Product and method for detecting urothelial carcinoma based on DNA methylation

Abstract

The invention provides a product and a method for detecting urinary tract epithelial carcinoma based on DNA methylation, and belongs to the technical field of urinary tract epithelial carcinoma detection, the product comprises reagents for detecting methylation of the following DNA regions: a PENK gene CpG island region Chr8: 56446618-56446724, a TMEM106A gene CpG island region Chr17: 43211685-43211847, and an OTX1 gene CpG island region Chr2: 63056808-63056956. Through joint detection of the target gene areas, accurate prediction of urothelial carcinoma is realized, and the kit has comprehensive coverage (bladder cancer, pelvis cancer and ureteral cancer) and detection advantages of early and low-grade cancers, can be used for early screening, postoperative recurrence monitoring, prognosis evaluation and other stages, and has a wide application prospect in the fields of early screening, postoperative recurrence monitoring, prognosis evaluation and the like. The positive significance is realized on the whole disease course management of the urothelial carcinoma.

Description

Technical Field

[0001] This invention relates to the field of urothelial carcinoma detection technology, specifically to a product and method for detecting urothelial carcinoma based on DNA methylation. Background Technology

[0002] Urothelial carcinoma (UC) is one of the most common malignant tumors of the urinary system, including upper urinary tract urothelial carcinoma and bladder urothelial carcinoma. Upper urinary tract urothelial carcinoma is further subdivided into renal pelvis carcinoma and ureteral carcinoma. Because the clinical manifestations of UC are often nonspecific, approximately 15-25% of patients already have muscle layer invasion or distant metastasis at initial diagnosis, resulting in an overall 5-year survival rate of less than 30%. While early-stage (non-muscle layer invasion) patients can achieve good short-term efficacy with transurethral resection of bladder tumor (TURP), the recurrence rate within 5 years is as high as 50-70%, with 10-20% progressing to muscle layer invasion. Therefore, establishing a highly sensitive, highly specific, and early-detection non-invasive or minimally invasive method for dynamically monitoring the risk of recurrence and progression is crucial for improving the prognosis of urothelial carcinoma.

[0003] Currently, cystoscopy and urine cytology remain the gold standard for early screening and recurrence monitoring of urothelial carcinoma in clinical practice. While cystoscopy has high sensitivity, it is an invasive procedure with poor patient compliance and high cost. Urine cytology is non-invasive, but its sensitivity is low (especially for low-grade tumors) and is affected by factors such as inflammation and stones. In recent years, the US FDA has approved several urine molecular diagnostic products, including UroVysion (FISH), BTA-stat, NMP22, and UroSEEK (based on TERT mutation and copy number), for auxiliary diagnosis. However, these methods have limitations such as single detection targets, insufficient sensitivity / specificity, or complex experimental procedures, and have not yet achieved widespread adoption. Therefore, there is an urgent need to find new biomarkers that are more stable, easier to standardize, and cover the entire course of urothelial carcinoma.

[0004] Epigenetic alterations, particularly abnormal DNA methylation, are among the early events in tumorigenesis and development, characterized by their heritability, reversibility, and ease of detection in bodily fluids. Previous studies have shown that hypermethylation of promoter regions of multiple genes, including CDKN2A, RASSF1A, APC, SOX1, and TWIST1, occurs frequently in ulcerative colitis (UC) tissues and urine, and is associated with tumor stage, grade, and prognosis. However, the sensitivity and specificity of single methylation markers are limited, failing to meet clinical needs; significant discrepancies exist between reported studies, and a unified, reproducible detection panel is lacking.

[0005] With the development of technologies such as high-throughput methylation capture sequencing (MC-seq), whole-genome bisulfite sequencing (WGBS), and 850K / 935K methylation chips, researchers can now obtain whole-genome methylation maps of UC at single-base resolution. Further analysis reveals that DNA methylation abnormalities not only involve promoter regions but are also widely present in enhancers, insulators, gene bodies, and open chromatin regions, making their regulatory network far more complex than traditionally understood. Nevertheless, how to screen and validate combinations of methylation biomarkers with clinical translational potential from massive datasets, and how to establish a standardized, quality-controlled, and scalable detection system, remain pressing technical bottlenecks that need to be addressed.

[0006] In summary, there is an urgent need in this field to systematically identify DNA methylation sites closely related to tumor occurrence, progression, and recurrence in clinical cohorts of urothelial carcinoma; and to develop a combined biomarker and supporting detection method covering key methylation regions to achieve non-invasive or minimally invasive early screening, postoperative recurrence monitoring, and prognostic assessment, ultimately improving the clinical management of urothelial carcinoma. Summary of the Invention

[0007] In view of the shortcomings of existing non-invasive detection technologies for urothelial carcinoma, such as insufficient sensitivity, limited specificity, single biomarker, and difficulty in dynamic monitoring, this invention aims to provide a product and method for detecting urothelial carcinoma based on DNA methylation, so as to achieve high sensitivity, high specificity, non-invasive or minimally invasive, and standardized full-course management of UC (i.e., able to meet all needs of early screening, postoperative recurrence monitoring, and prognostic assessment).

[0008] The specific technical solution of the present invention is as follows: In a first aspect, the present invention provides a composition for detecting urothelial carcinoma based on DNA methylation, comprising a reagent for detecting methylation of the following DNA regions: PINK CpG island region Chr8: 56446618-56446724; TMEM106A CpG island region of gene Chr17:43211685-43211847; OTX1 Gene CpG island region Chr2: 63056808-63056956.

[0009] Preferably, the composition comprises the following primers and probes: Detection PINK The primers and probes for the CpG island region Chr8: 56446618-56446724 of the gene are shown in SEQ ID NO.1-3, respectively. Detection TMEM106AThe primers and probes for the CpG island region Chr17:43211685-43211847 of the gene are shown in SEQ ID NO.4-6, respectively. Detection OTX1 The primers and probes for the CpG island region Chr2: 63056808-63056956 of the gene are shown in SEQ ID NO.7-9, respectively.

[0010] In a second aspect, the present invention provides the use of the above composition in the preparation of a kit for detecting urothelial carcinoma, wherein urothelial carcinoma includes bladder cancer, ureteral cancer and renal pelvis cancer.

[0011] Thirdly, the present invention provides a kit for detecting urothelial carcinoma, which, in addition to the composition described above, may also include primers and probes for detecting internal reference genes, DNA extraction reagents, bisulfite conversion reagents, and related reagents for PCR reactions.

[0012] In some embodiments of the present invention, the internal reference gene is ACTB, and the sequences of the primers and probes used to detect the internal reference gene are shown in SEQ ID NO.10-12, respectively.

[0013] It is understood that all probes in the above kit have luminescent and quenching groups attached to their 5' and 3' ends, respectively, and those skilled in the art can attach appropriate luminescent and quenching groups to each probe according to actual needs.

[0014] Fourthly, the compositions and kits provided by this invention are suitable for detecting the following in vitro samples: including but not limited to urine, feces, blood, saliva, or other bodily fluids. Based on the determination of the methylation positivity or antagonism of the in vitro samples according to the test results, the risk of urothelial carcinoma can be predicted.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) High accuracy, with sensitivity and specificity both above 90%, especially effective in detecting early (Ta stage) and low-grade urothelial carcinoma.

[0016] (2) Comprehensive coverage: It is applicable not only to bladder cancer, but also to upper urothelial carcinomas such as renal pelvis cancer and ureteral cancer. Clinically, the diagnosis of upper urothelial carcinoma is more challenging. On the one hand, urine cytology is less sensitive for upper urothelial carcinoma than for bladder cancer; on the other hand, ureteroscopy is prone to tumor implantation, which severely limits its use in the diagnosis of upper urothelial carcinoma. This invention provides a non-invasive and highly accurate alternative and supplementary method for upper urothelial carcinoma.

[0017] (3) Technical accessibility: After collecting 20-30 ml of urine and mixing it with urine preservation solution, only 1 ml of the mixture needs to be taken for DNA extraction. The detection cycle is ≤6 hours, which is suitable for promotion in primary hospitals. Attached Figure Description

[0018] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly described below. Obviously, the drawings described below are merely some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0019] Figure 1 This is a comparison of the Delta Ct values ​​of the PENK, TMEM106A, and OTX1 genes in urothelial carcinoma and interference samples in Example 4. Figure 2 The ROC curves are for the individual and combined detection of the PENK, TMEM106A and OTX1 genes in Example 4. Detailed Implementation

[0020] The technical solution of the present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the invention.

[0022] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0023] Example 1 This example provides a primer and probe composition for detecting urothelial carcinoma based on DNA methylation, comprising primers and probes that specifically detect methylation in the following regions: PINK CpG island region Chr8: 56446618-56446724; TMEM106A CpG island region of gene Chr17:43211685-43211847; OTX1 Gene CpG island region Chr2: 63056808-63056956.

[0024] In this example, the composition also includes a reference gene for detection. ACTB The primers and probes are as follows. Specifically, the sequences of each primer and probe are shown in Table 1.

[0025] Table 1. Sequence information of primers and probes

[0026] The probe has a luminescent group and a quencher group attached to its 5' and 3' ends, respectively, and the appropriate luminescent and quencher groups can be selected according to actual needs. For example, for the probe described above, PINK The luminescent and quenching groups of the probe can be CY5 and BHQ1, respectively. TMEM106A The luminescent and quenching groups of the probe can be ROX and BHQ2, respectively. OTX1 The luminescent and quenching groups of the probe can be FAM and BHQ3, respectively. ACTB The luminescent and quenching groups of the probe can be VIC and BHQ3, respectively.

[0027] Example 2 Based on the primer and probe composition provided in Example 1, this example provides a kit for detecting urothelial carcinoma, which, in addition to containing the composition in Example 1, also includes at least the following reagents: DNA extraction reagents are used to extract DNA from samples to be tested.

[0028] Bisulfite conversion reagent is used to convert unmethylated cytosine in a DNA sequence into uracil (in this conversion process, methylated cytosine remains unchanged); after bisulfite conversion, methylated DNA and unmethylated DNA are converted into very different sequences.

[0029] PCR reaction-related reagents, including PCR buffers, dNTPs, DNA polymerase, etc., are used to prepare PCR reaction systems containing the DNA to be tested, primers, and probes, enabling the specific detection of methylated DNA.

[0030] Example 3 Based on the primer and probe composition provided in Example 1, this example uses a urine sample to provide a method for predicting urothelial carcinoma, which includes the following steps: (1) Collection and processing of urine samples.

[0031] When collecting urine samples, 20 mL to 30 mL of fresh midstream urine should be collected (it must be the first or second urine after waking up on the same day; women should avoid sampling during their menstrual period).

[0032] Urine samples can be further preserved using urine sample preservation solution (catalog number AA18, containing preservatives) from Wuhan Aimeisen Life Technology Co., Ltd., preferably mixed at a volume ratio of 10:1. After mixing the urine and preservation solution, DNA can be extracted directly from the mixture without centrifugation.

[0033] (2) DNA extraction and bisulfite conversion.

[0034] Extract DNA from urine samples and perform bisulfite conversion. Specifically, use the Urine DNA Extraction and Bisulfite Conversion Kit from Wuhan Aimeisen Life Science Co., Ltd. (Wuhan, China, Catalog No. AA19). First, extract cellular genomic DNA and circulating cell-free DNA from a mixture of 1 ml of urine and preservation solution, then perform bisulfite conversion. In the final elution step of bisulfite conversion, use 30 μL of elution buffer.

[0035] (3) Methylation-specific PCR and result analysis.

[0036] Prepare the qPCR reaction system according to Table 2 and perform the detection according to the procedure shown in Table 3. After the qPCR reaction is complete, the baseline and appropriate threshold need to be manually adjusted. The baseline is usually the fluorescence signal of 3-15 cycles. First, confirm that the baseline fluorescence curve of each channel is stable, without drift or abnormal rise. If drift is found, manually advance the baseline termination cycle by 1-2 cycles until the curve is level. For samples with standard amplification curves, the automatic preset threshold set by the PCR instrument manufacturer can generally be used. The automatic threshold is generally set at 10% of the maximum fluorescence intensity.

[0037] Table 2. Methylation-Specific PCR Reaction System

[0038] Table 3 TaqMan PCR Program

[0039] It is important to note that positive and negative controls should be measured simultaneously with each clinical sample test to ensure the accuracy of the PCR testing process. In this invention, the positive control may use a mixture containing 10... 3 copies / μL containing ACTB plasmids containing amplicon sequences, 10 3 copies / μL containing PINK plasmids containing amplicon sequences, 10 3 copies / μL containing TMEM106A plasmids containing amplicon sequences, 10 3 copies / μL containing OTX1The plasmids containing the amplicon sequences are mixed in equal volumes. All plasmids are constructed using pMD-18T plasmid as the base vector. The construction method can be referred to existing technology and will not be described in detail here. Purified water can be used as the negative control.

[0040] To analyze the results of the qPCR reaction, the following requirements must be met: ① No amplification in the negative control PCR tube; ② A clear exponential growth phase in the positive control PCR tube, with Ct values ​​for both the internal reference gene and the target gene between 26 and 30; ③ A Ct value of the internal reference gene in the test sample less than or equal to 33. If the positive control, negative control, and internal reference gene all meet the above requirements, the test results of the test sample can be analyzed and interpreted. Otherwise, the experiment is invalid and must be repeated.

[0041] Receiver operating curve (ROC) analysis: The difference between the Ct values ​​of the target gene and the internal reference gene (Delta Ct, ∆Ct) was used as an indicator for ROC analysis. First, the Delta Ct values ​​of the three genes were integrated using a logistic binary regression model to output probability values. ROC analysis was performed on urothelial carcinoma samples and interference samples using probability values. The value at which the Youden index was maximized was taken as the cut-off value to analyze the sensitivity and specificity at the maximum Youden index.

[0042] Example 4 In this example, urine samples from two types of patients were used to verify the performance of the detection scheme of the present invention. One type was urine samples from patients with pathologically confirmed urothelial carcinoma (a total of 73 cases, details of which are shown in Table 4), including bladder cancer, ureteral cancer and renal pelvis cancer. The other type was urine samples from patients with interfering diseases confirmed by comprehensive clinical diagnostic criteria (a total of 97 cases), including benign diseases of the urinary system (such as inflammation, hyperplasia, stones, etc.) and benign tumors of the urothelial tract.

[0043] Table 4 Clinical information of urothelial carcinoma samples

[0044] Following the method in Example 3, the above-mentioned urothelial carcinoma and interfering samples were used as training set samples. DNA extraction, bisulfite conversion, and methylation-specific PCR were performed according to the method in Example 2, and the samples were analyzed respectively. PINK , TMEM106A and OTX1 The distribution of Ct values, area under the ROC curve (AUC value), cutoff value, sensitivity, and specificity of the three target gene regions in different samples.

[0045] PINK , TMEM106A and OTX1 Ct values ​​of the gene in urothelial carcinoma samples and interfering samples, as shown Figure 1 As shown, the Delta Ct values ​​of the three target gene regions differed significantly in the cancer samples (P < 0.0001, ****). This result indicates that the methylation levels of the three target gene regions have the potential to distinguish urothelial carcinoma samples from interfering samples.

[0046] In addition, referring to the method in Example 3, the effect of individual detection of the three target gene regions PENK, TMEM106A and OTX1 was analyzed (for individual detection, ROC analysis of Delta Ct of urothelial carcinoma sample and interference sample can be performed directly).

[0047] ROC curves obtained from individual or combined detection are as follows: Figure 2 As shown, from Figure 2 It can be known that: PINK When the gene region is used alone as a biomarker, the AUC value is 0.9057 (95% confidence interval: 0.8475-0.9630), the cutoff value at the maximum Youden index is delta Ct=12.3, the sensitivity at the maximum Youden index is 87.67%, and the specificity is 94.85%. TMEM106A When the gene region is used alone as a biomarker, the AUC value is 0.8937 (95% confidence interval: 0.8398-0.9476), the cutoff value at the maximum Youden index is delta Ct=11.41, the sensitivity at the maximum Youden index is 78.08%, and the specificity is 96.91%. OTX1 When the gene region is used alone as a biomarker, the AUC value is 0.9014 (95% confidence interval: 0.8494-0.9533), the cutoff value at the maximum Youden index is delta Ct=11.73, the sensitivity at the maximum Youden index is 84.93%, and the specificity is 92.78%. PINK , TMEM106A and OTX1 When the three target gene regions are used together as biomarkers, the AUC value is 0.9903 (95% confidence interval: 0.9808-0.9997), the cutoff value at the maximum Youden index is the probability value = 0.7646, the sensitivity at the maximum Youden index is 97.26%, and the specificity is 92.78%.

[0048] This shows that the combination of the three genes significantly improves the diagnostic ability for urothelial carcinoma.

[0049] Furthermore, the detection sensitivity of the three gene regions, both individually and in combination, for different types of urothelial carcinoma samples was analyzed. The results are shown in Table 5. The combined detection of the three genes achieved sensitivities of 90.00% and 91.30% for early Ta stage and low-grade urothelial carcinoma, respectively, demonstrating that the non-invasive detection method of this invention can effectively overcome the shortcomings of existing traditional methods.

[0050] Table 5. Statistical analysis of detection sensitivity for different types of urothelial carcinoma samples.

[0051] Example 5 In this case, urine samples were collected from 104 patients with urothelial carcinoma and 178 patients with interfering diseases, using the same collection criteria as in Example 4. DNA extraction, bisulfite conversion, and methylation-specific PCR were performed on the urine samples according to the method described in Example 3. Then, based on the cutoff values ​​determined in the training set of Example 4 for single-gene and triple-gene combined detection, the methylation positivity / negativity of the samples was determined, and sensitivity and specificity were calculated. Sensitivity is the proportion of methylation-positive patients in urothelial carcinoma, and specificity is the proportion of methylation-negative patients in interfering diseases. The detection results are shown in Table 6.

[0052] Table 6 Statistical Analysis of Verification Results

[0053] As shown in Table 6, except for T3 and T4 stage urothelial carcinoma samples, the sensitivity of the combined three-gene detection was higher than that of single-gene detection in all other urothelial carcinoma samples. The overall sensitivity of the combined three-gene detection for urothelial carcinoma was 90.38%, and the overall specificity was 91.57%. The sensitivity of the combined detection exceeded 90% for all three types of urothelial carcinoma, and was also greater than 90% for low-grade urothelial carcinoma. The sensitivity for Ta stage samples was 88%. These results indicate that the combined three-gene detection also has the advantages of full coverage, early detection, and accuracy in the validation set.

[0054] In summary, the present invention is based on PINK , TMEM106A and OTX1 By using specific regions on the gene as a combination of methylation markers, the combined detection of this combination enables accurate prediction of urothelial carcinoma. It also has the advantages of comprehensive coverage (applicable to bladder cancer, renal pelvis cancer, and ureter cancer) and effective detection of early and low-grade cancers, which is of positive significance for improving the clinical management of urothelial carcinoma.

[0055] It should be noted that the present invention is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of the present invention are included within the scope of the present invention. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of the present invention, are also included within the scope of the present invention.

Claims

1. A composition for detecting urothelial carcinoma based on DNA methylation, characterized in that, Includes reagents for detecting methylation in the following DNA regions: PENK CpG island region Chr8: 56446618-56446724; TMEM106A CpG island region of gene Chr17:43211685-43211847; OTX1 Gene CpG island region Chr2: 63056808-63056956.

2. The composition according to claim 1, characterized in that, Includes the following primers and probes: Detection PENK The primers and probes for the CpG island region Chr8: 56446618-56446724 of the gene are shown in SEQ ID NO. 1-3, respectively. Detection TMEM106A The primers and probes for the CpG island region Chr17:43211685-43211847 of the gene are shown in SEQ ID NO.4-6, respectively. Detection OTX1 The primers and probes for the CpG island region Chr2: 63056808-63056956 are shown in SEQ ID NO. 7-9, respectively.

3. Use of the composition according to claim 1 or 2 in the preparation of a kit for detecting urothelial carcinoma.

4. The application according to claim 3, characterized in that, The urothelial carcinoma includes bladder cancer, ureteral cancer, and renal pelvis cancer.

5. A reagent kit for detecting urothelial carcinoma, characterized in that, It contains the primers and probes as described in claim 2.

6. The reagent kit according to claim 5, characterized in that, The kit also includes DNA extraction reagents, bisulfite conversion reagents, and reagents for PCR reactions.

7. The application according to claim 5, characterized in that, The kit contains primers and probes for detecting the internal reference gene.

8. The application according to claim 7, characterized in that, The internal reference gene is ACTB, and the sequences of its corresponding primers and probes are shown in SEQ ID NO.10-12, respectively.

9. The application of the kit according to any one of claims 5-8 in in vitro sample detection.

10. The application according to claim 9, characterized in that, The in vitro samples include urine, feces, blood, saliva, or other bodily fluids.

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