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WO2014139979A1 - Hybridation in situ de points quantiques - Google Patents

Hybridation in situ de points quantiques Download PDF

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Publication number
WO2014139979A1
WO2014139979A1 PCT/EP2014/054633 EP2014054633W WO2014139979A1 WO 2014139979 A1 WO2014139979 A1 WO 2014139979A1 EP 2014054633 W EP2014054633 W EP 2014054633W WO 2014139979 A1 WO2014139979 A1 WO 2014139979A1
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Prior art keywords
sample
quantum dot
buffer
antibody
probe
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Inventor
Antony HUBBARD
Lei Tang
Wenjun Zhang
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F Hoffmann La Roche AG
Ventana Medical Systems Inc
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F Hoffmann La Roche AG
Ventana Medical Systems Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors

Definitions

  • 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.
  • ISH in situ hybridization
  • QD quantum dot
  • 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.
  • FISH Fluorescence in situ hybridization
  • Quantum dots 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.
  • 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.
  • 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.
  • 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).
  • Disclosed embodiments of the present invention address the deleterious results associated with these prior quantum dot in situ hybridization methodologies.
  • 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.
  • 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.
  • probes are added to the sample.
  • These probes comprise a hapten conjugated to a specific binding moiety designed to bind to a selected target.
  • these specific binding moieties are nucleic acid probes.
  • These probes can be targeted at DNA, including genomic DNA, and RNA, including mRNA.
  • 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
  • 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.
  • 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.
  • 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
  • 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.
  • the linker was 4-(N-maleimidomethyl)-cyclohexane- 1 -carboxylic acid N-hydroxysuccinimide ester (SMCC).
  • SMCC 4-(N-maleimidomethyl)-cyclohexane- 1 -carboxylic acid N-hydroxysuccinimide ester
  • 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.
  • the chelating agent was EDTA, or an analog thereof.
  • the sample is exposed to light.
  • 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.
  • 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.
  • 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.
  • a chelating agent 0.5 mM EDTA in this exemplary example
  • FIG. 9 provides fluorescent intensity graphs versus various chelating agent concentrations (e.g. EDTA; added at 500 ⁇ , 100 ⁇ , 20 ⁇ , 4 ⁇ 0.8 ⁇ and 0.16 ⁇ ) used for the exemplary automated protocol of FIG. IB, accompanied by the addition of 0.1 ⁇ , 1 ⁇ and 10 ⁇ Cu 2+ ion, establishing that 20 ⁇ EDTA is an effective concentration for QD ISH assays.
  • various chelating agent concentrations e.g. EDTA; added at 500 ⁇ , 100 ⁇ , 20 ⁇ , 4 ⁇ 0.8 ⁇ and 0.16 ⁇
  • 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
  • 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
  • FIG. 20 is a photographic image illustrating 3pALK/QD565 (green) and
  • 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.
  • a composition that includes a monoclonal antibody that specifically binds HER2, by any effective route.
  • 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.
  • the agent is a therapeutic agent, such as a therapeutic agent for the treatment of cancer.
  • 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).
  • ALK Anaplastic lymphoma kinase
  • 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.
  • NSCLC non-small-cell lung cancer
  • the standard test used to detect this gene in tumor samples is fluorescence in situ hybridization (FISH.
  • 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.
  • 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).
  • 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.
  • 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
  • Biological sample A biological specimen containing biological molecules, including genomic DNA, RNA (including mRNA and microRNA), nucleic acids, proteins, peptides, and/or combinations thereof.
  • the biological sample is obtained from a subject.
  • 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.
  • 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.
  • 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;
  • 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,
  • 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, NH 4 - acetate, potassium glutamate, NH 4 C1, 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 MgCl 2 , 50 mM
  • 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.
  • Tris buffer This buffer comprises water, Tris, EDTA disodium dehydrate, ProClin 950, Tween-20 and boric acid.
  • citrate buffer 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.
  • 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.
  • any polydentate molecule that can bind to a metal ion may be used as a chelator.
  • proteins, polysaccharides, and polynucleic acids are excellent polydentate ligands for many metal ions.
  • 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.
  • EDTA ethylenediaminetetraacetic acid
  • dimercaprol 2,3-dimercapto- 1 -propanol
  • porphine porphine
  • 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.
  • a chemotherapeutic agent is a radioactive compound.
  • Another example includes tyrosine kinase inhibitors, such as lapatinib.
  • 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).
  • 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,
  • 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.
  • a relatively small number of chromogenic substances have been identified that legitimately possess all of these qualities.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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
  • 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.
  • an agent such as an ERG specific antibody or a conjugate including an ERG specific antibody, or an ERG inhibitor
  • 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.
  • ETS erythroblast transformation-specific
  • 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.
  • TMPSSR2-ERG and NDRG1-ERG in prostate cancer
  • EWS-ERG in Ewing's sarcoma
  • FUS-ERG in acute myeloid leukemia.
  • Multiple alternatively spliced transcript variants encoding different isoforms have been identified.
  • TMPRSS2-ERG fusion is the most prevalent, occurring in approximately 50% of localized prostate cancers and 30% of androgen independent metastatic cancers.
  • 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.
  • ERG rearrangement has been observed in 10% to about 20% of HGPIN.
  • FISH Fluorescence in situ hybridization
  • FFPE Formalin fixed paraffin embedded sample.
  • Hapten is a molecule, typically a small molecule that can combine specifically with an antibody, but typically is substantially incapable of being
  • 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.
  • Plural different haptens may be
  • HER Human epidermal growth factor receptor
  • 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.
  • 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.
  • HER2 has no known ligand and assumes an open conformation, with its dimerization domain exposed for interaction with other ligand-activated HER receptors.
  • HER2 has no known ligand and assumes an open conformation, with its dimerization domain exposed for interaction with other ligand-activated HER receptors.
  • Herbst Int. J. Radiat. Oncol. Biol. Phys., 59:21-6, 2004; Zhang et al, J. Clin. Invest. 117 (8): 2051-8, 2007.
  • 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.
  • 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).
  • tumors e.g., tumors associated with breast cancer
  • 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
  • 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.
  • HER1 amino acid sequence examples include NCBI/Genbank accession Nos.
  • 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.
  • HER3 amino acid sequences include NCBI/Genbank accession Nos.
  • 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
  • Immunological binding properties of selected antibodies may be quantified using methods well known in the art.
  • ISH In situ hybridization
  • a probes such as a labeled complementary DNA or RNA strand
  • 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.
  • 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.
  • 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.
  • solid tumors such as sarcomas (connective tissue cancer) and carcinomas (epithelial cell cancer)
  • sarcomas connective tissue cancer
  • carcinomas epidermal cell cancer
  • 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,
  • CNS tumors such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma
  • hemangioblastoma 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.
  • 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 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.
  • 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
  • Oligonucleotide A linear polynucleotide sequence of between 5 and 100 nucleotide bases in length.
  • PTEN phosphatase and tensin homolog deleted on chromosome 10.
  • PIP3 Phosphatidylinositol (3,4,5)-triphosphate
  • AKT 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 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.
  • PSA serum prostate specific antigen
  • Proximal refers to the qualitative or quantitative distance between two molecules; for example, the distance between two proteins in a tissue sample.
  • 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.
  • two targets 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 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).
  • 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.
  • 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.
  • 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
  • TOP/TOPO phosphine/trioctyl phosphine oxide
  • the total size of the nanocrystal may vary, but typically is about 20-40nm, with a molecular weight of approximately 150KD.
  • 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.
  • 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.
  • Bio samples can be obtained from a subject using any method known in the art.
  • 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.
  • the non- cancerous tissue sample used as a control is obtained from a cadaver.
  • 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.
  • 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.
  • 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.
  • malignant tumor tissue samples are compared to a control.
  • the control is a benign tumor tissue sample obtained from a different subject.
  • 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.
  • 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.
  • a tumor e.g., a carcinoma.
  • 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.
  • 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.
  • homologs 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.
  • 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.
  • 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.,
  • 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.
  • 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.
  • 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.
  • 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.
  • specific binding moieties are antibodies, as well as binding fragments and derivatives or mimetics thereof.
  • the specific binding moiety may be either a monoclonal or polyclonal antibody.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 is an issue associated with QD analyses whereby relative large spots are observed in an image, and may result from QD aggregation.
  • Subject Any mammal, such as humans, non- human primates, pigs, sheep, cows, rodents and the like.
  • the term "subject” includes both human and veterinary subjects.
  • a subject is one known or suspected of having a HER+ tumor.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 for example by prolonging the life of a subject having tumor
  • 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
  • Tumor burden The total volume, number, metastasis, or combinations thereof of tumor or tumors in a subject.
  • a phrase that is used to describe any environment that permits a desired activity is formation of an immune complex.
  • 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.
  • 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.
  • the reliable and reproducible interpretation of the replicate slides demonstrates the feasibility of using the assay in clinical applications.
  • 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
  • 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.
  • haptens were then detected with anti-hapten antibodies conjugated with different QDs having sufficiently different emission wavelengths to allow the signals to be distinguished.
  • 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.
  • DIG-labeled probes 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,
  • Certain embodiments also provide an improved tissue pretreatment protocol.
  • pretreatment conditions are an important consideration for robust and reliable QD ISH staining.
  • QDs may require different tissue permeabilization procedures than other fluorophores to achieve optimum labeling as they are structurally large particles (often more than 150kD).
  • 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.
  • 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
  • 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.
  • a cell conditioning composition such as Tris buffer, pH 9.0, ImM EDTA significantly improved multi-color QD ISH assay robustness.
  • 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.
  • 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.
  • TMPRSS2 21q22.3
  • ETS oncogenic transcription factors family member ERG 21q22.2
  • TMPRSS2-ERG has been frequently associated with more aggressive prostate cancers and a poorer prognosis.
  • 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.
  • PTEN/PI3K/AKT pathway as preventive and therapeutic approaches in the future.
  • knowledge of ERG and PTEN gene status allows a substantially more accurate patient stratification and prognostication, and provides information leading to potential targeted therapies.
  • 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.
  • 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.
  • specific binding moiety hapten conjugates
  • 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.
  • 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.
  • a linker can include plural subunits or be formed from various subcomponents.
  • 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.
  • conjugates comprise an anti-hapten antibody, directed to a specific hapten included in a specific binding moiety-hapten conjugate, and a quantum dot.
  • the anti-hapten antibody can be directly conjugated to the quantum dot.
  • 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.
  • a linker can include plural subunits or be formed from various subcomponents.
  • 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.
  • 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.
  • 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 are Podophyllotoxin and Podophyllotoxin derivatives; and combinations thereof.
  • Specific binding moieties are defined above. Of particular interest are specific binding moieties that act as nucleic acid probes.
  • the specific binding moiety is a probe, comprising single or double stranded DNA from cell-based cloning or PCR.
  • 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.
  • the probe is targeted to viral DNA.
  • the probe is double stranded DNA or RNA which is then denatured into single stranded DNA or RNA for hybridization to a target sequence.
  • Both conjugates can have their respective moieties directly bonded to each other.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 PEG 4 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
  • 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.
  • 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.
  • the linker is a pair of molecules, having high affinity for one another.
  • high-affinity molecules include, for example, streptavidin and biotin, histidine and nickel (Ni), and GST and glutathione.
  • linkers include: ethylene glycol, polyalkylene glycols such as PEG 2 , PEG 3 , PEG 4 , PEG 5 , PEG 6 , PEG 7 , PEG 8 , PEG 9 , PEGio, PEGn, PEGi 2 , PEG13, PEG14, PEGi 5 , PEGi6, PEGn, PEG 1 8, PEG 1 9, PEG 2 0, 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-formy
  • PFP azidobenzoyl hydrazide, N-[4-(p-azidosalicylamino)butyl]-3'-[2'- pyridyldithio]propionamid), bis-sulfosuccinimidyl suberate, dimethyladipimidate, disuccinimidyltartrate, N-maleimidobutyryloxysuccinimide ester, N-hydroxy
  • probes useful for practicing disclosed embodiments of the present invention can be any probe now known or hereafter developed.
  • the specific binding moiety is a DNA probe, particularly a DNA probe that targets human DNA.
  • the probe target is RNA.
  • the target is messenger RNA (mRNA), such as Kappa or Lambda mRNA.
  • the probe targets viral DNA, such as the human papilloma virus (HPV), with a particular example including HPV 16.
  • HPV human papilloma virus
  • 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.
  • Exemplary conjugates of the probe-hapten type include ERG5'-DIG conjugate, ERG3'-DNP conjugate, PTEN-thiazole sulfonamide (TS) conjugate, and CEN10- nitropyrazole (NP) conjugate.
  • 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.
  • the quantum dots are selected such that the fluorescent signals are distinguishable by a system using filters to isolate the different 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.
  • 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.
  • 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.
  • MOPS 3-(N-Morpholino)propanesulfonic acid
  • 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.
  • 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., Arlington, AZ, USA).
  • DIG digoxigenin
  • DNP 2,4 dinitrophenyl
  • 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).
  • TS thiazole sulfonamide
  • 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.
  • NP dUTP conjugated nitropyrazole
  • Disclosed embodiments of the present invention used quantum dot-conjugated antibodies.
  • the quantum dot was conjugated to an antibody using a linker, such as an SMCC linker.
  • 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%.
  • DMSO suitable solvent
  • 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).
  • MES equilibration buffer
  • 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
  • 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.
  • the quantum do antibody conjugate was diluted to a final concentration of 1.0 ⁇ with borate buffer solution and mixed gently.
  • 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.
  • 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.).
  • FIGS. 1A and IB are provided by FIGS. 1A and IB, each of which is discussed in more detail below
  • the first step 12 concerns preparing an FFPE tissue sample. This is followed by deparaffinization using a mild detergent in step 14.
  • the tissue samples can undergo a pretreatment regimen at step 16.
  • 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.
  • the tissue samples were heated in the presence of one or more of the solutions.
  • the tissue samples were first treated with an acid buffer, then a protease solution.
  • 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.
  • 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.
  • 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.
  • 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.
  • the quantum dot: antibody conjugates are applied in step 26 and allowed to incubate.
  • the samples are washed multiple times with a buffer comprising water, Tris, acetic acid, Brij35 solution, ProClin 300 and sodium hydroxide.
  • the samples are washed with the above buffer and a basic buffer containing a chelating agent, such as EDTA.
  • the basic buffer comprised water, Tris, EDTA disodium dehydrate, ProClin 950, Tween-20 and boric acid.
  • 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.
  • 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.
  • 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.
  • TS Thiazole sulfonamide
  • NP nitropyrazole
  • 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.
  • 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.
  • 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.
  • 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.
  • CC1, Ventana Tris buffer-based solution
  • mouse anti-TS-QD605 antibodies were incubated in certain reaction buffer solutions and QD605 fluorescence was measured using a QD Fluorescence
  • 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 Cu 2+ , Zn 2+ , Fe 3+ , Hg 2+ , 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.
  • Zarkowsky et al. (Cytometry Part A, 79 (201 1), pp. 84-89) found that 0.0 luM Cu 2+ quenched 50% of QD655 fluorescence; andl ⁇ of Fe 3+ and Zn 2+ , and 0.1 ⁇ of Cu 2+ , completely eliminated QD 655 fluorescence.
  • a sharp intensity decrease is observed when Cu 2+ concentration is greater than about 0.03 uM. Fluorescence intensity is reduced to 50% of its initial value with about 0.5 mM Cu 2+ .
  • 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).
  • ligand exchange such as exchange with bifunctional ligands (i.e. thiol or phosphine mono or multidentate ligands)
  • amphiphilic polymers that contain both a hydrophobic segment (e.g. hydrocarbons) and a hydrophilic segment (e.g. polyethylene glycol [PEG] or multiple carboxylate groups).
  • 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.
  • 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 ⁇ added cupric ions.
  • a 0.07 ⁇ 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 ⁇ ) almost completely quenched QD fluorescence (approximately 10-20 absorbance units).
  • Metal/metal ion chelating agents provide another method for addressing metal/metal ion impaired QD fluorescence.
  • 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.
  • acidic moieties such as carboxylate, sulphonate, and/or phosphate groups
  • EDTA ethylenediaminetetraacetic acid
  • 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 ( ⁇ ), more typically from about 10 to about 100 ⁇ , with certain working embodiments using 20 ⁇ 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 ⁇ EDTA.
  • marginal reduction of QD fluorescence (approximately 2- fold) has been observed with EDTA in the absence of Cu 2+ . 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 Cu 2+ , potential metal ions (manifested as reduced
  • EDTA 20uM
  • staining protocols such as for HER2, ALK, and HPV16 QD ISH assays.
  • 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.
  • 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.
  • 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.
  • the addition of the chelating agent resulted in at least about 0.5 increase is signal intensity.
  • 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 ⁇ , 100 ⁇ , 20 ⁇ , 4 ⁇ 0.8 ⁇ and 0.16 ⁇ ) 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.
  • 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 Cu 2+ ion as indicated by the recitation of the concentration above the cluster.
  • the addition of 0.1 ⁇ , 1 ⁇ and 10 ⁇ Cu 2+ was tested.
  • the data of FIG. 9 establishes that 20 ⁇ was effective for all concentrations tested.
  • the addition of 20 ⁇ chelating agent increased the pass rate for an automated device from 71% to 100%.
  • 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.
  • 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.
  • 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.
  • 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.
  • certain disclosed embodiments of the present invention reduce spotting by maintaining a low ionic strength in QD ISH assay process solutions.
  • prior procedures were modified by reducing, or least substantially eliminating, salts from buffer process solutions.
  • certain disclosed embodiments employ a QD incubation step using a QD diluent-borate buffer comprising about 50mM [Na + ] or less.
  • 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.
  • BSA bovine serum albumin
  • casein casein
  • halide ion particularly fluoride
  • Bovine serum albumin (BSA) in saline solution can eliminate or substantially reduce non- covalent interactions between nanoparticles and efficiently improve colloidal stability.
  • BSA bovine serum albumin
  • 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.
  • 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.
  • QD background reduction can be achieved using any one of the following process
  • 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;
  • 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;
  • 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.
  • 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 ⁇ ⁇ of this solution was added to a microcentrifuge tube.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • SMCC-DMSO SMCC-DMSO required (mL) ⁇ (QD655 Vol after spin (mL) + SMCC Vol required (mL)) x 100%
  • 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.
  • 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).
  • MES equilibration buffer
  • 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:
  • the concentration of the functionalized QD655 was determined as follows.
  • the volume of antibody required (for single antibody lot) was calculated.
  • the DTT was dissolved in the amount of DI water calculated below.
  • 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).
  • MES equilibration buffer
  • 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
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 1 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.
  • 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).
  • 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).
  • 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
  • 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.
  • 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).
  • At least one set of red and green signals are two or more signal diameters apart (FIG. 3A&3B).
  • the same nucleus may have fused signals, broken apart signals and
  • PTEN/CEN10 is expected close to 1.
  • ERG3p, ERG5p, PTEN and CEN10 probes specifically bind to their target chromosomes
  • FIG. 4 shows a representative image of a normal metaphase spread stained with ERG/PTEN 4-color QD ISH on a
  • 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.
  • 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.
  • probe hybridization conditions e.g. probe concentration, denaturation temperature and time, stringency wash conditions, etc.
  • quantum dot- conjugated antibody detection conditions e.g. antibody concentration, incubation time, blocking steps, etc.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 5A - D 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • FFPE tissues may have some heavy metal contamination that can cause slide-to- slide variation of QD ISH signals.
  • a chelating agent such as EDTA
  • 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.
  • 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).
  • DIG digoxigenin
  • DNP 2, 4 dinitrophenyl
  • TS PTEN probe with thiazole sulfonamide
  • NP nitropyrazole
  • 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).
  • 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).
  • 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).
  • 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).
  • 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.
  • 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.
  • tissue sections were incubated in a basic Tris buffer-based solution (CC1, VMSI) at 90 °C for 92 minutes.
  • tissue sections were incubated in an acidic Citrate buffer-based solution (CC2, Ventana) at 82 °C for 36 minutes.
  • 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.
  • 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.
  • VMSI Hybrizol
  • 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.
  • 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 with experience interpreting QD ISH stained slides reviewed and scored the slides.
  • 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
  • FIG. 18C provides HPV16/QD605 FISH results.
  • 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.

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Abstract

L'invention concerne un procédé amélioré d'hybridation in situ de points quantiques pour la détection fiable et reproductible de cibles. Certains modes de réalisation décrits concernent un procédé de détection in situ utilisant un procédé modifié d'élimination de paraffine ainsi qu'une étape de prétraitement avant l'addition de sondes. Certains modes de réalisation utilisent une protéase et soit un traitement par un tampon acide, soit un traitement par un tampon basique, soit les deux. Le procédé peut également comprendre l'utilisation d'un agent de chélation dans des solutions de traitement pour éliminer des contaminants métalliques/d'ions métalliques. Après le prétraitement, au moins une sonde comprenant un haptène conjugué à un fragment de liaison particulier est ajoutée à l'échantillon. Après l'hybridation de la sonde sur la cible, un deuxième conjugué comprenant un anticorps anti-haptène et un point quantique est appliqué sur l'échantillon. Après lavage, l'échantillon est exposé à la lumière pour exciter le(s) point(s) quantique(s). Les signaux de fluorescence provenant du/des point(s) quantique(s) sont ensuite détectés et identifiés par l'obtention d'une série d'images monochromes, par exemple à l'aide de filtres pour détecter sélectivement chaque signal de point quantique. Ces images sont ensuite colorisées et empilées pour produire une image combinée finale. Des modes de réalisation décrivent un procédé de QD ISH solide et fiable présentant un bruit de fond de lame réduit permettant de faciliter l'analyse.
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CN105886596A (zh) * 2016-04-26 2016-08-24 南京师范大学 一种宫颈癌细胞检测试剂盒
WO2016190236A1 (fr) * 2015-05-28 2016-12-01 コニカミノルタ株式会社 Réactif sonde pour hybridation in situ
EP3198037A4 (fr) * 2014-09-22 2018-03-21 The Regents of the University of California Détection de molécule unique d'arn
JP2019523000A (ja) * 2016-07-18 2019-08-22 セル アイディーエックス， インコーポレイテッド 抗原をカップリングさせたハイブリダイゼーション試薬
CN115443507A (zh) * 2020-02-28 2022-12-06 格里尔公司 鉴定可鉴别或指示癌症病状的甲基化模式
US11867696B2 (en) 2015-02-06 2024-01-09 Cell Idx, Inc. Antigen-coupled immunoreagents
US11959838B2 (en) 2015-11-06 2024-04-16 Ventana Medical Systems, Inc. Representative diagnostics

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EP3198037A4 (fr) * 2014-09-22 2018-03-21 The Regents of the University of California Détection de molécule unique d'arn
US11867696B2 (en) 2015-02-06 2024-01-09 Cell Idx, Inc. Antigen-coupled immunoreagents
WO2016190236A1 (fr) * 2015-05-28 2016-12-01 コニカミノルタ株式会社 Réactif sonde pour hybridation in situ
JPWO2016190236A1 (ja) * 2015-05-28 2018-03-15 コニカミノルタ株式会社 インサイチュハイブリダイゼイション用のプローブ試薬
US11959838B2 (en) 2015-11-06 2024-04-16 Ventana Medical Systems, Inc. Representative diagnostics
CN105886596A (zh) * 2016-04-26 2016-08-24 南京师范大学 一种宫颈癌细胞检测试剂盒
JP2019523000A (ja) * 2016-07-18 2019-08-22 セル アイディーエックス， インコーポレイテッド 抗原をカップリングさせたハイブリダイゼーション試薬
EP3485275A4 (fr) * 2016-07-18 2020-06-17 Cell IDX, Inc. Réactifs d'hybridation couplés à un antigène
CN115443507A (zh) * 2020-02-28 2022-12-06 格里尔公司 鉴定可鉴别或指示癌症病状的甲基化模式

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