CN112229998A

CN112229998A - Prognostic diagnosis marker Claudin22 for ovarian cancer and application thereof

Info

Publication number: CN112229998A
Application number: CN202010904429.9A
Authority: CN
Inventors: 施亮; 傅志勤; 杨悦; 周陈西; 俞婷婷; 鲁超; 黄萍
Original assignee: Zhejiang Cancer Hospital
Current assignee: Zhejiang Cancer Hospital
Priority date: 2020-09-01
Filing date: 2020-09-01
Publication date: 2021-01-15
Anticipated expiration: 2040-09-01
Also published as: CN112229998B

Abstract

The invention relates to a prognostic diagnosis marker Claudin22 of ovarian cancer and application thereof, which firstly discovers that the prognosis of ovarian cancer is related by the expression level of an ovarian cancer drug-resistant marker Claudin22, and particularly discloses that in prognostic indexes such as progression-free survival and total survival and the like when Claudin22 is highly expressed, ovarian cancer cases show poor prognosis, namely the discovery of the invention provides prognostic prompt for the clinical treatment of ovarian cancer including chemotherapy and surgical treatment.

Description

Prognostic diagnosis marker Claudin22 for ovarian cancer and application thereof

Technical Field

The present invention is in the field of molecular biology, particularly in the field of diagnosis/staging and prognosis of cancer, and more particularly in the field of ovarian cancer. The invention also relates to the field of ovarian cancer diagnosis, grading, staging and prognosis involving biomarker expression, and provides related analytical reagents, diagnostic models, test kits and clinical assays.

Background

Ovarian cancer is the gynecological tumor with the highest disease death rate of women in China, and according to the annual statistics of Chinese cancers, about 5.21 ten thousand new patients live in 2015, and about 2.25 ten thousand people die of ovarian cancer in the same year. With the increasing progress of the aging of the population in China, the incidence rate of ovarian cancer is increased year by year, and the incidence age tends to be younger. Globally, the incidence of ovarian cancer is third in gynecological tumors, second only to cervical and endometrial cancers, and the mortality is second in gynecological tumors; in developed countries, the mortality rate of ovarian cancer is at the first place of gynecological tumors.

Ovarian cancer is most common in perimenopausal women, with about 60% to 70% of patients having developed stage III-IV or having developed abdominal metastases when they found disease. Because early symptoms of patients are not obvious and symptoms and signs such as abdominal pain, abdominal distension, abdominal mass, digestive system symptoms and tumor compression are usually shown in later stages, most ovarian cancer patients can be diagnosed in the middle and later stages of the onset. Despite the progress of surgical treatment techniques and chemoradiotherapy approaches for ovarian cancer, the 5-year productivity of the disease is still as low as 30%, and the prognosis is poor.

Ovarian cancer is a common gynecological malignant tumor, has high morbidity, and has high mortality at the top due to the lack of specific symptoms and effective early diagnosis methods, and most patients are diagnosed at a late stage. Although the operation technology and the treatment means are improved continuously, the overall survival rate of ovarian cancer patients is improved compared with the prior art, most patients still face chemotherapy resistance, and the treatment failure is one of the important causes of death.

Since the 21 st century, with the development of molecular biology, several diagnostic markers for ovarian cancer were developed, such as a systematic review of Dayyani F et al, 2016, which was included in 5 documents, 1975 subjects, and the results of analysis showed that the sensitivity and specificity of CA125 for ovarian cancer diagnosis were 0.796 (95% CI: 0.663-0.885) and 0.825 (95% CI: 0.662-0.919), respectively. However, the CA125 detection is not well-characterized in the diagnosis of patients with early ovarian cancer, and is greatly influenced by factors such as menstruation and pregnancy; . The united states Food and Drug Administration (FDA) has approved HE4 and CA125 for the detection of ovarian cancer. The detection means of HE4 is now mainly by ELISA technology. One study by Dayyani F et al showed that serum HE4 had sensitivity and specificity for detecting ovarian cancer of 0.817 (95% CI: 0683-0.902) and 0.851 (95% CI: 0.716-0.928), respectively; simon I and the like design a double-antibody sandwich enzyme-linked immunosorbent assay for detecting the level of a co-stimulatory molecule B7 homolog 4(B7-H4) in blood, and find that the level of B7-H4 in the blood of an ovarian cancer patient is obviously increased, the sensitivity and specificity of B7-H4 and CA125 joint diagnosis ovarian cancer can reach 65% and 97% respectively, and are higher than the single diagnosis of CA 125; in addition, researches show that chitin-40 ((YKL-40) has higher expression in breast cancer, ovarian cancer, prostate cancer and lung cancer and plays an important role in the generation and development of tumors, Resnick KE and the like detect serum samples of 28 serous ovarian cancer patients and serum samples of 15 normal control groups to find that miR-21, miR-29b, miR-92, miR-93 and miR-126 in the serum of ovarian cancer patients have higher levels, wherein miR-21, miR-92 and miR-93 have obvious up-regulation before CA125 is increased, which indicates that the three microRNAs can be used for early diagnosis of the ovarian cancer, and in addition, researches show that a plurality of non-coding long-chain RNAs also have abnormal expression in the ovarian cancer, for example, a plurality of lncRNAs show disorder in the ovarian cancer, and the researches show that compared with healthy tissues, multiple lncRNA are upregulated in ovarian tumor tissues, such as ANRIL, AB073614, BCYRN1, CCAT1, FALI, and the like.

Claudins belongs to the family of tight junction proteins and plays an important role in maintaining the polarity of epithelial cells and the bypass permeability of cells. Recent studies have found that Claudins can regulate cell proliferation, differentiation and lead to tumorigenesis and progression by recruiting cell signaling proteins. It has now been found that there are 25 Claudins of the members, of which claudin1-14 is overexpressed in a variety of tumors including prostate, uterine and breast cancers and is significantly associated with poor prognosis. However, at present, no research related to the occurrence and development of other family members and tumors exists, and particularly, no research related to the prognosis of Claudin22 and gynecological tumors exists, so that the relation between Claudin22 and gynecological tumors and the clinical application value are researched and developed based on the basis of the prior research.

Disclosure of Invention

The definition of the tumor related terms appearing in the present invention is as follows:

tumor recurrence: imaging found tumor progression and/or 2 consecutive serum CA125 times or more than 2-fold the upper limit of normal.

Degree of tumor differentiation: it refers to the degree of tumor cells close to normal cells, wherein the highly differentiated cancer and tumor tissues are similar to normal tissues, and have high maturity and low malignancy. The tumor tissues of low-differentiation cancer and undifferentiated cancer are greatly different from normal tissues, the maturity is low, and the malignancy is high.

Progression Free Survival (PFS): time from initial surgery to disease recurrence or progression.

Overall Survival (OS): from the time of initial surgery to the time of death or termination of follow-up.

All studies were approved and documented by the ethical committee of the tumor hospital, zhejiang (IRB-2019-3).

In order to overcome the defects in the prior art, the invention aims to provide a marker for prognosis of ovarian cancer and application thereof.

One aspect of the present invention is to provide a prognostic diagnostic marker for tumors, which is Claudin22, wherein Claudin22 has poor prognosis when highly expressed in tumor tissues. In a specific embodiment, the high expression refers to: the section has no impurity stain, takes the brown yellow or brown particles appearing on the normal stained part of the antibody as positive cells, and randomly selects 5 fields with more tumor cells under a 40 multiplied by 10 light microscope for observation. The proportion and distribution of the positive staining cells are determined as follows: claudin22 is stained in cytoplasm, and the proportion of Claudin22 positive cells is as follows: 0-25% ═ 1 part, 26-50% ═ 2 parts, 51-75% ═ 3 parts, 76-100% ═ 4 parts; target protein staining intensity: negative 0, weak positive 1, moderate positive 2, high positive 3, strong positive 4. The percentage of positively stained cells in each field was multiplied by the staining intensity score for the protein of interest and summed. Scores of 0-40 were classified as low expression Claudin22, and scores of 41-80 were classified as high expression Claudin 22.

In a particular embodiment, the tumor is a gynecological tumor, preferably ovarian cancer; in another specific embodiment, the prognosis includes the degree of tumor differentiation; in another specific embodiment, the prognosis includes Progression Free Survival (PFS); in another specific embodiment, the prognosis includes Overall Survival (OS).

The invention also provides an application of the reagent for detecting the tumor marker in preparing a tumor prognosis product, wherein the marker is Claudin22, the prognosis is related to the expression level of Claudin22, the high expression level indicates poor prognosis of the tumor, and the low expression level indicates good prognosis of the tumor. In a specific embodiment, the high expression refers to: the section has no impurity stain, takes the brown yellow or brown particles appearing on the normal stained part of the antibody as positive cells, and randomly selects 5 fields with more tumor cells under a 40 multiplied by 10 light microscope for observation. The proportion and distribution of the positive staining cells are determined as follows: claudin22 is stained in cytoplasm, and the proportion of Claudin22 positive cells is as follows: 0-25% ═ 1 part, 26-50% ═ 2 parts, 51-75% ═ 3 parts, 76-100% ═ 4 parts; target protein staining intensity: negative 0, weak positive 1, moderate positive 2, high positive 3, strong positive 4. The percentage of positively stained cells in each field was multiplied by the staining intensity score for the protein of interest and summed. Scores of 0-40 were classified as low expression Claudin22, and scores of 41-80 were classified as high expression Claudin 22. In a particular embodiment, the tumor is a gynecological tumor, preferably ovarian cancer; in another specific embodiment, the prognosis is the degree of tumor differentiation; in another specific embodiment, the prognosis includes Progression Free Survival (PFS); in another specific embodiment, the prognosis includes Overall Survival (OS).

In another aspect of the invention, a kit is provided, said kit comprising reagents for detecting Claudin 22. In a specific embodiment, the kit is a kit for immunohistochemical application, and in another specific embodiment, the kit comprises a reagent for detecting the expression of Claudin22, which is an antibody or a fragment thereof, preferably a monoclonal antibody.

The invention has the beneficial effects that: the gene expression of claudin22 is significantly related to the prognosis of ovarian cancer patients, tumor recurrence and tumor differentiation degree thereof, and is a biomarker for predicting the prognosis of ovarian cancer patients, tumor recurrence and tumor differentiation degree thereof.

Drawings

FIG. 1: the expression of Claudin22 in ovarian cancer tissues was (a) low expression and (b) high expression.

FIG. 2: the expression of Claudin22 in ovarian cancer tissues correlates with OS and PFS.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations.

EXAMPLE 1 sample preparation

1. Sample collection

Patients diagnosed with epithelial ovarian cancer in the tumor hospital in zhejiang province, 10 months to 2014 months in 2008, 38 patients were collected in this study, and all patients received standard treatment regimens: primary tumor cell debulking or primary staging plus post-operative 6-8 course first-line regimen chemotherapy (platinum in combination with paclitaxel) is classified according to tumor tissue type: 1. serous adenocarcinoma; 2. mucinous adenocarcinoma; 3. endometrioid adenocarcinoma; 4. clear cell carcinoma; 5. unclassified adenocarcinoma; 6. a metastatic cell carcinoma; 7. mixed cancers of two or more kinds. The clinical and pathological data of each patient are carefully collected, and the patients are followed up through the telephone after the treatment is finished. The last follow-up time is 4 months in 2020, and the follow-up time of the patient reaches 11-134 months.

2. The definition of the relevant indexes of ovarian cancer patient recurrence, chemotherapy sensitivity, chemotherapy resistance and the like in the research is as follows:

Degree of differentiation: it refers to the degree of tumor cells close to normal cells, wherein the highly differentiated cancer and tumor tissues are similar to normal tissues, and have high maturity and low malignancy. The tumor tissues of low-differentiation cancer and undifferentiated cancer are greatly different from normal tissues, the maturity is low, and the malignancy is high.

3. General data for patients with ovarian cancer

The general condition of the collected cases according to the invention is shown in Table 1

TABLE 1 general data for ovarian cancer patients

Example 2 Experimental methods

1. Immunohistochemistry

1) After the paraffin sections were retended by 2 physicians in the intensive disease department, the subsequent experiments were performed.

2) Dewaxing and hydrating: the sections were soaked in xylene solution 3 times for 10 minutes each. Taking out, soaking in 95% alcohol for 5 min, and soaking in 75% alcohol for 5 min. Finally soaking in 50% alcohol for 5 min. Taking out the slices, and soaking in distilled water.

3) Blocking endogenous peroxidase: the dewaxed and hydrated sections were soaked in a methanol solution containing 3% hydrogen peroxide for 15 minutes. The cells were then washed 3 times with PBS for 5 minutes each.

4) Microwave antigen retrieval: immersing the slices in a buffer solution containing EDTA and citrate, and placing the slices in a microwave oven, wherein 100% of power is maintained for 8 minutes, 80% of power is maintained for 6 minutes, and 60% of power is maintained for 6 minutes; buffer was replenished in time after each microwaving. After cooling, the sections were removed and washed 3 times with PBS for 5 minutes each.

5) Blocking of non-specific antigens: sections were placed in 10% calf serum for 30 minutes at room temperature.

6) Primary antibody incubation: dropping 1: 100(Claudin22) diluted primary antibody working solution, in a refrigerator at 4 ℃ overnight. After removal, the cells were washed 3 times with PBS for 5 minutes each.

6) And (3) secondary antibody incubation: and (3) dropwise adding an Envision reinforced secondary antibody on the section, incubating for half an hour at room temperature, taking out, and washing with PBS (phosphate buffer solution) for 3 times, each time for 5 minutes.

7) 50 μ l of DAB color developing solution freshly prepared was dropped on the slices, and observed under a microscope for 5 to 10 minutes.

8) Hematoxylin counterstaining: the slices were soaked in hematoxylin stain for 2 minutes, and then taken out and washed with running water for 10 minutes.

9) And (3) slicing and dehydrating: placing the slices into alcohol with different concentrations for dehydration and drying: 80% alcohol for 5 minutes; 90% alcohol for 5 minutes; absolute ethanol 2 times for 5 minutes each. Taking out, using dimethylbenzene to make transparency, dripping neutral gum into the slice, covering with glass slide, and observing result under microscope.

10) Grading standard:

and (3) positive cell judgment: the slices were free of contaminating material, and dark brown or yellowish particles were observed in the normally stained areas, and 5 fields with more tumor cells were randomly selected and observed under a 40X 10-fold microscope. The proportion and distribution of the positive staining cells are determined as follows: dyeing strength: negative 0, weak positive 1, moderate positive 2, high positive 3, strong positive 4. The proportion of positive cells is 1 minute for 0-25%, 2 minutes for 26-50%, 3 minutes for 51-75% and 4 minutes for 76-100%; the percentage of positively stained cells in each field was multiplied by the staining intensity score for the protein of interest and summed. Taking the median of 40, low expression is defined as a score of 40 or less, and high expression is defined as a score of 41 or more.

2. Experimental reagent

3. Statistical method

Data analysis used SPSS 20.0 statistical software. Expressing the measurement data by using the mean +/-standard deviation, and comparing the difference between groups by using Student t test; the rate (%) of use represents the counting data, the Fisher's exact test was used to compare the differences between groups, and the Kaplan-Meier method and Log-rank test were used to analyze the differences in survival data between groups. Statistical differences were considered for test levels α of 0.05 and P < 0.05. Wherein ". x" represents P <0.05, ". x" represents P <0.01, ". x" represents P < 0.001.

Example 3 Claudin22 expression correlates with poor prognosis

The expression of Claudin22 in the cases was subjected to immunohistochemical experiments and scored for statistics according to the above definitions, and the specific results are shown in Table 2

TABLE 2 correlation of Claudin22 expression with clinicopathological parameters

Claudin22 was mainly expressed in the cytoplasm of ovarian cancer cells, and was colored either tan or tan (FIG. 1). We found that 56.3% (18/32) of the patients with low expression of Claudin22 and low differentiation of the pathology were higher than 43.7% (14/32) of the patients with high differentiation (P0.047), and that PFS and OS of the patients with low expression of Claudin22 were significantly better than those with high expression of Claudin22 from the viewpoint of survival prognosis (P0.02, fig. 2).

Claims

Use of Claudin22 for the preparation of prognostic diagnostic markers for tumors, characterized in that Claudin22 is highly expressed in tumor tissues, said tumor patients having a poor prognosis.
2. Use according to claim 1, wherein the tumour is a gynaecological tumour, preferably ovarian cancer.
3. The use of any of claims 1-2, wherein the prognosis comprises Progression Free Survival (PFS), Overall Survival (OS), or degree of tumor differentiation.
4. The use of any one of claims 1-3, wherein the high expression is observed in a reading frame after immunohistochemical detection, wherein the reading frame is free from cross-staining, and the positive cells are brown yellow or brown particles on the normal staining part of the antibody, and 5 fields with more tumor cells are randomly selected under a 40 x 10 light microscope for observation. The proportion and distribution of the positive staining cells are determined as follows: claudin22 is stained in cytoplasm, and the proportion of Claudin22 positive cells is as follows: 0-25% ═ 1 part, 26-50% ═ 2 parts, 51-75% ═ 3 parts, 76-100% ═ 4 parts; target protein staining intensity: negative 0, weak positive 1, moderate positive 2, high positive 3 and strong positive 4; multiplying and summing the percentage score of positively stained cells and the staining intensity score of the protein of interest for each field; scores of 0-40 were classified as low expression Claudin22, and scores of 41-80 were classified as high expression Claudin 22.
5. The application of a reagent for detecting a tumor marker in preparing a tumor prognosis product, wherein the marker is Claudin22, and high expression indicates that a tumor patient has poor prognosis.
6. Use according to claim 5, wherein the tumour is a gynaecological tumour, preferably ovarian cancer.
7. The use of claim 5 or 6, wherein the prognosis comprises Progression Free Survival (PFS), Overall Survival (OS), or degree of tumor differentiation.
8. The use of any one of claims 5-7, wherein the agent is a monoclonal antibody.
9. The use of any one of claims 5-8, wherein the high expression is observed in a reading frame after immunohistochemical detection, wherein the reading frame is free from cross-staining, and the observation is performed by randomly selecting 5 fields with more tumor cells under a 40 x 10 light microscope, wherein the positive cells are brown yellow or brown particles in the normal staining part of the antibody. The proportion and distribution of the positive staining cells are determined as follows: claudin22 is stained in cytoplasm, and the proportion of Claudin22 positive cells is as follows: 0-25% ═ 1 part, 26-50% ═ 2 parts, 51-75% ═ 3 parts, 76-100% ═ 4 parts; target protein staining intensity: negative 0, weak positive 1, moderate positive 2, high positive 3 and strong positive 4; multiplying and summing the percentage score of positively stained cells and the staining intensity score of the protein of interest for each field; scores of 0-40 were classified as low expression Claudin22, and scores of 41-80 were classified as high expression Claudin 22.