JP2007531512A - Gynecological cell proliferation disorder analysis method - Google Patents

Abstract

【Task】
The present invention relates to a method for the detection, differentiation and prognosis of gynecological cell proliferation disorders, the following steps: a) obtaining a cervicovaginal secretion specimen from an individual, b) at least one or more CpGs Measuring the methylation status of the position, c) determining from this methylation status the presence, classification and / or prognosis of a gynecological cell proliferation disorder in the individual. Furthermore, the present invention relates to a kit for carrying out the method.

Description

  The present invention relates to a method for the detection, discrimination and prognosis of gynecological cell proliferation disorders, the following steps: a) obtaining a cervicovaginal specimen from an individual; b) methylation of at least one or more CpG positions Measuring the condition; c) determining from this methylation status the presence, classification and / or prognosis of a gynecological cell proliferation disorder in this individual. Furthermore, the present invention relates to a kit for carrying out the method.

Endometrial cancer Endometrial cancer is one of the most common reproductive cancers of women around the world. The highest incidence has been observed in Western and North America. Known risk factors for endometrial cancer include obesity, type II diabetes and hypertension. In addition, prolonged use of estrogens in ovulation arrest and hormone replacement therapy increases the risk of endometrial cancer. The genetic cause of endometrial cancer is rare despite the involvement of hereditary nonpolyposis colorectal cancer syndrome, and the individual risk increases to a cumulative incidence of 40% in the 70s. In 2001, the American Cancer Society established a screening for endometrial cancer in women with an average or increased risk of long-term estrogen therapy, inexperienced birth, infertility or ovulation failure, obesity, diabetes or hypertension. We concluded that the evidence is insufficient to recommend. Trial studies of screening methods for endometrial carcinoma in asymptomatic postmenopausal women use endometrial thickness measured by ultrasonography as an indication of risk. Transvaginal ultrasonography compared to endometrial biopsy for detecting endometrial disease has a sensitivity of 90% and specificity of 48% and only 9% positive to detect anomalies Predicted values (Langer, RD, Pierce, JJ, O'Hanlan, KA, Johnson, SR, Espeland, MA, Trabal. JF, Barnabei, VM, Merino, MJ, and Scully, RE "Detection of endometrial disease Transvaginal ultrasonography compared to endometrial biopsy in "Non-Patent Document 1". There is a need for sensitive and specific screening tests in high-risk women. It has been shown that genetic abnormalities can be used to detect endometrial cancer. Changes in the DNA methylation state are the most common molecular changes in human abnormal growth (Jones, PA “DNA methylation errors and cancer”, Non-Patent Document 2). The CpG islands of many genes that are not methylated in normal tissues have been remarkably recognized over the last 4-5 years that they are methylated to varying degrees in multiple types of human cancers ( Jones, PA, and Laird, PW “Post-cancer biology is well developed”, Non-Patent Document 3). Aberrant methylation of CpG islands in the promoter region of several genes such as E-cadherin, adenomatous polyp (APC), MLH1, p16, estrogen receptor, progesterone receptor and PTEN (MMAC1) It has been confirmed in endometrial cancer tissues. To date, no studies have been conducted to assess the methylation status of DNA obtained from cervicovaginal secretions of endometrial cancer patients.

  Long-term tamoxifen users with an increased risk of endometrial cancer have a poor prognosis in such cancers. This seems to be due to the lower prognosis group and higher stage. This indicates in particular that there is an urgent need for simple and non-invasive means of early detection of endometrial cancer in this subgroup of women.

Cervical Cancer Cervical cancer is an important cause of death in women around the world. Since the introduction of PAP smears into screening programs, the incidence and mortality of cervical cancer has decreased dramatically. However, successful screening methods are highly dependent on population coverage and the sensitivity and specificity of screening tests. A meta-analysis of studies investigating the pap test for the detection of cervical cancer and its precursors reveals sensitivity ranging from 30% to 87% and specificity ranging from 86% to 100% did. Collecting evidence from epidemiological and molecular studies in one point suggests that reproductive human papillomavirus (HPV ³ ) infection is accidentally linked to the development of cervical cancer. Therefore, tests for HPV DNA are being evaluated to improve cervical cancer screening. Many studies have shown high sensitivity in HPV tests to detect cervical cancer and its precursors, but the specificity is usually low compared to histology. In order to reduce the hassle and expense of repeatedly visiting a doctor, it has been proposed that women themselves collect cervical and vaginal specimens for HPV DNA analysis and possibly increase the coverage of screening programs.

  Several studies investigated the HPV DNA detection rate between self-collected samples and physician-collected samples, varying the point of concordance between the two different collection methods. Since many patients with HPV-related lesions do not progress to invasive cancer, it is clear that in addition to HPV infection, other factors are also involved in cervical carcinogenesis. Changes in DNA methylation status are the most common molecular denaturation in human overgrowth. Recently, abnormal methylation patterns have been found in multi-stage pathogenesis of cervical cancer with a tendency to increase methylation as pathological changes increase (Virmani, AK, Muller, C., Rathi, A., Zoechbauer-Mueller, S., Mathis, M., and Gazdar, AF "Abnormal methylation during cervical carcinogenesis", Non-Patent Document 4).

  In the Kinney et al. Study, over 60% of women diagnosed with invasive cervical cancer were not screened despite enrollment in a health maintenance organization. In order to reduce the hassle and expense of repeatedly attending a doctor, it has been proposed that women collect cervical and vaginal specimens themselves, and it is desirable to increase the application range of screening programs if possible. Previous studies investigating HVP DNA detection rates between self-collected and physician-collected samples described various agreements between two different collection methods. Recently, doctor-stained smears have been shown to detect up to 28% HPV-positive women compared to specimens collected by tampon. Collecting specimens with tampon is not a suitable method for HPV DNA detection, as many studies have demonstrated nearly 100% sensitivity in detecting SIL and cervical cancer by HVP DNA testing of doctor-collected samples It seems.

  In addition to HPV infection, it has been proposed that genetic or epigenetic mutations are necessary to maintain a malignant phenotype. Changes in DNA methylation status are the most common molecular mutation in human tumorigenesis. Recently, abnormal methylation has been suggested to play a role in cervical carcinogenesis (Virmani, AK, Muller, C., Rathi, A., Zoechbaur-Mueller, S., Mathis, M., And Gazdar, AF, “Abnormal methylation during cervical carcinogenesis”, Non-Patent Document 5).

  Cervical cancer is the leading cause of cancer death in women. The 5-year survival rate is in the range of 15-80%, depending on the extent of the disease. Recently, several studies have shown a significant reduction in the risk of cervical cancer recurrence and death. This was achieved by the simultaneous use of chemotherapy and radiation therapy. New predictive markers of recurrence will increase survival by improving the treatment of patients at high risk of recurrence.

  Several clinical and pathological features have been shown to be prognostic factors for recurrent disease, namely tumor stage, lymph node metastasis and vascular invasion. However, new and biochemical approaches for the identification and treatment of high-risk patients are needed to improve survival and avoid overtreatment of low-risk patients. The gene products of CDH1 and CDH13, ie E-cadherin and H-cadherin, play an important role in cell-cell adhesion. Changes in cell-cell and cell-matrix adhesion are accompanied by a change from benign tumor to invasive malignant cancer, followed by metastatic dissemination of tumor cells. Reduction or loss of E-cadherin expression is a common finding in many human epithelial cancers, including cervical cancer. The cadherin-mediated cell adhesion system can be inactivated by several mechanisms. Abnormal methylation of the E-cadherin (CDH1) and H-cadherin (CDH13) promoter or 5′-region CpG islands has been reported to result in reduced E-cadherin and H-cadherin expression. Many studies have shown findings with tumor-specific changes in DNA recovered from plasma or serum of patients with various grades, ie, molecular diagnostics and prognostic potential.

  Abnormal expression of cell adhesion molecules such as E-cadherin (CDH1) and H-cadherin (CDH13) occurs in various neoplastic diseases, and these abnormalities are meaningful in the progression of certain tumors including cervical cancer . Several mechanisms have been proposed in cadherin downstream regulation, such as tumor hypoxemia and necrosis, stimulation of epidermal growth factor receptor (EGFR) by EGF or TGF-α and mutation of the CDH1 gene. Recently, aberrant promoter methylation of CDH1 and CDH13 has been described as one of the mechanisms that result in loss or reduction of E-cadherin and H-cadherin expression. A decrease in E-cadherin expression has been shown to be associated with enhanced metastatic activity or more aggressive malignancies.

  Methylated DNA has been studied as a potential screening marker for neoplastic disease in several body fluids (Muller, HM and Widschwendter, M. “As a potential screening marker for neoplastic disease in several body fluids. Methylated DNA ", Non-Patent Document 6). However, no studies have so far been conducted to evaluate the methylation status of DNA obtained from cervical secretions in the evaluation of patients with gynecological cell proliferation disorders.

  Compared to endometrial histology for detecting endometrial disease, current methods of uterine cancer diagnosis, ie transvaginal ultrasonography, is 90% sensitive and 48% specific, It is estimated to show only 9% positive predictive value for detecting sex. There is a need in the art for sensitive and specific screening tests for women at high risk.

  The standard screening test for cervical cancer is a PAP smear. Effective screening is strongly dependent on the population coverage ratio and the sensitivity and specificity of the screening test. A meta-analysis of studies investigating the PAP test for detection of cervical cancer and its precursors revealed a sensitivity of 30% -87% and specificity of 86% -100%. However, the success of screening programs aimed at detecting precancerous conditions (dysplasia) and treating them before they progress is often limited by socio-economic factors. It is estimated that only about 5% of women in the developing world have been screened in the developed countries compared to nearly 40% to 50% of women in the developed countries who have been screened for cervical dysplasia. Successful screening of cervical cancer populations, especially high-risk populations with low socioeconomic status, requires a sensitive, specific, cost-effective and self-managing cervical cancer test.

  Cervical cancer is often a treatable disease, but in these patients with poor prognosis, a significant reduction in the risk of recurrence and death can be achieved by the combined use of chemotherapy and radiation therapy. However, current prognostic markers, primarily histological markers, show only limited signs of patient prognosis. Furthermore, treatment of patients who are not at risk of recurrence with adjuvant treatment can be an unwanted side effect. Thus, there is a need in the art for improved self-managing tools for the detection and prognosis of cervical cancer.

  As used herein, the term “prognosis” is taken to mean the prediction of disease progression. This is measured by appropriate parameters including, but not limited to, survival rate. This is preferably used to help define the level of patient death risk resulting from the inherent heterogeneity of the disease process.

As used herein, the term “survival” refers to survival to death (where the death is not related to a gynecological cell proliferation disorder-related cause associated); survival without recurrence (Where the term recurrence includes both localized recurrence and distant recurrence); survival without metastasis and disease-free survival (where the term disease is associated with gynecologic cell proliferation disorders and this) Including disease). These survival lengths are calculated with reference to the user's defined starting point (eg, at the start of diagnosis or treatment) and end point (eg, death, recurrence or metastasis).
As described herein, the term “cervicovaginal secretion” is taken to mean all substances secreted from cells, glands or organs into the cervical or vaginal region.
Postmenopausal Estrogen / Progestin Interventions Trial. N. Engl. J. Med., 337: 1792-1798, 1997 Cancer Res., 56; 2463-2467, 1996 Nat. Genet., 21; 163-167, 1999 Clin. Cancer Res., 7: 584-589, 2001 Clin. Cancer Re., 7: 584-589, 2001 Expert Rev Mol Diagn, 3: 443-458, 2003

  The present invention relates to a method for the detection, discrimination and prognosis of gynecological cell proliferation disorders, the following steps: a) obtaining a cervicovaginal specimen from an individual; b) methylation of at least one or more CpG positions Measuring the condition; c) determining from this methylation status the presence, classification and / or prognosis of a gynecological cell proliferation disorder in this individual. Furthermore, the present invention relates to a kit for carrying out the method.

  The method of the present invention provides a novel sensitive and specific means for the detection, discrimination and prognosis of gynecological cell proliferation disorders by analysis of cervicovaginal secretions. This allows the method to collect specimens by the patient without the assistance of a doctor. In a further aspect, the present invention provides a method for analyzing said specimen, particularly in the endometrium or cervix, for genomic methylation aspects associated with various gynecological cell proliferation disorders. In a further aspect, the present invention provides novel nucleic acids for the analysis of cervicovaginal secretion specimens that allow detection, discrimination and prognosis of gynecological cell proliferation disorders. Furthermore, the present invention provides a kit for the analysis of cervicovaginal secretions for the analysis of methylation aspects allowing the detection, discrimination and prognosis of gynecological cell proliferation disorders.

The method of the present invention comprises the following steps:
a) obtaining a cervical vaginal discharge specimen from an individual;
b) measuring the methylation status of at least one or more CpG positions;
c) determining the presence, classification and / or prognosis of the individual's gynecological cell proliferation disorder from its methylation status.

  Cervical vaginal secretions are obtained by standard means in the art including, but not limited to, gynecological smears, aspiration and cervicovaginal lavage. However, in the most preferred embodiment of these methods, the specimen is collected by a tampon inserted into the individual's vaginal passage. The tampon is then preferably stored in a buffer or other suitable solution. In a particularly preferred embodiment of the invention, this step of the method is performed by the individual without the assistance of a physician or other health professional. The solute containing DNA obtained from the cervical vagina is then used in the next step of the method.

  The use of self-managed cervical vaginal specimen collection methods is well established as a test for HPV (human papillomavirus) and cervical cancer indicators. In a study in the US (Harper DM, Hildesheim A, Cobb JL, Greenberg M, Vaught J, Lorincz AT, "Human Papillomavirus Collection Device" J Fam Pract. 1999, Jul; 48 (7): 531-5) A diagnostic concordance rate of 80% was shown between the sample collected by the doctor using the tampon and the sample collected by the patient. However, the use of this technique in the analysis of the affected tissue itself has not been published so far.

  The cervicovaginal specimen or these solutions are then analyzed by methylation analysis to detect abnormal methylation patterns involved in the development of gynecological cell proliferation disorders. This is possible by analyzing one or more CpG positions, whose abnormal methylation is an aspect of gynecological cell proliferation disorder or a prognostic sign thereof. In the final step of the method, the presence, classification and / or prognosis of the gynecological cell proliferation disorder is determined from the measured methylation status of one or more CpG positions.

  In a preferred embodiment of the method, the gynecological cell proliferative disorder is dysplastic or low-grade squamous intraepithelial lesion, high-grade squamous intraepithelial lesion, cervical cancer, endometrial cancer and grade 1-3 cervical epithelial Selected from the group consisting of abnormal growth.

  In one embodiment of the method, the CpG position is CDH1, CDH13, RASSF1A, hMLH1, HSPA2, SOCS1, SOCS2, GSTP1, DAPK, TIMP3, hTERT, SFRP2, SFRP4, SFRP5 and CCND2 and / or their promoters; It is selected from one or more genes selected from the group consisting of introns, first exons, regulatory elements and / or enhancers. It is another aspect of the present invention that the sequences of these genes are selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 11 and SEQ ID NO: 64 to SEQ ID NO: 67 listed in Table 7.

  In one embodiment of this method, endometrial cancer is detected in other gynecological cell proliferation disorders by analysis of one or more CpG positions of genes SFRP2, SFRP4, SFRP5, CCND2, RASSF1A, hMLH1, CDH13, HSPA2 and SOC2. Detected or determined. In a further embodiment of the method, the sequence of these genes is selected from SEQ ID NOs: 2, 3, 4, 5, 7, 64, 65, 66 and 67 listed in Table 7.

  In one embodiment of the method, the cervical cancer is one or more of the genes SFRP2, SFRP4, SFRP5, CCND2, SOCS1, CDH1, TIMP3, GSTP1, DAPK, hTERT, CDH13, HSPA2, MLH1, RASSF1A and SOCS2. It is detected or discriminated from other gynecological cell proliferation disorders by analysis of the CpG position. In a further embodiment of the method, the sequences of these genes are selected from SEQ ID NO: 1 to SEQ ID NO: 11 and SEQ ID NO: 64 to SEQ ID NO: 67 listed in Table 7.

  In a further embodiment of the method, the prognosis of a patient with cervical cancer is determined by analysis of one or both of the genes CDH1 and CDH13. In a further embodiment of the method, the sequence of the gene is selected from SEQ ID NOs: 1 and 2 listed in Table 7. Gene hypermethylation is associated with poor prognosis with poor life expectancy.

  As soon as the cervicovaginal secretion specimen is obtained from the patient in step a) of the method, genomic DNA is isolated. This is done by standard means in the art including the use of commercially available kits. Briefly, the DNA of interest is encapsulated in the cell membrane, and the biological sample must be destroyed or lysed by enzymatic, chemical or mechanical means. The DNA solution is then removed of proteins or other impurities, for example, by digestion using proteinase K. The genomic DNA is then recovered from the solution. This may be done by a variety of methods including salting out, organic solvent extraction or binding of DNA to a solid phase support. The choice of method will be influenced by several factors including time, expense and the amount of DNA required.

  The isolated genomic DNA is then analyzed for aberrant methylation of CpG dinucleotides. This is done by standard means in the art including the use of methylation sensitive restriction enzymes. However, since only a small amount of DNA obtained from the affected tissue is present in the sample, it is required to use a method with high sensitivity. Thus, step b) of the present method comprises genomic DNA or its reagents with one or more reagents that convert the unmethylated cytosine base at position 5 into uracil or other bases that are detectably different from cytosine in hybridization properties. Process and run the fragment. This treatment is preferably carried out with a bisulfite reagent (bisulfite, disulfite, bisulfite or combinations thereof) followed by alkaline hydrolysis. It is another embodiment of the method that the sequence to be analyzed is selected from the group consisting of SEQ ID NO: 12 to SEQ ID NO: 55 and SEQ ID NO: 68 to SEQ ID NO: 83.

  The treated genomic DNA or the treated fragment is then preferably complementary to the amplification enzyme and the respective target nucleic acid, or of moderately stringent or length that hybridizes under stringent conditions. Contact with at least two primers comprising a contiguous sequence that is at least nine nucleotides. Furthermore, it is another embodiment of the method that the sequence to be analyzed is selected from the group consisting of SEQ ID NO: 12 to SEQ ID NO: 55 and SEQ ID NO: 68 to SEQ ID NO: 83. In the final step of the method, an average methylation state or a value reflecting the average methylation state of at least one CpG dinucleotide sequence or a plurality of CpG dinucleotide sequences is measured based on the presence or absence of the amplification product or its characteristics. Thereby at least one of detecting gynecological cell proliferative disorders, determining gynecological cell proliferative disorders, or at least partially providing a prognosis.

  This is done by standard methods in the art that allow the detection of small amounts of methylated DNA against a high background of unmethylated DNA. In particular, MSP, HeavyMethyl (blocking with oligonucleotides) and RealTimes analysis (including but not limited to Lightcycler and MethyLight analysis) and all possible combinations thereof are preferred.

  The term “MSP” (methylation specific PCR) refers to an art recognized methylation analysis described in Herman et al., Proc. Natl. Acad. Sci. USA 93: 9821-9826, 1996. Say. In MSP application, methylated and unmethylated nucleic acids can be distinguished by the use of methylation state specific primers in the amplification of bisulfite treated DNA. The MSP primer pair includes at least one primer that hybridizes to a predetermined methylated bisulfite-treated CpG dinucleotide. Accordingly, the sequences of these primers include at least one CpG, TpG or CpA dinucleotide. MSP primers specific for unmethylated DNA contain a “T” at the 3 ′ position of the C-position of the CpG dinucleotide. The presence of methylated nucleic acid can be determined from the detection of the amplified product. Thereby, the use of MSP allows the detection of a nucleic acid in a predetermined methylated state amplified against the background of the other methylated nucleic acid (see FIG. 8 and its description herein).

The HeavyMethyl ^R technique, polymerase amplification assisted by the use of blocking oligonucleotides. This technique is used to block amplification of one methylated state target nucleic acid and increases the relative proportion of other methylated state amplification product nucleic acids. Primers are methylation specific (“MSP”) or specific for non-CpG treated target nucleic acids. The methylation status of bisulphite-treated CpG dinucleotides is not displaced by the action of polymerase and is thus measured by oligonucleotide blocking probes that block sequence amplification (FIG. 9). Blocking oligonucleotides can be unsubstituted by using polymerases that lack 5′-3 ′ exonuclease activity, using peptide nucleic acid oligomers, or suitable modified oligonucleotides (eg, DNA oligomers lacking a free 3′-hydroxyl group). May be used.

Figure 9 shows a polymerase-mediated amplification analysis of bisulfite treated DNA corresponding to the CpG rich genomic sequence using HeavyMethyl ^R technology ( "3"). Amplification of the treated DNA (“3”) is not possible if the blocking oligonucleotide (“5”) anneals to the treated DNA as shown in Example Case “B”. The arrow (“1”) indicates the primer, and the black circle marker position (“2”) on the bisulfite-treated nucleic acid strand (“3”) indicates the methylated bisulfite converted CpG position. On the other hand, the white circle marker position (“4”) indicates an unmethylated bisulfite conversion position. Blocking oligonucleotides (blockers) are indicated by black arrows (“5”). In Example Case A, all subjects' genomic CpG positions are both methylated, and both forward and reverse primers anneal to amplify without interference with the corresponding treated nucleic acid ("3"). Bring. In the second example case “B”, all of the subject's genomic CpG positions are not methylated and both the forward and reverse primers anneal to the treated DNA sequence (“3”), but amplify the sequence. I can't. This is because the synthesis of the complementary strand is blocked by a blocking oligonucleotide ("5") that anneals to the complementary position containing the unmethylated CpG sequence of the subject's genomic DNA.

“Real-time” based methods use detection probes that hybridize to the target nucleic acid processed during PCR amplification, thereby allowing detection of amplified product nucleic acids. The detection probe may be designed to hybridize to a methylation specific target sequence by including one or more CpG, TpG or CpG dinucleotides. Suitable real-time PCR-based methods include the art-recognized fluorescent real-time PCR technology MethyLight ^™ (Eads et al., Cancer Res. 59: 2302-2306, 1999; Laird et al., US Pat. No. 6,331,393; and Heid Et al., Genome Res. 6: 986-994, 1996) and TaqMan analysis. A particularly preferred embodiment is a fluorescence-based real-time quantitative PCR (Heid et al., Using a dual-labeled fluorescent oligonucleotide probe (TaqMan ^™ PCR using the ABI Prism 7700 sequencing system (Perkin Elmer Applied Biosystems, Forster City, California)). Genome Res. 6: 986-994, 1996). The TaqMan ^™ PCR reaction uses a non-extendable interrogating oligonucleotide called the TaqMan ^™ probe that is designed to hybridize to a CpG rich sequence located between the forward and reverse amplification primers. . The TaqMan ^™ probe further includes a fluorescent “reporter molecule” and a “quencher molecule” covalently linked to a linker molecule (eg, a phosphoramidite) that binds to the nucleotides of the TaqMan ^™ oligonucleotide. The methylation analysis of the nucleic acid following the bisulfite treatment, methylation specific, as probes are preferably described in the prior an is U.S. Patent No. 6,331,393 (inserted by reference herein) as MethylLight ^R Analysis Is. Modifications in the TaqMan ^™ detection method suitable for use in the described invention include the use of double probe technology (Lightcycler ^™ ) or fluorescence amplification primers (Sunrise ^™ technology).

  The described invention provides processed nucleic acids derived from genomic SEQ ID NO: 1 to SEQ ID NO11 and SEQ ID NO: 64 to SEQ ID NO: 67. Here, the treatment is suitable for converting at least one unmethylated cytosine base of the genomic DNA sequence into uracil or other base that is detectably different from cytosine in hybridization. The genomic sequence in question may contain one or more consecutive or random methylated CpG positions. This treatment preferably comprises the use of a reagent selected from the group consisting of bisulfite, bisulfite, disulfite and combinations thereof. In a preferred embodiment of the present invention, the object is a non-naturally modified nucleic acid comprising a contiguous nucleotide base sequence of at least 16 lengths selected from the group consisting of SEQ ID NO: 12 to SEQ ID NO: 55 and SEQ ID NO: 68 to SEQ ID NO: 83. Includes analysis. Here, this sequence comprises at least one CpG, TpA or CpA dinucleotide and sequences complementary thereto. The sequences of SEQ ID NO: 12 to SEQ ID NO: 55 and SEQ ID NO: 68 to SEQ ID NO: 83 are non-naturally occurring nucleic acids described in SEQ ID NO: 1 to SEQ ID NO: 11 and SEQ ID NO: 64 to SEQ ID NO: 67 and SEQ ID NO: 64 to SEQ ID NO: 67. Provide denatured product. Here, the denaturation of each genomic sequence results in the synthesis of a nucleic acid that is special and has a sequence that is discriminated from this genomic sequence as follows. For each sense strand genomic DNA, eg SEQ ID NO: 1, four transformed versions are described. The first version converts “C” to “T”, but “CpG” remains “CpG” (ie, in the genomic sequence, all “C” residues of the CpG dinucleotide sequence are methylated) And therefore corresponds to the case of no conversion). The second version describes the complementary strand (ie, the antisense strand) of the described genomic DNA sequence, where “C” is converted to “T”, but “CpG” remains “CpG” ( That is, it corresponds to the case where all “C” residues of the CpG dinucleotide sequence are methylated and therefore not converted). The “upmethylated” converted sequences of SEQ ID NO: 1 to SEQ ID NO: 11 and SEQ ID NO: 64 to SEQ ID NO: 67 correspond to SEQ ID NO: 12 to SEQ ID NO: 33 and SEQ ID NO: 68 to SEQ ID NO: 75. A third chemical conversion version of each genomic sequence is provided, where “C” is converted to “T” in all “C” residues, including that of the “CpG” dinucleotide sequence (ie, In the genomic sequence, this corresponds to the case where all “C” residues of the CpG dinucleotide sequence are unmethylated), the last chemically transformed version of each sequence is the complementary strand of the described genomic DNA sequence (ie , Antisense strand), where “C” is converted to “T” in all “C” residues including those of the “CpG” dinucleotide sequence (ie, complementary to each genomic sequence) In the strand (antisense), all “C” residues of the CpG dinucleotide sequence are unmethylated). The “downmethylated” converted sequences of SEQ ID NO: 1 to SEQ ID NO: 11 and SEQ ID NO: 64 to SEQ ID NO: 67 correspond to SEQ ID NO: 34 to SEQ ID NO: 55 and SEQ ID NO: 76 to SEQ ID NO: 83.

  In another preferred embodiment, such analysis comprises cytosine methylation in the genome or processed (chemically denatured) DNA as set forth in SEQ ID NO: 12-SEQ ID NO: 55 and SEQ ID NO: 68-SEQ ID NO: 83. Including the use of oligonucleotides or oligomers to detect the condition. These oligonucleotides or oligomers are processed nucleic acid sequences set forth in SEQ ID NO: 12-SEQ ID NO: 55 and SEQ ID NO: 68-SEQ ID NO: 83 and / or their complementary sequences, or SEQ ID NO: 1-SEQ ID NO: 11 And moderately stringent or stringent conditions (as defined above) to the genomic sequences set forth in SEQ ID NO: 64 to SEQ ID NO: 67 and SEQ ID NO: 64 to SEQ ID NO: 67 and / or sequences complementary thereto. And a nucleic acid sequence having a length of at least 9 nucleotides.

  That is, the present invention hybridizes under moderately stringent and / or stringent conditions to all or a part of the sequences of SEQ ID NO: 1 to SEQ ID NO: 55 and SEQ ID NO: 64 to SEQ ID NO: 83 or their complementary strands. Nucleic acid molecules such as oligonucleotides and peptide nucleic acid (PNA) molecules (PNA-oligomers). The hybridizing portion of the hybridizing nucleic acid is usually at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, at least 30 or 35 nucleotides. However, longer molecules also have utility and are therefore included within the scope of the present invention.

  Preferably, the hybridization portion of the hybridizing nucleic acid of the present invention comprises at least 95% or at least the sequence of SEQ ID NO: 1 to SEQ ID NO: 55 and SEQ ID NO: 64 to SEQ ID NO: 83 or a part thereof, or their complementary strands. 98% or 100% identical.

  Hybridizing nucleic acids of the type described herein can be used, for example, as a primer (eg, a PCR primer), or a diagnostic and / or prognostic probe or primer. Preferably, hybridization of the oligonucleotide probe to the nucleic acid sample is performed under stringent conditions, and the probe is 100% identical to the target sequence. The stability of a nucleic acid duplex or hybrid is expressed as the melting point or Tm. This is the temperature at which the probe dissociates from the target DNA. This melting point is used to define the required stringent conditions.

  In a preferred embodiment, the present invention is complementary or moderately stringent to a sequence selected from the group consisting of SEQ ID NO: 12 to SEQ ID NO: 55 and SEQ ID NO: 68 to SEQ ID NO: 83 and their complementary strands. Or a nucleic acid molecule comprising in each case a contiguous sequence of at least 9 nucleotides that hybridize under stringent conditions, wherein the nucleic acid molecules are SEQ ID NO: 1 to SEQ ID NO: 11 and SEQ ID NO: 64 to SEQ ID NO: 67 The corresponding unprocessed nucleic acid molecule described in 1 contains at least one TpA or CpA dinucleotide at the position containing the CpG dinucleotide. Preferred lengths of such nucleic acid molecules are 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29, 30, 35 nucleotides.

  A target sequence (allelic variant) that is related to or substantially identical to the corresponding sequence of SEQ ID NO: 1 to SEQ ID NO: 11 and SEQ ID NO: 64 to SEQ ID NO: 67 and SEQ ID NO: 64 to SEQ ID NO: 67. In SNP or the like, it is useful to first determine the minimum temperature at which only homogeneous hybridization occurs at a particular concentration of salt (eg, SSC or SSPE). Then, assuming that 1% mismatching results in a 1 ° C. decrease in Tm, the final wash temperature of the hybridization reaction is reduced accordingly (eg, if a sequence with> 95% identity with the probe is desired) Reduce final wash temperature by 5 ° C). Actually, the change in Tm is 0.5 to 1.5 ° C. per 1% mismatch.

For example, as shown by the polynucleotide position referring to SEQ ID NO: 1, an oligonucleotide of the present invention of length X (nucleotide) includes a set of consecutive overlapping oligonucleotides of length X (sense and antisense) The thing corresponding to is mentioned. The oligonucleotides in each consecutive overlapping set (corresponding to a given X value) are defined as a finite set of Z oligonucleotides from the nucleotide position:
n to (n + (X-1));
Here, n = 1, 2, 3,. . . (Y- (X-1));
Where Y is equal to the length of SEQ ID NO: 1 (nucleotide or base pair) (3190);
Where X is the common length (nucleotide) of each oligonucleotide in the set (eg, X = 20 for a set of consecutive duplicate 20-mers); and where long at a given sequence number of length Y The number of consecutive overlapping oligomers of length X (Z) is equal to Y- (X-1). For example, either the sense or antisense set of SEQ ID NO: 1 is Z = 3190-19 = 3171, where X = 20.
Preferably, this set is limited to those oligomers comprising at least one CpG, TpG or CpA dinucleotide.

Examples of 20-mer oligonucleotides of the invention include the following oligomers indicated by the position of the polynucleotide of SEQ ID NO: 1: 1-20, 2-21, 3-22, 4-23, 5-24,. . . 3171-3190 sets (and antisense sets complementary to them).
Preferably, this set is limited to those oligomers comprising at least one CpG, TpG or CpA dinucleotide.
Similarly, examples of 25-mer oligonucleotides of the present invention include the following oligomers indicated by the position of the polynucleotide of SEQ ID NO: 1: 1-25, 2-26, 3-27, 4-28, 5-29 ,. . . . . A set of 3164-3190 (and an antisense set complementary thereto).
Preferably, this set is limited to those oligomers comprising at least one CpG, TpG or CpA dinucleotide.

  The invention encompasses multiple consecutive overlapping sets of oligonucleotides of length X or degenerate oligonucleotides in each of SEQ ID NO: 1 to SEQ ID NO: 55 and SEQ ID NO: 64 to SEQ ID NO: 83 (sense or antisense). Here, for example, X = 9, 10, 17, 20, 22, 23, 25, 27, 30 or 35 nucleotides.

  The oligonucleotide or oligomer of the present invention is used for confirming genetic and epigenetic parameters of the genome sequence corresponding to SEQ ID NO: 1 to SEQ ID NO: 11 and SEQ ID NO: 64 to SEQ ID NO: 67 and SEQ ID NO: 64 to SEQ ID NO: 67. Construct useful and effective tools.

  Particularly preferred oligonucleotides or oligomers of the invention are those in which the cytosine of the CpG dinucleotide sequence (or the corresponding converted TpG or CpA dinucleotide) is present in the middle of one third of the oligonucleotide. That is, when the oligonucleotide is, for example, 13 bases in length, the CpG, TpG or CpA dinucleotide is located within the 5th to 9th nucleotides from the 5′-end.

The oligonucleotides of the invention can also be modified to chemically attach the oligonucleotide to one or more molecules or conjugates to enhance the activity, stability or detection of the oligonucleotide. Such molecules or conjugates include chromophores, fluorophores, cholesterol and other lipids, cholic acid, thioethers, aliphatic chains, phospholipids, polyamines, polyethylene glycol (PEG), palmityl molecules, and the like. The probe may also be present in the form of a PNA (peptide nucleic acid) with particularly favorable pairing properties. That is, the oligonucleotide may contain other attachment groups such as peptides, such as hybridization-triggered cleavage reagents (Krol et al., BioTechniques 6: 958-976, 1988), or insertion reagents (Zon, Pharm. Res. 5: 539-549, 1988). For this purpose, the oligonucleotide may be linked to other molecules, such as chromophores, fluorophores, peptides, hybridization-induced cross-linking agents, transport reagents, hybridization-induced cleavage reagents, and the like.
Oligonucleotides may also include at least one art-recognized modified sugar and / or base molecule, or may include a modified backbone or non-natural nucleoside intranucleoside bridges.

  In certain embodiments of the invention, the oligonucleotide or oligomer is typically a CpG dinucleotide of the genomic sequence of SEQ ID NO: 1 to SEQ ID NO: 11 and SEQ ID NO: 64 to SEQ ID NO: 67 or a sequence complementary thereto, or SEQ ID NO: 12 At least one oligomer for the respective analysis of the corresponding CpG, TpG or CpG dinucleotide in the sequence of the processed nucleic acids set forth in SEQ ID NO: 55 and SEQ ID NO: 68-SEQ ID NO: 83 or their complementary sequences Used in a “set” containing However, due to economic or other factors, it is preferred to analyze the limited selection of CpG dinucleotides within these sequences, and the contents of the set of oligonucleotides are therefore expected to vary.

  Thus, in certain embodiments, the present invention relates to processed genomic DNA (SEQ ID NO: 12 to SEQ ID NO: 55 and SEQ ID NO: 68 to SEQ ID NO: 83), or genomic DNA (SEQ ID NO: 1 to SEQ ID NO: 11 and SEQ ID NO: 64 to Provided is a set of at least two (oligonucleotide and / or PNA-oligomer) that are useful for detecting the cytosine methylation status of SEQ ID NO: 67 and sequences complementary thereto. These probes allow diagnosis and / or classification of genetic and epigenetic parameters of lung cell proliferation disorders. The set of oligomers can be processed genomic DNA (SEQ ID NO: 12 to SEQ ID NO: 55 and SEQ ID NO: 68 to SEQ ID NO: 83) or genomic DNA (SEQ ID NO: 1 to SEQ ID NO: 11 and SEQ ID NO: 64 to SEQ ID NO: 67 and complementary to these) May be used to detect single nucleotide polymorphisms (SNPs).

In a preferred embodiment, at least one or more of the set of oligonucleotides, preferably all are bound to a solid phase.
In another embodiment, the present invention provides a “primer” oligo for amplifying one DNA sequence of SEQ ID NO: 1 to SEQ ID NO: 55 and SEQ ID NO: 64 to SEQ ID NO: 83 and a sequence complementary thereto, or a segment thereof. A set of at least two oligonucleotides used as nucleotides is provided.

  Oligonucleotides are expected to constitute all or part of an “array” or “DNA chip” (ie, a sequence of different oligonucleotides and / or PNA-oligomers bound to a solid phase). Such arrays with different oligonucleotide- and / or PNA-oligomer sequences are characterized, for example, by being arranged on a solid phase in a rectangular or hexagonal lattice. The solid surface is composed of silicone, glass, polystyrene, aluminum, steel, iron, copper, nickel, silver or gold. Nitrocellulose as well as plastics such as nylon, which are present in pellet form or as a resin matrix, are also used. An overview of the state of the art of oligomer array production can be gathered from a special issue of Nature Genetics (Nature Genetics Supplement, Volume 21, January 1999, and references cited therein). Fluorescently labeled probes are often used for scanning immobilized DNA arrays. The simple binding of specific probes of Cy3 and Cy5 dyes to 5′-OH is particularly suitable for fluorescent labeling. Detection of the fluorescence of the hybridized probe is performed, for example, with a confocal microscope. Cy3 and Cy5 dyes, as well as many others, are commercially available.

  Oligonucleotides or their specific sequences are also expected to constitute all or part of a “virtual array”. In doing so, oligonucleotides or their specific sequences are used as `` specifiers '', for example, as part of or in combination with a diverse population of specialized labeled probes for analyzing complex mixtures of analytes. Is done. Such a method is described, for example, in US Patent Publication No. 2003/0013091 (U.S. Application No. 09 / 898,743, publication date Jan. 16, 2003). In such a method, sufficient labels are generated so that each nucleic acid in a complex mixture (ie, each analyte) is uniquely bound to a unique label and is therefore detected (each label directly Counted to give a digital output of each molecular species in the mixture). In the final step of the method, the presence, classification, or prognosis is determined by the methylation status of the CpG position analyzed. This is done with reference to an existing data set that defines the features of the methylation pattern to each disease phenotype. Preferably, the correlation between the methylation status of the marker CpG position and the phenotypic parameter is substantially observed without human intervention. Machine learning algorithms automatically analyze experimental data, find systematic structures in them, and discriminate between non-informative and related parameters.

  Machine learning predictors are trained on the known phenotyping and methylation patterns of the examined CpG sites in the samples. CpG positions that prove discriminatory are used to define the reference data set. This method has been successful in cancer classification (Model, F., Adorjan, P., Olek, A., and Piepenbrock, C., Bioinformatics, 17 Suppl 1: 157-164, 2001).

Another aspect of the present invention is a kit useful for discriminating and detecting gynecological cell proliferation disorders in a subject, comprising at least one bisulfite reagent, or a methylation sensitive restriction enzyme; The sequence selected from the group consisting of SEQ ID NO: 12 to SEQ ID NO: 55 and SEQ ID NO: 68 to SEQ ID NO: 83, and those complementary to the complementary strand, or moderately stringent or stringent conditions It comprises at least one nucleic acid molecule or peptide nucleic acid molecule comprising a contiguous sequence of at least 9 nucleotides that hybridize underneath. Kit further comprises MSP, MethylLight ^R, the standard reagents for performing a methylation assay selected from the HeavyMethyl and combinations thereof. Additional components are at least two oligonucleotides, in each case these sequences are SEQ ID NO: 1 to SEQ ID NO: 55 and SEQ ID NO: 64 to SEQ ID NO: 83, preferably SEQ ID NO: 12 to SEQ ID NO: 55 and Of the sequences of SEQ ID NO: 68 to SEQ ID NO: 83, it is complementary to a 16 bp long segment, or hybridizes under stringent or highly stringent conditions.

Further aspects of the invention include, for example, a bisulfite-containing reagent; sequences of SEQ ID NO: 1 to SEQ ID NO: 55 and SEQ ID NO: 64 to SEQ ID NO: 83 (most preferably SEQ ID NO: 12 to SEQ ID NO: 55 and SEQ ID NO: 68). To SEQ ID NO: 83) of a primer oligonucleotide comprising at least two oligonucleotides that are complementary to a 16-base long segment or hybridize under stringent or highly stringent conditions A kit comprising a set; oligonucleotides and / or PNA-oligomers; and instructions for performing and evaluating the described method. In a further preferred embodiment, the kit further comprises standard reagents for achieving a CpG position-specific methylation analysis. Here, the analysis of one or more of the following techniques: including MSP, MethyLight ^R, the HeavyMethyl ^TM. However, the kit according to the present invention may contain only a part of the components.

MethyLight Typical reagents for ^R analysis (e.g., will be found in a typical MethyLight ^R system kit), PCR primers for specific gene (or methylation-modified DNA sequence or CpG island); TaqMan ^R Probes; optimized PCR buffers and deoxynucleotides; and Taq polymerase, but are not limited to these.

  Typical reagents for MSP analysis (eg, found in typical MSP-based kits) are methylated or unmethylated PCR for specific genes (or methylated denatured DNA sequences or CpG islands) Including but not limited to primers, optimized PCR buffers and deoxynucleotides, and specific probes.

HeavyMethyl Typical reagents for ^R analysis (e.g., will be found in a typical HeavyMethyl ^R system kit), PCR primers for specific gene (or methylation-modified DNA or CpG island), HeavyMethyl blocking oligo Includes but is not limited to nucleotides, optimized PCR buffers and deoxynucleotides, and polymerases.

In addition, the bisulfite conversion reagent includes a DNA denaturation buffer; a sulfonation buffer; a DNA recovery reagent or kit (eg, precipitation, ultrafiltration, affinity column); a desulfonation buffer; and a DNA recovery component. May be.

  The invention will be further described in the following examples with reference to the accompanying drawings and sequence protocols, but is not limited thereto. All publications cited herein are hereby incorporated by reference.

The next study was conducted to analyze whether methylated DNA in cervical vaginal secretions could be detected to detect endometrial cancer.
Patients and Samples 124 patients were recruited for this study: 15 patients had endometrial cancer, while non-endometrial cancer group had 5 invasive cervical cancer patients, 35 cervicovaginal Patients with intraepithelial neoplasia (CINI, 3 cases, CIN II, 19 cases, CIN III, 13 cases) and 69 patients with benign uterine disease were included. Sampling took place between 1 January 2003 and 31 May 2003 at the University of Innsbruck, Austria, Department of Obstetrics and Gynecology. All patients who were scheduled to undergo uterine surgery the next day, including histological diagnosis, were asked to participate in the study. After obtaining patient consent, samples and clinical data were collected. To ensure standardized sampling, a tampon was inserted into the patient by the physician after the microscopic examination and held in the vagina for 30 minutes. Sample preparation is shown in FIG.

DNA isolation and methylation analysis Uses High Pure Viral Nucleic Acid Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's protocol with several modifications of the DNA extraction column to obtain sufficient amounts of DNA Thus, genomic DNA was isolated from the sample. Sodium bisulfite treated genomic DNA was analyzed by MethyLight, fluorescent real-time PCR analysis as described above. Briefly, two sets of primers and probes specifically designed for bisulfite converted DNA were used: methylation set of the gene of interest and reference set, β-actin ( ACTB) was used. The reaction specificity of methylated DNA was confirmed separately using SssI (New England Biolabs) -treated human leukocyte DNA (highly methylated). The percentage of fully methylated molecules at a specific locus was calculated by dividing the GENE: ACTB ratio of the sample by the GENE: ACTB ratio of SssI-treated leukocyte DNA and multiplying by 100. The abbreviation PMR (percent of fully methylated basis) indicates this measurement. If PMR is> 0, the gene is judged to be methylated. Primers and probes that are specific for methylated DNA and used in the MethyLight reaction are shown in Table 1.

  Reaction conditions The reaction was carried out under the following analytical conditions: the reaction solution contained 300 nM forward primer, 300 nM reverse primer and 100 nM probe; plus magnesium chloride; taq polymerase, dNTP and DNA in a final reaction volume of 30 μl; cycling conditions: 95 ℃ 10 minutes, then 95 ℃, 15 seconds 50 times, 60 ℃, 1 minute.

  Statistical analysis Associations between categorical variables were tested using the Chi-Square test. Differences in median age were tested using the Mann-Whitney U test, and differences between two or more groups were tested using the Kruskal Wallis test. Due to the significant age difference between the endometrial cancer patient group and the non-endometrial cancer patient group, the age-matched group of the non-endometrial cancer patient group was randomly selected with a matching ratio of 1: 2. Match group calculations were performed using MATLAB R12 (www.mathworks.com). To determine diagnostic accuracy, non-parametric receiver operating curve (ROC) analysis using one-dimensional interpolation was performed. A P-value of 0.05 or less was considered statistically significant. All statistical calculations were performed using SPSS, Windows version 11.0.

Results Analyzing the aberrant methylation of 38 genes of DNA obtained from the vaginal secretions of the first 5 patients with endometrial cancer and the first 4 patients with benign disease to determine suitable genes for further study It was determined. The most appropriate gene for our further analysis was determined to reveal the largest difference in PMR values between benign uterine patients and endometrial cancer patients (FIG. 2). Five genes were selected for further study, namely RASSFIA, hMLH1, CDH13, HSPA2 and SOCS2. Three or more DNA methylations of these five genes were observed in the cervicovaginal secretions of all five patients with endometrial cancer. On the other hand, all 4 patients who did not have endometrial cancer showed no methylated genes or 3 or fewer genes. Therefore, we determined that 3 or more positive methylations out of 5 investigated genes were cut-off values between non-uterine and endometrial cancers.

  The overwhelming majority of patients with no endometrial cancer (99 out of 109) showed no methylated genes or no more than three. On the other hand, all 15 endometrial cancer patients had three or more methylated genes in their vaginal secretions (FIG. 3, P <0.001, Chi-2 test). The histological examination of 10 patients in the non-endometrial cancer patient group with 3 or more methylation genes was as follows: invasive cervical cancer (4 cases), CIN III (1 case), endometrial polyps (4 Example) and uterine fibroids (1 case). 16 samples out of 124 patients were collected after the primary surgery (curettage, cervical biopsy or hysteroscopic surgery) and before the secondary surgery (hysterectomy); 9/16 patients were endometrial cancer patients, 3/16 were CIN III, and 4/16 were benign endometrial diseases. All nine endometrial cancer patients have more than two methylation genes, three CIN III patients do not show methylation genes, and one patient with benign disease has one methylation The gene was shown. In a group of patients with vaginal secretions collected prior to surgery, one patient showed complete stenosis of the cervical uterine fallopian tube with a serometra detected by tomography. This patient showed 3 methylation out of 5 genes tested. Of the five genes identified, DNA methylation is not statistically significant (data not shown) but appears to increase with age. Using all 15 cases of endometrial cancer and 109 controls, the area under the ROC curve was 0.973 (data not shown). To rule out the possibility that aberrant DNA methylation is merely a substitute for age rather than cancer-specific markers, we randomly matched two non-endometrial cancer controls in each endometrial cancer case did. An investigation of DNA methylation in the cervicovaginal secretions of these 45 patients could still distinguish between endometrial cancer patients and non-endometrial cancer patients who still showed 100% sensitivity and 80% specificity ( P <0.001, Chi 2 test).

Analyzing all patients aged 50 to 75 years except for patients with CIN III or cervical cancer, the sensitivity was 100% and the specificity increased to 97.2% (FIG. 4). In this group, only one of 35 samples was false positive.
In our study, all 5 endometrial cancer patients revealed 3 or more of the 5 surveyed genes, while 99 out of 109 patients with no endometrial cancer were completely methylated No genes were shown, or no more than 3 methylated genes. In the non-uterine cancer group with 3 or more methylation genes, 4 out of 10 patients had invasive cervical cancer. These cases show that some cervical cancer patients can also be confirmed by this analysis.

  In some cases (16 out of 124), samples were taken after the primary surgery and before the secondary surgery. In this group, all endometrial cancer patients had 3 or more methylated genes. These results demonstrate that abnormal methylation analysis can detect endometrial cancer even after primary surgery.

  Since endometrial cancer is more prevalent in older women and abnormal methylation of non-malignant tissue increases with age (18), we have worked hard to solve this problem specifically within this project. did. Comparison of DNA methylation between cervicovaginal secretions of uterine body cancer patients and age-corresponding non-uterine body cancer control rotator revealed large and significant differences between these two groups.

  Endometrial cancer occurs in most cases after menopause. Therefore, we analyzed all patients aged 50 to 75 years except those with CIN III or invasive cervical cancer. These patients represent a group that would benefit from endometrial cancer screening tests. In these groups, the sensitivity and specificity for detecting patients with endometrial cancer were 100% and 97.2%, respectively.

  Long-term tamoxifen users who have an increased risk of endometrial cancer have a poor prognosis for such cancers. This seems to be due to the fact that the good tissue image is small and the stage is high. This indicates that there is an urgent need for a simple and non-invasive method of early detection of endometrial cancer, especially for women in this subgroup.

The purpose of this study was to determine whether DNA methylation analysis from HPV DNA tests and cervical vaginal samples collected by tampon can detect patients with invasive cervical cancer, and these changes are precursor lesions To test whether it already exists.
Patients and Samples 34 patients with vaginal intraepithelial overgrowth and 5 patients with invasive cervical cancer were included in this study. The patient was referred to our hospital for further treatment because of abnormal PAP smears or obvious vaginal lesions. The patient underwent vaginal surgical organization to obtain a histological diagnosis, or a cervical histology in cases of obvious cancerous lesions. In addition, 10 patients who did not have vaginal malformation but who underwent hysterectomy due to hysteromyoma were investigated. All patients were analyzed in a previous study conducted to determine whether it was possible to detect endometrial cancer by analyzing methylated DNA in cervicovaginal specimens collected by tampon (Example 1). ).

Sampling Samples and clinical data were collected after obtaining informed consent. To ensure standard sampling, the tampon was inserted into the vagina by the physician after the microscopic examination prior to surgery (the previous day) and held in the vagina for 30 minutes. Sample preparation was described in Example 1 and FIG. Briefly, after removing the tampon, it was transferred to a 50 ml test tube and 1.2 ml PBS was added to the tampon. Centrifugation at 1000 g for 10 minutes produced supernatant and pellet. An aliquot (0.2 ml) of the supernatant was mixed with 0.2 ml of working solution from High Pure Viral Nucleic Acid Kit (Roche Diagnostics, Mannheim, Germany) and stored at −30 ° C. until DNA isolation.

DNA isolation and methylation analysis
Single genomic DNA from the sample using the High Pure Viral Nucleic Acid Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's protocol with some modification at multiple loads of the DNA extraction column to obtain sufficient amounts of DNA. Released. After sodium bisulfite conversion, 11 genes (SOCS1, CDH1, TIMP3, GSTP1, DAPK, hTERT, CDH13, HSPA2, MLH1, RASSF1A and SOCS2) were methylated by fluorescence-based real-time PCR MethyLight analysis as described above It was attached to. Briefly, two sets of primers and probes specifically designed for bisulfite converted DNA: a methylation set for the gene of interest and a reference set for normalizing the input DNA, β-actin (ACTB) used. The reaction specificity of methylated DNA was confirmed separately using SssI (New England Biolabs) -treated human leukocyte DNA (highly methylated). The percentage of fully methylated molecules at a specific locus was calculated by dividing the GENE-ACTB ratio of the sample by the GENE-ACTB ratio of SssI-treated DNA and multiplying by 100. The result is shown as PMR (percent of fully methylated reference). If the PMR value is> 0, the gene is considered methylated. The primers and probes used for the MethyLight reaction are shown in Table 2.

MethyLight reaction conditions
The reaction was performed under the following analytical conditions: reaction solution containing 300 nM forward primer, 300 nM reverse primer and 100 nM probe; plus magnesium chloride, taq polymerase, dNTP and DNA in a final reaction volume of 30 μl; cycling conditions: 95 ° C., 10 Minutes, then 95 ° C., 50 times 15 seconds; 60 ° C., 1 minute.

HPV DNA analysis HPV PCR Enzyme Immunoassay (PCR-EIA) was performed in accordance with Jacobs, MV, Snijders, PJ, van den Brule, AJ, Helmerhorst, TJ, Meijer, CJ, and Walboomers, JM, 14 6 General Primers GP + 5 / GP6 (+)-mediated PCR Enzyme Immunoassay for Rapid Detection of Low-Risk Human Papillomavirus Genotypes ”, J. Clin. Microbiol., 35: 791-795, 1997 Was carried out. Briefly, 10 μl of purified total cellular DNA was used for PCR with consensus primers GP5 + / bioGP6 +. The reaction mixture was 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 3.5 mM MgCl2, 200 μM of each dNTP, 1 U of thermostable DNA polymerase (Taq polymerase, Roche, Vienna, Austria) and 50 pmol of GP5 + (5 -TTTGTTACTGTGGTAGATATACTAC-3, SEQ ID NO: 56) and bioGP6 + primer (5-GAAAAATAAACTGTAAATCATATT-3, SEQ ID NO: 56) primer (MWG-Biotech, Ebersberg, Germany). After amplification 40 times in a PCR cycler (Gene Amp PCR Cystem 9600, Perkin Elmer, Norwalk, USA), a denaturation step at 94 ° C. for 4 minutes was performed. To determine HPV types, PCR products were subjected to enzyme immunoassay (EIA) that recognizes 14 different high-risk HPV types as described by Jacobs et al. (As described above).

Statistical analysis
Associations between categorical variables were tested using the Pearson's chi square test (or Fischer's exact test) and the Mantel-Haenzel test. Correlation between ordinal variables was evaluated using Spearman's rank correlation coefficient. In clinical cases and unsupervised hierarchical clustering of genes, we used the fully linked aggregate method and the Manhattan distance function.
All statistical calculations were performed using SPSS, version 11.0 for Windows and gene expression similarity package software (http://genome.tugraz.at/Software/Genesis/Genesis.html).

Results This study investigated the abnormal methylation of HPV DNA and 11 genes in cervical and vaginal specimens collected by intravaginal tampon application to patients without cervical dysplasia, patients with low and high SIL, and patients with invasive cervical cancer. did. High-risk HPV DNA was detected in 2/3, 21/31 and 3/5 in low-grade SIL, high-grade SIL and invasive cervical cancer, respectively (Table 3). Patients without cervical dysplasia showed no HPV infection. The three HPV-positive cervical cancers were large cell squamous cell carcinomas, while both HPV-negative cases were small cell cervical cancers. The difference in methylation of the investigated genes between the non-dysplastic group and the low SIL group was not statistically significant. Therefore, the results obtained from these two groups were compared in combination with the advanced SIL and invasive cancer groups. A summary of methylated gene frequencies is shown in Table 3. All investigated genes except GSTP1 and SOCS2 were significantly more frequently methylated in advanced SIL and / or invasive cancer compared to the non-dysplastic / low-grade SIL group. CDH1 and SOCS2 were judged to be methylated in approximately 1/4 of non-dysplastic / low SIL patients, whereas hTERT was exclusively methylated in specimens from cervical cancer patients. Unmethylated genes were found in 61% and 42% of cervicovaginal specimens from patients with dysplasia / low SIL and high SIL, respectively, while cervical cancer patients were 5% in each study sample. One or more methylated genes were revealed. The percentage of methylation positive samples tended to increase significantly from non-dysplastic / low SIL to invasive cervical cancer as shown in FIG. No significant correlation between HPV positive and abnormal hypermethylation was observed (p = 0.295, Fischer's exact test). HPV DNA-positive samples and / or at least one methylated gene is 46% (7/13; non-dysplastic / low SIL), 94% (29/31; high SIL) and 100% (5/5) of the sample ; Invasive cancer) (Table 3).
Two clusters were formed by unsupervised hierarchical cluster analysis using exclusively information about the DNA methylation of the 11 genes tested. Of the two clusters, one contained all five invasive cancers as well as two advanced SILs (Figure 3).

  Although hTERT was not judged to be methylated due to abnormal cervical intraepithelial proliferation, 80% of specimens obtained from cervical cancer patients were methylated. This finding suggests that hTERT methylation is a late event in cervical carcinogenesis. SOCS2 and CDH1 are methylated in approximately ¼ of patients in the dysplasia / low SIL group, indicating that methylation of these genes is an early event of cervical carcinogenesis. Our results clearly show that the increasing percentage of methylation positive samples is associated with large pathological changes in the cervix (Figure 5), in addition to HPV infection, methylation is an important factor in cervical carcinogenesis Suggest a factor. HPV DNA-positive samples and / or at least one methylated gene was found in over 90% of patients with advanced SIL and in 100% of patients with invasive cervical cancer. No significant correlation was observed between HPV positivity and abnormal hypermethylation. Abnormal methylation in women with or without HPV infection can be speculated to help identify subgroups with a high risk for pathological progression or cancer development. Our study shows that it is possible to detect HPV and aberrant hypermethylation of various genes in DNA obtained from tampon samples. We were able to identify invasive cervical cancer. The combined method detects advanced cervical lesions.

In the following study, we investigated the methylation status of CDH1 and CDH13 in serum samples of cervical cancer patients for use as prognostic markers.
Patients and Samples A total of 93 patients with invasive cervical cancer treated at the Department of Obstetrics and Gynecology at Innsbruck University Hospital from 1990 to 1998 were included in this study. Serum samples were taken on the day of diagnosis and prior to initial treatment. These serum samples were obtained from previous studies investigating in the presence of serum human papillomavirus DNA from cervical cancer patients.

  Patients with major clinical and pathological features are shown in Table 4. Treatment followed international standards. None of the patients received concurrent chemotherapy and radiation therapy. All patients were followed up after primary treatment in our department, i.e. from 3 months to 1 year with increasing intervals until the end of the study. The follow-up period ranged from 1 month to 12.4 years (average of 3.5 years).

DNA isolation and methylation analysis Serum samples (300 μl) were treated with SDS and proteinase K (300 μl of 1% SDS, 500 μg / ml proteinase K) overnight at 55 ° C., followed by phenol / chloroform extraction and ethanol precipitation. . DNA was resuspended in 80 μl LoTE buffer (30 mM Tris and 0.3 mM EDTA). Genomic DNA was converted to sodium bisulfite as described above. ^Twelve sodium bisulfite treated genomic DNA was analyzed by MethyLight, fluorescence-based real-time PCR analysis as described above. Briefly, two sets of primers and probes designed to be specific for bisulfite converted DNA: a methylation set for the gene of interest and a reference set for normalizing the input DNA, β-actin (ACTB) was used. The specificity of the methylated DNA reaction was confirmed separately using SssI (New England Labs) -treated human leukocyte DNA (highly methylated). The percentage of molecules fully methylated at a specific locus was calculated by dividing the GENE: ACTB ratio of the sample by the GENE: ACTB ratio of SssI-treated leukocyte DNA and multiplying by 100. The abbreviation PMR (percentage of fully methylated reference) indicates this measurement. In each MethyLight reaction, 10 μl of bisulfite treated genomic DNA was used. If the PMR value is> 0, it is determined that the gene is methylated. In order to demonstrate the reproducibility of each analysis, the reference value of the standard (Gene: ACTB) was compared between the different PCR streams. The following primers and probes were used for the MethyLight reaction:
CDH1:
5'-AATTTTAGGTTAGAGGGTTATCGCGT-3 'SEQ ID NO: 58 (forward primer)
5'-TCCCCAAAACGAAACTAACGAC-3 'SEQ ID NO: 59 (reverse primer)
5´-FAM-CGCCCACCCGACCTCGCAT-BHQ-1-3 'SEQ ID NO: 60 (probe)
CDH13:
5´-AATTTCGTTCGTTTTGTGCGT-3 'SEQ ID NO: 61 (forward primer)
5′-CTACCCGTACCGAACGATCC-3 ′ SEQ ID NO: 62 (reverse primer)
5′-FAM-AACGCAAAACGCGCCCGACA-BHQ-1-3 ′ SEQ ID NO: 63 (probe)

MethyLight reaction conditions The reaction was carried out under the following analytical conditions: reaction solution containing 300 nM forward primer; 300 nM reverse primer and 100 nM probe; plus magnesium chloride; taq polymerase, dNTP and DNA in a final reaction volume of 30 μl; cycling conditions: 95 ° C. 10 minutes, then 95 ° C, 15 seconds 50 times, 60 ° C, 1 minute.

Statistical analysis Associations between categorical variables were tested using Pearson's chi double test. The Kaplan-Meier method was used for univariate survival analysis and the log rank test was used to assess the differences between survival curves. Cox proportional hazard analysis was used to evaluate the prognostic effect of various variables. A P value of 0.05 or less was considered statistically significant. These statistical calculations were performed using SPSS, version 11.0 for Windows.

Results Abnormal promoter hypermethylation of CDH1 and CDH13 was observed in 42% (39 out of 93) and 4% (4 out of 93), respectively (Table 4). Of the CDH13 methylation positive serum samples, 3 also revealed CDH1 methylation. Therefore, we disrupted CDH1 and / or CDH13 methylation for further analysis. CDH1 methylation was observed mainly in FIGO stage III, but the distribution of CDH1 and CDH13 methylation within other clinical and pathological parameters showed no significant differences (Table 4).

  In order to determine whether prognostic significance is associated with differences in CDH1 and / or CDH13 methylation, we compared the clinical outcome of cervical cancer patients with the CDH1 / CDH13 methylation status. In patients with methylated CDH1 / CDH13, a trend towards lower overall survival was observed (P = 0.09) (FIG. 7B). Cervical cancer patients who do not have methylated CDH1 / CDH13 in serum samples taken before treatment revealed a statistically significant better disease-free survival compared to patients with methylated CDH1 / CDH13 (P = 0.03) (FIG. 7A). The average disease free survival of CDH1 / CDH13 methylation negative and positive patients was 4.3 years and 1.2 years, respectively.

  To assess the significance of independent prognosis, Cox proportional hazard model analysis was performed including tumor stage, histology, degree of differentiation, age and CDH1 / CDH13 methylation status. In addition to tumor stage and age, only the CDH1 / CDH13 methylation status (P = 0.005) was found to have independent prognostic significance in disease-free and overall survival of cervical cancer patients ( Tables 5 and 6). Serum CDH1 / CDH13 methylation positive patients were more than twice as likely to have recurrence and death as CDH1 / CDH13 methylation negative patients.

  The distribution of CDH1 methylation and CDH13 methylation within clinical and histological parameters showed no significant difference except that CDH1 methylation was observed mainly in the progressive FIGO stage. Therefore, no difference in CDH1 methylation at the FIGO stage was observed. In our study, these results confirmed the association of CDH1 methylation and CDH13 methylation in serum samples with higher CDH1 methylation frequency and increased recurrence frequency with increasing tumor stage. To date, it has not been investigated whether either the above-described mechanism alone or a combination of these mechanisms causes a loss of cadherin expression. DNA recovered from plasma or serum of patients with various malignancies reflects tumor-specific genetic and epigenetic changes such as methylation of primary tumors (Ziegler A, Zangemeister-Wittke U, Stahel RA. DNA: a treasure trove of new diagnostics? "Cancer Treat REv 2002; 28: 255-71). Simple blood collection techniques combined with high-throughput analysis such as MethyLight open up a feasible entrance for the potential routine use of these markers. Inactivation of the cadherin-mediated cell adhesion system caused by aberrant methylation is a common finding in human cancer. Therefore, the investigation of CDH1 and CDH13 methylation as a prognostic parameter in serum samples obtained from patients with various malignancies would be interesting. Our study revealed that CDH1 / CDH13 methylation is an independent prognostic parameter for both disease-free and overall survival in patients with cervical cancer.

  The next study will analyze whether methylated DNA in cervicovaginal secretions of the genes SFRP2, SFRP4, SFRP5 and CCND2 can be analyzed to detect endometrial cancer, ovarian cancer and invasive cervical cancer Carried out to determine.

Patient and sample
A total of 24 patients were recruited for this study: 4 patients had endometrial cancer, 2 patients had ovarian cancer, and 6 patients had cervical cancer. To ensure standardized sampling, a tampon was inserted into the patient by the physician after microscopic examination and held in the vagina for 30 minutes. Sample preparation is shown in FIG.

DNA isolation and methylation analysis
Genomic DNA from the sample using the High Pure Viral Nucleic Acid Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's protocol, which modifies several loadings of the DNA extraction column to obtain sufficient amounts of DNA. Was isolated. Sodium bisulfite treated genomic DNA was analyzed by MethyLight, fluorescent real-time PCR analysis as described above. Briefly, two sets of primers and probes designed specifically for bisulfite converted DNA were used: a methylation set of the gene of interest and a reference set to normalize the input DNA, β-actin (ACTB )It was used. The reaction specificity of methylated DNA was confirmed separately using SssI (New England Biolabs) -treated human leukocyte DNA (highly methylated). The percentage of fully methylated molecules at a specific locus was calculated by dividing the GENE: ACTB ratio of the sample by the GENE: ACTB ratio of SssI-treated leukocyte DNA and multiplying by 100. The abbreviation PMR (percent of fully methylated basis) indicates this measurement. If the PMR value is> 0, the gene is judged to be methylated. Primers and probes specific for methylated DNA and used in the MethyLight reaction are shown in Table 8.

MethyLight reaction conditions
The reaction was performed under the following analytical conditions: the reaction solution contained 300 nM forward primer, 300 nM reverse primer and 100 nM probe; plus magnesium chloride; taq polymerase, dNTP and DNA in a final reaction volume of 30 μl; cycling conditions: 95 ° C. 10 minutes, then 95 ° C, 15 seconds 50 times, 60 ° C, 1 minute.

Statistical analysis
Associations between categorical variables were tested using the Chi-Square test. Differences in median age were tested using the Mann-Whitney U test, and differences between two or more groups were tested using the Kruskal Wallis test. Due to the significant age difference between the endometrial cancer patient group and the non-endometrial cancer patient group, the age-matched group of the non-endometrial cancer patient group was randomly selected with a matching ratio of 1: 2. Match group calculations were performed using MATLAB R12 (www.mathworks.com). To determine diagnostic accuracy, non-parametric receiver operating curve (ROC) analysis using one-dimensional interpolation was performed. A P-value of 0.05 or less was considered statistically significant. All statistical calculations were performed using SPSS, Windows version 11.0.

result
See Table 9 for results. All ovarian cancer patients showed methylation of at least one gene of panels SFRP2, SFRP4, SFRP5 and CCND2. All cervical cancer patients showed methylation of at least two genes of panels SFRP2, SFRP4, SFRP5 and CCND2, and two patients showed methylation of all genes. All endometrial cancer patients showed methylation of at least two genes of panels SFRP2, SFRP4, SFRP5 and CCND2.

FIG. 1 shows a flowchart of the tampon insertion and sample preparation procedure. A-Tampon insertion B-Transfer of tampon into 50 ml test tube C-Addition of 1.2 ml PBS buffer onto tampon D-10 min centrifugation at 1000 g E- (1) shows supernatant ( 2) shows a pellet. An aliquot of 0.2 ml of the F-supernatant fraction was each mixed with 0.2 ml of the working solution of the High Pure Viral Nucleic Acid kit and stored at −30 ° C. until DNA isolation. FIG. 2A-2J shows the PMR values of 38 genes of patients with no endometrial cancer (N = 4) and those with endometrial cancer (N = 5). The genes RASSF1A, hMLH1, CDH13, HSPA2 and SOCS2 are highlighted with arrows. Same as above Same as above Same as above Same as above Same as above Same as above Same as above Same as above Same as above 3A-K show the methylation status (complete box = methylation, empty box = unmethylation), status (endometrial or non-uterine cancer) and age of the five investigated genes. * Indicates cervical intraepithelial abnormal growth grade III (CIN III); † indicates invasive cervical cancer. Same as above Same as above Same as above Same as above Same as above Same as above Same as above Same as above Same as above Same as above FIG. 4 shows ROC in the detection of endometrial cancer by methylation analysis of DNA obtained from vaginal secretions of patients aged 50-75 years (excluding patients with CIN III or cervical cancer). The area under the curve is 0.988. FIG. 5 shows an increasing methylation trend from low SIL to invasive cervical cancer. A statistically significant relationship (r = 0.47, P = 0.001) was found between histological type and number of methylated genes. There were no examples of 10 or 11 methylated genes. FIG. 5B shows the altitude SIL (n = 31). FIG. 5A shows low / abnormal SIL (n = 13) and FIG. 5C shows invasive cervical cancer (n = 5). The X-axis scale shows the percentage of methylated positive samples, and the Y-axis shows the number of methylated genes. FIG. 6 shows the DNA methylation status of 11 genes in 49 cervical vaginal samples analyzed using MethyLight technology. A gene is considered methylated if the PMR value is> 0 (materials and methods, white and pink indicate unmethylated and methylated, respectively). Patient groups were determined using unsupervised clustered cluster analysis (mean binding, Manhattan distance) for sample groups and CpG regions. The gene name is listed at the top of the figure. The inventors observed two clusters: 1 (red) and 2 (blue). All patients with cervical cancer are grouped together in one cluster (blue). FIG. 7 shows disease free (A) and overall survival (B) by CDH1 / CDH13 methylation status in serum samples. The lower dotted line indicates the methylated sample, and the dashed upper line indicates the unmethylated sample. FIG. 8 shows polymerase-mediated amplification of CpG-rich sequences using four methylation specific primers on four representative bisulfite treated DNA strands (sample cases “A”-“D”) (“MSP amplification”). "). The methylation-specific forward and reverse primers (“1”) in each case can anneal to the bisulfite-treated DNA strand (“3”) if the corresponding genomic CpG sequence of interest is methylated. it can. The bisulfite treated DNA strand ("3") can be amplified if the forward and reverse primers ("1") anneal, as shown in the representative case [A] at the top of the figure. Figure 9 shows a polymerase-mediated amplification analysis of bisulfite treated DNA corresponding to the CpG- rich genomic sequences using MethyLight ^R technology ( "3"). Amplification of the treated DNA (“3”) is ruled out if the blocking oligonucleotide (“5”) anneals to the treated DNA, as shown in sample case “B”.

Claims (13)

The following process:
a) obtaining a cervical vaginal discharge specimen from an individual;
b) measuring the methylation status of at least one or more CpG positions;
c) determining the presence, classification and / or prognosis of said individual's gynecological cell proliferation disorder from its methylation status;
A method for the detection, discrimination and prognosis of gynecological cell proliferation disorders. The CpG position is selected from one or more genes selected from the group consisting of SFRP2, SFRP4, SFRP5, CCND2, CDH1, CDH13, RASSF1A, hMLH1, HSPA2, SOCS1, SOCS2, GSTP1, DAPK, TIMP3 and hTERT The method of claim 1. 2. The method of claim 1, wherein the CpG position is selected from one or more sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 11 and SEQ ID NO: 64 to SEQ ID NO: 67. In step a), the specimen is obtained by one or more of the following methods: gynecological swabs, aspiration, cervical vaginal lavage and tampon collection. The gynecological cell proliferative disorder is selected from the group consisting of dysplasia or low-grade squamous intraepithelial injury, high-grade squamous intraepithelial injury, cervical cancer, endometrial cancer and grade 1-3 cervical intraepithelial abnormal growth, The method according to claim 1 or 2. The method of claim 1, wherein hypermethylation of the gene is associated with poor prognosis. Step b) includes the following steps:
a) treating genomic DNA or fragments thereof with one or more reagents that convert the 5-position unmethylated cytosine base into uracil or other bases that are detectably different from cytosine in hybridization properties;
b) In each case, the processed genomic DNA or processed fragment thereof is complementary to the amplification enzyme and the target nucleic acid, or hybridizes under moderately or stringent conditions. Contacting at least two primers comprising a contiguous sequence of at least 9 nucleotides in length;
c) measuring the methylation state of at least one CpG dinucleotide sequence or the average methylation state of a plurality of CpG dinucleotide sequences or a value reflecting the same based on the presence or absence of the amplification product or its characteristics; At least one of detecting a gynecological cell proliferative disorder, or determining a gynecological cell proliferative disorder, or at least partially providing a prognosis;
The method of claim 1, comprising: 8. The method of claim 7, wherein in step b), the target nucleic acid is selected from the group consisting of SEQ ID NO: 12 to SEQ ID NO: 55 and SEQ ID NO: 68 to SEQ ID NO: 83. In each case, it is complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 55 and SEQ ID NOs: 64 to 83, or their complementary strands, or is moderately stringent or stringent conditions A nucleic acid or peptide nucleic acid molecule comprising a contiguous sequence of at least 9 nucleotides that hybridizes underneath. The nucleic acid according to claim 9, wherein the sequence is selected from the group consisting of SEQ ID NO: 12 to SEQ ID NO: 55 and SEQ ID NO: 68 to SEQ ID NO: 83. The nucleic acid molecule comprises at least one TpA or CpA dinucleotide at a position where the corresponding unprocessed nucleic acid molecule set forth in SEQ ID NO: 1 to SEQ ID NO: 11 and SEQ ID NO: 64 to SEQ ID NO: 67 contains a CpG dinucleotide. The nucleic acid according to claim 10. At least one bisulfite reagent in each case, or a methylated sensitive restriction enzyme: and a sequence selected from the group consisting of SEQ ID NO: 12 to SEQ ID NO: 55 and SEQ ID NO: 68 to SEQ ID NO: 83 or a complementary strand thereof Gynecology of a subject comprising at least one nucleic acid molecule or peptide nucleic acid molecule comprising a contiguous sequence of at least 9 nucleotides that are complementary to, or moderately stringent to, or hybridize under stringent conditions A kit useful for detecting or distinguishing cell proliferation disorders. 13. The kit of claim 12, further comprising a standard reagent for performing a methylation analysis selected from the group consisting of MSP, MethyLight and HeavyMethyl and combinations thereof.

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