US20070128599A1 - Method for improved diagnosis of dysplasias - Google Patents

Method for improved diagnosis of dysplasias Download PDF

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US20070128599A1
US20070128599A1 US10/533,384 US53338403A US2007128599A1 US 20070128599 A1 US20070128599 A1 US 20070128599A1 US 53338403 A US53338403 A US 53338403A US 2007128599 A1 US2007128599 A1 US 2007128599A1
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marker
ink4a
cells
staining
detection
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Ruediger Ridder
Anja Reichert
Marcus Trunk-Gehmacher
Richard Batrla
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Roche MTM Laboratories AG
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Roche MTM Laboratories AG
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Priority claimed from EP02024030A external-priority patent/EP1416278B1/en
Application filed by Roche MTM Laboratories AG filed Critical Roche MTM Laboratories AG
Assigned to MTM LABORATORIES AG reassignment MTM LABORATORIES AG CORRECTIVE ASSIGNMENT TO CORRECT THE DOCKET NUMBER; ASSIGNOR; ASSIGNEE; SERIAL NUMBER; FILING DATE; TITLE PREVIOUSLY RECORDED ON REEL 017754 FRAME 0832. ASSIGNOR(S) HEREBY CONFIRMS THE ASSIGNMENT OF ASSIGNOR'S INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: REICHERT, ANJA, RIDDER, RUEDIGER, TRUNK-GEHMACHER, MARCUS, BATRLA, RICHARD
Publication of US20070128599A1 publication Critical patent/US20070128599A1/en
Priority to US12/289,487 priority Critical patent/US8975036B2/en
Priority to US12/381,098 priority patent/US8367353B2/en
Assigned to ROCHE MTM LABORATORIES AG reassignment ROCHE MTM LABORATORIES AG CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: MTM LABORATORIES AG
Priority to US13/798,998 priority patent/US9528159B2/en
Priority to US13/799,093 priority patent/US20130189686A1/en
Priority to US16/579,495 priority patent/US20200017923A1/en
Priority to US17/086,852 priority patent/US20210071267A1/en
Priority to US17/086,817 priority patent/US20210047697A1/en
Abandoned legal-status Critical Current

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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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57411—Specifically defined cancers of cervix
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites

Definitions

  • the present invention relates to a method for improved diagnosis of dysplasias based on simultaneous detection of INK4a gene products and at least one marker for cell proliferation.
  • the present invention provides a method for discriminating dysplastic cells over-expressing INK4a gene products from cells over-expressing INK4a gene products without being dysplastic by detection of a marker suitable for characterising the proliferation properties of the respective cell.
  • the characterisation of the proliferation properties may comprise the detection of a marker or a set of markers characteristic for active cell proliferation and/or a marker or a set of markers characteristic for retarded or ceased cell proliferation.
  • p16 INK4a The detection of the over-expression of p16 INK4a in biological samples has proven as a useful marker in the detection of anogenital lesions, such as carcinoma of the uterine cervix (see WO00/01845; Klaes et al., Int. J. Cancer: 92, 276-284 (2001)).
  • the method based on p16 INK4a -specific immuno-chemical staining allows for a sensitive and specific identification of dysplastic cells in tissue sections and in cytological samples.
  • dysplastic and neoplastic cells can be stained using a p16 INK4a specific antibody mediated staining procedure.
  • the histological diagnosis of neoplastic lesions can thus be supported by a staining procedure based on a molecular marker characteristic for transformation of cells in anogenital lesions.
  • the diagnosis, whether or not cells are neoplastic, in these procedures is not solely based on the p16 INK4a specific staining, but does also rely on the histological information.
  • metaplastic cells show some immuno-reactivity with p16 INK4 a specific antibodies, and thus are stained in the course of the procedures. Yet the staining pattern of these metaplastic cells differs from the pattern of neoplastic lesions.
  • Metaplastic cells give rise to a patchy or focal staining pattern, whereas neoplastic lesions give rise to diffuse staining pattern. Moreover, the staining intensities of metaplastic cells are predominantly less than that of neoplastic cells.
  • the percentage of cells showing over-expression of p16 INK4a increases in the course of emergence of dysplasias. So, in neoplastic or pre-neoplastic stages, when only a restricted population of neoplastic or pre-neoplastic cells is present in samples, the immunoreactivity of p16 INK4a may be weak. This weak immuno-reactivity may be of about the level as the level caused by metaplastic cells. In later stages of dysplasias, the overall immunoreactivity of p16 INK4a is stronger, so neoplastic lesions are easily discernible from metaplasias even in a cytological testing format. This might lead to cases where the presence of metaplastic cells expressing p16 INK4a might be confused with the presence of neoplastic cells, and thus produce a false positive result.
  • a method for discriminating the metaplasias from neoplastic and dysplastic lesions would be desirable.
  • a method for the discrimination of metaplasias from neoplastic and pre-neoplastic lesions is provided within the embodiments claimed according to the present invention.
  • a marker molecule For supporting the discrimination of metaplasias from neoplastic lesions in testing procedures based on the over-expression of p16 INK4a , a marker molecule would be desirable that is expressed in neoplastic and/or pre-neoplastic cells and tissues and which is not expressed simultaneously with INK4a gene products in one single metaplastic cell.
  • a solution to the problem present in the art is provided by the methods claimed according to this invention.
  • the inventors have found that a combination of detection of the presence or absence and/or the level of p16 INK4a , in combination with at least one marker for cell proliferation, such as e.g. Ki67, Ki-S2, mcm5 or mcm2, may solve the problem present in the art.
  • at least one marker for cell proliferation such as e.g. Ki67, Ki-S2, mcm5 or mcm2
  • EP1217377 a method for automated detection of cervical malignancies is disclosed that is mediated by detection of more than one marker molecules.
  • Some defined marker combinations are named within the document. There is no disclosure in this document relating to the choice of suitable markers for a combination. The purpose of the combination in this application is improved fidelity of the automated analysis of staining patterns in biological cytological specimens. This document mentions combination of p16 INK4a with other tumor markers.
  • WO02059616 discloses a method for detection of cell-cycle disturbances for improved diagnosis of cervical malignancies.
  • the document discloses that dysplastic cells exhibit disturbances in cell cycle control, and may thus be identified by means of detection of cyclin E type proteins together with post G1 substances in cells.
  • the inventors of the present invention sought to overcome the drawback present in the art that p16 INK4a , over-expressed in various dysplasias, may also be detected in some other non-dysplastic cells.
  • the discrimination between non-dysplastic p16 INK4a positive cells and dysplastic cells over-expressing p16 INK4a may be based on the proliferation characteristics of the respective cells. In normal control cells, p16 INK4a inhibits cdk4, and thus inhibits proliferation. In contrast, in dysplastic cells, this regulation is impaired. Thus, p16 INK4a does not lead—despite its uncommonly high expression level—to an inhibition of the cell proliferation.
  • dysplastic cells may be discriminated from cells exhibiting control cell proliferation by the simultaneous detection of p16 INK4a with a marker characteristic for cell proliferation. Due to the fact that, in normal cells, elevated levels of p16 INK4a inhibit cell proliferation, cells over-expressing p16 INK4a may be classified as being dysplastic provided they are exhibiting the characteristics of active cell proliferation.
  • FIG. 1 is an example of fluorescent staining of a histological specimen of the cervix uteri.
  • FIG. 1 shows staining of a severe dysplasia using antibodies directed against p16 INK4a .
  • Immunoreactivity for p16 INK4a renders green fluorescence. Almost all cells of the lesion are diffusely positive stained in the cytoplasm and in the nuclei.
  • FIG. 2 is an example of fluorescent staining of a histological specimen of the cervix uteri.
  • FIG. 2 shows staining of a severe dysplasia using antibodies directed against Ki67. For experimental details, see Example 1. Immunoreactivity for Ki67 renders red fluorescence. Many cells of the dysplasia show nuclear positivity for Ki67.
  • FIG. 3 is an example of fluorescent double staining of a histological specimen of the cervix uteri.
  • FIG. 3 shows staining of a severe dysplasia using antibodies directed against Ki67 and those directed against p16 INK4a .
  • Immunoreactivity for Ki67 renders red fluorescence
  • immunoreactivity for p16 INK4a renders green fluorescence
  • the overlay of red and green fluorescence renders yellow fluorescence.
  • Many cells of the dysplasia show nuclear positivity for Ki67, as well as positivity for p16 INK4a , and thus give rise to yellow fluorescence.
  • FIG. 4 is an example of fluorescent staining of a histological specimen of the cervix uteri.
  • FIG. 4 shows staining of a squamous metaplasia using antibodies directed against p16 INK4a .
  • Immunoreactivity for p16 INK4a renders green fluorescence.
  • Some cells of the metaplasia stain diffusely positive in the cytoplasm and in the nuclei.
  • FIG. 5 is an example of fluorescent staining of a histological specimen of the cervix uteri.
  • FIG. 5 shows staining of a squamous metaplasia using antibodies directed against Ki67.
  • Immunoreactivity for Ki67 renders red fluorescence. Many cells of the squamous metaplasia show nuclear positivity for Ki67. Areas that have been identified as positively expressing p16 INK4a do not show any positive staining for Ki67, indicating lack of expression of the antigen in these areas.
  • FIG. 6 is an example of fluorescent double staining of a histological specimen of the cervix uteri.
  • FIG. 6 shows staining of a squamous metaplasia using antibodies directed against Ki67 and against p16 INK4a .
  • Immunoreactivity for Ki67 renders red fluorescence
  • immunoreactivity for p16 INK4a renders green fluorescence
  • the overlay of red and green fluorescence renders yellow fluorescence.
  • Areas that are positively expressing p16 INK4a do not show any positive staining for Ki67, indicating lack of expression of the antigen in these areas. No cells in the specimen give rise to yellow fluorescence.
  • FIG. 7 is an example of chromogenic double staining of a histological specimen of the cervix uteri.
  • FIG. 7 shows staining of a severe dysplasia using antibodies directed against Ki67 and against p16 INK4a .
  • Immunoreactivity for Ki67 renders red nuclear staining
  • immunoreactivity for p16 INK4a renders brownish staining over the whole cell
  • double staining renders brown cells with red nuclei.
  • Many cells of the dysplasia show nuclear positivity for Ki67, as well as positivity for p16 INK4a , and thus, give rise to a pattern of brown cells with red nuclei.
  • FIG. 8 is an example of chromogenic double staining of a cytological specimen of the cervix uteri.
  • FIG. 8 shows staining of a severe dysplasia using antibodies directed against Ki67 and against p16 INK4a .
  • Immunoreactivity for Ki67 renders red nuclear staining
  • immunoreactivity for p16 INK4a renders brownish staining over the whole cell
  • double staining renders brown cells with red nuclei.
  • Many cells of the dysplasia show nuclear positivity for Ki67 as well as positivity for p16 INK4a , and thus, give rise to a pattern of brown cells with red nuclei.
  • the present invention relates to a method for discrimination of neoplastic, pre-neoplastic and/or dysplastic lesions from non-dysplastic cells showing elevated levels of p16 INK4a , in biological samples in a histological or cytological testing procedure based on the detection of the presence or absence of cells expressing p16 INK4a gene simultaneously with cell proliferation markers in said biological samples.
  • Suitable markers for cell proliferation may be, e.g., Ki67, Ki-S2, Ki-S5, mcm5, or mcm2.
  • protein or mRNA of the markers for cell proliferation may serve as a marker for discrimination of metaplasias from early dysplastic or pre-neoplastic lesions in samples.
  • One aspect of the present invention is to provide a method for discriminating dysplastic cells over-expressing INK4a gene products from other cells expressing INK4a gene products at a detectable level in biological samples comprising determining in a cytological or histological testing procedure the co-expression of at least two marker molecules in at least one single cell, wherein at least one marker molecule is an expression product encoded by the INK4a gene and at least one further marker molecule is a cell proliferation marker, wherein the over-expression of at least one INK4a gene product and expression of at least one marker for active cell proliferation at an immuno-chemically detectable level within said single cell is indicative of the dysplastic state of the cell, and wherein the over-expression of at least one INK4a gene product and expression of at least one marker for senescence, terminal differentiation of cells, apoptosis, or cell cycle arrest at a detectable level within said single cell is indicative of the non-dysplastic state of the cell.
  • a second aspect of the present invention relates to a test kit for determination of dysplasias in samples according to the method disclosed herein.
  • non-dysplastic cells may show immuno-reactivity for p16 INK4a .
  • the inventors found that these cells exhibiting ordered control of cell proliferation in response to the elevated levels of the INK4a gene products are subject to growth arrest. Thus, these individual cells do not show immuno-reactivity for markers of cell proliferation.
  • transformed cells over-expressing p16 INK4a exhibit dysregulated control of cell proliferation, and do not respond to the elevated level of p16 INK4a by cessation of proliferation.
  • these dysplastic cell show simultaneous expression of p16 INK4a with markers for cell proliferation.
  • the inventors thus found that a simultaneous detection of p16 INK4a with markers for cell proliferation may serve to discriminate dysplastic cells from arrested cells over-expressing p16 INK4a such as, e.g., metaplastic cells.
  • the method disclosed herein is based on the fact that dysplastic cells may be discriminated from cells exhibiting normal control cell proliferation by the simultaneous detection of an INK4a gene product such as, e.g., p16 INK4a with a marker characteristic for cell proliferation.
  • an INK4a gene product such as, e.g., p16 INK4a with a marker characteristic for cell proliferation.
  • marker as well as marker molecules in general, shall be used herein to pertain to proliferation marker gene expression products as well as to INK4a gene expression products.
  • the denominations given throughout this text for genes may, in part, relate to the genes or proteins as they have been discovered from any organism.
  • this denomination shall confer to the respective homologue of the named markers in the organism which is particularly in question for a method as disclosed herein.
  • this organism is a mammal and, in one embodiment, may be a human being.
  • the named markers shall be the human homologues of the respective denominated ones.
  • (cell) proliferation marker or “marker for cell proliferation” in the various grammatical forms is used to denominate proteins as well as nucleic acid markers.
  • protein name of a marker such as e.g. “replication protein” is used herein, this use shall be understood metonymically and pertain as well to the protein as to the nucleic acid marker molecules encoding the particular protein
  • a marker useful according to the present invention may be any molecule transcribed from a gene or any molecule translated from such a transcript.
  • gene product as used in the context of the present invention, may comprise polynucleotides such as, e.g., DNA or RNA and polypeptides such as proteins, proteoglycans, peptides, etc.
  • “Expression product(s)” as used in the context of the present invention shall comprise any transcript of a gene locus in forward or reverse direction including any reading frames and splicing variants. “Expression products” as used herein shall thus comprise any alternative products encoded by the nucleic acids of a particular gene locus.
  • INK4a encoded gene-products shall be any mRNA transcribed from the INK4a gene locus or any polypeptide translated from such an mRNA.
  • the expression products encoded by the INK4a gene may exhibit molecular weights of about 5 to 40 kDa or any value in between, and preferably of about 10 to 20 kDa or any value in between, and most preferably of about 14 to about 19 kDa or any value in between, respectively.
  • INK4a gene-products suitable for the method according to the present invention may comprise, e.g., gene-products such as, e.g., p16 INK4a and p14ARF.
  • (cell) proliferation marker or “marker for cell proliferation”, as used in the context of the present invention, shall comprise any marker molecule known in the art to be characteristic for the proliferation status of cells.
  • the proliferation status may be, e.g., a status of actively proliferating cells, of retarded cell proliferation, of arrested cell proliferation, of senescent cells, of terminally differentiated cells, of apoptosis, etc.
  • the cell proliferation marker is a marker molecule characteristic for active cell proliferation.
  • the proliferation marker molecule may be a molecule characteristic for arrested, terminally differentiated, senescent, or apoptotic cells.
  • proliferation markers for use in the context of the present invention may comprise genes engaged in the DNA replication, such as e.g., proteins of the pre-initiation complex or of the replication fork.
  • Such molecules may comprise, e.g., helicases, such as eucaryotic helicase or MCM proteins (MCM2, MCM3, MCM4, MCM5, MCM6, MCM7), protein TP as disclosed in WO0050451 and WO0217947 (also denominated HELAD1, Pomfil2, Unc-53), kinases or phosphatases engaged in the replication process such as, e.g., CDC6, CDC7 protein kinase, Dbf4, CDC14 protein phosphatase, CDC45 and MCM10.
  • proliferation markers may comprise proteins engaged in the processive replication fork such as, e.g., PCNA or DNA polymerase delta, replication protein A (RPA), replication factor C (RFC), and FEN1.
  • the proliferation markers may comprise molecules necessary for the maintenance of cell proliferation such as Ki67, Ki-S5, or Ki-S2.
  • proteins may be present, e.g., throughout the whole cell cycle. They are useful for performing a method according to the present invention provided they are characteristic of active cell proliferation and are not significantly expressed in arrested, terminally differentiated, apoptotic, or senescent states of cells.
  • Ki67, Ki-S2, and Ki-S5 as used herein shall denominate the protein marker molecules detected by the respective antibodies as well as the nucleic acids encoding these antigens.
  • the cell proliferation markers for use in a method according to the present invention may be a marker molecule characteristic of retarded or ceased cell proliferation such as, e.g., a senescence marker, a cell cycle arrest marker, a marker characteristic for terminally differentiated cells, or an apoptosis marker.
  • a marker molecule characteristic of retarded or ceased cell proliferation such as, e.g., a senescence marker, a cell cycle arrest marker, a marker characteristic for terminally differentiated cells, or an apoptosis marker.
  • Such molecules comprise, e.g., p21, p27, Caspases, BAD, CD95, fas-ligand, parp-proteins, etc.
  • Discrimination as used in the context of the present invention shall comprise an assessment whether a sample is to be classified in one or another way.
  • the discrimination pertains to the assessment of a tissue, or components thereof, being dysplastic or being not dysplastic.
  • the discrimination as used herein is a judgement about the growth properties of cells in a biological sample.
  • the discrimination comprises the detection of expression of an INK4a gene product simultaneously with the detection of expression of a marker characteristic for active cell proliferation.
  • cells co-expressing both marker molecules are to be classified as being dysplastic.
  • the discrimination comprises the detection of an INK4a gene product simultaneously with the detection of expression of a marker characteristic for arrested, ceased, or retarded cell proliferation.
  • a marker characteristic for arrested, ceased, or retarded cell proliferation In this case, cells co-expressing both marker molecules are to be classified as being non-dysplastic.
  • one INK4a gene expression product will be detected in concert with two or more markers for cell proliferation. This may be useful to enhance the identification of proliferation properties of the INK4a gene product-expressing cells in samples. Some proliferation markers are restricted to specific phases of the cell cycle or are present in low abundance in cells. Due to this fact, in some cases, the detection of proliferating cells expressing INK4a gene products may be improved by detection of two or more proliferation markers. In such cases, e.g., one proliferation being expressed during the whole proliferative cell cycle may be detected simultaneously by markers characteristic for specific cells cycle phases.
  • Ki67, Ki-S5, or Ki-S2 may be detected together with mcm5, mcm2, PCNA, rpA, rfC, etc.
  • proteins engaged in the DNA replication may be detected together with Ki67, Ki-S5, or Ki-S2.
  • Ki67 may be detected together with Ki-S2. It must be understood that these examples are intended to exemplify combinatorial possibilities, and shall not be comprehensive, so that various other combinations of proliferation markers are likewise useful and suitable in the procedure of the method according to the present invention.
  • combination of two or more cell proliferation marker molecules may be applied in a method as disclosed herein.
  • two or more marker molecules detectable over larger stretches of the cell cycle or even over the whole cell cycle, or in an actively proliferating cell may be detected in concert in a method as disclosed herein.
  • a combination of more than one cell proliferation marker molecules may be useful generally for improving the sensitivity of the detection of the proliferation characteristics of cells.
  • a combination may furthermore comprise other marker molecules such as senescence marker molecules, markers for arrested cells, markers for terminally differentiated cells, markers for apoptotic cells, markers for viral infection or for viral activity in cells, or cell cycle regulatory protein markers.
  • a detection of HPV associated marker molecules or markers for viral activity may be of use for a detection of a dysplasia.
  • the methods useful for detection of detection of HPV infection in samples are known to those of skill in the art. These methods may comprise methods employing probes specific for HPV agents or may employ nucleic acid amplification reactions. The detection of a viral infection may be carried out simultaneously or subsequently to the detection of the INK4a and proliferation marker molecules.
  • Dysplastic as used in the context of the present invention, shall refer to dysplasias from mild to severe dysplasias and their precursory stages, as well as carcinoma such as carcinomas in situ or invasive carcinomas and disseminated tumor cells. Thus, dysplastic as used herein shall also comprise early and precursory stages of dysplasias and carcinomas.
  • Non-dysplastic as used herein
  • Cells over-expressing INK4a gene products without being dysplastic may comprise, e.g., metaplastic cells, senescent cells, terminally differentiated cells, or cells that in certain stages of the cell cycle exhibit elevated levels of the INK4a gene products. In certain cells, elevated levels of INK4a gene products may even be effected as a response to external signals such as hormones, transmitters, etc.
  • the non-dysplastic cells over-expressing INK4a gene products comprise, e.g., metaplastic cells, endometrial cells, etc.
  • the method for detection of the expression level of the INK4a encoded gene-products and/or the proliferation marker gene products according to the present invention is any method, which may (but need not) be, e.g., suited to detect even very small amounts of specific biological molecules in biological samples.
  • the detection reaction according to the present invention is a detection either on the level of nucleic acids or on the level of polypeptides.
  • a marker molecule is said to be detectable as used in the context of the present invention, provided the marker may be detected in the course of suitable detection procedure such as, e.g., in situ hybridization, immuno-chemical staining, hybrid capture assay, etc.
  • the level of expression of a marker molecule may be made detectable using suitable reporter reactions such as, e.g., a chromogenic or fluorescence based immuno-chemical staining or in-situ-hybridization procedure for microscopic or automated analysis.
  • suitable methods for enhancing the reporter signal known to those of skill in the art may be applied in the course of a method according to the present invention.
  • the marker is said to be detectable in a case where the staining supersedes the respective background staining inherently obtained in the immuno-chemical staining procedure so as to produce significant staining results.
  • the marker molecules may be detected using reagents that specifically recognize these molecules.
  • the detection reaction for the INK4a gene-products and/or the proliferation marker gene products may comprise one or more reactions with detecting agents either recognizing the initial marker molecules or recognizing the prior molecules used to recognize other molecules.
  • two or more probes may be used for the detection of one single marker molecule.
  • two or more different binding agents e.g., antibodies
  • oligonucleotide probes directed against one single marker molecule or, as the case may be, against different epitopes or different sequences
  • the detection of the different gene products may be performed in one reaction vessel or container or in different containers simultaneously or subsequently in time.
  • the different gene products may be detected simultaneously in one cell co-expressing both products.
  • cells co-expressing the gene products may be used for separated detection reaction (separated in space or in time) to detect each a single marker in the cells.
  • the detection of the marker molecules in the different cells may also be performed simultaneously or separately in time and/or space.
  • the detection reaction further may comprise a reporter reaction indicating the presence or absence and/or the level of the marker molecule gene-products.
  • the reporter reaction may be, for example, a reaction producing a coloured compound, a bioluminescence reaction, a fluorescence reaction, generally a radiation emitting reaction, etc.
  • different marker molecules may be recognized by agents that produce different reporter signals, so that the signals referring to marker molecules can be distinguished.
  • the detection of the expression of the two or more INK4a gene-products and/or proliferation marker gene products is carried out simultaneously.
  • the reporter reaction may employ, for example, different fluorescent labels for the different molecules detected.
  • the question is whether any proliferation marker and/or INK4a gene product is expressed.
  • a procedure may be chosen that gives the same fluorescence signal as an indication of the presence of a proliferation marker. This procedure is suitable to improve sensitivity of the detection of the cell proliferation characteristics (different markers characteristic for active cell proliferation). As the case may be, the procedure may be applied so as to render one detectable signal for three, four, or even more marker molecules characteristic for cell proliferation. Analogously, the same may under certain circumstances be true for the INK4a gene expression products. It must be understood that different staining signals for different proliferation marker molecules may be desirable. The procedures may be applied to the necessities of the respective experiment.
  • a combination of one or more (e.g., two different) INK4a gene products may be detected with a combination of one or more (e.g., a set of two, a set of three, a set of four, a set of five or a set of even more) markers for cells proliferation.
  • the detection of the marker molecules for cell proliferation may render only one reporter signal.
  • each single marker for cell proliferation may render a specific reporter signal or groups of marker molecules may render specific reporter signals.
  • Signals for the indication of the presence of immuno-reactivity may be chromogenic signals produced by various methods known in the art. Alternatively, or even in combination, fluorescent signals may be used. Suitable reporter signals comprise fluorescent labels such as fluorescein, rhodamine, etc.
  • Applicable formats for the detection reaction according to the present invention may be blotting techniques, such as Western-Blot, Southern-blot, Northern-blot, and immunocytochemical, or immunohistochemical procedures.
  • the blotting techniques are known to those of ordinary skill in the art, and may be performed as, for example electro-blots, semidry-blots, vacuum-blots or dot-blots.
  • Immunocyto/histochemical staining procedures are known to those of skill in the art, and may comprise binding agent-mediated detection of polypeptides as well as in situ hybridisation techniques. Both different techniques may even be applied simultaneously.
  • hybrid capture of nucleic acids may be used for the detection.
  • Amplification reactions may also be applicable for the detection of e.g. nucleic acid molecules.
  • the detection of the level of INK4a and/or proliferation marker gene-products is carried out by detection of the respective nucleic acids (e.g., mRNA) or fragments thereof present in the sample.
  • the means for detection of nucleic acid molecules are known to those skilled in the art.
  • the procedure for the detection of nucleic acids can be carried out by, for example, a binding reaction of the molecule to be detected to complementary nucleic acid probes, proteins with binding specificity for the nucleic acids, or any other entities specifically recognizing and binding to said nucleic acids.
  • in situ hybridisation of oligonucleotide probes to nucleic acids in a sample may be used for the detection of expression products or markers.
  • Probes as used in the context of the present invention may be any agent binding specifically to a molecule.
  • a probe may be an oligonucleotide hybridising to a particular sequence.
  • the probe may be, e.g., a primer.
  • the probe as used herein may be, e.g., a binding agent such as an antibody.
  • the probes may be detectably labelled.
  • the label may be selected from the group comprising a radioisotope, a bioluminescent compound, a chemiluminescent compound, a fluorescent compound, a metal chelate, or an enzyme.
  • Probes may be applied in any detection procedure known in the art as, for example, in the course of an in situ hybridisation procedure, in the course of hybrid capture assays, in the course of immuno-chemical staining reaction, in the course of blotting techniques, etc.
  • This method may be performed as well in vitro as directly in situ for example, in the course of a detecting staining reaction.
  • Another way of detecting the marker mRNAs in a sample performed in the method according to the present invention is an amplification reaction of nucleic acids, which can be carried out in a quantitative manner such as, for example, the polymerase chain reaction.
  • real time (RT) PCR may be used to quantify the level of marker mRNAs in samples of dysplasias or tumors (cells or tissue samples).
  • the detection of the level of INK4a and/or proliferation marker gene-products is carried out by determining the level of expression of a protein or fragments thereof.
  • the determination of the marker gene-product on the protein level can, for example, be carried out in a reaction comprising a binding agent specific for the detection of the particular marker polypeptide.
  • binding agents can be used in many different detection techniques including, for example, in western-blot, ELISA or immuno-precipitation.
  • polypeptide binding agent based detection can be carried out as well in vitro as directly in situ as, for example, in the course of an immuno-chemical staining reaction. Any other method for determining the amount of particular polypeptides in biological samples can be used according to the present invention.
  • the immuno-cytochemical imaging procedures for use in the context of the present invention may comprise, e.g., the staining of cytological or histological preparations with chromogenic or fluorescent dyes.
  • the staining may comprise, e.g., binding of the molecules to be detected by a first binding agent, which itself is detected by a secondary binding agent which may be labelled.
  • the first binding agent may in certain embodiments be a nucleic acid or a protein binding agent (e.g. an antibody) and the secondary binding agent may be, e.g., a secondary antibody recognizing the first binding agent.
  • Binding agents as used in the context of the present invention for the detection of the level of INK4a polypeptides such as p16 INK4a or p14ARF polypeptides and proliferation marker polypeptides such as, e.g., mcm5, mcm2, Ki67, Ki-S5, PCNA, or Ki-S2 polypeptides may comprise antibodies and antigen-binding fragments, bi-functional hybrid antibodies, peptidomimetics containing minimal antigen-binding epitopes, anti-cullines (anti-calineTM), etc.
  • antibodies according to the present invention may be monoclonal or polyclonal antibodies.
  • the term antibody or monoclonal antibody is meant to include intact molecules as well as antibody fragments.
  • antibodies of the present invention include chimeric, single chain, and humanized antibodies.
  • binding agents may be used isolated or in combination. By means of combination, it is possible to achieve a higher degree of sensitivity.
  • the term antibody preferably, relates to antibodies which consist essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations.
  • Monoclonal antibodies are made from antigen-containing fragments of the polypeptide of the invention using any of a variety of techniques known to those of ordinary skill in the art (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).
  • an immunogen comprising the antigenic polypeptide, or a synthetic part thereof, is initially injected into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep, and goats).
  • the polypeptides of this invention may serve as the immunogen without modification.
  • a superior immune response may be elicited if the polypeptide is joined to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin.
  • a carrier protein such as bovine serum albumin or keyhole limpet hemocyanin.
  • the immunogen is injected into the animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically.
  • Polyclonal antibodies specific for the polypeptide may then be purified from such antisera by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support.
  • the main question may be whether one or the other proliferation marker is expressed in the cells.
  • the main question may be whether any proliferation marker is expressed.
  • a procedure was chosen that gives the same fluorescence signal as an indication of the presence of a proliferation marker. This procedure is suitable to improve sensitivity of the detection of the cell proliferation characteristics. As the case may be, the procedure may be applied so as to render one detectable signal for three, four, or even more marker molecules characteristic for cell proliferation. Analogously, under certain circumstances, the same may be true for the INK4a gene expression products. It must be understood, that, as the case may be, different staining signals for different proliferation marker molecules may be desirable. The procedures may be applied to the necessities of the respective experiment.
  • the INK4a gene-products and/or proliferation marker gene products may, according to the present invention, be detected simultaneously.
  • simultaneously according to the present invention shall mean either literally at the same instant or within the same testing procedure, whereby the single detection steps are temporarily consecutive.
  • the detection procedure according to the present invention may, furthermore, comprise a cytochemical staining procedure rendering a chromogenic or fluorescent staining of cells or cell compartments.
  • cytochemical staining procedures are known to those of skill in the art and may comprise, for example, staining for acidophilic or basophilic structures, of subcellular regions (e.g. the nucleus, the mitochondria, the golgi, the cytoplasm, etc.), of specific molecules (the chromosomes, of lipids, of glycoproteins, of polysaccharides, etc.) in the cytological specimens.
  • Fluorescence dyes such as DAPI, Quinacrin, Chromomycin, etc. may be employed.
  • chromogenic dyes such as Azan, Acridin-orange, Hematoxylin, Eosin, Sudan-red, and Thiazin-stains (Toluidin-blue, Thionin) may be applied.
  • staining procedures such as Pap-staining, Giemsa-staining, Hematoxylin-Eosin staining, van-Gieson staining, Schiff-staining (using Schiff reagent), staining procedures employing precipitation of metals (such as, e.g., of silver in staining procedures employing Silver Nitrate), or insoluble stains such as, e.g., of Turnbulls-blue (or other insoluble metal cyanides), etc. may be used in the course of a method as disclosed herein. It must be understood, that the named dyes and staining methods shall be examples for the applicable methods, and that any other method known in the art may be applied to a method as disclosed herein.
  • the staining procedures may produce chromogenic stains for light microscopic inspection or fluorescent stains for inspection under fluorescence microscopic conditions.
  • radiation emitting procedures, procedures employing substances impairing the transmission of radiation, or other contrast media for imaging of the cytological conditions in a sample e.g., the generation of optical impression by means such as (micro-)autoradiographic or (micro-)radiographic picture generation
  • the generation of optical impression by means such as (micro-)autoradiographic or (micro-)radiographic picture generation may be of use for a method according to the present invention.
  • All the staining and imaging procedures may be used for analysis not only in microscopic procedures, but also in automated analysis procedures such flow cytometry, automated microscopic (computerized or computer aided) analysis, or any other method for analysis of stained cytological specimens.
  • the analysis of the staining or imaging results of the different procedures may be performed in a single analysis step or in different subsequent steps.
  • the light microscopic inspection of a specimen may be performed before or after fluorescence microscopic inspection of the specimen.
  • fluorescence microscopy the analysis of different stains with different excitation wavelengths may be analyzed simultaneously or subsequently.
  • Other imaging methods may be employed simultaneously or subsequently to the named procedures.
  • a sample according to the method of the present invention may comprise any sample comprising cells.
  • Samples may comprise, e.g., secretions, smears, body fluids, and cell- or tissue-samples.
  • samples comprise cells of the anogenital tract, of the respiratory tract, or of the skin and its appendages.
  • the cells may be cells of the uterine cervix, the vagina, the vulva, the penis, the anus, the rectum, the bronchic tree, the lung, the peritoneum, the peritoneal space, the naso-pharyngeal space, the oral cavity, or the skin.
  • the sample may be a histological sample, a biopsy, or a cytological sample such as, e.g., a smear, a swab, a wash, or a body fluid containing cells (sputum, a secretion, saliva, etc.).
  • the samples may comprise cells infected by papilloma virus.
  • the samples may, in certain embodiments, comprise cervical smears, bronchioalveolar lavages, etc.
  • the sample may be prepared as a monolayer or thin layer preparation of a cytological specimen.
  • the respective methods for preparation of monolayer or thin-layer preparation in cytology are known to those of skill in the art.
  • the preparation may comprise, e.g., the ThinPrep technology.
  • Other methods comprise conventional smears, or method employing suspensions of cells for preparation of the cytological specimens.
  • Preparation of a sample may comprise, e.g., obtaining a sample of a tissue, of a body fluid, or of cells from a patient.
  • preparation of the sample may also comprise several steps of further preparations of the sample, such as preparation of dissections, preparation of cell suspensions, spreading or applying the cells to be examined onto microscopic slides, preparation of tissue arrays, isolation of polypeptides or nucleic acids, preparation of solid phase fixed peptides or nucleic acids, or preparation of beads, membranes, or slides to which the molecules to be determined are coupled covalently or non-covalently.
  • the method may be performed in an automated manner.
  • the automation of the method may be achieved by automated staining and analysis of histological or cytological specimens on a solid surface by microscopic means.
  • the automation may comprise a flow-cytometric analysis of the staining of cells in solution.
  • the dysplastic lesions to which the method according to the present invention may be applied comprise any dysplastic lesions characterized by over-expression of INK4a gene products such as, e.g., p16 INK4a or p14ARF.
  • those lesions are dysplasias associated with infections by papilloma viruses such as, e.g., HPV.
  • the HPV may be a high risk HPV subtype such as HPV16, HPV18, HPV31, HPV 33, HPV35, HPV 39, HPV 45, HPV 51, HPV 52, HPV56, HPV 58, HPV 59, HPV 66, HPV 68, etc.
  • the dysplastic lesions that may be detected according to the present invention comprise anogenital lesions, lesions of the respiratory tract, lesions of the head and the neck, or lesions of the skin and its appendages.
  • Such lesions may comprise dysplasias, e.g., of the anus or rectum, of the vulva, the vagina, the cervix or the penis, of the bronchic tree, the lung, the oral cavity, or the nasopharyngeal space.
  • kits for performing the method according to the present invention.
  • the kit may be, for example, a diagnostic kit or a research kit.
  • a kit according to the present invention comprises at least an agent suitable for detecting the INK4a gene-products.
  • kit according to present invention may comprise:
  • the reagents for the detection of the marker gene-products may include any agent capable of binding to the marker gene-products.
  • Such reagents may include proteins, polypeptides, nucleic acids, peptide nucleic acids, glycoproteins, proteoglycans, polysaccharides, or lipids.
  • the INK4a gene-product and proliferation marker gene product samples for carrying out a positive control may comprise, for example, nucleic acids in applicable form, such as solution or salt, peptides in applicable form, tissue section samples, or positive cells.
  • the detection of the marker gene-products is carried out on the level of polypeptides.
  • the binding agents may be, for example, antibodies specific for the marker gene-products or fragments thereof.
  • the detection of the marker gene-products is carried out on the nucleic acid level.
  • the reagent for the detection may be, for example, a nucleic acid probe or a primer reverse-complementary to said marker nucleic acids.
  • the present invention provides a method for the discrimination of neoplastic and/or dysplastic and pre-neoplastic lesions, identifiable by assessment of the over-expression of p16 INK4a , from other cells cells, which also detectably express p16 INK4a , in the course of histological and/or cytological testing procedures.
  • the method is based on the detection of expressed gene-products of two or more INK4a gene products.
  • the problem to be solved was to provide a method for discrimination between dysplastic cells and other cells lacking malignant growth potential.
  • the method may be applied to any stage of dysplasias and may be especially useful in early stages, when cytological diagnostic methods based on the p16 INK4a over-expression need further information for the identification of metaplastic cells.
  • the present invention provides a kit for performing the method according to the present invention.
  • Sections of formalin fixed, paraffin embedded tissue samples of the cervix uteri were immunofluorescent stained using antibodies specific for p16 INK4a and Ki67.
  • tissue sections were rehydrated through incubation in xylene and graded ethanol, and transferred to Aqua bidest.
  • Antigen retrieval was carried out with 10 mM citrate buffer (pH 6.0) for p16 INK4a and Ki67. Therefore, the slides were heated in a waterbath for 40 min at 95-98° C. The slides were cooled down to RT for 20 minutes, and transferred to washing buffer (50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20/DakoCytomation: code no.: S3006).
  • the specimens were incubated with 10% goat serum for 30 min at RT.
  • the slides were then incubated with the primary antibodies, mouse anti-human p16 INK4a antibody (3.48 ⁇ g/ml) and rabbit anti Ki67 (1:25) for 30 min at RT, after which the slides were rinsed with washing buffer and placed in a fresh buffer bath for 5 min. Excess buffer was tapped off, and each specimen was covered with 200 ⁇ l of the secondary reagent containing goat anti-mouse antibody, AlexaFluor®488 conjugated and goat anti-rabbit antibody, and Alexa Fluor®546 conjugated, and then incubated for 30 min at RT. Then slides were washed two times as before and directly mounted with a special mounting medium for fluorescence.
  • FIGS. 1-6 show the staining results for a severe dysplasia of the cervix uteri and for a squamous metaplasia using antibodies directed against Ki67 and against p16 INK4a .
  • Immunoreactivity for Ki67 rendered red fluorescence
  • immunoreactivity for p16 INK4a rendered green fluorescence
  • Smears of the uterine cervix may be semi-quantitatively analysed for the mRNA level of p16 INK4a and mcm2 in an in situ staining reaction.
  • the staining reaction is performed as follows:
  • the spray-fixed smears are incubated in fresh 50% EtOH on a rocking device.
  • the PEG film produced by the fixation procedure is removed by intensive rinsing.
  • the smears are rinsed in Aqua bidest.
  • the smears are incubated with proteinese K (10 ⁇ g/ml in PBS) for 10 min at 37° C.
  • the slides are transferred to washing buffer (PBS/0.1% Tween20) and finally, the area containing the cells is surrounded with a lipid-pencil.
  • the hybridization mixture is prepared by mixing 50 ⁇ l of ready-to-use hybridization buffer (DAKO A/S, Glostrup, Danmark) with about 5-10 pmol of the probes.
  • the probes are biotin-and Digoxygenin-labelled oligonucleotides of sequences complementary to the respective mRNAs.
  • the hybridization mixture is heated to 95° C. and afterwards, equilibrated to 37° C. After the boiling procedure, the smears are incubated with each 50 ⁇ l of the hybridization mixture for 4 hours at 42° C. The samples are washed in excess volumes of the wash buffers two times in 2 ⁇ SSC at 37° C. for 15 min, and once in 1 ⁇ SSC at 37° C. for 15 min. Then, the smears are rinsed two times at room temperature in 2 ⁇ SSC.
  • the dissections are incubated for 30 min with blocking buffer (NEN, blocking buffer) at room temperature, followed by a 1 hour incubation with a 1:100 diluted (in blocking buffer, see above) Streptavidin-alkaline phosphatase and monoclonal mouse HRP-labeled anti-Digoxygenine antibodies (Molecular Probes).
  • the smears are then washed 2 times in 1 ⁇ PBS/0.1% Triton X-100 for 10 min at room temperature, followed by one wash step with 1 ⁇ PBS, 50 mM MgCl 2 (pH 9.2) for 10 min at room temperature. Then the staining reaction is performed with ELF 97 phosphate (Molecular Probes) for 10 sec to 7 min at room temperature.
  • Excess substrate is washed 3 times with 1 ⁇ PBS/0.1% Triton X-100 for 10 min at room temperature.
  • the section is incubated with Tyramides-Alexa-Fluor 594 for 10 sec to 7 min.
  • Excess substrate is washed 3 times with 1 ⁇ PBS/0.1% Triton X-100 for 10 min at room temperature.
  • the smears are dipped in H 2 O dest and embedded with Fluorescence mounting medium (DakoCytomation). Then the stained dissections can be analysed by fluorescence microscopy.
  • mice Microscopic examination of the slides reveals that cells positive for expression of p16 INK4a and mcm2 are only found in samples that are microscopically identified as samples of dysplastic lesions. Cells stained by the p16 INK4a specific reaction that are identifiable as metaplasias are not stained by the reaction specific for mcm2. The microscopic inspection of the mRNA hybridization shows that metaplastic cells over-expressing p16 INK4a do not significantly express mRNA of mcm2. Dysplastic cells, in contrast, are stained by in situ hybridization with probes specific for mcm2, and with probes directed against p16 INK4a . So, in contrast to dysplastic cells, in metaplastic cells, no double staining using the Ki67 and p16 INK4a specific probes is observed.
  • Merckofix®-fixed cytological samples (conventional smears and liquid-based cytology (ThinPreps®)) of the cervix uteri are immunofluorescent stained using antibodies specific for p16 INK4a and Ki-S2.
  • the specimens are incubated with 10% goat serum for 30 min at RT.
  • the slides are then incubated with the primary antibodies, mouse anti-human p16 INK4a antibody (clone E6H4) (3.48 ⁇ g/ml) and rabbit anti-Ki-S2 (1:25) for 30 min at RT, and then the slides are rinsed with washing buffer and placed in a fresh buffer bath for 5 min. Excess buffer is tapped off, and the specimen is covered with 200 ⁇ l of the secondary reagent containing goat anti-mouse antibody, AlexaFluor®488 conjugated and goat anti rabbit antibody, and Alexa Fluor®546 conjugated and then incubated for 30 min at RT. Then, slides are washed two times as before and directly mounted with a special mounting medium for fluorescence.
  • mice Microscopic examination of the slides reveals that cells immunoreactive with p16 INK4a and Ki-S2 may be identified microscopically as dysplastic cells.
  • Cells stained by the p16 INK4a specific reaction which are not stained by the reaction specific for Ki-S2, may be classified by an experienced pathologist as either being metaplastic or of endometrial origin.
  • Microscopic inspection of the cell proliferation marker staining shows that metaplastic cells over-expressing p16 INK4a are not immunoreactive with the antibodies directed against Ki-S2.
  • Dysplastic cells in contrast, are immunoreactive with Ki-S2 and with antibodies directed against p16 INK4a . So, in contrast to dysplastic cells, in metaplasias, no cells are double stained using the Ki-S2 and p16 INK4a specific antibodies.
  • bronchioalveolar lavage specimens of patients are prepared according to ThinPrep technology.
  • Merckofix®-fixed cytological samples of the lavages of patients diagnosed with small cell lung cancer are immunofluorescent stained using antibodies specific for p16 INK4a , Ki67, and PCNA.
  • tissue sections are rehydrated through incubation in xylene and graded ethanol, and then transferred to Aqua bidest.
  • Conventional smears and liquid-based cytological samples (ThinPreps®) are rehydrated in ethanol (50%) for 10 min, and transferred in Aqua bidest.
  • Antigen retrieval is carried out with 10 mM citrate buffer (pH 6.0) for p16 INK4a , Ki67, and PCNA. Therefore, the slides are heated in a waterbath for 40 min at 95-98° C.
  • the slides are cooled down to RT for 20 minutes, transferred to washing buffer (50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20/DakoCytomation: code no.: S3006), and finally, the samples are surrounded with a lipid-pencil.
  • washing buffer 50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20/DakoCytomation: code no.: S3006
  • the specimens are incubated with 10% goat serum for 30 min at RT.
  • the slides are then incubated with the primary antibodies, mouse anti-human p16 INK4a antibody (3.48 ⁇ g/ml), rabbit anti-Ki67, and rabbit anti-PCNA (each 1:25) for 30 min at RT.
  • the slides are then rinsed with washing buffer and placed in a fresh buffer bath for 5 min. Excess buffer is tapped off, and the specimen is covered with 200 ⁇ l of the secondary reagent (containing goat anti-mouse antibody, AlexaFluor®488 conjugated and goat anti rabbit antibody and, Alexa Fluor®546 conjugated) and then incubated for 30 min at RT. Then slides are washed two times as before and directly mounted with a special mounting medium for fluorescence.
  • Cytological samples (Cell suspensions in PBS, pH 7.4) of the cervix uteri were fluorescent stained using antibodies specific for p14ARF and Ki-67 and oligoprobes for mcm-5 and evaluated by three-color fluorescent FACS analysis.
  • Cells were centrifuged, the supernatant decanted and fixed and permeabilized with 100 ml Permeafix (Ortho Diagnostic, Raitan, N.J., USA) for 1 hour at room temperature. Cells were washed in sterile PBS, pH 7.4, pelleted, and re-suspended in 100 ml Permeafix for 1 hour at room temperature. Cells were washed in sterile PBS, pH 7.4, pelleted, and re-suspended in sterile PBS. They were incubated with PE-conjugated anti-p14ARF antibody and PE-Cy5-conjugated anti-Ki67 antibody for 1 h at +4° C.
  • PE-conjugated anti-p14ARF antibody and PE-Cy5-conjugated anti-Ki67 antibody for 1 h at +4° C.
  • Cells were washed in sterile PBS, pH 7.4, pelleted, and re-suspended in 100 ml Permeafix for 30 min at room temperature. Cells were washed in sterile PBS, pelleted by centrifugation, and then washed again in 2 ⁇ standard saline citrate (SSC). After centrifugation, the cell pellet was resuspended in hybridization solution (2 ⁇ SSC, 30% formamide, sonicated salmon sperm, and yeast transfer DNA) containing 500 ng of 5-carboxy-fluorescein double end-labelled, mcm5-specific oligonucleotides probes. The intercellular hybridization was performed at 42° C.
  • Sections of formalin-fixed, paraffin-embedded tissue samples of the cervix uteri were immunoenzymatically double-stained using antibodies specific for p16 INK4a and Ki67.
  • tissue sections were rehydrated through incubation in xylene and graded ethanol, and transferred to Aqua bidest.
  • Antigen retrieval was carried out with 10 mM citrate buffer (pH 6.0) for p16 INK4a and Ki67. Therefore, the slides were heated in a waterbath for 40 min at 95-98° C. The slides were cooled down to RT for 20 minutes and transferred to washing buffer (DakoCytomation).
  • the slides were incubated with the second primary antibody, rabbit anti-human Ki67 antibody (Dianova, clone Ab-3) for 30 min at RT, and were then rinsed with washing buffer and placed in a fresh buffer bath for 5 min. Excess buffer was tapped off, and each specimen was covered with 200 ⁇ l of the secondary reagent (goat anti rabbit—Alkaline Phosphatase labeled/DakoCytomation) and incubated for 30 min at RT. Then slides were washed three times as before. For the chromogenic visualization, FastRed (BioGenex) was used by incubating the slides with the substrate chromogen complex for 30 min at RT. The reaction was stopped in deionized water.
  • the secondary reagent goat anti rabbit—Alkaline Phosphatase labeled/DakoCytomation
  • FIG. 7 show that double staining of cells with reagents specific for Ki67 and p16 INK4a also produces a specific double-staining pattern in chromogenic staining procedures.
  • the slides were incubated with the first primary antibody, mouse anti-human p16 INK4a antibody, for 30 min at RT, and were then rinsed with washing buffer and placed in a fresh buffer bath for 5 min. Excess buffer was tapped off, and each specimen was covered with 200 ⁇ l of the secondary reagent (EnVision goat anti mouse—Peroxidase) and incubated for 30 min at RT. Then the slides were washed three times as before.
  • DAB DeepoCytomation
  • the slides were incubated with the second primary antibody, rabbit anti-human Ki67 antibody (Dianova, clone Ab-3) for 30 min at RT, and were then rinsed with washing buffer and placed in a fresh buffer bath for 5 min. Excess buffer was tapped off, and each specimen was covered with 200 ⁇ l of the secondary reagent (goat anti rabbit—Alkaline Phosphatase labeled/DakoCytomation) and incubated for 30 min at RT. Then the slides were washed three times as before. For the chromogenic visualization, Fast Red (BioGenex) was used by incubating the slides with the substrate chromogen complex for 30 min at RT. The reaction was stopped in deionized water.
  • the secondary reagent goat anti rabbit—Alkaline Phosphatase labeled/DakoCytomation
  • FIG. 8 show that the double staining of cells with reagents specific for Ki67 and p16 INK4a also produces a specific double staining pattern in chromogenic staining procedures.

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US13/798,998 US9528159B2 (en) 2002-10-28 2013-03-13 Method for improved diagnosis of dysplasias
US13/799,093 US20130189686A1 (en) 2002-10-28 2013-03-13 Method for Improved Diagnosis of Dysplasias
US16/579,495 US20200017923A1 (en) 2002-10-28 2019-09-23 Method for improved diagnosis of dysplasias
US17/086,817 US20210047697A1 (en) 2002-10-28 2020-11-02 Method for improved diagnosis of dysplasias
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