WO2011036173A1 - Detection and prognosis of cervical cancer - Google Patents

Detection and prognosis of cervical cancer 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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PCT/EP2010/063968
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French (fr)
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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

The present invention relates to methods and kits for identifying, diagnosing, prognosing, and screening for cervical cancer. These methods include determining the methylation status or the expression levels of the EPB41L3 gene, and of panels comprising said gene.

Description

DETECTION AND PROGNOSIS OF CERVICAL CANCER
FIELD OF THE INVENTION The present invention relates to the area of cancer diagnostics and therapeutics. In particular, 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.
BACKGROUND TO THE INVENTION 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.
During the process of cervical cancer development, normal cervical cells gradually develop pre-cancerous changes that turn into cancer. 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]. 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.
colposcopy) and unnecessarily treatment with cryotherapy or loop electro surgical excision procedure, which permanently alters the cervix and have unknown
consequences on fertility and pregnancy. To improve early detection, the combination of HPV and PAP tests is now approved by the FDA for screening women 30 years of age and older. However, co-testing substantially increases the cost of screening. Recently preventive vaccines against hr-HPV-16 and hr-HPV-18 have been introduced in the Western world, which will reduce the incidence of cervical neoplasia significantly. However, these vaccines do not cover 100% of cervical cancers and it will take over decades before HPV vaccination affects cervical neoplasia incidence. Therefore, screening needs to be continued, while simultaneously efficiency of cytological screening in population based screening programs will be reduced by vaccination, due to a gradual decline of cervical neoplasia.
For all the above reasons, other markers are needed especially to improve the positive predictive value for population based screening of cervical neoplasia. 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]. 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 ( Oncomethy lo me Sciences S.A.) 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
comparable results in cervical tissue samples and cervical scrapings. 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. However, the finding of epigenetic inactivation in a specific tissue does not suggest a similar event for other tissues. For example, in the study performed by Agathanggelou et al. [25], RASSF1 A was methylated in majority of lung cancers but not in any of the cervical tumors tested.
WO2009/ 1 15615 (Oncomethylome Sciences S.A.) 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.
It is an object of the present invention to provide markers that allow improved detection of cervical cancer.
It is an object of the present invention to provide methylation markers or panels thereof for cervical cancer, more sensitive or more specific when compared to those known in the state of the art.
It is an object of the present invention to provide methylation markers for cervical cancer that present comparable results in cervical tissue samples and cervical scrapings. It is an object of the present invention to provide a validated methylation marker panel for triage of hr-HPV positive patients.
It is an object of the present invention to provide a method for screening of cervical cancer.
SUMMARY OF THE INVENTION 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. In one embodiment, in the method presented above, 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) providing a test sample comprising cervical cells or nucleic acids from cervical cells;
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;
d) if the gene or panel of genes in c) is methylated, refer the woman for colposcopy; e) if the gene or panel of genes in c) is unmethylated, refer the woman to a more frequent screening for the presence of hr-HPV. 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:
- a reagent that modifies non-methylated cytosine residues but not methylated
cytosine residues; and
- (a) at least one primer that hybridizes to a sequence comprising a modified non- methylated CpG dinucleotide motif but not to a sequence comprising an unmodified methylated CpG dinucleotide motif, thereby forming amplification products; or (b) 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; wherein the sequence is selected from those of the genes: 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. DETAILED DESCRIPTION OF THE INVENTION
We describe a novel high-throughput application for identifying new specific
(cervical) cancer methylation markers: the OpenArray™ (Biotrove, Inc) based MSP experiments. This methodology allows real time, 33 nL polymerase chain reactions on a 3072 holes microarray with the size of a microscope slide [14].
Using the aforementioned technique, we have identified 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
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.
Accordingly, 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.
In one embodiment, in the method presented above, 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.
In one embodiment, in the methods presented above, the additional genes are selected from JAM3, TERT, and C130RF18. In one embodiment, in the methods presented above, 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 screening protocols for the screening of woman for cervical cancer and the precursors thereof. Traditionally 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. The introduction of methylation markers consequently adds a new dimension to the screening for and treatment of cervical 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.
Accordingly, 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) providing a test sample comprising cervical cells or nucleic acids from cervical cells;
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;
d) if the gene or panel of genes in c) is methylated, refer the woman for colposcopy; e) if the gene or panel of genes in c) is unmethylated, refer the woman to a more frequent screening for the presence of hr-HPV.
The following 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).
Table 1
official official full NCBI Reference Sequences
symbol name Genomic mRNA and Protein(s)
NC_000018.9 Genome Reference NM_012307.2→NP_036439.2 erythrocyte
EPB41L3 Consortium Human Build 37 erythrocyte membrane protein membrane
(GRCh37), Primary Assembly band 4.1 -like 3
protein band 4.1 -like 3 NT_010859.14
NC_000011.9 Genome Reference
junctional Consortium Human Build 37 NM_032801.3→NP_116190.2
JAM3 adhesion (GRCh37), Primary Assembly junctional adhesion molecule 3 molecule 3 precursor
NT_033899.8
NM_198253.2→NP_937983.2 telomerase reverse transcriptase telomerase isoform 1
TERT reverse NG_009265.1 RefSeqGene
transcriptase NM_198255.2→NP_937986.1 telomerase reverse transcriptase isoform 2
NC_000013.10 Genome
chromosome Reference Consortium Human
13 open Build 37 (GRCh37), NM_025113.2→NP_079389.2
C13orfl8
reading frame Primary Assembly hypothetical protein LOC80183 18
NT_024524.14
The listed accession numbers above may be found in the publicly available gene database at http://www.ncbi.nlm.nih.gov.
The genomic molecules, transcripts, and protein products presented herein are not limited to the particular sequences referred to above, but also comprise variants thereof.
The term "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.
The term "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. Preferably, 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.
In one embodiment, in the methods of the present invention, 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.
The term "lavage" 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.
The term "self-sampling device" refers to any device suitable for the collection of a cervicovaginal sample. The term "lavage self-sampling device" refers to any device suitable for the collection of a cervicovaginal sample by irrigation of the
cervicovaginal tract as defined above, and which device can be operated by the subject herself. Examples of these lavage self-sampling devices are provided in patent publication numbers US20050020937, WO2006081621, WO2003066131,
WO1988005669, and EP502485, all incorporated herein by reference. A preferable lavage self-sampling device is the Pantarhei Screener Mermaid™.
The term "precursor", when used in the context of "cervical cancer", refers to those precancerous conditions of the cervix. In particular, when presented in relation to a cervical cancer cell, refers to that cell that is committed to a differentiation pathway, in particular any embryonic stem cell committed to a cervical cancer cell lineage. Examples of precursor cells encompass progenitor cells, stem cells, duct cells, lobules cells, and the like.
There are many systems in use in different parts of the world for classifying and naming precancerous conditions of the cervix, based on cytology and histology (see Table 2). The classification system of cervical intraepithelial neoplasia (CIN) is used in many countries for cytological reports, although it is more conveniently used for histological reports (results of microscopic examination of tissue samples). The Bethesda system, developed at the United States National Cancer Institute, is more appropriate for use in cytological reports (i.e. on microscopic examination of a smear). Table 2. Classification of precancerous conditions of the cervix recommended by the World Health Organization (WHO)
Figure imgf000010_0001
The term "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.
The term "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. Conversely, 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. Another means to determine 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. In one embodiment of the present invention, 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.
The term "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. Typically these reside near the transcription start site (TSS), for example, within about 10 kbp, within about 5 kbp, within about 3 kbp, within about 1 kbp, within about 750 bp, within about 500 bp, within about 200 bp, or within about 100 bp. Once a gene has been identified as the target of epigenetic modification in tumor cells, determination of reduced or enhanced expression can be used as an indicator of epigenetic modification.
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.
microarray technology, in situ hybridization, Northern blots). Messenger 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).
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
modification is assayed by detecting expression of mRNA of the EPB41L3 gene, of the other genes listed in Table 1 , or of any combination thereof.
In one embodiment of the present invention, 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. In one embodiment, the oligonucleotide primer is designed not to contain cytosines and amplifies modified and unmodified sequences. In some embodiments, 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. Alternatively, the amplification may be combined with restriction cutting by using methylation sensitive enzymes; only the methylated region is amplified in this case.
In one embodiment of the present invention, 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
transcription start site. In one embodiment, 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. Alternatively, the amplification may be combined with restriction cutting by using methylation sensitive enzymes; only the methylated region is amplified in this case.
Alternatively, 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. Alternatively, 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. In one embodiment of the present invention, 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. In a further embodiment, the chemical reagent comprises bisulfite ions. In a further embodiment, the method further comprises treating with alkali the bisulfite ion-contacted portion of the gene.
Other means for detection that are reliant on specific sequences can be used, including but not limited to electrophoresis, hybridization, amplification, quantitative
methylation-specific PCR (QMSP), sequencing, ligase chain reaction,
chromatography, mass spectrometry. Combinations of such techniques may also be used.
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).
One form of chromatography, high performance liquid chromatography, is used to separate components of a mixture based on a variety of chemical interactions between a substance being analyzed and a chromatography column. DNA is first treated with sodium bisulfite, which converts an unmethylated cytosine to uracil, while methylated cytosine residues remain unaffected. One may amplify the region containing potential methylation sites via PCR and separate the products via denaturing high performance liquid chromatography (DHPLC). 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. Similarly 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
sequentially added to the reaction mix in a predetermined order. If the nucleotide is complementary to the template base and thus incorporated, 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.
A variety of 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).
Sequence variation that reflects the methylation status at CpG dinucleotides in the original genomic DNA offers two approaches to PCR primer design. In the first approach, 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. In the second approach, the primers are designed to anneal specifically with either the methylated or unmethylated version of the converted sequence. If there is a sufficient region of complementarity, e.g., 12, 15, 18, or 20 nucleotides, to the target, then the primer may also contain 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.
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. Thus 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 yield
amplification products which can be readily observed. This method is known as MSP (Methylation Specific PCR; Patent Nos 5,786,146; 6,017,704; 6,200,756). 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. Alternatively, oligonucleotide probes can be used which will hybridize to amplification products from both modified and non-modified DNA.
Thus, 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. In one embodiment, the step of detecting the generated product (i.e. after treatment with bisulfite and alkali) comprises
amplification with at least one primer that hybridizes to a sequence comprising a modified non-methylated CpG dinucleotide motif but not to a sequence comprising an unmodified methylated CpG dinucleotide motif, thereby forming amplification products. In one embodiment, 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. These include but are not limited to fluorescent moieties, radioisotope labeled moieties, bio luminescent moieties, luminescent moieties, chemiluminescent moieties, enzymes, substrates, receptors, or ligands.
Another way for the identification of 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
International) and Scorpion® DzyNA®, Plexor™ (Promega) etc. The TaqMan® system and Molecular Beacon® system have separate probes labeled with a fluorophore and a fuorescence quencher. In the Scorpion® system 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. β- 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.
Confirming the presence of target DNA at the end point stage may indeed be sufficient and it can use the same approaches as widely used for real time PCR. 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. 2004), Enzymatic Regional Methylation Assay (Galm et al., 2002), HeavyMethyl (Cottrell, SE et al, 2004), QBSUPT, MS-SNuPE (Gonzalgo and Jones, 1997), MethylQuant (Thomassin H. et al. 2004), Quantitative PCR sequencing, and
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. Of course, as appropriate, the skilled person shall appreciate that 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.
It is possible for the methods of the invention to be used in order to detect more than one gene of interest in the same reaction. Through the use of several specific sets of primers, 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.
The phrase "screening of cervical cancer" refers to organized periodic procedures performed on groups of people for the purpose of detecting cervical cancer.
The phrase "assaying for hr-HPV" refers to testing for the presence of hr-HPV. There are various 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
Technologies), Hybrid Capture tube testing, Hybrid Capture microtiter plate assays, FDA approved Hybrid Capture II assay (Digene Corp.) with a probe cocktail for 13 carcinogenic types, 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
(Inno genetics), PapilloCheck (Greiner Bio-One GmbH), PreTect HPV-Proofer (Norchip), NucliSENS EasyQ HPV (BioMerieux), F-HPV typing™ (molGENTIX, S.L.). Such examples are not meant to be exhaustive, but rather exemplary. The so-called "high risk" HPV types are those strains of HPV more likely to lead to the development of cancer, while "low-risk" viruses rarely develop into cancer. The list of strains considered high risk is being adapted with the time and the increase in epidemiological knowledge. As such, 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.
The phrase "HPV16 testing" refers to testing for the presence of hr-HPV type 16. Similarly, "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.
The 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. Alternatively the 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.
To attain high rates of tumor detection, it 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). Several HPV detection kits are known in the art and commercially available, for example those listed above.
In another aspect, 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:
- a reagent that modifies non-methylated cytosine residues but not methylated
cytosine residues; and
- (a) at least one primer that hybridizes to a sequence comprising a modified non- methylated CpG dinucleotide motif but not to a sequence comprising an unmodified methylated CpG dinucleotide motif, thereby forming amplification products; or (b) 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; wherein the sequence is selected from those of the genes: 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.
In one embodiment, in the kit described above, 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.
In one embodiment, the kits described above further comprise at least one
oligonucleotide probe that hybridizes to the amplicon generated by the primer(s). Preferably, this at least one oligonucleotide probe is selected from those with SEQ ID NO: 11-15.
In one embodiment, the 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.
Typically 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. According to a further aspect, 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. Related to this, 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 above disclosure generally describes the present invention. All references disclosed herein are expressly incorporated by reference. A more complete
understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.
EXAMPLES
Patients and Methods Patients
From 1993 until today patients were asked to participate in various studies on biomarkers in cervical neoplasia during their initial visit at the outpatient clinic in the University Medical Center Groningen (UMCG). Frozen tissue and cervical scrapings were prospectively collected and stored in our tissue bank from cervical cancer patients, patient with normal cervices planned to undergo a hysterectomy for non- malignant reasons and from patients referred with an abnormal Pap smear. Clinico- pathological data were retrieved from patient files and stored in a large anonymized database. For all cervical cancer patients an examination under general anesthesia was planned for staging in accordance with the International Federation of Gynecology and Obstetrics (FIGO) criteria. All patients from whom material was obtained gave written informed consent. This study was approved by and followed the ethical guidelines of the Institutional Review Board of the UMCG. For the OpenArray™ experiments tissue specimens from 84 cervical cancer and 106 normal cervices from our tissue bank were randomly selected. Stage of cervical cancer patients was: 2 (2%) FIGO stage IA1, 53 (63%) FIGO stage IB, 16 (19%) FIGO stage IIA, 9 (11%) FIGO stage IIB, 2 (2%) FIGO stage IIIB, 2 (2%) FIGO stage IV. The histological classification of cervical cancer patients was: 71 (85%>) squamous cell carcinoma, 9 (11%) adenocarcinoma and 4 (4%) adenosquamous carcinoma. Median age of the cervical cancer patients was 50 years (range 25-91). All normal cervices contained epithelium without dysplasia. Median age of the women with normal cervices was 49 years (range 32-83). QMSP analysis was performed on cervical scrapings randomly selected from our tissue bank (74 cervical cancer patients, 69 normal cervices and 148 patients referred with an abnormal Pap smear). Stage of cervical cancers patients was: 38 (51%) FIGO stage IB, 10 (14%) FIGO stage IIA, 17 (23%) FIGO stage IIB, 7 (10%) FIGO stage IIIB and 2 (2%) FIGO stage IV. Histological classification of the cervical cancer patients was: 61 (82%) squamous cell carcinoma and 13 (18%) adenocarcinoma. Patients referred with an abnormal Pap smear (n=148) were divided in: 39 (26%>) without dysplasia (CINO), 39 (26%) CINl, 30 (20%) CIN2, 38 (26%) CIN3 and 2 (2%) micro-invasive cervical carcinoma. Median age of cervical cancer patients was 47 years (range 27-85) and controls 47 years (range 30-68). Median age of patients referred with an abnormal Pap smear was 35 years (range 20-65).
Sample collection and DNA isolation
Ten frozen tissue sections (10 μιη) were cut and for normal cervices macrodissection was performed to enrich for epithelial cells. Before and after cutting a hematoxylin and eosin slide was made per tumor and normal cervices. All slides were checked for proportion of cervical cancer cells or presence of normal epithelium. Cervical scrapings were collected using an Ayre's spatula and endocervical brush. The collected cervical cells were suspended in 5 ml of phosphate buffered saline (PBS: 6.4 mM Na2HP04; 1.5 mM KH2P04; 0.14 M NaCl; 2.7 mM KC1 (pH 7.2) and kept on ice until further processing. Of these 5 ml cell suspensions, 1 ml was used to make cytospins for cytomorpho logical assessment and 4 ml was centrifuged, washed and the cell pellet was snap-frozen in liquid nitrogen and stored at -80°C as described previously [8]. DNA isolation was performed using standard salt-chloroform extraction and isopropanol precipitation. Precipitated DNA was resuspended in 150 μΐ of Tris-EDTA buffer (10 mM Tris; 1 mM EDTA, pH 8.0). Genomic DNA was amplified in a multiplex PCR according to the BIOMED-2 protocol, to check the DNA quality [15].
OpenArray™ and LightCycler® experiments
The OpenArray™ 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 OpenArray™ qPCR analysis software. The LightCycler® MSPs were based on SYBR® Green I chemistry, the total reaction volume was 10 μΐ. 5 OpenArray™ data analysis
The first data analysis task was to rank candidate methylation markers (n=213) on the basis of methylation specific PCR experiments with OpenArray™. For this purpose, a novel biomarker scoring strategy based on a weighted combination of a marker's discriminating power and its robustness was developed. The OpenArray™ system 0 relates the presence of an amplification product to Ct and Tm. A sample was
considered to be methylated if Ct was less than 42 and Tm fell within an automatically derived marker-specific Tm interval. For a given candidate methylation marker, the Tm- interval was chosen such that cancer samples tended to be classified as
"methylated" and the controls as "unmethylated". More specifically, a 1 -sided 5 binomial test was used to estimate how unlikely it was to have at least the observed proportion of cancers within subset "methylated", given the frequency of cancers in the total set. For each methylation marker, we retained the Tm- interval with the lowest score for this test. One extra constraint was that the Tm- interval had to include the IVM samples (in vitro methylated). Next, we challenged the robustness of the Tm 0 interval by injecting increasing amounts of noise in the dataset to deteriorate less than a non-robust one. For that purpose, we took into account Tm variance (TmVar) as observed per methylation marker during quality control with IVM samples. We generated a series of datasets in which each Tm was replaced by a random value selected from the normal distribution with mean Tm and TmVar/NLev, where NLev is a noise level ranging from 10 to 1. For each of the 10 noise levels 1000 such datasets were generated for each marker, and each time the quality of the Tm- interval was recorded using the above binomial scoring function. Finally, we computed for each biomarker the average quality of the Tm- interval over all 10,001 datasets (including the original noise-free one) and used that average to sort biomarkers in ascending order. In the resulting ranking, methylation markers that combined discriminating power with robustness were sorted on top.
LightCycler® MSP and marker selection
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 OpenArray™ 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 was performed after bisulfite treatment on denatured genomic DNA. Bisulfite treatment was performed with the EZ DNA methylation kit according to
manufacturer's protocol (Zymogen, BaseClear, Leiden, the Netherlands). To correct for total DNA input, the housekeeping gene β-actin was used as a reference. QMSP was carried out in a total volume of 20 μΐ in 384 well plates in an Applied Biosystems 7900 Sequence Detector (Applied Biosystems, Nieuwekerk a/d IJsel, the Netherlands). Each sample was analyzed in triplicate. The final reaction mixture consisted of 300 nM of each primer, 200 nM probe, lx QuantiTect Probe PCR Kit (Qiagen, Leiden, the Netherlands) and 50 ng of bisulfite converted genomic DNA. As a positive control, serial dilutions of genomic leukocyte DNA, in vitro methylated with the CpG methyltransferase(M &s I) (New England Bio labs. Inc., Beverly, MA), were used in each experiment. A DNA sample was considered to be methylated if an exponential curve was visible with a Ct-value below 50 and DNA input of at least 225 pg β-actin. All amplification curves were visualized and scored without knowledge of the clinical data. The previous identified methylation marker C130RF18 was also tested on cervical scrapings, because this marker showed no methylation in cervical scrapings from CINO and CINl patients.
HPV detection and typing In all samples, presence of hr-HPV was analyzed by PCR using HPV 16 and HPV 18 specific primers. For all HPV 16 and HPV 18 negative cases a general primer- mediated PCR using primer set GP5+/6+ was performed, with subsequent nucleotide sequence analysis, as described previously [8]. Statistical analysis
All analyses were carried out using the SPSS software package (SPSS 16.0, Chicago, IL, USA). Difference in detection rates between normal scrapings, cervical cancers scrapings and CIN scrapings by methylation markers were analyzed using the χ2 test. Diagnostic performance for methylation markers and hr-HPV DNA testing was expressed in sensitivity and specificity with a cut-off for CIN2 or higher (CIN2+) or CIN3 or higher (CIN3+), respectively. Observed differences with a P value <0.05 were considered statistically significant.
Scenario analysis for population based screening program
Scenario analysis was performed in a virtual population of 100.000 women. The assumptions were based on two population based studies concerning hr-HPV testing and cytomorpho logical assessment [3,4]. In this population a total number of 1.100 CIN2+ patients were presumed, divided in 363 CIN2, 704 CIN3 and 33 cervical cancers. Hr-HPV testing was supposed to have a sensitivity of 95% and specificity of 94%. For conventional Pap smear as a triage test after hr-HPV testing, sensitivity for CIN2/CIN3 was estimated to be 70%> and for cervical cancer 80%>. The specificity of conventional Pap smear for CIN2+ after hr-HPV testing was estimated to be 82%. An analysis was performed for 2 scenarios: 1) primary hr-HPV testing followed by Pap smear, 2) primary hr-HPV testing followed by the methylation test. The scenario analysis was performed for one screening round without follow-up taken into account. Results
OpenArray™ results and methylation marker selection
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
OpenArray™. The first 3 markers; JAM 3, EPB41L3, and TERT were selected for further clinical validation (sequences in table 4). QMSP and hr-HPV on cervical scrapings from cervical cancer patients and normal cervices
QMSP was performed on 143 selected cervical scrapings (74 cervical cancer patients and 69 normal cervices). Methylation markers were positive in cervical cancer 5 scrapings in 83% to 90%> and in normal cervices only in 5% to 14% (p<0.0001, see table 5). Our methylation panel (J AM 3, EPB41L3, TERT and C130RF18) detected 94% of cervical cancers. Hr-HPV was detected in 88% of cervical cancer scrapings and 3% of normal cervical scrapings (table 5).
QMSP and hr-HPV on cervical scrapings from CIN patients
10 In total 148 cervical scrapings from patients referred with an abnormal Pap smear were included in this study. Due to low DNA input 5 patients were excluded from further analysis. Table 5 summarizes the results of QMSP for the 4 genes separately and our methylation panels. Methylation of EPB41L3 and JAM3 showed the highest detection level (65-68%) in CIN3 patients, while the methylation panel of 4 markers detected
15 81% of CIN3 patients. Methylation markers separately or the combination of 4
markers were positive in discriminative between CINl or lower and CIN2+ patients (pO.0001). 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). The results of QMSP for the 4 genes separately and the combination panels were also analyzed in hr-
20 HPV positive patients (Table 5). The detection level of the panel of 4 markers
increased to 83% in CIN3 hr-HPV positive patients.
Diagnostic performance of methylation markers and hr-HPV
In table 6 the diagnostic performance of methylation markers are shown. The sensitivity of methylation markers separately and as a panel for CIN2+ varies between
25 37% and 65% and for CIN3+ between 54% and 82%. The specificity of methylation markers separately and as a panel varies between 79% and 100%), depending on definition. Sensitivity of hr-HPV test was 85% for CIN2+ with a low specificity 43%- 53%. The diagnostic performance of the methylation markers separately and as a panel were also analyzed in hr-HPV positive patients. The sensitivity of our panel
30 increased to 71% for CIN2+ patients and 84% for CIN3+ patients.
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.
Overall the detection of CIN2+ was almost equal between both scenarios (p=0.934), although detection of CIN3 and cervical cancers was higher for hr-HPV testing in combination with our methylation panel (p=0.021). The percentage correct referrals were higher in the methylation test scenario (p<0.001) and there were less patient- doctor contacts (p<0.001). Discussion
In this study OpenArray™ technology in combination with LightCycler® MSP experiments and QMSP identified three new methylation markers {JAMS, EPB41L3 and TERT), highly specific for detection of cervical neoplasia. Together with
C130RF18, previously identified by our group, these genes were incorporated in a methylation panel that had a better diagnostic performance as a triage test after hr-HPV testing, than currently widely applied liquid based cytology.
Our scenario analysis estimated the strategy of combining hr-HPV testing with the methylation test. This screening approach might result in a higher detection of CIN3 and cervical cancers and a higher positive predictive value in a population based cohort. An advantage of the methylation test is that it could be performed on the same liquid based sample as used for hr-HPV testing. The methylation test seems also to be very interesting in the context of self-sampling. Since self-sampling is suitable for hr- HPV testing although less appropriate for cytological examination, the methylation test could be an alternative in this approach. Although, the methylation test should be examined in a large population based cohort, this shed a new light upon triage after hr-
HPV testing for population based screening programs of cervical neoplasia.
Since methylation of the promoter region of the genes tested in our study are so specific for cervical cancer and increasing with the severity of the lesion, it would be interesting to know the function of these genes in cervical carcinogenesis. JAM3 belongs to the family of junctional adhesion molecules, but how these molecules are exactly involved in carcinogenesis is not known so far [17]. Expression of TERT results in telomerase activity and telomerase is observed in particularly 79% of cervical cancers patients [18]. Therefore, methylation of the gene promoter of TERT seems to be a contradiction in the concept of gene silencing due to methylation. However, it has been shown that binding of the CTCF protein to the first exon of the promoter region of TERT resulted in repression and methylation of the CTCF binding site resulted in expression of TERT [19]. The EPB41L3 gene, also known as DAL-1, is a tumor suppressor gene [20], therefore loss of EPB41L3 might be involved in tumor progression [21]. C130RF18 is an open reading frame and currently we are investigating the role of C130RF18 in cervical carcinogenesis.
Interestingly, the markers SLITI, SLIT2, SOXI, LMXIA, WTl and TFPI2 ranked in the top 20 after OpenArray™ MSP experiments were also identified in other studies as discriminative methylated genes between cervical cancer and normal specimens
[10,13,22,23]. This finding confirmed the accuracy of OpenArray™ data analysis by a novel biomarker ranking strategy based on discriminating power and robustness.
In conclusion, we showed the robustness of a methylation test in detection of cervical neoplasia. This methylation test looks promising after hr-HPV testing in a population based screening program. Table 3. LightCycler MSP experiments
Figure imgf000030_0001
*AUC=area under the curve (ROC analysis) Table 4. Primer and probe sequences of the methylation markers
Figure imgf000031_0001
Figure imgf000031_0002
Figure imgf000031_0003
Table 5. Methylation markers and hr-HPV positive in cervical scrapings
Gene Cancer Normal CIN O CIN 1 CIN 2 CIN 3 Mi cancer
50/69 2/59 0/38 0/37 4/29 21/37 2/2
C130RF18
(73%) (3%) (0%) (0%) (14%) (57%) (100%)
57/69 3/59 2/38 6/37 7/29 24/37 2/2
JAM3
(83%) (5%) (5%) (16%) (24%) (65%) (100%)
62/69 5/59 2/38 6/37 4/29 19/37 2/2
TERT
(90%) (8%) (5%) (16%) (14%) (51%) (100%)
57/69 8/59 2/38 6/37 8/29 25/37 1/2
EPB41L3
(83%) (14%) (5%) (16%) (28%) (68%) (50%)
62/69 5/59 2/38 6/37 8/29 27/37 2/2
C13/JAM3
(90%) (9%) (5%) (16%) (28%) (73%) (100%)
64/69 7/59 2/38 6/37 5/29 25/37 2/2
C13/TERT
(93%) (12%) (5%) (16%) (17%) (68%) (100%) 62/69 10/59 2/38 6/37 8/29 29/37 2/2
C13/EPB
(90%) (17%) (5%) (16%) (28%) (78%) (100%)
65/69 6/59 4/38 9/37 9/29 25/37 2/2
JAM3/TERT
(94%) (10%) (11%) (24%) (31%) (68%) (100%)
61/69 10/59 4/38 10/37 12/29 26/37 2/2
JAM3/EPB
(88%) (17%) (11%) (27%) (41%) (70%) (100%)
65/69 12/59 3/38 9/37 8/29 27/37 2/2
TERT/EPB
(94%) (20%) (8%) (24%) (28%) (73%) (100%)
65/69 8/59 4/38 9/37 9/29 28/37 2/2
C13/JAM3/TERT
(94%) (14%) (11%) (24%) (31%) (76%) (100%)
63/69 12/59 4/38 10/37 12/29 29/37 2/2
C13/JAM3/EPB
(91%) (20%) (11%) (27%) (41%) (78%) (100%)
65/69 14/59 3/38 9/37 8/29 30/37 2/2
C13/TERT/EPB
(94%) (24%) (8%) (24%) (28%) (81%) (100%)
65/69 13/59 5/38 11/37 12/29 27/37 2/2
JAM3/TERT/EPB
(94%) (22%) (13%) (30%) (41%) (73%) (100%)
65/69 15/59 5/38 11/37 12/29 30/37 2/2
C13/JAM3/TERT/EPB
(94%) (25%) (13%) (30%) (41%) (81%) (100%)
61/69 2/59 18/38 25/37 21/29 35/37 2/2
HPV
(88%) (3%) (47%) (68%) (72%) (95%) (100%)
Only hr-HPV positive patients
45/61 0/2 0/18 0/25 2/21 21/35 2/2
C130RF18
(74%) (0%) (0%) (0%) (10%) (60%) (100%)
50/61 0/2 1/18 5/25 5/21 24/35 2/2
JAM3
(82%) (0%) (6%) (20%) (24%) (69%) (100%)
54/61 0/2 1/18 4/25 3/21 18/35 2/2
TERT
(89%) (0%) (6%) (16%) (14%) (51%) (100%)
51/61 1/2 0/18 5/25 6/21 25/35 2/2
EPB41L3
(84%) (50%) (0%) (20%) (29%) (71%) (100%)
55/61 0/2 1/18 5/25 6/21 27/35 2/2
C13/JAM3
(90%) (0%) (6%) (20%) (29%) (77%) (100%)
56/61 0/2 1/18 4/25 3/21 24/35 2/2
C13/TERT
(92%) (0%) (6%) (16%) (14%) (69%) (100%)
56/61 1/2 0/18 5/25 6/21 29/35 2/2
C13/EPB
(92%) (50%) (0%) (20%) (29%) (83%) (100%)
57/61 0/2 2/18 7/25 7/21 24/35 2/2
JAM3/TERT
(93%) (0%) (11%) (28%) (33%) (69%) (100%)
54/61 1/2 1/18 8/25 10/21 26/35 2/2
JAM3/EPB
(89%) (50%) (6%) (32%) (48%) (74%) (100%)
57/61 1/2 1/18 6/25 6/21 26/35 2/2
TERT/EPB
(93%) (50%) (6%) (24%) (29%) (74%) (100%)
57/61 0/2 2/18 7/25 7/21 27/35 2/2
C13/JAM3/TERT
(93%) (0%) (11%) (28%) (33%) (77%) (100%)
56/61 1/2 1/18 8/25 10/21 29/35 2/2
C13/JAM3/EPB
(92%) (50%) (6%) (32%) (48%) (83%) (100%)
57/61 1/2 1/18 0625 6/21 29/35 2/2
C13/TERT/EPB
(93%) (50%) (6%) (24%) (29%) (83%) (100%)
57/61 1/2 2/18 8/25 10/21 26/35 2/2
JAM3/TERT/EPB
(93%) (50%) (11%) (32%) (48%) (74%) (100%)
57/61 1/2 2/18 8/25 10/21 29/35 2/2
C13/JAM3/TERT/EPB
(93%) (50%) (11%) (32%) (48%) (83%) (100%) Table 6: Diagnostic performance for patients referred with an abnormal Pap smear.
Marker Sensitivity Specificity
CIN2+ CIN3 + CIN O CIN 0/1
40% 59% 100% 100%
C130RF18
(27/68) (23/39) (38/38) (75/75)
49% 67% 95% 89%
JAM3
(33/68) (26/39) (36/38) (67/75)
37% 54% 95% 89%
TERT
(25/68) (21/39) (36/38) (67/75)
50% 67% 95% 89%
EPB41L3
(34/68) (26/39) (36/38) (67/75)
54% 74% 95% 89%
C13/JAM3
(37/68) (29/39) (36/38) (67/75)
47% 69% 95% 89%
C13/TERT
(32/68) (27/39) (36/38) (67/75)
57% 74% 95% 89%
C13/EPB
(39/68) (31/39) (36/38) (67/75)
53% 69% 89% 83%
JAM3/TERT
(36/68) (27/39) (34/38) (62/75)
59% 72% 89% 81%
JAM3/EPB
(40/68) (28/39) (34/38) (61/75)
54% 74% 92% 84%
TERT/EPB
(37/68) (29/39) (35/38) (63/75)
57% 77% 89% 83%
C13/JAM3/TERT
(39/68) (30/39) (34/38) (62/75)
63% 79% 89% 81%
C13/JAM3/EPB
(43/68) (31/39) (34/38) (61/75)
59% 82% 92% 84%
C13/TERT/EPB
(40/68) (32/39) (35/38) (63/75)
60% 74% 87% 79%
JAM3/TERT/EPB
(41/68) (29/39) (33/38) (59/75)
65% 82% 87% 79%
C13/JAM3/TERT/EPB
(44/68) (32/39) (33/38) (59/75)
Hr-HPV 85% 95% 53% 43%
(58/68) (37/39) (20/38) (32/75)
Only hr-HPV positive patients
43% 62% 100% 100%
C130RF18
(25/58) (23/37) (18/18) (43/43)
53% 70% 94% 86%
JAM3
(31/58) (26/37) (17/18) (37/43)
40% 54% 94% 88%
TERT
(23/58) (20/37) (17/18) (38/43)
55% 70% 100% 88%
EPB41L3
(32/58) (26/37) (18/18) (38/43)
60% 78% 94% 86%
C13/JAM3
(35/58) (29/37) (17/18) (37/43)
50% 70% 94% 88%
C13/TERT
(29/58) (26/37) (17/18) (38/43)
64% 84% 100% 88%
C13/EPB
(37/58) (31/37) (18/18) (38/43)
57% 70% 89% 79%
JAM3/TERT
(33/58) (26/37) (16/18) (34/43) 66% 76% 94% 79%
JAM3/EPB
(38/58) (28/37) (17/18) (34/43)
59% 76% 94% 84%
TERT/EPB
(34/58) (28/37) (17/18) (36/43)
62% 78% 89% 79%
C13/JAM3/TERT
(36/58) (29/37) (16/18) (34/43)
71% 84% 94% 79%
C13/JAM3/EPB
(41/58) (31/37) (17/18) (34/43)
64% 84% 94% 84%
C13/TERT/EPB
(37/58) (31/37) (17/18) (36/43)
66% 76% 89% 77%
JAM3/TERT/EPB
(38/58) (28/37) (16/18) (33/43)
C13/JAM3/TERT/EPB 71% 84% 89% 77%
(41/58) (31/37) (16/18) (33/43)
mi-Ca=micro-invasive carcinoma,
Table 7. Scenario analysis population based screening program
Figure imgf000034_0001
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Claims

1. 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.
2. The method according to claim 1, wherein 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.
3. The method according to claim 2, wherein the additional genes are selected from JAM3, TERT, and C130RF18.
4. The method according to claim 2 or 3 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.
5. 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) providing a test sample comprising cervical cells or nucleic acids from cervical cells;
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;
d) if the gene or panel of genes in c) is methylated, refer the woman for
colposcopy; e) if the gene or panel of genes in c) is unmethylated, refer the woman to a more frequent screening for the presence of hr-HPV.
6. The method according to any one of claims 1-5, wherein the test sample comprises squamous cells, nucleic acids from squamous cells, adenocarcinoma cells, nucleic
5 acids from adenocarcinoma cells, adenosquamous carcinoma cells, nucleic acids from adenosquamous carcinoma cells, epithelium without dysplasia, or any combination thereof.
7. The methods according to any one of claims 1-6, wherein the test sample is a
cervical scraping.
10 8. The methods according to any one of claims 1-6, wherein the test sample is
collected with a lavage self-sampling device.
9. The method according to any one of claims 1-8, wherein epigenetic modification is assayed by detecting expression of mR A of the applicable gene.
10. The method according to any one of claims 1-8, wherein epigenetic modification 15 is assayed by detecting methylation of a CpG dinucleotide motif in the gene, or a promoter region thereof.
11. The method according to claim 10, wherein 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
20 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.
12. The method according to claim 11, wherein the chemical reagent comprises
bisulfite ions.
25 13. The method according to claim 12, further comprising treating with alkali the bisulfite ion-contacted portion of the gene.
14. The method according to any one of claims 11-13, wherein the product is detected by a method selected from the group consisting of electrophoresis, hybridization, amplification, quantitative methylation-specific PCR (QMSP), sequencing, ligase
30 chain reaction, chromatography, mass spectrometry, and any combination thereof.
15. The method according to any one of claims 11-13, wherein the step of detecting a product comprises amplification with at least one primer that hybridizes to a sequence comprising a modified non-methylated CpG dinucleotide motif but not to a sequence comprising an unmodified methylated CpG dinucleotide motif, thereby forming amplification products.
16. The method according to any one of claims 11-13, wherein the step of detecting a 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.
17. The method according to any one of claims 1-8, wherein 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.
18. The method according to any one of claims 1-8, wherein 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 transcription start site.
19. 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:
- a reagent that modifies non-methylated cytosine residues but not methylated cytosine residues; and
- (a) at least one primer that hybridizes to a sequence comprising a modified non- methylated CpG dinucleotide motif but not to a sequence comprising an unmodified methylated CpG dinucleotide motif, thereby forming amplification products; or (b) 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; wherein the sequence is selected from those of the genes: 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.
20. The kit according to claim 19, wherein the at least one primer is selected from the primers with SEQ ID NO: 1-10.
5 21. 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 10 C130RF18; EPB41L3, JAM3, and TERT; EPB41L3, JAM3, and C130RF18; EPB41L3, TERT, and C130RF18; and EPB41L3, JAM3, TERT, and C130RF18; wherein the region is within about 10 kb of said gene's transcription start site.
22. The kit according to any one of claims 19-21, further comprising at least one oligonucleotide probe that hybridizes to the amplicon generated by the primer(s).
15 23. The kit according to claim 22, wherein the probe is selected from those with SEQ ID NO: 11-15.
24. The kit according to any one of claims 19-23, further comprising a DNA
polymerase for amplifying DNA.
25. An isolated polynucleotide comprising a nucleotide sequence selected from the 20 group consisting of SEQ ID NO : 1 - 15.
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CN105177163A (en) * 2015-10-21 2015-12-23 山东大学齐鲁医院 Kit for early screening cervical cancer
CN105177164A (en) * 2015-10-21 2015-12-23 山东大学齐鲁医院 Molecular marker for early screening cervical cancer and detecting primers
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