KR20160092807A - Prediction and diagnosis method of cervical cancer using HPV test and MKRN1 expression pattern - Google Patents

Abstract

The present invention relates to a method for predicting and diagnosing uterine cervical cancer with respect to a HPV test and a MKRN1 expression pattern. In the case of using the diagnosis method of the present invention, the diagnosis method has the increased accuracy of the diagnosis in uterine cervical cancer, thereby being significantly used for predicting and diagnosing uterine cervical cancer in an effective manner. The method for providing information related to the diagnosis of HPV infection-associated diseases comprises the steps of: confirming whether to DNA or mRNA of HPV is present in a biological sample separated from a patient; and measuring an expression level of MKRN1 mRNA.

Description

[0001] The present invention relates to a method for predicting and diagnosing cervical cancer according to the HPV test and MKRN1 expression pattern,

The present invention relates to a method for predicting and diagnosing cervical cancer according to the HPV test and MKRN1 expression pattern.

Cervical cancer is the second leading cause of malignancy in women. More than 350,000 patients worldwide die from cervical cancer each year, and the number continues to increase (Pisani P, et al., Int J Cancer 2002; 97 (1): 72-81.). In general, cervical cancer is diagnosed using Pap smear (Liquid based cytology specimen). Cervical smear cytology examines the cells from the cervical surface, which are then collected on glass slides and examined microscopically for the presence or absence of abnormal cells. Due to the nature of the test, it is advantageous to examine many samples within a short time. However, it is necessary to detect only a few abnormal cells among 300,000 ~ 500,000 cells included in one patient's sample. Problems are pointed out.

Recently, cervical cancer diagnosis using human papillomavirus (HPV) has been developed. HPV is known to be the main cause of cervical cancer, and more than 100 HPV types have been found to work in female reproductive organs (de Villiers EM, et al., Virology 2004; 324: 17-27). When the HPV is infected with normal cells, the viral oncogene is inserted into the host cell gene and expresses various viral proteins. Recently, the viral protein of the HPV or HPV Predictive and diagnostic methods for cancer of the neck are being developed (European Register EP1253934, Korean Publication KR2013-0012512).

However, since the diagnosis of cervical cancer using HPV is very poor in the negative samples, it is difficult to use it for the early diagnosis of cervical cancer. Therefore, a new method of diagnosing cervical cancer combined with HPV and various cervical cancer genetic markers Development is required.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made in order to solve the above-mentioned problems in the prior art, and relates to a method for predicting and diagnosing cervical cancer according to HPV testing and MKRN1 expression pattern.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

Hereinafter, various embodiments described herein will be described with reference to the drawings. In the following description, for purposes of complete understanding of the present invention, various specific details are set forth, such as specific forms, compositions and processes, and the like. However, certain embodiments may be practiced without one or more of these specific details, or with other known methods and forms. In other instances, well-known processes and techniques of manufacture are not described in any detail, in order not to unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "embodiment" means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Accordingly, the appearances of the phrase " in one embodiment "or" an embodiment "in various places throughout this specification are not necessarily indicative of the same embodiment of the present invention. In addition, a particular feature, form, composition, or characteristic may be combined in any suitable manner in one or more embodiments.

Unless defined otherwise in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In one embodiment of the present invention, a suspected disease of a human papilloma virus is caused by infection of a human papillomavirus (HPV), a double helical DNA virus belonging to the Papoviridae, which causes warts and cervical cancer in a human infection It is a suspected disease. Of the 100 known HPV types, 40 have been found in the reproductive tract and are known to cause pathological changes in the cervical epithelium. Of these, the high-risk group, carcinogenic HPV, is known to be associated with cervical cancer, with 16,18 of the HPV types being the most important and more than 70% of the cervical cancers worldwide. In addition, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 69 and 73 belong to the high risk virus. The low risk group viruses found in the warts of the cervix were 6, 11, 34, 40, 42, 3, 44, 54, 61, 70, 72, 81 times.

In one embodiment of the present invention, cervical cancer is a malignant tumor that occurs in the cervix, also called uterine cancer. Cervical dysplasia, the pre-cervical cancer stage, takes about 7 years to progress to cervical intraepithelial neoplasia, and it takes about 14 years to progress from intraepithelial cancer to microinvasive cancer. In the case of microinvasive cancer, it takes about 3 years for the cancer to be visible to the naked eye because of its rapid progression rate. Early diagnosis is possible because of the relatively long time spent in the pre-cancer stage, and early diagnosis is important because early diagnosis is possible. It is mostly seen in multiparous women aged 45 to 55 years. The mean age of this cancer is 35 years old at 0, and the average age at diagnosis is 45 years old or older. Recently, cancer diagnosis tends to be diagnosed at young age. This disease is the 4th among the total cancer in Korea and the first among the cancer that occurs in women. Sexually transmitted infections are the most common cause of cervical cancer. In addition, early intercourse, multiple sex partners, male factors, HPV infection, and HIV infection are known to be risk factors, among which HPV infection is the most likely causative factor . The main symptom of cervical cancer is vaginal bleeding after intercourse, which can occur intermittently or after postmenopausal bleeding. In patients with advanced lesions, there may be persistent, massive vaginal bleeding, vaginal discharge with odor, and pain in the lateral abdomen or lower leg when metastasized to the ureter, pelvic wall, or sciatic nerve. Also, when transferred to the bladder or rectum, symptoms such as difficulty urinating, hematuria, rectal bleeding, and constipation may occur. Complications occur mainly after surgery. Acute complications include bleeding, ureteral fistula, bladder fistula, pulmonary embolism, small bowel obstruction, and febrile mood swings. Of these, pulmonary embolism is a complication that can lead to death after surgery. Subacute complications include bladder dysfunction, lymphatic cysts, and chronic complications include bladder atrophy and ureteral stenosis. Complications after radiation therapy include, but are not limited to, uterine perforation, fever, abdominal pain, hematuria, stool, rectal colitis, rectal fistula, bladder fistula, bladder fistula, The primary treatment is surgery or radiation therapy, and radiation therapy can be performed in all stages. Surgery usually involves radical hysterectomy and pelvic lymphadenectomy. Surgery is performed only in the early stages of cervical cancer and is appropriate in the following cases. (1) to preserve ovarian function among young women, (2) to preserve sexual function, (3) to cervical cancer complicated by pregnancy, (4) to accompany inflammatory diseases of the intestine, (5) ⑥ Pelvic organ prolapse accompanied by ⑦ uterine adnexal tumor. Radiotherapy may be performed adjunctively to reduce the risk of recurrence if the surgical margin is proximate to cancerous lesions, invasive cancer, or pelvic lymph node metastasis. Chemotherapy, on the other hand, has the advantage of being able to treat not only local cancers but also disseminated cancers, but it has no effect on squamous cell carcinoma, which accounts for 95% of cervical cancer. Women who have begun sexual intercourse are able to prevent cervical cancer by examining the presence or absence of cervical cancer by conducting a cervical cytology test at least every 6 months, even if there are no special symptoms.

In the present specification, the progression of cervical dysplasia is classified into normal (normal or negative), low-level squamous intraepithelial lesion (LSIL), high level intraepithelial lesion (normal or negative), flat condyloma, cervical intraepithelial neoplasia (cervical intraepithelial neoplasia), cervical intraepithelial neoplasia (CIN), cervical intraepithelial neoplasia It is classified into six stages: tumor 1 (CIN 1), cervical intraepithelial neoplasia 2 (CIN 2), cervical intraepithelial neoplasia 3 (CIN 3), and invasive cancer. Based on dysplasia according to Reagan classification, normal ), Flat condyloma, mild dysplasia, moderate dysplasia, severe dysplasia, carcinoma in situ, and invasiveness Cancer can be divided into seven stages. (CIN 2), cervical intraepithelial neoplasia 3 (CIN 3), and cervical intraepithelial neoplasia 1 (CIN 1) are included in low level intraepithelial lesions (LSIL) Are included in high levels of flat intra-epithelial lesions (HSIL). In addition, cervical intraepithelial neoplasia 1 (CIN 1) represents mild dysplasia, cervical intraepithelial neoplasia 2 (CIN 2) represents moderate dysplasia, and cervical intraepithelial neoplasia 3 (CIN 3) Including dysplasia (Severe dysplasia) and carcinoma in situ. This is shown in Fig.

In some cases, we can add atypical squamous cells of undetermined significance (ASCUS) as an intermediate stage between normal (negative or negative) and low-level intraepithelial lesions (LSIL) , Which indicates abnormalities in cell morphology in Papanicola staining but not clearly distinguished. (LSIL) is a stage in which the size, shape, and number of cervical surface cells change in Papanicola staining. A high level of flat intraepithelial lesion (HSIL) is associated with a low level of flat intraepithelial lesion (LSIL) It means a state in which the abnormal region is increased but the infiltration is not deep. This is illustrated in FIG.

In one embodiment of the present invention, the test for cervical cytology is a diagnostic test for detecting the occurrence, causes, or characteristics of a disease using cells removed from the cervix. The most commonly used goal is cytology for the diagnosis of cervical cancer. Cells from the cervix are harvested using a specially designed brush (brush) to monitor for abnormal cells. Upon observation, Papanicolaou's stain is additionally performed to facilitate cell sorting. Immediately after collection, the cell smears are fixed with an equal volume of ether or alcohol, passed through 70% ethanol, 50% ethanol, distilled water and stained with Harris hematoxylin for 4 minutes. It is washed with water and fractionated with a 0.5% hydrochloric acid solution. Again rinsed in oil water for 7 minutes and passed through 50% to 95% ethanol solution. OG6 for 1 min 30 sec, pass through 95% ethanol twice, stain for 2 min at EA 50, dehydrate with 95-100% ethanol and seal with Kisilro. The nucleus is bluish purple, cytoplasm is green or red. The composition of the reaction solution and the reaction time may vary depending on the condition of the cell sample and the test environment. In the late 1990s, liquid cell cytology techniques were developed to reduce errors that could occur when taking cell tests and to produce better cell samples. This is because the clinician uses a brush to collect cells from the cervix and then tosses the sole into a specimen tube containing a special preservative solution. In this way, concerns about cell loss are reduced, and the cell state can be preserved well before slide, contributing to the improvement of cancer detection rate.

In one embodiment of the present invention, MKRN1 is a ubiquitin ligase also referred to as Makorin ring finger protein 1 (MKRN1), which is a ubiquitin ligase for the ubiquitination of tumor suppressor p53 and p14ARF proteins, It is known to contribute to cancer development by inducing degradation.

In one embodiment of the present invention, p16INK4a is a tumor suppressor protein that is encoded by the CDKN2A gene. Although p16INK4a was not originally expressed in normal cells, it begins to be expressed when aging substances such as benzene enter the body. When the gene is expressed, the cells stop dividing, release various signaling substances that cause inflammation, and die. Therefore, many of them are found in natural cells or cancer cells rather than normal cells, and cervical cancer is used as a marker gene for diagnosis of uterine cancer.

In one embodiment of the present invention, the term "sensitivity" refers to the rate at which a person actually afflicted with a disease is judged to be positive in the test.

(See Table 1 for true positive and false negative definitions)

Highly sensitive tests usually have a low false negative rate, which can be taken to mean a good catch. That is, when the test is judged as negative, the probability of being normal or being normal is high.

A patient with a disease A normal person without disease Positive test results True positive (A) False positive (B) Test result voice False negative (C) True voice (D)

In one embodiment of the present invention, the specificity refers to the rate at which a normal person who has not been infected with a disease is judged as negative by the test, and can be expressed by the following equation.

(See Table 1 for true speech and false positive definitions)

High-specificity tests usually have a high percentage of normal tests, and if tested positive, the actual disease is likely to occur or is likely to occur in the future.

In one embodiment of the present invention, the prediction rate means a statistical value inferring the value of another variable (reference variable) based on a certain variable or a group of variables used as a predictive variable. Usually, the regression analysis is used to obtain the prediction equation and the prediction parameter is substituted into the prediction equation. In this case, when the data is collected again in the same situation, there is a nuance of 'guessing' based on the predictive equation of the predictive variables to determine a certain value of the reference variable. At this time, we are interested in how close the predicted value of the reference variable is to the actual value.

In the present specification, the prediction rate means a statistical value inferring the value of a genuine cervical cancer patient in a patient group suspected of having cervical cancer, and can be divided into a positive predictive value and a negative predictive value.

The positive predictive value means the percentage of the actual disease when the test result is positive and can be expressed by the following equation.

(See Table 1 for true positive and false positive definitions)

The voice prediction rate means a ratio of a normal test result when the test result is negative, and can be expressed by the following equation.

(See Table 1 for true speech and false negative definitions)

In one embodiment of the present invention, a peptide is a polymer of amino acids, and the linkage between amino acids means a polymer composed of an amide bond or a peptide bond.

In one embodiment of the present invention, the nucleic acid is a polymer organic substance in which a nucleotide is polymerized in a long chain shape, and is genetic information encoding a specific peptide in the present specification.

The peptides and the nucleic acid encoding the peptide of the present invention may originate from the HPV virus or may be a peptide against the peptide originated from the HPV virus. A peptide against peptides originating from HPV may be used as a preventive or therapeutic vaccine in a subject known to be infected by HPV, susceptible to HPV infection, or infected by HPV . Other suitable subjects include those who exhibit symptoms of, or susceptibility to, a disease associated with HPV. The peptides of the present invention are useful for the treatment of diseases associated with infection of the HPV virus such as bowenoid papulosis, anal dysplasia, respiratory or conjunctival papilloma, cervical dysplasia, cervical cancer, vulval cancer, , Or as a vaccine to treat or prevent prostate cancer. The ability of the peptides and nucleic acids of the invention to elicit an immune response can be assayed using methods known in the art for measuring immune responses. For example, the production of cytotoxic T cells can be measured using MHC tetramers or by measuring intracellular cytokine expression or by standard 51Cr release assays. Standard assays such as ELISA or ELISPOT can also be used to measure cytokine profiling that contribute to T cell activation. T cell proliferation can also be measured using assays such as 3H-thymidine uptake and other assays known in the art. B cell responses can be measured using an assay such as ELISA.

In one embodiment of the present invention, diagnosis refers to confirming the presence or characteristic of a pathological condition. For the purpose of the present invention, the diagnosis is to confirm whether or not the human papillomavirus is infected or the incidence of cervical cancer. In the present invention, the diagnosis of cervical cancer can be made by a method of confirming the presence of at least one species in the group consisting of DNA, mRNA or protein of HPV, and measuring the expression level of mRNA or protein encoding MKRN1 .

The term "diagnostic marker, diagnostic marker or diagnostic marker" in the present invention refers to a substance capable of diagnosing cancer cells by distinguishing cancer cells from normal cells, and includes polypeptides that show an increase or decrease in cancer- And organic biomolecules such as nucleic acids (e.g., mRNA), lipids, glycolipids, glycoproteins or sugars (monosaccharides, disaccharides, oligosaccharides and the like). For the purpose of the present invention, the cancer diagnostic marker of the present invention is a mRNA of the MKRN1 gene and a protein encoded thereby, which specifically expresses a high level of expression in a disease caused by HPV infection, as compared with cells of a normal target tissue.

The above-mentioned "measurement of mRNA expression level " is a process of confirming the presence and expression level of mRNA of cancer marker genes in a biological sample in order to diagnose cancer by measuring the amount of mRNA. (RT-PCR), Competitive RT-PCR, Real-time RT-PCR, RNase protection assay (RPA) and Northern blotting Northern blotting, or DNA chip, but are not limited thereto.

The agent for measuring the mRNA expression level of the gene means a molecule which can be used for detecting the marker by confirming the expression level of the MKRN1 gene whose expression is increased in cancer cells, Or a primer or a probe that binds to the target. Based on the polynucleotide sequence of MKRN1, one skilled in the art can design primers or probes that specifically amplify specific regions of these genes.

The term "primer" in the present invention refers to a nucleotide sequence having a short free 3 'hydroxyl group, capable of forming a base pair with a template complementary to the template, ≪ / RTI > The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i.e., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. In the present invention, PCR can be performed using a sense and antisense primer of MKRN1 polynucleotide to diagnose cancer through the production of a desired product. The PCR conditions, the lengths of the sense and antisense primers can be modified based on what is known in the art.

The term "probe " means a nucleic acid fragment such as RNA or DNA corresponding to a few nucleotides or hundreds of nucleotides that can specifically bind to mRNA, and the presence or absence of a specific mRNA can be confirmed by labeling The probe may be prepared in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, etc. In the present invention, a probe complementary to the MKRN1 polynucleotide Hybridization can be carried out using the hybridization method to diagnose cancer through hybridization. The selection and hybridization conditions of suitable probes can be modified based on those known in the art.

The primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, "capping ", replacement of natural nucleotides with one or more homologues, and modifications between nucleotides, such as uncharged linkers, such as methylphosphonate, Phosphoamidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).

The term " measurement of protein expression level " in the present invention is a process for confirming the presence and expression level of a protein expressed from a cancer marker gene in a biological sample in order to diagnose cancer. Preferably, The amount of protein can be confirmed using an antibody that binds to the protein. Examples of the assay methods include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis But are not limited to, tissue immuno staining, immunoprecipitation assays, complement fixation assays, fluorescence activated cell sorters (FACS), protein chips, and the like.

The agent for measuring the expression level of the protein means a molecule which can be used for detecting a marker by confirming the expression level of the protein expressed from the MKRN1 gene, which is a marker for increasing expression in cancer cells, May comprise an antibody specific for the protein. Based on the amino acid sequence of the protein expressed from the MKRN1 gene, one skilled in the art can design an antibody specific for said protein.

In one embodiment of the invention, "antibody" refers to a specific protein molecule as described above, directed against an antigenic site. For the purpose of the present invention, the antibody refers to an antibody that specifically binds to the HPV peptide of the present invention, and the monoclonal antibody to the protein is produced through a method for producing a monoclonal antibody common in the art Or a commercially available one can be used. In addition, a polyclonal antibody recognizing the protein may be used instead of the monoclonal antibody, or it may be produced and used through an antiserum preparation method customary in the art. The antibody of the present invention also includes partial peptides that can be made from the protein. Partial peptides of the present invention include at least 7 amino acids, preferably 9 amino acids, more preferably 12 amino acids do. The form of the antibody of the present invention is not particularly limited, and any polyclonal antibody, monoclonal antibody or antigen-binding antibody thereof may be included in the antibody of the present invention and include all immunoglobulin antibodies. Furthermore, the antibodies of the present invention include special antibodies such as humanized antibodies. Antibodies used in the detection of cancer diagnostic markers of the invention include functional fragments of antibody molecules as well as complete forms with two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.

In one embodiment of the present invention, a diagnostic kit refers to a set of compositions and accessories necessary for a specific purpose. For the purpose of the present invention, the diagnostic kit of the present invention is to confirm the incidence of cervical cancer due to HPV infection. The kit of the present invention includes a primer, a probe, an antibody that selectively recognizes a peptide, or an antibody that recognizes a specific peptide whose expression is specifically improved when infected with HPV, Or other component compositions, solutions or devices.

In one embodiment of the present invention, there is provided a method for diagnosing a human papilloma virus infection-related disease, comprising the steps of: confirming the presence of human papilloma virus DNA or mRNA from a biological sample separated from a patient; and measuring the expression level of MKRN1 mRNA The method comprising the steps of: providing a method of providing information, identifying the presence of HPV DNA or mRNA from a biological sample isolated from a patient, and measuring the level of expression of the MKRN1 protein. And determining the presence of the HPV protein from the biological sample isolated from the patient, and measuring the level of expression of MKRNl mRNA. The present invention provides a method of providing information about a human papilloma virus infection-related disease, Provides a way to provide information, Comprising the steps of: confirming the presence of a human papillomavirus protein from a biological sample separated from the human papilloma virus infection-related disease, and measuring the expression level of the MKRN1 protein, Wherein the human papilloma virus is at least one of subtypes 6, 11, 16, 18, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 56, 58, 59 and 68 The present invention provides a method for providing information on the diagnosis of an infection-related disease, wherein the HPV infection-related disease is selected from bowenoid papulosis, anal dysplasia, respiratory or conjunctival papilloma, cervical dysplasia, The present invention provides a method for providing information on the diagnosis of a HPV infection-related disease, which is characterized by being a vulval cancer or prostate cancer .

In another embodiment of the present invention, there is provided a diagnostic kit for a human papilloma virus infection-related disease, comprising a primer or a probe specifically binding to HPV DNA or mRNA and a primer or a probe specifically binding to MKRN1 mRNA, Provided is a diagnostic kit for a HPV infection-related disease, comprising a primer or a probe specifically binding to HPV DNA or mRNA and an antibody specifically binding to MKRN1 protein, wherein the antibody specifically binding to HPV protein And a primer or a probe that specifically binds to MKRN1 mRNA, and comprises an antibody that specifically binds to the HPV protein and an antibody that specifically binds to the MKRN1 protein Human papillomavirus Wherein the human papillomavirus is selected from subtypes 6, 11, 16, 18, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 56, 58, 59, 68, wherein the HPV infection-related disease is selected from the group consisting of bowenoid papulosis, anal dysplasia, respiratory or conjunctival papilloma, cervical lesion, The present invention provides a kit for diagnosing a human papilloma virus infection-related disease, which is characterized by a dysplasia, a cervical cancer, a vulval cancer, or a prostate cancer.

Hereinafter, the present invention will be described in detail.

Although cervical cancer is the second leading cause of malignancy in women and the cause of the disease is HPV (human papilloma virus), the accuracy of the negative samples is so low that it is difficult to use for early diagnosis of cervical cancer . However, since the diagnosis of cervical cancer according to the HPV test and the MKRN1 expression pattern of the present invention is more accurate than that of a single use of cervical cytology, HPV, and cervical cancer gene markers, It is expected to be useful for the prediction and diagnosis of cancer of the neck.

FIG. 1 illustrates the progress of cervical dysplasia according to an embodiment of the present invention.
2 shows a cervix of the pre-cancer stage according to a tissue lesion according to an embodiment of the present invention.
FIG. 3 shows an example of the collection status of cervical cytology specimens according to an embodiment of the present invention.
FIG. 4 is a cytological and histological examination result of a cervical liquid cell test according to an embodiment of the present invention.
FIG. 5 shows immunocytochemical staining results of negative and HISL specimens according to an embodiment of the present invention.
6 shows immunocytochemical staining results according to an embodiment of the present invention.
FIG. 7 is a graph illustrating the results of the analysis of the accuracy of diagnosis of cervical cancer in all samples according to an embodiment of the present invention.
FIG. 8 shows the results of an analysis of the improvement in the accuracy of diagnosis of cervical cancer in an ASCUS / LSIL sample according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example 1: Collection of specimens to be examined for cervical cancer

She was admitted to Gangnam Severance Hospital, Department of Obstetrics and Gynecology, Samsung Medical Center, and Department of Pathology, Samsung Medical Center, Seoul, Korea for 12 months. She underwent cervical cytology and HPV testing for women over 18 years of age. Age was calculated using the following formula for age at first screening.

Mann age calculation = current year - birth year (-1: if birthday has not passed)

An example of the status of collecting cervical cytology specimens according to the above is shown in FIG.

Women who are younger than 18 years of age, women who have undergone hysterectomy, women who have been diagnosed with other cancers, cardiovascular disease, kidney failure, cardiovascular disease except hypertension, other chronic illnesses with impaired immunity, psychiatric disorders Women and alcoholics with a history of treatment were excluded from the study.

In addition, all subjects participating in the study were informed in writing that they would participate in the test themselves before starting the study. If the subject withdraws or refuses consent, if the subject and the researcher do not follow the research procedure, and When the researcher decided that the study was necessary to stop the study, the research was stopped.

All the procedures of this study were planned according to the basic spirit of the KGCP and the Helsinki Declaration, and the examiner and the person in charge conducted the tests according to the above plan.

Example 2: Test for diagnosis of cervical cancer

Example 2-1. Prospective cervical cytology

Cervical specimens were collected from patients who were suspected of having abnormal cervical cells among the subjects selected by the method of Example 1, and 187 specimens collected were subjected to a cervical cytology.

Cervical cells were collected using a cervix-brush for cervical smear cytology, and specimens were prepared by mixing the specimens with a preservative solution. Cellular markers on the slides were subjected to Papanicolaou staining. The results of the staining and the presence or absence of the disease of the specimen were read by a pathologist. According to the cytologic criteria, 187 specimens were classified into 6 stages of negative, ASCUS or AGUS, LSIL, ASC-H, HSIL and cancer. According to histologic criteria, negative, CIN1, CIN2, CIN3 and 5 . As a result, there were 47 negative patients according to the cytological criteria, and 32 negative patients according to the histological criteria. This implies a low accuracy which is pointed out as a problem of cervical fluid cell examination. Cytological and histological examination results of cervical fluid cytology are shown in FIG.

Example 2-2. HPV DNA test

The 187 samples were subjected to HPV DNA testing.

The method used for the detection of HPV is a diagnostic kit (HC2 HPV DNA Test, QIAGEN) approved by the US FDA for the diagnosis of human papilloma virus infection. 13 HPV subtypes classified as high-risk HPV types (16/18/31/33/35/39/45/51/52/56/58/59/68; Book-Robbins basic pathology 8 ed. Chapter 19 and Vaccine 24 (3): S1-S10, Chapter 1) and five HPV subtypes classified as low-risk (6/11/42/43/44; Book-Robbins basic pathology 8 ed. Can be detected. Using the kit, a specific and complementary RNA probe was bound to the human papillomavirus target DNA, and a DNA-RNA hybrid was prepared. Then, the DNA-RNA hybrid was detected using an antibody specific to a solid phase coated DNA-RNA hybrid. Then, the antibody was bound to an antibody conjugated with alkaline phosphatase and the luminescence was measured using chemiluminescence method to confirm presence or absence of HPV.

Examples 2-3. Protein expression test using immunocytochemistry

Cellular paraffin blocks were prepared from the remaining surrogate specimens after cervical smear cytology and HPV DNA testing. Immunocytochemical staining for protein expression of MKRN1, p16INK4a, p21, p57 and Ki-67 was performed Respectively. The results of immunocytochemical staining in negative and HISL specimens are shown in Fig. Immunocytochemical staining results were read by a pathologist and the results are shown in Fig. As shown in FIG. 6, in the case of the liquid cytology test and the HPV DNA test, which are the primary screening test for cervical cancer, the accuracy of cancer is very high, 92.9% in case of cancer, but the accuracy is low at 42.9% Lt; / RTI > However, the expression of MKRN1 protein increased to 21.4%, 31.6%, 62.5%, 74.2% and 92.9% as CIN1, CIN2, CIN3, CIS and cancer progressed. This suggests that the MKRN1 protein, like the p16INK4a protein currently being studied for diagnostic purposes, may play an auxiliary role in increasing the accuracy of cervical smear cytology and HPV DNA testing in patients with suspected cervical cancer.

Examples 2-4. Analysis of MKRN1 Protein to Improve Diagnosis Accuracy of Cervical Cancer

Sensitivity and Specificity of Cervical Cancer Diagnostic Sensitivity and Specificity in Cervical Cancer Cell Lysis, Human Papillomavirus DNA Test, MKRN1 Protein Expression Test, p16INK4a Protein Expression Test, specific positive predictive value (PPV), and negative predictive value (NPV) were compared with those of normal controls (normal cervical cytology and HPV DNA test). The results for the whole sample of the above analysis are shown in Fig. 7, and the results for the ASCUS / LSIL sample are shown in Fig. As shown in FIG. 7, when two or more kinds of tests were performed as compared with the single cervical cytology test, HPV DNA test, MKRN1 protein expression test and p16INK4a protein expression test, The accuracy of the diagnosis was improved. In addition, the combination of MKRN1 protein expression test showed the highest diagnostic accuracy. The combination of cervical smear cytology and MKRN1 protein expression test, HPV DNA test and MKRN1 protein expression test, and p16INK4a protein expression test and MKRN1 protein expression test were 74.3%, 78.1% (71.3%), papillary carcinoma (67.4%), HPV DNA (74.3%) and p16INK4a protein expression test (63.6%), respectively. Especially, the combination of HPV DNA test and MKRN1 protein expression test showed the same accuracy as the combination of conventional cervical smear cytology and HPV DNA test with 78.1% diagnostic accuracy.

In the case of ASCUS / LSIL samples (FIG. 8), the diagnostic accuracy was 73.1% in the combination of HPV DNA test and MKRN1 protein expression test. This represents a much higher accuracy than the combination of all other cases implemented.

Claims (12)

Identifying the presence of human papillomavirus DNA or mRNA from a biological sample isolated from the patient, and measuring the level of expression of MKRNl mRNA.
Identifying a presence of human papillomavirus DNA or mRNA from a biological sample separated from the patient, and measuring the level of expression of the MKRN1 protein.
Identifying a presence of a human papilloma virus protein from a biological sample isolated from a patient, and measuring the level of expression of MKRNl mRNA.
Identifying a presence of the HPV protein from the biological sample isolated from the patient, and determining the level of expression of the MKRN1 protein.
5. The method according to any one of claims 1 to 4,
Wherein the human papilloma virus is at least one of subtypes 6, 11, 16, 18, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 56, 58, A method of providing information about the diagnosis of a disease associated with a virus infection.
5. The method according to any one of claims 1 to 4,
Wherein said HPV infection-related disease is selected from the group consisting of Bowenoid papulosis, anal dysplasia, respiratory or conjunctiva papilloma, cervical dysplasia, cervical cancer, vulval cancer, or prostate cancer. A method of providing information about the diagnosis of a disease associated with a virus infection.
A primer or a probe that specifically binds to HPV DNA or mRNA, and a primer or a probe that specifically binds to MKRN1 mRNA.
A kit for diagnosing a human papilloma virus infection-related disease, comprising a primer or a probe specifically binding to HPV DNA or mRNA, and an antibody specifically binding to MKRN1 protein.
An antibody that specifically binds to HPV protein and a primer or a probe that specifically binds to MKRN1 mRNA.
An antibody that specifically binds to the HPV protein and an antibody that specifically binds to the MKRN1 protein.
10. The method according to any one of claims 7 to 10,
Wherein the human papilloma virus is at least one of subtypes 6, 11, 16, 18, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 56, 58, Diagnostic kit for viral infection related diseases.
10. The method according to any one of claims 7 to 10,
Wherein said HPV infection-related disease is selected from the group consisting of Bowenoid papulosis, anal dysplasia, respiratory or conjunctiva papilloma, cervical dysplasia, cervical cancer, vulval cancer, or prostate cancer. Diagnostic kit for viral infection related diseases.

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