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WO1998002581A1 - Dosage de la telomerase pour les lesions precancereuses du col uterin - Google Patents

Dosage de la telomerase pour les lesions precancereuses du col uterin Download PDF

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Publication number
WO1998002581A1
WO1998002581A1 PCT/US1997/012193 US9712193W WO9802581A1 WO 1998002581 A1 WO1998002581 A1 WO 1998002581A1 US 9712193 W US9712193 W US 9712193W WO 9802581 A1 WO9802581 A1 WO 9802581A1
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WO
WIPO (PCT)
Prior art keywords
tissue
telomerase activity
cancer
telomerase
cervical
Prior art date
Application number
PCT/US1997/012193
Other languages
English (en)
Inventor
Brian Liu
David E. Burstein
Elisabeth Kavaler
Original Assignee
Brian Liu
Burstein David E
Elisabeth Kavaler
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 Brian Liu, Burstein David E, Elisabeth Kavaler filed Critical Brian Liu
Priority to AU37989/97A priority Critical patent/AU3798997A/en
Publication of WO1998002581A1 publication Critical patent/WO1998002581A1/fr

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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
    • 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
    • G01N33/57442—Specifically defined cancers of the uterus and endometrial
    • 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
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/91245—Nucleotidyltransferases (2.7.7)

Definitions

  • the present invention relates to a method of detecting or diagnosing a precancerous or cancerous lesion of the cervix, endocervix, vagina or vulva comprising measuring the telomerase activity of cells obtained from said tissue(s), wherein an increase in telomerase activity relative to the activity present in cells of non-cancerous tissue bears a positive correlation with the presence of a cancerous or precancerous lesion.
  • Carcinoma of the uterine cervix is the third most frequent of the female genital cancers. Although there were only 13,000 cases of invasive cervical cancer predicted for 1996, this does not include more than 50,000 cases of carcinoma in situ and several times that number of cases of preinvasive dysplasias of the cervix. Of all the female genital cancers, only cervical cancer can be reliably prevented by use of an effective, inexpensive screening technique that allows detection of precancerous conditions that can be treated effectively so as to prevent the development of invasive cancer. Thus, the vast majority of deaths due to cervical cancer each year can be said to be preventable if women avail themselves of routine screening with cervical cytology.
  • Preinvasive cervical carcinoma is usually described by one of two different classifications, both of which are common usage.
  • the first system divides lesions into dysplasias (mild, moderate and severe) and carcinoma in s u.
  • the second system uses three divisions of the term cervical intraepithelial neoplasia (CIN-1 , CIN-2 and CIN-3).
  • CIN-1 cervical intraepithelial neoplasia
  • CIN-2 cervical intraepithelial neoplasia
  • This precursor stage of cervical carcinoma begins with minimal morphologic changes (CIN-1 or mild dysplasia) and progresses to the point that the entire epithelium from the basement membrane to the surface is composed of malignant cells (CIN-3 or carcinoma in situ).
  • Preinvasive cervical carcinoma is detected by the Papanicolaou ("Pap") smear.
  • the purpose of periodic cytologic screening by means of the Pap smear is to prevent invasive cervical cancer.
  • microinvasive and early invasive cervical cancers will be detected and this early detection is valuable in decreasing the death rate from cervical cancer, the idea is to detect all cervical abnormalities in the premalignant stage and thus prevent invasive cervical cancer.
  • the Pap smear for precursors to cervical cancer has a number of serious flaws. These include: 1) a false negative rate which ranges from 6 to 55%, depending on the study cited (Singleton et al., 1995, CA-A Cancer Journal for Clinicians 45:305), 2) a high rate of equivocal diagnoses (i.e., Atypical Squamous Cells of Undetermined Significance (“ASCUS”)) and (3) suboptimal sampling or staining technique. Only 10-45% of patients with ASCUS diagnosis were found to have squamous intra-epithelial lesion on follow-up, leading to unnecessary colposcopy and/or biopsies.
  • ASCUS Atypical Squamous Cells of Undetermined Significance
  • ASCUS equivocal diagnoses
  • Candida or Trichomonas infections from Candida or Trichomonas, nonspecific inflammatory responses, post-surgical and post-partum conditions, radiation or chemotherapy, and nonspecific changes in post-menopausal women.
  • Telomerase is an enzyme which functions to maintain the length of telomeres, specialized regions of chromosomes necessary for maintaining chromosomal stability. The cell can only afford to lose a finite number of these telomeres before sequences of the parent DNA are lost, resulting in chromosomal instability and subsequent cell death (Harley, 1991, Mutation Res. 256:271). In contrast, when cancer cells expressing telomerase divide, telomere length is maintained, rendering cancer cells resistant to cell senescence. Thus, telomerase activation may play a key role in transforming a mortal somatic cell into an immortal tumor cell (Haber, 1995, N. Engl. J. Med. 332:955.-
  • the present invention relates to a method for detecting or diagnosing a precancerous or cancerous lesion of the cervix, endocervix, vagina or vulva, based on the presence of increased telomerase activity. It is based, at least in part, on the discovery that assaying cell samples from the surface of the cervix, endocervix, vagina and vulva for telomerase activity may be used as a sensitive alternative to the Pap smear for the detection of precancerous changes leading to cancer in these tissues. In particular, samples demonstrating a squamous intraepithelial precancerous lesion were found to express telomerase.
  • the present invention comprises the use of a molecular methodology for measuring telomerase activity (known as the "TRAP assay") for screening women for precancerous and cancerous lesions of the cervix, endocervix, vagina and vulva.
  • the use of the TRAP assay avoids the slow, labor intensive, manual microscopic screening presently required by the Pap smear.
  • the average Pap smear may contain epithelial cells, inflammatory cells, and microorganisms. It is thus not surprising that the average daily quota of Pap smears screened per day ranges from only 50 to a legal maximum of 100 smears per day per cytotechnologist. In contrast the TRAP assay for telomerase activity is objective. 4.
  • telomere activity is measured in cervical, endocervical, vaginal or vulvar cells harvested from a patient, for example, using a wooden spatula, cotton tipped swab, or brush-like device such as a cytobrush or cyto- broom, whereby an increase in telomerase activity bears a strong correlation to the presence of a precancerous or cancerous lesion of the cervix, endocervix, vagina or vulva.
  • Telomerase may be measured by any method known in the art, including the method set forth in the working example below.
  • Assay of cervical, endocervical, vaginal and/or vulvar cells may be used, for example, and not by way of limitation, as a primary general population screen for precursors to cancer of these tissues; as a non-issue based, cytologic screen for such conditions; as a method for assessing the efficacy of treatment of squamous intraepithelial precancerous or cancerous lesions; as an alternative to Pap smear testing or as an ancillary test used in conjunction with Pap smears to assess patient subpopulations with high rates of atypias; and/or as an ancillary test for evaluating patients with an ASCUS diagnosis on Pap smear.
  • sample extract The supernatant collected.
  • sample extract The supernatant, referred to as the "sample extract" was then used in the TRAP assay.
  • TRAP Telomeric repeat amplification protocol
  • Assay tubes were prepared by lyophihzing 0.1 ⁇ g of CX primer (see below) onto the bottom of the tube and sealing it with Ampliwax (Perkin-Elmer). Fifty microliter TRAP reactions above the wax barrier contained 20 mM tris-HCl (pH 8.3), 1.5 mM MgCL, 63 mM KCl.
  • Tween20 1 mM EGTA, 50 ⁇ M dNTP, 0.1 ⁇ g of TS primer (see below), 2 U Ampl.Taq DNA polymerase (Perkin-Elmer), 1 ⁇ g of T4g32 protein (Boehringer Mannheim), bovine serum albumin (0.1 mg/ml), 1 to 15 ⁇ l of sample extract, and 0.2 ⁇ l of ⁇ 32 P[dCTP] (Amersham) was added as described by Kim et al., 1994, Science 266:2011).
  • tubes were transferred to a thermal cycler for 27 rounds at 94°C for 30 seconds, 50°C for 30 seconds and 72 °C for 1.5 minutes as previously described (Kim et al., 1994, Science 266:2011 ).
  • the reaction was analyzed by electrophoresis in 0.5X tris-borate EDTA on 12% polyacrylamide nondenaturing gels and autoradiographed as previously described (Kim et al., 1994, Science 26£:2011).
  • the TS primer is: 5 '-AATCCGTCGAGCAGAGTT-3 '
  • the CX primer is: 5'-(CCCTTA) 3 CCCTAA-3'
  • telomerase assays were run on cell samples from 18 patients. Sixteen patients had cell samplings for telomerase immediately following routine Pap smears; two had cell sampling for telomerase after surgical removal of cervical tissue (from hysterectomy specimens). Of the eighteen samples assayed, thirteen showed positive telomerase activity, four showed negative telomerase activity, and one was equivocal. Of the thirteen positive telomerase samples, five had abnormal Pap smears (showing squamous intraepithelial lesion or SIL); seven had abnormal but non-definitive Pap smears (diagnosis ranging from atypical squamous cells of undetermined significance or ASCUS to atypia and suspicious for SIL). A single telomerase positive sample (#P11) had a negative Pap smear; however, this patient (#P11) had a history of SIL on biopsy followed by a recent abnormal Pap smear.
  • telomere samples Of the four non-positive telomerase samples, all were negative on Pap smear and/or were negative with cervical tissue diagnosis.
  • the one equivocal telomerase sample was from a patient with an abnormal but nondefmitive Pap smear (cannot rule out SIL or adenocarcinoma).
  • telomerase assay is able to detect the earliest precancerous changes detectable by Pap smear (i.e. low grade SIL including mild dysplasia, condyloma, CIN 1 ; 2) telomerase assay thus far has no proven false negative results; 3) telomerase assay thus far has no proven false positive results; and 4) a positive telomerase assay may be indicative of the presence of a true precancerous lesion that is not defined by Pap smear.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Urology & Nephrology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Reproductive Health (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne une méthode permettant de détecter ou de diagnostiquer un lésion précancéreuse ou cancéreuse du col utérin, de l'endocol, du vagin ou de la vulve, selon laquelle on mesure l'activité télomérase de cellules provenant desdits tissus. Une activité télomérase accrue par rapport à l'activité présente dans les cellules des tissus non cancéreux indique la présence d'une lésion cancéreuse ou précancéreuse.
PCT/US1997/012193 1996-07-17 1997-07-15 Dosage de la telomerase pour les lesions precancereuses du col uterin WO1998002581A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU37989/97A AU3798997A (en) 1996-07-17 1997-07-15 Telomerase assay for precancerous lesions of the uterine cervix

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US2332496P 1996-07-17 1996-07-17
US60/023,324 1996-07-17

Publications (1)

Publication Number Publication Date
WO1998002581A1 true WO1998002581A1 (fr) 1998-01-22

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AU (1) AU3798997A (fr)
WO (1) WO1998002581A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19804372A1 (de) * 1998-02-04 1999-08-05 Michael W Dr Dr Dahm Verfahren zur quantitativen Bestimmung von Tumorzellen in einer Körperflüssigkeit und dazu geeignete Testkits
US6582904B2 (en) 1995-11-16 2003-06-24 Michael W. Dahm Method of quantifying tumour cells in a body fluid and a suitable test kit
US7390891B1 (en) 1996-11-15 2008-06-24 Amgen Inc. Polynucleotides encoding a telomerase component TP2
US20190388453A1 (en) * 2016-03-24 2019-12-26 The Board Of Regents Of The University Of Texas System Treatment of drug resistant proliferative diseases with telomerase mediated telomere altering compounds

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5489508A (en) * 1992-05-13 1996-02-06 University Of Texas System Board Of Regents Therapy and diagnosis of conditions related to telomere length and/or telomerase activity

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5489508A (en) * 1992-05-13 1996-02-06 University Of Texas System Board Of Regents Therapy and diagnosis of conditions related to telomere length and/or telomerase activity

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SCIENCE, 23 December 1994, Vol. 266, KIM et al., "Specific Association of Human Telomerase Activity with Immortal Cells and Cancer", pages 2011-2014. *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6582904B2 (en) 1995-11-16 2003-06-24 Michael W. Dahm Method of quantifying tumour cells in a body fluid and a suitable test kit
US7390891B1 (en) 1996-11-15 2008-06-24 Amgen Inc. Polynucleotides encoding a telomerase component TP2
DE19804372A1 (de) * 1998-02-04 1999-08-05 Michael W Dr Dr Dahm Verfahren zur quantitativen Bestimmung von Tumorzellen in einer Körperflüssigkeit und dazu geeignete Testkits
US6821726B1 (en) 1998-02-04 2004-11-23 Michael W. Dahm Method for quantitatively analyzing tumor cells in a body fluid and test kits suited therefor
US20190388453A1 (en) * 2016-03-24 2019-12-26 The Board Of Regents Of The University Of Texas System Treatment of drug resistant proliferative diseases with telomerase mediated telomere altering compounds
US12070472B2 (en) * 2016-03-24 2024-08-27 The Board Of Regents Of The University Of Texas System Treatment of drug resistant proliferative diseases with telomerase mediated telomere altering compounds

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Publication number Publication date
AU3798997A (en) 1998-02-09

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