissue Sample Management group at Ventana Medical Systems, Inc. Cut slides from three test effusion specimens were also provided for assay development. Twenty-four formalin fixed paraffin embedded effusions blocks (9 positive pleural effusions, 10 positive peritoneal effusions and 5 negative cases), obtained from Thomas Jefferson University, Department of Pathology Anatomy and Cell Biology laboratory, were stained and evaluated by four pathologists.

[0098] Example 2— Procedure for Automated Immunohistochemistry

[0099] Brightfield DAB-based immunohistochemistry (IHC) was performed on 4μηι specimen sections using a Ventana Medical Systems, Inc. Benchmark XT platform (Ventana Medical

Systems, Inc, Tucson, AZ). DAB detection of each antibody listed in Table 1 was performed using the ultraView Universal DAB Detection Kit (VMSI Cat# 760-500) according to the manufacturer's instructions.

Table 1: Antibodies utilized in the assay.

[00100] All detections were performed after automated deparaffinization in EZPrep buffer

(VMSI Cat# 950-102) and cell conditioning of specimens in Cell Conditioning 1 (CC1) buffer (VMSI Cat#950-124) for 32 minutes. Chromogenic multiplexed detection was accomplished as described in Tables 4 and 5 (appended herein), in the following order: Ep-CAM, then Calretinin, then CD 163. Heat denaturation of enzymes and primary antibody melt-offs were performed between Ep- CAM and Calretinin, and subsequently between Calretinin and CD 163 at about 90°C for about 8 minutes to prevent cross-reactivity. The rationale for the antibody order was as follows: DAB-based detection according to manufacturer's instruction for Ep-CAM and Calretinin is to use Mild CC1 (about 32 minutes). A standard CC1 cell conditioning (about 64 minutes) is recommended for CD 163. Considering that additional cell conditioning is to occur during the heat denaturation steps (about 90°C for about 8 minutes) and that additional cell conditioning is suggested for CD 163, we decided to detect CD 163 last. A list of reagents used is shown in Table 2.

Table 2: Listing of reagents used in the assay.

[00101] Example 3— Results

[00102] Appendix specimens stained positive for all three antibodies by DAB according to the manufacturer's instructions. All effusion specimens were also processed for single antibody DAB detection as done for the appendix specimens for comparison to chromogenic multiplexed stains (Figures 4A - 4F). A contrast centric color selection and feasibility protocol (Table 4) was used to evaluate multiplexing where Ep-CAM was detected using Cy5, Calretinin was detected using Dabsyl and CD 163 was detected using Rhodamine 110 to aid in the visual differentiation of cell types. In addition, Ep-CAM-Cy5 and Calretinin-Dabsyl detection were selected in the event that biological co- expression was observed to generate a third, distinct color (green). Examples of an appendix and effusion specimen with this color scheme are shown in Figures 5A and 5B (appendix) and Figures 6A and 6B (effusion). It was notable that detection of Ep-CAM was not sensitive compared to DAB. At this stage, we wanted to determine if we could enhance Ep-CAM detection by altering the order of antibody detections. Accordingly, the order of detection was switched as follows: (a) Calretinin, (b) CD163, and (c) Ep-CAM.

[00103] It was noted here that the order of antibodies was crucial to a successful multiplexing, as evidenced by the chromogenic overlap of Dabsyl and Cy5 (Figures 7A and 7B). As such, it was decided to continue with the original order of antibody detection and instead switch the detection of Ep-CAM by using TAMRA, a more sensitive chromogen. This color switch allowed greater detection sensitivity over Cy5. In addition, it was noted that rare overlaps in Ep-CAM and Calretinin co-expressions could not justify a color selection bound by potential overlap (yellow + blue = green). Therefore, CD163 detection was switched to Cy5 (Figure 7A, 7B, 8A, and 8B).

[00104] After further evaluation, it was determined that a color switch of the Calretinin and

CD 163 detections would be contrast beneficial. Therefore, the following detection scheme using the protocol (Table 5) was employed: Ep-CAM-TAMRA, Calretinin-Cy5 and CD163-Dabsyl (Figures 10A - 10D).

[00105] This detection scheme was used to stain 24 formalin fixed paraffin embedded effusions specimens (9 positive pleural effusions, 10 positive peritoneal effusions and 5 negative cases) obtained from Thomas Jefferson University, Department of Pathology Anatomy and Cell Biology Laboratory. The multiplex panel was evaluated by four pathologists. These were compared to single DAB stains for each marker. Seventeen of nineteen cases of metastatic carcinoma stained strongly positive for Ber-EP4 (magenta membranous stain) and 2 of 19 showed focal weak positivity (Table 3). In all 24 cases the mesothelial cells stained strongly with calretinin (blue cytoplasmic/nuclear stain, labeled "a") and the macrophages with CD 163 (yellow cytoplasmic/membranous stain, labeled "c") (Figures 11A and 11B). The colors were distinct, easily differentiated and recognizable. There was no discrepancy between the expression of the individual antibodies and the expression of the same antibodies in the multiplex setting. This novel multiplex offers the capability to unequivocally diagnose metastatic carcinoma even when the tumor content is very low using a single four-micron section. This multiplex panel also allows the conservation of precious specimen for additional molecular testing.

STATEMENT OF INDUSTRIAL APPLICABILITY

[00106] The present disclosure has industrial applicability in the field of diagnostics.

[00107] All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.

[00108] Although the disclosure herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present disclosure. It is therefore understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present disclosure as defined by the appended claims.

Claims

1. A method of detecting metastatic carcinoma in a patient by analyzing a single tissue sample for the presence or absence of malignant cells by measuring the presence or absence of EP- Cam, calretinin, and CD 163 markers, wherein the patient is diagnosed with metastatic carcinoma if the malignant cells are detected.

2. The method of claim 1, wherein the method comprises the steps of applying a first primary antibody specific for one of the EP-Cam, calretinin, or CD 163 markers; applying first detection reagents to detect the first primary antibody; applying a second primary antibody specific for another one of the EP-Cam, calretinin, or CD 163 markers; applying second detection reagents to detect the second primary antibody; applying a third primary antibody specific for a third one of the EP-Cam, calretinin, or CD 163 markers; and applying third detection reagents to detect the third primary antibody.

3. The method of claim 3, wherein each of the first, second, and third detection reagents comprise (i) a secondary antibody specific to each of the primary antibodies, wherein the secondary antibody is conjugated to an enzyme such that the secondary antibody labels the marker with an enzyme; and (ii) a chromogenic substrate; and wherein each of the first, second, and third detection reagents comprise different components.

4. The method of claim 3, wherein a first chromogenic substrate comprises one of Dabsyl, Cy5, or TAMRA; a second chromogenic substrate comprises another one of Dabsyl, Cy5, or TAMRA; and a third chromogenic substrate comprises a third one of Dabsyl, Cy5, or TAMRA.

5. The method of claim 4, wherein the first primary antibody is specific for EP-Cam; the second primary antibody is specific for calretinin; and the third primary antibody is specific for CD163.

6. The method of claim 5, wherein the first chromogenic reagent comprises TAMRA; the second chromogenic reagent comprises Cy5; and the third chromogenic reagent comprises Dabsyl.

7. The method of claim 3, wherein the chromogenic substrates are selected such that a peak wavelength for each is separated by at least about 50nm.

8. The method of claim 2, wherein the primary antibodies are detected simultaneously.

9. The method of claim 1, wherein the tissue sample is a serous effusion or a pleural effusion.

10. The method of claim 1, wherein a size of the tissue sample ranges from about 3 microns to about 6 microns.

11. The method of claim 1 , wherein the metastatic carcinoma is selected from the group consisting of adenocarcinomas, basal cell carcinomas, squamous cell carcinomas, and transitional cell carcinomas.

12. A method for the diagnosis metastatic cancer in a human subject, wherein the metastatic cancer is characterized by the presence or absence of EP-Cam, calretinin, and CD 163 markers comprising:

(a) applying a first primary antibody specific for one of the EP-Cam, calretinin, or CD 163 markers to a tissue sample, wherein a presence of one of the markers creates a first antibody-marker complex;

(b) applying first detection reagents that detect the first antibody-marker complex;

(c) applying a second primary antibody specific for another of the EP-Cam, calretinin, or CD 163 markers to the tissue sample, wherein a presence of another one of the markers creates a second antibody-marker complex;

(d) applying second detection reagents that detect the second antibody-marker complex;

(e) applying a third primary antibody specific for a third one of the EP-Cam, calretinin, or CD 163 markers to the tissue sample, wherein a presence of a third one of the markers creates a third antibody-marker complex;

(f) applying third detection reagents that detect the third antibody-marker complex; and

(g) diagnosing metastatic cancer where the first, second, and third detection reagents are detected.

13. The method of claim 12, wherein each of the first, second, and third detection reagents comprise (i) a secondary antibody specific to each of the primary antibodies, wherein the secondary antibody is conjugated to an enzyme such that the secondary antibody labels the marker with an enzyme; and (ii) a chromogenic substrate; and wherein each of the first, second, and third detection reagents comprise different components.

14. The method of claim 13, wherein a first chromogenic substrate comprises one of Dabsyl, Cy5, or TAMRA; a second chromogenic substrate comprises another one of Dabsyl, Cy5, or TAMRA; and a third chromogenic substrate comprises a third one of Dabsyl, Cy5, or TAMRA.

15. The method of claim 14, wherein the first primary antibody is specific for EP-Cam; the second primary antibody is specific for calretinin; and the third primary antibody is specific for CD 163.

16. The method of claim 15, wherein the first chromogenic reagent comprises TAMRA; the second chromogenic reagent comprises Cy5; and the third chromogenic reagent comprises Dabsyl.

17. The method of claim 13, wherein the chromogenic substrates are selected such that a peak wavelength for each is separated by at least about 70nm.

18. The method of claim 12, wherein the primary antibodies are detected simultaneously.

19. The method of claim 12, wherein the tissue sample is a serous effusion.

20. The method of claim 12, wherein a size of the tissue sample ranges from about 3 microns to about 6 microns.

21. The method of claim 12, wherein the metastatic carcinoma is selected from the group consisting of adenocarcinomas, basal cell carcinomas, squamous cell carcinomas, and transitional cell carcinomas.

22. A method for diagnosing metastatic cancer in a human subject, wherein the metastatic cancer is characterized by the presence or absence of EP-Cam, calretinin, and CD 163 markers comprising, in any order, at least the steps of:

(a) applying a first primary antibody specific for EP-Cam to a tissue sample, wherein a presence of EP-Cam creates an antibody-EP-Cam marker complex;

(b) applying first detection reagents that detect the antibody-Ber-EP4 complex;

(c) applying a second primary antibody specific for calretinin to the tissue sample, wherein a presence of calretinin creates an antibody-calretinin marker complex;

(d) applying second detection reagents that detect the antibody-calretinin marker complex;

(e) applying a third primary antibody specific for CD 163 to the tissue sample, wherein a presence of CD 163 creates an antibody-CD 163 marker complex;

(f) applying third detection reagents that detect the antibody-CD 163 marker complex; and

23. The method of claim 22, wherein each of the first, second, and third detection reagents comprise (i) a secondary antibody specific to each of the primary antibodies, wherein the secondary antibody is conjugated to an enzyme such that the secondary antibody labels the marker with an enzyme; and (ii) a chromogenic substrate; and wherein each of the first, second, and third detection reagents comprise different components.

24. The method of claim 23, wherein a first chromogenic substrate comprises one of Dabsyl, Cy5, or TAMRA; a second chromogenic substrate comprises another one of Dabsyl, Cy5, or TAMRA; and a third chromogenic substrate comprises a third one of Dabsyl, Cy5, or TAMRA.

25. The method of claim 24, wherein the first chromogenic reagent comprises TAMRA; the second chromogenic reagent comprises Cy5; and the third chromogenic reagent comprises Dabsyl.

26. A method of detecting metastatic carcinoma in a patient by analyzing a single tissue sample for the presence or absence of malignant cells, comprising the steps of:

(a) contacting a biological sample with a first detection probe, the first detection probe comprising a first primary antibody selected from one of an antibody specific for an epithelial marker target of non-mesothelial origin, an antibody specific for mesothelial cells, or an antibody specific for macrophages;

(b) contacting the biological sample with first chromogenic detection reagents comprising a first enzyme;

(c) inactivating the first enzyme;

(d) contacting the biological sample with a second detection probe, the second detection probe comprising a second primary antibody selected from another of an antibody specific for an epithelial marker target of non-mesothelial origin, an antibody specific for mesothelial cells, or an antibody specific for macrophages;

(e) contacting the biological sample with second chromogenic detection reagents comprising a second enzyme;

(f) inactivating the second enzyme;

(g) contacting the biological sample with a third detection probe, the third detection probe comprising a third primary antibody selected from a third one of an antibody specific for an epithelial marker target of non-mesothelial origin, an antibody specific for mesothelial cells, or an antibody specific for macrophages;

(h) contacting the biological sample with third chromogenic detection reagents comprising a third enzyme; (i) detecting signals from the first, second, and third detection reagents, wherein each of the first, second, and third detection reagents comprise a different chromogenic moiety, and wherein the patient is diagnosed with metastatic carcinoma if the malignant cells are detected.

27. The method of claim 26, wherein the epithelial target marker target is EP-Cam.

28. The method of claim 26, wherein the marker for mesothelial cells is calretinin.

29. The method of claim 26, wherein the marker for macrophages is CD163.

30. The method of claim 26, wherein each of the first, second, and third detection reagents comprise a chromogenic substrate; and wherein each of the first, second, and third detection reagents comprise different components.

31. The method of claim 30, wherein a first chromogenic substrate comprises one of Dabsyl, Cy5, or TAMRA; a second chromogenic substrate comprises another one of Dabsyl, Cy5, or TAMRA; and a third chromogenic substrate comprises a third one of Dabsyl, Cy5, or TAMRA.

32. The method of claim 31 , wherein the first primary antibody is specific for EP-Cam; the second primary antibody is specific for calretinin; and the third primary antibody is specific for CD 163.

33. The method of claim 32, wherein the first chromogenic reagent comprises TAMRA; the second chromogenic reagent comprises Cy5; and the third chromogenic reagent comprises Dabsyl.

34. The method of claim 30, wherein the chromogenic substrates are selected such that a peak wavelength for each is separated by at least about 70nm.

35. The method of claim 26, wherein the antibodies are detected simultaneously.

36. The method of claim 26, wherein the antibodies are detected sequentially.

37. The method of claim 26, wherein the tissue sample is a serous effusion.

38. The method of claim 26, wherein a size of the tissue sample ranges from about 3 microns to about 6 microns.

39. The method of claim 26, wherein the metastatic carcinoma is selected from the group consisting of adenocarcinomas, basal cell carcinomas, squamous cell carcinomas, and transitional cell carcinomas.

40. A method of detecting metastatic carcinoma in a patient by analyzing a single tissue sample for the presence or absence of malignant cells, comprising the steps of: (a) contacting a biological sample with a first detection probe, the first detection probe comprising a first primary antibody selected from one of an antibody specific for an epithelial marker target of non-mesothelial origin, an antibody specific for mesothelial cells, or an antibody specific for macrophages;

(b) contacting the biological sample with a first labeling conjugate that specifically binds to the first detection probe, wherein the first labeling conjugate comprises a first enzyme;

(c) contacting the biological sample with a first signaling conjugate comprising a first latent reactive moiety and a first chromogenic moiety;

(d) inactivating the first enzyme;

(e) contacting a biological sample with a second detection probe, the second detection probe comprising a second primary antibody selected from another one of an antibody specific for an epithelial marker target of non-mesothelial origin, an antibody specific for mesothelial cells, or an antibody specific for macrophages;

(f) contacting the biological sample with a second labeling conjugate that specifically binds to the second detection probe, wherein the second labeling conjugate comprises a second enzyme;

(g) contacting the biological sample with a second signaling conjugate comprising a second latent reactive moiety and a second chromogenic moiety;

(h) inactivating the second enzyme;

(i) contacting a biological sample with a third detection probe, the third detection probe comprising a third primary antibody selected from a third one of an antibody specific for an epithelial marker target of non-mesothelial origin, an antibody specific for mesothelial cells, or an antibody specific for macrophages;

(j) contacting the biological sample with a third labeling conjugate that specifically binds to the third detection probe, wherein the third labeling conjugate comprises a third enzyme;

(k) contacting the biological sample with a third signaling conjugate comprising a third latent reactive moiety and a third chromogenic moiety;

(1) inactivating the third enzyme;

(m) detecting signals from the first, second, and third chromogenic moieties, wherein each of the first, second, and third chromogenic moieties are each different, and wherein the patient is diagnosed with metastatic carcinoma if the malignant cells are detected.

41. The method of claim 40, wherein a first chromogenic moiety comprises one of Dabsyl, Cy5, or TAMRA; a second chromogenic moiety comprises another one of Dabsyl, Cy5, or TAMRA; and a third chromogenic moiety comprises a third one of Dabsyl, Cy5, or TAMRA.

42. The method of claim 41 , wherein the first primary antibody is specific for EP-Cam; the second primary antibody is specific for calretinin; and the third primary antibody is specific for CD 163.

43. The method of claim 42, wherein the first chromogenic reagent comprises TAMRA; the second chromogenic reagent comprises Cy5; and the third chromogenic reagent comprises Dabsyl.

44. A method of detecting metastatic carcinoma in a patient by analyzing a single tissue sample for the presence or absence of EP-Cam, calretinin, and CD 163, wherein the patient is diagnosed with metastatic carcinoma if EP-Cam, calretinin, and CD163 are detected.

45. A kit for detecting metastatic carcinoma comprising a first primary antibody specific to EP- Cam; a second primary antibody specific to calretinin; and a third primary antibody specific to CD163.

46. The kit for detecting metastatic carcinoma of claim 45, wherein the kit further comprises a first labeling conjugate specific to the first primary antibody; a second labeling conjugate specific to the second primary antibody; and a third labeling conjugate specific to the third primary antibody.

47. The kit for detecting metastatic carcinoma of claim 46, wherein the kit further comprises first, second, and third chromogenic substrates.

48. The kit for detecting metastatic carcinoma of claim 47, wherein the kit further comprises one or more enzyme inactivation compositions.