Target combination for detecting upper urinary tract urothelial carcinoma and application thereof

Abstract

The invention discloses a target combination for detecting upper urinary tract urothelial carcinoma and application of the target combination. The target combination comprises a chromosome unstable region; the chromosome instability region comprises any one or a combination of at least two of 2p, 3q 26.2 to 29, 5q 11.2 to 15, 8p, 8q 11.21 to 23.3, 9q 21.12 to 31.2, 11p 13 to 15.5, 13q 13.3 to 21.2 and 20q 13.13 to 13.33; the target combination or the kit provided by the invention can more accurately diagnose the upper urinary tract and urinary tract epithelial cancer, is not interfered by bladder cancer and urinary system benign diseases, can detect the area AUC under the ROC curve of the upper urinary tract and urinary tract epithelial cancer to 0.9679, can more comprehensively guide the diagnosis and prognosis of the upper urinary tract and urinary tract epithelial cancer, and has a wide application prospect. And a scientific basis is provided for clinical early diagnosis of the upper urinary tract epithelial cancer and formulation of an individualized treatment scheme by applying a next-generation sequencing technology.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a target combination for detecting upper urinary tract urothelial carcinoma and an application thereof. Background Art

[0002] Upper tract urothelial carcinoma (UTUC) is a malignant tumor that occurs in the renal pelvis and ureteral urothelial tissue. UTUC is mostly unilateral, and the probability of bilateral upper tract urothelial carcinoma is 1.6% to 4.37%. About 7% to 17% of UTUC may be combined with bladder cancer. UTUC is a highly malignant tumor that is prone to recurrence. Currently, it is clinically recognized that radical nephroureterectomy combined with sleeve bladder resection is the "gold standard" for the treatment of UTUC. Therefore, early detection is crucial for upper tract urothelial carcinoma. Ureteroscopy combined with pathological biopsy is currently the "gold standard" for the diagnosis of upper tract urothelial carcinoma. However, this examination method is an invasive surgery, and patient compliance is relatively low. In addition, some currently approved non-invasive diagnostic methods for upper tract urothelial carcinoma based on urine, such as urine exfoliated cell examination and FISH, have problems such as low sensitivity (especially for low-grade tumors and upper tract urothelial carcinoma) and poor accuracy. To further realize the early detection and early treatment of UTUC, it is particularly important to seek non-invasive biomarkers with high sensitivity and strong specificity for early diagnosis.

[0003] Chromosomal instability is often associated with tumors, including the loss or amplification of copies of entire chromosomes or chromosome fragments. Amplification and loss of chromosomes or chromosome fragments related to tumorigenesis are often unique to tumorigenesis. Detecting chromosomal instability regions in tumors is crucial for studying tumorigenesis and developing tumor diagnostic techniques. Currently, in situ fluorescence hybridization methods are used clinically to detect the instability of certain regions on chromosomes, but the distribution characteristics of chromosomal instability in patients with upper tract urothelial carcinoma are lacking.

[0004] The second generation sequencing can detect multiple regions on the genome at the same time, and can be applied to the simultaneous detection and analysis of the whole genome or a large number of regions on the genome. Therefore, it is urgent to provide a target combination with high sensitivity and high specificity for detecting upper urinary tract urothelial carcinoma.

[0005] Through searching the existing patent documents, it was found that CN202410379205.9 discloses a target combination for detecting urothelial carcinoma, wherein the target combination includes chromosomal unstable regions and / or genetic unstable regions; the chromosomal unstable regions include: 1q, 3q, 5p15.3, 6p23-p25.3, 7p, 7q, 8q, 9p, 9q, 13q14.11-q14.3, 17p or 17q, any one or a combination of at least two; the genetic unstable regions include: TERT and / or FGFR3. It is also pointed out that: the target combination, probe combination or kit can be used to more accurately diagnose urothelial carcinoma, and the area under the ROC curve AUC for detecting urothelial carcinoma reaches 0.9556, which can more comprehensively guide the diagnosis and prognosis of urothelial carcinoma, and provide a scientific basis for the application of second-generation sequencing technology to the clinical early diagnosis of urothelial carcinoma and the formulation of individualized treatment plans. However, in clinical practice, there are significant differences in the diagnosis and treatment pathways for bladder cancer and upper tract urothelial carcinoma. Bladder cancer can be effectively diagnosed by cystoscopy. Although upper tract urothelial carcinoma can be diagnosed by ureteroscopy, ureteroscopy is more traumatic and can cause tumor metastasis, so it cannot be widely used in clinical practice. Therefore, the diagnosis method of upper tract urothelial carcinoma is more urgently needed clinically. Urine flows through the upper urinary tract and bladder and is excreted from the body. Both upper tract urothelial carcinoma and bladder cancer will have tumor cells shed into the urine. When detecting whether upper tract urothelial carcinoma occurs, it will be affected by bladder cancer, which will cause trouble for the accurate diagnosis and treatment of upper tract urothelial carcinoma. However, the combination disclosed in CN202410379205.9 cannot distinguish between bladder cancer and upper tract urothelial carcinoma. Summary of the invention

[0006] In view of the deficiencies of the prior art and actual needs, the present invention provides a target combination for detecting upper tract urothelial carcinoma and its application. The whole genome instability information of upper tract urothelial carcinoma is analyzed by second-generation sequencing technology, 9 regions suitable for characterizing upper tract urothelial carcinoma are screened out, and a detection kit is designed. The target combination or kit of the present invention can specifically detect upper tract urothelial carcinoma without being affected by bladder cancer, thereby providing support for precision medicine of upper tract urothelial carcinoma.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a target combination for detecting upper urinary tract urothelial carcinoma, wherein the target combination includes a chromosomal unstable region;

[0009] The chromosomal unstable region includes any one of 2p, 3q26.2-29, 5q11.2-15, 8p, 8q11.21-23.3, 9q21.12-31.2, 11p13-15.5, 13q13.3-21.2, and 20q13.13-13.33, or a combination of at least two of them.

[0010] The present invention analyzes the whole genome instability information of upper tract urothelial carcinoma by second generation sequencing technology, and screens out 9 regions suitable for characterizing upper tract urothelial carcinoma. These 9 regions are highly correlated with the occurrence and development of upper tract urothelial carcinoma, and the 9 regions rarely occur in bladder cancer and benign diseases of the urinary system. The mutations in the 9 regions are detected for specific characterization of upper tract urothelial carcinoma. The mutation types of each region when characterizing upper tract urothelial carcinoma are: 2p amplification, 3q26.2-29 amplification, 8q11.21-23.3 amplification, 20q13.13-13.33 amplification, 5q11.2-15 deletion, 8p deletion, 9q21.12-31.2 deletion, 11p13-15.5 deletion, 13q13.3-21.2 deletion, etc. At the same time, a corresponding detection kit is designed. The target combination or kit of the present invention can more accurately diagnose upper tract urothelial carcinoma with high sensitivity and specificity, and can more comprehensively guide the diagnosis and prognosis of upper tract urothelial carcinoma, providing a scientific basis for the application of second-generation sequencing technology to the clinical early diagnosis of upper tract urothelial carcinoma and the formulation of individualized treatment plans.

[0011] The numbering of the chromosomes and gene unstable regions involved in the present invention is defined according to the conventional numbering rules in the art. For example, the unstable region 3q refers to the long arm of chromosome 3, and the unstable region 2p refers to the short arm of chromosome 2 in the conventional rules in the art.

[0012] The chromosomal instability described in the present invention includes the deletion or amplification of the entire chromosome or chromosome fragment copy.

[0013] In a second aspect, the present invention provides the use of the detection reagent of the target combination for detecting upper urinary tract urothelial carcinoma described in the first aspect in the preparation of an upper urinary tract urothelial carcinoma detection product.

[0014] In a third aspect, the present invention provides a kit for detecting upper urinary tract urothelial carcinoma, the kit comprising the target combination described in the first aspect.

[0015] Preferably, the kit further comprises any one of a positive reference, a negative reference, a buffer, an enzyme, a library adapter, a nucleic acid extraction reagent or a nucleic acid purification reagent, or a combination of at least two thereof.

[0016] Preferably, the enzyme comprises any one of a fragmentation enzyme, a DNA polymerase, and a DNA ligase, or a combination of at least two of them.

[0017] Preferably, the positive reference comprises a variant cell line containing the target combination described in the first aspect; and the negative reference comprises a cell line without chromosomal variation.

[0018] In a fourth aspect, the present invention provides use of the target combination described in the first aspect in the preparation of a product for the treatment or auxiliary treatment of upper urinary tract diseases.

[0019] Preferably, the upper urinary tract disease comprises upper urinary tract urothelial carcinoma.

[0020] Preferably, the upper urinary tract urothelial carcinoma includes: any one of renal pelvis cancer and ureteral cancer or a combination of both.

[0021] In a fifth aspect, the present invention provides a device for detecting upper urinary tract urothelial carcinoma, the device comprising a detection unit and a determination unit;

[0022] The detection unit is used to perform the following steps:

[0023] Mixing the sample to be tested with the reagents in the kit described in the third aspect, and detecting the target combination described in the first aspect after mixing;

[0024] The determination unit is used to perform the following steps:

[0025] Screening, diagnosis, treatment plan selection, monitoring and prognosis evaluation of upper urinary tract urothelial carcinoma are performed based on the detection results of the detection unit.

[0026] Preferably, the sample to be tested includes any one of body fluids, tissues or cells.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1) The present invention develops the flow chart as follows Figure 1 As shown, the training set includes upper tract urothelial carcinoma, bladder cancer and benign urinary system diseases. The chromosomal instability information is analyzed by the second-generation sequencing technology, and 9 regions that specifically characterize upper tract urothelial carcinoma without being interfered by bladder cancer and benign urinary system diseases are screened out, and the corresponding detection kit is designed.

[0029] 2) The target combination or kit of the present invention can more accurately diagnose upper tract urothelial carcinoma with high sensitivity and specificity, and can more comprehensively guide the diagnosis and prognosis of upper tract urothelial carcinoma, providing a scientific basis for the application of second-generation sequencing technology for the clinical early diagnosis of upper tract urothelial carcinoma and the formulation of individualized treatment plans. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0031] Figure 1 A flow chart for the development of target combinations of the present invention;

[0032] Figure 2 It is the ROC curve diagram of the detection method of the present invention. DETAILED DESCRIPTION

[0033] The present invention is described in detail below in conjunction with embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, some adjustments and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0034] The present invention develops the flow chart as follows Figure 1 As shown, the training set includes upper tract urothelial carcinoma, bladder cancer and benign urinary system diseases. The chromosomal instability information is analyzed by the second-generation sequencing technology, and 9 regions that specifically characterize upper tract urothelial carcinoma without being interfered by bladder cancer and benign urinary system diseases are screened out, and the corresponding detection kit is designed.

[0035] If no specific techniques or conditions are specified in the following examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0036] Example 1

[0037] Urine genomic DNA extraction

[0038] The kit used in this example was purchased from QIAGEN (Cat. No. 80204).

[0039] a. Take 50 ml of urine and centrifuge it at 1600 g for 10 min. Carefully pour out the supernatant and then carefully discard the remaining supernatant with a pipette.

[0040] b. Add 350 μL 1× Buffer RL / DTT to a new 1.5 mL centrifuge tube and pipette to break up the cells.

[0041] c. Place the DNA purification column in a 2 mL collection tube. Transfer the cell lysate to the gDNA filter column. Centrifuge at 14,000 g for 2 min.

[0042] d. Place the DNA purification column in a 2 mL collection tube. Add 500 μL of Buffer DW1 to the column and let stand for 2 minutes. Centrifuge at 10,000 g for 30-60 seconds.

[0043] e. Discard the effluent and put the column back into the collection tube. Add 500 μL Buffer RW2 to the column. Centrifuge at 10,000 g for 30-60 seconds.

[0044] f. Discard the effluent and put the column back into the collection tube. Add 500 μL Buffer RW2 to the column. Centrifuge at 10,000 g for 30-60 seconds.

[0045] g. Discard the flow-through, put the column back into the empty collection tube, and centrifuge at 13000g for 2 minutes.

[0046] h. Place the DNA column in a 1.5 mL centrifuge tube. Add 30-50 μL of nuclease-free water preheated to 65°C to the center of the column membrane. Let stand at room temperature for 3 minutes. Centrifuge at 13,000 g for 1 minute.

[0047] i. Discard the DNA column and store the DNA at 2-8°C or -20°C.

[0048] Example 2

[0049] Whole genome sequencing.

[0050] 1. Genome fragmentation: Take 10 ng of human genomic DNA, prepare the enzyme digestion reaction system shown in Table 1, and perform the reaction according to the procedure in Table 2. In this example, the concentration of human genomic DNA is 2 ng / μL, and 10 μL needs to be added to the reaction system.

[0051] Table 1

[0052] DNA shearing reaction system Genomic DNA 10μL Breaking enzyme (KAPA, kk8602) 3μL Fragmentation reaction buffer 7μL Nucleic acid-free water Make up to 35 μL

[0053] After vortexing and centrifugation (avoid bubbles), run the following program on the PCR instrument:

[0054] Table 2

[0055]

[0056] 2. Adapter ligation reaction: According to the requirements of the quick ligation module (manufacturer New England Biolabs, catalog number: E6056L), the adapter ligation premix (15 μL / sample), adapter ligation enhancer (0.5 μL / sample), and adapter (1.5 μL / sample) were added to the above reaction solution, oscillated to mix, and centrifuged. Finally, the reaction system shown in Table 3 below was formed.

[0057] Table 3

[0058]

[0059]

[0060] Place the above system in a PCR instrument and run the program: 20°C, 30 min, with the heated lid closed.

[0061] 3. Purification steps after ligation reaction:

[0062] a. Take out the library purification magnetic beads in advance, let them stand at 25℃ for 30 minutes, and mix them before use.

[0063] b. Transfer the adapter ligation reaction solution in the above step to a 1.5 mL centrifuge tube with the corresponding number, add 46 μL of resuspended library purification magnetic beads, pipette 20 times at a constant speed, and incubate at 25°C for 5 min.

[0064] c. Place the centrifuge tube on a magnetic rack and discard the supernatant after the solution becomes clear.

[0065] d. Add 200 μL of freshly prepared 80% ethanol, let it stand for 30 seconds, and then discard the supernatant.

[0066] e. Repeat step d once.

[0067] f. Remove the centrifuge tube from the magnetic rack, centrifuge for 3 seconds, put the centrifuge tube back on the magnetic rack, and discard the remaining 80% ethanol. Be careful not to absorb the magnetic beads. Open the tube cover and dry it at 25℃ for 10 minutes.

[0068] g. When the magnetic beads turn matte, add 16 μL Low TE buffer to the centrifuge tube, shake gently to resuspend the magnetic beads, and incubate at 25°C for 5 min.

[0069] h. Place the centrifuge tube on a magnetic rack and let it stand for 2 minutes. After the solution is clear, take 15 μL of the supernatant for the next amplification reaction.

[0070] 4. PCR amplification: Add the corresponding reagents to the PCR tube according to Table 4 below:

[0071] Table 4

[0072]

[0073] The sequences of universal primers and index primers are shown in Table 5:

[0074] Table 5

[0075]

[0076] Place the mixed PCR tube into the PCR instrument and run the following program in Table 6.

[0077] Table 6

[0078]

[0079] 5. Purification of PCR products: Refer to step 3 for purification steps, where:

[0080] The amount of library purification magnetic beads resuspended in step b is 22.5 μL;

[0081] In step g, the amount of Low TE buffer added was 31 μL;

[0082] Take 30 μL of the supernatant in step h and save it for the next step.

[0083] 6. Library quantification on the machine

[0084] The purified library was analyzed for fragment size using the Agilent BioAnalyzer biochip analysis system. The dsDNA HS Assay Kit was used to measure the library mass concentration, and the library molar concentration was calculated by mass concentration and fragment size. The sequencing was performed using Illumina Hiseq X-ten according to the sequencer instructions.

[0085] Results: According to Examples 1 and 2, a total of 103 cases of upper tract urothelial carcinoma, 67 cases of bladder cancer, and 138 cases of non-upper tract urothelial carcinoma patients were tested for whole genome. By statistics, 11 chromosomal unstable regions in the present invention were screened out, specifically including: 2p, 3q26.2-29, 5q11.2-15, 8p, 8q11.21-23.3, 9q21.12-31.2, 11p13-15.5, 13q13.3-21.2, 20q13.13-13.33. The overall incidence of these 9 targets in upper tract urothelial carcinoma reached 93.2%, and the incidence of each target in upper tract urothelial carcinoma was not less than 20%. Among 67 cases of bladder cancer, the above targets were detected in 5 cases, with an incidence of 7.5%, and a specificity of 92.5% relative to bladder cancer; among 138 patients with non-upper tract urothelial carcinoma, the above 9 targets were detected in only 4 cases, and the specificity relative to benign urinary system diseases reached 97.1%; the overall specificity relative to bladder cancer and benign urinary system diseases was 95.5%.

[0086] The performance of the 9 selected regions in characterizing upper tract urothelial carcinoma was further verified according to Examples 1 and 2. The verification group completed the detection of 107 patients with upper tract urothelial carcinoma, 83 patients with bladder cancer and 141 patients with benign urinary system diseases. According to statistics, chromosomal mutations were detected in 98 patients with upper tract urothelial carcinoma, with a sensitivity of 91.6%; 7 of the 83 patients with bladder cancer were detected, with a detection rate of 8.4% and a specificity of 91.5% relative to bladder cancer; among the 141 patients with non-upper tract urothelial carcinoma, only 5 were detected with the above 9 targets, with an overall specificity of 96.4%; the overall specificity of the 9 selected regions relative to bladder cancer and benign urinary system diseases reached 94.6%. ROC curve of the verification data ( Figure 2 ) reached an AUC area of ​​0.9679, which fully demonstrates that the 9 regions in the present invention can be well used for the diagnosis of upper tract urothelial carcinoma. The validation group also analyzed the test results according to the combination disclosed in CN202410379205.9. Its sensitivity for upper tract urothelial carcinoma was 86.0%, and its detection sensitivity for bladder cancer was 94.0%. The specificity for bladder cancer was only 6.0%, and the specificity for non-urothelial carcinoma was 95.7%. From the above data, it can be found that the present invention can specifically detect upper tract urothelial carcinoma, eliminate the interference of bladder cancer, and better meet the diagnosis and treatment needs of upper tract urothelial carcinoma.

[0087] In summary, the present invention analyzes the whole genome instability information of upper tract urothelial carcinoma through the second-generation sequencing technology, screens out 9 regions suitable for characterizing upper tract urothelial carcinoma, and designs a corresponding detection kit. The target combination or kit of the present invention can more accurately diagnose upper tract urothelial carcinoma with high sensitivity and specificity, and can more comprehensively guide the diagnosis and prognosis of upper tract urothelial carcinoma, providing a scientific basis for the application of second-generation sequencing technology for the clinical early diagnosis of upper tract urothelial carcinoma and the formulation of individualized treatment plans.

[0088] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A target combination for detecting upper urinary tract urothelial carcinoma, characterized in that: The target combination includes chromosomal unstable regions; The chromosomal unstable region includes any one of 2p, 3q26.2-29, 5q11.2-15, 8p, 8q11.21-23.3, 9q21.12-31.2, 11p13-15.5, 13q13.3-21.2, and 20q13.13-13.33, or a combination of at least two of them.

2. Use of a detection reagent for the target combination as claimed in claim 1 in the preparation of an upper urinary tract urothelial carcinoma detection product.

3. A kit for detecting upper urinary tract urothelial carcinoma, characterized in that: The kit comprises the target combination as claimed in claim 1.

4. The kit according to claim 3, characterized in that The kit further comprises a positive reference, a negative reference, a buffer, an enzyme, a library adapter, and any one or a combination of at least two of a nucleic acid extraction reagent or a nucleic acid purification reagent; Preferably, the enzyme comprises any one of a fragmentation enzyme, a DNA polymerase, and a DNA ligase, or a combination of at least two thereof.

5. The kit according to claim 3 or 4, characterized in that The positive reference includes a cell line containing a variation of the target combination as described in claim 1; the negative reference includes a cell line without chromosomal variation.

6. Use of the target combination as claimed in claim 1 in the preparation of a product for the treatment or auxiliary treatment of urinary tract diseases.

7. The use according to claim 6, characterized in that: The urinary tract diseases include upper tract urothelial carcinoma.

8. The use according to claim 7, characterized in that: The upper urinary tract urothelial carcinoma includes: any one of renal pelvis cancer and ureteral cancer or a combination of both.

9. A device for detecting upper urinary tract urothelial carcinoma, characterized in that: The device comprises a detection unit and a determination unit; The detection unit is used to perform the following steps: Mixing the sample to be tested with the reagent in the kit as described in any one of claims 3 to 5, and detecting the target combination as described in claim 1 after mixing; The determination unit is used to perform the following steps: Screening, diagnosis, treatment plan selection, monitoring and prognosis evaluation of upper urinary tract urothelial carcinoma are performed based on the detection results of the detection unit.

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