CN118600006A - Application of gene methylation in the detection of cervical cancer and endometrial cancer - Google Patents

Abstract

The present invention provides an application of gene methylation in the detection of cervical cancer and endometrial cancer, and relates to the field of biotechnology. The present invention can quickly and effectively realize the auxiliary diagnosis of cervical cancer and endometrial cancer, realize the auxiliary grading of cervical squamous intraepithelial lesions, and realize the auxiliary identification of endometrial atypical hyperplasia and endometrial cancer by detecting the methylation levels of RASSF1A, SHOX2 and SEPTIN9 genes, and has good sensitivity and specificity, and can also identify the tissue sources of the two tumors.

Description

Application of gene methylation in the detection of cervical cancer and endometrial cancer

Technical Field

The present invention relates to the field of biotechnology, and in particular to an application of gene methylation in the detection of cervical cancer and endometrial cancer.

Background Art

Cervical cancer is a common gynecological malignancy, and its incidence is second only to breast cancer. If cervical precancerous lesions and early invasive cancer can be diagnosed, treated and managed promptly and effectively, the incidence of recurrent or metastatic cervical cancer can be reduced and the survival rate of patients can be improved.

Early diagnosis and treatment of cervical lesions and cervical cancer are mainly achieved through cervical cancer screening. Cervical cytology, HPV DNA testing, and visual inspection with acetic acid and iodine (VIA/VILI) are the current mainstream methods. From the perspective of clinical practice, although cytology can screen out most cervical intraepithelial neoplasia (CIN), the interpretation of cytology results mainly depends on the experience and skills of cytopathologists, and this technology is still relatively difficult to popularize in developing countries or grassroots areas. Past research data show that more than 30% of CIN2, CIN3 and invasive cancer in cytology examinations are difficult to distinguish, and may lead to clinical overdiagnosis and treatment or excessive anxiety in women, especially for young women of childbearing age, which may lead to the loss of fertility opportunities. HPV is a contagious carcinogenic virus. According to current research results, almost all cervical cancer tissue biopsy results show HPV infection. HPV DNA testing is one of the most widely used technologies for screening and follow-up monitoring. The high sensitivity and limited specificity of HPV testing still cannot distinguish between short-term self-limiting infections and persistent infections that may develop into neoplastic lesions. It may also produce more false-positive results than cytology, causing panic in patients. So far, neither cytology nor HPV DNA testing can provide sufficient specificity and sensitivity as a primary screening technology. Therefore, there is an urgent need to find new biomarkers and technologies for early screening or auxiliary diagnosis based on objective, easy-to-interpret, highly sensitive and highly specific non-invasive or minimally invasive sampling to assess the risk of CIN2/CIN3 developing into invasive cancer and conduct follow-up management.

Endometrial cancer is also a common gynecological malignancy. Early identification of high-risk patients and precancerous lesions, early cancer screening, and effective detection of cancer progression and treatment are one of the major issues in the current diagnosis and treatment of endometrial cancer. The common symptom of endometrial cancer is vaginal bleeding, but only 0.33% of women with abnormal uterine bleeding before menopause are eventually diagnosed with endometrial cancer, and only 5%-10% of patients with vaginal bleeding after menopause are eventually diagnosed with endometrial cancer. Currently, the commonly used clinical detection methods, such as diagnostic curettage and hysteroscopic endometrial biopsy, are invasive operations, which bring great physical, psychological and economic burdens to patients. At present, there is no non-invasive screening program recommended for clinical practice at home and abroad. Therefore, it is of great significance to find reliable markers for early detection of endometrial cancer.

Ascites (including peritoneal washings) cytology plays an important role in the diagnosis of cervical cancer and endometrial cancer. It is not only the basis for tumor staging, but also has important significance for the diagnosis, treatment, monitoring and prognosis of tumors. Ascites cytology has been used in gynecological tumor management since 1956. One of the staging criteria for stage IA and stage IB in stage I is "no malignant tumor cells in the ascites", and one of the staging criteria for stage IC is "malignant tumor cells in the ascites". Because cervical cancer and endometrial cancer have no specific symptoms in the early stage, most patients are in the late stage when they seek medical treatment. Many patients already have ascites, and some cases have ascites as the first symptom. At this time, ascites cytology can make a clear diagnosis as early as possible, so that clinicians can formulate treatment plans for patients as early as possible and give effective treatment. During the treatment process, ascites cytology can also be used to monitor the treatment situation. In addition, ascites cytology can also help clinicians judge the prognosis of patients. Intraoperative ascites is generally obtained by clinicians when they open the abdominal cavity during surgery and before any exploration. If ascites is found, even if the amount is very small, it must be sucked out and an anticoagulant must be added for immediate examination; if no obvious ascites is found, the abdominal cavity should be flushed with disinfected warm saline mixed with an anticoagulant, especially in areas where tumor cells are prone to implantation, such as the rectouterine pouch. The flushing fluid should be sucked out with a syringe and sent for examination immediately. Before closing the abdominal cavity at the end of the operation, the abdominal cavity should be flushed again and the peritoneal flushing fluid should be sent for examination to observe whether there are any tumor cells that have overflowed after tumor resection. Non-surgical ascites is generally extracted by clinicians through abdominal puncture according to the patient's specific condition, and then sent for examination after adding an anticoagulant. After the pathology department or cell room receives the sample, the centrifuged enriched cells are directly smeared or a liquid-based cell smear is prepared. After fixation and staining, the cytopathologist will read the film and diagnose the results. However, traditional cytological diagnosis mainly relies on pathologists to find cancer cells under a microscope. The diagnostic sensitivity is low and cannot meet the needs of clinical diagnosis. In recent years, with the development of science and technology and the progress of medicine, new medical technologies have continued to emerge. In addition to immunohistochemistry examinations, molecular biology techniques have also been applied to ascites/peritoneal lavage fluid cytology, making its diagnosis more accurate and providing better pathological support for the diagnosis and treatment of patients with cervical cancer and endometrial cancer.

If there are some markers that can not only detect cervical cancer and endometrial cancer with high sensitivity, but also identify the type of tumor, it will have great clinical application value.

In view of this, the present invention is proposed.

Summary of the invention

The first purpose of the present invention is to provide a reagent for detecting methylation of RASSF1A, SHOX2 and SEPTIN9 genes for use in preparing a kit for detecting cervical cancer and endometrial cancer, so as to solve the problem of lack of effective means for detecting cervical cancer and endometrial cancer in the prior art.

The second object of the present invention is to provide a reagent for detecting methylation of RASSF1A, SHOX2 and SEPTIN9 genes for use in preparing a kit for indicating the risk of cervical squamous intraepithelial neoplasia and guiding high-risk triage.

The third object of the present invention is to provide the use of a reagent for detecting methylation of RASSF1A, SHOX2 and SEPTIN9 genes in the preparation of a kit for detecting atypical endometrial hyperplasia and endometrial cancer.

In order to achieve the above objectives, the following technical solutions are adopted.

In a first aspect, the present invention provides the use of a reagent for detecting methylation of RASSF1A, SHOX2 and SEPTIN9 genes in the preparation of a kit for detecting cervical cancer and endometrial cancer.

As a further technical solution, the cervical cancer includes cervical adenocarcinoma and cervical squamous cell carcinoma.

As a further technical solution, if the sample is positive for methylation of any gene of RASSF1A, SHOX2 or SEPTIN9, it indicates an increased possibility of cervical or endometrial malignancy, and the patient is advised to undergo further clinical examination in a timely manner;

If the sample RASSF1A gene methylation is negative, and either SHOX2 or SEPTIN9 gene methylation is positive, it indicates that the possibility of the malignant lesion being cervical squamous cell carcinoma increases, and the patient is advised to undergo further clinical examination in a timely manner;

If the sample RASSF1A gene methylation is positive, and either SEPTIN9 or SHOX2 gene methylation is positive, it indicates that the possibility of the malignant lesion being cervical adenocarcinoma or cervical squamous cell carcinoma with adenocarcinoma increases, and the patient is advised to undergo further clinical examination in a timely manner;

If the sample's SEPTIN9 gene methylation is negative, but either SHOX2 or RASSF1A gene methylation is positive, it indicates that the possibility that the malignant lesion is endometrial cancer increases, and the patient is advised to undergo further clinical examination in a timely manner.

In a second aspect, the present invention provides the use of a reagent for detecting methylation of RASSF1A, SHOX2 and SEPTIN9 genes in the preparation of a kit for indicating the risk of cervical squamous intraepithelial neoplasia and guiding high-risk triage.

As a further technical solution, the reagent for detecting methylation of RASSF1A, SHOX2 and SEPTIN9 genes combined with the human papillomavirus test results indicates the risk of cervical squamous intraepithelial neoplasia and guides the high-risk triage of HPV-positive women before colposcopy;

If the sample has positive methylation of any gene of RASSF1A, SHOX2 or SEPTIN9, women with positive HPV test results are recommended to undergo colposcopy.

In a third aspect, the present invention provides the use of a reagent for detecting methylation of RASSF1A, SHOX2 and SEPTIN9 genes in the preparation of a kit for detecting atypical endometrial hyperplasia and endometrial cancer.

As a further technical solution, the samples to be detected include cervical exfoliated cells, endometrial exfoliated cells, pleural effusion, ascites, peritoneal lavage fluid, tissues of cervical cancer patients, or tissues of endometrial cancer patients.

As a further technical solution, the reagent or kit includes a primer set for detecting RASSF1A gene methylation, a primer set for detecting SHOX2 gene methylation, and a primer set for detecting SEPTIN9 gene methylation;

The primer set for detecting RASSF1A gene methylation comprises: a forward primer, a reverse primer and a probe, and the nucleotide sequences are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.7 respectively;

The primer set for detecting SHOX2 gene methylation comprises: a forward primer, a reverse primer and a probe, and the nucleotide sequences are shown in SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.8 respectively;

The primer set for detecting SEPTIN9 gene methylation includes: a forward primer, a reverse primer and a probe, and the nucleotide sequences are shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.9 respectively.

As a further technical solution, the two ends of the probe contain a fluorescence quenching group and a fluorescence reporter group respectively;

The fluorescence quenching group includes BHQ1 or BHQ2;

The fluorescent reporter groups include FAM, VIC, HEX or CY5.

As a further technical solution, the kit also includes bisulfite, a primer set for detecting an internal reference gene, and a PCR amplification reaction solution.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention can quickly and effectively realize the auxiliary diagnosis of cervical cancer and endometrial cancer, realize the auxiliary grading of cervical squamous intraepithelial lesions, and realize the auxiliary identification of endometrial atypical hyperplasia and endometrial cancer through the detection of the methylation levels of RASSF1A, SHOX2 and SEPTIN9 genes, and has good sensitivity and specificity, and can also identify the tissue origins of the two tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

Figure 1 shows the gene methylation expression in cervical adenocarcinoma, cervical squamous cell carcinoma, and cervical chronic inflammation tissues;

Figure 2 shows the methylation expression of each gene in benign endometrial lesions and cancer tissues;

Figure 3 is a diagnostic flowchart for screening cervical cancer and endometrial cancer using RASS1A/SHOX2/SEPTIN9 methylation detection in cervical exfoliated cells.

DETAILED DESCRIPTION

The embodiments of the present invention will be described in detail below in conjunction with the embodiments and examples, but it will be appreciated by those skilled in the art that the following embodiments and examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified, proceed according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

DNA methylation is a form of chemical modification of DNA that can change genetic expression without changing the DNA sequence. The so-called DNA methylation refers to the covalent bonding of a methyl group to the 5th carbon position of cytosine in the genomic CpG dinucleotide under the action of DNA methyltransferase. A large number of studies have shown that DNA methylation can cause changes in chromatin structure, DNA conformation, DNA stability, and the way DNA interacts with proteins, thereby controlling gene expression. In the precancerous lesion stage before the occurrence of malignant tumors, the genome of diseased tissue cells has undergone extensive DNA methylation modification compared to normal tissue cells. Therefore, compared with DNA mutations, DNA methylation modification is the earliest event in tumor occurrence, has higher sensitivity and specificity, and is more suitable as a diagnostic marker.

Ras-associated region family 1 (RASSF1) gene has five different transcripts: RASSF1A to E, among which RASSF1A is the most common. The full length of RASSF1A gene is 1 873 bp, with an open reading frame of 340 amino acids, encoding a protein polypeptide with a relative molecular mass of 388 000. Its N-terminus is highly homologous to the cysteine-rich diacylglycerol or fluorophore binding region, so it is also called the conserved region of protein kinase C. Studies have found that abnormal methylation and loss of expression of the RASSF1A promoter region exist in various tumor tissues, suggesting that RASSF1A plays an important role in the occurrence and development of various tumors as a tumor suppressor gene. The mechanism by which RASSF1A acts as a tumor suppressor gene is still unclear.

SHOX2, the full name of Short Stature Homobox 2, is a member of the homeobox gene family, whose gene expression regulation is closely related to organ development. This gene is a member of the homeobox family that encodes a protein containing a 60-amino acid residue sequence that represents a DNA binding domain. Homeobox genes have been extensively characterized as transcriptional regulators involved in pattern formation in both invertebrate and vertebrate species. Some human genetic diseases are caused by aberrations in human homeobox genes. This locus represents a pseudo-homologous gene that is thought to be responsible for idiopathic short stature, which is associated with the short stature phenotype in patients with Turner syndrome.

Septin is a family of conserved skeleton protein genes with GTPase activity widely distributed in all eukaryotic organisms except plants, and is related to cell division. In humans, it consists of 14 family members, named Septin1 to 14, of which SEPTIN9 is located at 17q25.3, contains 17 exons, and encodes SEPTIN9 protein, which has multiple subtypes (such as Septin 9-vl, v2, v3, v4, etc.), and each subtype has structural similarities, consisting of a central P-loop GTP binding domain, variable N-terminal and C-terminal domains, and is related to cell functions such as chromosome separation, DNA repair, migration, and apoptosis. Studies have found that SEPTIN9 is directly related to the occurrence of tumors, and its expression and function are different in different tumors. Herein, methods for detecting DNA methylation are well known in the art, such as PCR based on bisulfite conversion (e.g., methylation-specific PCR (Methylation-specific PCR, MSP)), DNA sequencing (such as bisulfite sequencing (Bisulfitesequencing, BS), whole-genome methylation sequencing (Whole-genome bisulfitesequencing, WGBS), reduced representation bisulfite sequencing (Reduced Representation Bisulfite Sequencing, RRBS)), chips, methylation-sensitive restriction enzyme analysis (Methylation-Sensitive Dependent Restriction Enzymes), fluorescence quantitative method, methylation-sensitive high-resolution melting curve method (Methylation-sensitivity High-resolution Melting, MS-HRM), chip-based methylation mapping analysis, mass spectrometry (e.g., flight mass spectrometry). In one or more embodiments, detection includes detecting any one chain at a gene or site.

Methods for calculating methylation levels are known in the art. For example, in embodiments where methylation is detected by PCR, methylation level = 2-ΔCt sample to be tested/2-ΔCt positive standard × 100, where ΔCt = Ct target gene - Ct reference gene. Alternatively, in embodiments where methylation is detected by sequencing, methylation level = number of methylated bases/total number of bases.

"DNA" or "DNA molecule" as used herein refers to deoxyribonucleic acid. The bases (bp) of DNA mainly include adenine (A), guanine (G), cytosine (C) and thymine (T). DNA forms include cDNA, genomic DNA, fragmented DNA or artificially synthesized DNA. DNA can be single-stranded or double-stranded.

"Uracil" or "U" as described herein is a component of RNA. "RNA" or "RNA molecule" refers to ribonucleic acid. RNA is a long chain molecule formed by condensation of ribonucleotides via phosphodiester bonds. There are four main bases in RNA, namely adenine (A), guanine (G), cytosine (C), and uracil (U). In the base pairing of RNA, U replaces the position of T in DNA, that is, A and U are paired via hydrogen bonds, and G and C are paired via hydrogen bonds.

In a first aspect, the present invention provides the use of a reagent for detecting methylation of RASSF1A, SHOX2 and SEPTIN9 genes in the preparation of a kit for detecting cervical cancer and endometrial cancer.

The present invention can quickly and effectively realize auxiliary diagnosis of cervical cancer and endometrial cancer by detecting the methylation levels of RASSF1A, SHOX2 and SEPTIN9 genes, and has good sensitivity and specificity.

In some optional embodiments, the cervical cancer includes cervical adenocarcinoma and cervical squamous cell carcinoma.

In some optional embodiments, ΔCt≤9 is used as the positive threshold. If the sample has positive methylation of any gene of RASSF1A, SHOX2 or SEPTIN9, it indicates an increased possibility of cervical or endometrial malignancy, and the patient is advised to undergo further clinical examination in a timely manner;

If the sample RASSF1A gene methylation is negative, and either SHOX2 or SEPTIN9 gene methylation is positive, it indicates that the possibility of the malignant lesion being cervical squamous cell carcinoma increases, and the patient is advised to undergo further clinical examination in a timely manner;

If the sample RASSF1A gene methylation is positive, and either SEPTIN9 or SHOX2 gene methylation is positive, it indicates that the possibility of the malignant lesion being cervical adenocarcinoma or cervical squamous cell carcinoma with adenocarcinoma increases, and the patient is advised to undergo further clinical examination in a timely manner;

If the sample's SEPTIN9 gene methylation is negative, but either SHOX2 or RASSF1A gene methylation is positive, it indicates that the possibility that the malignant lesion is endometrial cancer increases, and the patient is advised to undergo further clinical examination in a timely manner.

In a second aspect, the present invention provides the use of a reagent for detecting methylation of RASSF1A, SHOX2 and SEPTIN9 genes in the preparation of a kit for indicating the risk of cervical squamous intraepithelial neoplasia and guiding high-risk triage.

The inventors have found that the detection results of the methylation levels of RASSF1A, SHOX2 and SEPTIN9 genes can indicate the risk of cervical squamous intraepithelial neoplasia and guide high-risk triage.

In some optional embodiments, the reagent for detecting methylation of RASSF1A, SHOX2 and SEPTIN9 genes combined with the human papillomavirus test results indicate the risk of cervical squamous intraepithelial neoplasia and guide the high-risk triage of HPV-positive women before colposcopy;

Taking ΔCt≤9 as the positive threshold, if any gene methylation of RASSF1A, SHOX2 or SEPTIN9 in the sample is positive, women with positive HPV test results are recommended to undergo colposcopy.

In a third aspect, the present invention provides the use of a reagent for detecting methylation of RASSF1A, SHOX2 and SEPTIN9 genes in the preparation of a kit for detecting atypical endometrial hyperplasia and endometrial cancer.

The detection of methylation levels of RASSF1A, SHOX2 and SEPTIN9 genes can quickly and effectively assist in the identification of atypical endometrial hyperplasia and endometrial cancer with good sensitivity and specificity.

In some optional embodiments, the samples to be tested include, but are not limited to, cervical exfoliated cells, endometrial exfoliated cells, pleural effusion, ascites, peritoneal lavage fluid, cervical cancer patient tissue, or endometrial cancer patient tissue.

In some optional embodiments, the reagent or kit includes a primer set for detecting RASSF1A gene methylation, a primer set for detecting SHOX2 gene methylation, and a primer set for detecting SEPTIN9 gene methylation;

The primer set for detecting RASSF1A gene methylation includes: a forward primer, a reverse primer and a probe, and the nucleotide sequences are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.7 respectively:

Forward primer (SEQ ID NO. 1): CGGGGTTCGTTTTGTGGTTTC;

Reverse primer (SEQ ID NO. 2): CCGATTAAATCCGTACTTCG;

Probe (SEQ ID NO.7): TCGCGTTTGTTAGCGTTTAAAGT;

The primer set for detecting SHOX2 gene methylation comprises: a forward primer, a reverse primer and a probe, and the nucleotide sequences are shown in SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.8 respectively:

Forward primer (SEQ ID NO. 3): TTGTTTTTGGGTTCGGGTT;

Reverse primer (SEQ ID NO. 4): CATAACGTAAACGCCTATACTCG;

Probe (SEQ ID NO. 8): ATCGAACAAACGAAACGAAAATTACC;

The primer set for detecting SEPTIN9 gene methylation includes: a forward primer, a reverse primer and a probe, and the nucleotide sequences are shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.9 respectively:

Forward primer (SEQ ID NO. 5): GCGTTTTTTCGTCGTTGTT;

Reverse primer (SEQ ID NO. 6): CGCGTTAACCGCGAAATC;

Probe (SEQ ID NO.9): TCGCGCGATTCGTTGTTT.

The above primer sets have strong specificity and high sensitivity, and can accurately detect the methylation levels of RASSF1A, SHOX2 and SEPTIN9 genes.

In the present invention, the two ends of the probe contain a fluorescence quenching group and a fluorescence reporter group respectively;

The fluorescence quenching group includes but is not limited to BHQ1 or BHQ2, or other fluorescence quenching groups known to those skilled in the art;

The fluorescent reporter groups include, but are not limited to, FAM, VIC, HEX or CY5, or other fluorescent reporter groups known to those skilled in the art.

In some optional embodiments, bisulfite, a primer set for detecting an internal reference gene, and a PCR amplification reaction solution are also included.

In some optional embodiments, the internal reference gene is β-actin;

The primer set for detecting the internal reference gene includes: a forward primer, a reverse primer and a probe, and the nucleotide sequences are shown in SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12 respectively:

Forward primer (SEQ ID NO. 10): AAGATAGTGTTGTGGGTGTAGGT;

Reverse primer (SEQ ID NO. 11): TACTTAATACACACTCCAAAACCG;

Probe (SEQ ID NO. 12): ACACCAACCTCATAACCTTATCACAC.

In some optional embodiments, the PCR amplification reaction solution includes Taq DNA polymerase, PCR buffer, dNTPs and Mg ²⁺ .

In some optional embodiments, the Taq DNA polymerase is a hot-start Taq DNA polymerase;

And/or, the final concentration of Mg ²⁺ is 1.0-10.0 mM; the final concentration of each primer is 200-700 nM; the final concentration of each probe is 100-400 nM.

In some optional embodiments, the PCR reaction conditions are:

a) Stage 1: 95°C, 10 min, 1 cycle;

b) Second stage: 95°C, 15 seconds, 60°C, 30 seconds, 5 cycles;

c) Stage 3: 95°C, 15 sec, 57°C, 30 sec, 40 cycles;

Signal collection: In the third stage, FAM, VIC (or HEX) and CY5 signals were collected at 57°C.

In some optional embodiments, positive quality control products and negative quality control products of RASSF1A, SHOX2 and SEPTIN9 genes are also included.

By setting positive and negative controls, false negative or false positive results can be effectively avoided, further ensuring the accuracy of the test.

In a specific embodiment, the method for detecting methylation of RASSF1A, SHOX2 and SEPTIN9 genes using the above kit comprises:

(1) Sample preparation: including DNA extraction and quality inspection; (2) DNA conversion: the DNA obtained in step (1) is subjected to bisulfite conversion, and unmethylated cytosine (cytosine, C) is converted into uracil (uracil, U); methylated cytosine does not change after conversion; (3) Reaction mixture preparation: including PCR reaction solution, primer mixture, and probe mixture; (4) PCR mixture preparation: adding the template DNA converted with bisulfite in step (2) and the positive standard, negative control or non-template control (NTC) to step (3); (5) PCR reaction: performing PCR reaction and collecting fluorescence.

The PCR reaction solution includes: Taq DNA polymerase, PCR buffer, dNTPs, Mg ²⁺ ; Taq DNA polymerase is a hot start Taq DNA polymerase; the final concentration of Mg ²⁺ is 1.0-10.0 mM. The primer mixture is a mixture of primers of the gene to be amplified, wherein the final concentration of each primer is 200-700 nM. The probe mixture is a mixture of probes of the gene to be amplified, wherein the final concentration of each probe is 100-400 nM.

The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

Experimental methods

1. Preliminary sample processing

1. Pre-treatment of tissue paraffin sections:

1.1 Take 2-3 paraffin sections and place them in a centrifuge tube, add 1 mL of xylene, cover the tube tightly, and vortex for 10 seconds.

1.2 Centrifuge at 12000rpm for 2min, carefully discard the supernatant, but be careful not to discard the precipitate.

1.3 Add 1 mL of anhydrous ethanol and vortex to mix. Centrifuge at 12000 rpm for 2 min and carefully discard the supernatant, taking care not to discard the precipitate.

1.4 Open the tube cap and incubate at room temperature or 37°C for 10 min until no ethanol remains.

1.5 Add 180 μL of lysis buffer 1, resuspend the pellet, add 20 μL of proteinase K, and vortex to mix.

1.6 Incubate at 56°C for 1 hour until the sample is completely dissolved.

1.7 Incubate at 90℃ for 1 hour. Centrifuge briefly to collect the solution on the tube wall to the bottom of the tube.

1.8 Add 200 μL of lysis buffer 2 and vortex to mix thoroughly. Add 200 μL of anhydrous ethanol and vortex to mix thoroughly. Centrifuge briefly to collect the solution on the tube wall to the bottom of the tube.

1.9 Transfer all the solution obtained in 1.8 to the adsorption column, centrifuge at 12000rpm for 1min, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

2. Pre-treatment of cervical exfoliated cells, endometrial exfoliated cells, pleural effusions, and pleural and abdominal lavage fluid samples:

2.1 Transfer the sample to a 15 mL centrifuge tube (the sampling volume varies slightly for different sample types, including 10 mL for bronchoalveolar lavage fluid and ascites, and 5 mL for pleural effusion), centrifuge at 2000 rpm for 5 min, discard the supernatant, and resuspend the cell pellet with the remaining liquid (100-200 μL).

2.2 Transfer the above liquid to a 1.5 mL EP tube, centrifuge at 2000 rpm for 5 min, and discard the supernatant.

2.3 Add 200 μL of lysis buffer 1 and shake until the sample is thoroughly suspended.

2.4 Add 20 μL of proteinase K and 200 μL of lysis buffer 2 to the sample, and vortex to mix thoroughly.

2.5 Incubate in a 56℃ metal bath for 10 min, mixing every 2-3 min.

2.6 Centrifuge briefly, add 200 μL of anhydrous ethanol, vortex to mix thoroughly, and centrifuge briefly.

2.7 Transfer the liquid in the EP tube to the adsorption column of the collection tube. Be careful not to let the gun tip touch the filter membrane to avoid puncturing it.

2. Washing, eluting and quantifying nucleic acid samples from adsorption columns

1. Centrifuge at 12000rpm for 1min, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

2. Add 500 μL of washing buffer 1 to the adsorption column, centrifuge at 12000 rpm for 1 min, and discard the waste liquid in the collection tube.

3. Add 500 μL of rinsing buffer 2 to the adsorption column, 12000 rpm, 1 min, and pour out the waste liquid in the collection tube.

4. Repeat step 3 once to improve DNA purity.

5. Centrifuge at 12000rpm for 2min (the purpose of this step is to remove residual ethanol, which will inhibit PCR amplification, so this step cannot be omitted), prepare a new 1.5ml EP tube, place the adsorption column into the EP tube, discard the collection tube, and leave the adsorption column open at room temperature for 5min.

6. Add 50 μL of elution buffer to the middle of the filter membrane of the adsorption column and let it stand at room temperature for 5 minutes. Centrifuge at 12000 rpm for 1 minute to collect the DNA solution.

7. Take a new EP tube to prepare the reagent for detecting DNA concentration (Qubit kit), prepare the corresponding reagent according to the number of samples (199μL Buffer + 1μL dye per person), and vortex.

8. Take 199 μL of the prepared reagent and 1 μL of DNA, place them in the 0.5 ml matching tube that comes with the kit, shake lightly, shake off the bubbles, and protect from light for 3 minutes.

9. Use Qubit or Osun Fluo-100 fluorometer to detect DNA concentration. The DNA concentration should be greater than 10ng/μL. (If the DNA concentration is <10ng/μL, it is recommended to re-extract). If the measured DNA concentration is out of the detection range (higher than 100ng/μL), take 2μL DNA stock solution + 8μL elution solution, vortex, centrifuge instantly, and re-measure the concentration.

10. Use the elution buffer to dilute the sample DNA to 10 ng/μL and normalize the samples. (The subsequent sulfite modification step requires 10 ng/μL DNA, 20 μL).

3. Bisulfite modification of DNA:

1. Prepare PCR reaction tubes according to the number of DNA samples to be processed; add 130μL of CT conversion reagent to each PCR reaction tube, then add 20μL of DNA sample in sequence, mark, shake gently, and centrifuge briefly.

2. Place the PCR reaction tube on the reaction plate of the PCR instrument, cover the hot cover, and set the reaction program according to the following steps:

98℃, 8min;

64℃, 3.5h;

4℃, maintain.

3. Prepare purification columns and collection tubes (provided with the kit) according to the number of DNA samples to be processed, mark the sample number on the purification column, and place the purification column in the collection tube.

4. Take 600 μL of binding buffer and add it to the purification column.

5. Transfer all the products from step 2 to the purification column (containing 600 μL binding buffer), cover it, invert it several times (more than 10 times) to mix the sample, then centrifuge it at 12,000 rpm for 30 seconds and discard the liquid in the collection tube.

6. Add 100 μL of washing solution to the purification column and centrifuge at 12,000 rpm for 30 seconds.

7. Add 200 μL of desulfonated solution to the purification column, incubate at room temperature for 20 min (the PCR kit can be taken out from -20°C and thawed at room temperature), and centrifuge at 12,000 rpm for 30 s.

8. Add 200 μL of washing solution to the purification column, centrifuge at 12,000 rpm for 30 seconds, then add 200 μL of washing solution, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and centrifuge at 12,000 rpm for 1 minute.

9. Place the purification column in a new 1.5 ml EP tube (mark it), add 20 μL of elution buffer directly to the purification column matrix (must be added on the matrix), centrifuge at 12,000 rpm for 30 seconds to elute the DNA.

IV. PCR Amplification

1. Take out the sulfite-modified sample DNA to be tested.

2. Thaw the kit at room temperature for 30 minutes, remove the PCR reaction solution and DNA polymerase from the kit. The PCR reaction solution needs to be shaken and mixed for 20 seconds (fully shaken), and then briefly centrifuged together with the enzyme.

Note: The enzyme needs to be kept at low temperature, prepared immediately before use, and no shaking is allowed.

3. Prepare PCR reaction solution: According to the number of test specimens (number of samples + positive and negative quality control), prepare PCR mixed solution according to the following reagent ratio: PCR reaction solution: DNA polymerase = 15:0.3 (one person). Add each component to a new EP tube according to the ratio, invert and mix, and centrifuge instantly.

4. Place the PCR reaction tubes in order on the PCR tube rack, add 15 μL of the prepared PCR reaction solution to each well of the PCR reaction tube, then add 5 μL of the DNA sample, carefully cover the reaction tube, and mark it (mark on the handles at both ends of the tube cover).

5. Centrifuge briefly to remove bubbles in the reaction system and place the PCR reaction tube into the instrument.

6. Open the instrument settings window and set up the PCR amplification and signal collection procedures.

a) Stage 1: 95°C, 10 min, 1 cycle;

b) Second stage: 95°C, 15 seconds, 60°C, 30 seconds, 5 cycles;

c) Stage 3: 95°C, 15 sec, 57°C, 30 sec, 40 cycles;

Signal collection: In the third stage, FAM, VIC (or HEX) and CY5 signals were collected at 57°C.

7. Save the file and run the program.

Example 1 Screening of methylation diagnostic indicators for cervical cancer

Screen out the high methylation expression in cervical cancer tissues, and there should be no or low methylation in benign lesions. The specific screening method is as follows:

A tertiary hospital in Nantong provided 10 cervical adenocarcinomas, 20 cervical squamous cell carcinomas, and 20 chronic inflammatory tissue samples for 7 gene methylation tests. The test results are shown in Table 1 and Figure 1 (A is the methylation expression of the RASSF1A gene, B is the methylation expression of the SHOX2 gene, C is the methylation expression of the Septi9 gene, D is the methylation expression of the HOXA9 gene, E is the methylation expression of the p16 gene, F is the methylation expression of the CDH13 gene, and G is the methylation expression of the RARβ gene. The PCR detection curve did not jump at all, there was no target detection object in the sample, the PCR result was "Noct", and ΔCt was calculated as 20).

Table 1 Results of methylation detection of 7 genes in cervical adenocarcinoma tissue, cervical squamous cell carcinoma tissue and chronic inflammatory tissue

*Noct: The abbreviation of “no Ct” indicates that the PCR detection curve does not jump at all and there is no target detection substance in the sample.

The results show that:

Methylation of RASSF1A, SHOX2, and SEPTIN9 has good clinical value in detecting cervical adenocarcinoma. When the cutoff value ΔC≤9 is positive, the positive rates of the three for detecting cervical adenocarcinoma are 40%, 50%, and 40%, respectively, and the specificity for chronic inflammation is 100%, 85%, and 100%. The sensitivity of the three combined for diagnosing cervical adenocarcinoma is 80%.

SHOX2 and SEPTIN9 methylation have good clinical value in detecting cervical squamous cell carcinoma. When the cutoff value ΔCt≤9 is positive, the positive rates of the two for detecting cervical squamous cell carcinoma are 95% and 90% respectively, and the specificity for chronic inflammation is 85% and 100%. The sensitivity of the combined diagnosis of cervical squamous cell carcinoma is 100%.

RASSF1A methylation is completely absent in cervical squamous cell carcinoma, but has a diagnostic sensitivity of 40% in cervical adenocarcinoma. RASSF1A positivity can indicate the possibility of cervical adenocarcinoma or cervical squamous cell carcinoma with adenocarcinoma.

HOXA9 is expressed in chronic inflammation and cervical cancer samples, and its methylation status cannot be used for differential diagnosis of benign and malignant cervical samples. The methylation expression of other methylated genes P16, CDH13, and RARβ in benign and malignant cervical samples is very low, and their clinical value is low.

Conclusion: Methylation of RASSF1A, SHOX2, and SEPTIN9 has good clinical value in the detection of cervical cancer. First, it can be used for the differential diagnosis of benign and malignant cervical samples. Secondly, RASSF1A can indicate the diagnosis of cervical adenocarcinoma or cervical squamous cell carcinoma with adenocarcinoma.

Example 2 Screening of Methylation Diagnostic Indicators for Endometrial Cancer

Screen out those with high methylation expression in endometrial cancer tissue and no or low methylation in benign lesions. The specific screening method is as follows:

A tertiary hospital in Shanghai provided 20 endometrial cancer and 10 benign endometrial lesion tissue samples for 7 gene methylation tests. The test results are shown in Table 2 and Figure 2 (the PCR test curve does not jump at all, there is no target test object in the sample, the PCR result is "Noct", and ΔCt is calculated as 20).

Table 2 Results of methylation detection of 7 genes in endometrial cancer and benign endometrial lesions

*Noct: The abbreviation of “no Ct” indicates that the PCR detection curve does not jump at all and there is no target detection substance in the sample.

The results show that:

RASSF1A and SHOX2 methylation have high clinical value in detecting endometrial cancer. When the cutoff value ΔCt≤9 is positive, the positive rates of the two gene methylation in detecting endometrial cancer are 75% and 55%, and the specificity for benign endometrial lesions is 100%. RASSF1A and SHOX2 methylation can complement each other, with a diagnostic sensitivity of 85% and a specificity of 100% for endometrial cancer.

SEPTIN9 is completely absent in both endometrial cancer and benign tissues, and can be used as an indicator of endometrial tissue characteristics. There is no significant difference in the methylation expression of other indicators in endometrial cancer and benign lesions.

HOXA9 is expressed in chronic inflammation and cervical cancer samples, and its methylation status cannot be used for differential diagnosis of benign and malignant cervical samples. The methylation expression of other methylated genes P16, CDH13, and RARβ in benign and malignant cervical samples is very low, and their clinical value is low.

Conclusion: Combining the data of cervical cancer and endometrial cancer, the combination of RASSF1A, SHOX2, and SEPTIN9 can be selected for auxiliary diagnosis of cervical cancer and endometrial cancer, and can also assist in distinguishing tissue origin to a certain extent.

Example 3 Construction and use of methylation diagnostic kit for cervical cancer and endometrial cancer

Through experiments, it was finally determined that the genes RASSF1A, SHOX2, and SEPTIN9 were selected as detection targets for use in the diagnosis of cervical cancer and endometrial cancer.

1. Kit composition:

a), RASSF1A, SHOX2, SEPTIN9 gene methylation detection reaction solution;

Forward primer for RASSF1A (SEQ ID No. 1): CGGGGTTCGTTTTGTGGTTTC;

Reverse primer for RASSF1A (SEQ ID No. 2): CCGATTAAATCCGTACTTCG;

Forward primer for SHOX2 (SEQ ID No. 3): TTGTTTTTGGGTTCGGGTT;

Reverse primer for SHOX2 (SEQ ID No. 4): CATAACGTAAACGCCTATACTCG;

Forward primer for SEPTIN9 (SEQ ID No. 5): GCGTTTTTTCGTCGTTGTT;

Reverse primer for SEPTIN9 (SEQ ID No. 6): CGCGTTAACCGCGAAATC;

b) RASSF1A, SHOX2, SEPTIN9 positive quality control products;

c) Negative quality control products;

d), PCR reaction solution (Taq DNA polymerase, PCR buffer, dNTPs, Mg ²⁺ ; Taq DNA polymerase is hot start Taq DNA polymerase; Mg ²⁺ final concentration is 1.0-10.0 mM. Primer mixture is a mixture of primers of the gene to be amplified, wherein the final concentration of each primer is 200-700 nM. Probe mixture is a mixture of probes of the gene to be amplified, wherein the final concentration of each probe is 100-400 nM);

e), bisulfite;

f) Probe:

The nucleotide sequence of the probe for RASSF1A (SEQ ID No. 7): TCGCGTTTGTTAGCGTTTAAAGT;

The probe nucleotide sequence for SHOX2 (SEQ ID No. 8): ATCGAACAAACGAAACGAAAATTACC;

The probe nucleotide sequence for SEPTIN9 (SEQ ID No. 9): TCGCGCGATTCGTTGTTT;

g) Reference genes:

β-actin:

AC-forward primer (SEQ ID No. 10): AAGATAGTGTTGTGGGTGTAGGT;

AC-reverse primer (SEQ ID No. 11): TACTTAATACACACTCCAAAACCG;

AC-probe (SEQ ID No. 12): ACACCAACCTCATAACCTTATCACAC.

2. How to use the kit

For details, please refer to the above method section. Among them, PCR conditions:

a) Stage 1: 95°C, 10 min, 1 cycle;

b) Second stage: 95°C, 15 seconds, 60°C, 30 seconds, 5 cycles;

c) Stage 3: 95°C, 15 sec, 57°C, 30 sec, 40 cycles;

Signal collection: In the third stage, FAM, VIC (or HEX) and CY5 signals were collected at 57°C, where FAM was the probe fluorescence signal for detecting RASSF1A and SEPTIN9, VIC was the probe fluorescence signal for SHOX2, and CY5 was the probe fluorescence signal for internal reference detection.

3. Judgment of the results of each test indicator

RASSF1A is very low in methylation in benign lesions, and its index specificity is very good, with the cutoff value set within the stable detection range of the index PCR, ΔCT = 9.0;

The methylation level of SHOX2 in benign lesions is very low, and its indicator specificity is very good. The cutoff value is set within the stable detection range of the indicator PCR, ΔCT = 9.0.

SEPTIN9 was not expressed in all benign lesions; the cutoff value was set within the stable detection range of the index PCR at ΔCT = 9.0;

The Ct value of the fluorescence quantitative PCR detection signal reflects the concentration of the gene template to be tested in the sample. The lower the Ct value, the higher the concentration of the gene to be tested. The internal reference gene detects the housekeeping gene β-actin contained in all cells, and the detection signal comes from all cells. The methylation target gene detection is the highly methylated target gene contained in tumor cells, such as RASSF1A, SHOX2, SEPTIN9, and the detection signal only comes from tumor cells. Methylation detection ΔCt = ΔCt target gene - ΔCt internal reference. When ΔCt = 9, it indicates that the proportion of tumor cells containing methylation signals relative to the total number of cells is 1/2 ⁹ = 0.2%. At this time, if the internal reference fluctuates between 18-23, the Ct value of the target gene is between 27-32. Ct within 32 is the stable range of PCR amplification, so for methylation indicators, generally ΔCt is controlled within 9, which is the stable detection range of PCR and the maximum sensitivity. If the specificity of the detection indicator is poor, the cutoff value needs to be adjusted downward based on the detection results of the indicator in benign lesions to meet the specificity requirements.

IV. Judgment of the test results of the kit:

1) If any gene of RASSF1A, SHOX2, or SEPTIN9 is methylated positive in the sample, it indicates an increased possibility of cervical or endometrial malignancy, and the patient is advised to undergo further clinical examination in a timely manner;

2) If the sample is RASSF1A negative, and either SHOX2 or SEPTIN9 is methylated positive, it indicates that the possibility of the malignant lesion being cervical squamous cell carcinoma increases, and the patient is advised to undergo further clinical examination in a timely manner;

3) If the sample is RASSF1A positive, and either SEPTIN9 or SHOX2 is methylated, it indicates that the possibility of the malignant lesion being cervical adenocarcinoma increases, and the patient is advised to undergo further clinical examination in a timely manner;

4) If the sample is SEPTIN9 negative, but either SHOX2 or RASSF1A is methylated positive, it indicates that the possibility of the malignant lesion being endometrial cancer increases, and the patient is advised to undergo further clinical examination in a timely manner;

Example 4 Diagnostic sensitivity and specificity of methylation diagnostic kit for cervical squamous cell carcinoma and precancerous lesion tissue samples

A tertiary hospital in Nantong provided 100 cervical tissue samples of different pathological types, including 20 cases of cervical squamous cell carcinoma, 20 cases of CIN3, 20 cases of CIN2, 20 cases of CIN1 and 20 cases of chronic inflammation. The methylation test results are shown in Table 3.

Table 3 Results of methylation detection of three genes in cervical squamous cell carcinoma and precancerous lesion tissue samples

CIN3: Cervical intraepithelial neoplasia grade 3; CIN2: Cervical intraepithelial neoplasia grade 2; CIN1: Cervical intraepithelial neoplasia grade 1. Noct: The abbreviation of “no Ct” indicates that the PCR test curve does not jump at all and there is no target test substance in the sample.

The combined detection of SHOX2 and SEPTIN9 methylation, when the cutoff value is ΔCt = 9, the diagnostic sensitivity for cervical squamous cell carcinoma, CIN3, CIN2, CIN1 is 100%, 95%, 60%, 25%, and the specificity for chronic inflammation is 85%. When the cutoff value is ΔCt = 5.0, the diagnostic sensitivity for cervical cancer, CIN3, CIN2, CIN1 is 100%, 15%, 20%, 5%, and the specificity for chronic inflammation is 100%.

This study shows that the degree of SHOX2 and SEPTIN9 gene methylation is significantly different in cervical squamous cell carcinoma and different degrees of cervical precancerous lesions and chronic inflammation. The high methylation of SHOX2 and SEPTIN9 genes is closely related to the occurrence of cervical squamous cell carcinoma and high-grade cervical lesions. The detection rates of SHOX2 combined with SEPTIN9 methylation detection for benign cervical lesions and CIN 1, CIN2+ were 17.5% and 85%, respectively, and the diagnostic sensitivity for CIN3+ was 97.5%. Compared with the existing HPV DNA detection and E6/E7 mRNA detection, DNA methylation detection as a high-risk triage scheme can not only be used for the initial triage of human papillomavirus-positive women; it can also be used for the secondary triage of women with mild cytological abnormalities to determine the high risk of cervical intraepithelial neoplasia (CIN) grade 3 (CIN 3) or cancer; it can also prevent the missed diagnosis of HPV-negative cervical cancer and the screening of HPV-negative cervical cancer patients due to HPV virus integration.

Example 5 Clinical application of the kit 1: High-risk triage for HPV-positive patients

155 female patients with positive HPV test in the Affiliated Hospital of Nantong University were collected before colposcopy, and methylation test of three genes was performed. Pathological tissue was obtained by colposcopy to confirm the pathological diagnosis. The three genes RASSF1A, SHOX2, and SEPTIN9 were combined with internal references to calculate ΔCt, and ΔCt≤9 was judged as positive. All samples were negative for RASSF1A, and any one of SHOX2 and SEPTIN9 was positive, and the methylation test was judged to be positive. The test results are summarized in Table 4.

Table 4 Results of methylation detection of cervical exfoliated cells

The test results showed that among women who were HPV positive and planned to undergo colposcopy, the positive rates of methylation in cervical exfoliated cells were 5.9% and 17.3% in patients who were eventually diagnosed with chronic inflammation and CIN 1, respectively, while the positive rates of methylation in patients diagnosed with CIN2, CIN3 and cervical cancer were significantly increased to 84.2%, 96.2% and 100%. Clinical colposcopy is effective if CIN2 and above lesions (CIN2+) are found, and the colposcopy referral rate of chronic inflammation and CIN 1 should be reduced. In Table 5, the positive predictive value and negative predictive value of methylation for the diagnosis of CIN2+ lesions were calculated to be 84.5% and 91.5%, respectively.

Table 5 Diagnostic efficacy of cervical exfoliated cell methylation detection for CIN2+ lesions

Cervical squamous intraepithelial lesions (SIL) are also called cervical intraepithelial neoplasia (CIN). It is a squamous cell proliferation driven by HPV infection, showing maturation abnormalities and/or viral cell changes, but not extending beyond the basement membrane. It includes grades 1, 2, and 3, and grade 3 includes carcinoma in situ. Among them, low-grade squamous intraepithelial lesions include the original CIN grade 1 and histological changes with clear HPV virus infection. High-grade squamous intraepithelial lesions include the original CIN grades 2 and 3. Generally speaking, low-grade squamous intraepithelial lesions of the cervix can be followed up, while high-grade lesions require clinical intervention. The main purpose of cervical cancer screening is to first detect treatable precancerous lesions CIN 2, CIN 3, and adenocarcinoma in situ (AIS) that can develop into invasive cancer, thereby reducing the incidence, mortality, and treatment-related morbidity of cervical cancer.

According to calculations in this study, HPV-positive women can significantly reduce the referral rate of ineffective colposcopy through methylation testing, that is, 87.2% of patients with chronic inflammation and CIN1 can be prevented from undergoing colposcopy. At the same time, women who are HPV-positive and methylation-positive should undergo colposcopy immediately, because the probability of finding CIN2+ lesions is 84.9%. RASSF1A, SHOX2, and SEPTIN9 gene methylation testing can be used for high-risk triage before colposcopy in HPV-positive women, greatly avoiding unnecessary colposcopy referrals, effectively identifying high-risk CIN2+ patients, and refining the later clinical management of HPV-positive women.

Example 6 Clinical application of the kit 2: Improving the differential diagnosis of HPV-negative cervical adenocarcinoma

Case sharing from a tertiary hospital in Shanghai:

Basic information: The patient was a 62-year-old female who came to the hospital for treatment due to vaginal bleeding after menopause for about 10 days.

Gynecological examination: bleeding on the surface of the cervix, unclear outline. Triple examination: no thickening of the bilateral sacral cardinal ligaments. Cervical exfoliated cells were collected for laboratory testing.

Cervical exfoliated cell test: HPV negative, TCT negative, methylation test positive (RASSF1A positive ΔCt=7.3; SHOX2 negative; SEPTIN9 positive ΔCt=6.5).

Based on the positive methylation test, the patient underwent colposcopy and biopsy.

Biopsy tissue test: suspected adenocarcinoma, pathological morphology suspected endocervical adenocarcinoma, and cannot be well distinguished from endometrioid adenocarcinoma. Methylation test SEPTIN9 positive indicates non-endometrial adenocarcinoma, and immunohistochemical staining (vim ERp16 and monoclonal CEA) is used to distinguish endocervical cancer from endometrial cancer. The patient's sample showed positive expression of mCEA and p16, without vim and ER expression, which is consistent with the diagnosis of endocervical adenocarcinoma.

Cervical adenocarcinoma is a "rising star" of cervical cancer. Its incidence rate has reached 15-20% in cervical cancer. The cells where the lesions occur are different from those of squamous cell carcinoma. Cervical adenocarcinoma is more favored by young women in clinical practice. About 30% of the cases occur in women younger than 35%. Its clinical symptoms are hidden and lack specificity, making it difficult to be detected early. Compared with cervical squamous cell carcinoma, cervical adenocarcinoma is divided into HPV infection-dependent and HPV-independent types. The efficiency of HPV screening for cervical squamous cell carcinoma is significantly higher than that of cervical adenocarcinoma. Secondly, TCT is not sensitive to squamous epithelial lesions when diagnosing glandular epithelial lesions, which causes a considerable proportion of missed diagnosis of cervical adenocarcinoma in conventional HPV+TCT combined screening. Cervical adenocarcinoma is similar to endometrial glands during biopsy diagnosis and is also easy to misdiagnose. In summary, methylation detection of cervical exfoliated cells can be used as a supplement to HPV and TCT screening to prevent missed diagnosis of high-grade lesions and above.

Example 7 Clinical application of the kit 3: Application in differential diagnosis of endometrial atypical hyperplasia and endometrial cancer

Theoretically, atypical hyperplasia refers to morphological atypia of endometrial glandular cells. Because endometrial glandular cells show extremely active proliferation under the influence of estrogen in normal cycles, many morphological features overlapping with tumor conditions may appear under continuous estrogen stimulation; while in some well-differentiated endometrial cancers, only very slight cell atypia may be shown. Therefore, it is inevitable that there are large differences in the subjective judgment of atypicality, which is the primary difficulty in the diagnosis of atypical endometrial hyperplasia. Although there are difficulties in differential diagnosis between atypical endometrial hyperplasia and well-differentiated endometrial cancer, it is still the key basis for clinical selection of different treatment methods. Gene methylation detection has very good diagnostic specificity and sensitivity for endometrial cancer, and can be used in the differential diagnosis of atypical endometrial hyperplasia and endometrial cancer.

Twenty-five endometrial cancer specimens were clinically confirmed by surgery and/or small biopsy morphology, pathology and immunohistochemistry in a tertiary hospital in Shanghai. Among them, 10 cases were diagnosed with endometrial cancer by small biopsy morphology and pathology, 10 cases were diagnosed with atypical hyperplasia and the possibility of tumor was not ruled out, and 5 cases were diagnosed with no cancer cells in morphology. There were also 20 cases of benign lesions. All were paraffin-embedded samples. The results of three-gene methylation detection are recorded in Table 6.

Table 6 Methylation test results of endometrial paraffin tissue samples

The test results showed that no high methylation of SEPTIN9 was detected in all endometrial samples. The overall diagnostic sensitivity of RASSF1A combined with SHOX2 for endometrial cancer was 88% (22/25). The sensitivity of methylation detection in samples diagnosed as endometrial cancer by small biopsy was 100%, and the sensitivity of methylation detection in samples diagnosed as atypical hyperplasia was 90%. Among the five samples in which no cancer cells were found in pathology, methylation detection was positive in three cases. At the same time, the specificity of RASSF1A and SHOX2 combined diagnosis of endometrial cancer was 95%. The experimental results show that the methylation detection of RASSF1A and SHOX2 genes has very good diagnostic specificity and sensitivity for endometrial cancer, and can be used in the differential diagnosis of atypical endometrial hyperplasia and endometrial cancer.

Example 8 Clinical application of the kit 4: application in early screening and diagnosis of endometrial cancer

Endometrial cancer is one of the most common female reproductive system tumors. The common symptom of endometrial cancer is vaginal bleeding, but only 0.33% of women with abnormal uterine bleeding before menopause are eventually diagnosed with endometrial cancer, and 5%-10% of patients with vaginal bleeding after menopause are eventually diagnosed with endometrial cancer. Currently, the commonly used clinical detection methods, such as diagnostic curettage and hysteroscopic endometrial biopsy, are invasive operations, which bring great physical, psychological and economic burdens to patients. If a relatively non-invasive cytological sampling of patients is performed using a uterine cavity brush or a cervical brush, and then a high-sensitivity methylation test is performed, the screening sensitivity of endometrial cancer can be greatly improved. This study selected 150 patients suspected of endometrial lesions from a tertiary hospital in Shanghai who were to undergo diagnostic curettage or hysteroscopic endometrial biopsy. The final pathological diagnosis of 44 cases was endometrial cancer, and 106 cases were normal controls. Before biopsy, uterine cavity brush and cervical brush were used to collect cells, and the methylation of RASSF1A, SHOX2, and SEPTIN9 genes was detected. The diagnostic efficacy of methylation detection was calculated using the pathological results of endometrial biopsy as the diagnostic gold standard. All SEPTIN9 test results were negative, and the experimental test results are recorded in Table 7.

Table 7 Evaluation of the diagnostic efficacy of RASSF1A combined with SHOX2 for endometrial cancer under two different methodologies

The results of this study showed that the combined detection of dual gene methylation by uterine brush sampling for endometrial cancer had a sensitivity of 86.4%, a specificity of 94.2%, a positive predictive value of 86.4%, and a negative predictive value of 94.3%. At the same time, the results showed that the use of cervical brushes to collect cervical exfoliated cells can also achieve a certain screening efficiency, with a sensitivity of 65.9%, a specificity of 96.2%, a positive predictive value of 87.9%, and a negative predictive value of 87.2% for screening endometrial cancer.

Uterine brush sampling for DNAx methylation testing can be used as a non-invasive, highly accurate auxiliary diagnostic method for endometrial cancer, which is expected to reduce invasive endometrial biopsy operations, effectively improve patient compliance, and alleviate the shortage of medical resources.

Cervical cancer screening by collecting cervical exfoliated cells is very popular in clinical practice. Although the diagnostic sensitivity of cervical brush sampling for endometrial cancer is not as high as that of uterine cavity brush sampling, it is more convenient and has higher compliance. While screening for cervical cancer, RASSF1A/SHOX2/SEPTIN9 gene methylation testing can be used to screen for cervical squamous cell carcinoma, cervical adenocarcinoma and endometrial cancer at the same time. The positive combination of methylation also has certain differential diagnosis suggestions for the three tumors. The screening and diagnosis pathway is shown in Figure 3.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims (10)

1. Application of reagent for detecting RASSF1A, SHOX and SEPTIN9 gene methylation in preparation of kit for detecting cervical cancer and endometrial cancer.

2. The use according to claim 1, wherein the cervical cancer comprises cervical adenocarcinoma and cervical squamous carcinoma.

3. The use according to claim 1, wherein if either gene of RASSF1A, SHOX or SEPTIN9 of the sample is methylation positive, an increased likelihood of cervical or endometrial malignancy is indicated, suggesting that the patient is promptly subjected to further clinical examination;

If the sample RASSF1A gene methylation is negative, either the SHOX2 gene or the SEPTIN9 gene methylation is positive, the possibility that malignant lesions are cervical squamous carcinoma is increased, and the patient is suggested to be subjected to further clinical examination in time;

If the methylation of the RASSF1A gene of the sample is positive, and the methylation of any one of the SEPTIN9 and SHOX2 genes is positive, the possibility that malignant lesions are cervical adenocarcinoma or cervical squamous carcinoma with adenocarcinoma is increased, and the patient is suggested to be subjected to further clinical examination in time;

if the sample SEPTIN9 gene methylation is negative, either of SHOX2 and RASSF1A genes methylation is positive, suggesting that the likelihood of malignancy is endometrial cancer is increased, suggesting that the patient is receiving further clinical examination in time.

4. Use of a reagent for detecting RASSF1A, SHOX and SEPTIN9 gene methylation in the preparation of a kit for indicating risk of cervical squamous intraepithelial neoplasia and for guiding high-risk diversion.

5. The use according to claim 4, wherein the agent for detecting RASSF1A, SHOX and SEPTIN9 gene methylation in combination with human papillomavirus detection results is indicative of risk of cervical squamous intraepithelial neoplasia and directs high-risk pre-vaginoscope diversion of HPV-positive women;

if either gene methylation of RASSF1A, SHOX or SEPTIN9 is positive in the sample, colposcopy is recommended for women with positive HPV detection.

6. The application of the reagent for detecting RASSF1A, SHOX and SEPTIN9 genes in preparing a kit for detecting endometrial dysplasia and endometrial cancer.

7. The use according to any one of claims 1 to 6, wherein the sample detected comprises cervical detached cells, endometrial detached cells, hydrothorax, ascites, peritoneal irrigation fluid, cervical cancer patient tissue or endometrial cancer patient tissue of an organism.

8. The use according to any one of claims 1 to 6, wherein the reagent or kit comprises a primer set for detecting methylation of the RASSF1A gene, a primer set for detecting methylation of the SHOX2 gene and a primer set for detecting methylation of the SEPTIN9 gene;

The primer set for detecting RASSF1A gene methylation comprises: the nucleotide sequences of the forward primer, the reverse primer and the probe are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 7;

The primer set for detecting the methylation of the SHOX2 gene comprises: the nucleotide sequences of the forward primer, the reverse primer and the probe are respectively shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 8;

the primer set for detecting the methylation of the SEPTIN9 gene comprises the following components: the nucleotide sequences of the forward primer, the reverse primer and the probe are respectively shown as SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO. 9.

9. The use according to claim 8, wherein the probe comprises a fluorescence quenching group and a fluorescence reporting group at each end;

The fluorescence quenching group comprises BHQ1 or BHQ2;

the fluorescent reporter group comprises FAM, VIC, HEX or CY5.

10. The use according to claim 8, wherein the kit further comprises bisulphite, a primer set for detecting a reference gene and a PCR amplification reaction solution.