CN107765013B - Early ovarian cancer screening method and kit - Google Patents

Abstract

The invention discloses an early ovarian cancer screening method and a kit, wherein the method comprises the following steps: screening for early ovarian cancer was performed using triple detection of keratanase protein (YKL-40) in combination with cancer antigen 125(CA125) and human epididymis protein 4(HE 4). Through the mode, the accuracy of predicting early ovarian cancer can be improved.

Description

Early ovarian cancer screening method and kit

Technical Field

The invention relates to the technical field of tumor screening, in particular to an early ovarian cancer screening method and a kit.

Background

Ovarian cancer is one of the common malignant tumors of female reproductive organs, the incidence rate of the ovarian cancer ranks the third place in various gynecological cancers of women, but the mortality rate is the first place, and the ovarian cancer seriously threatens the life health of women. Because the embryo development, tissue dissection and endocrine function of the ovary are complex, the pathogenesis of the ovary is not clearly researched at present. In addition, because ovarian cancer has no obvious symptoms in the early clinical stage, most patients are found to be in the late stage, and can only receive conservative treatment. Research reports suggest that in laparotomy for ovarian cancer, the tumor is limited to 30% of ovaries, most of the ovaries have spread to bilateral attachments of the uterus, the organs of the omentum majus and the pelvic cavity, and more than 70% of patients have been diagnosed in a late stage, so that the ovarian cancer is a big problem in both diagnosis and treatment, and experts have conducted many studies on pathological forms, clinical occurrence and development rules and treatment schemes of ovarian malignant tumors for many years, and the statistics of clinical data at home and abroad show that: the 5-year survival rate of the ovarian cancer in the stage I can reach 90 percent, and the survival rate of the ovarian cancer in the advanced stage is only 2.3 to 20 percent, so that the early screening and diagnosis of the ovarian cancer have important influence on the prognosis. Because the cause of the ovarian cancer is unclear and difficult to prevent, how to discover the ovarian cancer at an early stage becomes a hot spot for domestic and foreign research.

Currently, there are two main methods for detecting ovarian cancer that are clinically used: one is Transvaginal Ultrasonography (TUV), which, although very commonly used, is not highly sensitive to detecting benign or malignant tumors. In addition, the method has high requirements on the experience and the technology of the examining physician. The other is the conventional auxiliary diagnosis method for detecting blood tumor markers: comprises cancer antigen 125(CA125), human epididymis protein 4(HE4) and a combined detection method of the cancer antigen 125 and the human epididymis protein 4. CA125 is a more common tumor marker, but CA125 has lower specificity and sensitivity, and is easy to have false negative or false positive. Compared with CA125, HE4 has higher sensitivity and stronger specificity, but has little significance for early auxiliary diagnosis of ovarian cancer, and the sensitivity of single HE4 to auxiliary diagnosis of stage I ovarian cancer is only 45.9%. The detection of two biomarkers simultaneously can improve the detection sensitivity, but the false positive rate is higher, and early auxiliary diagnosis still cannot be realized. Neither CA125 nor HE4 accurately predicted early stage ovarian cancer.

Disclosure of Invention

The invention mainly solves the technical problem of providing an early ovarian cancer screening method and a kit, which can improve the accuracy of early ovarian cancer prediction.

In order to solve the technical problems, the invention adopts a technical scheme that: provides the application of keratan enzyme protein (YKL-40) combined with cancer antigen 125(CA125) and human epididymis protein 4(HE4) in screening early ovarian cancer.

In order to solve the technical problem, the invention adopts another technical scheme that: there is provided a method of screening for early stage ovarian cancer, the method comprising: detecting the content of YKL-40, CA125 and HE4 in the sample;

if the content of CA125 and HE4 is within the respective critical value range and the content of YKL-40 is greater than the critical value range, the patient providing the sample is preliminarily judged to have a greater risk of ovarian cancer, and if the content of CA125 and HE4 is greater than the respective critical value range and the content of YKL-40 is equal to or less than the critical value range, the patient providing the sample is preliminarily judged to have a lesser risk of ovarian cancer.

Wherein the sample is a blood sample.

Wherein the critical value range is the content range of normal people.

In order to solve the technical problem, the invention adopts another technical scheme that: provides an in vitro diagnostic kit for joint detection of keratan enzyme protein (YKL-40), cancer antigen 125(CA125) and human epididymis protein 4(HE4), which comprises: a labeled anti-YKL-40 first antibody, a labeled anti-CA 125 first antibody, and a labeled anti-HE 4 first antibody coated on the first chromatographic material; unlabeled anti-YKL-40 secondary antibody, unlabeled anti-CA 125 secondary antibody, and unlabeled anti-HE 4 secondary antibody coated on a second chromatographic material.

Wherein the labeled anti-YKL-40 first antibody, the labeled anti-CA 125 first antibody and the labeled anti-HE 4 first antibody are labeled by quantum dots or fluorescent microspheres.

Wherein, the in vitro diagnosis kit is used for screening early ovarian cancer.

In order to solve the technical problem, the invention adopts another technical scheme that: provides a keratan enzyme protein (YKL-40) kit for screening early ovarian cancer, and the keratan enzyme protein (YKL-40) kit is used together with a CA125 kit and an HE4 kit.

The invention has the beneficial effects that: in contrast to the prior art, the present invention uses triple detection of keratanase protein (YKL-40) in combination with cancer antigen 125(CA125) and human epididymis protein 4(HE4) for early stage ovarian cancer screening. Because the sensitivity of the keratan enzyme protein (YKL-40) is higher than that of the cancer antigen 125(CA125) and human epididymis protein 4(HE4), the accuracy of predicting early ovarian cancer can be improved in the mode.

Drawings

FIG. 1 is a schematic diagram of experimental data analysis of CA125 content in clinical samples;

FIG. 2 is a schematic diagram of experimental data analysis of HE4 content in clinical samples;

FIG. 3 is a schematic diagram of experimental data analysis of the YKL-40 content in clinical samples;

FIG. 4 is a schematic representation of clinical experimental data of clinical samples;

FIG. 5 is another schematic of clinical experimental data for clinical samples.

Detailed Description

The present invention will be described in detail below with reference to the accompanying drawings and embodiments.

The invention provides application of keratan enzyme protein (YKL-40) and cancer antigen 125(CA125) as well as human epididymis protein 4(HE4) in early ovarian cancer screening.

CA125 is a glycoprotein from columnar epithelial cells, and in 1983 Bast et al first determined the content of CA125 in serum, and then researchers analyzed the level of CA125 in patients with ovarian cancer and determined the role of this molecule in aiding the diagnosis of ovarian cancer. CA125 is the main serum marker for screening early ovarian cancer at present, has been widely applied to clinical auxiliary diagnosis, curative effect observation and disease condition monitoring, but has lower sensitivity when being used for auxiliary diagnosis of early ovarian cancer alone. CA125 is increased in serum of only part of patients in stage I in early stage ovarian cancer (>35U/ml), and abnormal expression of CA125 can be caused by physiological conditions (menstrual period or gestational period) and some benign diseases (endometriosis, ovarian cyst and the like) as well as liver cirrhosis, heart failure and other diseases, which all indicate that the tumor marker has low detection rate and insufficient sensitivity and specificity in early auxiliary diagnosis of ovarian cancer. HE4 is another ovarian cancer tumor marker with which chinese physicians are less familiar than CA 125. The HE 4-encoding gene was first isolated from the epididymal epithelium by Kichhoff et al in 1991, and is located on chromosome 20q, with a total length of about 12 kb. Schummer et al, 1999, found high expression of HE4 (also known as WFDC2) mRNA in ovarian cancer tissue, but not in paracarcinoma tissue, by cDNA microarray analysis. The protein coded by the gene has high homology with an extracellular protease inhibitor, but the HE4 has no protease inhibition effect. Hellstrom et al combined the gene with proteome technology to screen HE4 and demonstrated that HE4 is the product of WFDC2 gene, consisting of 4 disulfide bond cores (WFDC) of the WAP type, and that this gene encodes a secreted protein, confirmed as a glycated protein. The research group also finds that most of ovarian cancer patients have obviously increased serum HE4 protein and low content of normal tissues and benign diseases by comparing the amount of HE4 protein in the serum of ovarian cancer patients and normal human serum, so that the HE4 has the value of auxiliary diagnosis of ovarian cancer and is superior to CA125 in early diagnosis. Because the new serum marker is extremely low in serum of benign tumor patients and normal people and is positively expressed in 93% of ovarian serous carcinoma and 100% of ovarian endometrioid carcinoma, the HE4 is obviously superior to 43.3% of CA125 (with the specificity of 95%) in identifying benign and malignant properties of ovarian tumor, and the sensitivity of HE4 reaches 72.9% (with the specificity of 95%). The sensitivity of HE4 alone to stage I ovarian cancer diagnosis was 45.9% (with 95% specificity) and also significantly better than that of CA125 alone to stage I15.1% (with 95% specificity).

Researchers at the university of Brown in the United states in 2007 report that HE4 helps in early-stage assisted diagnosis of ovarian cancer, and prediction accuracy of combined detection of HE4 and CA125 is higher than that of either marker alone. The Moore et al research also shows that the detection sensitivity of the CA125 combined with HE4 reaches 76.4%, the specificity is 95%, and is obviously higher than the sensitivity of the single detection of HE4 or CA125, and on the basis, the research of the Moore et al further researches that the detection of CA125 combined with other markers has no obvious change. Later, Montaqnana et al showed that serum CA125 and HE4 levels were significantly higher in ovarian cancer patients than in normal persons, as well as in benign tumors of the ovary, cervical cancer and endometrial tumor patients, and that serum HE4 was more sensitive and specific than CA 125. More experiments also prove that the combination of the two can increase the diagnosis accuracy of ovarian cancer, and can distinguish patients in a high-risk group and a low-risk group of ovarian cancer, but the joint detection of the two cannot accurately predict early ovarian cancer.

The inventor of the application researches and finds that the keratanase YKL-40 is an inflammatory factor and a tumor marker, and the content of the keratanase YKL-40 in blood increases along with the degree of disease from inflammation to canceration. The use of keratanase alone as a marker is not sufficiently pinpointing and cardiovascular diseases and diabetes also lead to elevated keratanase in the blood. But when it is detected together with other cancer markers, it can help the clinical early warning of cancer more effectively because its content in blood is significantly increased from inflammation to cancer.

In order to accurately localize ovarian cancer, the inventor of the application proposes a triple detection method, namely, on the basis of two markers (dual detection) of CA-125 and HE4 for auxiliary diagnosis of ovarian cancer, the detection of keratanase is increased.

Referring to fig. 1 to 3, blood samples of 110 clinical patients are detected, fig. 1 is used for detecting CA125 in the blood samples of the patients, the abscissa represents the stage of ovarian cancer, the ordinate represents the content of CA125 in U/ml, fig. 2 is used for detecting HE4 in the blood samples of the patients, the abscissa represents the stage of ovarian cancer, the ordinate represents the negative logarithm of the content of HE4, fig. 3 is used for detecting YKL-40 in the blood samples of the patients, the abscissa represents the stage of ovarian cancer, the ordinate represents the content of YKL-40 in ng/ml. For specific clinical data, please refer to fig. 4-5. As can be seen from fig. 1, the content of CA125 is low from the Healthy stage (health) to the ovarian cancer stage II, and the content of CA125 is not significantly increased until the ovarian cancer stage III; as can be seen from fig. 2, the content of HE4 was low in the Healthy stage (health), the inflammatory or benign tumor stage (Bengin) and the ovarian cancer stage I, and HE4 was elevated compared to the Healthy stage, but the inflammatory or benign tumor stage (Bengin) and the ovarian cancer stage I, and HE4 were comparable in content, and the inflammatory or benign tumor stage (Bengin) and the ovarian cancer stage I could not be distinguished according to the content of HE4, and the content of HE4 was significantly elevated compared to the inflammatory or benign tumor stage (Bengin) and the ovarian cancer stage I in the ovarian cancer stage II; as can be seen from fig. 3, the content of YKL-40 was substantially equivalent in the Healthy stage (health) and the inflammatory or benign tumor stage (Bengin), and the content of YKL-40 was significantly increased in the ovarian cancer stage I as compared to the Healthy stage (health) and the inflammatory or benign tumor stage (Bengin).

Thus, in conjunction with FIGS. 1-3, it can be seen that the sensitivity of YKL-40 is clearly higher than that of CA125 and HE 4. Moreover, in the normal control group, two blood samples of which both CA-125 and HE4 are out of the normal range, but both of which have YKL-40 in the normal range, were determined to be negative by the application of the combination of YKL-40 and CA-125 and HE4 if the CA-125 and HE4 two-link assay was positive, and were indeed negative by confirmed diagnosis. Therefore, the triple assay of YKL-40 in combination with CA-125 and HE4 of the present application is indeed superior to the original dual assay of CA-125 and HE 4.

The present invention also provides a method of screening for early stage ovarian cancer, the method comprising:

A. detecting the content of YKL-40, CA125 and HE4 in the sample;

B. if the content of CA125 and HE4 is within the respective critical value range and the content of YKL-40 is greater than the critical value range, the patient providing the sample is preliminarily judged to have a greater risk of ovarian cancer or other inflammations, and if the content of CA125 and HE4 is greater than the respective critical value range and the content of YKL-40 is equal to or less than the critical value range, the patient providing the sample is preliminarily judged to have a small risk of ovarian cancer, and other indexes need to be checked.

Wherein the sample is a blood sample, such as: serum, plasma, whole blood, and the like.

Wherein the critical value range is the content range of normal people.

In the initial experimental data, it has been found that the addition of YKL-40 to form a triple detection has the following two significant advantages: first, when both CA-125 and HE4 criteria are still within the cut-off range (Borderline Risk, the cut-off range is not sufficient to be positive), but if it is accompanied by a significant increase in YKL-40, this will help to qualify as positive. Thus, the positive rate of early auxiliary diagnosis of ovarian cancer is increased from 40% of the original CA-125 and HE4 two-link method to more than 60% of the current positive rate. In the above 110 test cases, further evidence was obtained for this. Second, the current clinical combination of CA-125 and HE4 tests still have a relatively high false positive rate. From the experimental data in this application, it was found that when the dual detection of CA-125 and HE4 showed an abnormal increase, while YKL-40 did not show a significant increase, the probability of cancer lesions was much reduced. Therefore, addition of YKL-40 as a triple assay can help to reduce the false positive rate. The existence of the advantage is also proved again in the blood sample detection of 110 patients: in the normal control group, two blood samples were found to have both CA-125 and HE4 outside the normal range, but the YKL-40 in these blood samples was found to be in the normal range, and thus was found to be negative by the triple detection of the present application, as determined to be positive by the tandem method. Thus, the triple assay with the addition of YKL-40 was indeed superior to the original dual CA-125 and HE4 assays.

The above 110 clinical samples, 22 normal population samples, 10 benign ovarian tumor patient samples, 78 ovarian cancer patient samples, 18 stage I patient samples, 19 stage II patient samples, 22 stage III patient samples, and 19 stage IV patient samples. Ovarian cancer stage depends mainly on the results of clinical tests. In the embodiment of the invention, the stage of the ovarian cancer sample is staged according to the diagnosis result of clinical images such as B-ultrasonic, CT scanning or MRI examination and the like and the diagnosis result of cytology such as cast-off cytology or fine needle aspiration method examination. Normal (i.e., healthy) population is women without gynecological tumors, benign tumor patients refer to ovarian cysts and non-malignant tumors, stage I patients refer to lesions confined to the ovaries, including lesions confined to one ovary, intact envelopes, no tumors on the surface, no ascites and lesions confined to two ovaries, intact envelopes, no tumors on the surface, no ascites and lesions that have passed out of the ovarian surface, or ruptured envelopes, or malignant cells found in ascites or peritoneal wash. The stage II patient refers to one side or two sides of ovary affected by pathological changes, accompanied with pelvic cavity metastasis, including pathological changes expanding or metastasizing to uterus or oviduct and pathological changes expanding to other pelvic cavity tissues and tumors penetrating out of the surface of the ovary, or rupture of the envelope; or malignant cells found in ascites or peritoneal washes. Stage III patients refer to patients with pathological changes affecting one or two ovaries, with implantation outside the pelvic cavity or retroperitoneal lymph node metastasis: the lesions are mainly seen in the pelvic cavity and are lymph node negative, but the peritoneoscope abdominal cavity has a planting tumor on the peritoneum surface and the diameter of the peritoneoscope abdominal cavity planting tumor is less than 2cm, the lymph node negative and peritoneoscope abdominal cavity planting tumor has the diameter of more than or equal to 2cm, or the retroperitoneal or inguinal lymph node metastasis is accompanied, but the metastasis is not transferred to other distant organs. Stage IV refers to metastasis of cancer cells to distant organs, such as the liver.

The detection reagents for CA125, HE4, and YKL-40 were provided by Shenzhen Hai Ge German Biotech. In the CA125 test, the Cutoff value (Cutoff) was set at 35, 4 of 22 normal population samples exceeded the Cutoff value, and 3 of 10 benign tumor cases exceeded the Cutoff value, with a false positive rate of 21.9%. In 18 samples of the patients with stage I ovarian cancer, only 4 samples exceed the critical value, and the detection rate is only 22.2 percent, and in 19 samples of the patients with stage II ovarian cancer, only 6 samples exceed the critical value, and the detection rate is only 31.58 percent, so the detection rate of the early stage ovarian cancer assisted diagnosis of CA125 is lower than 35 percent, the sensitivity is very low, and the specificity is also low. In the detection of HE4, the cut-off value (Cutoff) was determined to be 70, 4 of 22 normal population samples exceeded the cut-off value, 3 of 10 benign tumors exceeded the cut-off value, and the false positive rate was 21.88%. The detection rate is only 50% when 9 of 18 samples of the patients with stage I ovarian cancer exceed the critical value, and the detection rate is only 57.89% when 11 of 19 samples of the patients with stage II ovarian cancer exceed the critical value, so that the detection rate of HE4 for assisting in diagnosing early stage ovarian cancer is lower than 60%. In the detection of YKL-40, the cut-off value (Cutoff) was determined to be 100, 3 samples out of 22 normal population samples exceeded the cut-off value, 1 sample out of 10 benign tumors exceeded the cut-off value, and the false positive rate was 12.5%. However, in combination with the levels of CA125 and HE4, false positives of YKL-40 can be excluded. In 18 samples of the patients with stage I ovarian cancer, 16 samples exceeded the critical value, the detection rate was 88.89%, only 2 samples were below the critical value, and the omission factor was only 11.1%, and in 19 samples of the patients with stage II ovarian cancer, 17 samples exceeded the critical value, the detection rate was also 89.47%, and the omission factor was 10.53%. Therefore, the detection rate of the YKL-40 for diagnosing early ovarian cancer reaches 89 percent, namely the sensitivity reaches 89 percent, and the specificity is high. In patients with elevated control YKL-40, neither CA125 nor HE4 levels were high, and it was considered that the patients had the potential for other inflammation. Elevated YKL-40 in benign tumor patients, but not high levels of CA-125 and HE4, we can consider the patients to have other inflammations. In three of the advanced patients the YKL-40 levels were below cutoff, but the CA125 and HE4 levels were high, so that further investigation was required if 2 of the three indices exceeded the cutoff value a lot.

At present, the definite diagnosis of ovarian cancer is still carried out clinically, and the pathological method is mainly used. All blood sample tests clinically used at present, including the triple detection method provided by the application, have an auxiliary diagnosis effect on clinicians, and mainly have an effect of initial screening or early warning. According to the methods of the present application, the test results are positive and not equal to absolute ovarian cancer positivity. However, in the case of positive blood test, the incidence of ovarian cancer is ten times or more, compared to the case of negative blood test, which is enough to raise the alertness of the clinician and give the patient further necessary examinations. Many gynecologists reflect that current expensive scans (e.g., CT scan, MRI, or ultrasound of neotype) cannot be used on a large scale for initial disease screening. Therefore, for ovarian cancer which is not easy to make early auxiliary diagnosis, a primary auxiliary diagnosis method which is rapid, simple, low in cost, high in sensitivity and relatively reliable in result is particularly needed. The methods and products of the present application meet exactly one such clinical need.

The invention also provides an in vitro diagnostic kit for joint detection of keratan enzyme protein (YKL-40), cancer antigen 125(CA125) and human epididymis protein 4(HE4), which comprises:

a labeled anti-YKL-40 first antibody, a labeled anti-CA 125 first antibody, and a labeled anti-HE 4 first antibody coated on the first chromatographic material;

unlabeled anti-YKL-40 secondary antibody, unlabeled anti-CA 125 secondary antibody, and unlabeled anti-HE 4 secondary antibody coated on a second chromatographic material.

It is obvious that the in vitro diagnostic reagent of the triple-joint test adopts the principle of chromatography. The in vitro diagnostic reagent of the three-joint test can detect YKL-40, CA125 and HE4 in a sample.

Wherein, the labeled anti-YKL-40 primary antibody, the labeled anti-CA 125 primary antibody and the labeled anti-HE 4 primary antibody are labeled by quantum dots or fluorescent microspheres. Of course, colloidal gold may be used for labeling.

Wherein, the in-vitro diagnosis kit is used for screening early ovarian cancer.

Quantum Dots (Quantum Dots) are nanoparticles composed of semiconductor materials such as cadmium selenide (CdSe), and can release fluorescence which is ten times stronger than that of common fluorescein after being excited by common light, and have the characteristics of wide excitation spectrum and narrow emission spectrum. The emission spectrum can be changed and adjusted according to the difference of semiconductor materials and the difference of nano-particle sizes, and the emitted light has high stability and cannot be quenched. Due to the advantages of quantum dot fluorescence, the luminous intensity of the antibody marked by the quantum dot nano particles can be increased by hundreds of times compared with that of the antibody marked by common fluorescein, so that the signal sensitivity of the reagent reaches the level equivalent to that of chemiluminescence.

The development technical route of the kit is roughly divided into the following steps:

screening or self-developing monoclonal antibodies for detecting tumor markers;

determining and optimizing coupling conditions of the antibody and the quantum dot nano particles or the fluorescent microspheres;

determining and optimizing the condition for detecting the tumor marker by an immunochromatography method;

determining and optimizing performance technical parameters of the tumor marker detection kit;

the determination and optimization of the pilot test and mass production conditions and processes of the detection kit;

and (3) research, development and production of a fluorescence detection instrument matched with the kit.

The process flow for developing the kit adopts the following steps: preparing working liquid for coating and marking, coupling an antibody and quantum dot nano-particles or fluorescent microspheres, preparing a fluorescent pad, preparing a sample pad, coating the antibody on an NC (numerical control) membrane, drying, assembling a reagent strip and the like.

The invention also provides a keratan enzyme protein (YKL-40) kit for screening early ovarian cancer, and the keratan enzyme protein (YKL-40) kit is used together with a CA125 kit and an HE4 kit.

For example, a keratanase protein (YKL-40) enzyme-linked (ELISA) kit for early ovarian cancer screening.

In summary, the process and product of the present application have the following distinct advantages:

(1) by detecting the content of YKL-40 in blood, normal people, inflammation patients and cancer patients can be clearly classified, and in the case of cancer, the level of the YKL-40 is increased along with the development of the disease.

(2) The joint detection of CA125, HE4 and YKL-40 can make up the defects of HE4 and CA125, and the sensitivity and specificity of early auxiliary diagnosis of ovarian cancer are obviously improved.

(3) The value of early auxiliary diagnosis of ovarian cancer is far higher than that of the detection method commonly used in clinic at present, and the method has wide application prospect and fills the blank in the field.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present specification and drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.

Claims (1)

1. The application of a reagent for detecting multiple tumor markers in the preparation of a kit for screening early ovarian cancer is characterized by comprising the following steps:

detecting the content of YKL-40, CA125 and HE4 in the sample;

if the content of CA125 and HE4 is within the respective threshold range and the content of YKL-40 is greater than the threshold range, the patient providing the sample is preliminarily judged to have a greater risk of ovarian cancer,

if the content of CA125 and HE4 is greater than the respective threshold range and the content of YKL-40 is equal to or less than the threshold range, preliminarily judging that the patient providing the sample has a lower risk of ovarian cancer;

the sample is a blood sample;

the critical value range is the content range of normal people.

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