Methylation marker combination for early screening of endometrial cancer

Abstract

The invention discloses a methylation marker combination for early screening of endometrial cancer, and belongs to the cross technical field of molecular diagnosis and bioinformatics. Three methylation markers CDO1, NKX2-6 and SORCS3 are screened out through bioinformatics analysis, NGS targeted sequencing and qPCR verification, specific primers SEQ ID NO.1-2, 4-5 and 7-8 and probes SEQ ID NO.3, 6 and 9 of the methylation markers are designed, and a fluorescent quantitative PCR detection system is constructed in combination with a reference gene BACT. According to the method, the methylation level of a three-gene promoter region in a sample is detected, the cutoff value is 0.36, the sensitivity of endometrial cancer diagnosis of cast-off cells is 90.37%, the specificity is 89.60%, AUC is equal to 0.9682, and benign and cancerous samples can be effectively distinguished. According to the invention, noninvasive / minimally invasive early screening of endometrial cancer is realized, diagnosis accuracy and safety are improved, and NKX2-6 and SORCS3 are newly discovered markers and have important scientific value and clinical transformation potential.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of molecular diagnostics and bioinformatics, and particularly relates to a combination of methylation biomarkers for early screening of endometrial cancer. Background Technology

[0002] Endometrial carcinoma (EC), one of the three major malignant tumors of the female reproductive system, is experiencing a significant increase in its global disease burden. Over the past thirty years, new cases worldwide have increased by 132%, and the mortality rate has increased by 98%. This rapid increase is closely related to population aging, rising obesity rates, and the prevalence of metabolic syndrome.

[0003] Endometrial cancer is a common malignant tumor of the female reproductive system, and its early diagnosis is closely related to improved prognosis. Statistics show that the 5-year survival rate for stage I patients can reach over 95%, while the survival rate drops sharply to 17%-45% when it progresses to stages III-IV. This significant difference in survival highlights the clinical value of accurate early diagnostic techniques.

[0004] Currently, clinical diagnostic procedures primarily rely on a combination of imaging and invasive examinations. Transvaginal ultrasound (TUV) is the preferred method for early screening and diagnosis. However, as a first-line screening method, its diagnostic efficacy is significantly hampered by physiological hyperplasia. A large cohort of over 1,000 patients showed that when endometrial thickness ≥5 mm was used as the diagnostic threshold for postmenopausal women, although the sensitivity was as high as 96.2% and the negative predictive value was 99.3%, the specificity was only 51.5%, resulting in nearly half of the patients requiring unnecessary invasive examinations. Hysteroscopy can directly sample suspicious lesions and is suitable for patients with positive TUV tests or recurrent symptoms, making it the gold standard for diagnosing endometrial cancer. Information obtained from endometrial biopsies is often used for preoperative disease staging, which is crucial for surgical management and guiding the scope of surgery. However, hysteroscopy also increases the risk of cancer spread, and discomfort and false negative results are also drawbacks of this method. Therefore, we need to develop safer, more efficient, and reliable screening and early diagnostic methods in high-risk populations to reduce the mortality rate of endometrial cancer and improve clinical outcomes.

[0005] In recent years, breakthroughs in molecular diagnostic technologies and the application of DNA methylation in early cancer diagnosis have provided new directions for the early detection of endometrial cancer. Abnormal DNA methylation mainly occurs in CpG islands in gene promoter regions, participating in tumorigenesis and development by regulating the expression of key tumor genes. As a stable epigenetic modification, DNA methylation shows characteristic changes in the early stages of endometrial cancer, making it an ideal early marker.

[0006] Currently, there are various methods for detecting DNA methylation, including whole-genome methylation detection technologies (WGBS, RRBS, Illumina EPIC Bead Chip microarray, MeDIP-seq, MethylRAD) and site-specific methylation detection technologies (pyrosequencing, Massarray, BSP, MSP). These mature detection technologies provide strong support for large-scale screening and validation of methylation biomarkers. Therefore, identifying and optimizing a set of DNA methylation biomarkers that exhibit high sensitivity and specificity in early endometrial cancer (EC) is of vital importance for developing efficient non-invasive or minimally invasive early screening methods for endometrial cancer, achieving accurate diagnosis, and improving patient prognosis. Summary of the Invention

[0007] This invention screened 184,915 differentially methylated sites (DMPs) and 1,215 differentially methylated regions (DMRs) through bioinformatics analysis. After defining DMPs and DMRs and annotating them, 145 promoter differentially methylated genes were identified. Finally, based on GO and KEGG enrichment analysis, 10 genes that may be associated with the progression of endometrial cancer and show high methylation levels were selected.

[0008] Methylation-specific primer sets were designed for the CpG islands in the promoter regions of the aforementioned 10 genes using the Ion AmpliSeq Designer platform. High-throughput sequencing was used to perform targeted sequencing on 50 benign endometrial samples and 50 endometrial cancer samples. Data analysis showed that five genes—CDO1, TRH, PENK, NKX2-6, and SORCS3—were at a low methylation level in benign endometrial samples, with an average methylation frequency below 5%; while they were at a high methylation level in endometrial cancer samples, with an average methylation frequency above 20%. This significant difference in methylation levels effectively differentiated between benign and endometrial cancer samples.

[0009] Primers and probes for the methylation sites of the five candidate genes were designed using Primer Express software. The internal control gene BACT was introduced as a quality control measure for sample quality and experimental procedures. The detection performance of the primers and probes was evaluated using 50 benign endometrial samples and 50 endometrial cancer samples. The results showed that the CDO1, NKX2-6, and SORCS3 genes could effectively distinguish between benign and endometrial cancer samples, and could serve as biomarkers for early screening of endometrial cancer, potentially for the development of methylation detection kits. Accuracy validation using clinical samples showed that the combined detection of the CDO1, NKX2-6, and SORCS3 genes performed optimally: at a cutoff value of 0.36, the sensitivity was 90.37% (95% C1: 85.98%, 93.99%), the specificity was 89.60% (95% C1: 85.20%, 92.80%), and the AUC was 0.9682. The corresponding primer and probe sequences are as follows: Table 1

[0010] In the primer set, SEQ ID NO. 1-2 are primer sequences for CDO1 methylation detection, SEQ ID NO. 3 is a probe sequence for CDO1 methylation detection, SEQ ID NO. 4-5 are primer sequences for NKX2-6 methylation detection, SEQ ID NO. 6 is a probe sequence for NKX2-6 methylation detection, SEQ ID NO. 7-8 are primer sequences for SORCS3 methylation detection, SEQ ID NO. 9 is a probe sequence for SORCS3 methylation detection, SEQ ID NO. 10-11 are primer sequences for BACT methylation detection (internal reference gene), and SEQ ID NO. 12 is a probe sequence for BACT methylation detection (internal reference gene).

[0011] Furthermore, the probe sequence is labeled with a fluorescent reporter group at the 5' end and a fluorescent quencher group at the 3' end. Specifically, the probes of the sequences shown in SEQ ID NO. 3, SEQ ID NO. 6, and SEQ ID NO. 9 are labeled with the fluorescent reporter group FAM at the 5' end and the fluorescent quencher group MGB at the 3' end; the probe of the sequence shown in SEQ ID NO. 12 is labeled with the fluorescent reporter group HEX at the 5' end and the fluorescent quencher group MGB at the 3' end.

[0012] Compared with the prior art, the present invention has the following beneficial effects: (1) Through bioinformatics analysis, NGS targeted sequencing screening, and qPCR detection verification, this invention screened and confirmed three methylation markers closely related to the progression of endometrial cancer: CDO1, NKX2-6, and SORCS3, from 10 candidate genes. Among them, NKX2-6 and SORCS3 are newly discovered methylation markers related to endometrial cancer in this invention, which have important scientific value and diagnostic potential.

[0013] (2) The present invention provides a methylation detection kit for early screening of endometrial cancer. The kit is based on real-time PCR technology. By jointly detecting key methylation sites in the promoter regions of three marker genes, CDO1, NKX2-6 and SORCS3, it can effectively improve the sensitivity of endometrial cancer detection and has high specificity, thus realizing early screening of endometrial cancer. Attached Figure Description

[0014] Figure 1 This shows the methylation frequency and relative gene location of some of the CpG sites in the selected genes in Example 1.

[0015] Figure 2 The results are from the q-PCR tissue sample test in Example 3.

[0016] Figure 3 The image shows the EDM fraction distribution and ROC curve in the exfoliated cell sample from the uterine cavity in Example 4. Detailed Implementation

[0017] Example 1 Methylation data from 33 endometrial cancer tissues and adjacent normal tissues were obtained from the TCGA database. PCA results showed significant differences between tumor and normal tissues, potentially reflecting variations in gene expression, methylation patterns, or other biological characteristics. In this study, we defined DMPs as CpG sites with a q-value < 0.05 and DMRs as regions containing ≥ 4 DMPs. A total of 184,915 DMPs and 1,215 DMRs were identified. During screening, we limited DMPs to those located within the TSS1500 region of gene promoter regions and within CpG islands. After screening and annotation, 145 gene promoter regions were identified as meeting these methylation criteria.

[0018] Subsequently, based on the relevant etiologies of endometrial cancer (such as the effects of estrogen and progesterone, cell proliferation, etc.) and currently trending genes, the study further screened 10 potential target genes, namely PENK, GHSR, NKAPL, PON3, SORCS3, NKX2-6, SPAG6, TRH, ZNF177, and CDO1. DMP methylation information for these genes can be found in [link to relevant documentation]. Figure 1 .

[0019] Example 2 This embodiment uses NGS technology to detect multiple methylation sites in the promoters of 10 bioinformatics markers obtained from bioinformatics analysis, providing a reference for subsequent qPCR screening. The specific steps are as follows: 1. Primer design Methylation NGS primers for the CpG islands of the promoters of the CDO1, GHSR, NKX2-6, PENK, PON3, SORCS3, TRH, NKAPL, ZNF177, and SPAG6 genes were designed using the Ion AmpliSeq Designer website.

[0020] Table 2

[0021] 2. DNA Extraction: One hundred uterine cavity exfoliated cell samples with known pathological diagnoses were selected, including 50 benign endometrial samples and 50 endometrial cancer samples. DNA was extracted from all 100 samples using a DNA extraction kit. The concentration and quality of the extracted DNA were determined using a micro-spectrophotometer, requiring a total DNA content ≥ 2000 ng and an OD value of [missing value]. 260 / OD 280 Between 1.7 and 2.2.

[0022] 3. Sample methylation treatment: 100 DNA samples were methylated using a methylation modification kit (genomic DNA bisulfite modification reagent, Fujian Xiamen Medical Device Registration No. 20200141) to obtain modified bisDNA products.

[0023] 4. Library enrichment reaction The designed primers were diluted to 200 μM using nuclease-free water, and the primers were mixed and diluted to 10 μM in equal volumes according to the formulas in Tables 3-6. Then, the enrichment reaction systems of pool1 and pool2 were prepared according to the formulas in Table 7.

[0024] Table 3 Pool1 Primer Mix1 Formulation

[0025] Table 4. Pool1 Primer Mix2 Formulation

[0026] Table 5 Pool2 Primer Mix1 Formulation

[0027] Table 6 Pool2 Primer Mix2 Formulation

[0028] Table 7 Formulation of enrichment reaction system

[0029] Note: The reaction systems in pool1 and pool2 are respectively mixed with Mix1 and Mix2.

[0030] Add 5 μL of modified bisDNA sample to each of the prepared reaction systems pool1 and pool2, and place them on a miniAMP PCR instrument for PCR reaction. The reaction program is: 98℃ for 2 min, (98℃ for 15 s, 65℃ for 4 min) × 15 cycles, and store at 4℃.

[0031] 5. Purification of enriched products 1) Mix the enriched products from pool1 and pool2 completely, transfer them to a new 1.5 mL centrifuge tube, add 20 μL of nuclease-free water, add 50 μL of magnetic beads and mix by pipetting, and incubate at room temperature for 5 min. 2) Incubate on a magnetic rack for 2 minutes until the solution is clear. Carefully aspirate and discard the supernatant, being careful not to disturb the magnetic beads. Note: The magnetic beads contain the amplification library, so do not discard them. 3) Add 150 µL of freshly prepared 70% ethanol solution, place the centrifuge tube on a magnetic rack, rotate it clockwise / counterclockwise 5 times, incubate on the magnetic rack for 2 minutes until the solution is clear, and discard the supernatant; 4) Repeat step 3) above to perform a second wash; 5) Ensure that the ethanol solution in the centrifuge tube has been completely discarded, place the centrifuge tube on a magnetic rack, and let it air dry at room temperature for 5 minutes, taking care to avoid over-drying; 6) Remove the centrifuge tube from the magnetic rack, add 35 μL of TE (pH 8.0) buffer to fully wet the magnetic beads, shake well to mix, and then centrifuge quickly to collect the liquid to the bottom of the tube (or use a pipette to aspirate more than half of the liquid and repeatedly blow up and down the liquid to mix it thoroughly), and incubate at room temperature for 5 minutes. 7) Place the centrifuge tube on a magnetic rack and let it stand for 2 minutes until the solution is clear. Take out the supernatant, which is the library. Store at -20±5℃ or proceed with subsequent operations.

[0032] 6. Library preparation reaction Take 5 μL of the purified product and add it to different UDI reaction strips. Place them on a miniAMP PCR instrument for PCR reaction. The reaction program is: 98℃ for 2 min, (98℃ for 15 s, 65℃ for 4 min) × 25 cycles, and store at 4℃.

[0033] 7. Purification of library products 1) Transfer the library product to a new 1.5 mL centrifuge tube, add 10 μL of nuclease-free water, then add 25 μL of magnetic beads and mix by pipetting. Incubate at room temperature for 5 min. 2) Incubate on a magnetic rack for 2 minutes until the solution is clear. Carefully aspirate and discard the supernatant, being careful not to disturb the magnetic beads. Note: The magnetic beads contain the amplification library, so do not discard them. 3) Add 150 µL of freshly prepared 70% ethanol solution, place the centrifuge tube on a magnetic rack, rotate it clockwise / counterclockwise 5 times, incubate on the magnetic rack for 2 minutes until the solution is clear, and discard the supernatant; 4) Repeat step 3) above to perform a second wash; 5) Ensure that the ethanol solution in the centrifuge tube has been completely discarded, place the centrifuge tube on a magnetic rack, and let it air dry at room temperature for 5 minutes, taking care to avoid over-drying; 6) Remove the centrifuge tube from the magnetic rack, add 35 μL of TE (pH 8.0) buffer to fully wet the magnetic beads, shake well to mix, and then centrifuge quickly to collect the liquid to the bottom of the tube (or use a pipette to aspirate more than half of the liquid and repeatedly blow up and down the liquid to mix it thoroughly), and incubate at room temperature for 5 minutes. 7) Place the centrifuge tube on a magnetic rack and let it stand for 2 minutes until the solution is clear. Take out the supernatant, which is the library. Store at -20±5℃ or proceed with subsequent operations.

[0034] 8. Sequencing The purified library was subjected to fragment quality control using a capillary electrophoresis instrument. The main fragments of the library should be distributed in the range of 200-350 bp. The fragments were then detected using the BGI G99 sequencer.

[0035] 9. Experimental Results The sequencing results were analyzed and statistically analyzed to calculate the methylation level of each CpG site, and then the average methylation frequency of each gene was calculated. The methylation status of 10 candidate genes in benign endometrial samples and endometrial cancer samples was statistically analyzed. It was found that five genes—CDO1, TRH, PENK, NKX2-6, and SORCS3—were at a low methylation level in benign endometrial samples, with an average methylation frequency below 5%; while in endometrial cancer samples they were at a high methylation level, with an average methylation frequency above 20%. This significant difference in methylation levels indicates that CDO1, TRH, PENK, NKX2-6, and SORCS3 have greater potential for endometrial cancer screening.

[0036] Table 8 Diagnostic efficacy of methylation frequency at specific sites of the target gene

[0037] Example 3 This embodiment uses qPCR technology to further confirm and screen five biomarkers after NGS initial screening, mainly including the following steps: 1. Primer and Probe Design: Based on the NGS detection results in Example 2, five methylation markers—CDO1, TRH, PENK, NKX2-6, and SORCS3—were selected. Primer and probe designs for methylation level detection in the promoter regions of these five genes were used using Primer Express software. An internal control, BACT, was also introduced. In the designed primer sequences, SEQ ID NOs 1-3 are the primer and probe sequences for CDO1, SEQ ID NOs 4-6 are the primer and probe sequences for NKX2-6, SEQ ID NOs 7-9 are the primer and probe sequences for SORCS3, and SEQ ID NOs 10-12 are the primer and probe sequences for the internal control, BACT. The primer and probe sequences for TRH and PENK are as follows: Table 9

[0038] 2. In the primers, SEQ ID NO. 59-60 are TRH methylation detection sequences, and SEQ ID NO. 61 is a TRH methylation detection probe sequence; SEQ ID NO. 62-63 are PENK methylation detection sequences, and SEQ ID NO. 64 is a PENK methylation detection probe sequence. The probe sequences SEQ ID NO. 61 and SEQ ID NO. 64 are labeled with a fluorescent reporter group FAM at the 5' end and a fluorescent quencher group MGB at the 3' end.

[0039] 3. Another 50 benign endometrial samples and 50 endometrial cancer samples were selected and DNA was extracted and methylated according to the method in Example 2 to obtain modified bisDNA products.

[0040] 4. Preparation of reaction solution Prepare qPCR reaction solutions for the five markers according to the following formulas.

[0041] Table 10 qPCR reaction solution formulation

[0042] Note: "F (marker)" represents the upstream primer of one of the five markers: CDO1, TRH, PENK, NKX2-6, and SORCS3; "R (marker)" represents the downstream primer of one of the five markers; and "P (marker)" represents the probe of one of the five markers.

[0043] 5. On-machine testing Add 5 μL of the modified bisDNA sample to each of the above 5 reaction solutions, and perform qPCR detection on the Hongshi instrument. The reaction program is: 95℃ for 5 min, (95℃ for 15 s, 60℃ for 1 min to collect fluorescence) × 40 cycles.

[0044] 6. Result Interpretation The internal reference BACT should show an amplification curve rise in the VIC (HEX) channel, and the signal Ct value should be <30.

[0045] The ΔCt values ​​of the five genes are calculated using the following formula: ΔCt = Ct(FAM) - Ct(VIC).

[0046] 7. Results Analysis The ΔCt values ​​for the five genes in 100 samples were calculated separately. Data were statistically analyzed in two groups: benign samples and endometrial cancer samples, and the average ΔCt value for each group was calculated. Figure 2 The experimental results showed that the average ΔCt values ​​of different groups of TRH and PENK genes were small, and they could not effectively distinguish between benign samples and endometrial cancer samples. In contrast, CDO1, NKX2-6 and SORCS3 genes could better distinguish between benign samples and endometrial cancer samples, and could be used as biomarkers for early screening of endometrial cancer and for the development of methylation detection kits.

[0047] Example 4 This embodiment is mainly used to test the performance of the CDO1, NKX2-6, and SORCS3 three-gene combined methylation detection kit provided by this invention. Four different types of exfoliated cells from the uterine cavity were selected as test samples, including 175 samples of normal endometrium and 75 samples of benign endometrial hyperplasia, 36 samples of atypical endometrial hyperplasia, and 169 samples of endometrial cancer, totaling 455 samples.

[0048] 1. The sample was pretreated according to the DNA extraction and methylation modification method in Example 2 to obtain the modified bisDNA sample.

[0049] 2. Prepare three qPCR reaction solutions, CDO1, NKX2-6 and SORCS3, according to the formula in Table 10 of Example 3. Add 5 μL of modified bisDNA sample to each of the three reaction solutions and place them on the Hongshi instrument for qPCR detection. The reaction program is: 95℃ for 5 min, (95℃ for 15 s, 60℃ for 1 min to collect fluorescence) × 40 cycles.

[0050] 3. Results Analysis Based on the sample diagnostic results and the ΔCt value data, a multi-gene combined detection logistic regression diagnostic model was constructed, and the cutoff value of the model was determined to be 0.36. The test results showed that the cancer group + atypical hyperplasia group was considered positive, and the low-risk group (normal endometrial samples + benign endometrial hyperplasia samples) was considered negative. Compared with the clinical reference standard, the clinical sensitivity of this invention was 90.37% (95% C1: 85.98%, 93.99%), the clinical specificity was 89.60% (95% C1: 85.20%, 92.80%), and the AUC was 0.9682. Figure 3 (The sensitivity was 72.22% for atypical hyperplasia cases.)

[0051] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A combination of methylation biomarkers for early screening of endometrial cancer, characterized in that, The biomarker combination consists of the CDO1, NKX2-6, and SORCS3 genes.

2. A primer and probe for detecting the methylation marker combination of claim 1, characterized in that, The primer and probe sequences for detecting the methylation of the CDO1 gene are shown in SEQ ID NO. 1-3, the primer and probe sequences for detecting the methylation of the NKX2-6 gene are shown in SEQ ID NO. 4-6, and the primer and probe sequences for detecting the methylation of the SORCS3 gene are shown in SEQ ID NO. 7-9.

3. The primers and probes according to claim 2, characterized in that, The probes of SEQ ID NO.3, SEQ ID NO.6, and SEQ ID NO.9 are labeled with a FAM fluorescent reporter group at their 5' end and an MGB quencher group at their 3' end.

4. The use of the primers and probes according to claim 2 or 3 in the preparation of reagents or kits for early screening of endometrial cancer.

5. A reagent or kit for early screening of endometrial cancer, characterized in that, The reagent or kit contains the primers and probes as described in claim 2 or 3.

6. The reagent or kit according to claim 5, characterized in that, The reagent or kit also contains primers and probes for detecting the internal reference gene BACT, and the sequences of the primers and probes for detecting the internal reference gene BACT are shown in SEQ ID NO.10~12.

7. The reagent or kit according to claim 6, characterized in that, The reagent or kit also contains bisulfite.

8. The reagent or kit according to claim 7, characterized in that, The bisulfite is sodium bisulfite.

9. A system for diagnosing endometrial cancer, characterized in that, The system is a computational device for obtaining a diagnostic conclusion based on the detection results of the methylation marker of claim 1 in a subject sample, the system comprising: (1) A sample collection and processing device for performing the following steps: collecting samples from subjects and processing the samples; (2) Methylation detection device; (3) A computing device for obtaining a diagnostic conclusion based on the detection results of methylation markers in subject samples.

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