CN114107502A

CN114107502A - A diagnostic marker for upper urothelial carcinoma and its application

Info

Publication number: CN114107502A
Application number: CN202111431742.6A
Authority: CN
Inventors: 廖邦华; 林逸飞; 林玲; 黄进
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2021-11-29
Filing date: 2021-11-29
Publication date: 2022-03-01

Abstract

The invention belongs to the technical field of biomarkers, and discloses an Upper urinary tract epithelial cancer (UTUC) diagnosis marker and application thereof, wherein the Upper urinary tract epithelial cancer diagnosis marker is DNA methylation biomarkers GDF15, TMEFF2 and VIM; the mRNA sequence NM-004864 of GDF15 is SEQ ID NO: 1; the mRNA sequence NM-001305134 of the TMEF 2 is SEQ ID NO: 2; the mRNA sequence NM-003380 of VIM is SEQ ID NO: 3. according to the invention, the data of the upper urothelial cancer is comprehensively mined from a multiomic layer, and the biomarker related to the upper urothelial cancer is searched, so that the limitation of the traditional candidate gene method can be overcome, and the result accuracy is improved; the clinical data are combined for integrated analysis, multiple biomarkers are jointly detected, a multi-index combined diagnosis model is established, and the diagnosis sensitivity and specificity are improved.

Description

Upper urinary tract epithelial cancer diagnosis marker and application thereof

Technical Field

The invention belongs to the technical field of biomarkers, and particularly relates to an upper urinary tract epithelial cancer diagnosis marker and application thereof.

Background

Epithelial cancer (UTUC) including renal pelvis cancer and ureteral cancer is a complex urinary system tumor that is commonly caused by environmental factors and genetic factors, and accounts for 5-10% of the urinary epithelial cancer (UC) in the european and american countries, with an annual incidence rate of about 2 cases/10 million residents. The incidence rate of UTUC in China is higher and accounts for about 20-30% of Transitional Cell Carcinomas (TCCs). The history of possible contact with aristolochic acid medicaments is a unique cause of UTUC patients in China. Compared with European and American countries, the proportion of women in UTUC patients in China is higher than that of men, namely about 1.3:1, and the proportion of metastasis of lymph nodes is higher because the male UTUC patients have later stages, larger tumors and higher.

Due to the special anatomical structure of the affected part of the UTUC and the complexity of the influencing factors, the UTUC is hidden in the onset and difficult to diagnose in early stage. The delay in diagnosis results in a higher proportion of late stage tumors at the time of diagnosis (56% muscle layer infiltration or local invasion), making their 5-year survival rate much lower than that of urinary bladder epithelial cancer (UCB). The research proves that: 5-year specific survival rate of the pT2/pT3 staged patient is lower than 50%, and 5-year specific survival rate of the pT4 staged patient is lower than 10%; the 5-year specific survival rate was also lower for patients graded G3-G4 compared to G1-G2. Meanwhile, the UTUC has high invasive ability and high recurrence rate, the recurrence rate can reach 16-58%, wherein the recurrence rate of the bladder is about 22-47%.

The life threat and the life quality reduction caused by UTUC can be effectively reduced by early diagnosis and early treatment, and the search of effective characteristic targets of early diagnosis and treatment gateways is a key point and a hot point of research. In recent years, clinicians and researchers have tried to find effective prediction methods from multiple dimensions, but the existing findings are not sufficient to meet the requirements of accurate diagnosis and treatment. The detection rate of the ureteral tumor by imaging examination such as CT, MRI and the like is relatively low, the positive rate of urine cast-off cytology detection is lower than 50%, urine FISH detection is also limited by specimen conditions and the tumor obstruction level, and ureteroscope/soft-scope examination is invasive and can increase the risk of bladder metastasis.

In addition, there are studies focused on epigenetic features of the UTUC. Although several susceptibility genes or biomarkers have been discovered that are associated with the development, progression and prognosis of UTUC, because of numerous molecular events accompanying the development of UTUC, the process of the related genes for exerting biological functions and the regulation mechanism are complex and are not caused by single factors or simple sets of single factors, but is formed by the interaction of various biomolecular approaches such as genes, regulatory factors, proteins and the like, and the candidate gene method is mostly adopted in the past research, meanwhile, the research result is influenced by factors such as research design, inclusion and exclusion standards, sample size, individual difference of patients (genetic heterogeneity and different environments), different disease grading stages and the like, the types of the discovered biomarkers are different, even the expression trends are opposite, therefore, the reliability of evaluating the disease state is poor, the sensitivity and specificity are not high when the method is used for diagnosis, and the method is difficult to be applied to clinical practice.

Therefore, the method finds the biomarkers with high sensitivity and strong specificity, establishes a multi-index combined diagnosis model, establishes a rapid, simple and noninvasive early diagnosis technology, and has important theoretical significance and potential transformation application value for the early diagnosis and prognosis judgment of the UTUC.

Through the above analysis, the problems and defects of the prior art are as follows:

(1) UTUC diagnosis relies primarily on imaging examinations, ureteroscopy, and urine cytology examinations. These diagnostic methods have limited sensitivity and specificity and high rates of misdiagnosis or missed diagnosis.

(2) The existing UTUC biomarker has single research level, rare types and poor repeatability, and can not completely meet the clinical diagnosis and treatment requirements only by a single-factor prediction model.

(3) Early diagnosis is difficult, and effective molecular diagnosis markers suitable for Chinese population are lacked.

(4) The prior art is lack of individual treatment means, and the prognosis of patients in middle and later stages is extremely poor.

The difficulty in solving the above problems and defects is:

(1) the gene expression between single cells has heterogeneity, which is not favorable for the tracing of cytopathic process and the research of biodiversity.

(2) How to correlate multiple sets of mathematical data with clinical data to jointly detect multiple biomarkers.

The significance of solving the problems and the defects is as follows:

(1) an efficient and accurate early diagnosis and early warning system is established, especially noninvasive detection is used for early diagnosis and disease state assessment, and the survival rate and the survival quality of patients can be greatly improved by treating antedisplacement of the gateway.

(2) The UTUC related biomarkers are searched and the pathogenic mechanism of related genes is deeply researched, so that a target is provided for individualized treatment.

(3) If a highly sensitive and highly specific biomarker combination can be found in urine or peripheral blood, the kit has great significance for UTUC prognosis monitoring and recurrence metastasis monitoring.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a diagnosis marker for upper urothelial cancer and application thereof.

The invention is realized by the implementation of the method, and the upper urinary tract epithelial cancer diagnostic marker is DNA methylation biomarkers GDF15, TMEFF2 and VIM.

Further, the mRNA sequence NM _004864 of GDF15 is SEQ ID NO: 1.

further, the mRNA sequence NM-001305134 of TMEFF2 is SEQ ID NO: 2.

further, the mRNA sequence NM-003380 of VIM is SEQ ID NO: 3.

another object of the present invention is to provide a method for identifying a diagnostic marker for upper urothelial cancer using the diagnostic marker for upper urothelial cancer, comprising the steps of:

step one, performing joint analysis on multiple groups of chemical data by comprehensively using a bioinformatics means through a biological sample library, and discovering potential pathogenic sites/genes of UTUC and various biomarkers;

establishing a model for the early diagnosis and prognosis evaluation of the UTUC, and deeply determining the clinical application value of the early diagnosis of the related biomarkers for multiple verification;

and step three, the function of the relevant biomarkers for regulating and controlling the UTUC is determined, the relationship between the differential expression level and the occurrence and development of the UTUC is clarified, and the biological functions and pathogenic mechanisms of the differential sites/genes are clarified and potential treatment targets are discovered through multi-level verification of molecular biology, cell biology and the like.

The invention also aims to provide application of the diagnosis marker for the upper urinary tract epithelial cancer in preparation of a kit for diagnosing the upper urinary tract epithelial cancer.

By combining all the technical schemes, the invention has the advantages and positive effects that: the development of UTUC is a dynamic process, and the biomarkers and their sensitivity and specificity are different for the assessment of different stages of the disease, so it is important to use the most suitable biomarkers in different stages of the disease. Due to its own characteristics, the application of the biomarkers in the disease diagnosis and treatment process is particularly important:

(1) early diagnosis, in particular non-invasive detection for early diagnosis and disease state assessment

UTUC diagnosis relies primarily on imaging examinations, ureteroscopy, and urine cytology examinations. These diagnostic methods have limited sensitivity and specificity and high rates of misdiagnosis or missed diagnosis. And ureteroscope examination belongs to the operation, needs to carry out under anesthesia, requires to the operator highly, has subjective difference, has the wound to the patient simultaneously, causes the misery easily, also has the probability to take place the complication. Most patients with UTUC are diagnosed at a middle or advanced stage, resulting in a very poor prognosis. If the UTUC high-risk population can be effectively early diagnosed in a non-invasive or minimally invasive mode, the risk of suffering from cancer is detected as early as possible, a high-efficiency and accurate early diagnosis and early warning system is established, the treatment gateway is moved forward, and the survival rate and the survival quality of patients can be greatly improved.

(2) Searching treatment target points and providing targets for individualized treatment

The current gold standard for treatment of non-metastatic UTUC is radical nephro-uretero-vesical intramural resection. However, there is an overtreatment risk in radical surgery for patients with early or low grade malignancy UTUC. For advanced or highly malignant patients, even if radical surgery is performed, tumors still have a high probability of relapse, the treatment means for patients with side urinary tract system relapse after radical surgery is more limited, if the urinary tract system is preserved, the treatment effect is not good, and if the kidney cannot be preserved, dialysis is needed after resection. In the aspect of comprehensive treatment, chemotherapy mainly comprising gemcitabine and cisplatin is developed in many centers, and an auxiliary chemotherapy study after the UTUC operation is carried out, but the curative effect of the chemotherapy drugs is still controversial because of the toxicity of the chemotherapy drugs. The efficacy of other treatment methods (such as endoscopic resection) is not exact, and high-quality evidence of different types of patients and disease subtypes is lacking. The targeted biomarkers can modulate angiogenesis, signaling, immune system, and apoptosis. At present, the available UTUC treatment target is lacking clinically, and individualized medical treatment cannot be realized, so that the search of related biomarkers lays a solid foundation for screening the UTUC treatment target.

(3) Prognostic monitoring, UTUC recurrence metastasis monitoring

Because UTUC mostly develops from the affected side and multiple centers and seldom affects the clinical characteristics of the opposite side, after pure nephroureterectomy, the peripheral residual urothelium is easy to recur again. Even though patients with UTUC still have a greater possibility of relapse or progression after radical treatment, a monitoring means capable of accurately assessing the possibility of relapse and metastasis of patients is urgently needed to obtain a more practical treatment scheme and follow-up strategy. If a highly sensitive and highly specific biomarker combination can be found in urine or peripheral blood, the significance of the UTUC prognosis monitoring is great.

The upper urothelial cancer diagnosis marker provided by the invention is DNA methylation biomarkers GDF15, TMEFF2 and VIM which can be used for combined inspection and diagnosis of the upper urothelial cancer; the methylation biomarkers GDF15, TMEFF2 and VIM were all associated with prognosis of upper urothelial Cancer, including Tumor recurrence (Tumor recurrence), Tumor progression (Tumor progression) and Cancer-specific mortality (Cancer-specific mortality).

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

Fig. 1 is a flowchart of a method for identifying a marker for diagnosing upper urothelial cancer according to an embodiment of the present invention.

FIG. 2 is a schematic diagram showing the pleiotropic effects of the methylated GDF15, TMEFF2 and VIM genes in different prognostic results provided by the examples of the present invention.

FIG. 3 is a schematic diagram of the survival analysis of the methylated GDF15 and TMEFF2 genes provided in the examples of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

In view of the problems in the prior art, the present invention provides a diagnostic marker for upper urothelial cancer and the application thereof, and the present invention is described in detail below with reference to the accompanying drawings.

The upper urinary tract epithelial cancer diagnosis markers provided by the embodiment of the invention are DNA methylation biomarkers GDF15, TMEFF2 and VIM.

As shown in fig. 1, the method for identifying a diagnosis marker for upper urothelial cancer provided by the embodiment of the present invention includes the following steps:

s101, performing joint analysis on multiple groups of chemical data by comprehensively using a bioinformatics means through a biological sample library, and discovering potential pathogenic sites/genes of UTUC and various biomarkers;

s102, establishing a model for early diagnosis and prognosis evaluation of UTUC, and deeply determining the clinical application value of early diagnosis of the related biomarkers and multiple verifications;

s103, the function of the relevant biomarkers for regulating UTUC is determined, the relationship between differential expression level and UTUC occurrence and development is clarified, the biological functions and pathogenic mechanisms of differential sites/genes are clarified through multi-level verification of molecular biology, cell biology and the like, and potential therapeutic targets are discovered.

The technical solution of the present invention is further described below with reference to specific examples.

Example 1

1. Applicants followed The requirements of The project report of first choice (PRISMA) and tumor marker prognostic research report Specification (REMARK) for systematic summary and meta analysis and searched thoroughly for evidence related to UTUC epigenetic inheritance, including DNA methylation, published in authoritative databases (PubMed, The Cochrane Library, EMBASE, Scopus) from 1/2004 to 12/2020/31. Based on inclusion exclusion criteria, a total of 11 papers related to DNA methylation were included, of which 8 reported the epigenetic status of 10 genes (GDF15, TMEFF2, VIM, RASSF1A, BRCA1, ABCC6, SALL3, HSPA2, CDH1, THBS1) in UTUC and their relationship to clinical outcome.

(see Table 1).

2. Role of methylated genes in UTUC

(ii) diagnostic action

Of the 8 studies described above, 2 reported that methylated genes were associated with the diagnosis of UTUC.

In one study, tissue samples of 57 cases of UTUC (G1/> G1 ═ 15/42) and 36 cases of control (control being normal upper urothelium from renal cell carcinoma patients); 22 cases of UTUC (G1/> G1 ═ 3/19) were compared with 20 urine samples from a control group (renal cell carcinoma: n ═ 10; prostate cancer: n ═ 7; healthy blood donors without personal or family history of cancer: n ═ 3).

Specific methylation PCR experiments found that the methylation levels of GDF15, TMEFF2, and VIM were higher in UTUC tissue and urine samples than in the control group (P ═ 0.022; P < 0.001; P < 0.001). The combined examination of tissue and urine with methylated GDF15, TMEFF2 and VIM accurately identified UTUC with sensitivity of 100% and 91%, respectively, corresponding to areas under the tissue and urine curves of 1.000 and 0.923, respectively, and specificity of 100%.

One study collected 98 samples of UTUC (G1/> G1 ═ 26/63) and 113 control groups of urine (control groups were benign urological patients, no cystoscopic or pathological diagnosis of urothelial cancer, and no history of urothelial cancer or other genitourinary malignancies).

Experiments show that the sensitivity of methylated GDF15, TMEFF2, VIM, CDH1, RASSF1A, HSPA2 and joint detection UTUC is 0.82 (0.72-0.88), the specificity is 0.68 (0.59-0.76), and the area under the curve is 0.836 (0.782-0.891); compared with urine cytology examination and fluorescence in situ hybridization technology, the detection accuracy of UTUC in urinary sediment is highest.

In summary, the following steps: the urine/tissue-DNA methylation biomarkers GDF15, TMEFF2 and VIM can be tested in combination to diagnose UTUC.

② prognostic effect

Methylation of 10 genes (GDF15, TMEFF2, VIM, RASSF1A, BRCA1, ABCC6, SALL3, HSPA2, CDH1, THBS1) was associated with relapse, progression and cancer-specific mortality of UTUC. (see Table 2)

3. Summary of the invention

(1) The DNA methylation biomarkers GDF15, TMEFF2 and VIM can be tested in combination to diagnose upper urothelial cancer.

(2) The methylation biomarkers GDF15, TMEFF2 and VIM were associated with prognosis of upper urothelial cancer, including tumor recurrence, progression and cancer-specific mortality.

Example 2

GDF15：growth differentiation factor 15

mRNA sequence:

(r) NM-0048641200 bp mRNA (see SEQ ID NO: 1)

Coding region (CDS): 33-959bp

TMEFF2：transmembrane protein with EGF like and two follistatin like domains

mRNA sequence:

(r) NM-0013051342856 bp mRNA (see SEQ ID NO: 2)

ORIGIN

Coding region (CDS): 410-1450bp

VIM：vimentin

mRNA sequence:

(r) NM-0033802154 bp mRNA (see SEQ ID NO: 3)

ORIGIN

Coding region (CDS): 432-1832 bp-

ORIGIN

The technical effects of the present invention will be described in detail with reference to experiments.

(meta) -merging the analytical results

GDF15, TMEFF2, and VIM methylation were all associated with relapse, progression, and cancer-specific mortality of UTUC. (see Table 3)

NA:notapplicable.

As shown in fig. 2, Meta pooled results show that methylated GDF15 is able to significantly reduce the risk of UTUC relapse (Z2.82; P0.005) and progression (Z4.17; P < 0.0001); methylated GDF15 and TMEFF2 were able to significantly increase the risk of cancer specific death (Z ═ 2.10; P ═ 0.04, Z ═ 4.71; P < 0.00001); methylated VIM can significantly increase the risk of UTUC (Z ═ 2.10; P ═ 0.04) progression.

(ii) survival analysis result

And (4) carrying out Kaplan-Meier survival analysis by using a UCB data set of a TCGA data base. UTUC is a urothelial malignancy, which has a more aggressive clinical phenotype than UCB. Although the UTUC data was not included in the TCGA database, the results of the analysis of the UCB dataset (434 patients) had important reference values for UTUC clinical care. As shown in FIG. 3, the Overall survival rate (OS) and Disease specific survival rate (DSS) of patients hypermethylated with GDF15 were significantly lower than those of patients hypomethylated with GDF15 (CpG cg12008047, P. 0.0052; cg12008047, P. 0.0323) among UCB patients. Patients with TMEFF2 hypermethylated UCB had significantly lower OS and DSS than patients with TMEFF2 hypomethylated (cg15003763, P ═ 0.0112; cg15003763, P ═ 0.0166).

The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.

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atgagtccct ggaacgccag atgcgtgaaa tggaagagaa ctttgccgtt gaagctgcta

actaccaaga cactattggc cgcctgcagg atgagattca gaatatgaag gaggaaatgg

ctcgtcacct tcgtgaatac caagacctgc tcaatgttaa gatggccctt gacattgaga

ttgccaccta caggaagctg ctggaaggcg aggagagcag gatttctctg cctcttccaa

acttttcctc cctgaacctg agggaaacta atctggattc actccctctg gttgataccc

actcaaaaag gacacttctg attaagacgg ttgaaactag agatggacag gttatcaacg

aaacttctca gcatcacgat gaccttgaat aaaaattgca cacactcagt gcagcaatat

attaccagca agaataaaaa agaaatccat atcttaaaga aacagctttc aagtgccttt

ctgcagtttt tcaggagcgc aagatagatt tggaatagga ataagctcta gttcttaaca

accgacactc ctacaagatt tagaaaaaag tttacaacat aatctagttt acagaaaaat

cttgtgctag aatacttttt aaaaggtatt ttgaatacca ttaaaactgc tttttttttt

ccagcaagta tccaaccaac ttggttctgc ttcaataaat ctttggaaaa actc

Claims

1. A diagnostic marker for upper urothelial cancer, which is DNA methylation biomarkers GDF15, TMEFF2 and VIM.

2. The diagnostic marker of upper urothelial cancer of claim 1, wherein the mRNA sequence NM _004864 of GDF15 is SEQ ID NO: 1.

3. the diagnostic marker of upper urothelial cancer of claim 1, wherein the mRNA sequence NM _001305134 of TMEFF2 is SEQ ID NO: 2.

4. the diagnostic marker of upper urothelial cancer according to claim 1, wherein the mRNA sequence NM _003380 of VIM is SEQ ID NO: 3.

5. a method for identifying a diagnostic marker for upper urothelial cancer using the diagnostic marker for upper urothelial cancer according to any one of claims 1 to 4, comprising the steps of:

step one, performing joint analysis on multiple groups of chemical data by comprehensively using a bioinformatics means through a biological sample library, and discovering potential pathogenic sites/genes and various biomarkers of the upper urothelial cancer;

establishing a model for early diagnosis and prognosis evaluation of the upper urinary tract epithelial cancer, and deeply determining and multiply verifying the clinical application value of early diagnosis of related biomarkers;

and step three, the function of regulating and controlling the upper urinary tract epithelial cancer by the related biomarkers is determined, the relationship between the differential expression level and the occurrence and development of the upper urinary tract epithelial cancer is clarified, the biological function and pathogenic mechanism of the differential sites/genes are clarified through multi-level verification in molecular biology, cell biology and the like, and the potential treatment targets are discovered.

6. Use of the upper urinary tract epithelial cancer diagnostic marker according to any one of claims 1 to 4 in the preparation of a kit for diagnosing upper urinary tract epithelial cancer.