WO2011036173A1 - Détection et pronostic du cancer du col de l'utérus - Google Patents

Détection et pronostic du cancer du col de l'utérus Download PDF

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WO2011036173A1
WO2011036173A1 PCT/EP2010/063968 EP2010063968W WO2011036173A1 WO 2011036173 A1 WO2011036173 A1 WO 2011036173A1 EP 2010063968 W EP2010063968 W EP 2010063968W WO 2011036173 A1 WO2011036173 A1 WO 2011036173A1
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epb41l3
tert
jam3
cervical
gene
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Valérie DEREGOWSKI
Wim Van Criekinge
Luc Dehaspe
Bea G. A. Wisman
E. M. D. Schuuring
Ate G. J. Van Der Zee
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Oncomethylome Sciences S.A.
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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
    • C12Q2600/154—Methylation markers
    • 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
    • C12Q2600/156—Polymorphic or mutational markers
    • 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
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention relates to the area of cancer diagnostics and therapeutics.
  • it relates to methods and kits for identifying, diagnosing, prognosing, and monitoring cervical cancer. These methods include determining the methylation status or the expression levels of particular genes, or a combination thereof.
  • Cervical cancer is the fifth most deadly cancer in women. Worldwide, approximately 500,000 cases of cervical cancer are diagnosed and about 250,000 women die from this disease annually.
  • Cervical cancer evolves from pre-existing noninvasive premalignant lesions referred to as cervical intraepithelial neoplasias (CINs), ranging from CIN 1 (mild dysplasia) to CIN 2 (moderate dysplasia) to CIN 3 (severe dysplasia/carcinoma in situ). This process usually takes several years but sometimes can happen in less than a year. For most women, pre-cancerous cells will remain unchanged and disappear without any treatment. Infection with high-risk human papillomavirus (hr-HPV) is causally linked to cervical carcinogenesis [1].
  • hr-HPV human papillomavirus
  • Cervical cancer incidence is reduced by cyto logical screening, although cytology assessment of cervical scrapings is not ideal since its sensitivity is only about 55% [2].
  • Hr-HPV testing of cervical scrapings has been shown to improve sensitivity of cervical screening [3,4], but is also associated with low specificity, especially in a young screening population [5]. This low specificity of HPV testing leads to a higher number of unnecessarily follow-up diagnostic workups (e.g.
  • DNA methylation is a chemical modification of DNA performed by enzymes called methyltransferases, in which a methyl group (m) is added to certain cytosines (C) of DNA.
  • This non-mutational (epigenetic) process (mC) is a critical factor in gene expression regulation [1 1].
  • Promoter methylation of tumor suppressor genes has been reported to be an early event in carcinogenesis [6].
  • Gene promoter methylation of several cervical cancer specific genes has been suggested as an alternative diagnostic tool for early detection of cervical neoplasia by Quantitative Methylation Specific PCR (QMSP) [7,8].
  • QMSP Quantitative Methylation Specific PCR
  • Various methylated gene promoters for cervical neoplasia have been tested [9], mainly based on previously reported methylation status in cervical neoplasia or other tumor types. None of these markers can be used for cervical cancer screening so far, due to low sensitivity and specificity. Hence, there is a need for more sensitive and specific methylation markers.
  • WO2004/087957 discloses a QMSP method for detecting cervical cancer in a scraping sample comprising the use of a panel of genes whose hypermethylation status was already linked to the incidence of cervical cancer.
  • the genes investigated were p i 6, MGMT, GSTP 1 , DAP-kinase. and APC. Only for the DAP-kinase gene, the frequency of hypermcthy lat ion in cervical scrapings was similar to the one found for tissue samples (64 % versus 61 %). For the other individual genes, there is a discrepancy in the percentage of hypermethylation between cervical tissue samples and cervical scrapings. Accordingly, there is a need for alternative markers of cervical cancer, in particular for markers that provide
  • Kikuchi s et al [24] discloses the involvement of EPB41 L3 (referred therein as DAL- 1/4. 1 B) methylation in the development and progression of non-small cell lung cancers (NSCLC), providing an indicator for poor prognosis in NSCLC.
  • NSCLC non-small cell lung cancers
  • the finding of epigenetic inactivation in a specific tissue does not suggest a similar event for other tissues.
  • RASSF1 A was methylated in majority of lung cancers but not in any of the cervical tumors tested.
  • WO2009/ 1 15615 discloses a method for diagnosing cervical cancer by determining the methylation status or the expression levels of a long list of particular genes including EPB41 L3. It is an object of the present invention to provide methylation markers or panels of methylation markers for cervical cancer.
  • the present invention relates to a method for identifying, in a test sample, cervical cancer or its precursor, or predisposition to cervical cancer, said method comprising: providing a test sample comprising cervical cells or nucleic acids from cervical cells; assaying said test sample for epigenetic modification of the gene EPB41L3; wherein epigenetic modification of said gene indicates the presence of cells or nucleic acids from cells that are neoplastic, precursor to neoplastic, or predisposed to neoplasia.
  • epigenetic modification of a panel of genes is also assayed; wherein the panel of genes is selected from EPB41L3 and JAM3; EPB41L3 and TERT; EPB41L3 and C130RF18; EPB41L3, JAM3, and TERT; EPB41L3, JAM3, and C130RF18; EPB41L3, TERT, and C130RF18; and EPB41L3, JAM3, TERT, and C130RF18.
  • the present invention also relates to a method for screening or detection of cervical cancer, cervical intra-epithelial neoplasia 2 (CIN2), or cervical intra-epithelial neoplasia 3 (CIN3) comprising the steps of:
  • a test sample comprising cervical cells or nucleic acids from cervical cells
  • step b) assaying the test sample of step a) for high-risk human papillomavirus (hr-HPV); c) if b) is positive for the presence of hr-HPV, assaying for epigenetic modification of a gene or a panel of genes selected from EPB41L3; EPB41L3 and JAM3;
  • EPB41L3 and TERT EPB41L3 and C130RF18; EPB41L3, JAM3, and TERT; EPB41L3, JAM3, and C130RF18; EPB41L3, TERT, and C130RF18; and EPB41L3, JAM3, TERT, and C130RF18;
  • the present invention further relates to a kit for identifying cervical cancer or its precursor, or predisposition to cervical cancer in a test sample comprising cervical cells or nucleic acids from cervical cells, said kit comprising:
  • cytosines within CpG dinucleotides of DNA from particular genes isolated from a test sample, which are differentially methylated in human cervical cancer tissue samples and normal cervical tissue control samples.
  • the cancer tissues samples are hypermethylated or
  • epigenetic modification hypomethylated with respect to the normal samples (collectively termed epigenetic modification).
  • the differential methylation has been found in genomic DNA of the EPB41L3 gene as well as in the panel of genes selected from EPB41L3 and JAM3; EPB41L3 and TERT; EPB41L3 and C130RF18; EPB41L3, JAM3, and TERT;
  • EPB41L3, JAM3, and C130RF18 EPB41L3, TERT, and C130RF18; and EPB41L3, JAM3, TERT, and C130RF18.
  • the present invention relates to a method for identifying, in a test sample, cervical cancer or its precursor, or predisposition to cervical cancer, said method comprising: providing a test sample comprising cervical cells or nucleic acids from cervical cells; assaying said test sample for epigenetic modification of the gene EPB41L3; wherein epigenetic modification of said gene indicates the presence of cells or nucleic acids from cells that are neoplastic, precursor to neoplastic, or predisposed to neoplasia.
  • epigenetic modification of a panel of genes is also assayed; wherein the panel of genes comprises at least EPB41L3 together with one, two or three additional genes wherein epigenetic modification of at least one of the genes in the panel indicates the presence of cells or nucleic acids from cells that are neoplastic, precursor to neoplastic, or predisposed to neoplasia.
  • the additional genes are selected from JAM3, TERT, and C130RF18.
  • the panel of genes is selected from EPB41L3 and JAM3; EPB41L3 and TERT; EPB41L3 and C130RF18;
  • the present invention also relates to screening protocols for the screening of woman for cervical cancer and the precursors thereof.
  • the Pap Smear has been the primary screening method for the detection of abnormality of the cervix, but its performance is suboptimal.
  • Human Papillomavirus has been associated with the development of cervical cancer. Five high-risk types, 16, 18, 31, 45, and 58, and in particular HPV types 16 and 18 account for approximately 70% of all cervical carcinomas. A small percentage of women showing persistent infection progress from Low-grade to High-grade lesions.
  • Method for cervical cancer screening may combine high-risk human papillomavirus (hr-HPV) testing and methylation testing; or hr-HPV testing and cyto logical evaluation and methylation testing.
  • hr-HPV high-risk human papillomavirus
  • the present invention also relates to a method for screening or detection of cervical cancer, cervical intra-epithelial neoplasia 2 (CIN2), or cervical intra- epithelial neoplasia 3 (CIN3) comprising the steps of:
  • a test sample comprising cervical cells or nucleic acids from cervical cells
  • step b) assaying the test sample of step a) for high-risk human papillomavirus (hr-HPV); c) if b) is positive for the presence of hr-HPV, assaying for epigenetic modification of a gene or a panel of genes selected from EPB41L3; EPB41L3 and JAM3;
  • EPB41L3 and TERT EPB41L3 and C130RF18; EPB41L3, JAM3, and TERT; EPB41L3, JAM3, and C130RF18; EPB41L3, TERT, and C130RF18; and EPB41L3, JAM3, TERT, and C130RF18;
  • Table 1 provides the standard nomenclature, as well as the accession numbers for the genomic and mRNA and protein reference sequences of the marker genes of the present invention, derived all from Homo Sapiens. Source: National Center for Biotechnology Information (NCBI).
  • NC_000018.9 Genome Reference NM_012307.2 ⁇ NP_036439.2 erythrocyte
  • accession numbers above may be found in the publicly available gene database at http://www.ncbi.nlm.nih.gov.
  • genomic molecules, transcripts, and protein products presented herein are not limited to the particular sequences referred to above, but also comprise variants thereof.
  • identifying when relating to cervical cancer or predisposition to cervical cancer is defined herein to include the activities of detecting by way of examination; screening for a disease or pre-stadia of a disease; monitoring staging and the state or progression of the disease; checking for recurrence of disease following treatment and monitoring the success of a particular treatment.
  • the identification may also have prognostic value, and the prognostic value of the tests may be used as a marker of potential susceptibility to cancer.
  • test sample refers to biological material obtained from a subject, preferably a mammalian subject, more preferably a human subject.
  • the test sample may be any tissue sample, body fluid, body fluid precipitate, or lavage specimen.
  • the test sample comprises tissue, cells, and nucleic acids -meaning DNA or RNA- of viral or mammalian origin.
  • Test samples for diagnostic, prognostic, or personalized medicine uses can be obtained from cytological samples, from surgical samples, such as biopsies, cervical conization or hysterectomy, from (formalin fixed) paraffin embedded cervix or other organ tissues, from frozen tumor tissue samples, from fresh tumor tissue samples, from a fresh or frozen body fluid such as blood, serum, lymph, or from cervical scrapings, cervical smears, cervical washings and vaginal excretions.
  • Such sources are not meant to be exhaustive, but rather exemplary.
  • a test sample obtainable from such specimens or fluids includes detached tumor cells and free nucleic acids that are released from dead or damaged tumor cells.
  • the test samples may contain cancer cells or pre-cancer cells or nucleic acids from them.
  • the test sample comprises squamous cells, nucleic acids from squamous cells, adenocarcinoma cells, nucleic acids from adenocarcinoma cells, adenosquamous carcinoma cells, nucleic acids from adenosquamous carcinoma cells, epithelium without dysplasia, or any combination thereof.
  • Samples may contain mixtures of different types and stages of cervical cancer cells.
  • the test sample is a cervical scraping. In one embodiment, the test sample is collected with a lavage self-sampling device. Lavage self-sampling devices suitable for use in the methods of the present invention have been described in WO2002/041785.
  • cervicovaginal refers to the irrigation of the cervicovaginal tract, i.e. by washing the cervicovaginal cavity or surface with a flowing solution that is inserted and then removed. Additives or drugs can be added to the irrigation solution to add function.
  • self-sampling device refers to any device suitable for the collection of a cervicovaginal sample.
  • lavage self-sampling device refers to any device suitable for the collection of a cervicovaginal sample by irrigation of the
  • a preferable lavage self-sampling device is the Pantarhei Screener MermaidTM.
  • precursor when used in the context of "cervical cancer”, refers to those precancerous conditions of the cervix.
  • cervical cancer refers to that cell that is committed to a differentiation pathway, in particular any embryonic stem cell committed to a cervical cancer cell lineage.
  • precursor cells encompass progenitor cells, stem cells, duct cells, lobules cells, and the like.
  • nucleic acids include RNA, genomic DNA, mitochondrial DNA, single or double stranded, and protein-associated nucleic acids. Any nucleic acid specimen in purified or non-purified form obtained from such specimen cell can be utilized as the starting nucleic acid or acids.
  • epigenetic modification refers to a stable alteration in gene expression potential that takes place during development and cell proliferation, mediated by mechanisms other than alterations in the primary nucleotide sequence of a gene. Three related mechanisms that cause alteration in gene expression are recognized: DNA methylation, histone code changes, and RNA interference.
  • Epigenetic modification of a gene can be determined by any method known in the art. One method is to determine that a gene which is expressed in normal cells or other control cells is less expressed or not expressed in tumor cells. Diminished gene expression can be assessed in terms of DNA methylation status or in terms of expression levels as determined by their methylation status, generally manifested as hypermethylation.
  • a gene can be more highly expressed in tumor cells than in control cells in the case of hypomethylation.
  • This method does not, on its own, however, indicate that the silencing or activation is epigenetic, as the mechanism of the silencing or activation could be genetic, for example, by somatic mutation.
  • One method to determine that silencing is epigenetic is to treat with a reagent, such as DAC (5'-deazacytidine), or with a reagent that changes the histone acetylation status of cellular DNA, or any other treatment affecting epigenetic mechanisms present in cells, and observe that the silencing is reversed, i.e., that the expression of the gene is reactivated or restored.
  • epigenetic modification is to determine the presence of methylated CpG dinucleotide motifs in the silenced gene or the absence of methylated CpG dinucleotide motifs in the activated gene.
  • epigenetic modification is assayed by detecting methylation of a CpG dinucleotide motif in the EPB41L3 gene or a promoter region thereof, in other genes listed in Table 1 , and in any combination thereof.
  • Methylation of a CpG island at a promoter usually prevents expression of the gene.
  • the islands can surround the 5' region of the coding region of the gene as well as the 3 ' region of the coding region. Thus, CpG islands can be found in multiple regions of a nucleic acid sequence.
  • region when used in reference to a gene includes sequences upstream of coding sequences in a regulatory region including a promoter region, in the coding regions (e.g., exons), downstream of coding regions in, for example, enhancer regions, and in introns. All of these regions can be assessed to determine their methylation status. When the CpG distribution in the promoter region is rather scarce, levels of methylation are assessed in the intron and/or exon regions.
  • the region of assessment can be a region that comprises both intron and exon sequences and thus overlaps both regions.
  • TSS transcription start site
  • Expression of a gene can be assessed using any means known in the art. Typically expression is assessed and compared in test samples and control samples which may be normal, non-malignant cells. Either mRNA or protein can be measured. Methods employing hybridization to nucleic acid probes can be employed for measuring specific mR As. Such methods include using nucleic acid probe arrays (e.g.
  • RNA can also be assessed using amplification techniques, such as RT-PCR.
  • Sequencing-based methods are an alternative; these methods started with the use of expressed sequence tags (ESTs), and now include methods based on short tags, such as serial analysis of gene expression (SAGE) and massively parallel signature sequencing (MPSS).
  • ESTs expressed sequence tags
  • MPSS massively parallel signature sequencing
  • Differential display techniques provide another means of analyzing gene expression; this family of techniques is based on random amplification of cDNA fragments generated by restriction digestion, and bands that differ between two tissues identify cDNAs of interest. Specific proteins can be assessed using any convenient method including immunoassays and immuno-cytochemistry but are not limited to that. Most such methods will employ antibodies, or engineered equivalents thereof, which are specific for the particular protein or protein fragments.
  • the sequences of the mRNA (cDNA) and proteins of the markers of the present invention are known in the art and publicly available. In one embodiment of the present invention, epigenetic
  • epigenetic modification is assayed by amplification of at least a portion of the applicable gene using an oligonucleotide primer that specifically hybridizes under amplification conditions to a region of said gene, wherein the region is within about 10 kb of said gene's transcription start site.
  • the oligonucleotide primer is designed not to contain cytosines and amplifies modified and unmodified sequences.
  • transformation of the sample to determine epigenetic modification comprises restriction enzyme cleavage and/or modifying sequences with chemical reagents, as explained in more detail later.
  • An additional amplification may be subsequently performed with primers hybridizing to the modified sequence, thereby indicating methylation; or alternatively a detection step with a specific probe may be performed, thereby indicating methylation.
  • the amplification may be combined with restriction cutting by using methylation sensitive enzymes; only the methylated region is amplified in this case.
  • epigenetic modification is assayed by amplification of at least a portion of the applicable gene using at least one pair of oligonucleotide primers that specifically hybridizes under amplification conditions to a region of said gene, wherein the region is within about 10 kb of said gene's
  • the at least one pair of oligonucleotide primers is designed not to contain cytosines and amplifies modified and unmodified sequences.
  • An additional amplification may be subsequently performed with primers hybridizing to the modified sequence, thereby indicating methylation; or alternatively a detection step with a specific probe may be performed, thereby indicating methylation.
  • the amplification may be combined with restriction cutting by using methylation sensitive enzymes; only the methylated region is amplified in this case.
  • methylation-sensitive restriction endonucleases can be used to detect methylated CpG dinucleotide motifs. Such endonucleases may either preferentially cleave methylated recognition sites relative to non-methylated recognition sites or preferentially cleave non-methylated relative to methylated recognition sites.
  • Non limiting examples of the former are Aat II, Acc III, Ad I, Acl I, Age I, AIu I, Asc I, Ase 1, AsiS I, Ban I, Bbe I, BsaA I, BsaH I, BsiE I, BsiW I, BsrV I, BssK 1, BstB I, BstN I, Bs I, Cla I, Eae I, Eag I, Fau I, Fse I, Hha I, mPl I, HinC II, Hpa 11, Npy99 I, HpyCAIV, Kas I, Mbo I, MIu I, MapA I, Msp I, Nae I, Nar I, Not 1, Pml I, Pst I, Pvu I, Rsr II, Sac II, Sap I, Sau3A I, Sfi I, Sfo I, SgrA I, Sma I SnaB I, Tsc I, Xma I, and Zra I
  • Non limiting examples of the latter are Acc II, Ava I, BssH II, BstU I, Hpa II, Not I, and Mho I.
  • chemical reagents can be used that selectively modify either the methylated or non-methylated form of CpG dinucleotide motifs, thereby transforming the CpG-dinucleotide motifs. Modified products can be detected directly, or after a further reaction which creates products that are easily distinguishable. Means which detect altered size and charge can be used to detect modified products, including but not limited to electrophoresis, chromatography, and mass spectrometry. Examples of such chemical reagents for selective modification include hydrazine and bisulfite ions.
  • Hydrazine-modified DNA can be treated with piperidine to cleave it.
  • Bisulfite ion- treated DNA can be treated with alkali.
  • methylation is detected by contacting at least a portion of the applicable gene or promoter region thereof with a chemical reagent that selectively modifies a non- methylated cytosine residue relative to a methylated cytosine residue, or selectively modifies a methylated cytosine residue relative to a non-methylated cytosine residue; and detecting a product generated due to said contacting.
  • the chemical reagent comprises bisulfite ions.
  • the method further comprises treating with alkali the bisulfite ion-contacted portion of the gene.
  • QMSP methylation-specific PCR
  • sequencing ligase chain reaction
  • the principle behind electrophoresis is the separation of nucleic acids via their size and charge. Many assays exist for detecting methylation and most rely on determining the presence or absence of a specific nucleic acid product. Gel electrophoresis is commonly used in a laboratory for this purpose. One may use MALDI mass spectrometry in combination with a methylation detection assay to observe the size of a nucleic acid product. The principle behind mass spectrometry is the ionizing of nucleic acids and separating them according to their mass to charge ratio. Similar to electrophoresis, one can use mass spectrometry to detect a specific nucleic acid that was created in an experiment to determine methylation (Tost, J. et al. 2003).
  • chromatography high performance liquid chromatography
  • DNA is first treated with sodium bisulfite, which converts an unmethylated cytosine to uracil, while methylated cytosine residues remain unaffected.
  • DHPLC has the resolution capabilities to distinguish between methylated (containing cytosine) and unmethylated (containing uracil) DNA sequences.
  • Deng, D. et al. describes simultaneous detection of CpG methylation and single nucleotide polymorphism by denaturing high performance liquid chromatography.
  • Hybridization is a technique for detecting specific nucleic acid sequences that is based on the annealing of two complementary nucleic acid strands to form a double-stranded molecule.
  • One example of the use of hybridization is a microarray assay to determine the methylation status of DNA. After sodium bisulfite treatment of DNA, which converts an unmethylated cytosine to uracil while methylated cytosine residues remain unaffected, oligonucleotides complementary to potential methylation sites can hybridize to the bisulfite-treated DNA.
  • the oligonucleotides are designed to be complementary to either sequence containing uracil (thymine) or sequence containing cytosine, representing unmethylated and methylated DNA, respectively.
  • Computer- based microarray technology can determine which oligonucleotides hybridize with the DNA sequence and one can deduce the methylation status of the DNA.
  • primers can be designed to be complementary to either sequence containing uracil (thymine) or sequence containing cytosine.
  • Primers and probes that recognize the converted methylated form of DNA are dubbed methylation- specific primers or probes (MSP).
  • An additional method of determining the results after sodium bisulfite treatment involves sequencing the DNA to directly observe any bisulfite-modifications.
  • Pyro sequencing technology is a method of sequencing-by- synthesis in real time. It is based on an indirect bio lumino metric assay of the pyrophosphate (PPi) that is released from each deoxynucleotide (dNTP) upon DNA-chain elongation. This method presents a DNA template-primer complex with a dNTP in the presence of an exonuclease-deficient Klenow DNA polymerase.
  • the four nucleotides are
  • PPi is released.
  • the PPi and other reagents are used as a substrate in a lucif erase reaction producing visible light that is detected by either a luminometer or a charge-coupled device.
  • the light produced is proportional to the number of nucleotides added to the DNA primer and results in a peak indicating the number and type of nucleotide present in the form of a pyrogram. Pyro sequencing can exploit the sequence differences that arise following sodium bisulfite-conversion of DNA.
  • amplification techniques may be used in a reaction for creating distinguishable products. Some of these techniques employ PCR. Other suitable amplification methods include the ligase chain reaction (LCR) (Barringer et al, 1990), transcription amplification (Kwoh et al. 1989; WO88/10315), selective amplification of target polynucleotide sequences (US Patent No. 6,410,276), consensus sequence primed polymerase chain reaction (US Patent No 4,437,975), arbitrarily primed polymerase chain reaction (WO90/06995), nucleic acid based sequence amplification (NASBA) (US Patent Nos. 5,409,818; 5,554,517; 6,063,603), micro satellite length polymorphism (MLP), and nick displacement amplification (WO2004/067726).
  • LCR ligase chain reaction
  • NASBA nucleic acid based sequence amplification
  • MLP micro satellite length polymorphism
  • MLP micro satellite length polymorphism
  • Sequence variation that reflects the methylation status at CpG dinucleotides in the original genomic DNA offers two approaches to PCR primer design.
  • the primers do not themselves cover or hybridize to any potential sites of DNA methylation; sequence variation at sites of differential methylation are located between the two primers.
  • Such primers are used in bisulfite genomic sequencing, COBRA, Ms-SNuPE.
  • the primers are designed to anneal specifically with either the methylated or unmethylated version of the converted sequence.
  • the primer may also contain additional nucleotide residues that do not interfere with hybridization but may be useful for other manipulations.
  • additional nucleotide residues may be sites for restriction endonuclease cleavage, for ligand binding or for factor binding or linkers or repeats.
  • the oligonucleotide primers may or may not be such that they are specific for modified methylated residues.
  • One way to distinguish between modified and unmodified DNA is to hybridize oligonucleotide primers which specifically bind to one form or the other of the DNA. After primer hybridization, an amplification reaction can be performed. The presence of an amplification product indicates that a sample hybridized to the primer. The specificity of the primer indicates whether the DNA had been modified or not, which in turn indicates whether the DNA had been methylated or not. For example, bisulfite ions convert non-methylated cytosine bases to uracil bases. Uracil bases hybridize to adenine bases under hybridization conditions.
  • an oligonucleotide primer which comprises adenine bases in place of guanine bases would hybridize to the bisulfite- modified DNA, whereas an oligonucleotide primer containing the guanine bases would hybridize to the non-converted (initial methylated) cytosine residues in the modified DNA.
  • Amplification using a DNA polymerase and a second primer yields
  • MSP Method of PCR
  • Primers are designed to anneal specifically with the converted sequence representing either the methylated or the unmethylated version of the DNA.
  • Preferred primers and primer sets for assessing the methylation status of the concerned gene by way of MSP will specifically hybridize to a converted sequence, or to its complement sequence.
  • Most preferred primers and primer sets are provided in Table 4 and are represented by SEQ ID NO. 1-10.
  • Sense primers comprise or consist essentially of SEQ ID NO. 1-5
  • antisense primers consist essentially of SEQ ID NO. 6-10.
  • the amplification products can be optionally hybridized to specific oligonucleotide probes which may also be specific for certain products.
  • oligonucleotide probes can be used which will hybridize to amplification products from both modified and non-modified DNA.
  • the present invention relates to a method for identifying, in a test sample, cervical cancer or its precursor, or predisposition to cervical cancer, said method comprising: contacting a at least a portion of the applicable gene or promoter region thereof, the gene selected from those listed in Table 1 , with bisulfite to convert unmethylated cytosines to uracils, and further washed with alkali; detecting the generated product by contacting the converted nucleic acid with oligonucleotide primers whose sequence discriminates between the bisulfite-treated methylated and unmethylated version of the converted nucleic acid; and identifying the test sample as comprising cells that are neoplastic, precursor to neoplastic, or predisposed to neoplasia, or as comprising nucleic acids from cells that are neoplastic, precursor to neoplastic, or predisposed to neoplasia.
  • the step of detecting the generated product i.e. after treatment with bisulfite
  • the step of detecting the generated product comprises amplification with at least one primer that hybridizes to a sequence comprising an unmodified methylated CpG dinucleotide motif but not to a sequence comprising a modified non-methylated CpG dinucleotide motif, thereby forming amplification products.
  • Modified and non-modified DNA can be distinguished with use of oligonucleotide probes which may also be specific for certain products. Such probes can be hybridized directly to modified DNA or to amplification products of modified DNA. Probes for assessing the methylation status of the concerned gene will specifically hybridize to the converted sequence but not to the corresponding non converted sequence. Probes are designed to anneal specifically with the converted sequence representing either the methylated or unmethylated version of the DNA. Preferred probes are provided in Table 4 and are those of SEQ ID NO. 1 1-15. Preferred probes anneal specifically with the converted sequence representing the methylated version of the DNA, or to the complement sequence thereof. Oligonucleotide probes can be labeled using detection systems known in the art.
  • fluorescent moieties include but are not limited to fluorescent moieties, radioisotope labeled moieties, bio luminescent moieties, luminescent moieties, chemiluminescent moieties, enzymes, substrates, receptors, or ligands.
  • methylated CpG dinucleotides utilizes the ability of the MBD domain of the McCP2 protein to selectively bind to methylated DNA sequences (Cross et al, 1994; Shiraishi et al, 1999). Restriction endonuclease digested genomic DNA is loaded onto expressed His-tagged methyl-CpG binding domain that is immobilized to a solid matrix and used for preparative column chromatography to isolate highly methylated DNA sequences. Variants of this method have been described and may be used in present methods of the invention.
  • Real time chemistry allows for the detection of PCR amplification during the early phases of the reactions, and makes quantitation of DNA and RNA easier and more precise.
  • a few variants of real-time PCR are well known. They include Taqman® (Roche Molecular Systems), Molecular Beacons®, Amplifluor® (Chemicon
  • the TaqMan® system and Molecular Beacon® system have separate probes labeled with a fluorophore and a fuorescence quencher.
  • the labeled probe in the form of a hairpin structure is linked to the primer.
  • Quantitation in real time format may be on an absolute basis, or it may be relative to a methylated DNA standard or relative to an unmethylated DNA standard.
  • the absolute copy number of the methylated marker gene can be determined; or the methylation status may be determined by using the ratio between the signal of the marker under investigation and the signal of a reference gene with a known methylation (e.g. ⁇ - actin), or by using the ratio between the methylated marker and the sum of the methylated and the non-methylated marker.
  • Real-Time PCR detects the accumulation of amplicon during the reaction, but alternatively end-point PCR fluorescence detection techniques may be used.
  • DNA methylation analysis has been performed successfully with a number of techniques which are also applicable in present methods of the invention. These include the MALDI-TOFF, MassARRAY (Ehrich, M. et al. 2005), MethyLight (Trinh B. et al. 2001), Quantitative Analysis of Methylated Alleles (Zeschnigk M. et al.
  • Oligonucleotide-based microarray systems (Gitan RS et al., 2006).
  • the number of genes whose modification is detected can vary: one, two, three, or four genes according to Table 1 can be tested. Detection of epigenetic modification of at least one, two, three, or four genes according to Table 1 can be used as an indication of cancer or pre-cancer or risk of developing cancer.
  • functionally relevant variants of each of the gene sequences may also be detected according to the methods of the invention. For example, the methylation status of a number of splice variants may be determined according to the methods of the invention.
  • Variant sequences preferably have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%>, or at least 99% nucleotide sequence identity with the nucleotide sequences in the database entries.
  • Computer programs for determining percentage nucleotide sequence identity are available in the art, including the Basic Local Alignment Search Tool (BLAST) available from the NCBI.
  • BLAST Basic Local Alignment Search Tool
  • the methods of the invention can be used in order to detect more than one gene of interest in the same reaction.
  • amplification of several nucleic acid targets can be performed in the same reaction mixture. This may be termed "multiplexing”. Multiplexing can also be utilized in the context of detecting both the gene of interest and a reference gene in the same reaction.
  • screening of cervical cancer refers to organized periodic procedures performed on groups of people for the purpose of detecting cervical cancer.
  • test for hr-HPV refers to testing for the presence of hr-HPV.
  • PCR based assays commercially available to measure hr-HPV copy number or viral load in clinical samples.
  • Many testing methods have been used to detect the presence of HPV in cervicovaginal specimens, including viral load quantification, Southern blot, polymerase chain reaction (PCR), ViraPap (Life
  • Hybrid Capture tube testing Hybrid Capture microtiter plate assays
  • FDA approved Hybrid Capture II assay Digene Corp.
  • CISH Digene® HPV Test (Qiagen)
  • AMPLICOR HPV Test Roche
  • HPV High-Risk Molecular Assay Third Wave Technologies
  • LINEAR ARRAY HPV Genotyping Test (Roche), I NO-LiPA HPV Genotyping
  • those hr-HPV types comprise, without being limited to, strains 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, and 69.
  • Preferred "high risk" HPV types are HPV 16 and HPV 18.
  • HPV16 testing refers to testing for the presence of hr-HPV type 16.
  • HPV 18 testing refers to testing for the presence of hr-HPV type 18.
  • the various methods allowing type-specific HPV testing are well known to the person skilled in the art and are applicable in the methods of present invention. For instance, testing for the presence of hr-HPV-16 may be accomplished by PCR amplification using primers specific for HPV type 16, which are known by the skilled in the art.
  • test sample for screening of cervical cancer will most of the time be obtained from a subject suspected of being tumorigenic or from a subject undergoing routine examination and not necessarily being suspected of having a disease.
  • sample is obtained from a subject undergoing treatment, or from patients being checked for recurrence of disease.
  • Testing can be performed diagnostically or in conjunction with a therapeutic regimen. Testing can be used to monitor efficacy of a therapeutic regimen, whether a chemotherapeutic agent or a biological agent, such as a polynucleotide. Epigenetic loss of function of at least one gene selected from the group consisting of genes according to Table 1 can be rescued by the use of DNA demethylating agents and/or DNA methyltransferase inhibitors. Testing can also be used to determine what therapeutic or preventive regimen to employ on a patient. Moreover, testing can be used to stratify patients into groups for testing agents and determining their efficacy on various groups of patients.
  • the methods of the present invention may be convenient to combine the methods of the present invention with established methods or markers for cervical cancer identification (Malinowski D, 2007), such as morphology-based detection methods, HPV methylation testing (Badal et al. 2004, Kalantari et al. 2004), KRAS and BRAF mutation detection (Kang et al. 2007), chromosomal amplification (Rao et al. 2004), protein expression (Keating et al. 2001), and HPV detection methods (Brink et al. 2007).
  • morphology-based detection methods such as morphology-based detection methods, HPV methylation testing (Badal et al. 2004, Kalantari et al. 2004), KRAS and BRAF mutation detection (Kang et al. 2007), chromosomal amplification (Rao et al. 2004), protein expression (Keating et al. 2001), and HPV detection methods (Brink et al. 2007).
  • HPV detection kits
  • the present invention further relates to a kit for identifying cervical cancer or its precursor, or predisposition to cervical cancer in a test sample comprising cervical cells or nucleic acids from cervical cells, said kit comprising:
  • At least one primer is selected from the primers with SEQ ID NO: 1-10.
  • the present invention further relates to a kit for identifying cervical cancer or its precursor, or predisposition to cervical cancer in a test sample comprising cervical cells or nucleic acids from cervical cells, said kit comprising at least one pair of oligonucleotide primers that specifically hybridize under amplification conditions to a region of the genes selected from EPB41L3; EPB41L3 and JAM3; EPB41L3 and TERT; EPB41L3 and C130RF18; EPB41L3, JAM3 and TERT; EPB41L3, JAM3 and C130RF18; EPB41L3, TERT and C130RF18 or EPB41L3, JAM3, TERT and C130RF18 wherein the region is within about 10 kb of said gene's transcription start site.
  • kits described above further comprise at least one
  • this at least one oligonucleotide probe is selected from those with SEQ ID NO: 11-15.
  • kits described above further comprise a DNA polymerase for amplifying DNA.
  • Kits according to the present invention are assemblages of reagents for testing methylation. They are typically in a package which contains all elements, optionally including instructions. The package may be divided so that components are not mixed until desired. Components may be in different physical states. For example, some components may be lyophilized and some in aqueous solution. Some may be frozen. Individual components may be separately packaged within the kit.
  • the kit may contain reagents, as described above for differentially modifying methylated and non- methylated cytosine residues.
  • the kit comprises both a forward and a reverse primer for a single gene or marker. If there is a sufficient region of complementarity, e.g., 12, 15, 18, or 20 nucleotides, then the primer may also comprise additional nucleotide residues that do not interfere with hybridization but may be useful for other manipulations. Exemplary of such other residues may be sites for restriction endonuclease cleavage, for ligand binding or for factor binding or linkers or repeats.
  • the oligonucleotide primers may or may not be such that they are specific for modified methylated residues.
  • the kit may optionally contain oligonucleotide probes.
  • the probes may be specific for sequences containing modified methylated residues or for sequences containing non-methylated residues.
  • the kit may optionally contain reagents for modifying methylated cytosine residues.
  • the kit may also contain components for performing amplification, such as a DNA polymerase and deoxyribonucleotides. Means of detection may also be provided in the kit, including detectable labels on primers or probes.
  • Kits may also contain reagents for detecting gene expression for one of the markers of the present invention. Such reagents may include probes, primers, or antibodies, for example. In the case of enzymes or ligands, substrates or binding partners may be used to assess the presence of the marker.
  • Kits may comprise 1, 2, 3, 4, 5, or more of the primers or primer pairs of the invention.
  • Kits that comprise probes may have them as separate molecules or covalently linked to a primer for amplifying the region to which the probes hybridize.
  • Other useful tools for performing the methods of the invention or associated testing, therapy, or calibration may also be included in the kits, including buffers, enzymes, gels, plates, detectable labels, vessels, etc.
  • the invention also employs or relies upon or utilizes oligonucleotide primers or probes to determine the methylation status of at least one gene or panel of genes selected from EPB41L3; EPB41L3 and JAM3; EPB41L3 and TERT; EPB41L3 and C130RF18; EPB41L3, JAM3 and TERT; EPB41L3, JAM3 and C130RF18; EPB41L3, TERT and C130RF18 or EPB41L3, JAM3, TERT and C130RF18.
  • Preferred probes and their sequences bind to at least one of the polynucleotide sequences listed in Table 1 or to the complement sequence thereof.
  • Preferred primers and probes are selected from the primers and probes comprising or consisting essentially of the nucleotide sequences set forth in Table 4, i.e. SEQ ID NO. 1-15.
  • the present invention also relates to an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO. 1-15.
  • the OpenArrayTM platform (Biotrove, Inc.) consists of 3072 through-holes loaded with 5 400nM of each different primer per hole. Assays were custom made; representing 424 primers of 213 cancer specific methylated genes, on average one gene was represented by 2 different primers. These genes were derived from previous studies by our group [12,16] and literature. For genes, see Tables 1 and 3, for primer pairs and probes, see Table 4. MSP was carried out in a total volume of 33 nL based on SYBR® Green I 0 chemistry in an Applied Biosystems 7900HT Sequence Detector System. Plates were cycled with the manufacturer protocol (www.biotrove.com). In the final step melting temperature (Tm) analysis was performed. Threshold cycle (Ct) were automatically calculated by the OpenArrayTM qPCR analysis software. The LightCycler® MSPs were based on SYBR® Green I chemistry, the total reaction volume was 10 ⁇ . 5 OpenArrayTM data analysis
  • the OpenArrayTM system 0 relates the presence of an amplification product to Ct and Tm. A sample was
  • Tm- interval was chosen such that cancer samples tended to be classified as
  • TmVar Tm variance
  • the top 20 of ranked methylation markers were validated on frozen tissue from 20 normal cervices and 27 cervical cancer patients by using LightCycler® MSP. These 47 specimens were selected from the specimens used for OpenArrayTM experiments. After LightCycler® MSP, methylation markers were ranked on the highest area under the curve by ROC analysis. The top 3 methylation markers were selected for further clinical validation. Quantitative Methlation Specific PCR (QMSP) on cervical scrapings
  • QMSP quantitative polymerase chain reaction
  • ROC analysis was performed for these 20 markers and in table 3 these 20 markers are ranked based on the area under the curve.
  • Some genes in table 3 are represented by 2 different primers, because on average 2 primers were used per gene on the
  • Hr-HPV Hr-HPV was detected in 47% of CINO, 68% of CINl, 72% of CIN2, 95% of CIN3 and 100% in micro -invasive cervical carcinoma (table 5).
  • Scenario analysis population based screening for detection cervical neoplasia Since the population attending for screening has a different prevalence of CIN2+ in comparison to our study population a scenario analysis was performed. This analysis provides information on the expected performance of the methylation test in a population based screening program. In table 7 the scenario analysis is shown.
  • HPV testing for population based screening programs of cervical neoplasia HPV testing for population based screening programs of cervical neoplasia.
  • Bazzoni G The JAM family of junctional adhesion molecules. Curr Opin Cell Biol 2003;15(5):525-530.
  • EPB41L3 protein 4. lB/Dal-1) as a target for treatment of advanced prostate cancer. Expert Opin Ther Targets 2008;12(7):845-853.

Abstract

La présente invention concerne des procédés et trousses pour identifier, diagnostiquer, pronostiquer et cribler un cancer du col de l'utérus. Ces procédés comprennent la détermination de l'état de méthylation ou des niveaux d'expression du gène EPB41L3, et de panels comprenant ledit gène.
PCT/EP2010/063968 2009-09-24 2010-09-22 Détection et pronostic du cancer du col de l'utérus WO2011036173A1 (fr)

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CN105177168A (zh) * 2015-10-27 2015-12-23 山东大学齐鲁医院 基于宫颈液基细胞学与dna甲基化筛查早期宫颈癌的试剂盒
CN105177164A (zh) * 2015-10-21 2015-12-23 山东大学齐鲁医院 一种用于宫颈癌早期筛查的分子标记和检测引物
CN105177163A (zh) * 2015-10-21 2015-12-23 山东大学齐鲁医院 一种用于宫颈癌早期筛查的试剂盒
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WO2016048138A1 (fr) 2014-09-22 2016-03-31 Rijksuniversiteit Groningen Biomarqueurs destinés au cancer du col de l'utérus
WO2018158589A1 (fr) * 2017-03-02 2018-09-07 Ucl Business Plc Procédés de diagnostic et de pronostic
WO2022057608A1 (fr) * 2020-09-18 2022-03-24 北京起源聚禾生物科技有限公司 Composition et kit de réactifs pour la détection précoce des lésions du col de l'utérus de haut grade et du cancer du col de l'utérus

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