WO2002015770A2 - Techniques de pronostic et d'evaluation de cancer - Google Patents

Techniques de pronostic et d'evaluation de cancer Download PDF

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WO2002015770A2
WO2002015770A2 PCT/US2001/041774 US0141774W WO0215770A2 WO 2002015770 A2 WO2002015770 A2 WO 2002015770A2 US 0141774 W US0141774 W US 0141774W WO 0215770 A2 WO0215770 A2 WO 0215770A2
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htert
htert mrna
mature
level
mrna
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WO2002015770A3 (fr
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Inna R. Williams
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Digene Corporation
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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
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development

Definitions

  • the invention relates to a method of diagnosis, staging and therapy management of pre-cancer and cancerous lesions by measuring levels of expression of the catalytic subunit of the human telomerase reverse transcriptase (hTERT) gene.
  • hTERT human telomerase reverse transcriptase
  • the detection and diagnosis of disease is of obvious importance for the treatment of disease. Many diseases are preceded by, and are characterized by, changes in the state of the affected cells. Changes can be found in the expression patterns of genes in affected cells, and changes in cell morphology. The detection, diagnosis, and monitoring of diseases can be aided by the assessment of such cell states.
  • Telomerase is a ribonucleoprotein enzyme that elongates the G-rich strand of chromosomal termini by adding telomeric repeats. This elongation occurs by reverse transcription of apart of the telomerase RNA component.
  • telomere enzyme activity is not present in normal adult tissues except of germline cells, i.e., testes/ovaries, and at very low levels in peripheral blood. Germline cells, whose chromosomal ends must be maintained through repeated rounds of DNA replication, do not decrease their telomere length with time, presumably due to the activity of telomerase. In contrast, somatic cells appear to lack telomerase, and their telomeres shorten with multiple cell divisions. The repression of telomerase activity in somatic cells is likely to be important in controlling the number of times they divide. The loss of telomeric DNA may signal to the cell the end of its replicative potential as part of an overall mechanism by which multicellular organisms limit the proliferation of their cells.
  • telomerase due to its role in controlling replication, telomerase has been implicated in oncogenesis. It is thought that late stage tumors probably require reactivation of telomerase in order to avoid total loss of their telomeres and massive destabilization of their chromosomes.
  • telomere enzyme activity is evident in over 85% of various types of cancer, including HP V-related cervical cancer and human ovarian carcinoma cells.
  • U.S. Patent No. 5,693,474 reports a method for cancer diagnosis and prognosis by analyzing a sample for telomerase activity, correlating the activity with a standard level of telomerase activity, and correlating a high telomerase activity with an indication of unfavorable prognosis and a low telomerase activity with a favorable prognosis.
  • the method reported suffers from low accuracy and sensitivity.
  • the method does not provide adequate guidance for determining what high telomerase activity and low telomerase activity are.
  • an accurate and sensitive method for dete ⁇ nining relative levels of telomerase activity are needed.
  • the present invention correlates hTERT mRNA expression with telomerase activity and telomerase activity with the possibility, inevitability or presence of cancer.
  • the invention relates to a novel diagnostic technique for direct quantitative determination of human hTERT mRNA level from tissues, biopsies, cells or total RNA samples. The level of hTERT mRNA is then used for pre-cancer or cancer diagnosis and prognosis.
  • the present invention can be used to assess the stage or risk of cancer as indicated by and correlated to the state of the cells.
  • the hTERT mRNA test can also be applied as a sensitive tool for post-therapeutic monitoring of a patient and be used to guide or assess the effectiveness of a therapy by identifying appropriate therapy based on the indicated cell state or by indicating any change in the state of cells subjected to the therapy.
  • a preferred embodiment involves the detection and quantification of alternatively spliced hTERT mRNA species and mature hTERT mRNA species.
  • This embodiment employs a ratio of total hTERT mRNA (mature hTERT mRNA and variant hTERT mRNA) to mature hTERT mRNA as a more accurate and sensitive method for cancer diagnosis and staging.
  • Mature hTERT is defined as the complete coding sequence for the catalytic subunit of human telomerase reverse transcriptase.
  • the ratio of total hTERT mRNA to mature hTERT mRNA in a sample is greater than approximately 10, the tissue from which the sample was taken is considered normal and predicted to remain normal.
  • the ratio of total hTERT mRNA to mature hTERT mRNA in a sample is between approximately 10 and approximately 3, the tissue from which the sample was taken is hyperplastic or dysplastic or the tissue has a propensity to develop hyperplasia or dysplasia.
  • the ratio of total hTERT mRNA to mature hTERT mRNA in a sample is below approximately 3, it is indicative of cancer such as in situ cancer or invasive cancer in the tissue from which the sample was taken or it is indicative of the propensity of the tissue to develop cancers such as in situ cancer or invasive cancer.
  • Figure 1 is a schematic representation of the relative ratio of total hTERT mRNA over mature hTERT mRNA.
  • a ratio of 10 and higher is obtained when the levels of total and mature hTERT mRNA are low or absent which indicates a normal or disease-free or low disease risk status of a specimen.
  • a ratio of approximately between 10 and 3 is obtained when the levels of total hTERT are significantly higher than levels of mature hTERT mRNA which indicates hyperplasia, dysplasia, or neoplasia, such as low grade cervical intraephithelial neoplasia (CIN) with primary nuclear abnormalities, warts, papillomas, or condylomas, or malignant disease risk.
  • a ratio of 3 or less is obtained when the levels of total hTERT are not significantly higher than mature hTERT mRNA which indicates high grade CIN, cancer such as in situ cancer, or invasive cancer.
  • Figure 2 depicts a method of determining levels of total (mature plus variant) hTERT mRNA species.
  • Single stranded DNA probes which are complementary to either mature hTERT mRNA or region(s) conserved between hTERT mRNA species are employed.
  • the single stranded DNA probes are modified for capture on a solid surface, such as by biotin-streptavidin covalent bonding.
  • the total (mature plus variant) hTERT mRNA species are hybridized to a specific DNA probe and captured on the solid surface. Detection of the captured RNA/DNA hybrids is performed utilizing anti-RNA/DNA antibody labeled with alkaline phosphatase.
  • the rate of dephosphorylation of the dioxetane derivative of CDP-Star ® is measured by luminometer.
  • RNA/DNA hybrids will not be formed. Therefore, a single stranded DNA probe only will be captured on a solid surface, but will not be recognized by an anti-RNA/DNA antibody labeled with alkaline phosphatase. Therefore, no signal will be detected by luminometer.
  • Figure 3 depicts a method of determining levels of hTERT mRNA species which are not spliced at exons 7 and 8. This method can be used to determine the level of hTERT mRNA which is not alternatively spliced by deletion at exons 7 and 8.
  • a single stranded DNA Probe cocktail comprises Probes a, b, and c which are complementary to mature hTERT mRNA. However, only Probe b, which is complementary to exons 7 and*8 of hTERT mRNA, is modified for capture on a solid surface, such as by biotin-streptavidin covalent bonding.
  • hTERT mRNA species not spliced at exons 7 and 8 will be hybridized to the Probe cocktail and be captured on a solid surface. Detection of the captured RNA/DNA hybrids is performed utilizing an anti-RNA/DNA antibody labeled with alkaline phosphatase. The rate of dephosphorylation of the dioxetane derivative of CDP-Star ® is measured by luminometer.
  • Figure 4 depicts an alternative method for determining the level of hTERT mRNA which is not alternatively spliced by deletion at exons 7 and 8.
  • RNA/DNA hybrids are formed with DNA Probes a and c only. These hybrids will not be captured on a solid surface but will be washed away prior detection with anti-RNA/DNA antibody.
  • DNA Probe b only will be captured on a solid surface which will not be recognized by anti- RNA/DNA antibody labeled with alkaline phosphatase. No signal will be measured by luminometer.
  • Figure 5 depicts a method of determining levels of hTERT mRNA species alternatively spliced by insertion of intron 14.
  • a single stranded DNA probe complementary to intron 14 regions of hTERT mRNA is modified for capture on a solid surface, such as by biotin-streptavidin covalent bonding.
  • hTERT mRNA species which are alternatively spliced by insertion of intron 14 will hybridized to the DNA probe and be captured on the solid surface. Detection of the captured RNA/DNA hybrids is performed utilizing anti-RNA/DNA antibody labeled with alkaline phosphatase. The rate of dephosphorylation of the dioxetane derivative of CDP-Star ® will be measured by luminometer.
  • Figure 6 depicts an alternative method for the detection of hTERT mRNA species alternatively spliced by insertion of intron 14.
  • hTERT mRNA spliced by insertion of intron 14 RNA/DNA hybrids are not formed with the DNA probe.
  • hTERT not spliced by insertion of intron 14 and other mRNA species will be washed away prior to detection with anti-RNA DNA antibody.
  • the DNA probe only will be captured on a solid surface which will not be recognized by anti- RNA/DNA antibody labeled with alkaline phosphatase. No signal will be measured by luminometer.
  • Figure 7 depicts detection of hTERT mRNA species utilizing a microarray platform.
  • DNA capture sequence probes complementary to regions of exons 2, 3, and 4 of hTERT mRNA (designed for total hTERT mRNA species detection), CSP complementary to regions of exons 6, 7, and 8 of hTERT mRNA (designed for alternatively spliced by deletion hTERT mRNA species detection), and CSP complementary to regions of introns 4, 11, and 14 of hTERT gene (designed for alternatively spliced by insertion hTERT mRNA species detection) are spotted on the solid surface of a microarray at specific positions.
  • CSP DNA capture sequence probes
  • hTERT mRNA species are hybridized with the spotted DNA probes and the hybrids detected with a single labeled anti-RNA/DNA antibody or with a double antibody "sandwich", where a secondary antibody is labeled. In the presence of hTERT mRNA species complementary to the specific DNA probes, a positive signal will be obtained.
  • FIG. 8 is a schematic diagram illustrating an embodiment of the invention.
  • CSP complementary to regions of exons 2, 3, and 4 of hTERT mRNA (designed for total hTERT mRNA species detection)
  • CSP complementary to regions of exons 6, 7, and 8 of hTERT mRNA (designed for alternatively spliced by deletion hTERT mRNA species detection)
  • CSP complementary to regions of introns 4, 11, and 14 of hTERT gene are spotted on a solid surface of microarray at specific positions.
  • hTERT mRNA species are hybridized with the spotted DNA probes and with signal sequence probes (SSP) either simultaneously or sequentially.
  • SSP signal sequence probes
  • SSP are designed to be complementary to regions adjacent to CSP on the hTERT mRNA, and are used for specific signal amplification, if necessary.
  • the formed DNA/RNA hTERT hybrids are detected with a single labeled anti-RNA/DNA antibody or with a double antibody "sandwich", where a secondary antibody is labeled. In the presence of hTERT mRNA species complementary to a specific CSP, a positive signal will be obtained.
  • Figure 9 is a graphical representation of hTERT mRNA. Mature hTERT mRNA with a complete coding region (16 exons) is shown in A. hTERT mRNA variants alternatively spliced by deletion of exons 6, 7 and 8 are shown in B. hTERT mRNA variants alternatively spliced by insertions of introns 4, 11, and 14 are shown in C.
  • the present invention relates to the identification and monitoring of diseased cells.
  • One method involves measuring the levels of expression of genes involved in a disease state, and comparing their expression to each other or to reference genes, as an indication of the state of the cells. Such measurements can be combined with other assays to increase the accuracy and reliability of the assessment of the disease state.
  • One method of the present invention can be used to assess the stage of a disease as indicated by the state of the cells. This method can also be used to monitor or assess the effectiveness of a therapy for a disease by identifying appropriate therapy based on the indicated disease state or by indicating any change in the state of cells subjected to the therapy.
  • neoplastic and cancerous cells generally exhibit certain distinctive morphologies and growth characteristics.
  • Molecular characteristics, such as gene mutations and gene expression patterns are also a good indicator of disease progression.
  • the characteristics of the cell state such as changes in cell morphology or expression of genes, can be predicted from a patient sample.
  • the characteristics to be detected are generally specific to the cell state of interest and the disease suspected of being present in the test sample. Such characteristics can be generally divided into two types, cytological characteristics and molecular characteristics.
  • cytological characteristics are characteristics such as, for example, overall cell shape and appearance. The primary identification and classification of many neoplastic and cancerous cells has traditionally been accomplished using cytological characteristics. Identification of cytological characteristics is generally slow, requires a relatively high level of training, and generally cannot be easily automated.
  • molecular characteristics are the presence and state of particular molecular species, such as proteins, nucleic acids, and metabolites. Such molecular characteristics are generally identified by detecting and measuring the particular molecules of interest.
  • the characteristics assayed can include additional or surrogate marker characteristics that are not a direct cause or result of the disease but that are related to certain disease and cell states.
  • additional markers include polymorphic markers, human leukocyte antigens (HLA) such as B7 that predispose women for cervical carcinomas, oncogenes, p53 mutations, other cancer markers, oncosupressors, cytokines, growth factor receptors, and hormones.
  • HLA human leukocyte antigens
  • Such markers can be present in, or absent from, cells exhibiting state- or disease-specific characteristics, and such presence or absence can be indicative of, for example, a more severe or less severe disease state.
  • markers can be used in conjunction with the disclosed method to infer either higher or lower risk of neoplastic disease depending on the number of abnormal scores or the magnitude of change in quantitative markers.
  • methods for detection and assessment of HPV infections ' which can lead to cancer, for example cervical cancer. Such methods are described in U.S. Patent Nos. 5,916,752; 5,891,639; 5,639,613; 5,415,995; 4,777,239; 5,484,699; 4,983,728; 5,527,898; 5,364,758; 5,639,871; 5,501,947; 5,665,533; 4,748,109; 5,623,932; 5,665,571 and 5,648,459.
  • nucleic acid detection assays that may be used in combination with the disclosed molecular assay.
  • many of the techniques described above can also be adapted for use in the disclosed assay for the detection of expression level of hTERT nucleic acid.
  • Examples of disease states for assessment using the present invention include, but are not limited to neoplasias and cancer.
  • cancers for prognosis and detection according to the methods of the present invention include cervical cancer, ovarian cancer, bladder cancer, head and neck cancer, laryngeal cancer, prostate cancer, breast .cancer, lung cancer, skin cancer.
  • HPV-based disease including, but not limited to, HPV infection, cervical intraepithelial neoplasia (CIN), atypical squamous cells of undetermined significance (ASCUS), squamous cell carcinoma (SCC), or carcinoma in situ, metastatic carcinoma, warts, condylomata, epidermodysplasia verruciformis and other skin diseases, laryngeal papilloma, oral papilloma and conjunctival papilloma.
  • CIN cervical intraepithelial neoplasia
  • ASCUS atypical squamous cells of undetermined significance
  • SCC squamous cell carcinoma
  • cancer refers to more than 100 forms of the disease. Tumor development occurs in stages and begins when a normal cell sustains a genetic mutation that increases its propensity to proliferate when it would normally rest. The altered cell and its descendants continue to look normal, but t ey exhibit a high proliferation rate — a condition termed hyperplasia. When one of these hyperplastic cells then suffers another mutation, controls on cell growth are further loosened. In addition to proliferating excessively, the offspring of this cell appear abnormal in shape and in orientation. The cells are now described as exhibiting dysplasia. One of these dysplastic cells may then suffer a rare mutation that further alters cellular behavior, and the affected cell becomes still more abnormal in growth and appearance.
  • the tumor has not yet broken through any boundaries between tissues, it is called in situ cancer.
  • the tumor may remain contained indefinitely; however, some cells may eventually acquire additional mutations. If the genetic changes allow the tumor to begin invading underlying tissue and to shed cells into the blood or lymph, the mass is considered to have become malignant.
  • hTERT mRNA is attributed to the early stages of cancer development such as the molecular abnormalities within phenotypically normal cells termed hyperplasia, and the appearance of abnormal cells termed dysplasia.
  • an assessment and/or prognosis of a patient can be obtained.
  • a sample is obtained from a patient and the levels of mature hTERT mRNA and variant hTERT mRNA are quantified.
  • the ratio of total hTERT mRNA (mature hTERT mRNA and variant hTERT mRNA) to mature hTERT mRNA is then determined.
  • Mature hTERT is defined as the complete coding sequence for the catalytic subunit of human telomerase reverse transcriptase without any intron sequences.
  • the ratio of total hTERT mRNA to mature hTERT mRNA in the patient sample is greater than about 10, the patient assessment is normal and the prognosis for the patient is also normal.
  • the ratio of total hTERT mRNA to mature hTERT mRNA in the patient sample is between about 10 and about 3, the sample may be normal or show signs of hyperplasia and/or dysplasia when examined cytological methods.
  • the patient assessment may be either normal or that the patient has hyperplasia and/or dysplasia.
  • FIG. 1 provides a graphical representation of the ratio of total hTERT mRNA to mature hTERT mRNA.
  • Useful techniques for measuring the level of expression of a gene of interest in a test sample include the Hybrid Capture® technology described in WO 93/10263 by Digene, PCR in situ hybridization techniques described by Nuovo, PCR In Situ Hybridization: Protocols and Applications, 3rd Edition, Lippencott-Raven Publishers, Philadelphia 1997, branched DNA assays by Chernoff et al. J. Clinical Microbiology 35(ll):2740-2744 (1997), transcription-mediated amplification (TMA) by Stoflet et al.
  • TMA transcription-mediated amplification
  • hTERT nucleic acid many of the techniques for the detection of hTERT nucleic acid in general can also be adapted for use in the disclosed assay for the detection of hTERT nucleic acid.
  • Some examples of these assays are the RT-PCR amplification of the hTERT region using specific primers in total RNA samples as described by Greenberg et al. Oncogene 18(5):1219-26 (1999).
  • Another method is the RNase protection assay as described in U.S. Patent No. 5,770,370.
  • hTERT detection assays which can be used in the disclosed method, are known, including assays involving Southern blots, dot blots, in situ hybridization, polymerase chain reaction, and solution hybridization.
  • Mant et al. J. Virol. Meth. 66(2): 169-178 (1997) describes PCR assays used to identify DNA from specific HPV types.
  • Cope et al. J. Clin Microbiol 35(9):2262-2265 (1997) describes a PCR-based test using a consensus primer and a Hybrid Capture assay (HCA) of detection of HPV types.
  • HCA Hybrid Capture assay
  • the Hybrid Capture assay is also described in WO 93/10263 by Digene.
  • the Hybrid Capture assay is a useful method for detection of HPV and for determining HPV type in combination with the disclosed assay.
  • the test sample may be a human or veterinary clinical specimen.
  • the test sample is a biological fluid such as urine, blood, plasma, serum, sputum or the like.
  • the test sample may be a tissue specimen or cellular scrape suspected of carrying a nucleic acid of interest.
  • the target nucleic acid in the test sample may be present initially as a discrete molecule so that the sequence to be detected constitutes the entire nucleic acid, or it may only be a component of a larger molecule. It is not necessary that the nucleic acid sequence to be detected be present initially in a pure form.
  • the test sample may contain a complex mixture of nucleic acids, of which the target nucleic acid may correspond to a gene of interest contained in total human genomic DNA or RNA or a portion of the nucleic acid sequence of interest.
  • a test sample as the term is used herein is primarily a collection of cells from a patient.
  • One method of obtaining cells is through non-invasive means, which is defined herein as obtained without the puncturing of a patient. Examples of non- invasive means are, for example, test samples obtained from urine or a nasal, epithelial, cervical or other cell surface scrape.
  • Patient cells can also be obtained by other means including, for example, needle biopsy or tissue biopsy.
  • the test sample can be preserved in a collection medium which allows for a combination of two or more assays of different characteristics related to a cell state of interest.
  • the assay or assays refer to detection or measurement of specific characteristics, the results of which may be combined with other such measurements of other characteristics to an overall assessment of a cell suspected of being infected with one or more diseases.
  • These assays may include, for example, a combination of morphological analysis and quantitation of a particular RNA or DNA or protein whose levels provide a specific indication of the presence or progression of a disease.
  • Test samples for use in the present invention can be collected and stored in liquid medium.
  • useful cell collection media are PreservCyt® (Cytyc) and CytoRichTM (Autocyte). These media (PreservCyt® and CytoRichTM) were developed for the collection of cytological samples but can be adapted for use with molecular assays.
  • a preferred cell collection media is a Universal Collection Media (UCM).
  • UCM Universal Collection Media
  • a water-based media comprising a preservative, a fixative and an anti-degradation agent is preferred.
  • a UCM comprising an alcohol as a preservative, poly(ethylene glycol) as a fixative, and EDTA as an anti-degradation agent is more preferred.
  • a UCM comprising 9% butanol, 0.05% NaN 3 , 5 niM EDTA, 2% PEG-1500, 10 mM NaOAc-HOAc at pH 4.5 is most preferred.
  • Test samples for use in the method of the present invention can be fixed or processed in any manner consistent with the assays to be performed.
  • both cytological and molecular assays can be performed using cells fixed on a solid substrate such as, for example, a slide.
  • the requirements of the assays to be performed will generally identify the sample processing to be used.
  • variant-specific hybrid capture Detection of a specific nucleic acid probe indicates the presence of a particular nucleic acid sequence in the test sample.
  • the variant-specific hybrid capture method employs a cocktail of capture sequences probes (CCSPs).
  • a CCSP comprises nucleic acid sequences which are capable of hybridizing to a complete transcribing region(s) of a target nucleic acid and being captured onto a solid phase.
  • the CSP used in the detection method can be DNA, RNA, peptide nucleic acids (PNAs) or other nucleic acid analogues.
  • PNAs are oligonucleotides in which the sugar-phosphate backbone is replaced with a polyamide or "pseudopeptide" backbone.
  • the CSP is DNA.
  • the CSP has a minimum length of 8 bases but preferably between 50 to 1500 bases long, and more preferably between 100 to 1000 bases long.
  • the CSP is substantially complementary to the sequence within a target nucleic acid to which it hybridizes.
  • the sequence of a CSP is preferably at least 75% complementary to the target hybridization region, more preferably, 100% complementary to this sequence.
  • the CSP contains less than or equal to 75% sequence identity, more preferably less than 50% sequence identity, to non-desired sequences believed to be present in a test sample.
  • the sequence within a target nucleic acid to which a CSP binds is preferably 50 to 1500 bases long and more preferably 100-1000 bases long. It is also preferred that the sequences to which the CSP hybridizes are unique sequences or variant-specific sequences. Variant-specific sequences are multiple related sequences that form discrete variants.
  • the CSP are used for detection of target nucleic acid alternatively spliced by insertion. The CSP is complementary to an inserted region of target nucleic acid.
  • the nucleic acid probes of the invention may be produced by any suitable method known in the art, including for example, by chemical synthesis, isolation from a naturally-occurring source, recombinant production and asymmetric PCR (McCabe, 1990 In: PCR Protocols: A guide to methods and applications. San Diego, CA., Academic Press, 76-83). It may be preferred to chemically synthesize the probes in one or more segments and subsequently link the segments, or use PCR and ⁇ Exonuclease digestion as described by Higuchi et al. (1989, Nucleic Acid Research, 17:5865). Several chemical synthesis methods are described by Narang et al. (1979 Meth. Enzymol. 68:90), Brown et al.
  • cloning methods may provide a convenient nucleic acid fragment which can be isolated for use as a promoter primer.
  • a double-stranded DNA probe is first rendered single-stranded using, for example, conventional denaturation methods prior to hybridization to the target nucleic acids.
  • Hybridization is conducted under standard hybridization conditions well known to those skilled in the art.
  • Reaction conditions for hybridization of a probe to a nucleic acid sequence vary from probe to probe, depending on factors such as probe length, the number of G and C nucleotides in the sequence, and the composition of the buffer utilized in the hybridization reaction.
  • Moderately stringent hybridization conditions are generally understood by those skilled in the art as conditions approximately 25 °C below the melting temperature of a perfectly base-paired double stranded DNA. Higher specificity is generally achieved by employing incubation conditions having higher temperatures, in other words more stringent conditions.
  • Hybridization is typically performed in a buffered aqueous solution, for which conditions such as temperature, salt concentration, and pH are selected to provide sufficient stringency such that the probes hybridize specifically to their respective target nucleic acid sequences but not any other sequence.
  • Hybridization of the CSP and the SSP to the target nucleic acid may be performed simultaneously or sequentially and in either order.
  • hybridization of the CSP and hybridization of the SSP to the target nucleic acid are performed simultaneously.
  • the hybrid formed is then captured onto a solid phase coated with a substrate to which ligand attached to the CSP binds with specificity.
  • hybridization of the SSP to the target nucleic acid is performed before or after the hybridization of the CSP to the target nucleic acid.
  • the CSP may be immobilized on a solid phase before or after hybridization.
  • both the CSP and the target may be bound to the solid phase during the SSP hybridization reaction.
  • a solid phase or matrix includes, for example, polystyrene, polyethylene, polypropylene, polycarbonate or any solid plastic material in the shape of plates, slides, dishes, beads, particles, cups, strands, chips and strips.
  • a solid phase also includes glass beads, glass test tubes and any other appropriate glass product.
  • a functionalized solid phase such as plastic or glass that has been modified so that the surface contains carboxyl, amino, hydrazide, aldehyde groups, nucleic acid or nucleotide derivatives can also be used.
  • Any solid phase such as plastic or glass microparticles, membranes, beads, strips, test tubes, capillaries, slides, strands, chips or micrc-titer plates can be used.
  • the present invention employs the use of total, mature, and variant hTERT mRNA levels to produce cancer diagnosis and prognosis.
  • Total hTERT mRNA is the sum total of mature, deletion variant, and insertion variant mRNA levels.
  • Mature hTERT mRNA is the complete hTERT mRNA coding sequence without insertions or deletions.
  • Total variant hTERT mRNA includes both insertion and deletion variants.
  • An insertion variant is a variant that is alternatively spliced by insertion of a coding or non-coding region(s). The insertion of a non-coding region is especially common in hTERT.
  • a deletion variant is a variant that is alternatively spliced by deletion of a coding region(s).
  • Total hTERT mRNA levels can be determined using a probe or probes complementary to the target nucleic acid region or regions preserved among total mRNA species. For example, total hTERT may be determined by detecting a region or regions of exons 2, 3, and/or 4. Insertion variant hTERT mRNA levels can be determined using a probe or probes complementary to an inserted region or regions of a target. For example hTERT insertion variants can be determined by detecting a region or regions of introns 4, 11, and/or 14. Mature hTERT mRNA levels can be determined using a probe or probes complementary to a region or regions of non-deletion species including mature mRNA, i.e.
  • non-deletion hTERT can be determined by detecting a region or regions of exons 6, 7, and/or 8 and insertion hTERT can be determined as described above.
  • the level of deletion variant hTERT mRNA can be determined indirectly by determining the total hTERT mRNA level and subtracting from it the level of mature hTERT mRNA. The result is the total variant hTERT mRNA level. From the total variant level the insertion variant hTERT mRNA level is subtracted to obtain the deletion variant hTERT mRNA level.
  • the level of mature and insertion variant species is determined simultaneously by the use of a probe or probes to a region or regions conserved throughout these two species. This mRNA is referred to as non-deletion hTERT mRNA.
  • hTERT mRNA levels are determined by the variant-specific hybrid capture method employing a CSP and a SSP.
  • a SSP comprises a nucleic acid sequence which is capable of hybridizing to regions within a target nucleic acid that are adjacent to the unique regions recognized by the CSP. The sequences of the CSP and the SSP are selected so that they will not hybridize to the same region of a target nucleic acid or to each other.
  • the hybridization reaction is run on microarray slides.
  • Microarray slides are known in the art and are commercially available, for example, from Corning (Corning, NY) and from SurModics, (Eden Prairie, MN).
  • the CSP is spotted individually onto a solid phase microarray slide prior hybridization.
  • Target nucleic acid with or without SSP are then applied to the same location as the CSP.
  • the slide is covered, placed in a hybridization cassette (Tele Chem International, Inc., Sunnyvale, CA) and incubated in a water bath for about 1 to 20 hours at about 50 to 65 °C.
  • the slide is then washed and then treated with anti- RNA/DNA antibodies.
  • the slide is then treated with labeled antibodies, such as anti- murine antibodies.
  • labeled antibodies such as anti- murine antibodies.
  • the resulting complex is then detected by methods specific for the label on the labeled antibody.
  • labels are chemiluminescent, fluorescent, and radioactive.
  • the anti-RNA DNA antibody can be labeled and this complex can be detected directly without the need for a secondary antibody.
  • Detection of captured hybrids is preferably achieved by binding a conjugated antibody to the hybrid during an incubation step. Surfaces are then washed to remove any excess conjugate.
  • manual washes may be performed using either an EppendorfTM Repeat Pipettor with a 50 ml CombitipTM (Eppendorf, Hamburg, Germany), a Corning repeat syringe (Corning, Corning, NY), a simple pump regulated by a variostat, or by gravity flow from a reservoir with attached tubing.
  • Commercially available tube washing systems (Source Scientific Systems, Garden Grove, CA), and Automated Plate Washing Systems (Tri-Continent Grass Valley, CA) can also be used.
  • Bound conjugate is subsequently detected by methods conventionally used in the art, for example, colorimetry or chentiluminescence as described in Coutlee, et al, J. Clin. Microbiol. 27:1002-1007 (1989).
  • bound alkaline phosphatase conjugate is detected by chemiluminescence by adding a substrate which can be activated by alkaline phosphatase. Chentiluminescent substrates that are activated by alkaline phosphatase are well known in the art.
  • detection can be performed utilizing mouse anti-goat antibodies labeled with Cy3 and/or Cy5 (Zymed Laboratories Inc., San Francisco, CA). Fluorescent signals from bound secondary antibodies can be detected using, for example, a GMS 418 Array Scanner (Affymetrix, Santa Clara, CA) or other types of scanners.
  • GMS 418 Array Scanner Affymetrix, Santa Clara, CA
  • total (mature plus total variant) hTERT mRNA is hybridized to CSP and SSP simultaneously or sequentially, preferably to CSP only.
  • the CSP and SSP are complementary to regions of hTERT exons 2, 3 and 4.
  • Total insertion hTERT mRNA species, alternatively spliced by insertions, are hybridized to CSP and SSP simultaneously, preferably to CSP only.
  • CSP and SSP are complementary to regions of hTERT introns 4, 11, and 14.
  • hTERT mRNA not spliced by deletion is hybridized to CSP and SSP simultaneously or sequentially, preferably to CSP only.
  • the CSP and SSP are complementary to regions of hTERT exons 6, 7 and 8.
  • the hybrids are detected using single anti-RNA/DNA antibodies or double antibody sandwiches as shown in Figure 7 and 8.
  • Mature hTERT mRNA is calculated by subtraction of hTERT spliced by insertion from hTERT not spliced by deletion. Then the ratio of total hTERT over mature hTERT is calculated and used for diagnosis, classification of cancer, and therapy management.
  • the SSP used in the detection method is a nucleic acid molecule which comprises a DNA-RNA duplex and a single stranded nucleic acid sequence which is capable of hybridizing " to the target nucleic acid. Detection may be accomplished, for example, by binding a labeled antibody capable of recognizing the DNA-RNA duplex portion of the signal sequence probe, thereby detecting the hybrid formed between the target nucleic acid, the capture sequence probe and the signal sequence probe.
  • the signal sequence probe used in the detection method is a molecule which does not contain sequences that are capable of hybridizing to the target nucleic acid.
  • Bridge probes comprising sequences that are capable of hybridizing to the target nucleic acid as well as sequences that are capable of hybridizing to the signal sequence probe are used.
  • the signal sequence probe comprises a DNA-RNA duplex portion and a single stranded DNA sequence portion containing sequences complementary to sequences within the bridge probe.
  • the bridge probe which hybridizes to both the target nucleic acid and the signal sequence probe, therefore serves as an intermediate for connecting the signal sequence probe to the target nucleic acid and the capture sequence probe hybridized to the target nucleic acid.
  • blocker probes comprising oligonucleotides complementary to the capture sequence probes are used in the method to eliminate excess capture sequence probe, thereby reducing the background signal in detection and increasing specificity of the assay.
  • the target specific hybrid capture method of the invention employs blocker probes in addition to the CSP and SSP.
  • a blocker probe comprises sequences that are complementary to the sequences of the CSP.
  • the sequence of a blocker probe is preferably at least 75% complementary to the sequence of the CSP, more preferably, 100% complementary to the CSP.
  • the addition of the blocker probes to the hybridization reaction mixture prevents non-hybridized CSP from hybridizing to cross-reactive nucleic acid sequences present in the target and therefore increases the specificity of the detection.
  • the blocker probe is generally at least 5 bases long, preferably 12 bases long.
  • the concentration of the blocker probe in the hybridization reaction is preferably in excess to that of the CSP and SSP.
  • the blocker probe is present in a 2- fold molar excess, although, it may be present in up to 10,000-fold molar excess.
  • the blocker probes can be DNA, RNA, peptide nucleic acids (PNAs) or other nucleic acid analogues.
  • the level of hTERT mRNA is determined using an aliquot of specimen or total RNA isolated from a specimen.
  • the aliquot of specimen is lysed using 150 units of Proteinase K and the mixture incubated at 37 °C for 30 minutes.
  • the total RNA from the specimen can be prepared using commercially available methods.
  • the number of cells subjected to lysis and/or the amount of total RNA can be determined by cell counting or RNA quantification, respectively.
  • ⁇ -actin DNA measurement utilizing probe hybridization from the same amount of specimen can be applied for cell quantification.
  • a first cocktail of biotinylated single stranded DNA probes complementary to the complete coding translated region of hTERT sense DNA strand (16 exons; approximately 4,000 base pairs), is used for total (mature plus variant hTERT mRNA) level detection.
  • a second cocktail of three single stranded DNA probes complementary to: a) exons 1 through 6; b) exons 7 and 8 (biotinylated single- stranded DNA) of hTERT sense DNA strand; and c) exons 9 through 16 (a and c non- biotinylated single-stranded DNA) is used for hTERT mRNA levels detection excluding mRNA alternatively spliced at exons 7 and 8.
  • spliced species represented by deletion and insertion mechanisms, are described, for example, in Ulaner et al. Cancer Research 58, 1998 and Meyerson et al. Cell 90, 1997.
  • a third biotinylated single stranded DNA probe (optional), complementary to intron 14 of hTERT DNA, is used for detection of alternatively spliced hTERT nucleic acid by insertion of intron 14 hTERT mRNA variant. All three DNA probes are used individually during hybridization.
  • the hTERT single stranded DNA probes are hybridized to specific hTERT mRNA from the lysates/total RNA at 65 °C for 2 hours.
  • hTERT DNA-RNA hybrids are then captured and detected with anti-DNA-RNA monoclonal antibodies labeled with - alkaline phosphatase on a streptavidin coated surface at 22-24 °C with agitation.
  • the unbound reactants are discarded and the surface bound complex is sequentially washed with Hybrid Capture II and Enhance wash buffers.
  • the detection of captured hTERT mRNA is performed by measuring the rate of dephosphorylation of the dioxetane derivative of CDP-Star® chemiluminescent reagent during the reaction with alkaline phosphatase after a period of time.
  • the measurement in Relative Lights Units (RLUs) is performed using a luminometer.
  • the levels of hTERT mRNA in a cell or in a sample can be used to make an assessment and prognosis concerning cancer.
  • an undetectable level of hTERT mRNA corresponds to absence of disease
  • a low level of hTERT mRNA corresponds to molecular abnormalities that will lead to pre-cancer development such as hyperplasia and dysplasia
  • a high level of hTERT mRNA corresponds to high- grade lesions and cancer development.
  • the present invention provides methods for determining levels of hTERT mRNA present in a cell or sample and methods for using these hTERT mRNA levels to make assessments and prognoses concerning cancer.
  • Figure 2 depicts one embodiment of the present invention for determining total (mature plus variant) hTERT nucleic acid levels.
  • a DNA probe which is complementary to the mature hTERT mRNA sequence of interest or a portion thereof is used. Typically, the probe is from about 3 kb to about 100 bases in size.
  • hTERT DNA-RNA hybrids are formed and captured through biotin-streptavidin bonding on the solid surface.
  • Anti-DNA-RNA antibodies labeled with alkaline phosphatase recognize double-helix formation and bind to it.
  • a positive RLU signal representing the rate of dephosphorylation of the dioxetane derivative of CDP-Star® reagent, is then measured by a luminometer.
  • Figure 3 depicts a method for determining the level of hTERT mRNA which is not spliced by deletion, in this case not spliced by deletion at exons 7 and 8.
  • a DNA probe cocktail which includes three DNA probes, probes a, b, and c, where only probe b, which is complementary to hTERT exons 7 and 8, is biotinylated.
  • the DNA probe cocktail forms hTERT DNA-RNA hybrids which are captured through biotin-streptavidin covalent bond on the solid surface.
  • Anti-DNA-RNA antibodies labeled with alkaline phosphatase recognize double-helix formation, and bind to it.
  • a positive RLU signal representing the rate of dephosphorylation of the dioxetane derivative of CDP-Star® reagent, is then measured by a luminometer.
  • Figure 4 depicts an alternative method for determining the level of hTERT mRNA which is not spliced by deletion.
  • hTERT mRNA which is alternatively spliced by deletion will produce no signal.
  • hTERT DNA-RNA hybrids are formed. However, they are not captured on the solid surface. The non-captured DNA-RNA hybrids are decanted prior the incubation with anti-DNA-RNA antibodies. The antibodies do not bind to the single stranded probe b, therefore no RLU signal is generated.
  • the signal intensity is inversely associated with the proportion of the spliced hTERT mRNA.
  • Figure 5 depicts a method of determining levels of hTERT mRNA which are alternatively spliced by insertion, for example insertion of intron 14.
  • the figure shows a biotinylated DNA probe which is complementary to a portion of intron 14 of hTERT.
  • the DNA probe forms hTERT DNA-RNA hybrids which are captured through biotin-streptavidin bonding on the solid surface.
  • Anti-DNA-RNA antibodies labeled with alkaline phosphatase recognize the double-helix formation and bind to it.
  • a positive RLU signal representing the rate of dephosphorylation of the dioxetane derivative of CDP- Star® reagent, is measured by a luminometer.
  • Figure 6 depicts an alternative method of determining levels of hTERT mRNA which are alternatively spliced by insertion, for example insertion of intron 14
  • hTERT DNA-RNA hybrids are not formed and, therefore, not captured on the solid surface.
  • the anti-DNA:RNA antibodies are washed from the surface and no RLU signal is generated.
  • Figure 7 depicts detection of hTERT mRNA species utilizing a microarray platform.
  • hTERT mRNA species are hybridized with the spotted DNA probes and the hybrids detected with a single labeled anti-RNA/DNA antibody or with a double antibody "sandwich", where a secondary antibody is labeled. In the presence of hTERT mRNA species complementary to the specific DNA probes, a positive signal will be obtained.
  • FIG. 8 is a schematic diagram illustrating an embodiment of the invention.
  • CSP complementary to regions of exons 2, 3, and 4 of hTERT mRNA (designed for total hTERT mRNA species detection), complementary to regions of exons 6, 7, and 8 of hTERT mRNA (designed for alternatively spliced by deletion hTERT mRNA species detection), and complementary to regions of introns 4, 11, and 14 of hTERT gene (designed for alternatively spliced by insertion hTERT mRNA species detection) are spotted on a solid surface of microarray at specific positions.
  • hTERT mRNA species are hybridized with the spotted DNA probes and with SSP probes either simultaneously or sequentially.
  • SSP are designed to be complementary to regions adjacent to CSP on the hTERT mRNA, and are used for specific signal amplification, if necessary.
  • the formed DNA/RNA hTERT hybrids are detected with a single labeled anti-RNA DNA antibody or with a double antibody "sandwich", where a secondary antibody is labeled. In the presence of hTERT mRNA species complementary to a specific CSP, a positive signal will be obtained.
  • Figure 9 is a graphical representation of hTERT mRNA. Mature hTERT mRNA with a complete coding region (16 exons) is shown in A. hTERT mRNA variants alternatively spliced by deletion of exons 6, 7 and 8 are shown in B. hTERT mRNA variants alternatively spliced by insertions of introns 4, 11, arid 14 are shown in C.
  • Quantification of an unknown sample can be performed by using in vitro transcribed hTERT RNA as a control at various concentrations in the methods described in Figures 2-8. Regression analysis can be used to quantify unknown samples based on the signal measured from the known RNA controls. In one embodiment, the results from a process similar to that depicted in Figure 5 or 6 are subtracted from those obtained from a process similar to Figure 3 to obtain levels of mature hTERT mRNA. The value for total hTERT mRNA obtained by a method according to Figure 2 can then be divided by the level of mature hTERT mRNA to obtain a value for cancer assessment and prognosis according to the invention.
  • the results obtained from a method according to Figure 3 are indicative of the level of mature hTERT mRNA.
  • the ratio obtained from a method according to Figure 2 over the level obtained from a method according to Figure 3 provides a value for cancer prognosis and assessment according to this invention.
  • the ratios obtained according to the methods described above can be used to make a cancer prognosis and assessment.
  • the ratio of total hTERT mRNA to mature hTERT mRNA in a sample is greater than about 10, the tissue from which the sample was taken is considered normal and predicted to remain normal.
  • the ratio of total hTERT mRNA to mature hTERT mRNA in a sample is between about 10 and about 3, the tissue from which the sample was taken is hyperplastic or dysplastic or the tissue has a propensity to develop hyperplasia or dysplasia.
  • the ratio of total hTERT mRNA to mature hTERT mRNA in a sample is below about 3, it is indicative of cancer such as in situ cancer or invasive cancer in the tissue from which the sample was taken or it is indicative of the propensity of the tissue to develop cancers such as in situ cancer or invasive cancer.
  • Specimens with low levels of hTERT mRNA by direct measurement can be subjected to RT-PCR (with limited cycles of amplification)-probe hybridization.
  • the RT step from lysed cells and or total RNA prepared by methods known in the art can be performed using commercially available methods.
  • PCR, using specific primer sets for the coding region of hTERT gene can be performed at 10-16 amplification cycles using commercially available reagents (Applied Biosystems, Boston, MA). Detection of PCR products can be performed utilizing probe hybridization methodology.
  • RT-PCR with limited cycles of amplification
  • hTERT mRNA species
  • RNA is analyzed directly by solution based procedures.
  • the cells are first lysed by adding a proteolytic enzyme to the cells contained in wells of a microtiter plate.
  • enzymes for use in the present invention include proteinase K or Pronase.
  • Cells can also be subjected to detergent lysis or osmotic lysis or a French Press.
  • biotinylated DNA probes or a mixture of biotinylated and non-biotinylated DNA probes are added to each well.
  • Alkaline phosphatase-conjugated antibodies to DNA-RNA hybrids are added to each well in the hybridization microplate.
  • the DNA-RNA hybrids are captured onto a solid phase by trarisferring to sfreptavidin coated microplates. Signals are generated by adding a chemiluminescent reagent such as CDP-Star ® with Emerald II (Applied Biosystems, Boston, MA) to each well. The signal is read from the microplate.
  • a chemiluminescent reagent such as CDP-Star ® with Emerald II (Applied Biosystems, Boston, MA)
  • each combination of assays is followed by an assessment, using the combined assay results of the cell state, disease state, patient status, patient prognosis, or other assessment as described herein.
  • results of initial assays are either equivocal or suggest a more severe stage of disease
  • further assays are useful to clarify and confirm the initial results.
  • the disclosed assay and other assays can also be sequentially combined. That is, first one type of assay can be performed, and then, depending on the results, another assay can be performed. For example, the disclosed molecular assay can be performed first, and if the results of the assay were indicative of hyperplasia, dysplasia, or cancer, a cytological assay or biopsy can be performed. Such sequential combinations are particularly useful for limiting more extensive testing to patients and samples that are identified as high risk.
  • the disclosed method can include the combination of a molecular assay as described above with any other assay for assessing a disease or state of cells in a test sample.
  • a molecular assay measuring the level or ratio of expression of hTERT nucleic acid can be combined with cytological assays, histological assays, assays detecting other cellular markers such as oncoproteins or tumor suppressors, or combinations of these assays.
  • cytological assays cytological assays, histological assays, assays detecting other cellular markers such as oncoproteins or tumor suppressors, or combinations of these assays.
  • Such assays are known and are used for the diagnosis of cancer and assessment of the stage of disease.
  • Results from a molecular assay and one or more additional assays can be combined to increase the reliability of any assessment, prognosis, diagnosis, or monitoring of cancer.
  • Useful combinations include a cytological assay and the disclosed molecular assay, and a combination of a cytological assay, an assay for detecting cancer markers, and the disclosed molecular assay.
  • Combined assays can be performed in any order and in any temporal relationship. For example, various assays can be performed in parallel or simultaneously. Such assays can be performed in any manner such as on the same apparatus by the same person, with different apparatus, or in the same or different locations.
  • cytological assays for use in assessing the stage of HPV-based disease are known and can be used in the disclosed method.
  • the well-established Pap smear and Hematoxylin & Eosin stains (H&E) are preferred examples.
  • the use and analysis of Pap smears and H&E stains are well known in the art.
  • kits that include one or more of the materials needed for the method, such as reagents and sample collection and handling materials.
  • the kit will contain probes for determining expression levels of mature hTERT mRNA and variant hTERT mRNA.
  • the kits can also include cell collection medium, sample preserving reagents, reagents for specific detection of DNA and/or expression products (RNA or proteins) of one or more portions of the hTERT gene and sample handling containers.
  • Useful reagents for detection of expression of hTERT nucleic acids are nucleic acid probes for those sequences.
  • a kit may also contain control samples or reagents, or reagents and materials for performing other assays to be combined with the disclosed assay.
  • kits can contain reagents for the separation of RNA and/or DNA from other cellular components.
  • the kit can also contain microarray(s) spotted with probes as described in this invention.
  • the kit may also contain instructions detailing the methods described herein to make a cancer diagnosis or assessment according to the specific ratios described.
  • the present invention can be performed using devices adapted to the method.
  • Numerous devices for performing similar assays are known and in use and can be adapted for use with the disclosed assays and method.
  • devices are known for automating all or a part of sample assays and sample handling in assays.
  • All or part of the disclosed method can be controlled or managed using special purpose computer programs.
  • the data collected from the disclosed method, and data from any other assay used in combination can be compiled, analyzed, and output in various forms and for various purposes using special purpose computer programs.
  • Such programs can be used with, or combined into, other patient or data management computer programs. The iisefulness of such a program increases with the number of measurements or assessments to be combined, and the relative importance of each type of measurement to the overall assessment.
  • Computer programs for use with the disclosed method can be used on general purpose computers, or can be incorporated into special purpose computers or computerized devices for controlling the disclosed method, handling and analyzing data from the disclosed method or both.
  • Variant-Specific Hybrid Capture® Clinical samples suspected of abnormal expression of hTERT are lysed with 150 units of Proteinase K at 37 °C for about 30 minutes. Alternatively, total RNA isolated from the same specimens can be used directly for hybridization without lysis. The DNA probes, approximately 180 pM each, are added to the lysates or RNA on a hybridization plate and incubated at 65 °C for 2 hours. The resulting RNA DNA hybrids are allowed to stand at room temperature for 5 to 10 minutes. Anti-RNA/DNA antibodies labeled with alkaline phosphatase are added.
  • the reaction mixture is transferred to a 96-well streptavidin capture plate (Digene Corporation, Gaithersburg, MD) and the plate is shaken at 1100 rpm for 1 hour at room temperature. The supernatant is then decanted and the plates washed four times with HCIi Wash buffer. The plates are filled with Enhance Wash Buffer and incubated for 45 minutes at 53 °C. The supernatant is then decanted and the plates washed four times with HCII Wash buffer. Following removal of the residual liquid, chemiluminescent substrate CDP-Star with Emerald (Applied Biosystems, Boston, MA) is added to each well and the plate is incubated for 15 to 30 minutes at room temperature.
  • CDP-Star with Emerald Applied Biosystems, Boston, MA
  • the individual wells are then read on a plate luminometer to obtain the relative light unit (RLU) signal.
  • Solutions containing no lysates or RNA are used as negative controls for the test samples.
  • the signal to noise ratio is calculated as the ratio of the average RLU obtained from a test sample to the average RLU of the negative control.
  • the signal to noise ratio is used as the basis for deterrnining the quantity of target nucleic acid.
  • the quantified signals from total hTERT mRNA, from non-deletion variants including mature hTERT mRNA, and from insertion variants are then obtained.
  • the level of mature hTERT mRNA is determined by subtracting the level of insertion variant mRNA from the level of non- deletion variant including mature hTERT mRNA.
  • the patient assessment is normal and the prognosis for the patient is also normal.
  • the ratio of total hTERT mRNA to mature hTERT mRNA in the patient sample is between about 10 and about 3
  • the sample may be normal or show signs of hyperplasia and/or dysplasia when examined cytological methods.
  • the patient assessment may be either normal or that the patient has hyperplasia and/or dysplasia.
  • the tissue appears to be normal by cytological methods, the patient nevertheless has a propensity to develop hyperplasia or dysplasia and should be treated or closely monitored.
  • the ratio of total hTERT mRNA to mature hTERT mRNA in a patient sample is below about 3, it is indicative of cancer such as in situ cancer or invasive cancer or the propensity to develop cancer such as in situ cancer or invasive cancer.
  • Variant-Specific Hybrid Capture® Microarray Protocol Specifically designed CSP are spotted or synthesized on a solid phase prior to the assay.
  • a mixture of CSP complementary to the regions of exons 2, 3, and 4 of hTERT is used for the determination of total (mature plus variant) hTERT mRNA level.
  • CSP complementary to regions of exons 6, 7, 8, and regions of introns 4, 11, 14 are used individually for the determination of hTERT mRNA species not spliced by deletion of exons and spliced by insertion of introns, respectively.
  • Clinical samples suspected of abnormal expression of hTERT are lysed with 150 units of Proteinase K at 37 °C for about 30 minutes.
  • total RNA isolated from the same specimens can be used directly for hybridization without lysis.
  • the lysates or total RNA samples are denatured at 95 °C for 5 minutes and applied to the same area on the solid phase previously spotted with the CSP.
  • either the lysates or total RNA samples are pre-hybridized with complementary SSP prior to their addition onto the spotted surface, or the sample and SSP ⁇ iixture is applied simultaneously to the same area on the solid phase previously spotted with the CSP.
  • the microarrays with a target and probe are hybridized for 15 to 20 hours at 50 to 65 °C.
  • the microarrays are washed with PBS and 0.1% Tween for 5 minutes at room temperature and washed with Enhance buffer for 1 minute at 50 °C.
  • the microarrays are incubated with anti- RNA/DNA antibodies for 10 to 30 minutes at room temperature and washed with PBS and 0.1% Tween.
  • Secondary goat anti-mouse antibodies labeled with Cy3 or Cy5, or others are used for detection if the primary anti-RNA/DNA antibodies are not labeled.
  • the secondary antibodies are incubated for 10 to 30 minutes at room temperature.
  • the microarrays are washed with PBS and 0.1% Tween followed by centrifugation to dry the surface. The signal is determined by scanning the microarray on a 418 GMS Scanner.
  • Mature hTERT mRNA levels are determined by subtraction of hTERT mRNA levels spliced by insertion of introns 4, 11, 14 from hTERT mRNA levels not spliced by deletion of exons 6, 7, 8. Ratio of total hTERT mRNA over mature hTERT mRNA is then calculated.

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Abstract

La présente invention concerne une technique permettant de prévoir et d'évaluer un cancer par la détermination des niveaux d'expression de la sous unité catalytique de la transcriptase inverse de télomérase humaine (TERTh). On utilise en particulier un rapport des niveaux d'expression d'ARNm de TERTh total sur l'ARNm de TERTh mature pour cette évaluation et ce pronostic.
PCT/US2001/041774 2000-08-18 2001-08-17 Techniques de pronostic et d'evaluation de cancer WO2002015770A2 (fr)

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Publication number Priority date Publication date Assignee Title
DE102006007249A1 (de) * 2006-02-15 2007-08-30 Universitätsklinikum Schleswig-Holstein Verfahren zur Bestimmung von Blasenkarzinomen des Differenzierungsgrades G2

Non-Patent Citations (3)

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Title
ULANER ET AL.: 'Regulation of telomerase by alternative splicing of human telomerase reverse transcriptase (hTERT) in normal and neoplastic ovary, endometrium and myometrium' INTERNATIONAL JOURNAL OF CANCER vol. 85, February 2000, pages 330 - 335, XP002907092 *
VILLA ET AL.: 'Possible regulation of telomerase activity by transcription and alternative splicing of telomerase reverse transcriptase in human melanoma' JOURNAL OF INVESTIGATIVE DERMATOLOGY vol. 116, no. 6, June 2001, pages 867 - 873, XP002907091 *
WICK ET AL.: 'Genomic organization and promoter characterization of the gene encoding the human telomerase reverse transcriptase (hTERT)' GENE vol. 232, May 1999, pages 97 - 106, XP002176112 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102006007249A1 (de) * 2006-02-15 2007-08-30 Universitätsklinikum Schleswig-Holstein Verfahren zur Bestimmung von Blasenkarzinomen des Differenzierungsgrades G2
DE102006007249B4 (de) * 2006-02-15 2008-07-31 Universitätsklinikum Schleswig-Holstein Verfahren zur Bestimmung von Blasenkarzinomen des Differenzierungsgrades G2

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