CN111172284A - Biomarker for monitoring and evaluating curative effect of head and neck cancer - Google Patents
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Abstract
The invention relates to the field of clinical diagnosis of head and neck cancer, in particular to a biomarker for monitoring and evaluating curative effect of head and neck cancer. The invention discloses application of m5C regulatory gene in preparing a reagent or a kit for diagnosing, monitoring, evaluating curative effect or monitoring metastasis and relapse of head and neck cancer. The invention has higher specificity for monitoring and evaluating the curative effect of head and neck cancer, and can be used for dynamically monitoring the recurrence and metastasis of head and neck cancer. The gene marker can be combined with other clinical indexes, and provides more accurate judgment for screening, diagnosis, treatment and prognosis of head and neck cancer.
Description
Technical Field
The invention relates to the field of clinical diagnosis of head and neck cancer, in particular to a biomarker for monitoring head and neck cancer and evaluating curative effect.
Background
Head and neck cancer is the seventh ranked malignant tumor type of neoplastic disease, accounting for approximately 7% of the overall human body's systemic malignant tumor disease, with more than 90% of Head and neck cancers belonging to the Head and Neck Squamous Cell Carcinosoma (HNSCC). More than 50 ten thousand patients are diagnosed with head and neck squamous cell carcinoma every year, the number of new cases in China accounts for about 1/5 of the new cases, and the number of death cases is about 5.6 ten thousand cases every year. Generally speaking, head and neck squamous cell carcinoma is classified into oral cancer, nasopharyngeal cancer, oropharyngeal cancer, laryngeal cancer and the like. Meanwhile, since about one fourth of patients with head and neck squamous cell carcinoma are associated with in vivo infection with human papillomavirus (particularly oropharyngeal cancer patients), head and neck squamous cell carcinoma can also be classified as human papillomavirus negative and positive tumors. Poor living habits such as smoking and drinking are generally considered to be the cause of head and neck squamous cell carcinoma. Meanwhile, due to the reasons that the diagnosis consciousness of patients is poor, the distant metastasis or recurrence symptoms in the bodies of patients are common and the like, the clinical early diagnosis and comprehensive treatment of head and neck squamous cell carcinoma diseases are still greatly limited, and the average life cycle of the head and neck squamous cell carcinoma patients is only 5 years.
Through the development of the last decades, the survival rate of the patients with head and neck cancer in 5 years is not significantly improved by the operation, radiotherapy and chemotherapy, and the surgical treatment and excision range of the head and neck cancer is severely limited due to the special anatomical parts, so that the life quality of the treated patients is generally reduced, and the patients are often damaged or disabled.
Recently, with the rapid development of various omics and bioinformatics analysis technologies, head and neck cancer has become one of the most ideal disease models internationally recognized for performing molecular typing studies with precise medical guidance, in combination with previous research results. Screening and verifying effective head and neck cancer analysis and classification markers and molecular spectrum patterns are helpful for promoting the conversion of disease classification of head and neck cancer from macro morphology to a new classification system based on molecular characteristics, the classification method has clear guiding significance for clinical treatment, finally realizes individualized accurate treatment for patients, and greatly improves the survival rate and the quality of life of the patients.
The biological marker is biological molecules capable of distinguishing the physiology and disease states of organisms, and the clinical treatment effect of patients can be greatly improved by screening the biological marker for early disease discovery and early diagnosis. tRNAs are very important non-coding RNAs in a class of organisms, and have the main functions of transferring amino acids and participating in protein synthesis.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a biomarker for monitoring and evaluating the curative effect of head and neck cancer.
(II) technical scheme
In order to achieve the purpose, the invention is realized by the following technical scheme:
the m5C regulatory gene is applied to preparing a reagent or a kit for diagnosing, monitoring, evaluating curative effect or monitoring metastasis and relapse of head and neck cancer.
Preferably, there is a significant association between changes in the m5C regulatory gene and changes in HRAS and RAC 1.
Preferably, expression of the Eraser and Reader genes in the m5C regulatory gene is significantly correlated with CNV.
Preferably, the m5C regulatory gene tends to be highly expressed in the case of high TNM Stage.
The application of the DNMT1 gene or/and the ALYREF gene in preparing a reagent or a kit for diagnosing, monitoring, evaluating the curative effect of head and neck cancer or monitoring metastasis and relapse.
Preferably, low expression of the DNMT1 gene is associated with a poor prognosis of patients with HNSCC, and high expression of the ALYREF gene is associated with a poor prognosis of patients with HNSCC.
Preferably, the reagent or kit comprises primers that specifically recognize the DNMT1 gene or/and the ALYREF gene.
Preferably, high expression of the ALYREF gene is associated with mitochondrial physiological function; high expression of the DNMT1 gene was associated with peptide cross-linking and humoral immunity.
(III) advantageous effects
The biomarker has higher specificity on head and neck cancer monitoring and curative effect evaluation, and can be used for dynamically monitoring head and neck cancer recurrence and metastasis. The marker of the invention can be combined with other clinical indexes to provide more accurate judgment for screening, diagnosis, treatment and prognosis of head and neck cancer.
Drawings
FIG. 1 is a mutation site diagram of DNMT3A and TET2 genes;
FIG. 2 is a graph of functional alterations in NSUN4 and NSUN7 genes in relation to patient survival;
FIG. 3 is a diagram of drive gene in HNSCC;
FIG. 4 is a graph of CNV versus expression level for the m5C regulatory gene;
FIG. 5 is a graph of different clinical ratings versus patient prognosis;
FIG. 6 is a cluster heatmap of m5C regulatory genes with different Stage cases;
FIG. 7 is a graph comparing the expression of the m5C regulatory gene in different clinical grade cases;
FIG. 8 is a plot of Survival curve and AUC for multifactor COX regression;
FIG. 9 is a graph of DNMT1 and ALYREF gene expression versus patient prognosis;
FIG. 10 is a graph of AUC versus risk analysis of the DNMT1 and ALYREF genes;
FIG. 11 is a graph of enriched genes in DNMT1 and ALYREF gene mRNA expression level samples;
FIG. 12 is a graph of AUC versus risk analysis of the DNMT1 and ALYREF genes in the validation dataset;
FIG. 13 is a graph demonstrating the expression of DNMT1 in the data set as a function of patient prognosis.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Screening of head and neck cancer gene markers
(1) Composition of m5C regulatory gene
Writers: NSUN1, NSUN2, NSUN3, NSUN4, NSUN5, NSUN6, NSUN7, DNMT1, DNMT2, DNMT3A, and DNMT3B genes;
erases: the TET2 gene;
readers: the ALYREF gene.
(2) Clinical information screening
All HNSCC clinical data, CNVs, mutations and mRNA expression data were retrieved from the TCGA website by TCGA-assembler and downloaded in 2019 for 12 months; the verification data set for the verification results is from the TCGA, and is retrieved by the TCGA-assembler from the TCGA website for download.
For HNSCC transcriptome data, 500 tumor samples were obtained and the downloaded data were TPM and FPKM. For SNV data of HNSCC, acquiring data of 508 tumor samples, and downloading level3 data after muTect processing; for CNV data, 521 tumor samples were total; for the clinical information data, 527 pieces of clinical information were in total.
After integrating the data, samples with incomplete clinical information and survival time less than 30 days were removed, and 490 samples were available for survival analysis.
(3) Results and analysis
1) mutations in the m5C regulatory gene and CNV
In the 508 HNSCC patient sequencing data, the m5C regulatory gene appeared in 82 independent samples.
TABLE 1 overview of m5C regulatory gene mutations in HNSCC samples
As can be seen from Table 1, DNMT3A of the "Writer" gene had the largest number of mutations, followed by TET2 of the "Eraser" gene; the number of mutations in the NSUN2, DNMT1 and DNMT3B genes was also high.
TABLE 2 functional annotation statistics for m5C regulatory genes in HNSCC samples
Statistically, 43 samples were functionally altered by the annotated m5C regulatory gene, and 11 genes among the m5C regulatory genes were annotated to be functionally altered; as can be seen from Table 2, all of the 11 genes were mismutated (MissenseNutation).
As shown in FIG. 1, the mismutation of the gene may cause the function of the gene to be damaged, and the m5C is influenced to regulate the transmission of the gene signal in the cell, so that the functional disorder is caused. The high mutation of the DNMT3A and TET2 genes in the tumor cells indicates that the function of the m5C regulatory gene in the tumor cells may be abnormal.
The survival of HNSCC patients was prognosed using 11 functionally altered genes, and the prognosis was worse in the 2-gene-mutated samples than in the non-mutated samples, as shown in FIG. 2, in which there was a significant correlation between the mutations of NSUN4 and NSUN7 genes and the prognosis.
TABLE 3 CNV statistical Table of m5C regulatory genes in HNSCC samples
In 521 HNSCC samples with CNV data, it was observed that the m5C regulatory gene has a high frequency of CNV. As can be seen from Table 3, the frequency of the "Writer" gene NSUN2 was 61.65%, followed by the "Writer" gene NSUN3, and the frequency of CNV events was 61.09%, while the frequency of the "Writer" gene DNMT3A was the lowest, 11.09%.
2) The change of m5C regulatory gene is related to clinical pathological characteristics and molecular characteristics
COX regression analysis was performed for each clinical profile and the results are shown in table 4.
TABLE 4 COX assay Table of clinical features and m5C regulatory Gene alterations
As can be seen from table 4, survival and age of HNSCC patients was significantly correlated with TNM Stage, but not with whether SNV or CNV was developed. From the perspective of global changes in the m5C regulatory gene, neither SNV nor CNV alone, nor both, are significant in their correlation with patient prognosis.
Since the driver gene that plays an important role in the onset of HNSCC is not clear, the driver gene is predicted using the oncode function of maftools. As shown in fig. 3, MAPK, HRAS, RAC1, HIST1H4K and HIST1H3C are potential driver genes in HNSCC, and alterations in m5C regulatory genes are not significantly associated with alterations in MAPK1, HIST1H4K and HIST1H3C, but significantly associated with alterations in HRAS and RAC 1.
TABLE 5 relationship of alterations in m5C regulatory genes to HNSCC-associated high frequency disease genes
As can be seen from table 5, in 27 patients with altered HRAS, alterations in the m5C regulatory gene were detected in 14 samples.
From the above analysis, CNV changes of m5C regulated genes were more significant than SNV changes, which could affect the expression levels of genes by dose-compensating effects. Next, the effect of the alteration of the m5C regulatory gene on mRNA expression was evaluated. First, the differentially expressed genes in HNSCC and normal tissue samples were analyzed, and no differential expression of the m5C regulatory gene was found between tumor and paracarcinoma.
Among 11 genes of the m5C regulatory genes, 10 gene mRNA expression levels of the m5C regulatory gene were significantly associated with different CNV patterns in 500 HNSCC samples. For these 10 genes, an increase in copy number of 10 genes was associated with higher mRNA expression; whereas deletion results in decreased mRNA expression (as shown in figure 4). These 10 genes are distributed throughout the regulatory process of the m5C regulatory gene.
3) Association between m5C regulatory genes and survival in HNSCC patients
As shown in FIG. 5, it can be seen that age and TNM Stage stratification correlate with the prognosis of HNSCC patients. TNM Stage I and II were considered as Low TNM Stage cases, while TNM Stage III and IV above were High TNM Stage cases. Based on this division, the expression of the m5C regulatory gene in different TNM Stage cases was clustered (as shown in fig. 6), and the results showed that the m5C regulatory gene tended to be highly expressed in high TNM Stage cases.
Next, the expression differences of m5C regulatory genes in different clinical grade cases were analyzed. The results show that the expression of only 2 genes was significantly correlated with the clinical grade of the patient and showed a positive correlation (as shown in fig. 7).
There is no significant correlation between CNV of m5C regulatory gene and patient prognosis, but there is a very strong positive correlation between the expression level of part of m5C regulatory gene and CNV. To this end, COX regression was used to explore the relationship of the expression levels of the various m5C regulated genes to patient prognosis for a single factor.
TABLE 6 COX single factor analysis Table
As can be seen from table 6, the expression values of 3 genes were significantly correlated with the prognosis of the patients (p < 0.05), and the expression levels of these genes were significantly correlated with their CNV changes. The risk of the patient is evaluated by using 11 gene expression information of the m5C regulatory gene, as shown in fig. 8, the expression of the m5C regulatory gene can obviously judge the risk of the patient, and the AUC curve areas of 1 year and 3 years are both more than 0.55; the results indicate that the expression of the m5C regulatory gene can be used as a prognostic marker of head and neck cancer.
TABLE 7 Lasso analysis results of m5C regulatory gene
Combining the results of 1000 Lasso regressions, it is known that there are more than 700 repeats in Lasso results and that their CNVs have a significant effect on expression levels, and that there are 2 genes whose expression has a significant impact on prognosis, namely DNMT1 and the ALYREF genes (as shown in table 7). It can be seen that these two genes are the Writer gene and the Reader gene, respectively, which are involved in the important functional regulation of the m5C regulatory gene.
Next, the relationship between gene expression and patient prognosis was analyzed using the expression levels of 2 genes as indices, and the results showed that high expression of ALYREF was associated with poor prognosis of the patients, while low expression of DNMT1 was associated with poor prognosis of the patients (as shown in FIG. 9).
These two genes were used to perform a COX regression analysis to calculate a patient risk value. The median risk value was used for patient risk prediction, and it was found that the expression of 2 genes was effective for analytical prediction of HNSCC patients (as shown in fig. 10). The AUC of 2 m5C regulated genes was greater than 0.57 for both 1 year and 3 years, and the p-value for the risk prediction of patients with them as markers was also less than 0.05.
4) Functional enrichment analysis of 2 Gene expression levels
Given that DNMT1 is one of the Writer genes in methylation; while ALYREF is one of the Reader genes in the methylation process, the role of m5C dysregulation in the pathogenesis of HNSCC is next explored. The enriched gene set in the 2 gene mRNA expression level samples was examined.
As shown in fig. 11, gene enrichment analysis indicates that high ALYREF expression is associated with various mitochondrial physiological functions); while high DNMT1 was associated with peptide cross-linking and humoral immunity. The high expression of ALYRF is related to high mitochondrial activity, which suggests that the high ALYRF promotes intracellular energy metabolism, maintains the continuous energy supply of cells and has positive significance for the growth of tumor cells. While high expression of DNMT1 was immunologically relevant, the value of DNMT1 in HNSCC immunotherapy was next explored.
Verification analysis of 2 genes as important head and neck cancer targeting molecules in verification data set
Next, we analyzed the relationship between 2 gene expression and patient survival using the validation dataset. Since there are few studies on HNSCC, we adopted as our validation data set other cancers of squamous cell carcinoma that are homologous to HNSCC. Among the squamous cell carcinomas of TCGA we selected CESC (cervical squamous cell carcinoma) as the validation dataset. Since CESC has a similar cellular origin and tumor tissue structure to HNSCC.
Based on COX regression analysis, the risk value of the sample was calculated using the 2 gene expression levels and the risk values. We found that 2 genes also have good risk prediction capability in the validation dataset TCGA _ CESC dataset. AUC was around 0.7 (as shown in fig. 12). Meanwhile, in the validation dataset, low expression of the DNMT1 gene was also associated with a poor prognosis (as shown in fig. 13).
Example 1
The m5C regulatory gene is applied to the preparation of a reagent or a kit for diagnosing, monitoring, evaluating the curative effect or monitoring the metastasis and recurrence of head and neck cancer; the change of m5C regulatory gene is significantly related to the change of HRAS and RAC 1; the m5C regulatory gene tends to be highly expressed in the case of high TNMStage.
The m5C regulatory genes include:
writers, methylases, RNA methylation modification processes, currently known components are NSUN1, NSUN2, NSUN3, NSUN4, NSUN5, NSUN6, NSUN7, DNMT1, DNMT2, DNMT3A and DNMT 3B;
erasers, demethylases, with TET 2;
readers, m5C binds to the protein, recognizes and binds to the m5C site on mRNA, with ALYREF.
Expression of the Eraser and Reader genes in the m5C regulatory gene was significantly associated with CNV.
Example 2
The application of the DNMT1 gene in preparing a reagent or a kit for diagnosing, monitoring, evaluating the curative effect or monitoring metastasis and relapse of head and neck cancer; high expression of the ALYREF gene is associated with a poor prognosis in HNSCC patients.
The reagent or kit comprises a primer specifically recognizing DNMT1 gene or a primer or probe specifically recognizing DNMT1 protein nucleic acid.
Preferably, low expression of the DNMT1 gene is associated with a poor prognosis in patients with HNSCC and high expression of the DNMT1 gene is associated with peptide cross-linking and humoral immunity.
Example 3
The ALYREF gene is applied to the preparation of a reagent or a kit for diagnosing, monitoring, evaluating the curative effect or monitoring metastasis and recurrence of head and neck cancer; high expression of the ALYREF gene is associated with a poor prognosis in patients with HNSCC; high expression of the ALYREF gene is associated with mitochondrial physiological function.
The reagent or the kit comprises a primer for specifically recognizing the ALYREF gene or a primer or a probe for specifically recognizing the ALYREF protein nucleic acid.
Example 4
The application of DNMT1 gene and ALYREF gene in preparing a reagent or a kit for diagnosing, monitoring, evaluating curative effect or monitoring metastasis and recurrence of head and neck cancer; the low expression of DNMT1 gene is related to the poorer prognosis of HNSCC patients, and the high expression of ALYREF gene is related to the poorer prognosis of HNSCC patients; high expression of the ALYREF gene is related to mitochondrial physiological function, and high expression of the DNMT1 gene is related to peptide cross-linking and humoral immunity.
The reagent or the kit comprises a primer for specifically recognizing DNMT1 gene and ALYREF gene or a probe for specifically recognizing DNMT1 and ALYREF protein nucleic acid.
The biomarker has higher specificity on head and neck cancer monitoring and curative effect evaluation, and can be used for dynamically monitoring head and neck cancer recurrence and metastasis. The marker of the invention can be combined with other clinical indexes to provide more accurate judgment for screening, diagnosis, treatment and prognosis of head and neck cancer.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (8)
- The application of the m5C regulatory gene in preparing a reagent or a kit for diagnosing, monitoring, evaluating the curative effect or monitoring the metastasis and recurrence of head and neck cancer.
- 2. The use of the m5C regulatory gene according to claim 1 in the preparation of a reagent or kit for head and neck cancer diagnosis, monitoring, efficacy assessment or monitoring of metastasis/recurrence, wherein: the alteration of the m5C regulatory gene was significantly associated with the alteration of HRAS and RAC 1.
- 3. Use of the m5C regulatory gene according to claim 1 or 2 for preparing a reagent or kit for head and neck cancer diagnosis, monitoring, efficacy assessment or monitoring of metastasis/recurrence, wherein: the expression of the Eraser and Reader genes in the m5C regulatory gene is obviously related to CNV.
- 4. The use of the m5C regulatory gene according to claim 3 for preparing a reagent or kit for head and neck cancer diagnosis, monitoring, efficacy assessment or metastasis/recurrence monitoring, wherein: the m5C regulatory gene tends to be highly expressed in the case of high TNM Stage.
- The application of the DNMT1 gene or/and the ALYREF gene in preparing a reagent or a kit for diagnosing, monitoring, evaluating curative effect or monitoring metastasis and recurrence of head and neck cancer.
- 6. The use of the DNMT1 gene or/and the ALYREF gene according to claim 5 for preparing a reagent or a kit for head and neck cancer diagnosis, monitoring, efficacy assessment or metastasis/recurrence monitoring, wherein: the low expression of the DNMT1 gene is related to the poorer prognosis of HNSCC patients, and the high expression of the ALYREF gene is related to the poorer prognosis of the HNSCC patients.
- 7. The use of the DNMT1 gene or/and the ALYREF gene according to claim 7 for preparing a reagent or a kit for head and neck cancer diagnosis, monitoring, efficacy assessment or metastasis/recurrence monitoring, wherein: the reagent or kit comprises a primer which specifically recognizes the DNMT1 gene or/and the ALYREF gene.
- 8. The use of the ALYREF gene of any one of claims 5 to 7 in the preparation of a reagent or kit for head and neck cancer diagnosis, monitoring, efficacy assessment or metastasis/recurrence monitoring, wherein: the high expression of the ALYREF gene is related to the physiological function of mitochondria; the high expression of the DNMT1 gene was associated with peptide cross-linking and humoral immunity.
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