WO2014139979A1 - Quantum dot in situ hybridization - Google Patents

Abstract

An improved quantum dot in situ hybridization method for reliably and reproducibly detecting targets is disclosed. Certain disclosed embodiments concern an in situ detection method utilizing a modified paraffin removal process, as well as a pretreatment step prior to the addition of probes. Some embodiments use a protease and either an acidic buffer treatment, a basic buffer treatment, or both. The method also may comprise using a chelating agent in process solutions to remove metal/metal ion contaminants. After pretreatment, at least one probe comprising a hapten conjugated to a specific binding moiety is added to the sample. After hybridizing the probe to the target a second conjugate comprising an anti-hapten antibody and quantum dot is applied to the sample. After washing, the sample is exposed to light to excite the quantum dot(s). Fluorescence signals from the quantum dot(s) are then detected and identified by obtaining a series of monochrome images, such as by using filters to selectively detect each quantum dot signal. These images are then colorized and stacked to produce a final combined image. Disclosed embodiments provide a robust and reliable QD ISH process having reduced slide background to facilitate analysis.

Description

QUANTUM DOT IN SITU HYBRIDIZATION CROSS REFERENCE TO RELATED APPLICATION

This application is related to subject matter disclosed in U.S. provisional application No. 61/777,613, filed on March 12, 2013, entitled Quantum Dot In Situ Hybridization Using Modified Tissue Conditioning, which is incorporated herein by reference in its entirety.

FIELD

The present invention concerns an in situ hybridization (ISH) method, and more particularly concerns a quantum dot (QD) ISH assay for detecting one or more targets in a sample.

BACKGROUND

Fluorescence in situ hybridization (FISH) techniques provide a direct visualization of the spatial location of specific nucleic acid sequences at a particular cellular site in a tissue section. Such morphological and topological information has been invaluable for clinical evaluation of human generic aberrances, gene expression levels, viral infections, etc. Although conventional FISH uses various organic fluorophores that allow

simultaneous visualization of multiple targets in distinct colors, these conventional FISH techniques face several limitations, including limited number of available fluorochromes, broad emission spectra, and rapid photo-bleaching.

Quantum dots (QDs) are semiconductor nanocrystals that are useful as novel fluorophores. They have narrow emission spectra that facilitate detection of multiple cellular targets simultaneously. They are ultra-bright fluorophores that are not as prone to photo-bleaching as organic fluorophores, which is advantageous for generating enhanced signal-to-noise ratio staining. Given these remarkable optical properties, quantum dots have recently emerged as potentially ideal fluorophores for use in FISH.

For example, quantum dots have been used to detect nucleic acids both indirectly (i.e. biotin-labeled probes with quantum dots-conjugated to streptavidin) and directly (i.e. synthesis of quantum dot-labeled short nucleotide probes). Single quantum dot in situ hybridization assays have been performed on human metaphase, mouse, plant chromosomes, E.coli., Epstein-Barr virus, and human papilloma virus. Moreover, biotinylated Her2 DNA probes with streptavidin-conjugated QD605 were applied to detect low copy HER2 gene in human lymphocytes and HER2 gene-amplification in SK-BR-3 breast cancer cells. Gene amplification on lung cancer specimens has been observed with the application of quantum dots conjugated to genomic DNA. Duplex quantum dot FISH techniques have been used to detect centromere-associated satellite sequences on cultured cell lines with oligonucleotides that directly attach QD592 and 655. Furthermore, several triplex quantum dot in situ hybridization assays have been developed as quantitative tools to measure gene expression profile. Chan et al. (Nucleic Acids Res, 33 (2005), p. el61) reported a multiplex cellular detection of mRNAs in mouse tissue. Two oligonucleotide probes were labeled with quantum dot fluorophores QD565 and 605, and two other probes labeled with two organic fluorophores Alexa^® 488 and Alexa^® 568. The multi-spectral simultaneous detection of up to four different mRNA targets in the same cells was achieved with confocal microscopy. Byers et al. (J of Mol Diag, 9 (2007), pp. 20-29) presented a triplex quantum dot in situ hybridization and immunohistochemistry (IHC) approach that enables simultaneous measurement of two gene expression profiles and one cell lineage marker on routinely processed tissue samples. Two oligonucleotide probes labeled with biotin or digoxigenin were detected with respective anti-hapten antibodies conjugated to QD525 or QD605, while the cell lineage marker was revealed by an antibody conjugated with QD655. Tholouli et al. (Biochem Biophys Res Commun, 425

(2012), pp. 333-339) described a triplex quantum dot in situ hybridization assay using two sets of three oligonucleotide probes, each of which was conjugated to quantum dots 605, 655, and 705, respectively. This assay was used for 6 gene expression profiling in an acute myeloid leukemia tissue microarray.

ERG rearrangement and PTEN deletion are two of the most common genomic events in human prostate cancer. Overexpression of the ERG protein caused by ERG rearrangement has been frequently associated with more aggressive prostate cancers and a poorer prognosis. PTEN genomic deletion and absence of PTEN expression are associated with unfavorable clinical outcome measures. The use of such molecular biomarkers in routine clinical practice is presently limited by technical difficulties associated with multivariate in situ hybridization (e.g. detection of multiple targets simultaneously).

Unfortunately, these prior quantum dot in situ hybridization assays have also been accompanied by reliability and/or reproducibility issues. Even when identical assays have been run in parallel, the results have still been unreliable. Ioannou and Griffin, for example, concluded that indirect quantum dot experiments were successful only approximately 25-35% of the time. The intermittent success has partially been explained by steric hindrance and accessibility issues based on the large-size nanocrystals. Quantum dots are larger than organic fluorophores and some scientists have noted that while they obtained bright signals from decondensed, interphase nuclei, they did not achieve comparable results from highly coiled metaphase chromosomes. Apparently, the steric hindrance was too great in compact areas.

Disclosed embodiments of the present invention address the deleterious results associated with these prior quantum dot in situ hybridization methodologies. SUMMARY

Disclosed embodiments concern an improved quantum dot in situ hybridization method for detecting multiple targets. With this improved methodology, multiple targets can be reliably and reproducibly detected simultaneously.

Certain disclosed embodiments of the present invention concern an in situ detection method utilizing quantum dots and a pretreatment step prior to the addition of the probes. Some pretreatments comprise using a protease and either an acidic buffer treatment, a basic buffer treatment, or both. In certain working embodiments the acidic buffer was a citrate buffer and the basic buffer was a Tris buffer. The sample typically was heated with the protease at an effective temperature between about 25 °C and 50 °C, and held there for an effective period of time, typically about 10 minutes or more. In some particular embodiments the temperature was about 37 °C. The sample also may be heated with the basic buffer at an effective temperature of from about 70 °C to about 95 °C, and for an effective period of time of between about 30 minutes and 1 hour. In some working embodiments the sample was heated with the basic buffer at a temperature above 82 °C, and for a period of about 48 minutes or longer.

After pretreatment the probes are added to the sample. These probes comprise a hapten conjugated to a specific binding moiety designed to bind to a selected target. In some embodiments these specific binding moieties are nucleic acid probes. These probes can be targeted at DNA, including genomic DNA, and RNA, including mRNA. For example the probes can be for a HER2 quantum dot in situ hybridization assay in a breast tissue sample, an ALK break-apart quantum dot in situ hybridization assay in a lung tissue sample, a Kappa and/or Lambda quantum dot in situ hybridization assay in a

hematological sample or a human papilloma virus (HPV) quantum dot in situ hybridization assay in a cervical tissue sample. Other exemplary targets for nucleic acid probes include ERG3', ERG5', PTEN and CEN10.

A hapten is conjugated to each specific binding moiety. The haptens are typically either digoxigenin, 2,4-dinitrophenyl, biotin, or avidin, or are selected from azoles, nitroaryl compounds, benzofurazans, triterpenes, ureas, thioureas, rotenones, oxazoles, thiazoles, coumarins, cyclolignans, heterobiaryl compounds, azoaryl compounds or benzodiazepines. In certain working embodiments the haptens were digoxigenin, 2,4- dinitrophenyl, nitropyrazole and thiazole sulfonamide. The haptens can be either directly conjugated to the specific binding moiety, or there can be a linker between the hapten and the specific binding moiety. In some working embodiments the haptens were conjugated to the nucleic acid probes by nick translation, using a hapten-labeled dUTP or dCTP. Exemplary disclosed conjugates include ERG5'-DIG, ERG3'- DNP, PTEN- TS and

CEN10- NP.

After hybridizing the probes to the target the second conjugates are applied to the sample. These conjugates comprise anti-hapten antibodies and quantum dots. Each anti- hapten antibody is matched to a hapten on a probe conjugate. Exemplary anti-hapten antibodies include rat anti-DNP, mouse anti-DIG, mouse anti-TS and mouse anti-NP. The quantum dots are selected so that the signal of each dot is identifiable from the other signals using filters. In certain working embodiments the quantum dots have an emission fluorescence separated by at least 40 nm. Exemplary quantum dots include QD655, QD605, QD565 and QD525. The quantum dots can be either directly conjugated to the anti-hapten antibody, or can be conjugated through a linker. In some working embodiments the linker was 4-(N-maleimidomethyl)-cyclohexane- 1 -carboxylic acid N-hydroxysuccinimide ester (SMCC). Certain disclosed exemplary antibody-quantum dot conjugates include rat anti-DNP-QD655, mouse anti-DIG-QD565, mouse anti-TS- QD605 and mouse anti-NP-QD525.

The antibody-quantum dot conjugates are applied to the sample and allowed to incubate. The sample is then washed. Certain working embodiments include preincubation before adding QD-conjugates, co-incubation with QD-conjugates, and/or a wash with a solution comprising a chelating agent to remove any metal contaminants that may negatively impact the strength of the quantum dot signal. These metal contaminants can originate from a number of sources including, for example, trace metal impurities from the reagents used in the assay or buffer preparation or from process tissue specimens. In certain working embodiments the chelating agent was EDTA, or an analog thereof.

After the washing step, the sample is exposed to light. In some embodiments the excitation wavelength was in the range of from about 355 nm to about 405 nm.

Fluorescence emission signals from the quantum dots are then detected and identified.

This is achieved by obtaining a series of monochrome images using filters to selectively detect each quantum dot signal, then colorizing and stacking the images to produce a final combined image. The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying FIGs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and IB are flow charts illustrating certain steps for exemplary embodiments of a disclosed QD ISH assay, particularly an automated QD ISH assay.

FIGS. 2A - 2C and 3A - 3D provide guidelines for signal enumeration and cell classification of ERG gene status.

FIG. 4 is a representative image of a normal metaphase spread stained with ERG/PTEN 4-color QD ISH on a BENCHMARK^® ULTRA instrument.

FIGS. 5A - D are photographs showing a benign prostate sample stained with an exemplary embodiment of an ERG/PTEN 4-color QD ISH assay.

FIG. 6A - F is a photograph illustrating a prostate cancer sample (VMSI- 106- C21D) stained with an exemplary embodiment of an ERG/PTEN 4-color QD ISH assay.

FIGS. 7A - D are Pareto charts illustrating the contributions of various changes to QD 525 intensity, QD 565 intensity, QD 605 intensity and QD 655 intensity, using an exemplary embodiment of a QD ISH procedure, where A is a 92 minute pretreatment using CC1, B is a temperature increase to 85 °C during denaturing of target DNA and applied probes, and C is an increased incubation time for applying QD conjugates, with alpha = 0.05.

FIG. 8A - D provide QD signal intensity provided by an automated staining protocol on a first exemplary staining device after addition of a chelating agent (0.5 mM EDTA in this exemplary example) during the process of FIG. IB.

FIG. 9 provides fluorescent intensity graphs versus various chelating agent concentrations (e.g. EDTA; added at 500 μΜ, 100 μΜ, 20 μΜ, 4 μΜ 0.8 μΜ and 0.16 μΜ) used for the exemplary automated protocol of FIG. IB, accompanied by the addition of 0.1 μΜ, 1 μΜ and 10 μΜ Cu²⁺ ion, establishing that 20 μΜ EDTA is an effective concentration for QD ISH assays.

FIG. 10 is a photographic image illustrating dusting effects.

FIG. 1 1 is a photographic image illustrating spotting effects.

FIG. 12 is an interval plot of fluorescence intensity for QD605 illustrating application of disclosed embodiments of the present invention for Her2 analysis.

FIG. 13 is an interval plot of staining intensity for QD565 illustrating application of disclosed embodiments of the present invention for Chrl7 analysis. FIG. 14 is a photographic image illustrating Her2/QD605 (red) and Chrl7/QD565 (green) QD ISH results obtained using disclosed embodiments of the present invention.

FIG. 15 is a photographic image illustrating for 5pALK/QD605 (red) and

3pALK/QD565 (green) QD ISH results obtained using disclosed embodiments of the present invention.

FIG. 16 is an interval plot of signal intensity results for 5pALK/QD605 obtained using disclosed embodiments of the present invention for 5pALK analysis.

FIG. 17 is an interval plot of signal intensity results for 3pALK/QD565 obtained using disclosed embodiments of the present invention for 3pALK analysis.

FIG. 18A - C are photographs showing (A) H&E staining, (B)

HPV16/diaminobenzidine (DAB) staining, and (C) HPV16/QD605 QD ISH results obtained using disclosed embodiments of the present invention.

FIG. 19 is a photographic image illustrating HER2/QD605 (red) and

CHR17/QD565 (green) QD ISH results obtained using disclosed embodiments of the present invention.

FIG. 20 is a photographic image illustrating 3pALK/QD565 (green) and

5pALK/QD605 (red) QD ISH results obtained using disclosed embodiments of the present invention.

FIG. 21 is a photographic image illustrating Kappa mRNA/QD605 (red) QD ISH results obtained using disclosed embodiments of the present invention.

FIG. 22 is a photographic image illustrating Lambda mRNA/QD605 (red) QD ISH results obtained using disclosed embodiments of the present invention.

DETAILED DESCRIPTION

I. Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and

Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term "comprises" means "includes."

All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. All GenBank Accession numbers are herein incorporated by reference as they appeared in the database on June 8, 201 1. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative and not intended to be limiting.

In order to facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided:

Administration: To provide or give a subject an agent, for example, a composition that includes a monoclonal antibody that specifically binds HER2, by any effective route. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal (e.g., topical), intranasal, vaginal and inhalation routes.

Agent: Any substance or any combination of substances that is useful for achieving an end or result; for example, a substance or combination of substances useful for decreasing or decreasing a protein-protein interaction. In some embodiments, the agent is a therapeutic agent, such as a therapeutic agent for the treatment of cancer.

ALK: ALK is a gene that encodes for the Anaplastic lymphoma kinase (ALK), also known as ALK tyrosine kinase receptor or CD246 (cluster of differentiation 246). The ALK gene can be oncogenic in three ways - by forming a fusion gene with any of several other genes, by gaining additional gene copies, or with mutations of the actual DNA code for the gene itself. One example is the EML4-ALK fusion gene that is responsible for approximately 3-5% of non-small-cell lung cancer (NSCLC). The vast majority of these cases are adenocarcinomas. The standard test used to detect this gene in tumor samples is fluorescence in situ hybridization (FISH. ALK lung cancers are more common in light cigarette smokers or nonsmokers, but a significant number of patients with this disease are current or former cigarette smokers. Antibody: A polypeptide ligand including at least a light chain or heavy chain immunoglobulin variable region which specifically binds an epitope of an antigen or a fragment thereof. Antibodies include intact immunoglobulins and the variants of them well known in the art, such as Fab', F(ab)'2 fragments, single chain Fv proteins (scFv), and disulfide stabilized Fv proteins (dsFv). A scFv protein is a fusion protein in which a light chain variable region of an antibody and a heavy chain variable region of an antibody are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains. The term also includes genetically engineered forms such as chimeric antibodies (for example, humanized murine antibodies) and heteroconjugate antibodies (such as, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3^rd Ed., W.H. Freeman & Co., New York, 1997.

The antibodies disclosed herein specifically bind a defined target (or multiple targets, in the case of a bispecific antibody). Thus, an antibody that specifically binds to HER2 is an antibody that binds substantially to HER2, for example cells or tissue expressing

HER2. It is, of course, recognized that a certain degree of non-specific interaction may occur between an antibody and a non- target {e.g., a cell that does not express HER2).

Typically, specific binding results in a much stronger association between the antibody and protein or cells bearing the antigen than between the antibody and protein or cells lacking the antigen. Specific binding typically results in greater than a 2-fold increase4, such as greater than 5-fold, greater than 10-fold, or greater than 100-fold increase, in amount of bound antibody (per unit time) to a protein including the epitope or cell or tissue expressing the target epitope as compared to a protein or cell or tissue lacking this epitope. Specific binding to a protein under such conditions requires an antibody that is selected for its specificity for a particular protein. A variety of immunoassay formats are appropriate for selecting antibodies or other ligands specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York (1988), for a description of immunoassay formats and conditions that can be used to determine specific

immunoreactivity.

Biological sample: A biological specimen containing biological molecules, including genomic DNA, RNA (including mRNA and microRNA), nucleic acids, proteins, peptides, and/or combinations thereof. In some examples, the biological sample is obtained from a subject. In other examples, the biological sample is a cell culture, including a cell culture grown from a biological sample obtained from a subject. Biological samples include all clinical samples useful for detecting disease (e.g., cancer) in subjects, including, but not limited to, cells, tissues, and bodily fluids, such as blood, derivatives and fractions of blood (such as serum); as well as biopsied or surgically removed tissue, for example tissues that are unfixed, frozen, or fixed in formalin or paraffin. In a particular example, a biological sample is obtained from a subject having or suspected of having a tumor; for example, a subject having or suspected of having breast cancer, ovarian cancer, stomach cancer or uterine cancer. In some embodiments, the subject has or is suspected of having a carcinoma.

Breast cancer: A neoplastic tumor of breast tissue that is or has potential to be malignant. Approximately 30% of breast cancers exhibit overexpression of HER2;

overexpression of HER2 is associated with increased disease recurrence and worse prognosis. The most common type of breast cancer is breast carcinoma, such as ductal carcinoma. Ductal carcinoma in situ is a non-invasive neoplastic condition of the ducts. Lobular carcinoma is not an invasive disease but is an indicator that a carcinoma may develop. Infiltrating (malignant) carcinoma of the breast can be divided into stages (I, IIA,

IIB, IIIA, IIIB, and IV). See, for example, Bonadonna et al, (eds), Textbook of Breast Cancer: A clinical Guide the Therapy. 3^rd; London, Tayloy & Francis, 2006.

Buffers: Buffer solutions are commonly used to maintain correct pH levels for biological and chemical systems. Many of the exemplary embodiments disclosed herein include using a buffer solution. Representative buffering agents or salts that may be present in the buffer include, but are not limited to, Tris, Tricine, HEPES, MOPS, TAPS, Bicine, TAPSO, TES, PIPES, Cacodylate, SSC, MES, KC1, NaCl, potassium acetate, NH₄- acetate, potassium glutamate, NH₄C1, ammonium sulphate, MgC^, magnesium acetate and the like. One preferred buffer solution is phosphate buffered saline (PBS). Another preferred buffer solution is BirA reaction buffer (0.1 M KC1, 5.5 mM MgCl₂, 50 mM

Tris-HCl (pH = 8.0), 0.05% Brij-35, 0.1 mM dithiothreitol (DTT), 3 mM ATP, and 60 mM biotin). The amount of buffering agent will typically range from about 5 to 150 mM, usually from about 10 to 100 mM, and more usually from about 20 to 50 mM, where in certain preferred embodiments the buffering agent will be present in an amount sufficient to provide a pH ranging from about 6.0 to about 9.5, more typically a pH range of from about 6.5 to about 7.4 at room temperature. Other agents that may be present in the buffer medium include chelating agents, such as EDTA, EGTA and the like.

One particular example of a buffer is the Tris buffer. This buffer comprises water, Tris, EDTA disodium dehydrate, ProClin 950, Tween-20 and boric acid. Another particular example of a buffer is the citrate buffer, comprising water, sodium acetate trihydrate, sodium metabisulfite, glacial acetic acid, sodium citrate, citric acid, magnesium chloride, sodium dodecyl sulfate, ethylene glycol and ProClin 300.

Chelator: A chelator, chelating agent or sequestering agent is a chemical that forms soluble, complex molecules with certain metal ions, inactivating the ions so that they cannot normally react with other elements or ions. A chelator, chelating agent or sequestering agent can be used individually or in combination. A person of ordinary skill in the art will appreciate that any polydentate molecule that can bind to a metal ion may be used as a chelator. Thus, proteins, polysaccharides, and polynucleic acids are excellent polydentate ligands for many metal ions. Also molecules with porphyrin rings such as hemoglobin and chlorophyll may be used as a chelator. Exemplary chelators include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), dimercaprol (2,3-dimercapto- 1 -propanol), porphine, diethylenetriaminepentaacetic acid (DTP A), N,N- bis(carboxymethyl)glycine (NTA), D-penicillamine (DPA), meso-2,3-dimercaptosuccinic acid (DMSA), sodium 2,3-dimercaptopropane sulfonate (DMPS), deferoxamine (DFO), l,2-dimethyl-3-hydroxypyrid-4-one(Ll), tetraethylenetetraamine (TETA), nitrilotriacetic acid (NTA) and derivatives and analogs thereof.

Chemotherapeutic agent: Any chemical agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. For example,

chemotherapeutic agents are useful for the treatment of cancer, including breast cancer. In one embodiment, a chemotherapeutic agent is a radioactive compound. Another example includes tyrosine kinase inhibitors, such as lapatinib. In particular examples, such chemotherapeutic agents are administered in combination with a treatment that decreases or reduces homo- or heterodimerization of HER proteins (for example before, during or after administration of a therapeutically effective amount of one or more antibodies that specifically bind to HER2 or conjugate thereof). One of skill in the art can readily identify a chemotherapeutic agent of use (see for example, Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al, Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2^nd ed., © 2000 Churchill Livingstone, Inc; Baltzer, L., Berkery, R. (eds): Oncology Pocket Guide to Chemotherapy,

2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer, D.S., Knobf, M.F., Durivage, H.J. (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993; Chabner and Longo, Cancer Chemotherapy and Biotherapy: Principles and Practice (4th ed.). Philadelphia: Lippincott Williams & Wilkins, 2005; Skeel. Handbook of Cancer Chemotherapy (6th ed.). Lippincott Williams & Wilkins, 2003). Combination chemotherapy is the administration of more than one agent to treat cancer.

Chromogenic Staining: Chromogenic substrates have been used widely for immunohistochemistry for many years and for in situ hybridization more recently.

Chromogenic detection offers a simple and cost-effective detection method. Chromogenic substrates have traditionally functioned by precipitating when acted on by the appropriate enzyme. That is, the traditional chromogenic substance is converted from a soluble reagent into an insoluble, colored precipitate upon contacting the enzyme. The resulting colored precipitate requires no special equipment for processing or visualizing. There are several qualities that successful IHC or ISH chromogenic substrates share. First, the substance should precipitate to a colored substance, preferably with a very high molar absorptivity. The enzyme substrate should have high solubility and reagent stability, but the precipitated chromogen products should be very insoluble, preferably in both aqueous and alcohol solutions. Enzyme turnover rates should be very high so as to highly amplify the signal from a single enzyme in a short amount of time. Until now, a relatively small number of chromogenic substances have been identified that legitimately possess all of these qualities. Reference is made to U.S. Provisional Patent Application Nos. 61/616,330 and 61/710,607, which are incorporated herein by reference in their entirety for disclosure related to chromogenic staining.

Conjugate: Two or more molecules coupled together, for example, by a covalent bond or non-covalent interaction. The two components comprising the conjugate can be directly coupled or indirectly coupled using a linker. In one example, a conjugate comprises a specific binding moiety linked to a biotinylating enzyme, such as an antibody coupled to biotin ligase. Another example of a conjugate is a specific binding moiety coupled to a biotin ligase substrate, such as an antibody linked to BTS either directly or indirectly by a linker.

Conjugate(ing), join(ing), bond(ing) or link(ing): Coupling a first molecule to a second molecule. This includes, but is not limited to, covalently bonding one molecule to another molecule, non-covalently bonding one molecule to another {e.g. , electrostatically bonding) (see, for example, U.S. Patent No. 6,921,496), hydrogen bonding, van der Waals forces, and any and all combinations of such couplings.

Contacting: Placement in direct association, for example solid, liquid or gaseous forms.

Control: A sample or standard used for comparison with a test sample, such as a biological sample, e.g., a biological sample obtained from a patient (or plurality of patients) or a cell culture. In some embodiments, a cell culture that is not incubated with a test agent serves as a control for a cell culture that is incubated with a test agent. In some embodiments, the control is a sample obtained from a healthy patient (or plurality of patients) (also referred to herein as a "normal" control), such as a normal breast sample. In some embodiments, the control is a historical control or standard value (i.e. a previously tested control sample or group of samples that represent baseline or normal values). In some embodiments the control is a standard value representing the average value (or average range of values) obtained from a plurality of patient samples.

Coupled: Two or more molecules joined together, either directly or indirectly. A first atom or molecule can be directly coupled or indirectly coupled to a second atom or molecule. A secondary antibody is indirectly coupled to an antigen when it is bound to a primary antibody that is bound to the antigen.

Deconvolution: Where emission signals from the quantum dots are not resolvable by using filters alone, filters can be used in combination with deconvolution software. When using deconvolution software, an apparatus using a scanning method of detection collects luminescent data from the sample relative to a microscope objective by moving either the sample or the objective. The resulting luminescence is passed thought a single monochromator, a grating or a prism to resolve the colors spectrally. Alternatively, filters could be used to resolve the colors spectrally. The software takes the composite spectra and isolates the individual contribution of each quantum dot population. The spectra of each individual quantum dot population (neat solution) can be fitted to a Gaussian profile. Then the composite emission spectrum from each sample mixture of quantum dots can be fit using a superposition of Gaussian-like profiles. Software then recreates the scanned image, resulting in a single picture (file) containing all the colors of the quantum dot in the sample.

Decrease or Reduce: To reduce the quality, amount, or strength of something, such as a decrease of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

Detecting: To identify the existence, occurrence, presence, or fact of something. General methods of detecting are known to a person of ordinary skill in the art and may be supplemented with the protocols and reagents disclosed herein. For example, included herein are methods of detecting a first target proximal to a second target in a biological sample.

Diagnosis: The process of identifying a disease by its signs, symptoms and/or results of various tests. The conclusion reached through that process is also called "a diagnosis." Forms of testing commonly performed include blood tests, medical imaging, genetic analysis, urinalysis, biopsy and analysis of biological samples obtained from a subject.

Diagnostically significant amount: An increase or decrease of a measurable characteristic that is sufficient to allow one to distinguish one patient population from another (such as distinguishing a subject having increased ERG immunoreactivity due to ERG gene rearrangement).

Dusting: Refers to background in QD ISH images comprising: fine and very- small-size dots. Specific signals are usually preserved well when the dusting occurs. Dusting is most often manifested when using QD525 when there is excessive probe and/or

QD-conjugated antibody. Dusting can appear anywhere on the slide, not necessarily solely on the tissue section, and tends to stay within the paraffin boundary on the slide.

Effective amount: The amount of an agent (such as an ERG specific antibody or a conjugate including an ERG specific antibody, or an ERG inhibitor) that alone, or together with one or more additional agents, induces the desired response.

ERG: The ERG gene is found on Chromosome 21 between about 38675670, at the 3' end, and 38792267, at the 5' end. This gene encodes a member of the erythroblast transformation-specific (ETS) family of transcriptions factors. All members of this family are key regulators of embryonic development, cell proliferation, differentiation, angiogenesis, inflammation, and apoptosis. The protein encoded by this gene is mainly expressed in the nucleus. It contains an ETS DNA-binding domain and a PNT (pointed) domain implicated in the self-association of chimeric oncoproteins. This protein is required for platelet adhesion to the subendothelium, inducing vascular cell remodeling. It also regulates hematopoesis, and the differentiation and maturation of megakaryocyte cells. This gene is involved in chromosomal translocations, resulting in different fusion gene products, such as TMPSSR2-ERG and NDRG1-ERG in prostate cancer, EWS-ERG in Ewing's sarcoma and FUS-ERG in acute myeloid leukemia. Multiple alternatively spliced transcript variants encoding different isoforms have been identified. Recently, recurrent gene fusions involving the ETS family of transcription factors, ERG, ETV1, ETV4, and ETV5, fused to TMPRSS2 or other upstream partners, have been identified in the majority of prostate cancers. Among these aberrations, TMPRSS2-ERG fusion is the most prevalent, occurring in approximately 50% of localized prostate cancers and 30% of androgen independent metastatic cancers. As TMPRSS2 and ERG are located approximately 3 Mb apart on chromosome 21, the rearrangement between them occurs either through translocation or by an interstitial deletion. Emerging data suggests that TMPRSS2-ERG fusion plays an important role in carcinogenesis in vitro and in vivo. Clinically, ERG rearrangement has been observed in 10% to about 20% of HGPIN.

Mosquera et al. showed that of 143 high grade prostatic intraepithelial neoplasia (HGPIN) cases, 16% (23 of 143) were ERG rearrangement positive, and in all cases the paired prostate cancer was ERG rearrangement positive through the same mechanism. These observations suggest that ERG rearrangement may be an early event in prostate cancer.

Fluorescence in situ hybridization (FISH): FISH is a technique used to detect and localize the presence or absence of specific DNA and/or RNA sequences on chromosomes. FISH uses fluorescently labeled probes that bind to only those parts of the chromosome to which they show a high degree of sequence similarity under defined reaction conditions. FISH also can be used to detect particular mRNA sequences within tissue samples.

FFPE: Formalin fixed paraffin embedded sample.

Hapten: A hapten is a molecule, typically a small molecule that can combine specifically with an antibody, but typically is substantially incapable of being

immunogenic except in combination with a carrier molecule. Many haptens are known and frequently used for analytical procedures, such as di-nitrophenyl, biotin, digoxigenin, fluorescein, rhodamine, or combinations thereof. Other haptens have been specifically developed by Ventana Medical Systems, Inc., assignee of the present application, including haptens selected from oxazoles, pyrazoles, thiazoles, nitroaryls, benzofurans, triterpenes, ureas, thioureas, rotenoids, coumarins, cyclohgnans, and combinations thereof, with particular hapten examples of haptens including benzofurazan, nitrophenyl, 4-(2- hydroxyphenyl)-lH-benzo[b][l,4]diazepine-2(3H)-one, and 3-hydroxy-2- quinoxalinecarbamide. Plural different haptens may be coupled to a polymeric carrier. Moreover, compounds, such as haptens, can be coupled to another molecule using a linker, such as an NHS-PEG linker.

Human epidermal growth factor receptor (HER): A family of structurally related proteins, including at least HERl, HER2, HER3 and HER4 (a.k.a. EGFRl, EGFR2, EGFR3 and EGFR4, respectively, or ErbB-1, ErbB-2, ErbB-3 and ErbB-4, respectively). HERl, HER2 and HER4 are receptor tyrosine kinases; although HER3 shares homology with HERl, HER2 and HER4, HER3 is kinase inactive. Included in the HER family is p95, a truncated form of HER2 lacking portions of the HER2 extracellular domain (see, e.g., Arribas et al. , Cancer Res. , 71 : 1515- 1519, 201 1 ; Molina et al. , Cancer Res. , 61 :4744- 4749, 2001). "HER protein" or "a HER protein" refers to the family of HER proteins, including at least HERl , HER2, HER3, HER4 and p95. HER proteins mediate cell growth and are dis-regulated in many types of cancer. For example HER1 and HER2 are upregulated in many human cancers, and their excessive signaling may be critical factors in the development and malignancy of these tumors. Receptor dimerization is essential for HER pathway activation leading to receptor phosphorylation and downstream signal transduction. Unlike HER1, -3 and -4, HER2 has no known ligand and assumes an open conformation, with its dimerization domain exposed for interaction with other ligand-activated HER receptors. (See, e.g., Herbst, Int. J. Radiat. Oncol. Biol. Phys., 59:21-6, 2004; Zhang et al, J. Clin. Invest. 117 (8): 2051-8, 2007.) Approximately 30% of breast cancers have an amplification of the Her2 gene or overexpression of its protein product. HER2 overexpression also occurs in other cancer types, such as ovarian cancer, stomach cancer, and biologically aggressive forms of uterine cancer, such as uterine serous endometrial carcinoma. See, e.g., Santin et al, Int. J.

Gynacol. Obstet., 102 (2): 128-31, 2008. HER2-containing homo- and hetero-dimers are transformation competent protein complexes. Trastuzumab, a humanized antibody that prevents HER2 homodimerization is used to treat certain HER2 overexpressing cancers, including breast cancer. Additionally, the level of HER2 expression in cancer tissue is predictive of patient response to HER2 therapeutic antibodies (e.g., Trastuzumab).

Because of its prognostic role as well as its ability to predict response to Trastuzumab, tumors (e.g., tumors associated with breast cancer) are routinely checked for

overexpression of HER2.

The HER pathway is also involved in ovarian cancer pathogenesis. Many ovarian tumor samples express all HER proteins. Co-expression of HER1 and HER2 is seen more frequently in ovarian cancer than in normal ovarian epithelium, and overexpression of both receptors correlates with poor prognosis. Preferred dimerization with HER2

(HER1/HER2, HER2/HER3) and subsequent pathway activation via receptor

phosphorylation have also been shown to drive ovarian tumor cell proliferation, even in the absence of HER2 overexpression. Pertuzumab, a humanized antibody that prevents HER2 dimerization (with itself and with HER3) has been shown to provide therapeutic benefit to patients with HER2 and/or HER3 expressing ovarian cancer.

Examples of HER1 amino acid sequence include NCBI/Genbank accession Nos.

NP_005219, CAA25240, AAT52212, AAZ66620, BAF83041, BAH1 1869, ADZ75461, ADL28125, BAD92679, AAH94761, all of which are incorporated by reference herein as provided in Genbank on October 27, 201 1. Examples of, HER2, amino acid sequences include NCBI/Genbank accession BAJ17684, P04626, AAI67147, NP 001005862, NP 004439, AAA75493, AAO 18082, all of which are incorporated by reference herein as provided in Genbank on October 27, 201 1. Examples of HER3 amino acid sequences include NCBI/Genbank accession Nos. NP 001973, P21860, AAH82992, AAH02706, AAA35979, all of which are incorporated by reference herein as provided in Genbank on October 27, 201 1. Examples of HER4 amino acid sequences include NCBI/Genbank accession Nos., AAI43750, Q15303, NP_005226, NP_001036064, AAI43748, all of which are incorporated by reference herein as provided in Genbank on October 27, 201 1.

Immune complex: The binding of antibody to a soluble antigen forms an immune complex. The formation of an immune complex can be detected through conventional methods known to the person of ordinary skill in the art, for instance

immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, immunob lotting (i.e., Western blot), magnetic resonance imaging, CT scans, X-ray and affinity chromatography. Immunological binding properties of selected antibodies may be quantified using methods well known in the art.

In situ hybridization (ISH): A type of hybridization that uses a probes, such as a labeled complementary DNA or RNA strand, to localize a specific DNA or RNA sequence in a portion or section of tissue (in situ), or, if the tissue is small enough (e.g., plant seeds, Drosophila embryos), in the entire tissue (whole mount ISH). DNA ISH can be used to determine the structure of chromosomes, such as for use in medical diagnostics to assess chromosomal integrity. RNA ISH (hybridization histochemistry) is used to measure and localize mRNAs and other transcripts within tissue sections or whole mounts.

Linker: The two components of a conjugate are joined together either directly through a bond or indirectly through a linker. Typically, linkers are bifunctional, i.e., the linker includes a functional group at each end, wherein the functional groups are used to couple the linker to the two conjugate components, either covalently or non-covalently. The two functional groups may be the same, i.e., a homobifunctional linker, or different, i.e., a heterobifunctional linker, but more typically are heterobifunctional. Where linkers are employed, suitable functional groups are selected to allow attachment of the two components of the conjugate, while not impairing the functionality of the components. Linkers of interest may vary widely depending on the components in the conjugate. In many embodiments the linker, when present, is biologically inert.

Neoplasia, cancer or tumor: A neoplasm is an abnormal growth of tissue, or cells, that results from excessive cell division. Neoplastic growth can produce a tumor. The amount of a tumor in an individual is the "tumor burden" which can be measured as the number, volume, or weight of the tumor. A tumor that does not metastasize is referred to as "benign." A tumor that invades the surrounding tissue and/or can metastasize is referred to as "malignant."

Tumors of the same tissue type are primary tumors originating in a particular organ (such as colon, skin, breast, prostate, bladder or lung). Tumors of the same tissue type may be divided into tumors of different sub-types. For example, lung carcinomas can be divided into an adenocarcinoma, small cell, squamous cell, or non-small cell tumors.

Examples of solid tumors, such as sarcomas (connective tissue cancer) and carcinomas (epithelial cell cancer), include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colorectal carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma,

adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, and CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma,

hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and retinoblastoma).

Nucleic acid: A polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic non- naturally occurring analogs thereof) linked via phosphodiester bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. Thus, the term includes nucleotide polymers in which the nucleotides and the linkages between them include non-naturally occurring synthetic analogs, such as, for example and without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. It will be understood that when a nucleotide sequence is represented by a

DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T."

Nucleotide: Term includes, but is not limited to, a base linked to a sugar, such as a pyrimidine, purine or synthetic analogs thereof, or a base linked to an amino acid, as in a peptide nucleic acid (PNA). A nucleotide is one unit in a polynucleotide. A nucleotide sequence refers to the sequence of bases in a polynucleotide.

Conventional notation is used herein to describe nucleotide sequences: the left- hand end of a single-stranded nucleotide sequence is the 5 '-end; the left-hand direction of a double-stranded nucleotide sequence is referred to as the 5 '-direction. The direction of 5' to 3 ' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the "coding strand;" sequences on the DNA strand having the same sequence as an mRNA transcribed from that DNA and which are located 5' to the 5 '-end of the RNA transcript are referred to as "upstream sequences;" sequences on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the coding RNA transcript are referred to as "downstream sequences."

Nucleic Acid Probe: A sequence of nucleotides, between about 10 and up to at least 500 nucleotides in length, used to detect the presence of a complementary sequence by molecular hybridization. Probes are generally contiguous nucleotides sequences, complementary to the target nucleic acid molecule, having from about 10 to about 500 nucleotides, more typically from about 30 to about 400 nucleotides, such as from about 100 to about 400 nucleotides or from about 200 to about 300 nucleotides. A person of ordinary skill in the art will appreciate that the specificity of a particular probe increases with its length. In particular examples, probes include a label that permits detection of probe:target sequence hybridization complexes. Typical labels include haptens, but can also include radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, and enzymes. These labels may be directly attached to the probe or attached via a linker. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed, for example, in Sambrook et ah, Molecular

Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989) and Ausubel et ah, Current Protocols in Molecular Biology Greene Publishing Associates and Wiley- Intersciences (1987). Methods suitable for attaching a linker to nucleic acid probe also are known to those of ordinary skill in the art. See, for example, U.S. Patent No. 5,733,523, and Hermanson, "Bioconjugate Techniques," Academic Press, San Diego, 1996.

Oligonucleotide: A linear polynucleotide sequence of between 5 and 100 nucleotide bases in length.

PTEN: PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a key tumor suppressor gene in prostate cancer. Loss of PTEN function results in increased PIP3 (Phosphatidylinositol (3,4,5)-triphosphate) levels and subsequent AKT phosphorylation and modulation of its downstream molecular oncogenic processes. A series of in vivo studies have demonstrated the role of PTEN in prostate carcinogenesis with prostate-specific deletion. Clinically, deletion or mutation of at least one PTEN allele may occur in 20-40% of localized cancers and up to 60% of metastases. Fluorescent in situ hybridization and immunohistochemical studies demonstrated that PTEN genomic deletion and absence of PTEN expression are associated with unfavorable clinical outcome measures. Recent studies also showed that PTEN inactivation plays an important role in prostate cancer during progression to androgen-independence.

Prostate Cancer: Prostate cancer is one of the most prevalent malignancies affecting men worldwide, and is the most frequent cancer among American men with an estimated incidence of approximately 220,000 (29% of all cancers in men) and a mortality estimated to be over 27,000 (9% of all male cancer deaths) in 2007. Most prostate cancers are slow growing; however, there are cases of aggressive prostate cancers. The cancer cells may metastasize from the prostate to other parts of the body, particularly the bones and lymph nodes. Prostate cancer may cause pain, difficulty in urinating, problems during sexual intercourse, or erectile dysfunction. Other symptoms can potentially develop during later stages of the disease. Treatment options include active surveillance, prostatectomy, radiation therapy and androgen ablation therapy, all influenced by the use of serum prostate specific antigen (PSA) levels. Nonspecific PSA tests result in a large number of false positives for prostate cancer, leading to a /awx-cancer burden and repeated biopsies.

More specific diagnostic modalities, prognostic indicators of progression and a better understanding of prostate cancer biology for treatment of hormone refractory disease are high priorities in prostate cancer research.

Proximal: Refers to the qualitative or quantitative distance between two molecules; for example, the distance between two proteins in a tissue sample. In some embodiments, molecules that are proximal to each other are within at least about 100 nm, at least about 75 nm, at least about 50 nm, at least about 35 nm, at least about 30 nm, at least about 25 nm, at least about 20 nm, at least about 15 nm, at least about 10 nm, at least about 5 nm or less distance of each other. Proximal may also provide a functional relationship. For examples, two targets (e.g., two proteins) may be considered proximal if the first target is within sufficient distance of the second target for a biotin ligase associated with the first target to allow biotinylation of a substrate associated with a second target.

Quantum dot: A nanoscale particle that exhibits size-dependent electronic and optical properties due to quantum confinement. Quantum dots (QD) are novel inorganic fluorochromes, which are photo-stable, show bright fluorescence with narrow symmetric emission spectra and are available in multiple resolvable colors. These remarkable optical properties promise potentially unprecedented resolution and strong signal intensities that have not been possible to attain using traditional fluorophores. QD have several distinct optical properties when compared with organic fluorophores. First, QD have a broad excitation spectra. Different QD can be excited by the same single wavelength. The long excitation state of a QD enables longer signal acquisition times. The distance between excitation and emission wavelengths is large (that is a large Stokes shift). Second, QD have narrow symmetric emission spectra. The constant electron shift for all atoms in the precise crystalline structure results in a tightly defined emission spectrum. Classical organic fluorophores, in contrast, have narrow excitation and broad emission that often results in spectrum overlap or red tailing. Third, QD produce significantly brighter fluorescence (2- 1 1 times) than organic dyes because of the extremely high fluorescence efficiency. High molar extinction coefficients are achieved because most, if not all, of the atoms in each crystal are excited simultaneously. Fourth, the inorganic composition makes

QD much more resistant (approximately lOOOx) to photo-bleaching than organic fluorophores. QD typically have a long fluorescence lifetime, for example of from about 10 to at least about 50 nanoseconds, when compared with organic dyes that typically decay in the order of a few nanoseconds. The combination of constant excitation wavelength, sharp and symmetrical emission spectrum, and large Stokes shift makes QD desirable fluorescent markers for multiplex target detection. These characteristics enable multiple spectra to be distinguished from each other with emission detection at wavelengths substantially different from the excitation. QD have, for example, been constructed of semiconductor materials (e.g., cadmium selenide and lead sulfide) and from crystallites (grown via molecular beam epitaxy), etc. QDs typically have a heavy metal core, such as a core comprising cadmium sulfide (CdS), cadmium selenide (CdSe), indium phosphate (InP) or lead selenide (PbSe). The core typically is coated with a shell of high band gap semiconductor, such as zinc sulfide, that improves quantum yields and optical properties. QD also may include an extra polymer coating, such as a mixture of thioctyl

phosphine/trioctyl phosphine oxide (TOP/TOPO), which serves as a site for conjugation with biomolecule moieties, such as proteins, antibodies, oligonucleotides, and streptavidin. The total size of the nanocrystal may vary, but typically is about 20-40nm, with a molecular weight of approximately 150KD. A variety of quantum dots having various surface chemistries and fluorescence characteristics are commercially available from Invitrogen Corporation, Eugene, Oregon. Quantum dots are also commercially available from Evident Technologies (Troy, N.Y.). Other quantum dots include alloy quantum dots such as ZnSSe, ZnSeTe, ZnSTe, CdSSe, CdSeTe, ScSTe, HgSSe, HgSeTe, HgSTe, ZnCdS, ZnCdSe, ZnCdTe, ZnHgS, ZnHgSe, ZnHgTe, CdHgS, CdHgSe, CdHgTe, ZnCdSSe, ZnHgSSe, ZnCdSeTe, ZnHgSeTe, CdHgSSe, CdHgSeTe, InGaAs, GaAlAs, and InGaN quantum dots (Alloy quantum dots and methods for making the same are disclosed, for example, in U.S. Application Publication No. 2005/0012182 and PCT Publication WO 2005/001889). See, also, U.S. Patent Nos. 6,815,064, 6,682,596 and 6,649,138, each of which patents is incorporated by reference herein.

Sample: Certain disclosed embodiments utilize biological samples. A biological sample is typically obtained from a mammalian subject of interest, such as a human. The sample can be any sample, including, but not limited to, tissue from biopsies, autopsies and pathology specimens. Biological samples also include sections of tissues, for example, frozen sections taken for histological purposes. Biological samples also include cell cultures or portions of cell cultures, for example, a cell culture grown from a biological sample taken from a subject.

Biological samples can be obtained from a subject using any method known in the art. For example, tissue samples can be obtained from breast cancer patients who have undergone tumor resection as a form of treatment. From these patients, both tumor tissue and surrounding non-cancerous tissue can be obtained. In some embodiments, the non- cancerous tissue sample used as a control is obtained from a cadaver. In some embodiments, biological samples are obtained by biopsy. Biopsy samples can be fresh, frozen or fixed, such as formalin- fixed and paraffin embedded. Samples can be removed from a patient surgically, by extraction (for example by hypodermic or other types of needles), by micro-dissection, by laser capture, or by any other means known in the art.

In some embodiments, the biological sample is a tissue sample, e.g., a tissue sample obtained from a subject diagnosed with a tumor, such as a malignant or benign breast cancer tumor, a malignant or benign lung tissue sample, a malignant or benign cervical tissue sample, a hematological sample, or a metaphase spread. In some cases, the tissue samples are obtained from healthy subjects or cadaveric donors. A "sample" refers to part of a tissue that is either the entire tissue, or a diseased or healthy portion of the tissue. In some embodiments, malignant tumor tissue samples are compared to a control. In some embodiments, the control is a benign tumor tissue sample obtained from a different subject. In some embodiments, the control is non-cancerous tissue sample obtained from the same subject, such as a benign tumor adjacent to the tumor. In other embodiments, the control is non-cancerous tissue sample obtained from the same subject, such as non-cancerous tissue surrounding the malignant tumor. In other embodiments, the control is non-cancerous tissue sample from a cadaver. In other embodiments, the control is a reference sample, such as standard or reference value based on an average of historical values.

In some embodiments, the biological sample is obtained from a subject that has, is suspected of having, or is at risk of developing, a tumor, e.g., a carcinoma. For example, the subject has, is suspected of having, or is at risk of developing breast, ovarian, uterine or stomach cancer.

Sensitivity and specificity: Statistical measurements of the performance of a binary classification test. Sensitivity measures the proportion of actual positives which are correctly identified (e.g., the percentage of samples that are identified as including nucleic acid from a particular virus). Specificity measures the proportion of negatives which are correctly identified (e.g., the percentage of samples that are identified as not including a target nucleic acid, such as a nucleic acid from a particular virus or bacteria).

Sequence identity: The similarity between two nucleic acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity, similarity, or homology; a higher percentage identity indicates a higher degree of sequence similarity.

The NCBI Basic Local Alignment Search Tool (BLAST), Altschul et al, J. Mol. Biol. 215:403-10, 1990, is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD), for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. It can be accessed through the NCBI website. A description of how to determine sequence identity using this program is also available on the website.

When less than the entire sequence is being compared for sequence identity, homologs will typically possess at least 75% sequence identity over short windows of 10- 20 amino acids, and can possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are described, for example on the NCBI website.

These sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided. An alternative indication that two nucleic acid molecules are closely related is that the two molecules hybridize to each other under stringent conditions. Stringent conditions are sequence-dependent and are different under different environmental parameters.

Generally, stringent conditions are selected to be about 5 °C to 20 °C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.

The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Conditions for nucleic acid hybridization and calculation of stringencies can be found in Sambrook et al; and Tijssen, Hybridization With Nucleic Acid Probes, Part I: Theory and Nucleic Acid Preparation, Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Ltd.,

1993.

Specific binding moiety(ies): A member of a specific-binding pair. Specific binding pairs are pairs of molecules that are characterized in that they bind preferentially to each other and/or to the substantial exclusion of binding to other molecules (for example, specific binding pairs can have a binding constant that is at least 10^"3 greater, 1 O^ greater or 10^"5 greater than a binding constant for either of the two members of the binding pair with other molecules in a biological sample). The specific binding moiety used to make the exemplary conjugates disclosed herein may be any of a variety of different types of molecules, so long as it exhibits the requisite binding affinity for the target.

The specific binding moiety may comprise a small molecule or large molecule. A small molecule will range in size from about 50 to about 10,000 daltons, more typically from about 50 to about 5,000 daltons, and even more typically from about 100 to about 1000 daltons. A large molecule is one whose molecular weight is typically greater than about 10,000 daltons. The small molecule may be any molecule, typically an organic molecule that is capable of binding with the requisite affinity to the target. The small molecule typically includes one or more functional groups allowing it to interact with the target, for example by hydrophobic, hydrophilic, electrostatic or covalent interactions. Where the target is a protein, lipid or nucleic acid, the small molecule typically will include functional groups allowing for structural interactions such as hydrogen bonding, hydrophobic-hydrophobic interactions, electrostatic interactions, etc. The small molecule ligand often includes an amine, amide, sulfhydryl, carbonyl, hydroxyl or carboxyl group, and preferably at least two of these functional groups.

The small molecules often comprise cyclic and/or heterocyclic non-aromatic structures, and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Also useful small molecules include structures found among biomolecules, including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

The small molecule may be derived from a naturally occurring or synthetic compound that may be obtained from a wide variety of sources, including libraries of synthetic or natural compounds. For example, numerous methods are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including the preparation of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known small molecules may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc., to produce structural analogs.

As such, the small molecule may be obtained from a library of naturally occurring or synthetic molecules, including a library of compounds produced through combinatorial means, i.e. a compound diversity combinatorial library. When obtained from such libraries, the small molecule employed will have demonstrated some desirable affinity for a target in a convenient binding affinity assay. Combinatorial libraries, as well as methods for their production and screening, are known in the art and are described in U.S. Patent Nos. 5,741 ,713 and 5,734,018, the disclosures of which are incorporated herein by reference. Additional information concerning specific binding moieties is provided by assignee's U.S. Patent No. 7,695,929, which also is incorporated herein by reference. The specific binding moiety may comprise a large molecule. Of particular interest as large molecule specific binding moieties are antibodies, as well as binding fragments and derivatives or mimetics thereof. As such, the specific binding moiety may be either a monoclonal or polyclonal antibody. Also of interest are antibody fragments or derivatives produced either recombinantly or synthetically, such as single chain antibodies or scFvs, or other antibody derivatives such as chimeric antibodies or CDR-grafted antibodies, where such recombinantly or synthetically produced antibody fragments retain the binding characteristics of the above antibodies. Such antibody fragments, derivatives or mimetics of the subject invention may be readily prepared using any convenient methodology, such as the methodology disclosed in U.S. Patent Nos. 5,851,829 and 5,965,371, the disclosures of which are incorporated herein by reference.

Also suitable for use as large molecule specific binding moieties are polynucleic acid aptamers. Polynucleic acid aptamers may be RNA oligonucleotides that selectively bind proteins, much in the same manner as a receptor or antibody (Conrad et ah, Methods Enzymol. (1996), 267(Combinatorial Chemistry), 336-367), or DNA oligomers that complement specific DNA target sequences.

In addition to antibody-based peptide/polypeptide or protein-based binding domains, the specific binding moiety may also be a lectin, a soluble cell-surface receptor or derivative thereof, an affibody or any combinatorially derived protein or peptide from phage display or ribosome display or any type of combinatorial peptide or protein library. Combinations of any specific binding moiety may be used.

Importantly, the specific binding moiety will be one that allows for coupling to the second component of the conjugate, or to a linker, without substantially affecting the binding affinity of the specific binding moiety to its target.

Particular examples of specific binding moieties include specific binding proteins (for example, antibodies, lectins, avidins such as streptavidins, and protein A). Specific binding moiety(ies) also includes the molecules (or portions thereof) that are specifically bound by such specific binding proteins.

Spotting: Spotting is an issue associated with QD analyses whereby relative large spots are observed in an image, and may result from QD aggregation.

Spotting is most often associated with QD655, and less frequently with QD605. Failures associated with QD655 specific staining is, at times, associated with

QD655 spotting.

Subject: Any mammal, such as humans, non- human primates, pigs, sheep, cows, rodents and the like. Thus, the term "subject" includes both human and veterinary subjects. In one example, a subject is one known or suspected of having a HER+ tumor. In another example, a subject is one who is being considered for treatment with an antibody that is specific for HER, such as pertuzumab or trastuzumab.

Target: Any molecule for which the presence, location and/or concentration is or can be determined. Examples of target molecules include proteins and haptens, such as haptens covalently bonded to proteins. Target molecules are typically detected using one or more conjugates of a specific binding molecule and a detectable label. Examples of specific targets include proteins, carbohydrates, or nucleic acid molecules. Exemplary protein targets include p95, HERl, HER2, HER3 or HER4. Target nucleic acid molecules include those molecules whose proximity, rearrangement, amplification, deletion, detection, quantitation, qualitative detection, or a combination thereof, is sought. For example, the target can be a defined region or particular portion of a nucleic acid molecule, for example a portion of a genome (such as a gene or a region of DNA or RNA containing a gene (or portion thereof) of interest). The nucleic acid molecule need not be in a purified form. Various other nucleic acid molecules can also be present with the target nucleic acid molecule. For example, the target nucleic acid molecule can be a specific nucleic acid molecule (which can include RNA or DNA), the amplification of at least a portion thereof (such as a portion of a genomic sequence or cDNA sequence) is intended. In some examples, a target nucleic acid includes a viral nucleic acid molecule, or a bacterial nucleic acid molecule, such as a nucleic acid molecule from Escherichia coli or Vibrio cholera. Purification or isolation of the target nucleic acid molecule, if needed, can be conducted by methods known to those in the art, such as by using a commercially available purification kit or the like. Exemplary tissue types that can targeted include, but are not limited to, Breast tissue, Lung tissue, hematological tissue, Cervical tissue and metaphase spread. Exemplary target types include genomic DNA (including, but not limited to, ERG, PTEN, CEN10, HER2 and ALK), mRNA (including, but not limited to, Kappa and Lambda) and virus, such as HPV16.

Treating or Treatment: A therapeutic intervention {e.g., administration of a therapeutically effective amount of an antibody that specifically binds HER2 or a conjugate thereof) that ameliorates a sign or symptom of a disease or pathological condition related to a disease (such as a tumor). Treatment can also induce remission or cure of a condition, such as cancer. In particular examples, treatment includes preventing a tumor, for example by inhibiting the full development of a tumor, such as preventing development of a metastasis or the development of a primary tumor. Prevention does not require a total absence of a tumor.

Reducing a sign or symptom associated with a tumor can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject (such as a subject having a tumor which has not yet metastasized), a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease (for example by prolonging the life of a subject having tumor), a reduction in the number of relapses of the disease, an improvement in the overall health or well-being of the subject, or by other parameters well known in the art that are specific to the particular tumor.

Tumor burden: The total volume, number, metastasis, or combinations thereof of tumor or tumors in a subject.

Under conditions sufficient for: A phrase that is used to describe any environment that permits a desired activity. In one example the desired activity is formation of an immune complex. In another example, the desired activity is peroxidase- catalyzed formation of a covalent bond between a tyramide and a phenol moiety, for example catalysis that occurs in the presence of hydrogen peroxide.

3' end (3p): The end of a nucleic acid molecule that does not have a nucleotide bound to it 3' of the terminal residue.

5' end (5p): The end of a nucleic acid sequence where the 5' position of the terminal residue is not bound by a nucleotide.

II. General Description of Method

A pervasive trend in modern pathology is to explore the origins of disease by characterizing complicated genetic abnormalities. Disclosed embodiments of the present invention satisfy this unmet need for rapid and reliable detection of at least one biomolecule, and typically multiple biomolecules, in routinely processed clinical tissues. Certain disclosed embodiments concern QD ISH assays, particularly automated process embodiments, and in certain embodiments multiplexed QD ISH assays for simultaneous detection of two or more targets. The robustness of this assay (resulting in a greater than 90% pass rate, often greater than 95% pass rate) alleviate, if not substantially eliminate, prior concerns of the "unreliable" nature of QDs for FISH. In addition, the reliable and reproducible interpretation of the replicate slides demonstrates the feasibility of using the assay in clinical applications.

Several features distinguish the disclosed embodiments from other QD ISH applications previously described in the literature. First, certain disclosed embodiments use an "in-direct" QD detection scheme whereby QDs are labeled with antibodies, but not on probes directly. This improves hybridization efficiency by hapten-labeled probes, in contrast to using QD-labeled probes that cause steric hindrance issues because of the QD size. It also avoids incubation of QDs at high temperature, which affects the stability of QDs. Various efforts have been made to address the possible steric hindrance of the

"direct" QD detection. Ma et a/.(Chromosoma, 1 17 (2008), pp. 181-187) tried to keep the oligonucleotide probe further away from the QD surface using a homo-polymer of thymidine sequence. The modified hydrodynamic diameter of the probes was deemed small enough to penetrate into maize chromosomes. Choi et al. (Small, 5 (2009), pp. 2085-2091) coupled DNA oligonucleotides via a l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) molecule coupled with polymer-coated QDs visualize gene targets in Drosophila. Chan et al. (Nucleic Acids Res, 33 (2005), p. el61) raised the issue of the multiple streptavidin sites on the QD molecule that could interfere with hybridization efficiency. They used biocytin, a competitive blocker of streptavidin, in the labeling process of oligonucleotide probes. On the other hand, Ioannou D and Griffin DK (Nano Rev, 1 (2010), p. 51 17) reported repeated failed attempts using direct conjugation of DNA to QDs. Disclosed embodiments of indirect QD labeling provide an alternative approach for effective hybridization efficiency.

Certain disclosed embodiments used two or more, and in certain embodiments at least four, genomic probes that were nick-translation labeled with 4 distinct haptens.

These haptens were then detected with anti-hapten antibodies conjugated with different QDs having sufficiently different emission wavelengths to allow the signals to be distinguished. Unlike the widely used DNP and DIG, two new haptens - NP and TS - lacked information on optimal labeling conditions, functionality of the NP and TS labeled probe on hybridization, as well as the latter immunologic detection. Optimal nick translation methods for NP- and TS-modified nucleotides were empirically developed. Functional staining results and hapten incorporation measurements confirmed that: (1) the 3: 1 ratio of hapten-labeled dUTP versus native dUTP offers optimal hybridization efficiency; (2) this labeling method allows optimal enzymatic incorporation of the modified nucleotide into the probes; and (3) the labeled haptens serve well as targets for anti-TS conjugated QD, such as QD605, and anti-NP conjugated QD, such as QD525, antibodies, and maintain low background noise as well. For DNP-labeled probes, DNP- dCTP or DNP-dUTP (with no native dCTP) may be used. For DIG-labeled probes, a 1 :2 ratio of DIG-dUTP versus native dUTP was used in working embodiments. No superior staining was observed with higher ratios of DIG-dUTP versus native dUTP. Similarly,

Ioannou D and Griffin DK (Nano Rev, 1 (2010), p. 51 17) observed no noticeable difference on biotin-labeled probes with different ratios of biotin-dUTP and unlabeled dUTP. Empirical optimization of probe labeling for each hapten can be an important consideration for QD conjugated anti-hapten antibody ISH assays. With the knowledge of optimal probe labeling conditions for DNP, DIG, TS and NP, hapten libraries and their respective anti-hapten antibodies provide an invaluable tool for robust multiplexing ISH assays.

Certain embodiments also provide an improved tissue pretreatment protocol. After systemically testing all the major components in the staining procedure with a Design of Experiment (DOE) approach, pretreatment conditions are an important consideration for robust and reliable QD ISH staining. Without being bound to a theory of operation, QDs may require different tissue permeabilization procedures than other fluorophores to achieve optimum labeling as they are structurally large particles (often more than 150kD).

Detecting hapten-labeled nucleotides using antibody: QD conjugates presents additional challenges for immunohistochemistry, potentially including: (1) removal of cellular proteins and nucleic acid associated proteins; (2) retention of nucleic acids in the tissues; and (3) penetration of labeled nucleic acid probes and QDs-conjugated antibodies. Chemical and physical pretreatments are the two main approaches, which are parallel to the antigen retrieval methodologies for immunohistochemistry. While proteinase digestion may be important for hybridization of nucleic acids, heat treatment is usually applied for superior sensitivity and reproducibility of immunostaining. Proteinase K and pepsin are the most described methods for QD ISH application. In addition, Xue et al. (Oral Pathol Med, 38 (2009), pp. 668-671) described a combined approach of microwave heat-induced treatment and proteinase K treatment for detecting HPV 16/18 by a biotin-probe with QD- streptavidin in oral squamous cell carcinoma. Moreover, Ioannou A et α/.'s (J Biomed

Biotech (2012) article ID: 627602) work on whole-mount tissue QD ISH provides direct visual evidence of the levels of tissue penetration by QDs upon various pretreatments. Proteinase K treatment for 25 minutes (x5 longer than on a chromogenic ISH) followed with 0.1% Tween significantly improved the penetration of QD705-streptavidin, QD655 anti-FITC, or QD655-anti-DIG, and resulted in specific QD staining in deep tissues (using

Biotin-, FITC, and DIG-labeled RNA probes).

The chemical composition of the buffer and the pH are factors to consider when choosing heat treatment. Citrate buffer at acidic pH and Tris-HCl at basic pH are the most often used buffers. Basic solutions seem to be more efficient in antigen retrieval.

Moreover, a basic buffer containing EDTA may help antigens "return" to their native states by chelating calcium in the cage-like structures formed by fixative crosslinking. Disclosed embodiments establish that heating with a cell conditioning composition, such as Tris buffer, pH 9.0, ImM EDTA significantly improved multi-color QD ISH assay robustness.

Furthermore, heavy metals in process solutions, such as formalin solutions during tissue fixation, can cause partial or total loss fluorescence quenching, even at very low metal concentrations. Accordingly, in certain embodiments a metal chelator or scavenger, exemplified in working embodiments by ethylenediaminetetracetic acid at an effective concentration, such as from about 0.1 to at least about 1 mM EDTA in a Tris buffer solution, can help prevent QD fluorescence quenching by chelating contaminated metals in variously prepared tissue samples.

Certain tissue pretreatment conditions included two courses of heating. Exemplary working embodiments used a Tris buffer pretreatment at 90 °C for 92 minutes, a citrate buffer pretreatment at 82 °C for 36 minutes, and/or a Protease 3 pretreatment at 37 °C for 28 minutes. Disclosed embodiments are highly reliable and therefore provide a diagnostic tool for particular diagnostic assays, such as ERG and PTEN gene status in prostate cancer. Prostate cancer is the second most common cancer and the second leading cause of cancer death in American men. As a slow-growing disease, some tumors grow so slowly that they likely never lead to death or any adverse symptoms. Molecular biomarkers that can distinguish indolent from clinically significant prostate cancer would have extremely high clinical utility, as patients could be stratified based on risk assessment. In the past several years, a recurrent fusion between the androgen regulated gene TMPRSS2 (21q22.3) and ETS oncogenic transcription factors family member ERG (21q22.2) has been reported as a common occurrence in prostate carcinoma. TMPRSS2-ERG has been frequently associated with more aggressive prostate cancers and a poorer prognosis. More recent studies find that genomic loss of PTEN and ERG genetic rearrangements are genetic events significantly associated with human prostate cancer. The two frequent critical events in human prostate cancer cooperate to promote tumor development and progression in the prostate. These findings may imply novel targeted therapies against ERG and

PTEN/PI3K/AKT pathway as preventive and therapeutic approaches in the future. Taken together, knowledge of ERG and PTEN gene status allows a substantially more accurate patient stratification and prognostication, and provides information leading to potential targeted therapies.

In summary, disclosed QD ISH assay embodiments, particularly multi-color QD

ISH multiplexed assays, have been developed, particularly embodiments for automated staining platforms. The new multi-color QD ISH assays enable multiplexed in situ detection of molecular biomarkers in routinely processed human clinical tissue. These new multi-color assays provide several benefits. First, they allow simultaneous detection of plural genomic targets on a single slide and therefore increase efficiency on a limited amount of sample. Second, they use routinely available fluorescence microscopes for signal interpretation, and certain embodiments are not dependent on spectral imaging software. Third, QD brightness and resistance to photo-bleaching make the signals easy to read. Finally, a fully automated assay, can be completed overnight, provides consistent and accurate results.

III. Conjugates

Compounds of the present invention, also referred to as conjugates, that have been used in certain disclosed exemplary embodiments can be separated into two general categories: specific binding moiety-hapten conjugates; and anti-hapten antibody-quantum dot conjugates. A. Specific binding moiety: hapten conjugates

These conjugates comprise a specific binding moiety, directed to a specific target of interest, directly or indirectly coupled to a hapten. The specific binding moiety typically includes functional groups allowing for structural interactions with the target such as hydrogen bonding, hydrophobic-hydrophobic interactions, electrostatic interactions, etc.

Exemplary tissue types that can be targeted include, but are not limited to, prostate tissue, breast tissue, lung tissue, hematological tissue, cervical tissue and metaphase spread. Exemplary target types include genomic DNA (including, but not limited to, ERG, PTEN, CEN10, HER2 and ALK), mRNA (including, but not limited to, Kappa and Lambda mRNA) and viruses, such as HPV16.

In some embodiments the specific binding moiety can be directly conjugated to the hapten. Thus, a first general formula describing certain embodiments of the present disclosure is specific binding moiety:hapten. Suitable specific binding moiety-hapten conjugates can also optionally include a linker that links the specific binding moiety to the hapten. Embodiments having a linker satisfy the formula specific binding moiety-linker- hapten. Furthermore, a linker can include plural subunits or be formed from various subcomponents. For example, both a specific binding moiety and a hapten can include attached linkers, wherein the linkers can then be reacted to couple the hapten and the specific binding moiety together to form the conjugate. B. Anti-hapten antibody: quantum dot conjugates

These conjugates comprise an anti-hapten antibody, directed to a specific hapten included in a specific binding moiety-hapten conjugate, and a quantum dot. In some embodiments the anti-hapten antibody can be directly conjugated to the quantum dot. Thus, a first general formula describing certain conjugate embodiments of the present disclosure is anti-hapten antibody:quantum dot. Such compounds can also optionally include a linker that links the anti-hapten antibody and the quantum dot. Embodiments having a linker satisfy the formula anti-hapten antibody-linker-quantum dot. Furthermore, a linker can include plural subunits or be formed from various subcomponents. For example, both an anti-hapten antibody and a quantum dot can include attached linkers, wherein the linkers can then be reacted to couple the quantum dot and the antibody together to form the conjugate.

Each component of these conjugates is described in more detail below.

1. Haptens

Disclosed embodiments of the present invention use haptens, and hapten: QD conjugates, to perform tissue diagnostics. A person of ordinary skill in the art will appreciate that any hapten now known or hereafter discovered can be used to practice the disclosed method. For example, certain haptens that are commonly used in diagnostic applications that can be used to practice disclosed embodiments include digoxigenin, avidin, streptavidin, nitrophenyl, acetylaminoflurene, biotin, and bromodeoxy uridine.

Ventana Medical Systems, Inc. also has developed additional classes of haptens that can be used to practice disclosed embodiments. These haptens are fully described in U.S. Patent No. 7,695,929, which is incorporated herein by reference. Briefly, disclosed embodiments of such haptens include pyrazoles, particularly nitropyrazoles; nitrophenyl compounds; benzofurazans; triterpenes; ureas and thioureas, particularly phenyl ureas, and even more particularly phenyl thioureas; rotenone and rotenone derivatives, also referred to herein as rotenoids; oxazole and thiazoles, particularly oxazole and thiazole sulfonamides; coumarin and coumarin derivatives; cyclolignans, exemplified by

Podophyllotoxin and Podophyllotoxin derivatives; and combinations thereof.

2. Specific Binding Moiety

Specific binding moieties are defined above. Of particular interest are specific binding moieties that act as nucleic acid probes. In one embodiment the specific binding moiety is a probe, comprising single or double stranded DNA from cell-based cloning or PCR. In some embodiments the probe is designed to be complementary to a target DNA sequence. The specific binding moiety also may comprise RNA, such as mRNA, generated by transcription. The probe also may be an oligonucleotide, both single stranded or double stranded. In another embodiment the probe is targeted to viral DNA. In some embodiments the probe is double stranded DNA or RNA which is then denatured into single stranded DNA or RNA for hybridization to a target sequence.

3. Linker

Both conjugates can have their respective moieties directly bonded to each other.

Conversely one or both of them can also include a linker that links the components, either the hapten to a specific binding moiety, or the antibody to a quantum dot.

Typically, linkers are bifunctional, i.e., the linker includes a functional group at each end, wherein the functional groups are used to couple the linker to the two conjugate components. The two functional groups may be the same, i.e., a homobifunctional linker, or different, i.e., a heterobifunctional linker, but more typically are heterobifunctional. Where linkers are employed, such groups may be chosen to allow for attachment of the two components of the conjugate, while not impairing their functionality. Such terminal functional groups, include by way of example and without limitation, amines, alcohols, thiols, hydrazides, carbonyl-reactive group (such as aldehydes, acids and esters), vinyl ketones, epoxides, isocyanates, maleimides), functional groups capable of cycloaddition reactions, forming disulfide bonds, binding to metals or photo-reactive groups. Specific examples include primary and secondary amines, hydroxamic acids, N- hydroxysuccinimidyl esters, N-hydroxysuccinimidyl carbonates, oxycarbonylimidazoles, nitrophenylesters, trifluoroethyl esters, glycidyl ethers, vinylsulfones, and maleimides.

These groups facilitate coupling to the specific binding moieties and other desired compounds.

In some embodiments, the linker is generally at least about 50 daltons, but more particularly at least about 100 daltons and may be as large as 500 daltons or larger. A first class of linkers suitable for forming disclosed conjugates is the aliphatic compounds, such as aliphatic hydrocarbon chains having one or more sites of unsaturation, or alkyl chains. The length of the chain can vary, but typically has an upper practical limit of about 30 atoms. Chain lengths greater than about 30 carbon atoms have proved to be less effective than compounds having smaller chain lengths. Thus, aliphatic chain linkers typically have a chain length of from about 1 carbon atom to about 30 carbon atoms. However, a person of ordinary skill in the art will appreciate that, if a particular linker has greater than 30 atoms, and the conjugate still functions as desired, then such chain lengths are still within the scope of the present invention.

In one embodiment, the linker is a straight-chain or branched alkyl chain functionalized with reactive groups, such as an amino- or mercapto-hydrocarbon, with more than two carbon atoms in the unbranched chain. Examples include aminoalkyl, aminoalkenyl and aminoalkynyl groups. Alternatively, the linker is an alkyl chain of 10-20 carbons in length, and may be attached through a Si-C direct bond or through an ester, Si-O-C, linkage (see U.S. Patent No. 5,661,028 to Foote, herein incorporated by reference). Other linkers are available and known to the person of ordinary skill in the art (see, e.g., U.S.

Patent Nos. 5,306,518, 4,71 1,955 and 5,707,804; all herein incorporated by reference).

A second class of linkers useful for practicing the present invention is the alkylene oxides. The alkylene oxides are represented herein by reference to glycols, such as ethylene glycols. Conjugates of the present invention have proved particularly useful if the hydrophilicity of the linker is increased relative to their hydrocarbon chains. A person of ordinary skill in the art will appreciate that, as the number of oxygen atoms increases, the hydrophilicity of the compound also may increase. Thus, linkers of the present invention typically have a formula of (-OCH2CH2-)n where n is from about 2 to about 20, but more particularly n is from about 2 to about 10, and even more typically from about 4 to about 8, which can be represented as PEG₄ to PEGg. Linkers, such as heterobifunctional polyalkyleneglycol linkers, useful for practicing certain disclosed embodiments of the present invention are described in assignee's co-pending applications, including "Nanoparticle Conjugates," U.S. Patent Application No.1 1/413,778, filed April 28, 2006; "Antibody Conjugates," U.S. Application No. 12/381,638, filed March 13, 2009; and "Molecular Conjugate," U.S. Patent

Application No. 12/687,564, filed January 14, 2010, and U.S. Patent No. 7,695,929; all of which are incorporated herein by reference. The linkers disclosed in these applications can be used to link specific binding moieties, biotin ligases, biotin ligase substrates, signal generating moieties and haptens in any and all desired combinations to form conjugates for use with disclosed embodiments of the present invention.

Other examples of linkers include, but are not limited to, peptides, carbohydrates, cyclic or acyclic systems that may possibly contain heteroatoms. Linker groups also may comprise ligands that bind to metals such that the presence of a metal ion coordinates two or more ligands to form a complex. In another embodiment, the linker is a pair of molecules, having high affinity for one another. Such high-affinity molecules include, for example, streptavidin and biotin, histidine and nickel (Ni), and GST and glutathione.

Specific exemplary linkers include: ethylene glycol, polyalkylene glycols such as PEG₂, PEG₃, PEG₄, PEG₅, PEG₆, PEG₇, PEG₈, PEG₉, PEGio, PEGn, PEGi₂, PEG13, PEG14, PEGi₅, PEGi6, PEGn, PEG₁8, PEG₁9, PEG₂0, 1 ,4-diaminohexane, xylylenediamine, terephthalic acid, 3,6-dioxaoctanedioic acid, ethylenediamine-N,N-diacetic acid, 1,1'- ethylenebis(5-oxo-3-pyrrolidinecarboxylic acid), 4,4'-ethylenedipiperidine, succinimidyl- 6-hydrazino-nicotinamide(S-HyNic, HyNic-NHS), N-succinimidyl-4-formylbenzoate (S- 4FB, 4-FB-NHS), maleimide HyNic (MHPH), maleimide 4FB (MTFB), succinimidyl- [(N-maleimidopropionamido)-octaethyleneglycol] ester (Mal-PEGg-NHS), succinimidyl- [(N-maleimidopropionamido)-tetraethyleneglycol] ester (Mal-PEG₄-NHS), 4-FB-PEG₄-

PFP, azidobenzoyl hydrazide, N-[4-(p-azidosalicylamino)butyl]-3'-[2'- pyridyldithio]propionamid), bis-sulfosuccinimidyl suberate, dimethyladipimidate, disuccinimidyltartrate, N-maleimidobutyryloxysuccinimide ester, N-hydroxy

sulfosuccinimidyl-4-azidobenzoate, N-succinimidyl[4-azidophenyl]- 1 ,3'-dithiopropionate, N-succinimidyl[4-iodoacetyl]aminobenzoate, glutaraldehyde, and succinimidyl-4-[N- maleimidomethyl]cyclohexane-l-carboxylate, 3-(2-pyridyldithio)propionic acid N- hydroxysuccinimide ester (SPDP), 4-(N-maleimidomethyl)-cyclohexane- l-carboxylic acid N-hydroxysuccinimide ester (SMCC), and the like. 4. Exemplary Working Embodiments of Specific Binding

Moiety: Hapten Conjugates

A person of ordinary skill in the art will appreciate that probes useful for practicing disclosed embodiments of the present invention can be any probe now known or hereafter developed. In particular embodiments the specific binding moiety is a DNA probe, particularly a DNA probe that targets human DNA. In yet another particular embodiment the probe target is RNA. In yet another aspect the target is messenger RNA (mRNA), such as Kappa or Lambda mRNA. In another aspect the probe targets viral DNA, such as the human papilloma virus (HPV), with a particular example including HPV 16.

For certain disclosed embodiments the probe is DNA. This DNA can be designed and synthesized to be complementary to a DNA target sequence. Exemplary probes of this type include, but are not limited to, probes for ERG5', ERG3' PTEN and CEN10.

The probes typically are haptenated. Haptens can be conjugated to a DNA probe using methods known to those of ordinary skill in the art. One such exemplary conjugation method is nick translation with dUTP. Another exemplary method uses dCTP. Exemplary haptens include, but are not limited to, digoxigenin (DIG), 2,4-dinitrophenyl (DNP), thiazole sulfonamide (TS), nitropyrazole (NP), biotin and avidin. Other suitable specific haptens developed by Ventana Medical Systems, Inc. include 5-nitro-3- pyrazolecarbamide, 2,4-dinitrophenyl, 2, l,3-Benzoxadiazole-5-cabamide, 3,5- bis(trifluoromethyl)phenyl thiourea, rotenone isoxazoline, 2-acetamido-4-methyl-5- thiazolesulfonamide, 7-(diethylamino)-2-oxo-2H-chromene-3-carboxylic acid, p- methoxyphenylpyrazopodophyllamide, 2-(3,4-dimethoxyphenyl)-quinoline-4-carbamide, 4-(dimethylamino)azobenzene-4'-sulfonamide, or (E)-2-(2-(2-oxo-2,3-dihydro-lH- benzo[b][l,4]diaepin-4-yl)phenoxy)acetamide.

Exemplary conjugates of the probe-hapten type include ERG5'-DIG conjugate, ERG3'-DNP conjugate, PTEN-thiazole sulfonamide (TS) conjugate, and CEN10- nitropyrazole (NP) conjugate.

5. Exemplary Embodiments of Anti-Hapten Antibody:QD Conjugates In particular embodiments of the present invention anti-hapten antibodies are selected for the haptens used for the hapten: specific binding moiety conjugates, such that there is one antibody for each hapten selected. Exemplary antibodies include, but are not limited to, rat anti-DNP, mouse anti-DIG, mouse anti-TS and mouse anti-NP.

In some embodiments the quantum dots are selected such that the fluorescent signals are distinguishable by a system using filters to isolate the different signals.

Alternatively, a combination of filters and deconvoluting software may be used to distinguish the fluorescent signals. Emission wavelengths of the quantum dots may be purposefully selected to be separated from each other by a particular wavelength, such as at least 10 nm, typically at least 20 nm, and even more particularly the wavelengths of the emission signals from the quantum dots are separated from each other by at least 40 nm. Exemplary quantum dots include, but are not limited to, QD655, QD565, QD605 and QD525. Exemplary quantum dot-antibody conjugates include rat anti-DNP-QD655, mouse anti-DIG-QD565, mouse anti-TS-QD605 and mouse anti-NP-QD525.

Different quantum dots give rise to signals with different strengths. As a person of ordinary skill in the art will appreciate, while any quantum dot can be matched to any specific binding moiety-hapten conjugate through the antibody the dot is conjugated to, some particular combinations are advantageous. For example, when the probe is designed to detect a deletion sequence, a strong QD signal is preferential to maximize the detection. Conversely, for an abundant target, a less strong signal will suffice.

IV. Methods of Making Exemplary Conjugates

A. Repeat-depleted probe production

A sample of target DNA is digested with a protease into fragments suitable for PCR, and the fragments separated by agarose gel electrophoresis. The PCR fragments isolated by gel electrophoresis are purified, quantitated and mixed together in equimolar concentration for ligation to generate large molecules each containing many PCR fragments in no particular order. The DNA generated is amplified using random priming reactions, such as random-priming amplification of DNA using highly processive DNA polymerase with strand displacement activity. Phi29 DNA has been used to produce sufficient material for probe manufacturing. Amplified DNA is then labeled with the respective hapten.

B. ERG3p, ERG5p, PTEN, and CEN10 Probes

Target DNA and the Nick Translation nucleotide mix for the hapten were obtained and stored on ice until needed. A calculated volume of DNA, nucleotide mix and water were added to labeled containers, mixed and placed in a water bath at 15 °C for about 1 hour. DNA polymerase I was added followed by DNase I enzyme mix. Once an appropriate incubation period was completed an aliquot of the reaction mixture was added to EDTA, mixed, frozen on dry ice and stored at -70 °C.

The size of the fragments in the aliquot was determined by gel electrophoresis on 4% agarose gel. If the target size was achieved EDTA was added to the bulk reaction mixture, followed by sodium chloride solution and a 3-(N-Morpholino)propanesulfonic acid (MOPS) buffer, at pH 7.0. The products were purified on QIAGEN columns, precipitated, isolated by centrifugation and stored at -15 °C to -25 ° C.

ERG3p and ERG5p probes were developed to assess the rearrangements of the ERG gene loci. These probes were designed to target the neighboring centromeric region (317 kb) and telomeric region (370 kb) of the ERG gene, which flank the known breakpoint region of ERG gene. Repeat-depleted probe production was used to generate two exemplary probes. An ERG5p probe was labeled by nick translation using dUTP conjugated to digoxigenin (DIG) (Roche Applied Sciences, Indianapolis, IN) (1 :2 ratio of DIG-dUTP: dUTP), while an ERG3p probe was labeled using dCTP conjugated to 2,4 dinitrophenyl (DNP) (Ventana Medical Systems, Inc., Tucson, AZ, USA). 30ug/ml each of a ERG3p-DNP-labeled probe and a ERG5p-DIG-labeled probe were formulated with 3mg/mL human placental DNA in a formamide-based buffer in a dispenser.

A working embodiment of a PTEN probe was designed to target a 765 kb region of PTEN location (10q23.31), and was generated by the same technology as the ERG probes. The PTEN probe was labeled by nick translation using dUTP conjugated to thiazole sulfonamide (TS) (3: 1 ratio of TS-dUTP: dUTP). 20ug/ml of the PTEN-TS- labeled probe was formulated with 3mg/mL human placental DNA in a formamide-based buffer. 20ug/ml of the CENlO-NP-labeled probe was formulated in a formamide-based buffer in a dispenser.

A chromosome 10-specific pAl 0RP8 plasmid (ATCC, Manassas, VA, USA) that contains the centromeric region was used to generate an exemplary CEN10 probe. The CEN10 probe was labeled by nick translation using dUTP conjugated nitropyrazole (NP) (3: 1 ratio ofNP-dUTP: dUTP). 20ug/ml of the CENlO-NP-labeled probe was formulated in a formamide-based buffer in a dispenser.

C. QD: antibody conjugates

1. Preparation of QD655-SMCC linker

Disclosed embodiments of the present invention used quantum dot-conjugated antibodies. In certain embodiments, the quantum dot was conjugated to an antibody using a linker, such as an SMCC linker. One method for making such conjugates proceeds as follows. The quantum dot and SMCC linker were equilibrated to ambient temperature prior to opening the reagent containers. Appropriate amounts of the quantum dot and linker were provided. The linker was placed in a light-occluding container and dissolved in a suitable solvent, such as DMSO, to make a solution of about 10 mg/ml. If applicable, the least amount of borate buffer required was added to the quantum dot solution to ensure that the percent volume of SMCC-DMSO was <10%. A selected amount of SMCC- DMSO solution was added to the quantum dot solution and reacted at ambient temperature with agitation for a period of from about 60 to about 75 minutes. The sample was then loaded onto an equilibrated Sephadex G25 column, as part of a desalting procedure, and the column was eluted with an equilibration buffer (MES).

2. Preparation of Antibody-DTT

A desired antibody amount was determined, as was the requisite amount of DTT. DTT was dissolved in deionized water, and the appropriate amount of the DTT solution was added to the required volume of antibody. The mixture was reacted at ambient temperature with agitation for a period of from about 25 to about 30 minutes. The sample was then desalted on a Sephadex G-25 column, with elution using an equilibration buffer

(MES).

3. Conjugation

Appropriate amounts of antibody and quantum dot solutions required for conjugation were determined and then mixed together in an appropriately sized, light- occluding container. The components were allowed to react with agitation for a period of from about 60 to about 75 minutes at ambient temperature.

4. Purification (Superdex 200 Column Chromatography)

Conjugates made according to disclosed embodiments were then purified using a

Superdex 200 purification column. The column was eluted with an equilibration buffer, such as a borate buffer. The conjugate peak corresponding to the FWHM (full width at half max) of the conjugate peak was determined and the appropriate fractions were pooled.

5. Final concentration adjustment and storage

The quantum do antibody conjugate was diluted to a final concentration of 1.0 μΜ with borate buffer solution and mixed gently.

Using these techniques, a rat monoclonal anti-DNP (Clone 1 C7- 1 C7, Ventana) was conjugated to QD655; a mouse anti-DIG monoclonal antibody (Clone 1- 171-256, Roche Applied Science) was conjugated to QD565; a mouse anti-TS monoclonal antibody (Clone 13A06-01E1 1, Ventana) was conjugated to QD605; a mouse anti-NP monoclonal antibody (Clone 27F09-02F08, Ventana) was conjugated to QD525. All QDs were custom-made by Life Technologies, Carlsbad, CA, USA. All antibody conjugations were conducted using 30n PEGylated QD (Life Technologies) and purified monoclonal antibodies²⁶. 25 nM each of the QD-conjugated antibodies were mixed in a borate buffer based diluent in a dispenser. V. Method of Using Conjugates

Disclosed embodiments of the present invention can be used for biological assays of specimen targets, for example, in situ hybridization assays, particularly fluorescence in situ hybridization, and even more particularly QD ISH assays. Disclosed embodiments also are quite useful for multiplexed assays, that is where two or more assays for different targets are performed on the same sample. One embodiment of a multiplexed QD ISH assay is for an automated 4-color ERG/PTEN QD ISH assay performed on a

BENCHMARK^® ULTRA automated slide stainer (Ventana Medical Systems, Inc.).

Flow diagrams illustrating steps of exemplary processes according to the present invention, such as might be performed on an automated instrument, such as a

BENCHMARK^® ULTRA instrument, are provided by FIGS. 1A and IB, each of which is discussed in more detail below

A. Exemplary Process Embodiment of FIG. 1 A

With reference to FIG. 1A, the first step 12 concerns preparing an FFPE tissue sample. This is followed by deparaffinization using a mild detergent in step 14.

1. Pretreatment

The tissue samples can undergo a pretreatment regimen at step 16. In some embodiments the pretreatment comprised contacting the tissue samples with one or more pretreatment solutions. The pretreatment solutions can include, but are not limited to, a basic buffer, an acidic buffer and a protease solution. In some embodiments the tissue samples were heated in the presence of one or more of the solutions.

In a particular embodiment the tissue samples were first treated with an acid buffer, then a protease solution. In a more particular embodiment the tissue samples were treated with a basic buffer, then an acidic buffer, then a protease solution. The acidic buffer comprised water, sodium acetate trihydrate, sodium metabisulfite, glacial acetic acid, sodium citrate, citric acid, magnesium chloride, sodium dodecyl sulfate, ethylene glycol and ProClin 300. The basic buffer comprised water, Tris, EDTA disodium dehydrate, ProClin 950, Tween-20 and boric acid.

The samples may be heated in the presence of the basic buffer at a temperature of from about 60 °C to about 90 °C, typically from about 80 °C to about 90 °C, with certain exemplary working embodiments heating at a temperature above 82 °C. The heating time period can vary, but typically is from about 30 minutes to about 1 hour, and more typically about 48 minutes. For acidic buffer treatments, the tissue samples also were heated, such as at a temperature of from about 60 °C to about 80 °C, more typically 70 °C to about 80 °C, for a period of time of greater than 30 minutes, such as from about 30 to about 45 minutes, with certain exemplary working embodiments heating for a period of about 36 minutes. For protease treatment, the tissue samples also typically were heated, such as by treating the sample with a protease at a temperature within the range of from about ambient to at least about 50 °C, and typically at a temperature of about 37 °C.

2. Apply and hybridize probes

After pretreatment the probes are applied to the tissue samples in step 18. Where the target was genomic DNA, the DNA and probes are denatured in step 20. The probes are hybridized to their targets in step 22, followed by multiple stringency washings in step 24. In some embodiments these washes are done with a sodium citrate sodium chloride buffer.

3. Apply antibody conjugates

After the stringency washes the quantum dot: antibody conjugates are applied in step 26 and allowed to incubate. In some embodiments the samples are washed multiple times with a buffer comprising water, Tris, acetic acid, Brij35 solution, ProClin 300 and sodium hydroxide. In particular embodiments the samples are washed with the above buffer and a basic buffer containing a chelating agent, such as EDTA. In more particular embodiments the basic buffer comprised water, Tris, EDTA disodium dehydrate, ProClin 950, Tween-20 and boric acid.

4. Staining and viewing

After washing, DAPI was applied online in step 28, to counter-stain nuclei for imaging. The stained slides were coverslipped in Cytoseal60^® (Richard- Allan Scientific) and viewed on a fluorescent microscope with filters appropriate for the quantum dots used.

5. Optimization of DNA probe concentrations and their

corresponding QD:antibody conjugates

A study was designed to evaluate the optimal concentrations of 3pERG, 5pERG,

PTEN and CEN10 probes and their corresponding QD-conjugated antibodies (aDNP- Qd655 Ab, aDIG-Qd565 Ab, aTS-Qd605 Ab, and aNP-Qd525 Ab, respectively) on the Ventana BENCHMARK^® ULTRA platform.

Four probe concentrations (10, 20, 30, and 40 ug/ml for the 3pERG and 5pERG probe, respectively; and 5, 10, 20, and 30 ug/ml for the PTEN and CEN10 probe, respectively), and four antibody QD conjugate concentrations (6.25, 12.5, 25, and 50 nM for each of aDNP-Qd655 Ab, aDIG-Qd565 Ab, aTS-Qd605 Ab, and aNP-Qd525 Ab) were evaluated. One hundred and sixty (4 X 4 X 5 X 2 replicates = 160) slides were randomized and stained on one BENCHMARK ULTRA instrument for a total of 6 runs. A board- certified pathologist and development team evaluated the slides. A systematic data analysis approach was applied. First, two sets of reads were independently collected from both a pathologist and the assay development team. The intent was to ensure the adequacy of the measurement system conducted by a human using microscopic observation. Second, factorial design analysis was implemented to determine which factors have a significant effect on a response, and optimal settings of these factors. Third, traditional descriptive data analyses also were performed to capture any unusual events and failures at given tested conditions. By using all these methods, the optimal range of relative concentrations for the probes and Q-dot conjugated antibodies were identified, as indicated below in Table 1.

Table 1

The recommended conditions provided the staining performances stated below in

Table 2.

Table 2

For the abovementioned recommended conditions, 82.5% of the 40 slides demonstrated the best possible results for each of the 4 targets simultaneously (92.5% for 525, 90% for 565, 85% for 605 and 655, respectively).

A confirmative run was conducted under the recommended conditions using 2 lots of the reagents on another instrument. Similar performance was achieved on the 28 slides. In summary, the optimal concentration ranges for the 3pERG, 5pERG, PTEN and CEN10 probes and their corresponding QD-conjugated antibodies (aDNP-Qd655 Ab, aDIG-Qd565 Ab, aTS-Qd605 Ab, and aNP-Qd525 Ab, respectively) were established. 6. Optimization of Nick Translation Labeling Conditions for

Exemplary 4 Color 3pERG, 5pERG, PTEN and CENIO Probe and QD ASR assay

The range of labeling density (or labeling efficiency) for a dsDNA probe is approximately one in every 10-25 bases, depending on the individual haptens and anti- hapten antibodies. The labeling efficiency was empirically determined so that it: (1) does not significantly interfere with the hybridization efficiency of the probe to its target; (2) allows optimal enzymatic incorporation of the modified nucleotide into the probe; and (3) offers the most sensitive targets for indirect (immunological) detection, and maintains the lowest background noise as well.

An exemplary 4 Color 3pERG, 5pERG, PTEN and CENIO Probe and QD ASR assay used 4 probes that require 4 different haptens. DNP and DIG haptens were chosen for 3p ERG probe and 5p ERG probe. Thiazole sulfonamide (TS) and nitropyrazole (NP) were selected for the PTEN probe and the CENIO probe. Preliminary work proved the feasibility of nick translation labeling of CENIO and PTEN probes with NP and TS at a 1 :2 ratio of modified dUTP versus dTTP nucleotide mix. These 4 haptens were detected using their respective anti-hapten antibodies conjugated with 4 QDs, which create optical signals that allow direct detection and localization via fluorescence microscopy.

In contrast to DIG and DNP, little data was available for nick translation using NP and TS modified nucleotides, functionality of the NP and TS labeled probe on

hybridization, and the latter immunologic detection. The QD525/CEN10-NP assay signal intensity was found 1.85±0.73, which is below the acceptable criteria (> 2 for intensity) in a multi-ULTRA instrument study. Further work was required to confirm or optimize the nick translation procedures with these two new haptens.

Trials were conducted at different ratios of labeled nucleotide :unmodified nucleotide to optimize the labeling density for each probe combination individually.

Assays were run at 1 :2, 1 : 1, 2: 1 and 3 : 1 ratios to determine which ratio resulted in the best signal intensity and stromal coverage while maintaining an acceptable background. A board-certified pathologist adequately trained on interpreting QD stained slides reviewed and scored the slides. A Zeiss fluorescent microscope and appropriate filters for each QD target (655 for 3p ERG, 565 for 5pERG, 605 for PTEN, and 525 for or CENIO) were used for slide evaluation. Each slide was scored for signal intensity, background, and overall coverage. Based on the results, a 3: 1 ratio of modified dUTP:dTTP was recommended for PTEN-TS and CEN10-NP probe labeling. These trials confirmed that the current hapten labeling conditions for DNP (All DNP-dCTP) and DIG (1 :2 ratio of modified dUTP:dTTP) were appropriate for 3p ERG and 5p ERG probes. 7. Improvement of assay reproducibility on automated staining platforms

An exemplary 4-color 3pERG, 5pERG, PTEN, CEN10, and Q-dots ASR assay was performed on 12 Ventana BENCHMARK^® ULTRAs to evaluate the variance of the assay performance across different ULTRA instruments by controlling other variables (e.g. readers, reagents, tissues, and staining procedures). A board-certified pathologist adequately trained on interpreting QD slides reviewed and scored the slides. A Zeiss fluorescent microscope and appropriate filters for each QD target (655 for 3p ERG, 565 for 5pERG, 605 for PTEN, and 525 for or CEN10) were used for slide evaluation. A total of 144 slides/stains were completed, among which 59 stains passed, and 85 stains failed. These data suggested that failures may occur at both system-level (instrument-related) and assay- level. The average pass rate across the 12 ULTRA instruments was 41%. All of the ULTRA instruments were within the calibration period and were presumably in-spec. The baseline values of the staining profile (e.g. intensity, background, and coverage) for each target (3pERG, 5pERG, PTEN, and CEN10) were established on the passed and/failed slides.

Based on the above findings, several changes were made to improve the assay robustness: (1) optimization of nick labeling conditions, which significantly improved CEN10 and PTEN staining performance; (2) optimization of the concentrations for individual probes and antibodies; and (3) optimization of staining procedure to facilitate large-size, QD-conjugated antibodies to access their targets in FFPE tissues.

Thirteen ULTRA instruments were included in this study. Three (3) replicates for each of 10 prostatectomy specimens were stained in a full run (approximately 30 slides) on each of the thirteen ULTRA instruments. Among these specimens, eight (8) were benign cases, 1 had ERG break-apart, and 1 had homozygous PTEN deletion. A total of 386 slides were evaluated for staining performance from the 389 stained slides. The same pathologist evaluated these slides by applying similar scoring criteria as used in the previous study.

Based on these changes, a significant improvement was found in the overall sample pass rate processed by the automated staining platforms. More specifically, the overall pass rate increased from an average of 41% to about 91% for all 4 targets, with a 97% pass rate for a PTEN/CEN10 assay, and a 91% pass rate for ERG 3p& 5p assay. For the 2 abnormal cases, 100% agreement was achieved on PTEN and ERG gene status on the evaluated slides. B. Exemplary Process Embodiment of FIG. IB

FIG. IB provides process steps associated with a second embodiment of an automated QD ISH assay according to the present invention. Common references numbers used for FIGS. 1A and IB refer to common process steps, although certain modifications to these steps also provided substantially improved assay results. Further optimizations, additions, etc. to the exemplary process of FIG. 1A are reflected in the process of FIG. IB. Primary concerns were to increase the reliability of assay results and to reduce, or substantially eliminate, background in QD FISH assay.

The first deparaffinization step can be important as residual paraffin can produce ISH background. Accordingly, to decrease the presence of residual paraffin, longer incubation times, such as from greater than 12 minutes to at least about 36 minutes, more typically from about 16 minutes to at least about 32 minutes, allowed thorough dissolution of paraffin from tissue sections. Moreover, each commercial paraffin composition used with tissue samples has its own particular melting temperature. Accordingly, the process heating temperature was increased from greater than 60 °C to at least about 75 °C, more typically greater than about 69 °C up to at least about 72 °C, to cover melting points of all currently available paraffin products on the market. Using these two approaches, substantially less paraffin residue remained on the slides relative to the exemplary embodiment of FIG. 1A; and correspondingly, less background was observed resulting from residual paraffin-covered areas.

FIG. IB also indicates the addition of several Tris buffer-based solution (CC1, Ventana) applications relative to the exemplary process of FIG. 1A. One reason for this is to reduce, or substantially eliminate, QD quenching.

1. Heavy Metals may Diminish QD Fluorescence Signals

Occasionally, prior QD ISH assay slides from the same tissues resulted in partial or total loss of QD fluorescence signals under the same assay conditions. The loss of QD fluorescent signals might be due to either: (1) the QD-conjugated anti-hapten antibodies failed to bind to their targets; or (2) the QD-conjugated anti-hapten antibodies bound to their targets but failed to illuminate. To determine which, six test slides were selected that were stained with a PTEN-TS probe and mouse anti-TS-QD605 but failed to show visible

QD605 signal. To determine whether the mouse anti-TS-QD605 antibody was present at the target sites in tissue, the six slides were incubated with an anti-mouse antibody to detect the mouse anti-TS-QD605 antibody. The anti-mouse secondary antibody was conjugated to alkaline phosphate for fast red detection. The specific nuclear red signals revealed presence of the QD605-conjugated antibody at the target sites. This established that the mouse anti-TS-QD605 antibody associated appropriately with the target but failed to illuminate.

To test the second possibility, that QDs associated appropriately with the desired target fail to illuminate, mouse anti-TS-QD605 antibodies were incubated in certain reaction buffer solutions and QD605 fluorescence was measured using a QD Fluorescence

96 Plate Assay. QD605 fluorescence was significantly decreased (from > 800 to approximately 20 absorbance unit) after incubating in certain reaction buffers. Similar results were obtained for the other QD-conjugated antibodies.

QD fluorescence is susceptible to quenching by various heavy metal ions, presumably by incorporation into the nanocrystal. These trace amounts of metal contaminants might originate from a number of sources including, for example from: trace metal impurities of reagents used in the assay or buffer preparation; processing of tissue specimens; from staining processes; etc. The luminescence properties of QD are closely related to the nature of their surface. Fluorescence quenching is believed to result from modification of atoms and molecules located near the QD surface. The QD quenching process turns "bright" QDs into "dark" QDs. Quenching can result from different processes, such as energy transfer, electron transfer, etc. Some heavy metal ions, such as Cu²⁺, Zn²⁺, Fe³⁺, Hg²⁺, and Ag⁺ quench QD fluorescence. This quenching may be attributed to the metal ions nonspecifically binding to the QD surface, thereby changing its surface chemistry. By titrating heavy metal cations into the staining solutions used for flow cytometry, Zarkowsky et al. (Cytometry Part A, 79 (201 1), pp. 84-89) found that 0.0 luM Cu²⁺ quenched 50% of QD655 fluorescence; andl μΜ of Fe³⁺ and Zn²⁺, and 0.1 μΜ of Cu²⁺, completely eliminated QD 655 fluorescence. A sharp intensity decrease is observed when Cu²⁺ concentration is greater than about 0.03 uM. Fluorescence intensity is reduced to 50% of its initial value with about 0.5 mM Cu²⁺. With copper concentrations of

>20 mM all accessible QD are quenched.

The inhibitory concentrations of most metal cations are too high to be a concern as contaminants in typical tissue staining solutions, such as those used in automated ISH protocols. One method for addressing the QD quenching effect is to alter QD surface chemistry. For example, hydrophobic QD can be made water soluble by ligand exchange, such as exchange with bifunctional ligands (i.e. thiol or phosphine mono or multidentate ligands), or coating with amphiphilic polymers that contain both a hydrophobic segment (e.g. hydrocarbons) and a hydrophilic segment (e.g. polyethylene glycol [PEG] or multiple carboxylate groups). For the former method, Wu CS et al, [see, "Highly Sensitive Multiplexed Heavy Metal Detection Using Quantum-Dot-Labeled DNAzymes," ACS Nano 2010] introduced surface silanization onto QD to form a silica cell so that the QD could be isolated and protected. Invitrogen's QD have an amphiphilic polymer coating, and the affinity reagent is coupled via a functionalized PEG linker.

Analysis of certain reaction buffers for automated systems indicated that the presence of certain metal ions, particularly copper, was responsible for the failure to generate visible QD fluorescence signals. Accordingly, the effect of different

concentrations of cupric ions on QD fluorescence was examined by incubating QD- conjugated antibodies in reaction buffer with 0, 0.01, 0.07, 0.2, or 0.5 μΜ added cupric ions. A 0.07 μΜ concentration of cupric ions caused more than a 4-fold reduction (from -450 to 100 absorbance unit) on QD fluorescence. Higher concentrations (0.2 and 0.5 μΜ) almost completely quenched QD fluorescence (approximately 10-20 absorbance units). The effect of different concentrations of cupric ions on QD ISH signals in FFPE tissues also was examined by incubating triplicate slides that were appropriately stained with ERG/PTEN 4-color QD ISH in certain reaction buffers with the addition of cupric ions at 0, 0.01, 0.07, 0.2, and 0.5 μΜ. QD signal was almost completely diminished in reaction buffer comprising 0.2 or 0.5 μΜ cupric ions. Non-uniform QD signal was observed in reaction buffer comprising 0.07 μΜ cupric ions. No QD signal effect was observed when the cupric ion concentration was 0.01 μΜ.

2. Addition of Chelating Agent(s) protects QD fluorescence in the presence of metal contamination

Metal/metal ion chelating agents provide another method for addressing metal/metal ion impaired QD fluorescence. For example, chelating agents comprising at least one, and typically multiple, acidic moieties, such as carboxylate, sulphonate, and/or phosphate groups, can be used for metal chelation in automated QD ISH process fluids. One example, without limitation, of a suitable chelating agent, is

ethylenediaminetetraacetic acid (EDTA).

EDTA has been found useful for decreasing QD fluorescence quenching when used in various concentrations ranging from greater than 0 up to at least 500 micromolar (μΜ), more typically from about 10 to about 100 μΜ, with certain working embodiments using 20 μΜ EDTA. The QD fluorescence protective effect of metal chelators has been repeatedly demonstrated, such as for a ERG/PTEN QD ISH assay with approximately 0.5 mM, as well as other QD ISH assays, such as HER2, ALK and HPV16 using 20 μΜ EDTA. Moreover, marginal reduction of QD fluorescence (approximately 2- fold) has been observed with EDTA in the absence of Cu²⁺. All ERG/PTEN QD ISH tests using 0.5mM EDTA showed improvement and/or no negative impact on fluorescence, no matter whether they had detectable Cu²⁺, potential metal ions (manifested as reduced

fluorescence) or no obvious abnormality. Thus, at least relatively small amounts of EDTA (20uM) can be used for staining protocols, such as for HER2, ALK, and HPV16 QD ISH assays.

Furthermore, little or no deleterious effects have been noted for slides produced using the protocol of FIG. IB even when stored for at least 5 months. These results establish that using chelating agents, such as EDTA, during the QD ISH assay helped preserve QD signal degradation.

Additional information concerning metal contamination and the positive effects of using a chelating agent, such as EDTA, is provided below in the working examples.

Based on the results discussed above, the exemplary process of FIG. IB includes the addition of CC1 at process step 16, process step 30 (5 times addition), 34, 38, and 42. Each of these steps occurs immediately after a rinse step, such as rinsing with reaction buffer. FIGS. 15 and 16 provide data establishing the beneficial results that are obtained by the addition of 0.5 mM EDTA to certain fluids used in the exemplary process of FIG.

IB. FIG. 8A - D provide QD signal intensity graphs obtained using an automated protocol on a first device after addition of a chelating agent (0.5 mM EDTA) for the exemplary process of FIG. IB. The scores associated with the addition of the chelation agent are represented with the (+) on the x-axis. The y-axis is the associated signal intensity score. The change was also considered on a second instrument and similar, although not as dramatic, changes were observed. In particular, the average intensity for the (-) runs were around 1.5 - 2 for all of the different QDs, while the average intensity for the (+) runs were around 2 - 2.5. For each QD tested, the addition of the chelating agent resulted in at least about 0.5 increase is signal intensity. While the magnitude of the improvement is considerable, one important aspect of the chelating agent is its ability to salvage a QD signal when it is completely quenched without (see FIG. 8A - B). It was discovered that the fluorescence intensity of each of QD525, QD565, QD605, and QD655 increased on two separate automated systems with the addition of a chelating agent, such as EDTA.

Example 21 and FIG. 9 provide additional data establishing the beneficial effects for disclosed assays, such as the exemplary process illustrated by FIG. IB. FIG. 9 is a graph of fluorescent intensity versus various concentrations of EDTA added at 500 μΜ, 100 μΜ, 20 μΜ, 4 μΜ 0.8 μΜ and 0.16 μΜ) during the exemplary automated protocol of FIG. IB. The graph has fluorescence intensity on the y-axis and various conditions tested on the x-axis. In particular, of each cluster of bars, the different EDTA concentrations are shown as a separate bar. The order of the concentrations is unchanged from cluster to cluster, despite only being labeled once. The clusters of bars each represent a different concentration of added Cu²⁺ ion as indicated by the recitation of the concentration above the cluster. In particular, the addition of 0.1 μΜ, 1 μΜ and 10 μΜ Cu²⁺ was tested. The data of FIG. 9 establishes that 20 μΜ was effective for all concentrations tested. It was also discovered that the addition of 20 μΜ chelating agent increased the pass rate for an automated device from 71% to 100%. It was also discovered that QD525 intensity, QD565 intensity, QD605intensity, and QD655 intensity increased using an automated staining protocol and system with the addition of chelating agent for all QD tested.

FIG. IB also indicates modifications to steps 16, 20 and 26 relative to FIG. 1A. FIGS. 7 A - D, which are Pareto charts, illustrate the contributions of various changes to QD 525, 565 605, and 655 intensity using one embodiment of a disclosed QD ISH procedure. The change indicated by "A" in FIG. IB is a 92 minute pretreatment using CC1, primarily for reasons discussed above concerning improving QD-to-target accessibility . B is a temperature increase from 80 °C up to at least about 85 °C during denaturing of target DNA and applied probes; and C is an increased incubation time of from 16minutes up to at least about 60minutes for applying QD conjugates. Without being limited to a theory of operation, step A appears to be the main contributor for improving the accessibility of the target to the QD conjugate, thereby increasing the reliability of QD ISH assays.

3. Reducing Background in Staining Images

a. Spotting and Dusting

Dusting and spotting are two problems that may result during an assay, such as a QD ISH assay. A dusting example is provided by FIG. 10. A spotting background example is provided by FIG. 1 1. Disclosed embodiments of the present application also reduce, or substantially eliminate, the deleterious problems associated with these types of background defects, thereby further increasing the efficacy and utility of QD ISH assays.

b. Reducing QD Aggregation

One potential cause for spotting is QD aggregation. The large-size QD655 background staining with no specific signal correlates well to fluorescence quenching phenomenon that water-soluble CdSe quantum dots exhibit upon aggregation. The dispersibility (or colloidal stability) of QD depends at least in part on coordination between the ligands and the semiconductor core surfaces. The dissociation and re-coordination of ligands to the core surface in solution is a dynamic process, in which "normal" aggregates can be detected with dynamic light scattering. Certain suboptimal conditions, such as low H, high salt concentration, gelatin, dextran sulfate, etc., may favor ligand dissociation, and hence more aggregates form. The aggregates precipitate when they are large enough.

Thus, certain disclosed embodiments of the present invention reduce spotting by maintaining a low ionic strength in QD ISH assay process solutions. For example, prior procedures were modified by reducing, or least substantially eliminating, salts from buffer process solutions. Accordingly, certain disclosed embodiments employ a QD incubation step using a QD diluent-borate buffer comprising about 50mM [Na⁺] or less.

Physical adsorption also can be used to stabilize nanoparticles. Certain stabilizing or dispersing agents, such as bovine serum albumin (BSA), casein and/or halide ion, particularly fluoride, can be used to help stabilize nanoparticles for use in QD ISH assays.

Bovine serum albumin (BSA) in saline solution can eliminate or substantially reduce non- covalent interactions between nanoparticles and efficiently improve colloidal stability. In trials, BSA did not block all general background as well as casein; however, casein formed QD aggregates in solution. Invitrogen notes that Casein can cause quenching of QD® conjugate". Inorganic ion [F ] disassembled CdTe QD aggregates and produced high colloidal stability, thereby providing hydrophilic aggregate-free QD. The F^" ions also greatly eliminated the nonspecific adsorption of QD on glass slides and cells.

For certain disclosed embodiments, QD aggregation in the presence of casein was addressed by using a pre -blocking step designed to allow incubation of the tissue section with casein for an effective period of time of greater than zero minutes to at least about 30 minutes, with certain working embodiments using a casein pre-blocking step of about 20 minutes. The casein was then washed away before introducing the QD-antibody conjugate. The incidence of QD background spotting, particularly QD655 spotting background, when using both low ionic strength QD solutions and a casein preblock is substantially reduced, if not entirely eliminated, relative to processes that do not use these two steps.

4. Blocking

Furthermore, in contrast to its performance in QD incubation, BSA demonstrated effective blocking in pre-hybridization and hybridization steps. The effectiveness of both blocking steps (probe and QD-antibody incubation) was confirmed for metaphase spread analysis. Background was largely reduced with these blocking steps. Metaphase chromosomes have been identified as difficult targets for QD ISH assays, presumably due to condensed chromatin structure in the nuclei. Disclosed embodiments of the present invention allow reliable QD ISH assays of metaphase spread. 5. Reduced Background of ERG/PTEN QD FISH staining on prostate tissue

The impact of several test conditions on QD background was tested on 30 consecutively cut slides from one prostate specimen. Based on these results, QD background reduction can be achieved using any one of the following process

modifications, or two or more of such modifications in combination: (1) using modified deparaffinization comprising increased deparaffinization times of from about 12 to at least about 32 minutes, more typically from about 16 minutes to at least about 28 minutes, and temperatures of from about greater than 60 °C to at least about 75 °C, more typically greater than about 69 °C up to at least about 72 °C, to improve efficient paraffin dissolution; (2) using one or more blocking agents, such as an effective amount of a BSA blocking buffer in an amount greater than 0% to at least about 5% BSA, during pre-probe hybridization, during probe hybridization, or both; (3) using a blocking buffer, such as a casein-blocking buffer, prior to QD detection; and (4) using borate buffer as a QD diluent during QD-antibody incubation.

Using disclosed embodiments of the present invention, 3 prostate specimens were stained using 4 BENCHMARK ULTRA instruments. Seventy-two (72) slides were tested. Disclosed embodiments of the present invention significantly improved background staining (p<0.05) of all 4 QD tested. There was a substantial elimination of dusting and of spotting using the procedure of FIG. IB.

VIII. Working Examples

The following examples are provided to illustrate certain features of working embodiments. A person of ordinary skill in the art will appreciate that the scope of the invention is not limited to the particular features exemplified by these examples.

Example 1

Synthesis of ERG5'-DIG conjugate

ERG 5 ' DNA and 10X Nick Translation nucleotide mix for DIG was thawed at room temperature (17°C to 28°C). The thawed reagents were held on ice until needed. A water bath was set to 15 °C, and the qualified reaction time for plasmid DNA was recorded from a container of DNase I enzyme mix.

Qualified Plasmid Time x 0.75 = Reaction Time for ERG5 ' Labeling Reaction Formulation: The calculated volume of DNA, nucleotide mix and water were added to the labeled container(s). After mixing the containers were placed in the 15 °C ± 1 °C water bath for 45-60 minutes.

DNA Polymerase I was added, followed by the calculated volume of DNase I Enzyme Mix. The reaction was mixed gently, then returned to the 15°C ± 1 °C water bath and incubated for the qualified reaction time.

1 mL of 20 mM EDTA was prepared by combining 0.04 mL of 0.5 M EDTA pH 8.0 and 0.96 ml of water, and mixed well. For each reaction container, 100 μΐ_^ of this solution was added to a microcentrifuge tube.

Once the incubation was complete, a 100 μΐ_^ aliquot was removed from each reaction container and added to one of the 100 μΐ_^ EDTA tubes, and mixed well. The remaining material from the incubation was frozen in a dry ice/alcohol mixture to stop the reaction.

In Process Test: 30 μΐ_^ of solution was transferred from each tube to a fresh and properly-labeled microcentrifuge tube. 3.3 μΐ_^ of 10X Gel Loading Solution was added to each tube.

After centrifugation in a microcentrifuge for approximately 10 seconds each, the size was determined by gel electrophoresis using a 4% agarose gel. The samples were run until the dye front had migrated 2/3 of the way down the gel or as long as necessary to separate the bands. The DNA was nicked so that the majority of the smear was between 100 bp and 400 bp for 5p ERG Probe DNA.

Reaction Termination and Purification of labeled DNA: 0.5 M EDTA at pH 8.0 was added to the bulk containers of frozen DNA solution. These were placed in a 25 °C water bath shaker and the total volume of labeled DNA was recorded. The DNA sample(s) were adjusted to 750 mM NaCl, 50 mM MOPS, pH 7.0 using the formulas provided below:

Vol. of labeled DNA (mL) ÷ 3 = 10X MOPS, pH 7.0 (mL) Vol. of labeled DNA (mL) + Vol. of 10X MOPS (mL) = Total Volume (mL)

Total Volume (mL) x 0.0584 x 0.75 = Amt. of NaCl (g) After the additions the solutions were mixed well. DNA was purified in QIAGEN- columns, precipitated and then centrifuged. The supernatant was removed, the pellets were washed and then stored at - 15 °C to -25 °C. Example 2

Synthesis of ERG3'-DNP conjugate

An ERG3'-DNP conjugate was synthesized by the same method as the ERG5'- DIG conjugate above, using an ERG3 ' DNA template and DNP Nick Translation mix containing all DNP-dCTP (no dCTP).

Example 3

Synthesis of PTEN-thiazole sulfonamide (TS) conjugate

A PTEN-thiazole sulfonamide (TS) conjugate was synthesized by the same method as the ERG5'-DIG conjugate above, using a PTEN DNA template and TS Nick Translation mix containing a 3: 1 ratio of TS-dUTP: dUTP.

Example 4

Synthesis of CENlO-nitropyrazole (NP) conjugate

A CENlO-nitropyrazole (NP) conjugate was synthesized by the same method as the ERG5'-DIG conjugate above, using a CEN10 DNA template and NP Nick Translation mix containing a 3: 1 ratio ofNP-dUTP: dUTP.

Example 5

Synthesis of rat anti DNP-QD655 conjugate

Equilibrate the Sephadex G-25 (Fine Grade Media) desalting columns with MES buffer. Record the equilibration volumes. Coordinate the timing of the QD655-SMCC and Ab-DTT reactions such that the antibody reduction is completed just prior to the completion of the nanocrystal activation reaction.

Preparation of QD655-SMCC Linker: QD655 and SMCC linker are equilibrated to ambient temperature prior to opening. The amount of QD655 starting material was calculated and the QD655 was centrifuged at approximately 800 x g (3000 rpm for standard microcentrifuge) for approximately 4 minutes. The supernatant was collected for conjugation in polypropylene tubes.

Calculate amount of SMCC linker: The least amount of SMCC linker required was weighed and placed into a light-occluding container. The SMCC was dissolved in the calculated volume of DMSO to make a solution of 1 Omg/ml, and then vortexed for 60 to 120 seconds.

The amount of SMCC-DMSO solution required was calculated [SMCC Linker Required (mg) ÷ 10 (mg/mL)] and it was determined if the SMCC-DMSO required exceeded 10% of the total volume. % Vol of SMCC-DMSO = SMCC-DMSO required (mL) ÷ (QD655 Vol after spin (mL) + SMCC Vol required (mL)) x 100%

If the value above (% Vol of SMCC-DMSO) exceeded 10% then the amount of borate buffer required to add to the QD655 solution was calculated. If the value was <10% then the calculation below was not applicable.

SMCC-DMSO required (mL) ÷ 0.1 - QD655 Vol after spin (mL) - SMCC- DMSO required (mL)

If applicable, the least amount of borate buffer was calculated for the required QD655 solution. The required volume of SMCC-DMSO was added to the QD655 solution, the reaction mixture was placed on an orbital shaker or rotator and reacted at ambient temperature for 60 to 75 minutes. Using equilibrated Sephadex G25 column, sample was loaded onto the column as part of the QD-SMCC desalting method, and the column eluted with the equilibration buffer (MES). The QD655 peak was identified and the appreciate fractions pooled corresponding to that peak.

A L IO dilution of the desalted QD655 in MES buffer was prepared. A spectrophotometer was blanked with MES buffer and absorbance values of the QD655 sample were determined at 638nm. If the reading did not fall between 0.1 and 1.0, a new dilution was prepared so that the max value fell within this range. The chosen dilution was confirmed by:

(mL sample per dilution + mL MES per dilution ) ÷ mL sample per dilution =

Chosen dilution factor

The concentration of the functionalized QD655 was determined as follows.

Avg. A638 for QD655 ÷ 800,000 x Chosen Dilution Factor x 1,000,000 =

Preparation of Antibody-DTT: The total amount (mg) of antibody (Ab) required was calculated.

Batch size (nmol) X 1.2 mg Ab/nmol Batch

The volume of antibody required (for single antibody lot) was calculated.

Total mg Antibody required (mg) ÷ Antibody concentration (mg/mL)

The required quantity of DTT was calculated.

Total mL Antibody required (mL) X 4.05 mg DTT / mL Antibody

The DTT was dissolved in the amount of DI water calculated below.

DTT required (mg) ÷ 77 mg/mL

The DTT solution was added to the required volume of antibody, and allowed to react at ambient temperature on an orbital shaker or rotator for 25 to 30 minutes. Sample was loaded onto an equilibrated Sephadex G-25 column as part of the Ab-DTT desalting method. The column was eluted with equilibration buffer (MES).

The antibody peak was determined, and appropriate fractions were pooled.

A 1 : 10 dilution of the desalted antibody was prepared in MES buffer. A spectrophotometer was blanked with MES buffer and absorbance values were measured at

280nm. If the reading did not fall between 0.1 and 1.0, a new dilution was prepared so that the absorbance fell within this range. The chosen dilution factor was confirmed as follows:

(mL Ab sample per dilution +mL MES per dilution ) ÷ mL Ab sample per dilution

= Chosen dilution factor

The concentration of the functionalized antibody was calculated.

Avg. A280 for Antibody ÷ 1.4 x Antibody Dilution Factor Conjugation: Using the volumes and concentrations of pooled QD655 and pooled antibody, the amounts of each activated material was calculated.

a. Volume Pooled QD655 (mL) X Concentration pooled QD655 (μΜ) b. Volume pooled antibody (mL) X concentration pooled antibody (mg/mL)

The amount of antibody required was calculated based on the total nmol of QD655 available as well as the amount of antibody available for conjugation.

a. Total QD655 Amount (nmol) X 4

b. Total Antibody Amount (mg) ÷ 0.15 (mg/nmol) If antibody was limiting (i.e. Antibody Available < Antibody Required), the process proceeded to step A. If the QD655 was limiting (i.e. Antibody Available > Antibody Required), the process proceeded to step B.

Step A. The amount of QD655 needed for conjugation was calculated, a. antibody available for conjugation x 0.25 nmol QD655/nmol Ab = amount of QD655 required (nmol)

b. amount of QD655 required (nmol) ÷ concentration of pooled QD655 (μΜ) = amount of

QD655 needed for conjugation (mL)

The volume of QD655 required for conjugation was added to the entire volume of antibody in an appropriately sized light-occluding container, and the process proceeded to Step C.

Step B. The amount of antibody needed for conjugation was calculated, a. antibody required for conjugation (nmol) x 0.15 mg Ab/ nmol Ab = mg antibody required for conjugation (mg)

b. mg antibody required for conjugation (mg) ÷ pooled antibody concentration (mg/mL) = amount of antibody needed for conjugation (mL) The entire volume of QD655 was adde4d to the volume of antibody required for conjugation in an appropriately sized light-occluding container.

Step C. The volume of QD655-Ab reaction mixture was calculated, and the reaction vial was placed on the orbital shaker or rotator and react for 60 to 75 minutes at ambient temperature.

Purification (Superdex 200 Column Chromatography): The reaction mixture may be concentrated using 50K molecular weight cut off centrifuge filters in order to reduce the sample size to the maximum Superdex 200 column load volume. Using the equilibrated Superdex 200 column, the sample volume was loaded onto the column as part of the QD655-Ab purification method, and the column eluted with the equilibration buffer (borate buffer).

Once purification was completed, the conjugate peak corresponding to the FWHM (full width at half max) of the conjugate peak was identified and appropriate fractions were pooled. A spectrophotometer was blanked with Borate buffer and the absorbance value of a neat sample at 638 nm was determined. If the reading did not fall between 0.1 and 1.0, a new dilution was prepared in borate buffer so that the absorbance fell within this range. Three samples at the chosen dilution or neat were prepared and the average absorbance value determined, and the concentration of the QD655-Ab Conjugate was calculated.

Avg. A638 for QD655-Ab ÷ 800,000 X Dilution Factor X 1,000,000

The conjugate yield was calculated:

Volume of pooled QD655-Ab (mL) X Concentration of pooled QD655-Ab (μΜ)

And the QD655 percent yield was calculated:

QD655-Ab conjugate final yield (nmol) ÷ QD655 required (nmol) x 100%

Final concentration adjustment and storage: The volume of borate buffer required to add to dilute the QD655-Ab conjugate to a final concentration of 1.0 μΜ was calculated, and the buffer added. The resulting composition was stored in foil at 2-8 °C and assigned an expiration date of one year from production.

Example 6

Synthesis of mouse anti DIG-QD565 conjugate

A mouse anti DIG-QD565 conjugate was synthesized by the same method as the rat anti DNP-QD655 conjugate above, using quantum dot QD565 and mouse anti DIG antibody. Example 7

Synthesis of mouse anti TS-QD605 conjugate

A mouse anti TS-QD605 conjugate was synthesized by the same method as the rat anti DNP-QD655 conjugate above, using quantum dot QD605 and mouse anti TS antibody.

Example 8

Synthesis of mouse anti NP-QD525 conjugate A mouse anti NP-QD525 conjugate was synthesized by the same method as the rat anti DNP-QD655 conjugate above, using quantum dot QD525 and mouse anti NP antibody.

Example 9

Tissue Samples

Ten (10) prostatectomy tissue samples were obtained from a tissue specimen archive maintained at Ventana Medical Systems, Inc. (Tucson, AZ, USA). Samples were redundant clinical specimens that had been de-identified and unlinked from patient information and therefore patient informed consent was not required²⁰. One hundred serial approximately 4 micron sections were cut for each sample, and #1 and #100 cuts were stained with H&E. Cases were included if both cuts had adequate epithelium and consistent pathological interpretation. Eight (8) cases were diagnosed as benign tissues, while 2 cases were prostate cancer. Thirty -nine (39) slides were first randomized selected from each of the 10 prostatectomy specimens, 3 slides from each of the 39 slides for the 10 cases were then randomly selected and distributed onto the 30 slide positions of 13 BENCHMARK^® ULTRA instruments (Ventana Medical Systems, Inc. Tucson, AZ, USA).

Example 10

Automated 4-Color ERG/PTEN QD In Situ Hybridization (ISH)

An automated 4 color ERG/PTEN quantum dot in situ hybridization (QD ISH) assay was performed on Ventana's BENCHMARK^® ULTRA instruments. The schematic diagram of assay configuration is illustrated in FIG. 1. FFPE tissue sections on slides were deparaffinized using a mild detergent (EZ-Prep^®, Ventana) at 69 °C for 72 minutes. Tissue pretreatment was conducted with a combination of heat- and proteolytic- induced epitope retrieval steps. First, tissue sections were incubated in the Tris buffer solution) at 90 °C for 92 minutes. Next, tissue sections were incubated in the citrate buffer solution at 82 °C for 36 minutes. Last, tissue sections were treated with a serine protease (Protease 3 , Ventana) at 37 °C for 28 minutes. After pretreatment, all four probes were added to the slides. Genomic DNA and the probes were denatured online at 85 °C for 8 minutes, followed by probe hybridization for 6 hours at 44 °C in Hybrizol^® (Ventana). After three stringency washes with SSC^® (Ventana) at 72 °C for 8 minutes each, the four QD- conjugated antibodies mixture was added and incubated at 37 °C for 60 minute. The slides were rinsed three times with a buffer comprising water, Tris, acetic acid, Brij 35 solution, ProClin 300 and sodium hydroxide. DAPI^® (Ventana) was then applied online to counter- stain nuclei for imaging. The stained slides were cover slipped in Cytoseal60^® (Richard- Allan Scientific).

Example 11

Automated 4-Color ERG/PTEN QD ISH for metaphase spreads

An automated 4 color ERG/PTEN QD ISH assay for metaphase spreads was performed on Ventana' s BENCHMARK^® ULTRA instruments. All four probes were added to FFPE tissue sections on slides that had not been deparaffinized or pretreated. Genomic DNA and the probes were denatured online at 85 °C for 8 minutes, followed by probe hybridization for 6 hours at 44 °C in Hybrizol^® (Ventana). After three stringency washes with SSC^® (Ventana) at 72 °C for 8 minutes each, the four QD-conjugated antibodies mixture was added and incubated at 37 °C for 60 minutes. The slides were rinsed three times with a buffer comprising water, Tris, acetic acid, Brij 35 solution, ProClin 300 and sodium hydroxide. DAPI ^® (Ventana) was then applied online to counter- stain nuclei for imaging. The stained slides were coverslipped in Cytoseal60^® (Richard- Allan Scientific).

Example 12

QD ISH Slide Review, Image Capture and Analysis

A Zeiss^® fluorescent microscope with appropriate filters was used for slide review. Custom- made filter sets (Chroma technology Corp., Bellows Falls, VT, USA) used in the study are listed in Table 3. Table 3

Filter configurations

The excitation wavelength for all the dots is 355-405 nm. Monochrome images were captured using a spectral imaging acquisition system (ASI; Applied Spectral Imaging, Israel), and photographs were taken using a SPOT CCD microscope digital camera (SN# 252371 ; Diagnostic Instruments, Inc., Sterling Heights, MI, USA). The layers of individual monochrome FISH signal were colorized and merged to provide overlay images for visualization of relative probe localizations using Image J (Wayne

Rasband, NIH).

Example 13

Analytical Slide Scoring Criteria

A board-certified pathologist with experience on interpreting the 4 color ERG/PTEN QD ISH stained slides reviewed and scored the slides. Each slide was scored for signal intensity, background, and coverage. An analytical slide scoring criteria was used to describe "Acceptable" or "Not Acceptable" staining. The "Acceptable" or "Not Acceptable" criteria correspond to the capability whether the ERG (or 565/655) or the PTEN/CEN10 (525/605) pairs of signals are enumerable in 50 cells on a slide. The scoring criteria were developed and used as a stringent analytical tool to optimize the assay.

Example 14

Signal Enumeration and Cell Classification of ERG

The signals were enumerated within the nuclear boundary of each selected epithelial cell according to the guidelines provided in FIG. 2 and FIG. 3. Nuclei containing signals of only one color should not be enumerated.

(1) Cells are considered negative for ERG gene rearrangement (FIG. 2): • When red (ERG3p/QD655) and green (ERG5p/QD565) signals are adjacent or fused (appearing yellow) (FIG. 2A; yellow signal indicated by an arrow).

• Red and green signals are less than two signal diameters apart (FIG. 2B). · There is a single green (ERG5p/QD565) signal without a corresponding red signal. (FIG. 2C; single green signal indicated by an arrow).

(2) Cells are considered positive for ERG gene rearrangement:

• At least one set of red and green signals are two or more signal diameters apart (FIG. 3A&3B).

· There is a single red (ERG3p/QD655) signal without a corresponding green signal in addition to fused and/or broken apart signals (FIG. 3C; single red signal indicated by an arrow).

• The same nucleus may have fused signals, broken apart signals and

deletions (FIG. 3D).

For each slide, 50 cells were enumerated and percentage of cells positive for ERG gene rearrangement was calculated.

Example 15

Signal Enumeration of PTEN and CEN10

For each slide, the number of PTEN and CEN10 signals was enumerated in 50 nuclei. The PTEN/CEN 10 signal ratio was calculated. For normal tissue, the ratio of

PTEN/CEN10 is expected close to 1.

Example 16

Verification of Specificity of the ERG3p, ERG5p, PTEN and CEN10 Probes

To verify that ERG3p, ERG5p, PTEN and CEN10 probes specifically bind to their target chromosomes, co-localization of ERG3p and ERG5p was demonstrated with an

Abbott/Vysis' LSP21 probe to the same chromosome (chromosome 21), and co- localization of PTEN and CEN10 probes with Abbott/Vysis CEP 10 to the same chromosome (chromosome 10). No extra signals of ERG3p, ERG5p, PTEN or CEN10 were observed on other chromosomes (data not shown). FIG. 4 shows a representative image of a normal metaphase spread stained with ERG/PTEN 4-color QD ISH on a

BENCHMARK^® ULTRA instrument. ERG3p-DNP labeled probe and ERG5p-DIG labeled probe were co-hybridized on the same chromosome, and detected with QD655 (red) and QD565 (green). Two pairs of red signals representing ERG3p were shown adjacent to the two pair of green signals representing ERG5p. PTEN-TS labeled probe and CEN10-NP labeled probe were co-hybridized on another chromosome, and detected with QD605 (pink) and QD525 (blue) respectively. No extra signals of ERG3p, ERG5p, PTEN or CEN10 probes were observed on other chromosomes.

Example 17

Efficient nuclear permeabilization for a 4-color ERG/PTEN QD ISH assay

The lack of reliability and/or reproducibility of the 4-color ERG/PTEN QD ISH assay initially was observed in a manner similar to that as described by others concerning quantum dot in situ hybridization assays. The intermittent experimental success was manifested as (1) identical experiments could often be perfectly successful on one day but unsuccessful on different days; or (2) one slide could be very well stained, but not the adjacently-cut slide identically processed in parallel. On average, the success rate of an initial 4-color ERG/PTEN quantum dot in situ hybridization assay was less than 50% of the time, based on the analytical scoring criteria. In attempts to improve the efficacy and reliability of the 4-color ERG/PTEN quantum dot in situ hybridization assay, the assay conditions were systematically investigated, including various pretreatment conditions (for nuclear permeabilization), probe hybridization conditions (e.g. probe concentration, denaturation temperature and time, stringency wash conditions, etc.) and quantum dot- conjugated antibody detection conditions (e.g. antibody concentration, incubation time, blocking steps, etc.). While optimization of probe hybridization or quantum dot detection conditions helps, pretreatment condition for nuclear permeabilization was most effective for reliable and reproducible quantum dot in situ hybridization staining.

Two pretreatment conditions were compared: (1) citrate buffer at 82 °C for 36 minutes followed by Protease 3 at 37 °C for 28 minutes; and (2) Tris buffer at 90 °C for 92 minutes followed by the citrate buffer at 82 °C for 36 minutes and then Protease 3 at 37 °C for 28 minutes. Thirty (30) consecutive 4 μηι sections were prepared from a prostatectomy case (VR-209- 1 1-3), and were stained with the 4-color ERG/PTEN quantum dot in situ hybridization on a BENCHMARK ULTRA instrument. Half of the slides were treated with Condition (1) and the other half slides were treated with Condition (2). The 15 slides treated with the citrate buffer and Protease 3 [Condition (1)] resulted in a wide range of signal intensity and staining coverage, in which none was acceptable for all the four targets. In contrast, the 15 slides treated with the Tris buffer in addition to the citrate buffer and Protease 3 [Condition (2)] resulted in consistently acceptable signal intensity (> 2) and staining coverage (>50%) for all the four targets on the 15 slides. Background and tissue morphology were acceptable for all the 30 slides and comparable between the two pretreatment conditions. Table 4 compares the average signal intensity and staining coverage for each QD staining between the two pretreatment conditions.

These results indicate that pretreatment with the citrate buffer and Protease 3 may not be sufficient for a reliable 4-color ERG/PTEN quantum dot in situ hybridization assay. Addition of the Tris buffer to the citrate buffer and Protease 3 pretreatment significantly improved QD ISH staining. It not only resulted in a greater average signal intensity and staining coverage for all the 4 targets, but also much less variances among the replicate slides.

Table 4

Signal intensity and staining coverage for the 4 targets

under the two pretreatment conditions

Example 18

Evaluation of the 4-color ERG/PTEN QD ISH assay performance on multiple

BENCHMARK ULTRA instruments

An exemplary 4-color ERG/PTEN quantum dot in situ hybridization assay with the improved pretreatment condition (the Tris buffer at 90 °C for 92 minutes followed by the citrate buffer at 82 °C for 36 minutes and then Protease 3 at 37 °C for 28 minutes) was evaluated on 13 exemplary BENCHMARK ULTRA instruments supplied by Ventana. A total of 389 slides from 10 prostatectomy specimens were stained. The 10 specimens were 8 benign prostate tissues and 2 prostate cancer cases. Triplicate slides of each case were placed on each instrument (total 30 slides per instrument). Out of the 389 slides 386 were evaluated for staining performance. Three slides were excluded from the analyses due to non-assay related causes. Overall 91% (350/386) of the slides resulted in acceptable staining for both ERG3p&5p and PTEN/CEN10. Thirty-six slides failed out of which 28 failures were due to "QD655 high background" and 8 failures were due to "weak or no signal".

The 350 slides with acceptable quantum dot in situ hybridization staining were evaluated for ERG and PTEN gene status. 280 slides were selected from the eight benign prostate cases, and 70 slides were from the two prostate cancer cases. Wild type ERG (1-2 fused signals per nucleus), PTEN (1-2 signals per nucleus) and CEN10 (1-2 signals) were consistently observed in all 280 slides from the eight benign prostate cases.

The representative images for a benign prostate case are shown in FIG. 5A - D.

FIG. 5A is the H&E staining of a normal gland. FIG. 5B is the ERG3p, ERG5p, PTEN and CEN10 4-color ERG/PTEN QD ISH staining. FIG. 5C is the 2-color image of ERG3p (red) and ERG5p (green) ISH staining. FIG. 5D is the 2-color image of PTEN (pink) and CEN10 (blue) ISH Staining. Of the two prostate cancer cases, ERG break-apart and PTEN deletion were consistently observed in the 36 slides of Case #VMSI106-C21D, while ERG break-apart and PTEN normal status were consistently observed in the 34 slides of Case #161 168T21D.

The representative images for VMSI-106-C21D are shown in FIG. 6A - F. FIG. 6A is the H&E staining. FIG. 6B is the 4-color image of ERG3p, ERG5p, PTEN and CEN10 QD ISH staining. FIG. 6C is the 2-Color image of ERG3p and ERG5p ISH staining. Single ERG 3p (red) and ERG5p (green) signals were observed in the nuclei of the tumor cells. FIG. 6D is the 2-Color image of PTEN (pink) and CEN10 (blue) ISH staining in both tumor and adjacent stromal tissue area. FIG. 6E and 6F are closer views of the PTEN and CEN10 signals from the tumor area or the adjacent stromal tissue area in FIG. 6D. PTEN signal is missing in tumor cells, only CEN10 signals are present (FIG. 6D

& E). Both PTEN signals and CEN10 signals are present in the adjacent stromal area (FIG. 6D & F). The PTEN staining at the stromal area serves as the internal control. Example 19

Detection of ERG and PTEN gene status

After the overall evaluation of the 350 slides, 19 slides were randomly selected from 2 benign and 2 prostate cancer cases for signal enumeration to determine ERG and PTEN gene status. They were stained on 3 BENCHMARK^® ULTRA instruments over 3 different days. For each slide, 50 nuclei were enumerated for ERG3p, ERG5p, PTEN and CENIO signals using a conventional fluorescent microscope. A triple-band filter (DAPI+565+655), a single-band filter (565), and a dual-band filter (DAPI+655) were used for ERG signal enumeration. PTEN and CEN 10 signals were enumerated using single- band filter (605) and dual-band filter (DAPI+525) respectively. The number of ERG3p and ERG5p fused signals, ERG3p single signals and ERG5p single signals were enumerated in each nucleus and ERG gene status was classified for each of the 50 cells (See FIGS. 2 and 3). Percent nuclei with ERG break-apart were calculated for each slide. The ratios of PTEN/CEN10 signals were calculated for 50 nuclei per slide.

The data demonstrates that the staining results of ERG and PTEN gene status are reproducible for the 4 cases stained in multiple days across 3 ULTRA instruments. For case VR-62-1 1-24 (benign prostate tissue), the percentages of ERG rearrangement positive cells was between 0-6% and the ratios of PTEN/ CENIO signals range between 0.94-1.0 for the 5 slides. For case 161168T21D (prostate cancer), the percentages of ERG rearrangement positive cells ranged between 78%-90% and the ratios of PTEN/ CENIO signals ranged between 0.8-0.9 for the 4 slides. For case VMSI106-C21D (prostate cancer), the percentages of ERG rearrangement positive cells ranged between 74%-88% and the ratios of PTEN/ CENIO signals are 0 for the 6 slides. For case VR1 1-330- 4(benign prostate tissue), the percentages of ERG rearrangement positive cells ranged between 0%-2.0% and the ratios of PTEN/ CENIO signals ranged between 0.9-1.1 for the 3 slides.

Example 20

Possible metal contaminants

One reason for some "weak or no signal" failures is the presence of trace metal contaminants. These metal contaminants may arise from a variety of sources, including reagents and solutions bought from suppliers. For example the MSDS for boric acid, supplied by Sigma- Aldrich, lists Iron < 5 ppm, magnesium < 5 ppm and heavy metals < 10 ppm. A metal contaminant such as copper at such low levels as 0.1 μΜ can significantly reduce the signal from the quantum dots. To overcome this possible contamination, slides were washed with a basic buffer containing EDTA before the DAPI staining. The EDTA improved the overall success rate, as illustrated below in Table 5.

Table 5

Example 21

The effect of ETDA on QD fluorescence in the absence or presence of cupric ions at different concentrations was considered using a QD Fluorescence 96 Plate Assay. QD605 fluorescence was measured of the mouse anti-TS-QD605 antibody in certain reaction buffers that contained cupric ions and/or EDTA at difference concentrations. In the absence of cupric ions, high level EDTA concentrations (100-500 μΜ of EDTA) caused moderate (approximately 55%) QD fluorescence loss (See FIG. 9). However, QD fluorescence decreased dramatically with increased cupric ion concentration (e.g. 1 or 1 OuM) when there was no EDTA in solution. The presence of a low amount of EDTA (e.g. 20uM) is effective to help prevent the loss of QD fluorescence caused by a high concentration (e.g. 1 or lOuM) of cupric ions (FIG. 9).

Example 22

This example examines whether the addition of EDTA during QD incubation on slide helped prevent QD ISH signal loss. This example also considered whether adding EDTA during QD incubation on slide would negatively impact QD ISH signal on automated instruments that otherwise had bright QD ISH signal.

On each of the several instruments, half numbers of slides were processed according to procedure (a) whereby QD-conjugated antibodies were incubated in the reaction buffer (no EDTA) on slide, and the other half numbers of slides were processed according to procedure (b) whereby QD-conjugated antibodies were incubated in CC1

(approximately 500 μΜ EDTA) on slide. Otherwise, the two staining procedures were identical. Complete or partial loss of QD ISH signals was observed for slides processed according to procedure (a) (no EDTA). In contrast, on the same instruments, slides processed according to procedure (b) provided visible and much stronger QD ISH signals. The addition of EDTA did not negatively impact QD ISH signals of the slides stained according to procedure b, and instead actually reduced variation among slides.

FFPE tissues may have some heavy metal contamination that can cause slide-to- slide variation of QD ISH signals. Using a chelating agent, such as EDTA, effectively chelated the metal ions in the tissue providing more consistent staining. Moreover, the QD ISH signals were still interpretable after the slides processed according to procedure b using EDTA were stored at room temperature for 5 months. These results establish that addition of a chelating agent, such as ETDA, during QD incubation on slides with FFPE tissues effectively protects QD signals from quenching.

Example 23

A. Tissue Samples

Six (6) prostate specimens, 5 breast cancer specimens, 5 lung cancer specimens, 3 tonsil specimens, and 3 cervical specimens with high grade CIN2,3 were obtained from a tissue specimen archive maintained at Ventana Medical Systems, Inc. (VMSI, Tucson, AZ). The other 10 prostate specimens were obtained from the same resource. The samples were redundant clinical specimens. Four (4) micron thick sections were used for ISH staining.

B. Probes

Information on probes and QD-conjugated antibodies are summarized below in Table 6. The ERG5p probe was labeled with digoxigenin (DIG), ERG3p probe with 2, 4 dinitrophenyl (DNP), PTEN probe with thiazole sulfonamide (TS), and CENIO probe with nitropyrazole (NP) (22). 30ug/ml each of the ERG3p-DNP-labeled probe and ERG5p- DIG-labeled probe were formulated with 3mg/mL human placental DNA in a formamide- based buffer in a dispenser. 20ug/ml each of PTEN-TS-labeled probe and CEN10-NP- labeled probe were formulated the same way in another dispenser. Table 6

ERG/PTEN 4-color QD FISH assay

The INFORM Her2 Dual ISH DNA Probe Cocktail dispenser was obtained from VMSI (Cat# 780-4422). The Her2 probe was labeled with DNP, while the chromosome 17 centromere was labeled with DIG (24) (Table 7).

Table 7

The ALK break-apart probe set was generated to hybridize the neighboring centromeric (5' probe labeled with DIG) and telomeric (3' probe labeled with DNP) sequence of the ALK gene. 15ug/ml each of the ALK5p-DIG-labeled probe and ALK3p- DNP-labeled probe were formulated in a formamide-based buffer in a dispenser (Table 8).

Table 8

Targets ALP3p ALK5p

Probe 15ug/ml of DNP-labeled 15ug/ml of DIG-labeled

ALK3p ALK5p

QD- 25nM of anti-DNP conjugated 25nM of anti-DIG conjugated conjugated to QD605 to QD565

antibody The HPV16 probe was DNP labeled from a plasmid with full length HPV16 genotype (GenBank #: K02718) cloned in vector pGEM-2. 30ug/ml of the HPV16 probe was formulated with 0.125mg/ml human placenta DNA in a formamide-based buffer in a dispenser (Table 9).

Table 9

HPV16 Single Color QD ISH Assay

The four (4) Kappa DNP Probes (Cat# 760-1201, 760-1202, 760-1203, and 760- 1204) and four (4) Lambda DNP Probes (Cat# 760-1205, 760-1206, 760-1207, and 760- 1208) were obtained from VMSI (Tucson, AZ). 3.2ug/ml of each of the 4 Kappa or Lambda DNP probes were mixed and formulated in a formamide-based buffer in a dispenser (Table 10).

Table 10

Kappa and Lambda mRNA Single Color QD ISH Assay

C. QD-conjugated antibodies

A rat monoclonal anti-DNP (Clone 1C7-1C7, Ventana) was conjugated to QD655 for the detection of ERG3p DNP probe (Table 1.1). The same anti-DNP antibody was also conjugated to QD605 for the detection of HER2 DNP probe (Table 7), ALK3p DNP probe (Table 8), Kappa and Lambda DNA probe (Table 10), and HPV 16 DNP probe (Table 9).

A mouse anti-DIG monoclonal antibody (Clone 1-171-256, Roche Applied Science) was conjugated to QD565 for the detection of CHR17 DIG probe (Table 7), ALK5p DIG probe (Table 8), and ERG5p DIG probe (Table 6). A mouse anti-TS monoclonal antibody (Clone 13A06-01E1 1, Ventana) was conjugated to QD605 for the detection of PTEN TS probe (Table 6). A mouse anti-NP monoclonal antibody (Clone 27F09-02F08, Ventana) was conjugated to QD525 for the detection of CEN10 NP probe (Table 6). All QDs were custom-made by Life Technologies, Carlsbad, CA, USA. All antibody conjugations were conducted using 30n PEGylated QD (Life Technologies) and purified monoclonal antibodies. For Her2/Chrl7 dual-color QD ISH assay and ALK break-apart dual color QD ISH assay, 25nM each of the two QD-conjugated antibodies were mixed in a borate buffer based diluent in a dispenser (Table 7). For the ERG/PTEN 4-color QD FISH assay, 25nM each of the four QD-conjugated antibodies were mixed in a borate buffer based diluent in a dispenser (Table 6). For the Kappa or Lambda single-color QD FISH assay (Table 10) and HPV16 single-color QD FISH assay (Table 9), 40nM of the respective QD-conjugated antibody was mixed in a borate buffer based diluent in a dispenser. D. Automated QD Fluorescence In Situ Hybridization

Genomic targets - ERG/PTEN, HER2/CHR17, ALK break-apart, and HPV16: The automated QD in situ hybridization (QD ISH) assay was performed on Ventana's BENCHMARK® ULTRA instruments. The staining protocol was further improved according to the process of FIG. IB. The modified deparaffinization step started with ULTRA LCS (Ventana) at 58°C for 24minutes, by which the organic oil molecules dissolved paraffin in the tissue section. The dissolved paraffin was then rinsed out with a mild detergent (EZ-Prep, Ventana) at 69°C for 32 minutes. Tissue pretreatment was conducted as described. First, tissue sections were incubated in a basic Tris buffer-based solution (CC1, VMSI) at 90 °C for 92 minutes. Next, tissue sections were incubated in an acidic Citrate buffer-based solution (CC2, Ventana) at 82 °C for 36 minutes. Last, tissue sections were treated with a serine protease (Protease 3, Ventana) at 37 °C. Both breast and lung tissues were treated for 24 minutes, while prostate and cervical tissues were treated for 28 minutes. After pretreatment, 0.5% BSA was incubated for 28 minutes as blocking reagent before the respective probe/probe sets were added on to the slides.

Genomic DNA and the probes were denatured at 85 °C for 8 minutes, followed by probe hybridization for 6 hours at 44 °C in Hybrizol (VMSI). After three stringency washes with SSC (Ventana) at 72 °C for 8 minutes each, the tissue section was then treated 5 times with CC1 that contained ImM EDTA.

Several modifications were then made for the purpose of background reduction and QD fluorescence protection. First, a phosphate-based buffer that contained 13.4mg/ml of casein was incubated for 20 minutes as blocking reagent. It was then washed away with Reaction Buffer rinse twice. Second, the QD-conjugated antibody (in borate buffer) was dispensed to the slide without contact with the blocking buffer. Third, CC1 was present in each rinse and incubation step while the QD-conjugated antibody was introduced onto the tissue section. The QD-conjugated antibody was incubated at 37 °C for 60 minutes. The slides were rinsed three times with Reaction Buffer (Ventana), DAPI was then applied online to counter-stain nuclei for imaging. The stained slides were coverslipped in Cytoseal60® (Richard-Allan Scientific). E. Kappa Lambda mRNA targets

The front portion of the staining protocol was tailored for optimal mRNA QD detection. The deparaffinization step started with ULTRA LCS (Ventana) incubation at 58 °C for 8 minutes, followed with EZ-Prep rinse 3 times at 69 °C for 12 minutes. After having prefixed with formalin for 8 minutes, tissue sections were incubated in CC1 at 90 °C for 32 minutes, followed with Protease 3 at 37 °C for 12 minutes. A post-fixation step was optional. 0.5% BSA was applied and incubated for 28 minutes before the probe was added. Denaturing occurred at 90 °C for 4 minutes, followed by a 5 °C temperature decrease every 4 minutes until 55 °C. The probe was then hybridized to its mRNA target for 2 hours at 37 °C, and washed 3 times with SSC at 60 °C for 8minutes each. The rest of the treatments were the same as described for the genomic targets.

F. QD ISH Slide Review, Image Capture and Analysis

A Zeiss® fluorescent microscope with appropriate filters was used for slide review. Custom- made emission filter sets (Chroma technology Corp., Bellows Falls, VT, USA) were used in the study.

The excitation wavelength for all the QD is 355-405 nm. All emission filter sets possessed the same excitation filter bandwidth. Monochrome images were captured using a Spectral Imaging acquisition system (ASI; Applied Spectral Imaging, Israel), and photographs were taken using a SPOT CCD microscope digital camera (SN# 252371 ; Diagnostic Instruments, Inc., Sterling Heights, MI, USA). The layers of individual monochrome FISH signal were colorized and merged to provide overlay images for visualization of relative probe localizations using Image J (Wayne Rasband, NIH).

A board-certified pathologist (P.B.) with experience interpreting QD ISH stained slides reviewed and scored the slides. For the genomic targets Her2/CHR17, ALK3p and ALK5p, and ERG3p, ERG5p, PTEN and CENIO, each slide was scored for signal intensity and background. Staining performance was evaluated for the presence of HPV16 and Kappa and Lambda mR A signals on the QD ISH stained slides.

FIG. 12 provides an interval plot of staining intensity for QD605; FIG. 13 is an interval plot of staining intensity for QD565; and FIG. 14 is a photographic image illustrating results for HER2/QD605 (red) and CHR17/QD565 (green). FIGS. 12-13 illustrate the reliable utility for HER2 and CHR17 QD ISH analysis using disclosed embodiments of the present invention.

FIG. 15 is a photographic image illustrating staining results for 5pALK/QD605 (red) and 3pALK/QD565 (green). FIG. 16 is an interval plot of staining intensity for 5pALK/QD605; FIG. 17 is an interval plot of staining intensity for 3pALK/QD565; and FIGS. 15-17 illustrate the reliable utility for 5pALK and 3pALK QD ISH analysis using disclosed embodiments of the present invention.

FIG. 18A - C are photographs showing the use of the disclosed methods for the analysis of HPV. FIG. 18A provides H&E staining, FIG. 18B provides the

HPV16/diaminobenzidine (DAB) staining, and FIG. 18C provides HPV16/QD605 FISH results. These figures illustrate the reliable utility for HPV 16 QD ISH analysis using disclosed embodiments of the present invention.

FIG. 19 is a photographic image illustrating QD ISH results for HER2/QD605 (red) and CHR17/QD565 (green) obtained using disclosed embodiments of the present invention. FIG. 20 is a photographic image illustrating QD ISH results for 3pALK/QD565 (green) and 5pALK/QD605 (red) using disclosed embodiments of the present invention. FIG. 21 is a photographic image illustrating QD ISH results for Kappa mRNA/QD605 (red) using disclosed embodiments of the present invention. FIG. 22 is a photographic image illustrating QD ISH results for Lambda mRNA/QD605 (red) using disclosed embodiments of the present invention. FIGS. 19-22 establish the reliable utility for QD ISH analysis of exemplary specimen targets. A person of ordinary skill in the art will appreciate that the presently disclosed embodiments can be used for QD ISH analysis of targets in addition to these exemplary targets.

In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

PATENT CLAIMS

1. A quantum dot in situ hybridization method, comprising:

pretreating a sample according to a pretreatment regimen comprising a protease and one or more pretreatment solutions selected from a basic buffer and an acidic buffer, where each of the protease and one or more pretreatment solutions is used for an effective period of time at an effective temperature;

contacting the sample with at least one haptenated probe that binds specifically to different targets, wherein each probe is labeled with a different hapten;

contacting the sample with at least one anti- hapten antibody:quantum dot conjugate comprising a quantum dot that can be detected selectively; and

detecting fluorescence signals from the quantum dots after excitation;

whereby the method provides an increased sample pass rate relative to a process that does not use the pretreatment regimen.

2. The method according to claim 1 or 2 further comprising using both the basic buffer and the acid buffer for the pretreatment regimen.

3. The method according to claim 1 further comprising adding a chelating agent to chelate metal impurities.

4. The method according to claim 3 where the chelating agent is added during the step of contacting the sample with anti-hapten antibody:quantum dot conjugates.

5. The method of any of claims 1 to 4 comprising treating the sample with the protease at a temperature within the range of from about ambient to at least about 50 °C.

6. The method according to claim 5 wherein the temperature is about 37 °C.

7. The method of claim 5 or 6 wherein the sample is treated with the protease for an effective period of time of greater than about 10 minutes.

8. The method of any of claims 1 to 7 wherein the sample is treated with the basic buffer solution at an effective treatment temperature of from about 60 °C to at least about 95 °C.

9. The method of any of claims 1 to 7 wherein the sample is treated with the basic buffer solution at an effective treatment temperature of from about 80 °C to about 90 °C.

10. The method of any of claims 1 to 7 wherein the sample is treated with the basic buffer solution at an effective treatment temperature above 82 °C.

1 1. The method of claim 9 wherein the effective period of time is 30 minutes or greater.

12. The method of any of claims 1 to 1 1 wherein the basic buffer is a Tris buffer solution.

13. The method of any of claims 1 to 1 1 wherein the pretreatment regimen comprises using a Tris buffer solution and a protease solution.

14. The method of claim 1 comprising using a protease, a basic buffer and an acidic buffer.

15. The method of any of claims 1 to 14 wherein the probes are nucleic acid probes.

16. The method according to any of claims 1 to 15 wherein the sample is a breast tissue sample, a lung tissue sample, a cervical tissue sample, a prostate sample, a hematological sample, or a metaphase spread.

17. The method according to claim 1 wherein the sample is breast tissue sample and the method comprises a Her2 quantum dot in situ hybridization assay, an ALK break-apart quantum dot in situ hybridization assay, a Kappa and Lambda quantum dot in situ hybridization assay, or a human papilloma virus (HPV) quantum dot in situ hybridization assay.

18. The method of any of claims 1 to 16 comprising using from about 2 to about 10 probes in a multiplexing assay.

19. The method of claim 18 comprising using from about 4 to about 8 probes in a multiplexing assay.

20. The method of any of claims 1 to 19 wherein the quantum dots have signal maxima with wavelengths separated by at least 40 nm.

21. The method of any of claims 1 to 20 wherein the anti-hapten antibodies are conjugated to the quantum dots through a linker.

22. The method of claim 21 wherein the linker is selected from PEG₂, PEG₃, PEG₄, PEG₅, PEG₆, PEG₇, PEG₈, PEG₉, PEGio, PEGn, PEGi₂, PEGi₃, PEGi₄,

PEGi5, PEGi6, PEGn, PEG18, PEG19, PEG₂o, 1 ,4-diaminohexane, xylylenediamine, terephthalic acid, 3,6-dioxaoctanedioic acid, ethylenediamine-N,N-diacetic acid, 1 , 1 '-ethylenebis(5-oxo-3-pyrrolidinecarboxylic acid), 4,4'-ethylenedipiperidine, succinimidyl-6-hydrazino-nicotinamide(S-HyNic, HyNic-NHS), N-succinimidyl-4- formylbenzoate (S-4FB, 4-FB-NHS), maleimide HyNic (MHPH), maleimide 4FB

(MTFB), succinimidyl-[(N-maleimidopropionamido)-octaethyleneglycol] ester (Mal-PEGg-NHS), succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol] ester (Mal-PEG₄-NHS), 4-FB-PEG₄-PFP, azidobenzoyl hydrazide, N-[4-(p- azidosalicylamino)butyl]-3'-[2'-pyridyldithio]propionamid), bis-sulfosuccinimidyl suberate, dimethyladipimidate, disuccinimidyltartrate, N- maleimidobutyryloxysuccinimide ester, N-hydroxy sulfosuccinimidyl-4- azidobenzoate, N-succinimidyl[4-azidophenyl]- 1 ,3 '-dithiopropionate, N- succinimidyl[4-iodoacetyl]aminobenzoate, glutaraldehyde, and succinimidyl-4-[N- maleimidomethyl]cyclohexane-l-carboxylate, 3-(2-pyridyldithio)propionic acid N- hydroxysuccinimide ester (SPDP), and 4-(N-maleimidomethyl)-cyclohexane-l- carboxylic acid N-hydroxysuccinimide ester (SMCC).

23. The method of claim 21 wherein the linker is succinimidyl-4-(N- maleimidomethyl)cyclohexane- 1 -carboxylate (SMCC).

24. The method according to any of claims 1 to 23 comprising exposing the sample to light within the wavelength range 355-405 nm prior to detecting fluorescence signals.

25. The method according to any of claims 1 to 24 comprising:

obtaining a series of monochrome images using filters to selectively detect each quantum dot signal; and

colorizing and stacking the images to produce a final combined image.

26. The method according to any of claims 1 to 25 comprising an automated method.

27. The method according to claim 1, comprising:

HER2/Chromosome 17 dual QD ISH to detect HER2 gene status and chromosome 17 in breast cancer;

ALK (Anaplastic lymphoma kinase) break-apart dual QD ISH to detect ALK gene status in non-small cell lung cancer;

HPV16 (Human papillomavirus 16) single-color QD ISH to detect the presence of high-risk HPV16 in high grade cervical intraepithelial neoplasias (CIN); or

Kappa and lambda mRNA single-color QD ISH to detect immunoglobulin (Ig) light chain mRNA expression in lymphoid tissue.

28. The method according to claim 3, wherein

pretreating a sample according to a pretreatment regimen includes conditions so that QD permeability and access to the target is increased as measured by fluorescence intensity; and

adding a chelating agent to chelate metal impurities includes conditions so that a QD metal quenching is reduced as measured by fluorescence intensity.

29. The method according to any of claims 1 to 28 comprising using process solutions comprising a low ionic strength.

30. The method according to claim 29 comprising using a borate buffer comprising 50 mM sodium ion or less.

31. The method according to any of claims 1 to 30 comprising using a casein blocking step in concentrations such that background spotting and dusting are reduced.

32. The method according to any of claims 1 to 31 comprising using deparaffinization times of from about 12 minutes to at least about 36 minutes, and deparaffinization temperatures of from about60 °C to at least about 75 °C.

33. The method according to any of claims 1 to 32 comprising using one or more blocking agents during pre -probe hybridization, during probe hybridization, or both.

34. The method according to claim 33 wherein the blocking agent is an effective amount of a BSA blocking buffer in an amount greater than 0% to at least about 5% BSA.

35. The method according to any of claims 1 to 34 comprising using a casein- blocking buffer prior to QD detection.

36. The method according to any of claims 1 to 35 comprising using borate buffer as a QD diluent during QD-antibody incubation.

37. A method, comprising:

providing a formalin fixed paraffin embedded sample selected from a prostate tissue sample, breast tissue sample, a lung tissue sample, a cervical tissue sample, a hematological sample, or a metaphase spread;

deparaffinizing the sample;

treating the sample with a protease and at least one of a Tris buffer and a citrate buffer;

contacting the sample with four or more haptenated probes that bind specifically to different targets, wherein the targets are selected from ERG3', ERG5', PTEN, CENIO, HER2, ALK, Kappa, Lambda, HPV16 and combinations thereof. contacting the sample with four or more anti-hapten antibody:quantum dot conjugates, each conjugate comprising a different quantum dot that can be detected selectively;

contacting the sample with a chelating agent to remove metal impurities; and detecting fluorescence signals from the quantum dots after excitation using filters selected for each particular quantum dot.

38. The method of claim 37, further comprising:

incubating the sample in a basic Tris buffer-based solution for an effective period of time at an effective temperature;

incubating the sample in an acidic citrate buffer-based solution for an effective period of time at an effective temperature;

treating with a protease for an effective period of time at an effective temperature; contacting a sample with DNA probes selected from ERG5'-PEG₃o-DIG, ERG3 '- PEG₃₀-DNP, PTEN-PEG₃₀-TS or CEN 10-PEG₃₀-NP;

contacting the sample with anti-hapten antibody:quantum dot conjugate selected from rat anti-DNP-QD655, mouse anti-DIG-QD565, mouse anti-TS-QD605, or mouse anti-NP-QD525;

exposing the sample to light within the wavelength range 355-405 nm;

obtaining a series of monochrome images using filters to selectively detect each quantum dot signal; and

colorizing and stacking the images to produce a final combined image.

39. The method according to claim 38 wherein the pretreatment solution is a basic buffer solution, and the sample is treated with the basic buffer solution at an effective treatment temperature above 82° C for an effective period of time of from about 30 minutes to about 1 hour.

40. The method of claim 38 comprising treating the sample with the protease at an effective temperature within the range of from about 35 °C to about 40 °C for an effective period of time greater than about 10 minutes.

41. A kit for practicing the method of claim 1.