WO2015107430A2 - Méthodes et biomarqueurs pour la détection et le pronostic du cancer du col de l'utérus - Google Patents

Méthodes et biomarqueurs pour la détection et le pronostic du cancer du col de l'utérus Download PDF

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WO2015107430A2
WO2015107430A2 PCT/IB2015/000567 IB2015000567W WO2015107430A2 WO 2015107430 A2 WO2015107430 A2 WO 2015107430A2 IB 2015000567 W IB2015000567 W IB 2015000567W WO 2015107430 A2 WO2015107430 A2 WO 2015107430A2
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methylation
cervical cancer
genes
subject
stc2
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PCT/IB2015/000567
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WO2015107430A3 (fr
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Heidi Lyng
Cathinka Halle JULIN
Malin Lando
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Oslo Universitetssykehus Hf
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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
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • 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
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to methods and biomarkers for detection of cervical cancer in biological samples, and in particular to methylation status markers associated with aggressiveness of cervical cancer.
  • Cervical cancer is one of the most common malignancies in females worldwide, and is the world's second highest cause of female cancer mortality. Improvement in the handling of this disease will thus affect a large number of women.
  • a promising and needed strategy for treatment improvement is a prognostic test to identify patients at high risk of failure. This allows for a reduced radiation dose to the low- risk patients and a higher cure probability of the high-risk patients, and be of benefit with respect to increased survival, reduction in side effects, increased life quality, and be a considerable saving for the community due to reduced need for rehabilitation after therapy.
  • the present invention relates to methods and biomarkers for detection of cervical cancer in biological samples, and in particular to methylation status markers associated with aggressiveness of cervical cancer.
  • the present invention provides methods for predicting a predisposition to cervical cancer in a subject, diagnosing a cervical cancer in a subject, predicting the likelihood of recurrence of cervical cancer in a subject, providing a prognosis for a subject with cervical cancer, or selecting a subject with cervical cancer for treatment with a particular therapy, comprising: a) obtaining DNA from a biological sample of the subject; and b) contacting the DNA with one or more methylation specific detection reagents to determine the level, presence, or frequency of methylation of a nucleic acid polymer corresponding to one or more genes selected from, for example, AK2, AK3L1, ALDOA, B3GNT4, CLK3, C14orf2, C4orfl, C20orf20, DDIT3, FGF11, GAPDH, ISG15, KCTD11, P4HA2, PFKFB4, PVR, PYGL, RHOC, RPL36A, S100A2, SCARB1, SH3GL
  • the one or more genes is two or more, three or more, four or more, five or more, or all of the genes.
  • the level or frequency of methylation of a nucleic acid polymer is compared to a reference level or frequency of methylation.
  • the method further comprises comparing the level, presence, or frequency of methylation of the nucleic acid polymer with a reference level, presence, or frequency of methylation, wherein an altered level, presence, or frequency of methylation for the patient relative to the reference provides an indication of, for example, predicting a predisposition to cervical cancer in a subject, diagnosing a cervical cancer in a subject, predicting the likelihood of recurrence of cervical cancer in a subject, providing a prognosis for a subject with cervical cancer, or selecting a subject with cervical cancer for treatment with a particular therapy.
  • the method further comprises the step of determining a treatment course of action based on the level, presence, or frequency of methylation.
  • the treatment is chemotherapy or radiation.
  • the method further comprises the step of administering the treatment.
  • the nucleic acid comprises a CpG island, a CpG island shore, or a CpG island shelf.
  • the CpG island, shore, or shelf is present in a coding region or a regulatory region such as, for example, a promoter.
  • the determining of the level of altered methylation of a nucleic acid polymer comprises determining the methylation frequency of the CpG island, shore, or shelf.
  • the determining of the level of a nucleic acid polymer with altered methylation is achieved by a technique selected from, for example, methylation-specific PCR, quantitative methylation- specific PCR, methylation-sensitive DNA restriction enzyme analysis, methylation - insensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, or bisulfite genomic sequencing PCR.
  • the methylation specific detection reagent is, for example, a pair of amplification primers that specifically hybridizes to said gene, an amplification primer that specifically hybridizes to said gene, a restriction enzyme, or sodium bisulfite, although the present invention is not limited to a particular methylation specific detection reagent.
  • the reagent e.g., probe
  • the biological sample is a tissue sample, a cell sample, a blood sample, a urine sample, or other biological fluid.
  • the present invention provides methods for predicting a predisposition to cervical cancer in a subject, diagnosing a cervical cancer in a subject, predicting the likelihood of recurrence of cervical cancer in a subject, providing a prognosis for a subject with cervical cancer, or selecting a subject with cervical cancer for treatment with a particular therapy, comprising: a) obtaining DNA from a biological sample of the subject; and b) contacting the DNA with one or more methylation specific detection reagents to determine the level, presence, or frequency of methylation of a nucleic acid polymer corresponding to one or more genes (e.g., two or more, three or more, four or more, five or more, or all of the genes) selected from, for example AK2, AK3L1, ALDOA, B3GNT4, C4orf2, FGFll, ISG15, KCTDll, P4HA2, PFKFB4, PVR, PYGL, RHOC, RPL36A, S100A2, SCARB
  • a methylation specific nucleic acid detection sequence corresponding to one or more genes selected from, for example, AK2, AK3L1, ALDOA, B3GNT4, CLK3, C14orf2, C4orfl, C20orf20, DDIT3, FGFll, GAPDH, ISG15, KCTDll, P4HA2, PFKFB4, PVR, PYGL, RHOC, RPL36A, S100A2, SCARB1, SH3GL3, STC2, TRAPPCl, or UPK1A for detecting or characterizing cervical cancer in a subject.
  • genes selected from, for example, AK2, AK3L1, ALDOA, B3GNT4, CLK3, C14orf2, C4orfl, C20orf20, DDIT3, FGFll, GAPDH, ISG15, KCTDll, P4HA2, PFKFB4, PVR, PYGL, RHOC, RPL36A, S100A2, SCARB1, SH3GL3, STC2, TR
  • an altered level, presence, or frequency of methylation for a patient relative to a reference provides an indication selected from, for example, predicting a predisposition to cervical cancer in a subject, diagnosing a cervical cancer in a subject, predicting the likelihood of recurrence of cervical cancer in a subject, providing a prognosis for a subject with cervical cancer, or selecting a subject with cervical cancer for treatment with a particular therapy.
  • a methylation specific nucleic acid detection sequence corresponding to one or more genes selected from, for example, AK2, AK3L1, ALDOA, B3GNT4, C4orf2, FGFll, ISG15, KCTDll, P4HA2, PFKFB4, PVR, PYGL, RHOC, RPL36A, S100A2, SCARB1, SH3GL3, STC2,or TRAPPCl for detecting or characterizing cervical cancer in a subject.
  • kits for detecting the presence of a cervical cancer in a mammal comprising methylation specific detection reagents useful, sufficient, or necessary for detecting and/or characterizing level, presence, or frequency of methylation of one or more genes selected from, for example, AK2, AK3L1, ALDOA, B3GNT4, CLK3, C14orf2, C4orfi, C20orf20, DDIT3, FGFll, GAPDH, ISG15, KCTDll, P4HA2, PFKFB4, PVR, PYGL, RHOC, RPL36A, S100A2, SCARB1, SH3GL3, STC2, TRAPPCl, or UPK1A.
  • kits for detecting the presence of a cervical cancer in a mammal comprising methylation specific detection reagents useful, sufficient, or necessary for detecting and/or characterizing level, presence, or frequency of methylation of one or more genes selected from, for example, AK2, AK3L1, ALDOA, B3GNT4, C4orf2, FGFll, ISG15, KCTDll, P4HA2, PFKFB4, PVR, PYGL, RHOC, RPL36A, S100A2, SCARBl, SH3GL3, STC2,ox TRAPPCI.
  • Yet other embodiments provide a system comprising a computer readable medium comprising instructions for utilizing information on the level, presence, or frequency of methylation of one or more genes selected from, for example, AK2, AK3L1, ALDOA, B3GNT4, CLK3, C14orf2, C4orfl, C20orf20, DDIT3, FGFll, GAPDH, ISG15, KCTDll, P4HA2, PFKFB4, PVR, PYGL, RHOC, RPL36A, S100A2, SCARBl, SH3GL3, STC2, TRAPPCI, or UPK1A to provide an indication selected from, for example, an indication of predicting a predisposition to cervical cancer in a subject, diagnosing a cervical cancer in a subject, predicting the likelihood of recurrence of cervical cancer in a subject, providing a prognosis for a subject with cervical cancer, or selecting a subject with cervical cancer for treatment with a particular therapy.
  • a system comprising a computer readable medium comprising instructions for utilizing information on the level, presence, or frequency of methylation of one or more genes selected from, for example, AK2, AK3L1, ALDOA, B3GNT4, C4orf2, FGFll, ISG15, KCTDll, P4HA2, PFKFB4, PVR, PYGL, RHOC, RPL36A, S100A2, SCARBl, SH3GL3, STC2,or TRAPPCI to provide an indication selected from, for example, an indication of predicting a predisposition to cervical cancer in a subject, diagnosing a cervical cancer in a subject, predicting the likelihood of recurrence of cervical cancer in a subject, providing a prognosis for a subject with cervical cancer, or selecting a subject with cervical cancer for treatment with a particular therapy.
  • Still further embodiments provide a complex comprising one or more genes selected from the group consisting of AK2, AK3L1, ALDOA, B3GNT4, CLK3, C14orf2, C4orfi, C20orf20, DDIT3, FGFll, GAPDH, ISG15, KCTDll, P4HA2, PFKFB4, PVR, PYGL, RHOC, RPL36A, S100A2, SCARBl, SH3GL3, STC2, TRAPPCI, or UPK1A , wherein each gene is complexed to a methylation status informative reagent.
  • a complex comprising one or more genes selected from the group consisting of AK2, AK3L1, ALDOA, B3GNT4, C4orf2, FGFll, ISG15, KCTDll, P4HA2, PFKFB4, PVR, PYGL, RHOC, RPL36A, S100A2, SCARBl, SH3GL3, STC2,or TRAPPCI , wherein each gene is complexed to a methylation status informative reagent.
  • Fig. 1 shows an overview of the 151 methylation probes which significantly
  • Fig. 2 shows Kaplan Meier curves for progression-free survival of patients with low (below median) and high (above median) levels of STC2 (A), PVR (B), and RPL36A (C) methylation, respectively.
  • Fig. 3 shows Kaplan Meier curves for progression-free survival of patients with low (below median) and high (above median) levels of the methylation score based on 22 probes in 158 cervical cancer patients. P-value from log-rank test is indicated.
  • Fig. 4 shows an overview of 84 methylation probes.
  • sensitivity is defined as a statistical measure of
  • performance of an assay e.g., method, test
  • performance of an assay calculated by dividing the number of true positives by the sum of the true positives and the false negatives.
  • performance of an assay e.g., method, test
  • performance of an assay calculated by dividing the number of true negatives by the sum of true negatives and false positives.
  • informative or “informativeness” refers to a quality of a marker or panel of markers, and specifically to the likelihood of finding a marker (or panel of markers) in a positive sample.
  • neoplasm refers to any new and abnormal growth of tissue.
  • a neoplasm can be a premalignant neoplasm or a malignant neoplasm.
  • neoplasm-specific marker refers to any biological material that can be used to indicate the presence of a neoplasm. Examples of biological materials include, without limitation, nucleic acids, polypeptides, carbohydrates, fatty acids, cellular components (e.g., cell membranes and mitochondria), and whole cells.
  • cervical neoplasm-specific marker refers to any biological material that can be used to indicate the presence of a cervical neoplasm (e.g., a premalignant cervical neoplasm; a malignant cervical neoplasm).
  • cervical neoplasm-specific markers include, but are not limited to, AK2, AK3L1, ALDOA, B3GNT4, CLK3, C14orf2, C4orfi, C20orf20, DDIT3, FGF11, GAPDH, ISG15, KCTD11, P4HA2, PFKFB4, PVR, PYGL, RHOC, RPL36A, S100A2, SCARB1, SH3GL3, STC2, TRAPPC1, and UPK1A.
  • methylation status informative reagent or “methylation specific detection reagent” refers to a reagent or reagents that are informative for
  • reagents are primers, probes or antibodies for detection of gene expression products (e.g., RNA transcripts or proteins) of the following genes: AK2, AK3L1, ALDOA, B3GNT4, C14orf2, C4orfl, CLK3, C20orf20, DDIT3, FGF11, GAPDH, ISG15, KCTD11, P4HA2, PFKFB4, PVR, PYGL, RHOC, RPL36A, S100A2, SCARB1, SH3GL3, STC2, TRAPPC1, and UPK1A.
  • gene expression products e.g., RNA transcripts or proteins
  • amplicon refers to a nucleic acid generated using primer pairs.
  • the amplicon may be, for example, double or single-stranded DNA (e.g., the result of asymmetric amplification), and either RNA or dsDNA.
  • amplifying or “amplification” in the context of nucleic acids refers to the production of multiple copies of a polynucleotide, or a portion of the polynucleotide, typically starting from a small amount of the polynucleotide ⁇ e.g. , a single polynucleotide molecule), where the amplification products or amplicons are generally detectable.
  • Amplification of polynucleotides encompasses a variety of chemical and enzymatic processes.
  • the generation of multiple DNA copies from one or a few copies of a target or template DNA molecule during a polymerase chain reaction (PCR) or a ligase chain reaction (LCR; see, e.g., U.S. Patent No. 5,494,810; herein incorporated by reference in its entirety) are forms of amplification.
  • Additional types of amplification include, but are not limited to, allele-specific PCR (see, e.g., U.S. Patent No. 5,639,611; herein incorporated by reference in its entirety), assembly PCR (see, e.g., U.S. Patent No. 5,965,408; herein incorporated by reference in its entirety), helicase-dependent amplification (see, e.g., U.S. Patent No.
  • hot-start PCR see, e.g., U.S. Patent Nos. 5,773,258 and 5,338,671; each herein incorporated by reference in their entireties
  • intersequence-specific PCR see, e.g., Triglia, et al. (1988) Nucleic Acids Res., 16:8186; herein incorporated by reference in its entirety
  • ligation-mediated PCR see, e.g., Guilfoyle, R. et al, Nucleic Acids Research, 25: 1854-1858 (1997); U.S. Patent No. 5,508,169; each of which are herein incorporated by reference in their entireties
  • methylation-specific PCR see, e.g., Herman, et al, (1996) PNAS 93(13) 9821-9826; herein incorporated by reference in its entirety
  • miniprimer PCR multiplex ligation-dependent probe amplification
  • multiplex PCR see, e.g., Chamberlain, et al., (1988) Nucleic Acids Research 16(23) 11141-11156; Ballabio, et al, (1990) Human Genetics 84(6) 571-573; Hayden, et al, (2008) BMC Genetics 9:80; each of which are herein incorporated by reference in their entireties
  • nested PCR overlap-extension PCR (see, e.g., Higuchi, et al, (1988) Nucleic Acids Research 16(15) 7351-
  • the terms “complementary” or “complementarity” are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules.
  • sequence “5'-A-G-T-3', M is complementary to the sequence "3'-T-C-A-5 ⁇ "
  • Complementarity may be "partial,” in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids.
  • the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods that depend upon binding between nucleic acids.
  • the term "primer” refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, that is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product that is complementary to a nucleic acid strand is induced (e.g., in the presence of nucleotides and an inducing agent such as a biocatalyst (e.g. , a DNA polymerase or the like) and at a suitable temperature and pH).
  • the primer is typically single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is generally first treated to separate its strands before being used to prepare extension products.
  • the primer is an inducing agent
  • the primer is sufficiently long to prime the synthesis of extension products in the presence of the inducing agent.
  • the exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.
  • the primer is a capture primer.
  • nucleic acid molecule refers to any nucleic acid containing molecule, including but not limited to, DNA or RNA.
  • the term encompasses sequences that include any of the known base analogs of DNA and RNA including, but not limited to, 4 acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5- (carboxyhydroxyl-methyl) uracil, 5-fluorouracil, 5-bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl-aminomethyluracil,
  • dihydrouracil inosine, N6-isopentenyladenine, 1 -methyladenine, 1-methylpseudo-uracil, 1- methylguanine, 1 -methylinosine, 2,2-dimethyl-guanine, 2-methyladenine, 2-methylguanine, 3-methyl-cytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5- methylaminomethyluracil, 5 -methoxy-amino-methyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N- isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thi
  • nucleobase is synonymous with other terms in use in the art including “nucleotide,” “deoxynucleotide,” “nucleotide residue,” “deoxynucleotide residue,” “nucleotide triphosphate (NTP),” or deoxynucleotide triphosphate (dNTP).
  • oligonucleotide refers to a nucleic acid that includes at least two nucleic acid monomer units (e.g., nucleotides), typically more than three monomer units, and more typically greater than ten monomer units.
  • nucleic acid monomer units e.g., nucleotides
  • the exact size of an oligonucleotide generally depends on various factors, including the ultimate function or use of the oligonucleotide. To further illustrate, oligonucleotides are typically less than 200 residues long (e.g., between 15 and 100), however, as used herein, the term is also intended to encompass longer
  • Oligonucleotides are often referred to by their length. For example a 24 residue oligonucleotide is referred to as a "24-mer".
  • the nucleoside monomers are linked by phosphodiester bonds or analogs thereof, including phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like, including associated counterions, e.g., H + , NH 4 + , Na + , and the like, if such counterions are present.
  • oligonucleotides are typically single-stranded.
  • Oligonucleotides are optionally prepared by any suitable method, including, but not limited to, isolation of an existing or natural sequence, DNA replication or amplification, reverse transcription, cloning and restriction digestion of appropriate sequences, or direct chemical synthesis by a method such as the phosphotriester method of Narang et al. (1979) Meth Enzymol. 68: 90-99; the phosphodiester method of Brown et al. (1979) Meth Enzymol. 68: 109-151 ; the diethylphosphoramidite method of Beaucage et al. (1981) Tetrahedron Lett. 22: 1859-1862; the triester method of Matteucci et al. (1981) J Am Chem Soc.
  • a “sequence” of a biopolymer refers to the order and identity of monomer units (e.g., nucleotides, etc.) in the biopolymer.
  • the sequence (e.g., base sequence) of a nucleic acid is typically read in the 5' to 3' direction.
  • the term “subject” refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment.
  • the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.
  • non-human animals refers to all non-human animals including, but are not limited to, vertebrates such as rodents, non-human primates, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, aves, etc.
  • gene refers to a nucleic acid (e.g., DNA) sequence that comprises coding sequences necessary for the production of a polypeptide, RNA (e.g., including but not limited to, mRNA, tRNA and rRNA) or precursor.
  • RNA e.g., including but not limited to, mRNA, tRNA and rRNA
  • the polypeptide, RNA, or precursor can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties (e.g. , enzymatic activity, ligand binding, signal transduction, etc.) of the full-length or fragment are retained.
  • the term also encompasses the coding region of a structural gene and the including sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb on either end such that the gene corresponds to the length of the full-length mRNA.
  • the sequences that are located 5' of the coding region and which are present on the mRNA are referred to as 5' untranslated sequences.
  • the sequences that are located 3' or downstream of the coding region and that are present on the mRNA are referred to as 3' untranslated sequences.
  • gene encompasses both cDNA and genomic forms of a gene.
  • a genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed "introns” or “intervening regions” or “intervening sequences”.
  • Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript;
  • introns therefore are absent in the messenger RNA (mRNA) processed transcript.
  • mRNA messenger RNA
  • the mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.
  • locus refers to a nucleic acid sequence on a chromosome or on a linkage map and includes the coding sequence as well as 5 ' and 3 ' sequences involved in regulation of the gene.
  • the present invention relates to methods and biomarkers for detection of cervical cancer in biological samples, and in particular to methylation status markers associated with aggressiveness of cervical cancer.
  • DNA methylation status of genes is a stable and heritable covalent modification which mostly occurs in cytosines in CpG dinucleo tides.
  • DNA methylation is a stable biomarker which may be analyzed on patient samples without stringent handling requirements, since it is far more stable than mRNA which is the most common material analyzed in gene expression biomarker tests.
  • the methylation profile of 31 genes of interest was evaluated using the Illumina Infinium 45 OK assay, with a view to identifying which methylation patterns, if any, are associated with disease aggressiveness and survival outcome, and thus may be useful as prognostic markers.
  • 22 were found to have potential as components of a prognostic methylated gene panel.
  • the present invention provides methods for predicting a predisposition to cervical cancer in a subject, diagnosing a cervical cancer in a subject, predicting the likelihood of recurrence of cervical cancer in a subject, providing a prognosis for a subject with cervical cancer, or selecting a subject with cervical cancer for treatment with a particular therapy.
  • the methods comprise
  • altered methylation status relative to the methylation status in a reference sample is indicative of a predisposition of the subject to cervical cancer, an indication that the subject has cervical cancer, an indication of the likelihood of recurrence of the cervical cancer in the subject, an indication of survival of the subject, and indication of the aggressiveness of the cervical cancer, an indication of the likely outcome of treatment of the cervical cancer or an indication that the subject is a candidate for treatment with a particular therapy.
  • detection utilizes methylation specific detection reagents specific for the detection of one or more gene products (e.g., RNA or proteins) or the methylation status of the gene products resulting from the expression of one or more of the following genes: AK2, AK3L1, ALDOA, B3GNT4, CLK3, C14orf2, C4orfl, C20orf20, DDIT3, FGFll, GAPDH, ISG15, KCTDll, P4HA2, PFKFB4, PVR, PYGL, RHOC, RPL36A, S100A2, SCARB1, SH3GL3, STC2, TRAPPC1, or UPK1A.
  • gene products e.g., RNA or proteins
  • the genes are selected from AK2, AK3L1, ALDOA, B3GNT4, C4orf2, FGFll, ISG15, KCTDll, P4HA2, PFKFB4, PVR, PYGL, RHOC, RPL36A, S100A2, SCARB1, SH3GL3, STC2,or TRAPPC1.
  • compositions, systems, and methods utilize reagents for detection of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or all of the described genes).
  • reagents include, but are not limited to, nucleic acid primers, probes, primer pairs, methylation specific restriction enzymes, bisulfite, etc.
  • the reagent e.g., probe
  • the reagent is one or more (e.g., two) of SEQ ID NOs: 1- 106.
  • the reagent is one or more of at least 8 (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or all nucleotides) of SEQ ID NOs: 1-106.
  • the reagent is one or more of at least 8 (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or all nucleotides of the complement of SEQ ID NOs: l- 106.
  • detection methods comprise a single probe as described above (e.g., probes indicated as Type II in Table 7).
  • detection methods comprise a pair of probes (e.g., as indicated as Type I in Table 7; e.g., one or more of SEQ ID NOs: 4:84; 8:85; 13:86; 14:87; 25:88; 26:89; 27:90; 46:91; 37:92; 38:93; 39:94; 44:95; 45:96; 48:97; 49:98; 60:99; 69: 100; 70: 101; 71 : 102; 72:103; 73: 104; 75: 105; or 79: 106).
  • probes e.g., as indicated as Type I in Table 7; e.g., one or more of SEQ ID NOs: 4:84; 8:85; 13:86; 14:87; 25:88; 26:89; 27:90; 46:91; 37:92; 38:93; 39:94; 44:95; 45:96; 48:97; 49:98; 60:99; 69: 100; 70: 101; 71 :
  • a marker includes, for example, nucleic acid(s) whose methylation status is characteristic of a cervical neoplasm or the aggressiveness or prognosis of a cervical neoplasm.
  • the statistical analysis will vary. For example, where a particular combination of markers is highly specific for cervical cancer, the statistical significance of a positive result will be high. It may be, however, that such specificity is achieved at the cost of sensitivity (e.g., a negative result may occur even in the presence of cervical cancer). By the same token, a different combination may be very sensitive (e.g., few false negatives, but has a lower specificity).
  • compositions and methods of the present disclosure find use in determining a treatment course of action of administering, for example, hypoxia-targeting and methylation modulating drugs.
  • Subjects may be monitored after a therapy and/or course of action to determine the effectiveness of that specific therapy and/or course of action.
  • the methods of the present invention are not limited to particular indicators of cervical neoplasm.
  • embodiments of the present invention provide diagnostic and screening methods that utilize the detection of methylation status of gene products resulting from the expression of one or more of the following genes: AK2, AK3L1, ALDOA, B3GNT4, CLK3, C14orf2, C4orfl, C20orf20, DDIT3, FGF11, GAPDH, ISG15, KCTD11, P4HA2, PFKFB4, PVR, PYGL, RHOC, RPL36A, S100A2, SCARBI, SH3GL3, STC2, TRAPPCl, and UPK1A. Exemplary, non- limiting methods are described below.
  • the sample may be a tissue sample (e.g., a cervical tumor biopsy sample or pelvic lymph node biopsy).
  • the patient sample is subjected to preliminary processing designed to isolate or enrich the sample for the gene products or cells that contain the gene products.
  • preliminary processing designed to isolate or enrich the sample for the gene products or cells that contain the gene products.
  • a variety of techniques known to those of ordinary skill in the art may be used for this purpose, including but not limited to: centrifugation; immunocapture; cell lysis; and, nucleic acid target capture (See, e.g., EP Pat. No. 1 409 727, herein incorporated by reference in its entirety).
  • methods, kits, and systems of the present invention involve determination of methylation state of a locus of interest (e.g., in human DNA) (e.g., in human DNA extracted from a blood sample, from a serum sample, from a plasma sample, from a cell sample, etc). Any appropriate method can be used to determine whether a particular DNA is hypermethylated or hypomethylated. Standard PCR techniques, for example, can be used to determine which residues are methylated, since unmethylated cytosines converted to uracil are replaced by thymidine residues during PCR.
  • PCR reactions can contain, for example, 10 ⁇ ⁇ ⁇ captured DNA that either has or has not been treated with sodium bisulfite, IX PCR buffer, 0.2 mM dNTPs, 0.5 ⁇ sequence specific primers (e.g., primers flanking a CpG island or CpG shore within the captured DNA), and 5 units DNA polymerase (e.g., Amplitaq DNA polymerase from PE Applied Biosystems, Norwalk, CT) in a total volume of 50 ⁇ .
  • DNA polymerase e.g., Amplitaq DNA polymerase from PE Applied Biosystems, Norwalk, CT
  • a typical PCR protocol can include, for example, an initial denaturation step at 94°C for 5 min, 40 amplification cycles consisting of 1 minute at 94°C, 1 minute at 60°C, and 1 minute at 72°C, and a final extension step at 72°C for 5 minutes.
  • sequences of PCR products corresponding to samples treated with and without sodium bisulfite can be compared.
  • the sequence from the untreated DNA will reveal the positions of all cytosine residues within the PCR product. Cytosines that were unmethylated will be converted to thymidine residues in the sequence of the bisulfite-treated DNA, while residues that were methylated will be unaffected by bisulfite treatment.
  • nucleic acid sequencing methods are utilized for detection.
  • the sequencing is Second Generation (a.k.a. Next Generation or Next- Gen), Third Generation (a.k.a. Next-Next-Gen), or Fourth Generation (a.k.a. N3-Gen) sequencing technology including, but not limited to, pyrosequencing, sequencing-by-ligation, single molecule sequencing, sequence-by-synthesis (SBS), semiconductor sequencing, massive parallel clonal, massive parallel single molecule SBS, massive parallel single molecule real-time, massive parallel single molecule real-time nanopore technology, etc.
  • SBS sequence-by-synthesis
  • Morozova and Marra provide a review of some such technologies in Genomics, 92: 255 (2008), herein incorporated by reference in its entirety. Those of ordinary skill in the art will recognize that because RNA is less stable in the cell and more prone to nuclease attack experimentally RNA is usually reverse transcribed to DNA before sequencing.
  • DNA sequencing techniques include fluorescence-based sequencing methodologies (See, e.g., Birren et al, Genome Analysis: Analyzing DNA, 1, Cold Spring Harbor, N.Y.; herein incorporated by reference in its entirety).
  • the sequencing is automated sequencing.
  • the sequenceing is parallel sequencing of partitioned amplicons (PCT Publication No: WO2006084132 to Kevin McKernan et al., herein incorporated by reference in its entirety).
  • the sequencing is DNA sequencing by parallel oligonucleotide extension (See, e.g., U.S. Pat. No. 5,750,341 to Macevicz et al., and U.S. Pat. No.
  • NGS Next-generation sequencing
  • NGS methods can be broadly divided into those that typically use template amplification and those that do not.
  • Amplification-requiring methods include pyrosequencing commercialized by Roche as the 454 technology platforms (e.g., GS 20 and GS FLX), Life Technologies/Ion Torrent, the Solexa platform commercialized by Illumina, GnuBio, and the Supported Oligonucleotide Ligation and Detection (SOLiD) platform commercialized by Applied Biosystems.
  • Non-amplification approaches also known as single-molecule sequencing, are exemplified by the HeliScope platform commercialized by Helicos
  • template DNA is fragmented, end- repaired, ligated to adaptors, and clonally amplified in-situ by capturing single template molecules with beads bearing oligonucleotides complementary to the adaptors.
  • Each bead bearing a single template type is compartmentalized into a water-in-oil microvesicle, and the template is clonally amplified using a technique referred to as emulsion PCR.
  • the emulsion is disrupted after amplification and beads are deposited into individual wells of a picotitre plate functioning as a flow cell during the sequencing reactions. Ordered, iterative
  • each of the four dNTP reagents occurs in the flow cell in the presence of sequencing enzymes and luminescent reporter such as luciferase.
  • luminescent reporter such as luciferase.
  • the resulting production of ATP causes a burst of luminescence within the well, which is recorded using a CCD camera. It is possible to achieve read lengths greater than or equal to 400 bases, and 10 6 sequence reads can be achieved, resulting in up to 500 million base pairs (Mb) of sequence.
  • sequencing data are produced in the form of shorter-length reads.
  • single- stranded fragmented DNA is end-repaired to generate 5'-phosphorylated blunt ends, followed by Klenow-mediated addition of a single A base to the 3' end of the fragments.
  • A-addition facilitates addition of T-overhang adaptor oligonucleotides, which are subsequently used to capture the template-adaptor molecules on the surface of a flow cell that is studded with oligonucleotide anchors.
  • the anchor is used as a PCR primer, but because of the length of the template and its proximity to other nearby anchor oligonucleotides, extension by PCR results in the "arching over" of the molecule to hybridize with an adjacent anchor oligonucleotide to form a bridge structure on the surface of the flow cell.
  • These loops of DNA are denatured and cleaved. Forward strands are then sequenced with reversible dye terminators.
  • sequence of incorporated nucleotides is determined by detection of post-incorporation fluorescence, with each fluor and block removed prior to the next cycle of dNTP addition. Sequence read length ranges from 36 nucleotides to over 250 nucleotides, with overall output exceeding 1 billion nucleotide pairs per analytical run.
  • Sequencing nucleic acid molecules using SOLiD technology also involves fragmentation of the template, ligation to oligonucleotide adaptors, attachment to beads, and clonal amplification by emulsion PCR.
  • beads bearing template are immobilized on a derivatized surface of a glass flow-cell, and a primer complementary to the adaptor oligonucleotide is annealed.
  • a primer complementary to the adaptor oligonucleotide is annealed.
  • this primer is instead used to provide a 5' phosphate group for ligation to interrogation probes containing two probe-specific bases followed by 6 degenerate bases and one of four fluorescent labels.
  • interrogation probes have 16 possible combinations of the two bases at the 3' end of each probe, and one of four fluors at the 5' end. Fluor color, and thus identity of each probe, corresponds to specified color-space coding schemes.
  • sequencing is nanopore sequencing (see, e.g., Astier et al, J. Am. Chem. Soc. 2006 Feb 8; 128(5): 1705-10, herein incorporated by reference).
  • the theory behind nanopore sequencing has to do with what occurs when a nanopore is immersed in a conducting fluid and a potential (voltage) is applied across it. Under these conditions a slight electric current due to conduction of ions through the nanopore can be observed, and the amount of current is exceedingly sensitive to the size of the nanopore.
  • As each base of a nucleic acid passes through the nanopore this causes a change in the magnitude of the current through the nanopore that is distinct for each of the four bases, thereby allowing the sequence of the DNA molecule to be determined.
  • sequencing is HeliScope by Helicos Biosciences
  • Template DNA is fragmented and polyadenylated at the 3' end, with the final adenosine bearing a fluorescent label.
  • Denatured polyadenylated template fragments are ligated to poly(dT) oligonucleotides on the surface of a flow cell.
  • Initial physical locations of captured template molecules are recorded by a CCD camera, and then label is cleaved and washed away.
  • Sequencing is achieved by addition of polymerase and serial addition of fluorescently-labeled dNTP reagents. Incorporation events result in fluor signal corresponding to the dNTP, and signal is captured by a CCD camera before each round of dNTP addition.
  • Sequence read length ranges from 25-50 nucleotides, with overall output exceeding 1 billion nucleotide pairs per analytical run.
  • the Ion Torrent technology is a method of DNA sequencing based on the detection of hydrogen ions that are released during the polymerization of DNA (see, e.g., Science
  • a microwell contains a template DNA strand to be sequenced. Beneath the layer of microwells is a hypersensitive ISFET ion sensor. All layers are contained within a CMOS semiconductor chip, similar to that used in the electronics industry.
  • a dNTP is incorporated into the growing complementary strand a hydrogen ion is released, which triggers a hypersensitive ion sensor. If homopolymer repeats are present in the template sequence, multiple dNTP molecules will be incorporated in a single cycle. This leads to a corresponding number of released hydrogens and a proportionally higher electronic signal.
  • the per-base accuracy of the Ion Torrent sequencer is -99.6% for 50 base reads, with -100 Mb to 100Gb generated per run.
  • the read- length is 100-300 base pairs.
  • the accuracy for homopolymer repeats of 5 repeats in length is -98%.
  • the benefits of ion semiconductor sequencing are rapid sequencing speed and low upfront and operating costs.
  • sequencing is the technique developed by Stratos Genomics, Inc. and involves the use of Xpandomers.
  • This sequencing process typically includes providing a daughter strand produced by a template-directed synthesis.
  • the daughter strand generally includes a plurality of subunits coupled in a sequence corresponding to a contiguous nucleotide sequence of all or a portion of a target nucleic acid in which the individual subunits comprise a tether, at least one probe or nucleobase residue, and at least one selectively cleavable bond.
  • the selectively cleavable bond(s) is/are cleaved to yield an Xpandomer of a length longer than the plurality of the subunits of the daughter strand.
  • the Xpandomer typically includes the tethers and reporter elements for parsing genetic information in a sequence corresponding to the contiguous nucleotide sequence of all or a portion of the target nucleic acid. Reporter elements of the Xpandomer are then detected. Additional details relating to Xpandomer-based approaches are described in, for example, U.S. Pat. Pub No. 20090035777, entitled "High Throughput Nucleic Acid Sequencing by Expansion," filed June 19, 2008, which is incorporated herein in its entirety.
  • methods of the present invention involve the determination (e.g., assessment, ascertaining, quantitation) of methylation level of an indicator of cervical neoplasm (e.g., the methylation level of a CpG island or CpG shore in the coding or regulatory region of a gene locus) in a sample (e.g., a DNA sample extracted from stool, bile or blood).
  • a sample e.g., a DNA sample extracted from stool, bile or blood.
  • a reference e.g., a reference level, a control level, a threshold level, or the like.
  • the term "elevated methylation” as used herein with respect to the methylation status (e.g., CpG DNA methylation) of a gene locus is any methylation level that is above a median methylation level in a sample from a random population of mammals (e.g., a random population of 10, 20, 30, 40, 50, 100, or 500 mammals) that do not have a cervical neoplasm (e.g., cervical cancer). Elevated levels of methylation can be any level provided that the level is greater than a corresponding reference level.
  • an elevated methylation level of a locus of interest e.g., methylation can be 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fold greater than the reference level methylation observed in a normal sample.
  • a reference level can be any amount.
  • the term "elevated methylation score" as used herein with respect to detected methylation events in a matrix panel of particular nucleic acid markers is any methylation score that is above a median methylation score in a sample from a random population of mammals (e.g., a random population of 10, 20, 30, 40, 50, 100, or 500 mammals) that do not have a cervical neoplasm.
  • An elevated methylation score in a matrix panel of particular nucleic acid markers can be any score provided that the score is greater than a corresponding reference score.
  • an elevated score of methylation in a locus of interest e.g., can be 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fold greater than the reference methylation score observed in a normal sample.
  • a reference score can be any amount.
  • the methods are not limited to a particular type of mammal.
  • the mammal is a human.
  • the cancer is premalignant.
  • the cancer is malignant.
  • the cancer is an aggressive cervical cancer.
  • the compositions and methods described herein differentiate between aggressive and non-aggressive cervical cancers.
  • the present invention also provides methods and materials to assist medical or research professionals in determining whether or not a mammal has a cervical cancer or to determine the aggressiveness of the cervical cancer.
  • Medical professionals can be, for example, doctors, nurses, medical laboratory technologists, and pharmacists.
  • Research professionals can be, for example, principle investigators, research technicians, postdoctoral trainees, and graduate students.
  • a professional can be assisted by (1) determining the ratio of particular markers in a sample, and (2) communicating information about the ratio to that professional, for example.
  • a medical professional can take one or more actions that can affect patient care. For example, a medical professional can record the results in a patient's medical record. In some cases, a medical professional can record a diagnosis of a cervical cancer, or otherwise transform the patient's medical record, to reflect the patient's medical condition. In some cases, a medical professional can review and evaluate a patient's entire medical record, and assess multiple treatment strategies, for clinical intervention of a patient's condition. In some cases, a medical professional can record a prediction of tumor occurrence with the reported indicators. In some cases, a medical professional can review and evaluate a patient's entire medical record and assess multiple treatment strategies, for clinical intervention of a patient's condition.
  • a medical professional can initiate or modify treatment of a cervical cancer after receiving information regarding the level (score, frequency) associated with markers in a patient's stool, blood, serum, bile or plasma sample.
  • a medical professional can compare previous reports and the recently communicated level (score, frequency) of markers, and recommend a change in therapy.
  • a medical professional can enroll a patient in a clinical trial for novel therapeutic intervention of cervical cancer neoplasm.
  • a medical professional can elect waiting to begin therapy until the patient's symptoms require clinical intervention.
  • patients identified as having aggressive cervical cancer are given adjuvant radiation or chemotherapy following surgical treatment while patient identified as having non-aggressive cervical cancer are not given adjuvant therapy.
  • the assay is repeated after adjuvant treatment.
  • the assay to determine methylation status of the described genes is performed one or more times before, during, or after, primary or adjuvant therapy.
  • a medical professional can communicate the assay results to a patient or a patient's family.
  • a medical professional can provide a patient and/or a patient's family with information regarding cervical neoplasia, including treatment options, prognosis, and referrals to specialists, e.g., oncologists and/or radiologists.
  • a medical professional can provide a copy of a patient's medical records to communicate assay results to a specialist.
  • a research professional can apply information regarding a subject's assay results to advance cervical neoplasm research. For example, a researcher can compile data on the assay results, with information regarding the efficacy of a drug for treatment of a cervical cancer to identify an effective treatment.
  • a research professional can obtain assay results to evaluate a subject's enrollment, or continued participation in a research study or clinical trial. In some cases, a research professional can classify the severity of a subject's condition, based on assay results. In some cases, a research professional can communicate a subject's assay results to a medical professional. In some cases, a research professional can refer a subject to a medical professional for clinical assessment of cervical neoplasia, and treatment thereof. Any appropriate method can be used to communicate information to another person (e.g., a professional). For example, information can be given directly or indirectly to a professional. For example, a laboratory technician can input the assay results into a computer-based record.
  • information is communicated by making a physical alteration to medical or research records.
  • a medical professional can make a permanent notation or flag a medical record for communicating a diagnosis to other medical professionals reviewing the record.
  • any type of communication can be used to communicate the information.
  • mail, e-mail, telephone, and face-to-face interactions can be used.
  • the information also can be communicated to a professional by making that information electronically available to the professional.
  • the information can be communicated to a professional by placing the information on a computer database such that the professional can access the information.
  • the information can be communicated to a hospital, clinic, or research facility serving as an agent for the professional.
  • a single sample can be analyzed for one cervical cancer-specific marker or for multiple cervical neoplasm-specific markers.
  • a single sample is analyzed for multiple cervical neoplasm-specific markers, for example, using multi-marker assays.
  • multiple samples can be collected for a single mammal and analyzed as described herein.
  • a sample is split into first and second portions, where the first portion undergoes cytological analysis and the second portion undergoes further purification or processing (e.g., sequence-specific capture step(s) (e.g., for isolation of specific markers for analysis of methylation levels).
  • the sample undergoes one or more preprocessing steps before being split into portions.
  • the sample is treated, handled, or preserved in a manner that promotes DNA integrity and/or inhibits DNA degradation (e.g., through use of storage buffers with stabilizing agents (e.g., chelating agents, DNase inhibitors) or handling or processing techniques that promote DNA integrity (e.g., immediate processing or storage at low temperature (e.g., -80 degrees C)).
  • stabilizing agents e.g., chelating agents, DNase inhibitors
  • processing techniques that promote DNA integrity (e.g., immediate processing or storage at low temperature (e.g., -80 degrees C)).
  • kits for the diagnosis or screening of cancer comprising one or reagents for detection of methylation status of the genes selected from, for example one or more those described herein.
  • the reagents comprise nucleic acids (e.g., oligonucleotides, primers, probes, etc.).
  • kits provide reagents useful, necessary or sufficient for detecting methylation status and/or providing a diagnosis or prognosis.
  • Compositions for use in the diagnostic methods described herein include, but are not limited to, kits comprising one or more methylation status informative reagents as described above.
  • kits comprise one or more methylation status informative reagents for detecting altered gene expression in a sample from a subject having or suspected of having cervical cancer, wherein the reagents are specific detection of one or more gene products from the following genes: AK3L1, ALDOA, B3GNT4, CLK3, C14orfi, C4orfl, C20orf20, DDIT3, FGF11, GAPDH, ISG15, KCTD11, P4HA2, PFKFB4, PVR, PYGL, RHOC, RPL36A, S100A2, SCARB1, SH3GL3, STC2, TRAPPC1, and UPK1A.
  • the reagents are specific detection of one or more gene products from the following genes: AK3L1, ALDOA, B3GNT4, CLK3, C14orfi, C4orfl, C20orf20, DDIT3, FGF11, GAPDH, ISG15, KCTD11, P4HA2, PFKFB4, PVR, PY
  • the present disclosure provides complexes of one or more of the above-described genes and a methylation status informative reagent.
  • the diagnostic kits may further comprise any reagent or media necessary, sufficient or useful to perform analyses, such as PCR analyses, such as methylation specific polymerase chain reaction (MSP) sequence analyses, bisulphite treatment, bisulphite sequencing, electrophoresis, pyrosequencing, mass spectrometry and sequence analyses by restriction digestion, next generation sequencing, quantitative and/or qualitative methylation, pyrosequencing, Southern blotting, restriction landmark genome scanning (RLGS), single nucleotide primer extension, CpG island microarray, SNUPE, COBRA, mass spectrometry, by use of methylation specific restriction enzymes or by measuring the expression level of said genes.
  • MSP methylation specific polymerase chain reaction
  • the kit may further comprise one or more methylation specific detection components selected from, for example, deoxyribonucleoside triphosphates, buffers, stabilizers, thermostable DNA polymerases, restriction endonucleases (including methylation specific endonucleases), and labels (including fiuorescent, chemiluminescent and radioactive labels).
  • the diagnostic assay according to the invention may further comprise one or more reagents required for isolation of DNA.
  • kits of the present invention include a means for containing the reagents in close confinement for commercial sale such as, e.g., injection or blow-molded plastic containers into which the desired reagent are retained.
  • a means for containing the reagents in close confinement for commercial sale such as, e.g., injection or blow-molded plastic containers into which the desired reagent are retained.
  • Other containers suitable for conducting certain steps of the disclosed methods also may be provided.
  • compositions and methods disclosed herein are useful in monitoring the treatment of cervical cancers.
  • the methods may be performed immediately before, during and/or after a treatment to monitor treatment success or determine a treatment course of action.
  • the methods are performed at intervals on disease free patients to ensure treatment success.
  • the present disclosure provides compositions and method for determining a treatment course of action and administering the treatment.
  • the methods are repeated and the results are used to determine a treatment course of action (e.g., to start, stop, or modify a treatment).
  • the present invention also provides a variety of computer-related embodiments. Specifically, in some embodiments the invention provides computer programming for analyzing and comparing a pattern of cervical cancer-specific marker detection results in a sample obtained from a subject to, for example, a library of such marker patterns known to be indicative of the presence or absence of a cervical cancer, or a particular stage or prognosis of a cervical cancer.
  • the present invention provides computer programming for analyzing and comparing a first and a second pattern of cervical cancer-specific marker detection results from a sample taken at least two different time points.
  • the first pattern may be indicative of a pre-cancerous condition and/or low risk condition for a cervical cancer and/or progression from a pre-cancerous condition to a cancerous condition.
  • the comparing provides for monitoring of the progression of the condition from the first time point to the second time point.
  • the invention provides computer programming for analyzing and comparing a pattern of cervical cancer-specific marker detection results from a sample to a library of cervical cancer-specific marker patterns known to be indicative of the presence or absence of a cervical cancer, wherein the comparing provides, for example, a differential diagnosis between an aggressively malignant cervical cancer and a less aggressive cervical cancer (e.g., the marker pattern provides for staging and/or grading of the cancerous condition).
  • the methods and systems described herein can be implemented in numerous ways. In one embodiment, the methods involve use of a communications infrastructure, for example the internet. Several embodiments of the invention are discussed below. It is also to be understood that the present invention may be implemented in various forms of hardware, software, firmware, processors, distributed servers (e.g., as used in cloud computing) or a combination thereof. The methods and systems described herein can be implemented as a combination of hardware and software.
  • the software can be implemented as an application program tangibly embodied on a program storage device, or different portions of the software implemented in the user's computing environment (e.g., as an applet) and on the reviewer's computing environment, where the reviewer may be located at a remote site (e.g., at a service provider's facility).
  • portions of the data processing can be performed in the user-side computing environment.
  • the user-side computing environment can be programmed to provide for defined test codes to denote platform, carrier/diagnostic test, or both; processing of data using defined flags, and/or generation of flag configurations, where the responses are transmitted as processed or partially processed responses to the reviewer's computing environment in the form of test code and flag configurations for subsequent execution of one or more algorithms to provide a results and/or generate a report in the reviewer's computing environment.
  • the application program for executing the algorithms described herein may be uploaded to, and executed by, a machine comprising any suitable architecture.
  • the machine involves a computer platform having hardware such as one or more central processing units (CPU), a random access memory (RAM), and input/output (I/O) interface(s).
  • the computer platform also includes an operating system and microinstruction code.
  • the various processes and functions described herein may either be part of the microinstruction code or part of the application program (or a combination thereof) which is executed via the operating system.
  • various other peripheral devices may be connected to the computer platform such as an additional data storage device and a printing device.
  • the system generally includes a processor unit.
  • the processor unit operates to receive information, which generally includes test data (e.g., specific gene products assayed), and test result data (e.g., the pattern of cervical neoplasm-specific marker detection results from a sample).
  • This information received can be stored at least temporarily in a database, and data analyzed in comparison to a library of marker patterns known to be indicative of the presence or absence of a pre-cancerous condition, or known to be indicative of a stage and/or grade of cervical cancer.
  • Part or all of the input and output data can also be sent electronically; certain output data (e.g., reports) can be sent electronically or telephonically (e.g., by facsimile, e.g., using devices such as fax back).
  • Exemplary output receiving devices can include a display element, a printer, a facsimile device and the like.
  • Electronic forms of transmission and/or display can include email, interactive television, and the like.
  • all or a portion of the input data and/or all or a portion of the output data are maintained on a server for access, e.g., confidential access.
  • the results may be accessed or sent to professionals as desired.
  • a system for use in the methods described herein generally includes at least one computer processor (e.g., where the method is carried out in its entirety at a single site) or at least two networked computer processors (e.g., where detected marker data for a sample obtained from a subject is to be input by a user (e.g., a technician or someone performing the assays)) and transmitted to a remote site to a second computer processor for analysis (e.g., where the pattern of cervical cancer-specific marker) detection results is compared to a library of patterns known to be indicative of the presence or absence of a pre-cancerous condition), where the first and second computer processors are connected by a network, e.g., via an intranet or internet).
  • a network e.g., via an intranet or internet
  • the system can also include a user component(s) for input; and a reviewer component(s) for review of data, and generation of reports, including detection of a pre-cancerous condition, staging and/or grading of a cervical cancer, or monitoring the progression of a pre-cancerous condition or a cervical cancer.
  • Additional components of the system can include a server component(s); and a database(s) for storing data (e.g., as in a database of report elements, e.g., a library of marker patterns known to be indicative of the presence or absence of a pre-cancerous condition and/or known to be indicative of a grade and/or a stage of a cervical cancer, or a relational database (RDB) which can include data input by the user and data output.
  • the computer processors can be processors that are typically found in personal desktop computers (e.g., IBM, Dell, Macintosh), portable computers, mainframes, minicomputers, tablet computer, smart phone, or other computing devices.
  • the input components can be complete, stand-alone personal computers offering a full range of power and features to run applications.
  • the user component usually operates under any desired operating system and includes a communication element (e.g., a modem or other hardware for connecting to a network using a cellular phone network, Wi-Fi, Bluetooth, Ethernet, etc.), one or more input devices (e.g., a keyboard, mouse, keypad, or other device used to transfer information or commands), a storage element (e.g., a hard drive or other computer-readable, computer-writable storage medium), and a display element (e.g., a monitor, television, LCD, LED, or other display device that conveys information to the user).
  • the user enters input commands into the computer processor through an input device.
  • the user interface is a graphical user interface (GUI) written for web browser applications.
  • the server component(s) can be a personal computer, a minicomputer, or a mainframe, or distributed across multiple servers (e.g., as in cloud computing applications) and offers data management, information sharing between clients, network administration and security.
  • the application and any databases used can be on the same or different servers.
  • Other computing arrangements for the user and server(s), including processing on a single machine such as a mainframe, a collection of machines, or other suitable configuration are contemplated. In general, the user and server machines work together to accomplish the processing of the present invention.
  • the database(s) is usually connected to the database server component and can be any device which will hold data.
  • the database can be any magnetic or optical storing device for a computer (e.g., CDROM, internal hard drive, tape drive).
  • the database can be located remote to the server component (with access via a network, modem, etc.) or locally to the server component.
  • the database can be a relational database that is organized and accessed according to relationships between data items.
  • the relational database is generally composed of a plurality of tables (entities). The rows of a table represent records (collections of information about separate items) and the columns represent fields (particular attributes of a record).
  • the relational database is a collection of data entries that "relate" to each other through at least one common field.
  • Additional workstations equipped with computers and printers may be used at point of service to enter data and, in some embodiments, generate appropriate reports, if desired.
  • the computer(s) can have a shortcut (e.g., on the desktop) to launch the application to facilitate initiation of data entry, transmission, analysis, report receipt, etc. as desired.
  • the present invention provides methods for obtaining a subject's risk profile for developing cervical cancer.
  • such methods involve obtaining a blood or blood product sample from a subject (e.g., a human at risk for developing cervical cancer; a human undergoing a routine physical examination), detecting the presence, absence, or level (e.g., methylation frequency or score) of one or more markers specific for a cervical cancer in or associated with the blood or blood product sample (e.g., specific for a cervical cancer) in the sample, and generating a risk profile for developing cervical cancer based upon the detected level (score, frequency) or presence or absence of the indicators of cervical cancer.
  • a generated risk profile will change depending upon specific markers and detected as present or absent or at defined threshold levels.
  • the present invention is not limited to a particular manner of generating the risk profile.
  • a processor e.g., computer
  • the processor uses an algorithm (e.g., software) specific for interpreting the presence and absence of specific markers as determined with the methods of the present invention.
  • the presence and absence of specific markers as determined with the methods of the present invention are imputed into such an algorithm, and the risk profile is reported based upon a comparison of such input with established norms (e.g., established norm for pre-cancerous condition, established norm for various risk levels for developing cervical cancer, established norm for subjects diagnosed with various stages of cervical cancer).
  • established norms e.g., established norm for pre-cancerous condition, established norm for various risk levels for developing cervical cancer, established norm for subjects diagnosed with various stages of cervical cancer.
  • the risk profile indicates a subject's risk for developing cervical cancer or a subject's risk for re-developing cervical cancer.
  • the risk profile indicates a subject to be, for example, a very low, a low, a moderate, a high, and a very high chance of developing or re-developing cervical cancer or having a poor prognosis (e.g., likelihood of long term survival) from cervical cancer.
  • a health care provider e.g., an oncologist
  • a course of treatment or intervention e.g., biopsy, wait and see, referral to an oncologist, referral to a surgeon, etc.
  • Relapse progressive disease was classified as locoregional (regression within the irradiated field), distant, or both.
  • locoregional regression within the irradiated field
  • distant distant
  • genomic DNA was isolated from the cervical cancer biopsies according to a standard protocol, including proteinase K, phenol, chloroform, and isoamylalcohol (Jones et al, 2012 Nat. Rev. Genet. 75:484-492).
  • DNA (1 ⁇ g) was digested overnight, using Dpnll endonuclease (New England Biolabs, Beverly, MA, USA), and purified using the QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA). Purified DNA quality and concentration were assessed with Quant-iTTM PicoGreen® dsDNA Assay Kit (Life Technologies, Paisley, UK) prior to bisulfite conversion.
  • the percentage of methylation of a given CpG locus was reported as a ⁇ -value, which is a continuous variable between 0 and 1. This corresponds to the ratio of intensities between methylated and unmethylated alleles.
  • Epigenomics 4:325-341) was implemented using R (version 2.15.1).
  • probes with less than three functional beads on the array were considered to be nonfunctional, and were assigned a detection p-value equal to 1.
  • "Good quality” samples were defined as having >80% high quality (detection p-value ⁇ 0.01) probes. All patient samples respected this criterion, and were thus kept for the further analyses. Further, all allosomal probes located on the Y chromosome were removed, since the samples were from female patients.
  • the endpoint was progression- free survival, where the time from diagnosis to cancer-related death or to the first event of relapse was used.
  • Cox proportional hazard analysis was used to evaluate the prognostic value of various parameters with respect to progression-free survival.
  • Kaplan-Meier curves were compared using log-rank test. Spearman rank correlation analyses were utilized to assess toe correlation between methylation and gene expression values. P-values ⁇ 0.05 or adjusted p-values ⁇ 0.1 were considered significant.
  • methylation probes There were 548 methylation probes annotated to one of the 31 hypoxia-associated genes, respectively. As illustrated in Figure 1, there were several methylation probes in both the promoter and gene body of the genes which showed a correlation with expression of the relevant/related gene. More detailed information about the methylation probes is listed in Table 1. Most of the promoter methylation and gene body methylation are negatively and positively correlated, respectively, with the gene expression.
  • STC2 Stanniocalcin 2
  • PVR Poliovirus receptor
  • RPL36A ribosomal protein L36a
  • methylation score Even if the probes for the remaining 15 genes did not significantly correlate with survival individually, they may contribute to predicting survival if assessed in combination with the other probes in a "methylation score". Thus, one representative probe for each of the 22 genes was included in the score, and the resulting combined score for the 22 genes was assessed for prognostic significance by Cox regression analysis. A methylation score was calculated to obtain a metric which took into account the methylation level of all these 22 genes. The methylation score was calculated from the methylation probes as described below.
  • a majority of the genes in the 31 gene signature were regulated by methylation. Furthermore, the methylation level of these genes could predict survival of cervical cancer patients in a highly significant manner, independent of other clinical variables. Thus, a test based on the methylation status of all or a subset of these genes could be valuable for identification of cervical cancer patients of high risk of recurrence who may benefit from more aggressive treatment.
  • STC2 stanniocalcin 2
  • KCTD11 was shown to be hypermethylated in multiple cancer types not including cervical cancer (Mancarelli et al, 2011 Mol Cancer 9 : 172), and P4HA2 was reported to be transcriptionally silenced by methylation in lymphoma (Hatzimichael et al, 2012 Br J Cancer 2012 Oct 9;107(8)).
  • the promoter oiSH3GL3 was aberrantly methylated in colorectal cancer patients (Fang et al, 2012 Asian Pac J Cancer Prev.
  • DDIT3 The expression level of DDIT3 has also been reported to have prognostic value in malignant mesothelioma (Dalton et al, 2013 Br J Cancer 108: 1340-1347), melanoma (Korabiowska et al, 2002 Histol Histopathol 17:805-811) and non-small cell lung cancer (Lee et al, 2012 Oncol Lett. 4:408-412), but without any association with methylation in the relevant malignancies.
  • DDIT3 was found to be methylated in chronic myeloid leukemia, but without reference to prognosis (Wang et al, 2010 J Exp Clin Cancer Res. 29:54).
  • Table 1 Information about the 151 probes which are significantly correlated (adj.p ⁇ 0.1) with their annotated gene.
  • Promoter is defined as +/- 2000 bp from the transcriptional start site of the gene, while “body” designates all other regions.
  • Table 3 The 22 genes for which gene expression was regulated by methylation levels.
  • TRAPPC1 Promoter 0.01 -0.25 0.159 5.13
  • ALDOA Promoter 0.05 -0.21 0.767 -0.48 a The methylation probe was defined as being located in the promoter of a gene if positioned ⁇ 2000bp from its transcriptional start site (TSS). If positioned >2000bp from the TSS, the probe was defined as being located in the body of the gene.
  • FIGO Confidence Interval
  • FIGO Federation International de Gynecologie et d'Obstetrique.
  • Tumor volume and methylation score were divided into two groups based on median values of all patients, respectively.
  • FIGO stage was divided into two groups; lb-2b and 3a-4a.
  • Example 1 gene expression data and clinical data from 46 patients was used to develop a prognostic hypoxia gene signature, while 109 independent patients were used for validation (Halle et al., Cancer Res., 72: 5285-5295, 2012).
  • the combined samples (designated cohort 1 for the purposes of this Example) were used to investigate correlation of methylation levels of the originally identified 31 hypoxia-associated genes with expression and clinical outcome.
  • data for the 109 validation patients was used to investigate the relevance of methylation of the signature genes, to be able to have an independent analysis of the gene methylation without interference from the previous analysis. Methylation data were available for 107 of these patients, which thus constitutes sub-cohort 1 in the current analysis.
  • the initial total number of methylation probes annotated to the 31 genes was 587. Of these probes, 39 were annotated to a different isoform than the one included in the hypoxia score in a previous study (Halle et al, supra), and were therefore excluded. The 32 probes that were located within 10 bp of a known SNP were not included in further analyses.
  • Methylation probes that showed little variation across patients, as defined by a standard deviation of ⁇ 0.06 were removed. After these selection steps were performed, 168
  • the methylation probes in the study that showed a correlation with expression of their annotated gene were located both in the promoter and in the body of the genes (Table 5). Moreover, most of the promoter methylation and gene body methylation were positively and negatively correlated with gene expression, respectively.
  • the Infinium 450K DNA methylation assay was performed on an independent cohort of 117 cervical cancer patients, and gene expression profiling was assessed by the Illumina bead arrays human HT12-v4.
  • a Spearman rank correlation analysis was performed to evaluate the correlation between methylation and gene expression for all the genes in the hypoxia score.
  • a correlation between methylation and gene expression was validated for all but three of the 21 genes from the test cohort (sub-cohort 1).
  • 70 of the methylation probes, representing 18 genes were significantly correlated with gene expression.
  • the correlations between gene expression and correlation for both cohorts are visualized in Figure 4, while the correlation data are listed in table 5.
  • Example 1 cohort 1 was used to assess the correlation between gene expression probes and methylation probes for the 31 genes. In the current analysis (Example 2), this correlation was re-assessed in an optimized sub-group of cohort 1 , called sub-cohort 1 , and further validated in cohort 2. Table 6 summarizes which genes had correlating gene expression and methylation probes in the various cohorts. Twenty genes had correlating probes in independent cohorts (at least one from cohort 1 and sub-cohort 1, plus cohort 2). These genes constitute are highlighted in table 6.
  • b Promoter was defined as +/- 2000 bp from the transcriptional start site, while gene body was defined as the remaining part of the transcript.
  • Table 6 Summary of genes displaying significant correlation between gene expression and methylation from the various analyses and cohorts
  • Table 7 shows probe exemplary probe sequences. Two Infinium design types were used. Type I design involves two probes per methylation locus (e.g., allele A and allele B), while type II design has one probe per locus.
  • AAATATACCCC II AAAATACAATA AAAAAATACCT AAAAATATTTA
  • AAACCTATATA II AC CCTACAAA ACTAAAACCTA ACRATAAAAAT
  • AAAAATCRATA II AAAACAATCAT AAAATATAAAA CTCAAC CAAC
  • CACCATCCCCA AACTCCTAAC ACTCCTAACCA CAAATCAACA AATCAACAAAC AACCAAAAA CAAAAAATAAT ATAATAACAC
  • CAAATACTACC II AAACAAAATAC CAAACCAACCT TTTAAAAACAA

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Abstract

Cette invention concerne des méthodes et des biomarqueurs pour la détection du cancer du col de l'utérus dans des échantillons biologiques, et en particulier des marqueurs d'état de méthylation associés à l'agressivité du cancer du col de l'utérus.
PCT/IB2015/000567 2014-01-16 2015-01-16 Méthodes et biomarqueurs pour la détection et le pronostic du cancer du col de l'utérus WO2015107430A2 (fr)

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CN108949970A (zh) * 2017-05-23 2018-12-07 中国科学院深圳先进技术研究院 基于多组学的***特征获取方法和***
RU2809330C1 (ru) * 2023-10-20 2023-12-11 Федеральное государственное бюджетное образовательное учреждение высшего образования "Курский государственный медицинский университет" Министерства здравоохранения Российской Федерации Способ генотипирования полиморфного локуса rs2277947 (G>A) гена C19orf53 у человека методом ПЦР в режиме "реального времени" с применением аллель-специфических флуоресцентных зондов

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