CN113718031A

CN113718031A - Establishment of ovarian cancer early diagnosis composition

Info

Publication number: CN113718031A
Application number: CN202110942676.2A
Authority: CN
Inventors: 程晓东; 王芬芬; 李阳; 陈晓静; 陆伶佳
Original assignee: Womens Hospital of Zhejiang University School of Medicine
Current assignee: Shanghai Yunsheng Medical Laboratory Co ltd
Priority date: 2021-08-17
Filing date: 2021-08-17
Publication date: 2021-11-30
Anticipated expiration: 2041-08-17
Also published as: CN113718031B

Abstract

The invention relates to the field of tumor diagnosis, and discloses an ovarian cancer early diagnosis composition, which can be used for predicting, diagnosing and/or prognostically evaluating ovarian cancer by measuring the cfDNA content, the cfDNA TP53 mutation abundance and the CA125 protein expression level, wherein the sensitivity, specificity and accuracy of the composition reach 91.11%, 94.34% and 93.38%, and the detection rate of early ovarian cancer reaches 78.9%. By adopting the marker combination and the model scoring method, 71.05% of patients can be detected in CA125 negative ovarian cancer patients, 74% of subjects can be correctly judged in CA125 positive non-tumor population, and the problems of CA125 omission and false detection can be well solved. The invention can provide effective ovarian cancer early diagnosis or screening markers for clinic, and is beneficial to improving the prediction accuracy and detection rate of early ovarian cancer.

Description

Establishment of ovarian cancer early diagnosis composition

Technical Field

The invention relates to the field of tumor diagnosis or screening, relates to establishment of an ovarian cancer early diagnosis composition, and particularly relates to an ovarian cancer biological standard composition and application thereof, a kit and an evaluation model.

Background

The annual incidence rate of ovarian cancer is at the 3 rd position of female reproductive system tumor, and the ovarian cancer is in the trend of rising year by year and is located behind cervical cancer and uterine body malignant tumor, while the mortality rate is located at the top of gynecological tumor, and the ovarian cancer is one of the malignant tumors seriously threatening female health. The 5-year survival rate of patients with stage I ovarian cancer can exceed 90%, while the 5-year survival rate of advanced ovarian cancer is less than 20%. The deep pelvic cavity of the ovary, when the ovarian lesion is in the early stage, often has no specific clinical symptoms, and when the disease is diagnosed, 70% of patients are in the late stage. Therefore, the early screening and diagnosis of the ovarian cancer have important significance on the prognosis.

Currently, clinically used methods for detecting ovarian cancer include: cancer Antigen 125(CA125), transvaginal ultrasound alone or in combination. In ovarian cancer, the positive rate of CA125 is related to the stage and histological type of tumors, and the positive rate of patients with advanced and serous ovarian cancer is significantly higher than that of patients with early and non-serous ovarian cancer (the positive rate of the early stage is about 43.50-65.70%, and the positive rate of the advanced stage is 84.10-92.40%). The expression level of CA125 is influenced by various factors, such as common benign diseases of female physiology, pregnancy status, endometriosis, pelvic inflammation and the like can cause the increase of CA125, and meanwhile, the increase of CA125 of all ovarian cancer patients is not enough. Although the application of the vaginal ultrasound is common, the vaginal ultrasound has higher requirements on the experience and the technique of an operating doctor, and the accuracy rate of judging the benign and malignant tumors is not high. Thus, neither CA125, transvaginal ultrasound screening alone, nor a combination of the two, have achieved satisfactory results.

Therefore, there is a need to develop new peripheral blood markers with diagnostic value to predict and diagnose ovarian cancer at an early stage sensitively and specifically in a minimally invasive manner.

Sequencing of free DNA (cfDNA) is a new technology that has emerged in recent years, and it has been demonstrated that the plasma cfDNA content of tumor patients is significantly higher than that of healthy people. Fluid biopsies based on cfDNA sequencing have proven to have potential application in a variety of clinical indications for a variety of tumors. Tissue sequencing showed that greater than 90% of ovarian cancer tissue samples carried the TP53 mutation. However, patients with early stage ovarian cancer have a low tumor burden, less cfDNA is released into the blood, and the mutation abundance of ctDNA is low, which requires extremely sensitive techniques to detect. Although ctDNA-NGS sequencing technology is continuously advancing, patients with early ovarian tumors still have the situation of no mutation detection or missed detection.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to establish a combination for screening or early diagnosis of ovarian cancer, provide a multi-molecular marker combination and an evaluation model for diagnosis of ovarian cancer, and prepare a chip and a kit for diagnosing ovarian cancer on the basis of the combination.

According to an aspect of the invention, there is provided a biomarker panel for the predictive, diagnostic and/or prognostic assessment of ovarian cancer. The biomarker panel comprises circulating free dna (cfDNA), cfDNA TP53 Mutant Abundance (MAF) and plasma cancer antigen 125(CA125) protein.

Another aspect of the invention is to provide the use of the biomarker population cfDNA, cfDNA TP53 mutation abundance and CA125 in the prediction, diagnosis and/or prognostic assessment of ovarian cancer. In the present invention, the prediction includes screening or early diagnosis of ovarian cancer in the individual to be tested. In a specific embodiment, the biomarker panel is used for ovarian cancer screening or early diagnosis.

Another aspect of the present invention is to provide the application of the biomarker group cfDNA, cfDNA TP53 mutation abundance and CA125 in the preparation of in vitro diagnostic products for the prediction, diagnosis and/or prognosis evaluation of ovarian cancer.

Another aspect of the present invention provides the use of a substance that specifically recognizes or detects a biomarker panel as described above for the preparation of an in vitro diagnostic product for the prediction, diagnosis and/or prognostic evaluation of ovarian cancer. The substances that specifically recognize or detect the biomarker groups as described above include substances that specifically recognize or detect cfDNA, substances that specifically recognize or detect the abundance of mutations of cfDNA TP53, and substances that specifically recognize or detect the expression level of CA125 protein.

In the application of the invention, the diagnosis refers to the diagnosis of whether the individual to be detected has ovarian cancer or whether the ovarian cancer of the individual to be detected progresses or recurs, wherein the diagnosis comprises early screening or diagnosis; the prediction refers to predicting the risk of the individual to be tested suffering from ovarian cancer; the evaluation refers to the evaluation of the degree of ovarian cancer of the individual to be tested, or the evaluation of the curative effect of the individual to be tested after receiving related treatment.

Wherein, the evaluation of the degree of ovarian cancer in the test individual refers to the evaluation of whether the ovarian cancer is stage I ovarian cancer, stage II ovarian cancer, stage I-II ovarian cancer, stage III ovarian cancer, stage IV ovarian cancer or stage III-IV ovarian cancer.

In the application of the invention, the ovarian cancer types are as follows: ovarian epithelial cell carcinoma, classified as serous adenocarcinoma, clear cell carcinoma, mucinous adenocarcinoma, endometrioid adenocarcinoma; ovarian malignant germ cell tumors, classified as yolk sac tumor, asexual cell tumor, immature teratoma; malignant solitary interstitial tumors, classified as supporting cell-interstitial cell tumor, granulocytic-interstitial cell tumor; metastatic tumor, which is the primary kukenberg tumor of gastrointestinal tract.

In the present invention, the biomarker panel may be used in combination with other ovarian cancer-associated markers including, but not limited to, carcinoembryonic antigen, alpha-fetoprotein, human chorionic gonadotropin, lactate dehydrogenase, Prkdc protein, Rad54L protein, protein encoded by NPM1 gene, protein encoded by GNAS gene, protein encoded by P53 gene, protein encoded by FUBP1 gene, or protein encoded by KRAS gene, and the like.

In another aspect, the invention provides an ovarian cancer related in vitro diagnostic product, which comprises a reagent for specifically recognizing or detecting cfDNA, a reagent for specifically recognizing or detecting the abundance of TP53 mutation in cfDNA, and a reagent for specifically recognizing or detecting the expression level of CA125 protein.

In the invention, the in-vitro diagnosis product is a reagent, a chip or a kit. In one embodiment, the kit comprises the chip.

In the invention, the reagent specifically identifies or detects the content of plasma circulating free DNA (cfDNA), specifically identifies or detects the cfDNA TP53 Mutant Abundance (MAF) and specifically identifies or detects the cancer antigen 125(CA125) protein, and is used for predicting, diagnosing and/or prognostically evaluating the ovarian cancer by detecting the existence or the level of the ovarian cancer related cfDNA, the cfDNA TP53 mutant abundance and the CA125 in an individual to be detected. In another preferred embodiment, the agent is used for the assessment of the therapeutic efficacy of ovarian cancer, preferably using plasma as a sample.

The chip provided by the invention is used for diagnosing ovarian cancer by using the composite biomarker group for diagnosing ovarian cancer of a subject. In one embodiment, the chip provided by the invention comprises a reagent for specifically recognizing or binding to a dna (cfDNA) content, a reagent for specifically recognizing or binding to cfDNA TP53 Mutant Abundance (MAF), and a reagent for specifically recognizing or binding to plasma cancer antigen 125(CA125) protein.

According to another aspect of the present invention, there is provided a kit for diagnosing ovarian cancer using a composite biomarker group for the purpose of diagnosing ovarian cancer in a subject. In a specific embodiment, the invention provides a kit comprising an agent that specifically recognizes or binds to a dna (cfDNA) content, an agent that specifically recognizes or binds to cfDNA TP53 Mutant Abundance (MAF), and an agent that specifically recognizes or binds to plasma cancer antigen 125(CA125) protein. In some embodiments, the kit is a chemiluminescent kit. Preferably, the chemiluminescent kit comprises a capture antibody, a detection antibody and a chemiluminescent substrate.

Another aspect of the invention is to provide the use of an in vitro diagnostic product for the predictive, diagnostic and/or prognostic assessment of ovarian cancer. The in vitro diagnosis product is used for diagnosing whether an individual to be detected has ovarian cancer or not, predicting the risk of the individual to be detected having ovarian cancer, or evaluating the degree of the individual to be detected having ovarian cancer, or diagnosing whether the ovarian cancer of the individual to be detected progresses or recurs or not, or evaluating the curative effect of the individual to be detected after being treated related.

In the present invention, the evaluation of the ovarian cancer degree of the individual to be tested means the evaluation of whether the ovarian cancer is stage I ovarian cancer, stage II ovarian cancer, stage I to stage II ovarian cancer, stage III ovarian cancer, stage IV ovarian cancer or stage III to stage IV ovarian cancer. In another preferred embodiment, the diagnosis of ovarian cancer comprises: to distinguish between benign and malignant.

In the present invention, the stage of ovarian cancer is generally referred to NCCN guidelines and AJCC seventh edition. In some embodiments of the invention, the ovarian cancer is of the type: ovarian epithelial cell carcinoma, classified as serous adenocarcinoma, clear cell carcinoma, mucinous adenocarcinoma, endometrioid adenocarcinoma; ovarian malignant germ cell tumors, classified as yolk sac tumor, asexual cell tumor, immature teratoma; malignant solitary interstitial tumors, classified as supporting cell-interstitial cell tumor, granulocytic-interstitial cell tumor; metastatic tumor, which is the primary kukenberg tumor of gastrointestinal tract.

In the present invention, the sample of the subject to be tested is derived from whole blood, serum, plasma, saliva, buccal swab, lymph fluid, cerebrospinal fluid, ascites, cervical pap smear, bladder wash, uterine wash, stool, urine, tissue biopsy, etc.

In another aspect, the present invention provides a model for predicting, diagnosing and/or prognostically evaluating ovarian cancer, wherein the model comprises:

determining the content of cfDNA, the TP53 mutation abundance and the CA125 expression level of the individual to be tested; and

analyzing the determined content, abundance, or expression level to generate a risk score, wherein the risk score is used for the prediction, diagnosis, and/or prognostic assessment of ovarian cancer.

In one embodiment, the model is a model for ovarian cancer screening or early diagnosis.

In one embodiment, the expression analysis of the risk score is performed by statistical analysis, and the risk of ovarian cancer in the subject to be tested can be evaluated by the following mathematical function:

the Score value represents the risk of ovarian cancer in the test individual, a Score > 0.15 indicates a high risk of ovarian cancer, and a Score ≦ 0.15 indicates a low risk, i.e., healthy or benign disease.

The calculation method of the TP53 MAF abundance measured value is as follows: mutant copy number/(mutant copy number + wild type copy number) × 100%; the determination method can adopt any known mode, and the invention preferably adopts high-throughput sequencing, wherein the sequencing aims at the whole coding region of TP53, such as: a whole genome sequence or a whole exon sequence or other targeting sequence comprising the entire coding region of TP 53.

Wherein the unit of cfDNA content is ng/ml; the determination method is that the total amount (ng) of cfDNA extracted from blood plasma is divided by the volume (mL) of the blood plasma, wherein the total amount of cfDNA is obtained by determining the cfDNA in the solution, the determination method can be performed by a Qubit or Q-seq or other instruments, and the total amount of cfDNA can be obtained by multiplying the concentration of the obtained cfDNA by the volume of the solution.

Wherein the unit of the content of CA125 is U/ml; the determination method can adopt any known mode, including but not limited to electrochemical immune luminescence method, chemiluminescence immune assay, enzyme-linked immune assay (ELISA) and the like; the present invention is preferably an electrochemical immuno-luminescence method, a chemiluminescent immunoassay or an enzyme-linked immuno-sorbent assay (ELISA).

In the present invention, by selecting a certain value as a reference value/reference level, it is possible to distinguish between individuals that are good survival and individuals that are poor survival by comparing the measured value with the reference value/reference level; or to distinguish between individuals who would benefit from administration of a particular drug and those who would not benefit from administration of a particular drug, etc. By benefit of administration of a particular drug is meant improvement of the individual's condition by any measure, including those commonly used in the art, such as overall survival, long-term survival, relapse free survival, and long-term relapse free survival, among others.

In the present invention, the reference value/reference level of the test subject can be determined based on normal subjects in the population. The method of determination may be performed with reference to methods known in the art. In one embodiment, the critical value range of CA125 may be a clinically commonly used content range (35U/mL).

It is another aspect of the present invention to provide a device for the prediction, diagnosis and/or prognostic assessment of ovarian cancer, wherein the diagnosis includes early screening or diagnosis; the device comprises:

a data providing module: data for providing the content of cfDNA, the abundance of mutations of cfDNA TP53 and the expression level of CA125 in the individual to be tested;

an analysis module: for generating a risk score by analyzing the cfDNA content, cfDNA TP53 mutation abundance and CA125 expression level data in a test individual, wherein the risk score is used for prediction, diagnosis and/or prognosis evaluation of ovarian cancer.

In the analysis module, the risk score is calculated by the following mathematical function:

wherein the Score value represents the risk value of ovarian cancer of the individual to be tested, Score > 0.15 indicates high risk ovarian cancer, and Score < 0.15 indicates low risk, i.e. healthy or benign disease;

The inventor detects 156 ovarian cancer patients, 56 ovarian benign disease people and 315 asymptomatic healthy people by a targeted high-throughput sequencing technology (NGS) and an electrochemical immune luminescence method technology to determine the plasma cfDNA content, the cfDNA TP53 mutation and the mutation abundance and the expression level of plasma CA125 protein, further researches the efficiency of the three in ovarian cancer diagnosis independently and jointly, and finds that the sensitivity, the specificity and the accuracy of the CA125 used for ovarian cancer diagnosis independently are respectively as follows: 73.33%, 85.85 and 82.12%; the sensitivity, specificity and accuracy of the combined evaluation of the three marker indexes for ovarian cancer diagnosis are respectively as follows: 91.11%, 94.34 and 93.38%, all are significantly higher than the single index of CA125 and other existing detection methods. Wherein the sensitivity of the comprehensive scores of the three indexes to the ovarian cancer of stages I-III is respectively as follows: 70%, 88.9%, 100%, sensitivity to early phase (phase I + phase II): 78.9%, are all obviously higher than the single index of CA 125. Meanwhile, compared with the existing method, the composition scoring method has the advantage that the specificity and the sensitivity are also obviously improved. Particularly, in the ovarian cancer patients with CA125 negative, 71.05% of the patients can be successfully detected by the biomarker group and the model scoring method, which shows that the problem of CA125 omission can be well compensated by the method. In the non-tumor population with positive CA125, 74% of the subjects can be correctly judged by adopting the marker combination and the model scoring method, which shows that the invention can also make up the problem of CA125 false detection. Therefore, the value of early ovarian cancer diagnosis by combining the cfDNA content, the cfDNA TP53 mutation abundance and a comprehensive scoring model established by the expression level of the CA125 protein is revealed.

The ovarian cancer can be screened or diagnosed by using the model, and compared with the prior art, the invention has the advantages that

(1) According to the invention, the content of cfDNA, the mutation abundance of cfDNA TP53 and the cancer antigen 125(CA125) are combined to screen or diagnose ovarian cancer, and the detected indexes can be detected by a common clinical detection platform, so that the method has higher repeatability and operability, and can realize high-throughput automatic detection.

(2) The biomarker populations cfDNA, cfDNA TP53 mutation abundance and CA125 constitute a composite biomarker population with higher ovarian cancer diagnostic capability compared to existing biomarkers. The cfDNA indexes (cfDNA plasma content, TP53 mutation abundance) can make up the defects that CA125 has low specificity and is easily influenced by physiological states, and the accuracy, specificity and the like of predicting, screening or diagnosing early ovarian cancer can be improved through the technical scheme of the invention.

(3) The interpretation of the score results is objective and straightforward, and the efficiency of the kit for ovarian cancer diagnosis and the method for ovarian cancer diagnosis using the kit for ovarian cancer diagnosis can be further improved.

(4) The product/model of the invention is used for diagnosing ovarian cancer, has high sensitivity, specificity and accuracy which respectively reach 91.11 percent, 94.34 percent and 93.38 percent, and can reduce false positive; in addition, the detection rate of the ovarian cancer patients in early stage reaches 78.9 percent, which is obviously higher than that of the existing clinical common method (CA125, ROMA).

(5) The invention has the advantages of rapid detection, good stability, high-throughput detection and low cost.

(6) The invention can improve the survival rate of ovarian cancer patients through effective diagnosis, and can monitor the response of the patients to treatment, thereby changing the treatment mode according to the result. Provides a very effective detection means for clinical diagnosis and scientific research work, and provides a good auxiliary function for doctors to evaluate whether ovarian cancer exists.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

In order to make the purpose, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings:

FIG. 1 expression levels of CA125 in different groups of subjects.

FIG. 2 distribution of plasma free DNA (cfDNA) content of different groups of subjects.

FIG. 3 mutation abundance of the free DNATP53 gene mutation in different groups of subjects.

Figure 4 composite score distribution for different groups of subjects.

FIG. 5A ROC curves in the training set.

FIG. 5B validates the concentrated ROC curve.

FIG. 6 is a comparison of the sensitivity of different detection methods/models to different stages of ovarian cancer detection.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and those skilled in the art can easily understand the advantages and effects of the present invention from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention, in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ conventional techniques in the art of molecular biology, immunology, inspection, gene sequencing, bioinformatics, and related arts.

The present invention provides a biomarker panel for the predictive, diagnostic and/or prognostic assessment of ovarian cancer. The biomarker panel comprises circulating free dna (cfDNA), cfDNA TP53 Mutant Abundance (MAF) and plasma cancer antigen 125(CA125) protein.

cfDNA is plasma circulating free DNA (cfDNA) that is mostly released by the rupture of cells or white blood cells of the body. The content of circulating free DNA (cfDNA) refers to the amount of free DNA extracted per ml of plasma.

The TP53 gene, also known as P53, is a cancer suppressor gene that encodes a protein with a molecular weight of 53 kDa. The protein encoded by this gene is a transcription factor that controls the initiation of the cell cycle, and plays a crucial role from the beginning of cell division. The cfDNA TP53 mutation was a mutation reported in the COSMIC database or a mutation predicted to have an effect on the functional activity of TP 53. The assay for the abundance of the TP53 mutation contained the entire coding region of TP53, as: whole genome sequencing or whole exon sequences or other targeting sequences that contain the entire coding region of TP53, the assay method can be performed by high throughput sequencing.

"CA 125", carbohydrate antigen 125, also known as mucin-16, is a protein encoded by the MUC16 gene. CA125 is one of the markers of ovarian epithelial cancer, and is widely used for diagnosis and follow-up of ovarian epithelial cancer at present. In the present invention, "CA 125" means a polypeptide biomarker or fragment thereof having at least 85% sequence identity to NCBI accession No. NP _ 078966.2.

In the present invention, the "prediction" refers to the evaluation of the probability of ovarian cancer of the individual to be tested. Further, setting a reference level of the biomarker group, wherein when the biomarker level of the individual to be tested is lower than the reference level, the individual to be tested is a normal individual, and when the biomarker level of the individual to be tested is higher than the reference level, the individual to be tested is at risk of ovarian cancer.

In the present invention, "diagnosis" means the identification of the presence or nature of ovarian cancer in a test individual. The diagnosis includes early diagnosis of ovarian cancer (stage I, stage II or stage I + II) or differentiation between benign and malignant. In the invention, the ovarian cancer is specifically classified into stage I, stage II, stage III and stage IV tumors. Wherein, in the I stage: the tumor is limited in the ovary, and no metastasis is generated at other parts; and stage II: when the tumor affects the ovary, pelvic cavity diffusion occurs, such as transfer of uterus, oviduct and parauterine tissue; stage III: the tumor is subjected to extrapelvic peritoneal membrane implantation or local lymph node metastasis; and IV, period: distant metastases, such as liver, lung, brain, bone, etc. In general, the higher the stage, the higher the degree of malignancy, the worse the prognosis; conversely, the lower the staging, the better the prognosis. Additionally, "early stage of ovarian cancer" or "early stage ovarian cancer" means ovarian cancer that is in stage I or II.

In the present invention, the "sensitivity" of a diagnostic assay is the percentage of diseased individuals in the population that test positive to the total diseased individuals.

In the present invention, the "specificity" of the diagnostic assay is 1 minus the false positive rate, where "false positive rate" refers to the proportion of individuals who test positive but are not actually diseased.

In the present invention, the "accuracy" of a diagnostic assay, also known as the efficiency (efficiency), is expressed as the percentage of the total number of subjects tested, which is the sum of the number of subjects tested as true positive and true negative.

In the present invention, the "positive predictive value" in the diagnostic measurement is the percentage of the number of true positives to the number of test result positives, and indicates the probability that a test result positive belongs to a true case.

In the present invention, the "negative predictive value" in the diagnostic measurement is the percentage of the number of true negative persons to the number of negative persons in the test result, and indicates the probability that the negative persons in the test result belong to non-cases.

In the present invention, "detection rate/sensitivity to early stage ovarian cancer" refers to the percentage of the sum of individuals who have had the disease and who are tested to have stage I or stage II ovarian cancer, relative to the sum of individuals who actually have had the disease and who have had stage I or stage II ovarian cancer.

In the present invention, "sensitivity to stage I ovarian cancer" refers to the percentage of affected individuals who test for stage I ovarian cancer to those who actually have the disease for stage I ovarian cancer.

In the present invention, "sensitivity to stage II ovarian cancer" refers to the percentage of affected individuals who test as stage II ovarian cancer to those who actually have the disease.

In the present invention, "sensitivity to stage III ovarian cancer" refers to the percentage of affected individuals who test for stage III ovarian cancer to those who actually have the disease for stage III ovarian cancer.

In the present invention, "sensitivity to stage IV ovarian cancer" refers to the percentage of affected individuals who test for stage IV ovarian cancer to those who actually have the disease for stage IV ovarian cancer.

In the present invention, "prognostic evaluation" refers to the prognostic judgment of the course and/or outcome of ovarian cancer patients, or the evaluation or monitoring of the therapeutic effect of a therapeutic agent. For example, following treatment with a drug, the level of the biomarker population in the test individual remains above the reference level, indicating progression of ovarian cancer in the test individual.

In the present invention, the meaning of "ovarian cancer" should be well known to those skilled in the art. For the avoidance of doubt, ovarian cancer is a cancer that develops from the ovary. More than 90% of ovarian cancers are epithelial in origin, originating from the ovarian surface. However, it is believed that the fallopian tubes may also be the source of some ovarian cancers, and in this case, cancers that develop from the fallopian tubes are also encompassed by the term "ovarian cancer".

In the present invention, the types of ovarian cancer that are predicted, diagnosed and/or prognostically assessed are: ovarian epithelial cell carcinoma, classified as serous adenocarcinoma, clear cell carcinoma, mucinous adenocarcinoma, endometrioid adenocarcinoma; the ovarian malignant germ cell tumor is divided into yolk sac tumor, asexual cell tumor and immature teratoma; malignant solitary interstitial tumors, classified as supporting cell-interstitial cell tumor, granulocytic-interstitial cell tumor; metastatic tumors, such as kukenberg tumor, which originate in the gastrointestinal tract. Among them, epithelial tumors account for about 2/3 in ovarian cancer, and generally originate from germinal epithelium on the surface of the ovary. Germ cell tumors can account for 20% of ovarian cancer, second to epithelial tumors; genital tumors can be seen at any age, and are generally common in young women; ovarian tumors are a source of germ cells in 60% of children and adolescent women. Interstitial tumor of sex cord, it accounts for ovarian tumor 5% to 10% approximately; such ovarian tumors secrete hormones and develop the corresponding symptoms. Metastatic tumors, approximately 5% to 10% of ovarian tumors are metastatic. Preferably, the ovarian cancer that is predicted, diagnosed and/or prognostically assessed is epithelial ovarian cancer.

In the present invention, various techniques can be used to detect cfDNA, cfDNA TP53 mutation abundance, and CA125 levels, all of which can be included in the present invention. The detection can be performed by any known technique, including but not limited to: western blotting, SDS-PAGE, in situ hybridization, enzyme-linked immunosorbent assay, Polymerase Chain Reaction (PCR), Southern blotting, protein sequence analysis, mass spectrometry or DNA sequence analysis, etc.

In the present invention, the sample is whole blood, serum, plasma, saliva, buccal swab, lymph fluid, cerebrospinal fluid, ascites, cervical pap smear, bladder wash, uterine wash, stool, urine, tissue biopsy, etc. from the subject. Preferably, the present invention uses plasma as the sample to be tested. The sample may be fresh, frozen, or paraffin-fixed embedded cells.

In one embodiment, the sample is a plasma sample obtained from the test subject that is a yellow liquid component of blood in which normally blood cells in whole blood are suspended. It accounts for about 55% of the total blood volume. The majority of which is water (90 vol%) and contains dissolved proteins, glucose, coagulation factors, mineral ions, hormones and carbon dioxide. Plasma is prepared by centrifuging fresh blood in tubes in a centrifuge until the blood cells settle to the bottom of the tubes.

In one embodiment, the sample is a serum sample obtained from the subject. Serum is derived from plasma, which is plasma without fibrinogen or other clotting factors.

The present inventors have made an effort to improve sensitivity, specificity, accuracy, etc. of ovarian cancer diagnosis, as well as early diagnosis of ovarian cancer. The inventor collects a large number of clinical cases, analyzes a large sample size and deeply researches to disclose a group of ovarian cancer combined markers: cfDNA, cfDNA TP53 mutant abundance, and CA 125. The expression of these molecules was significantly higher in the serum/plasma of ovarian cancer patients than in healthy controls. Therefore, the markers can be used for preparing a diagnosis chip or a diagnosis kit which can efficiently diagnose ovarian cancer. The markers are used in clinical diagnosis to determine whether the individual to be tested suffers from ovarian cancer and the severity of ovarian cancer, so as to provide a basis for disease diagnosis or prognosis, or to evaluate the possibility of the individual to be tested suffering from ovarian cancer at an early stage, or to evaluate the effectiveness of treatment for the individual to be tested who has already been treated. Therefore, the combined application of the biomarker group can obviously improve the sensitivity, specificity, accuracy and the like of ovarian cancer diagnosis, and can efficiently detect patients with early ovarian cancer. The effect is significantly better than the case of using a single marker and also significantly higher than the case of using other markers in combination.

The invention also provides applications of the biomarker group cfDNA, cfDNA TP53 mutation abundance and CA125 in prediction, diagnosis and/or prognosis evaluation.

The invention provides an ovarian cancer detection reagent, which specifically identifies or detects cfDNA, specifically identifies or detects cfDNA TP53 mutation abundance, and specifically identifies or detects CA125 protein, and provides detection of the ovarian cancer related cfDNA, cfDNA TP53 mutation abundance and the existence or level of CA125 in an individual to be detected, and is used for prediction, diagnosis and/or prognosis evaluation of ovarian cancer. The detection aiming at cfDNA and the mutation abundance of cfDNA TP53 can be respectively a high-throughput sequencing method; in a preferred embodiment, the high throughput sequencing method for TP53 mutation detection comprises all exons of TP 53. The detection of CA125 may be by electrochemiluminescence.

In the present invention, the reagent may rely on the presence of a detectable label for detection purposes. The reagents are typically labeled as follows: the agent is covalently or non-covalently bound to a substance or ligand that provides or allows the generation of a detectable signal. Some examples include, but are not limited to, radioisotopes, enzymes, fluorescent substances, luminescent substances, ligands, microparticles, redox molecules, substrates, cofactors, inhibitors, magnetic particles, and the like. Examples of enzymes that can serve as detection markers include, but are not limited to, beta-glucuronidase, beta-glucosidase, urease, peroxidase or alkaline phosphatase, acetylcholinesterase, glucose oxidase, hexokinase and gdpase, rnase, glucose oxidase and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphoenolpyruvate decarboxylase, and beta-lactamase. Examples of fluorescent substances include, but are not limited to, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine (fluorescamin). Examples of luminescent substances include, but are not limited to, acridinium esters, luciferin, and luciferase. Examples of ligands include, but are not limited to, biotin and its derivatives. Examples of microparticles include, but are not limited to, colloidal gold and colored latex. Examples of redox molecules include, but are not limited to, ferrocene, ruthenium complexes, viologen, quinone, It ion, Cs ion, diimide, 1, 4-benzoquinone, hydroquinone.

The invention provides an ovarian cancer diagnosis chip using a composite biomarker group for diagnosing ovarian cancer of a subject, wherein the ovarian cancer diagnosis chip comprises reagents for detecting the content of plasma circulating free DNA (cfDNA), cfDNA TP53 Mutation Abundance (MAF) and the expression level of plasma cancer antigen 125(CA125) protein serving as biomarkers. The chip is used for screening early ovarian cancer in an individual to be tested, and/or used for evaluating the ovarian cancer progression in the individual to be tested, and/or used for evaluating the treatment effect of a medicament, and the like. In one embodiment, the chip provided by the invention comprises a reagent for specifically recognizing or binding to a dna (cfDNA) content, a reagent for specifically recognizing or binding to cfDNA TP53 Mutant Abundance (MAF), and a reagent for specifically recognizing or binding to plasma cancer antigen 125(CA125) protein.

In one embodiment, the invention provides an ovarian cancer detection chip, which comprises a DNA chip and a protein chip, wherein the DNA chip and the protein chip can be arranged separately or on the same chip. When the DNA chip and the protein chip are disposed on different chips, different types of adsorbents are present on different solid supports. When the DNA chip and the protein chip are disposed on the same chip, different types of adsorbents are present on the same solid support. The DNA chip hybridizes with a nucleic acid corresponding to the disclosure herein for specifically recognizing or detecting cfDNA, specifically recognizing or detecting cfDNA TP53 mutation abundance, and the protein chip is used for specifically recognizing or detecting CA125 protein.

In the present invention, the DNA chip can be prepared by a technique known in the art. In one embodiment, the DNA chip comprises a substrate loaded with nanometals, which are linked to raman signal molecules, which are linked to probes via linker molecules. The DNA chip is formed by attaching a high-density DNA fragment array on a material such as glass, nylon or the like in a certain arrangement manner by a microarray technology.

In the present invention, the protein chip can be prepared by a technique known in the art. Comprises a solid phase carrier, a capture antibody and a labeled detection antibody. The protein chip is a high-throughput monitoring system, and the interaction between protein molecules is monitored through the interaction between target molecules and capture molecules. In a preferred embodiment of the present invention, the protein chip is a liquid chip, and preferably, a double antibody sandwich method is used in combination with the liquid chip technology. The double antibody sandwich method is conventionally performed by immobilizing a capture antibody (also called a primary antibody or a primary antibody) on a carrier, reacting the capture antibody with a protein antigen, washing, reacting with a labeled detection antibody (also called a secondary antibody or a secondary antibody), washing, and finally performing chemiluminescence or enzyme-linked color reaction to detect a signal.

The capture antibody can be prepared by linking a monoclonal antibody against the target biomarker to magnetic microparticles in the presence of a coupling solution under appropriate reaction conditions. Further, the detection antibody may be a monoclonal antibody having an alkaline phosphatase label, and combinations thereof.

As an embodiment of the protein chip of the present invention, the capture antibody is immobilized on a microsphere carrying a specific detectable signal (molecule) to prepare a liquid phase protein chip, and the principle of detection by using the liquid phase protein chip is as follows: a single microsphere is passed through the detection channel and two lasers are used to simultaneously detect the microsphere identification signal and the detectable signal on the microsphere. One type of laser excitation is the identification signal of the microsphere, and the microsphere can be classified according to the different types of identification signals on the microsphere, thereby distinguishing different binding reactions. Another type of laser excitation is a detectable signal, in order to determine the amount of detectable signal bound to the microsphere, and thus the amount of molecule of interest bound to the microsphere. Therefore, by simultaneous detection of two lasers, the kind and amount of the bound detection object can be determined.

In one embodiment, the antibody is derived from a CA125 protein and is an antibody specific for the CA125 protein. In one embodiment, the agent is detectably labeled. The antibodies can be prepared by various techniques known to those skilled in the art. For example, the purified CA125 protein, or antigenic fragment thereof, can be administered to an animal (e.g., mouse, rat, rabbit, etc.) to induce the production of polyclonal antibodies, and various adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant, etc. Similarly, cells expressing the CA125 protein or antigenic fragments thereof can be used to immunize animals to produce antibodies. The antibody may also be a monoclonal antibody. Such monoclonal antibodies can be prepared using well-known hybridoma techniques.

The term "antibody" as used herein has its broadest meaning and encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (e.g., linear antibodies, single chain antibody molecules, Fc or Fc ' peptides, Fab ', F (ab ')2, and Fv fragments), single chain Fv (scfv) mutants, multispecific antibodies, e.g., bispecific antibodies produced from at least two intact antibodies, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site, so long as the antibody exhibits the desired biological activity. Antibodies can be one of any of five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses thereof (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), are based on the identification of their heavy chain constant domains (referred to as α, δ, ε, γ, and μ, respectively). The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. The antibody may be a naked antibody, or conjugated to other molecules (e.g., toxins, radioisotopes, etc.).

The "microsphere recognition signal" refers to a recognition signal for distinguishing different capture antibodies on the microsphere. For convenience, the microsphere recognition signals are preferably the same for microspheres immobilized with the same capture antibody. Preferably, the microsphere identification signal is a fluorescent signal, and the color of the fluorescence is different for microspheres immobilized with different species of capture antibodies, and preferably the same for microspheres immobilized with the same species of capture antibodies. The detailed procedures for immobilizing the capture antibody on the microspheres can be carried out by conventional methods, thereby obtaining conjugates of different microspheres with the corresponding capture antibody.

Wherein said detection antibody is optionally labeled with a plurality of detectable signals. For example, the detectable signal is selected from: FITC, CY3, CY5, phycocyanin, oregon green dye, texas red dye, and the like. The detection substance corresponding to the detectable signal is a molecule (reporter molecule) capable of binding to the detectable signal and capable of reporting the binding. When biotin is used to label the detection antibody, streptavidin-phycoerythrin may be used as the reporter molecule. Detectable signal labeling methods are well known to those skilled in the art.

In order to eliminate false positive and false negative, positive control and negative control are preferably set in the detection process to determine the expression condition and expression amount of the biomarkers. In addition, to obtain quantitative results, standards containing known concentrations of cfDNA, cfDNA TP53 mutation abundance, and CA125 can be set up in the detection process. The setting method of the reference or standard substance adopts a conventional method. Preferably, the specific probe or antibody carries an identifiable signal, so that the detection result can be conveniently and visually obtained.

The invention also provides the application of the biochip, which is used for predicting, diagnosing and/or prognostically evaluating ovarian cancer or preparing a kit for diagnosing ovarian cancer.

The invention also provides an ovarian cancer diagnosis kit which utilizes the composite biomarker population for diagnosing ovarian cancer of a subject, and is used for detecting cfDNA, cfDNA TP53 mutation abundance and CA125 level so as to carry out prediction, diagnosis and/or prognosis evaluation on ovarian cancer.

In the present invention, the kit comprises the reagent or biochip described above. The kit may also include various other reagents required for hybridization or enzyme-linked assays, etc., including but not limited to: PCR reaction solution, quality control solution, standard substance, enzyme, contrast solution, color development solution, washing solution and the like. Further, the kit may further comprise a container and instructions for use to instruct a person to perform the test according to the correct procedures, conditions and dosage, including how to collect the sample to be tested, how to wash the probe, etc. The standard substance is a DNA standard substance and a plasma cancer antigen 125(CA125) protein standard protein.

In the invention, the kit can be prepared into a chemiluminescence kit. "Chemiluminescent kit" means a kit for measuring the level of a biomarker using a Chemiluminescent enzyme immunoassay (cLEIA). Specifically, in a chemiluminescent enzyme immunoassay, an immunoreaction is carried out with an enzyme-labeled bioactive substance, the enzyme on the immunoreactive complex acts on a luminescent substrate, emits light under the action of a signal reagent, and is then subjected to luminescence measurement with a luminescence signal measuring instrument, thereby quantitatively analyzing the level of a biomarker in a sample from an individual to be tested.

The invention also provides the use of the kit for the predictive, diagnostic and/or prognostic assessment of ovarian cancer.

The invention also provides a model for predicting, diagnosing and/or prognostically assessing ovarian cancer, wherein the model comprises:

determining the levels of cfDNA, cfDNA TP53 mutation and CA125 in the individual to be tested; and

analyzing the expression level to generate a risk score, wherein the risk score is used for prediction, diagnosis and/or prognostic assessment of ovarian cancer.

When evaluating the risk of the individual to be tested for ovarian cancer, specifically, the expression analysis of the risk score is completed by statistical analysis, and the mathematical function is as follows:

The present invention also provides a device for the prediction, diagnosis and/or prognostic assessment of ovarian cancer, wherein the diagnosis includes early screening or diagnosis; the device comprises:

the TP53 MAF is calculated in the following way: mutant copy number/(mutant copy number + wild type copy number) × 100%;

wherein, the determination method of the cfDNA content is that the total amount of the cfDNA extracted from the blood plasma is divided by the volume of the blood plasma, and the unit is ng/ml;

wherein the unit of the content of CA125 is U/ml.

The invention also provides a method for detecting whether a to-be-detected individual suffers from ovarian cancer, which comprises the following steps: detecting the cfDNA, the mutation abundance of the cfDNA TP53 and the expression level in CA125 in the individual to be detected; analyzing the expression level to generate a risk score, wherein the risk score can be used to determine whether the subject suffers from ovarian cancer.

The invention also provides a method for screening whether a test sample is suffered from early ovarian cancer, which comprises the following steps: testing the cfDNA, the abundance of the mutation of cfDNA TP53 and the expression level in CA125 in the individual to be tested; analyzing the expression level to generate a risk score, wherein the risk score can be used to determine whether the test individual has early ovarian cancer.

In one embodiment, the early stage ovarian cancer is stage I cancer.

The identification of cfDNA, cfDNA TP53 mutation abundance, and CA125 associated with the aforementioned ovarian cancer also allows for methods of assessing the efficacy of a treatment for the cancer in a test individual. Analyzing the expression level to generate a risk score by determining the expression level in the test individual of cfDNA, the abundance of cfDNA TP53 mutation, and CA125, wherein the risk score can be used to determine whether the test individual will benefit from receiving the treatment.

The methods of the invention are employed to monitor the efficacy of a treatment regimen for ovarian cancer, ultimately with the aim of eliminating the disease. In such cases, success of treatment can be detected as a reduction or elimination of cfDNA, cfDNA TP53 mutation abundance, and CA125 from the diseased test individual. The aforementioned cfDNA, cfDNA TP53 mutation abundance and CA125 levels will be below the respective reference levels if the treatment is effective, whereas the aforementioned cfDNA, cfDNA TP53 mutation abundance and CA125 levels will be above or the same as the respective reference levels if the treatment is ineffective. Thus, in some embodiments, a level of cfDNA, cfDNA TP53 mutation abundance, and CA125 in the test individual that is lower than the respective reference level indicates that ovarian cancer has declined in the test individual and successful treatment is observed. The cfDNA, cfDNA TP53 mutation abundance, and CA125 or reference levels of each thereof may be obtained from an average of multiple normal samples.

Accordingly, the present invention also provides a method of assessing the progression or recurrence of ovarian cancer in a test individual, the method comprising the steps of: and detecting the cfDNA, the mutation abundance of the cfDNA TP53 and the CA125 in the individual to be detected. In some embodiments, if the level of ovarian cancer-associated cfDNA, cfDNA TP53 mutation abundance, and CA125 in the test individual is above their respective reference levels, then indicating that ovarian cancer is recurrent in the test individual; if the ovarian cancer-associated cfDNA, cfDNA TP53 mutation abundance, and CA125 levels in the test individual are below or near the respective reference levels, then indicating that ovarian cancer has not relapsed in the test individual.

In one embodiment, the method comprises:

(a) detecting the ovarian cancer related cfDNA, cfDNA TP53 mutation abundance and CA125 level in the individual to be tested;

(b) analyzing the expression level to generate a risk score, wherein the risk score can be used to determine the progression or recurrence of ovarian cancer in the test individual.

In one embodiment, the subject is undergoing treatment for ovarian cancer. In some embodiments, the ovarian cancer-associated cfDNA, cfDNA TP53 mutation abundance, and CA125 level in the test individual is greater than a reference level, indicating progression or recurrence of ovarian cancer in the test individual.

The present invention also provides a method of screening for a candidate therapeutic agent useful for treating ovarian cancer in a test individual, the method comprising the steps of: assessing the activity of the candidate therapeutic agent in reducing the ovarian cancer-associated cfDNA, cfDNA TP53 mutation abundance, and CA125 level in the test individual.

In one embodiment, the method comprises:

(a) administering said candidate therapeutic agent to said test subject;

(b) detecting the ovarian cancer-associated cfDNA, cfDNA TP53 mutation abundance and CA125 level in the individual to be tested; and

(c) comparing the ovarian cancer-associated cfDNA, cfDNA TP53 mutation abundance, and CA125 level in the test individual to the ovarian cancer-associated cfDNA, cfDNA TP53 mutation abundance, and CA125 level in an untreated test individual having ovarian cancer;

wherein the candidate therapeutic agent is useful for treating ovarian cancer if the level of ovarian cancer-associated cfDNA, cfDNA TP53 mutation abundance, and CA125 in the test individual is lower than the level of ovarian cancer-associated cfDNA, cfDNA TP53 mutation abundance, and CA125 in the untreated test individual.

The screening assay may be performed in vitro and/or in vivo. For example, prospective agents can be screened in cell-based assays to identify candidate therapeutic agents for treating ovarian cancer. In this regard, each of the contemplated agents is incubated with cultured cells (e.g., cells obtained from the ovary of a normal test individual or an individual having ovarian cancer test, or a cell line derived from a normal or diseased test individual) and then the cfDNA, cfDNA TP53 mutation abundance, and CA125 levels are measured.

The reference levels of each of the cfDNA, cfDNA TP53 mutation abundance, and CA125 can be determined as described above.

In some embodiments of the invention, the cfDNA, cfDNA TP53 mutation abundance, and CA125 presence or levels are detected at more than one time point. Such "continuous" sampling is well suited, for example, to monitoring the progression of ovarian cancer. Continuous sampling may be performed on any desired timeline, such as monthly, quarterly (i.e., once every three months), semi-annually, bi-annually, or less frequently.

In the biomarker groups, the uses or methods of the present invention, the biomarker groups may be used in combination with other ovarian cancer-associated markers that have been determined now or discovered and determined in the future for predicting or assessing the occurrence, development, risk of ovarian cancer, including, but not limited to, carcinoembryonic antigen, alpha-fetoprotein, human chorionic gonadotropin, lactate dehydrogenase, Prkdc protein, Rad54L protein, protein encoded by the NPM1 gene, protein encoded by the GNAS gene, protein encoded by the P53 gene, protein encoded by the FUBP1 gene, or protein encoded by the KRAS gene, and the like.

The term "subject to be tested" as used herein refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, dogs, cats, horses, cows, sheep, deer, pigs, rodents, and any other animal known to have ovarian cancer. Thus, while the above is described with respect to human cfDNA, cfDNA TP53 mutation abundance, and CA125 assay information, it is to be understood that the methods of the invention are not limited to humans.

In some embodiments, the detecting comprises detection of a biomarker population cfDNA, cfDNA TP53 mutation abundance, and CA125, wherein the biomarker population can also be used in combination with other markers associated with ovarian cancer.

Test object

The subjects mentioned in the examples are all women, including 315 asymptomatic healthy persons, 56 persons with benign ovarian disease, 156 persons with epithelial ovarian cancer; of these, 156 subjects with epithelial ovarian cancer and 56 subjects with benign ovarian disease were enrolled primarily in the affiliated obstetrics and gynecology hospital of Zhejiang university at 12 months to 2021 months in 2018.

Ovarian cancer subjects were diagnosed with epithelial ovarian cancer and did not receive any anti-cancer treatment prior to blood collection. As shown in Table 1, early ovarian cancer cases (stage I + II) accounted for 41% (64 cases), with the median age of the population being 55.5 years. The median age of benign ovarian disease population is 50 years. All patients filled informed consent to use their clinical data, and the present study was conducted strictly in accordance with the declaration of helsinki and was simultaneously approved by the ethical review committee of the affiliated obstetrical hospital of zhejiang university. The 315 asymptomatic healthy volunteer samples mentioned in the examples, with a median age of 56 years, were partially obtained from a previous biological sample bank, and were performed in small amounts by means of volunteer recruitment, all of which filled in informed consent to use their blood samples.

TABLE 1 case characteristics of the Subjects

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Example 1

1. Blood sample collection

Venous blood collection was performed using free DNA blood collection tubes (LBgard blood collection test tubes, biomanirca, usa) and samples were sent to sandiskei medical laboratory (shanghai, china) at room temperature (4-37 ℃) for plasma and leukocyte (WBC) separation. All subjects had a 10mL whole blood sample collection. For plasma and peripheral blood samples, were drawn prior to anesthesia on the day of surgery. Plasma of healthy volunteers in the biological sample bank is stored at-80 ℃ for at least 3mL within 2 years and transported to Shanhai Xicheng medical inspection institute through dry ice.

Extraction of cfDNA and genomic DNA (genomic DNA)

Peripheral blood (10 mL of each freshly collected sample, 3mL of the biological specimen bank sample) was centrifuged at 4 ℃ and 1600g for 10 minutes. Plasma (upper yellow layer) and leukocytes (middle layer) were collected, and then the upper plasma was subjected to a second centrifugation to remove residual cells or cell debris under the following centrifugation conditions: 16000g, 10 min at 4 ℃. 0.5mL of plasma was isolated and stored separately for subsequent protein marker detection, and the remaining plasma was used for cfDNA extraction. Plasma and leukocyte sample markers correspond to subject number and separation date and are stored at-80 ℃ until use.

cfDNA was extracted from 2.5-5mL plasma using the whole-format gold-free DNA extraction kit, according to the kit supplier's instructions. gDNA was extracted from leukocyte samples using a Tiangen blood genomic DNA (genomic DNA) extraction kit, according to the kit supplier's instructions.

cfDNA was quantified and fragment analyzed by a Qubit fluorometer (life technologies, Carlsbad, CA) and cfDNA content (ng/mL plasma) was calculated, and the cfDNA content distribution for each set of samples is shown in figure 2. DNA samples were stored at-20 ℃ prior to use.

The formula for calculating cfDNA content is:

3. protein marker detection

Detection of the protein marker CA125 was performed using the cobas e411 system (Roche). The detection was performed using a Roche carbohydrate antigen 125 detection kit (electrochemiluminescence), and the experimental procedure was performed according to the instructions of the kit supplier.

CA125 was used as a single evaluation index, and 35U/mL, which is commonly used, was used as a plasma CA125 expression cutoff value. Of the 156 subjects with epithelial ovarian cancer, 38 (24.4%) of CA125 were within the normal range, 118 (75.6%) of CA125 were above the cutoff value, and the average of 156 of CA125 was 572.8U/mL; of the 56 subjects with benign ovarian disease, 38 (67.9%) had CA125 in the normal range, 18 (32.1%) had plasma CA125 above the cutoff value, and 56 had CA125 with an average value of 70.6U/mL; of the 315 asymptomatic healthy people, 283 (89.8%) CA125 were within the normal range, 32 (10.2%) plasma CA125 were out of range, and the average value of 315 CA125 was 17.7U/mL (see table 1, fig. 1 for details). Overall, the false positive rate for a single CA125 index is: 13.48% (i.e., (18+32)/(56+ 315)).

High throughput sequencing and identification of somatic mutations by cfDNATP53

High-throughput sequencing-based detection of cfDNA TP53 mutations was performed by exon sequencing based on capture pooling, both leukocyte gDNA and cfDNA sequencing were performed using this technique. Specific methods for cfDNA and gDNA library construction can be performed with reference to relevant guidelines of the KAPA library construction kit (KAPA Biosystems, usa). The NGS library was sequenced on MGISEQ-2000 (wisdom hua), the sequencing results were compared to the hg19/GRCh38 human reference sequence, background noise caused by random NGS errors was removed, and then true mutants could be identified, and the mutation frequency was calculated by comparing the number of unique sequencing results containing the mutants to the number of all sequencing results used to map variant positions. Germline mutations and hematopoietic clonal mutations can be filtered by white blood cell sequencing by subtracting the mutations detected in leukocytes to obtain the true cfDNATP53 somatic mutations.

The positive rate of TP53 somatic mutation in 156 epithelial ovarian cancer subjects was 77.6% (mutation abundance median value of mutation positive samples: 0.35%, range: 0.01% -33.92%); in 315 asymptomatic health cohorts, the positive rate of TP53 was 10.2% (median mutation abundance in mutation-positive samples: 0.05%, range: 0.01% -0.54%); of the 56 benign ovarian disease subjects, 16 (28.6%) had the cfDNATP53 mutation (median mutation abundance: 0.28%, range: 0.02% -12.78%) (see FIG. 3 for details).

5. Construction of a predictive model

Each set of samples was run as 2.5: 1 was randomly assigned to the training set and validation set, and the final 111 samples of epithelial ovarian cancer, 40 samples of benign ovarian disease, and 225 samples of healthy subjects were included in the training set, and the detailed information of the population characteristics is shown in table 1. Models were constructed by logistic regression using SPSS 26.0 software, and P values below 0.05 were considered statistically significant. Age, cfDNA content, CA125 expression level, TP53 mutation abundance (TP53 wild-type mutation abundance is defined as 0) are included in the analysis, age has no statistical significance, and finally a model with CA125 expression level, TP53 mutation abundance and cfDNA content as variables is determined, and the mathematical function of the model is as follows:

the function value (i.e.the Score value) represents the risk value for ovarian cancer in the subject, with Score > 0.15 indicating a high risk (ovarian cancer) and Score ≦ 0.15 indicating a low risk (healthy or benign disease).

The composite scores of all the samples are calculated according to the mathematical function, and the distribution of the composite scores of each group of samples is shown in figure 4. The ROC curve for the training set is shown in FIG. 5-A.

6. Model performance verification

Sample data from 45 epithelial ovarian cancer samples, 16 benign ovarian disease samples and 90 healthy subjects were used as the validation set, and detailed information on the population characteristics is shown in Table 1. The ROC curve for the validation set is shown in FIG. 5-B. According to the validation set data of this example, the sensitivity, specificity and accuracy of CA125 alone for ovarian cancer diagnosis were: 73.33%, 85.85 and 82.12%; the sensitivity, specificity and accuracy of the comprehensive evaluation of the three indexes for diagnosing the ovarian cancer are respectively as follows: 91.11%, 94.34 and 93.38% (table 2). Wherein, the sensitivity of the comprehensive evaluation of the three indexes to the ovarian cancer stage I-III is respectively as follows: 70%, 88.9%, 100%, sensitivity to early stage (stage I + stage II) ovarian cancer: 78.9%, all significantly higher than the single index of CA125 (FIG. 6).

Meanwhile, compared with the literature report method, the specificity and the sensitivity of the composition scoring method are also obviously improved (Table 3). In particular, the problems of missed and false detections can be well avoided by the compositions and models of the present invention, see table 4; for example, 27 of 38 CA 125-negative ovarian cancer patients can be successfully detected (71.05%) by the composition and the model scoring method, which shows that the invention can well compensate the problem of CA125 omission; in 50 non-tumor populations positive for CA125, 74% (37/50) of the populations can be correctly judged by the composition and the model scoring method, which shows that the invention can well compensate the problem of CA125 false detection.

TABLE 2 comparison of the Complex Scoring model with the CA125, TP53 independent index for evaluating ovarian cancer Performance

TABLE 3 comparison of Performance of the composite Scoring model with the clinically common methods

^*Ovarian cancer diagnosis and treatment standard of the national health committee of the people's republic of China (2018 edition) [ J]Tumor complex treatment electronic journal, 2019,5(2):87-96 link http:// www.jmcm2018.com/CN/Y2019/V5/I2/87

TABLE 4 comprehensive scoring method to remedy the deficiencies of CA125

In conclusion, the biomarker group and the evaluation model of the invention have excellent performance, such as higher than the existing common clinical CA125 and ROMA indexes, and the sensitivity, accuracy and the like are improved while the specificity is obviously improved; particularly, the detection rate of CA125 negative ovarian cancer patients reaches 71.05%, and 74% of non-tumor populations with CA125 positive ovarian cancer patients can be correctly judged, so that the kit has high clinical value for early diagnosis of ovarian cancer and high industrial utilization value.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims

1. A biomarker population associated with ovarian cancer, the biomarker population comprising cfDNA, cfDNA TP53 mutation and CA 125.

2. Use of a substance that specifically recognizes or detects the biomarker panel according to claim 1 for the preparation of an in vitro diagnostic product for the prediction, diagnosis and/or prognostic assessment of ovarian cancer.

3. The use according to claim 2, wherein the substances that specifically recognize or detect the biomarker panel according to claim 1 comprise substances that specifically recognize or detect cfDNA, substances that specifically recognize or detect the abundance of mutations in cfDNATP53, and substances that specifically recognize or detect the level of CA125 protein expression.

4. The use of claim 2, wherein the diagnosis is of whether the subject has ovarian cancer or whether the ovarian cancer has progressed or has recurred, wherein the diagnosis comprises early screening or diagnosis; the prediction refers to predicting the risk of the individual to be tested suffering from ovarian cancer; the evaluation refers to the evaluation of the degree of ovarian cancer of the individual to be tested, or the evaluation of the curative effect of the individual to be tested after receiving related treatment.

5. The use of claim 3, wherein assessing the extent to which the test subject has ovarian cancer is assessing whether the ovarian cancer is stage I ovarian cancer, stage II ovarian cancer, stage I-II ovarian cancer, stage III ovarian cancer, stage IV ovarian cancer, or stage III-IV ovarian cancer.

6. The use according to claim 2, wherein the ovarian cancer is of the type: ovarian epithelial cell carcinoma, classified as serous adenocarcinoma, clear cell carcinoma, mucinous adenocarcinoma, endometrioid adenocarcinoma; ovarian malignant germ cell tumors, classified as yolk sac tumor, asexual cell tumor, immature teratoma; malignant solitary interstitial tumors, classified as supporting cell-interstitial cell tumor, granulocytic-interstitial cell tumor; metastatic tumor, which is the primary kukenberg tumor of gastrointestinal tract.

7. The use of claim 4, wherein the sample from the subject is derived from whole blood, serum, plasma, saliva, buccal mucosal swab, lymph, cerebrospinal fluid, ascites, cervical pap smear, bladder wash, uterine wash, stool, urine, and tissue biopsy.

8. The ovarian cancer-associated biomarker panel of claim 1 or the use of any of claims 2 to 7, wherein the biomarker panel is used in combination with other ovarian cancer-associated markers.

9. An ovarian cancer-associated in vitro diagnostic product comprising a substance that specifically recognizes or detects cfDNA, a substance that specifically recognizes or detects the abundance of mutations of cfDNA TP53, and a substance that specifically recognizes or detects the level of CA125 protein expression.

10. The in vitro diagnostic product of claim 9, wherein the in vitro diagnostic product is a reagent, chip or kit; wherein the kit comprises the chip.

11. A device for the prediction, diagnosis and/or prognostic assessment of ovarian cancer, wherein the diagnosis includes early screening or diagnosis; the device comprises:

12. The apparatus of claim 11, wherein in the analysis module, the risk score is calculated by the mathematical function:

wherein the unit of the content of CA125 is U/ml.