+

WO1997016731A1 - Marquage immunicytochimique d'echantillons de cellules cervicales exfoliees - Google Patents

Marquage immunicytochimique d'echantillons de cellules cervicales exfoliees Download PDF

Info

Publication number
WO1997016731A1
WO1997016731A1 PCT/US1996/017399 US9617399W WO9716731A1 WO 1997016731 A1 WO1997016731 A1 WO 1997016731A1 US 9617399 W US9617399 W US 9617399W WO 9716731 A1 WO9716731 A1 WO 9716731A1
Authority
WO
WIPO (PCT)
Prior art keywords
sample
cells
antibodies
squamous
smears
Prior art date
Application number
PCT/US1996/017399
Other languages
English (en)
Inventor
Charles J. Dunton
Karen H. Van Hoeven
Albert J. Kovatich
Original Assignee
Thomas Jefferson University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Thomas Jefferson University filed Critical Thomas Jefferson University
Publication of WO1997016731A1 publication Critical patent/WO1997016731A1/fr

Links

Classifications

    • 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

Definitions

  • the invention relates to identification of pre ⁇ neoplastic and neoplastic squamous cell lesions of the human uterine cervix by evaluating immunocytochemically-stained samples of exfoliated cervical tissue, looking for abnormally proliferating squamous cells.
  • Pap smear Cytomorphologic interpretation of a Papanicolaou- stained cervical smear (Pap smear) is the mainstay of cytologic evaluation of the human cervix.
  • the predictive value of the cytologic diagnosis of squamous intraepithelial lesion (SIL) is quite high (DiBonito L, et al . 1993 Cancer 72:3002-6) .
  • SIL squamous intraepithelial lesion
  • cervical cytology serves as an excellent tool for identifying patients who need colposcopic evaluation and biopsy.
  • ASCUS cytologic diagnosis of squamous atypia, or "atypical squamous cells of undetermined significance"
  • ASCUS typically squamous cells of undetermined significance
  • Patients with ASCUS may be managed by performing a repeat Pap smear - that may be subject to the same interpretive and sampling problems of the earlier smear (Busseniers A.E. and Sidawy M.K. 1991 J. Reprod. Med. 36:85-88 and Soutter W.P., et al . 1986 Br. J. Obstet . Gynaecol . 93:70- 74) - or by colposcopy (Darnell Jones D.E., et al . 1987 Am. J. OJbstet.
  • the present invention relates to a method of identifying an individual with a squamous intraepithelial lesion.
  • the method comprises the steps of first depositing a sample of exfoliated cervical cells from an individual onto a solid support and then fixing cells of the sample by non- immunologically neutralizing desiccation or a non- immunologically neutralizing fixative.
  • the sample is then incubated with specific antibodies that bind to antigens that are expressed by proliferating epithelial cells for a time sufficient and under conditions suitable for the antibodies to form complexes with antigens present in the sample. Unbound antibodies are removed from the sample and the sample is inspected to detect the presence of antibodies bound to antigen in epithelial cells in the sample.
  • the presence of antibodies bound to antigen in squamous epithelial cells with enlarged nuclei in the sample indicates a proliferating cell population.
  • the detection of this proliferating cell population indicates a high probability that a squamous intraepithelial lesion or squamous carcinoma is present.
  • the present invention relates to a method of identifying an individual with a squamous intraepithelial lesion.
  • the method comprises the steps of: preparing corresponding parallel samples that contain exfoliated cervical cells.
  • a first sample of exfoliated cervical cells from said individual is deposited onto a solid support and a second sample of exfoliated cervical cells from said individual is deposited onto a solid support.
  • the first sample of exfoliated cervical cells from said individual is deposited onto a first solid support and a second sample of exfoliated cervical cells from said individual is deposited onto a second solid support.
  • the first sample of exfoliated cervical cells from said individual is deposited onto a solid support and a second sample of exfoliated cervical cells from said individual is deposited onto an adjacent part of the solid support.
  • the cells of the first sample are fixed with a Pap smear fixative that prepares them for Papanicolaou staining.
  • the cells of the second sample are fixed by non-immunologically neutralizing desiccation or a non-immunologically neutralizing fixative.
  • the cells of the first sample are contacted with Papanicolaou stain for sufficient time for the cells to incorporate stain and become stained.
  • the excess stain is removed by washing to remove unincorporated stain and the sample is evaluated to detect staining patterns of cells consistent of Pap smear analysis to determine if the sample contains (1) normal cells only or (2) a squamous intraepithelial lesion or (3) atypical squamous cells of undetermined significance. If the Pap smear analysis results indicates atypical squamous cells of undetermined significance, the second sample is evaluated by the immunocytochemical assay.
  • the immunocytochemical assay comprises the further steps of contacting the second sample with detectable antibodies that bind to antigens that are expressed by proliferating epithelial cells for a time sufficient and under conditions suitable for said antibodies to form complexes with antigens present in the second sample.
  • the unbound antibodies are removed and the sample is inspected to detect the presence of antibodies bound to the antigen in epithelial cells.
  • the presence of antibodies bound to antigen in squamous cells with enlarged nuclei in the second sample indicates a proliferating cell population.
  • the detection of this proliferating cell population indicates a high probability that a squamous intraepithelial lesion or squamous carcinoma exists.
  • a method which can identify proliferating cells in an exfoliated cervical tissue sample.
  • the present invention provides methods of analyzing an exfoliated cervical tissue sample to obtain information with respect to the nature the cell proliferation, the molecular basis of the cell proliferation, the presence of malignant disease, the extent of development of disease.
  • an exfoliated cervical tissue sample may be used to identify individuals with abnormally proliferating cells, to identify whether the proliferation is a benign hyperplasia or a malignancy, what the molecular nature and basis of the malignancy is and/or the extent of disease development.
  • the invention provides the means to identify proliferation of cells in exfoliated cervical samples and to make qualitative assessments of them.
  • the present invention provides methods of identifying SIL in an exfoliated cervical tissue sample.
  • SIL in exfoliated cervical tissue samples is provided.
  • the method may be used independent of Pap smear analysis, parallel with Pap smear analysis on separate samples taken from the same patient or as a follow up analysis after ASCUS diagnosis.
  • Pap smear methodology is well known, widely used and readily available to those having ordinary skill in the art. Briefly, to take a Pap smear, exfoliated cells are removed from the exocervix and endocervix with a swab, brush, spatula, or other sampling device. The cells are wiped onto one or two glass slides, and thereafter immediately sprayed with one of a number of commercially available fixatives (such as Surgipath Cytology Fixative, Surgipath Medical Industries, Inc., Grayslake, IL) and transported to the laboratory. There, the slide is stained with one of a variety of commercially available Papanicolaou stains or modifications thereof.
  • fixatives such as Surgipath Cytology Fixative, Surgipath Medical Industries, Inc., Grayslake, IL
  • Slides are examined microscopically by a cytotechnologist to determine adequacy of the specimen, and to screen for the presence of any cellular abnormality. If the cytotechnologist does not find any cellular abnormality, the case is diagnosed as negative, a report is generated, and signed by the cytotechnologist. If the cytotechnologist detects a cellular abnormality, the slide is reviewed by a pathologist who interprets the cellular abnormality on the smear, a report is issued, and signed by the pathologist.
  • the currently accepted reporting system for Pap smears (also known as cervical smears or Papanicolaou smears) is the 1991 Bethesda System (Kurman RJ, Solomon D.
  • Atypical squamous cell of undetermined significance (ASCUS) : cellular abnormalities qualitatively or quantitatively insufficient for a definitive diagnosis of a squamous intraepithelial lesion or squamous carcinoma.
  • LGSIL Low grade squamous intraepithelial lesion
  • High grade squamous intraepithelial lesion criteria of LGSIL but with a higher nuclear/cytoplasmic ratio, resulting in a cell size that is usually lower than seen in LGSIL.
  • Nucleoli are generally absent and nuclear outlines are irregular.
  • the cytoplasm may appear either lacy or keratinized.
  • the invention provides an immunocytochemical method of diagnosing SIL.
  • antibodies which bind to antigens characteristically expressed by proliferating cells are contacted with samples of exfoliated cervical tissue for a time sufficient and under conditions in which the antibodies will bind to the antigens present in cells of the tissue sample.
  • the antibodies that are used bind to antigens that are characteristically expressed by proliferating cells and that are not expressed by normal squamous intraepithelial cells.
  • the antibodies are usually detectable by light microscopy.
  • the antibodies are usually detectable by light microscopy due to a label conjugated directly to them or by contacting them with a second antibody that is specific for the first antibody and that is detectable such as by a conjugated label.
  • the presence of antigens in cells in the sample may be determined by detecting antibodies bound to cells of the sample.
  • the sample is examined to determine whether squamous intraepithelial cells in the sample contain the antigen.
  • samples are obtained in the manner similar to that which is used to obtain Pap smear samples. That is, samples are collected using a swab, brush or spatula and cells in the sample are deposited on microscope plates by standard techniques well known to those skilled in the art.
  • a cytobrush is used to obtain endocervical samples that are smeared onto one half of a slide, and Ayre spatulas are used to obtain cervical samples smeared onto the other half of the same slide.
  • Air-dried samples are obtained smearing collected exfoliated cells onto uncoated or NEOPRENE"" adhesive cement- coated glass slides.
  • NEOPRENETM adhesive cement polychloroprene, Aldrich Chemical Company, Milwaukee, Wisconsin
  • Other types of coated slides include poly-L-lysine coated slides, silanated slides and positively charged slides.
  • Uncoated glass slides may be used for smears provided that the subsequent steps are not excessively disruptive to result in t h e de t a c hme nt o f t he c e l l s .
  • samples may first be incorporated in a liquid medium for transport for example. Prior to analysis, the samples are transferred to a solid support.
  • the sample In the case of a smear to be stained with the Papanicolaou stain, the sample is first fixed to the slide after it is deposited on a slide.
  • the fixative used denatures proteins.
  • the samples used in the immunocytochemical assay of the present invention are not fixed in the manner used in Pap smear methodology. Rather, samples used in the immunocytochemical assay of the present invention are air dried and/or desiccated with a non-immunologically neutralizing fixative such as acetone.
  • non-immunologically neutralizing fixative is meant to refer to fixatives which do not render antigenic proteins non- antigenic by disruption of the conformation of recognized epitopes or otherwise.
  • antibodies specific for a protein will recognize a protein after it has been exposed to a non-immunologically neutralizing fixative.
  • Samples are air dried by exposing them to open air, a controlled gaseous environment and/or a vacuum or pressure deficient environment. Air dried samples are exposed to such environments until sufficient evaporation of moisture in the sample takes place. Samples prepared without immunologically neutralizing fixative are referred to herein as "non- immunologically neutralized samples" .
  • non- immunologically neutralizing fixatives examples include acetone and alcohol such as ethanol.
  • antibody is meant to refer to complete, intact antibodies, and Fab fragments and F(ab) 2 fragments thereof that binds to an antigen that is characteristically expressed by proliferating cells and that is not expressed by normal squamous intraepithelial cells.
  • Complete, intact antibodies include monoclonal antibodies such as murine monoclonal antibodies, chimeric antibodies and humanized antibodies.
  • the production of antibodies and the protein structures of complete, intact antibodies, Fab fragments and F(ab) 2 fragments and the organization of the genetic sequences that encode such molecules are well known and are described, for example, in Harlow, E. and D.
  • proliferating cell-specific antibodies is meant to refer to antibodies that bind to antigens that are characteristically expressed by proliferating cells and that are not expressed by normal squamous intraepithelial cells.
  • the samples fixed with non-immunologically neutralizing fixative are contacted with proliferating cell specific antibodies.
  • the conditions and amount of time sufficient for immunocomplex formation between the antibodies and antigens present in the non-immunologically neutralized samples can be determined using routine experimentation.
  • the techniques for detecting and visualizing any antibody-protein complexes of the immunocytochemically stained samples are well known.
  • the conditions and times are dependent on the location of the antigen in the cell, i.e. cytoplasmic, cell membrane, nuclear membrane, nucleus.
  • Incubation time is also a function of the affinity of the primary antibody for the antigen, the concentration of the antibody, and the concentration of the antigen.
  • antigens that are characteristically expressed by proliferating cells and that are not expressed by normal squamous intraepithelial cells include: Ki-67, cyclins, mutant p53 and other oncoproteins.
  • assays are performed to identify other antigens after initial identification of the presence of Ki-67.
  • the presence of other antigens can indicate the nature of the proliferation, the type of cancer which the patient may have and the extent of development of disease.
  • examples of other antigens that may be useful after triaging SILs by Ki-67 staining are cyclins, mutant p53 and other oncoproteins.
  • Ki-67 is a nuclear nonhistone antigen expressed in cells that are proliferating (Gerdes J. , et al . 1984 J. Immunol . 133:1710-1715) .
  • Proliferating cell specific antibodies may be commercially available.
  • monoclonal antibody MIB-1 which binds to Ki-67, is commercially available from Immunotech Inc. (Westbrook, ME) .
  • Proliferating cell specific antibodies must be detectable either by labelling them such as conjugating them to a detectable material, or by first contacting samples with proliferating cell specific antibodies and then, after removing any uncomplexed antibodies, contacting the sample with detectable antibodies that bind to the proliferating cell specific antibodies.
  • labelling methods must be compatible with the samples to reduce or eliminate false labelling and ensure that labelled antibodies bound to antigen can be detected.
  • labels for making antibodies detectable include avidin-biotin peroxidase (ABC) , immunoalkaline phosphatase anti-alkaline phosphatase (APAAP) , streptavidin peroxidase, streptavidin alkaline phosphate, glucose oxidase, fluorescein conjugates and immunogold to name a few.
  • ABSC avidin-biotin peroxidase
  • APAAP immunoalkaline phosphatase anti-alkaline phosphatase
  • streptavidin peroxidase streptavidin alkaline phosphate
  • glucose oxidase fluorescein conjugates
  • immunogold to name a few.
  • the immunocytochemical assay is performed routinely.
  • Proliferating cell specific antibodies are contacted with the smear a time sufficient and under conditions suitable for binding of the antibodies to any antigen present in the cells in the smear. After sufficient time has elapsed, the smear is washed to remove unbound antibody.
  • the smears are examined by a trained evaluator or an automated evaluator. Squamous cells in the smear are identified by their cytomorphologic characteristics. Examples of automated evaluators include the SAMBA 4000 (Imaging Products International Inc., Chantilly, IL) and Papnet (Neuromedical Systems, Inc. Suffern, New York) . The squamous cells on the smear are evaluated for nuclear labelling by the chromogen detector as determined by the immunocytochemical methodology that was used. For example, diaminobenzidine ⁇ H 2 0 2 . (DAB) is commonly used as the chromogen for ABC.
  • DAB diaminobenzidine ⁇ H 2 0 2 .
  • DAB decorates the abnormally enlarged squamous nuclei a dark brown color.
  • the presence of abnormally enlarged squamous nuclei that stain positively e.g. are brown in color
  • the preferred embodiment of the invention provides the immunocytochemical assay as a secondary screening of samples following primary Pap smear evaluation. That is, two samples are taken from a patient: one is processed for Pap smear analysis while the other part of a processed for immunocytochemical analysis.
  • the Pap smear sample is fixed with a denaturing fixative while the second smear is either not fixed (air dried) or fixed with a non-immunologically neutralizing fixative.
  • the first smear and second smear are labelled so that they can be matched to each other as corresponding samples should parallel analysis be undertaken.
  • the first smear is fixed and stained as per standard Pap smear methodology and evaluated by a trained evaluator. Initially, the smear is evaluated by a cytotechnologist. If no cellular abnormality is identified, the smear is diagnosed as negative, a report is generated, and is signed by the cytopathologist. If a cellular abnormality is identified by the cytotechnologist, then the smear is evaluated by a pathologist.
  • a second slide containing cervical cells may be evaluated by the immunocytochemical assay of the invention to diagnose SIL.
  • Ki-67 nuclear antigen is expressed in upper epithelial levels of intraepithelial neoplasia of the cervix and vulva, variably in condyloma, and in basal and parabasal cells of normal squamous mucosa in histologic preparations.
  • the possible application of this pattern of immunoreactivity to cervical smears was explored using either 1) air-dried acetone-fixed cervical smears obtained from 106 consenting patients or 2) a single slide from archival 2-slide cases of SIL. MIB-1 monoclonal antibody to Ki-67 was tested using two immunocytochemical techniques.
  • cervical smears presented unique difficulties that were not anticipated.
  • H 2 0 2 /PBS blockade was found to be essential to facilitate a reliable interpretation of the slides. This step had not been performed in prior descriptions of Ki-67 immunostaining of cytologic material.
  • the reliability of uncoated glass smears for cervical immunocytochemistry had not been tested. Very few investigators have applied immunocytochemical stains to Pap smears. Immunocytochemical detection of MN tumor-associate antigen was studied in decolorized cervical smears.
  • the study protocol was approved by the institutional review board of Thomas Jefferson University. All patients signed written consent for air-dried Pap smears to be taken and used for experimental purposes. Patients were enrolled for the study at the time of routine gynecologic examination, or at the time of colposcopic examination.
  • Neoprene polychloroprene, Aldrich Chemical Company, Milwaukee, Wisconsin
  • a cytobrush was used to obtain endocervical samples that were smeared onto one half of a slid
  • Ayre spatulas were used to obtain cervical samples smeared onto the other half of the same slide. Additional smears were prepared from each patient using the same spatula and brush sample from the first smear, without additional swabbing of the cervix.
  • Air-dried smears labelled only with the patient's name, were desiccated, acetone-fixed for 10 minutes, and then frozen at -70 degrees centigrade or immediately stained. Slides are stored in sealed slid boxes for up to three months before staining and the immunoreactivity is retained.
  • the monoclonal antibody to Ki-67 antigen was MIB-1 which was obtained from Immunotech (Westbrook, ME) .
  • the antibody was used in a dilution of 1:50 to 1:100. Slides were incubated with the antibody for sixty minutes.
  • Shandon Cadenza Immunostainers (Shandon, Pittsburgh, Pennsylvania) were used to perform the immunostaining, which was done at room temperature. This instrument has proven to be effective for immunocytochemical staining of cytospin preparations, peripheral blood smears, and touch preparations. Two different immunocytochemical detection studies were tested on cervical smears.
  • the second immunocytochemical detection system used an immunoalkaline phosphatase anti-alkaline phosphatase (APAAP) method for detection of antigen-antibody complexes (Cordell J.L., et al . 1984 J. Hi ⁇ tochem. Cytochem . 32:219-229), employing new fuchsin as the chromogen.
  • APAAP immunoalkaline phosphatase anti-alkaline phosphatase
  • any nuclear decoration in squamous epithelial cells were considered immunoreactive.
  • Abnormal squamous epithelial cells were those with enlarged nuclei, and often with irregular nuclear borders. Since air-drying artifact is commonly associated with artifactual nuclear swelling, the increased size of abnormal nuclei was assessed visually by comparison to neighboring normal superficial and intermediate cells.
  • the optimal protocol for identifying nuclear expression of Ki-67 using the monoclonal antibody MIB-1 utilized air-dried smears that were acetone-fixed, blocked with H 2 0 2 /PBS, and stained using ABC. Although most smears were collected on Neoprene-coated slides, uncoated glass slides performed equally well.
  • the wash protocols of the Shandon Candenza immunostainer are not very rigorous, so coated slides did not make a difference. Manual techniques or other instruments may be more rigorous; there might be cellular loss on uncoated slides due to lengthy incubations in an aqueous environmen .
  • MATERIALS AND METHODS Approval for the study was obtained from the institutional R colposcopy because of abnormal cytology Review Board of Thomas Jefferson University Hospital. Patients referred for were offered participation and signed informed consent. Colposcopic referral was for all HGSIL, LGSIL, AGUS cytology. ASCUS smears qualified as favoring neoplasia according to the 1991 Bethesda classification were also referred and underwent Colposcopic examination. ASCUS smears qualified as representing reactive changes are followed by repeat cytology. Smears were obtained from 124 non-pregnant patients at the time of colposcopy. The referral cytology was used as the basis for final comparison to K-l-67 and histology.
  • Neoprene-coated slides Air-dried cervical/endocervical smears were obtained from each patient, and placed onto either Neoprene-coated slides, or, in a few patients, uncoated glass slides.
  • Wisconsin is a chemical that increases cellular adhesion.
  • Exfoliated endocervical material was smeared onto half of the slide from a CYTOBRUSH * sample collection device (MedScand AB, Hollywood FL ) and the exocervical sample was smeared onto the other half of the slide from an Ayre spatula.
  • the test was positive when MIB-l staining was identified in abnormally enlarged squamous nuclei.
  • Nuclear enlargement was defined as a nuclear diameter at least two and half times that of a normal intermediate squamous cell nucleus. Immunoreactivity confined to the nucleolus, endocervical reactivity, or decoration of naked nuclei, devoid of cytoplasm were considered insufficient for positivity. When nuclear decoration was identified in rare cells with minimal nuclear enlargement (1.5 to 2.5 times normal intermediate cell nucleus), the test was considered equivocal. These equivocal results were treated as positive in statistical analysis.
  • Referral cytology was ASCUS, favoring neoplasia (42) , AGUS (3) , LGSIL (56) , and HGSIL (23) .
  • the association between referral cytology and final histologic diagnoses are seen in Table 1.
  • the average age of these 124 patients was 27.5 (13- 62) years.
  • Thirty-five patients were Caucasian, 84 were African-American and 5 were Hispanic.
  • 62 (50%) were referred for first abnormal cytology and 62 (50%) had a history of abnormal pap smears or treatment for dysplasia.
  • Forty-three (34.7%) were smokers and 81 (65.3%) were non-smokers.
  • Patients with greater than CIN 2 lesions were treated with loop excision procedures and the final pathology is reported as the worst lesion encountered on either colposcopically directed biopsy or loop excision.
  • Ki-67 immunoreactivity in relation to final histologic diagnosis is seen in Table 2. All smears were considered satisfactory for evaluation, although some were limited by a scant or absent endocervical component and others were limited by scant cellularity. No smears were considered limited by obscuring inflammatory cells, because these cells were not immunoreactive. Enlarged squamous cell nuclei stained dark brown and were identified without difficulty, even in the presence of numerous light blue neutrophils.
  • Ki-67 immunoreactivity had a sensitivity (.89), a specificity (.65), a positive predictive value (.60) and a negative predictive value (.91) in detection of high grade CIN.
  • the prevalence of high grade CIN was 0.37.
  • Ki-67 immunostaining demonstrated sensitivity (.96), specificity (.67), positive predictive value (.49) and negative predictive value (.98) in detection of high grade CIN.
  • the prevalence of high grade CIN was 0.24.
  • Ki-67 immunostaining was a significant predictor in multivariate analysis (odds ratio
  • Ki-67 nuclear staining is observed in normal mucosa only in parabasal and basal cells, and its expression is seen in higher epithelial levels in SIL. Areas of mature and immature metaplasia, while usually staining in a pattern like that of normal mucosa, may exhibit small numbers of cells in the upper epithelial levels. Ki-67 staining of a few cells in smears from patients with squamous metaplasia is generally regarded as negative, since nuclei in squamous metaplasia are not abnormally enlarged. Abnormal nuclear enlargement in cells with high nuclear to cytoplasmic ration can at times be seen in immature metaplasia. We attribute some of our "false positives" to this phenomenon. The difficulty of distinguishing immature metaplastic cells from high grade SIL by morphologic means is a problem with Papanicolaou staining that could, in some smears, be resolved by Ki-67 staining.
  • the evaluation of MIB-l stained slides is a two-step process, involving both morphologic identification of an enlarged squamous nucleus and MIB- I immunoreactivity using a chromogen to mark those cells.
  • the biology of an ASCUS cell is unknown by morphologic evaluation alone; only approximately 30% of ASCUS diagnoses reveal SIL on biopsy.
  • Ki-67 staining provides data on the biology (i.e. proliferative capacity) of the cells. From our data, an adjunctive test such as Ki-67 staining could potentially reduce the number of Colposcopic procedures in biopsy-negative patients by more than 50%. Ki-67 staining using the MIB-l antibody correlates more strongly with the presence of HPV in SIL than does biopsy alone.
  • HPV/CIN1 lesions were considered as negative for disease in the statistical analysis. While it is recognized that a small percentage of these patients may develop high precursors or cancer, observation of these lesions is an acceptable modality. If no treatment of these patients is planned then detection of such lesions is less important.
  • Ki-67 staining may demonstrate less predictive value if used in a population with large numbers of inflammatory lesions.
  • low rates of high grade precursors were discovered allowing for follow-up with cytology only. Adjunctive testing is probably not necessary in this group of patients with such low incidence of CIN.
  • MIB-l stains squamous intraepithelial lesions (SIL) in both histologic and cytologic preparations.
  • SIL squamous intraepithelial lesions
  • MTB-1 stained smears were submitted for analysis by PAPNET. Routine microscopic evaluation revealed MIB-1+ squamous cells in 57 smears; 49 were biopsied: all but one revealed SIL. Smears from the other 43 patients were devoid of MIB-l positive squamous cells by routine evaluation; 35 patients had previous abnormal Pap smears - all but six had followup smears or biopsies that were negative. Of the latter six, three had persistent atypical smears, and three had SIL on biopsy.
  • PAPNET detected MIB-1+ squamous cells in all 57 smears where they had been identified by manual screening.
  • PAPNET identified MIB-1+ squamous cells in four of 43 smears considered negative by manual screening; three of these four were from the group of six with persistent atypical smears or SIL on biopsy.
  • the PAPNET system detected MIB-1+ cells in immunostained cervical smears.
  • This automated detection system combined with MIB-l immunostaining for SIL, offers a largely unexplored technology to analyze gynecologic smears.
  • slides were scanned at Neuromedical Systems, Inc., by their computer system which uses both algorithmic and neural network processing.
  • the computer selects 128 cell scenes, identified as potentially abnormal, which are projected onto a high-resolution monitor at a PAPNET review station and interpreted by the operator. Slide coordinates are provided so that manual microscopic review of suspicious areas can be performed.
  • PAPNET could be utilized to identify MIB-l positive cells on cervical smears stained by immunocytochemical methods.
  • MIB-l reactive squamous nuclei in all 57 smears that had been manually screened as positive.
  • many of the cell scenes on the computer monitor displayed MIB-l reactive cells. Only a few MIB-l reactive cells were captured on the monitors from smears with few MIB-l reactive cells Thus, the number of cell scenes showing MIB-l positive cells was proportional to the number seen manually.
  • MIB-l negative koilocytotic cells morphologically consistent with human papillomavirus cytopathic effect, were captured by the computerized network and projected onto the monitor.
  • Biopsies were available from 49 of these 57 patients. All but one revealed a squamous intraepithelial lesion (SIL) .
  • SIL squamous intraepithelial lesion Table 5 lists clinical and pathologic data analyzed according to MIB-l reactivity as detected by PAPNET.
  • MIB-l negative ASCUS was often captured via the neural network and projected as abnormal cells on the monitor screen. MIB-l negative ASCUS is interpreted as negative using this immunocytochemical staining method.
  • the PAPNET system was able to select MIB-l reactive cells for analysis and interpretation. In addition to identifying MIB-l positive cells in all 57 smears where they were found manually, it found rare reactive cells in four smears where they had been missed manually. Because cervical smears contain at least 300,000 cells, and only a few cells in any smear may be abnormal, the PAPNET system has the capacity to ferret out these rare cells and display them for the operator.
  • MIB-l expression is seen in histologic sections of SIL.
  • Low grade SIL derived from HPV 16/18 and 31/33/35/novel types provide higher densities of MIB-l positive cells than HPV 6/11 lesions.
  • Resnick M., et al. Hum. Pathol. 1996 27:234-239. These data provide support for a hypothesis that MIB-l reactivity is a marker of the capacity for biologic aggression.
  • MIB-l- negative koilocytotic cells that were detected to low densities of MIB-l reactivity associated with low risk HPV types.
  • MIB-l labels the nuclear antigen Ki-67, a proliferation marker that stains nuclei in the GI, S, G2 and mitotic phases of the cell cycle. Because its expression is a marker of the biology of the cell, i.e. its proliferative capacity, MIB-l immunoreactivity provides more useful information than morphology alone.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention porte sur un procédé d'identification d'un individu porteur de lésions intraépithéliales squameuses consistant à analyser par un moyen immunocytochimique des échantillons de cellules cervicales exfoliées. Lesdits échantillons sont séchés à l'air ou desséchés à l'aide d'un fixatif neutralisant non-immunologique puis mis en contact avec des anticorps détectables se fixant aux antigènes exprimés par les cellules épithéliales proliférantes. Après incubation des anticorps et des échantillons et élimination des anticorps non fixés, les échantillons sont examinés pour y déceler la présence d'anticorps liés aux antigènes des cellules épithéliales, cette présence étant révélatrice de lésions intraépithéliales squameuses dans ces mêmes échantillons. L'invention porte en outre sur un procédé d'identification d'un individu porteur de lésions intraépithéliales squameuses par analyse immunocytochimique s'effectuant en parallèle avec un test de Papanicolaou ou suite à l'identification, sans signification déterminée, d'individus porteurs de cellules squameuses atypiques, obtenue par le test de Papanicolaou.
PCT/US1996/017399 1995-11-02 1996-11-01 Marquage immunicytochimique d'echantillons de cellules cervicales exfoliees WO1997016731A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US617195P 1995-11-02 1995-11-02
US60/006,171 1995-11-02

Publications (1)

Publication Number Publication Date
WO1997016731A1 true WO1997016731A1 (fr) 1997-05-09

Family

ID=21719642

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/017399 WO1997016731A1 (fr) 1995-11-02 1996-11-01 Marquage immunicytochimique d'echantillons de cellules cervicales exfoliees

Country Status (1)

Country Link
WO (1) WO1997016731A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997043646A1 (fr) * 1996-05-10 1997-11-20 University Of Limburg Procede et kit pour determiner le potentiel de malignite de cellules tissulaires
DE19936618A1 (de) * 1999-08-04 2001-03-15 Monotec Gmbh Diagnostisches Kit und dessen Verwendung
US7056690B2 (en) * 1997-10-21 2006-06-06 Cancer Research Technology Limited Detection of dysplastic or neoplastic cells using anti-MCM2 antibodies
US20080261266A1 (en) * 2004-12-17 2008-10-23 Ventana Medical Systems, Inc. Methods and compositions for a microemulsion-based tissue treatment
WO2009113859A1 (fr) * 2008-03-13 2009-09-17 Vereniging Voor Christelijk Hoger Onderwijs, Wetenschappelijk Onderzoek En Patiëntenzorg Algorithmes de dépistage cervical
CN113607534A (zh) * 2021-08-20 2021-11-05 河南赛诺特生物技术有限公司 一种染色方法、试剂盒及应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4782015A (en) * 1984-03-30 1988-11-01 Syntex (U.S.A.) Inc. Method for determining the presence of malignant cells

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4782015A (en) * 1984-03-30 1988-11-01 Syntex (U.S.A.) Inc. Method for determining the presence of malignant cells

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CANCER RESEARCH, November 1985, Vol. 45, KOPROWSKA et al., "Common Antigenic Sites on Exfoliated Cells Derived from Cervical Carcinoma and in Tumor Cells of Nonuterine Origin as Demonstrated by Monoclonal Antibodies in Immunoperoxidase Assay", pages 5964-5968. *
JOURNAL OF CLINICAL INVESTIGATION, November 1985, Vol. 76, MORHENN et al., "A Monoclonal Antibody Against Basal Cells of Human Epidermis, Potential Use in the Diagnosis of Cervical Neoplasia", pages 1978-1983. *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997043646A1 (fr) * 1996-05-10 1997-11-20 University Of Limburg Procede et kit pour determiner le potentiel de malignite de cellules tissulaires
US8470544B2 (en) 1997-10-21 2013-06-25 Cancer Research Technology Limited Detection of dysplastic or neoplastic cells using anti-MCM3 antibodies
US7056690B2 (en) * 1997-10-21 2006-06-06 Cancer Research Technology Limited Detection of dysplastic or neoplastic cells using anti-MCM2 antibodies
US7459157B2 (en) 1997-10-21 2008-12-02 Cancer Research Technology Limited Detection of dysplastic or neoplastic cells using anti-MCM2 antibodies
US8609351B2 (en) 1997-10-21 2013-12-17 Cancer Research Technology Limited Detection of dysplastic or neoplastic cells using anti-MCM4 antibodies
US8148087B2 (en) 1997-10-21 2012-04-03 Cancer Research Technology Limited Detection of dysplastic or neoplastic cells using anti-MCM6 antibodies
DE19936618A1 (de) * 1999-08-04 2001-03-15 Monotec Gmbh Diagnostisches Kit und dessen Verwendung
US20080261266A1 (en) * 2004-12-17 2008-10-23 Ventana Medical Systems, Inc. Methods and compositions for a microemulsion-based tissue treatment
US8288121B2 (en) * 2004-12-17 2012-10-16 Ventana Medical Systems, Inc. Methods and compositions for a microemulsion-based tissue treatment
US8512978B2 (en) 2004-12-17 2013-08-20 Ventana Medical Systems, Inc. Methods and compositions for a microemulsion-based tissue treatment
US8652803B2 (en) 2004-12-17 2014-02-18 Ventana Medical Systems, Inc. Methods and compositions for a microemulsion-based tissue treatment
WO2009113859A1 (fr) * 2008-03-13 2009-09-17 Vereniging Voor Christelijk Hoger Onderwijs, Wetenschappelijk Onderzoek En Patiëntenzorg Algorithmes de dépistage cervical
CN113607534A (zh) * 2021-08-20 2021-11-05 河南赛诺特生物技术有限公司 一种染色方法、试剂盒及应用

Similar Documents

Publication Publication Date Title
AU2003262554B2 (en) Method for solution based diagnosis.
EP1025444B1 (fr) Determination d'anomalies de la croissance cellulaire
US8513398B2 (en) Human papilloma virus probes for the diagnosis of cancer
Coon et al. Detection ofABH Tissue Isoantigens by Immunoperoxidase Methods in Normal and Neoplastic Urothelium: Comparison with the Erythrocyte Adherence Method
AU770729B2 (en) Protein quantitation with cell imaging densitometry
JP2002296275A (ja) 少なくとも２種の異なる分子マーカーを同時に測定することによって腫瘍およびそれらの前駆体段階を検出する場合に臨床的特異性を増強する方法
US9551700B2 (en) Device and methods for the detection of cervical disease
US20040002125A1 (en) Detection of abnormal cells
EP3214445B1 (fr) Procédé diagnostique
JP2001522983A (ja) 子宮頸異形成及び癌を検出するための生化学的方法
CA2700635A1 (fr) Procedes avances de criblage de cellules du col de l'uterus
WO1997016731A1 (fr) Marquage immunicytochimique d'echantillons de cellules cervicales exfoliees
US9182403B2 (en) Kits for and methods of differential staining of cervical cancer cells and/or tissues
EP0934530B1 (fr) Composition immunologique de detection du cancer de la vessie, et son mode d'utilisation
US11054426B2 (en) Diagnostic method
Aslani et al. Re-evaluation of Negative Cone Biopsy Results with Ki-67 and p16 Immunostaining following Positive Cervical Biopsy
JPH07502112A (ja) 悪性及び前悪性状態の検出
CA2149617A1 (fr) Diagnostic differentiel du cancer par methode immunocytochimique reposant sur des anticorps ou des antiserums contre hcg.beta.
Edwards et al. Transitional cell cytokeratins as a second parameter in flow cytometry of bladder cancer

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 97512998

Format of ref document f/p: F

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载