CN105671187B

CN105671187B - Group of genes for molecular typing of head and neck squamous cell carcinoma and application thereof

Info

Publication number: CN105671187B
Application number: CN201610212589.0A
Authority: CN
Inventors: 宁云山; 李妍
Original assignee: Southern Medical University
Current assignee: Southern Medical University
Priority date: 2016-04-08
Filing date: 2016-04-08
Publication date: 2020-06-05
Anticipated expiration: 2036-04-08
Also published as: CN105671187A

Abstract

The invention provides a group of genes for HNSCC molecular typing, which comprise a TP53 gene, a CDKN2A gene, a FAT1 gene, a CASP8 gene, an AJUBA gene, a PIK3CA gene, a NOTCH1 gene, a KMT2D gene, an NSD1 gene, an HLA-A gene, an HRAS gene, an FBXW7 gene, an RB1 gene, a PIK3R1 gene, a TRAF3 gene, an NFE2L2 gene, a CUL3 gene and a PTEN gene. The invention also provides application of the gene in preparation of a kit and a gene chip for HNSCC molecular typing, and the kit and the gene chip for HNSCC molecular typing prepared by using the gene. The method can enhance the rationality and accuracy of HNSCC typing, thereby providing key technical support for realizing early diagnosis, effective blocking and individualized treatment of HNSCC.

Description

Group of genes for molecular typing of head and neck squamous cell carcinoma and application thereof

Technical Field

The invention relates to a group of genes for molecular typing of head and neck squamous cell carcinoma and application thereof in preparing a kit for molecular typing of head and neck squamous cell carcinoma.

Background

Head and Neck Squamous Cell Carcinoma (HNSCC) is one of the most common malignant tumors of the head and neck, mainly including lip cancer, gum cancer, tongue cancer, soft and hard palate cancer, jaw cancer, mouth floor cancer, oropharyngeal cancer, salivary gland cancer, and maxillary sinus cancer, and cancers occurring in facial skin mucosa, and the like, and generalized oral cancer includes cancers occurring in the range below the eye orbit and above the neck, mostly belonging to squamous cell carcinoma, collectively referred to as head and neck squamous cell carcinoma, and in developing countries of china is a high-incidence area of HNSCC. Global HNSCC mortality is ranked sixth in cancer mortality, with less than 50% survival 5 years after diagnosis, and is a poor prognosis, devastating disease (Petersen, P.E, (2009) Oral cancer prevention and control-the improvement of the World Health organization. Oral Oncol 45, 454-460). Despite The long-term development of tumor therapies such as surgery, radiotherapy and chemotherapy over The last thirty years, The survival rate of patients with HNSCC has been limited, and this persistent high mortality rate has been attributed primarily to metastasis and recurrence of HNSCC (Leemans, c.r., Braakhuis, b.j., and Brakenhoff, r.h. (2011) The molecular biology of liver and neural Cancer. nat Rev Cancer 11, 9-22). HNSCC is the result of combined action of internal and external factors such as somatic gene mutation, tobacco, alcohol, HPV infection and the like, has high heterogeneity, and causes great difference in clinical treatment effect and prognosis.

Currently, histological grading methods described by Broders and adopted by WHO are still used clinically for typing of HNSCC. The method is divided into 3 grades according to the tumor cell keratinization degree, the cell and nucleus polymorphism and the subjective evaluation of nuclear division: grade 1 (high differentiation), grade 2 (medium differentiation), grade 3 (low differentiation), the highest grade determining the final classification when the tumor shows different grades (Barns L, et al (2005.) Pathology and genetics of head and channel tumor: IARCPress, 168-. However, most authorities and scholars currently believe that the relevance of the Broders/WHO histology grading system to prognosis, treatment response is poor in individual patients (Woolgar JA (2006.) histopathologist inorganic and radiopharmaceutical tissue and tissue cancer. oral Oncol 42: 229-: the differentiation of the tumor is not careful, and the tissue allotype exists, so that the specimen is limited; grading depends on the structural characteristics of the tumor cells rather than on function; tumor cells are not evaluated in connection with their supporting stroma and host tissues, etc. Therefore, establishing a new and more accurate HNSCC typing is an urgent method for the clinical treatment of head and neck tumors, and is also the basis and precondition for developing individualized treatment.

In recent years, the rapid development of high-throughput gene sequencing technology provides possibility for HNSCC molecular typing and precise treatment. In 2013, the International Cancer Genome Consortium (International Cancer Genome Consortium) utilizes an exon sequencing technology to sequence the gingival-cheek squamous cell carcinoma in the oral Cancer, and finds that the gingival-cheek squamous cell carcinoma has other specific variant genes besides common variant genes TP53, HRAS and the like of head and neck malignant tumors, further proves the difference of gene variation among different head and neck malignant tumors, and provides a wide prospect for the gene typing of HNSCC and guiding the clinical treatment of the HNSCC. Meanwhile, targeted drug therapy based on molecular typing also offers the possibility of individualized treatment of HNSCC.

Cetuximab (Cetuximab) was the first FDA-approved molecular target drug for head and neck squamous cell carcinoma, acting primarily on the extracellular domain of EGFR. One compelling outcome from a multicenter clinical trial (N424) has shown that cetuximab in combination with Radiotherapy effectively reduces the local recurrence rate of head and neck tumors, extends patient survival, and significantly improves patient five-year survival compared to Radiotherapy alone (Bonner, j.a., Harari, p.m., Giralt, j., azania, n., Shin, d.m., Cohen, r.b., Jones, c.u., Sur, r., Raben, d., Jassem, j., et al (2006). In another multicenter Phase III clinical trial (N117), cetuximab in combination with cisplatin treatment significantly improved chemotherapy sensitivity compared to cisplatin single-drug chemotherapy (Burtness, B., Goldwasser, M.A., Flood, W., Mattar, B., Forastiere, A.A., and Easter Cooperative Oncology, G (2005), Phase III random trial of clinical usage board complex with central clinical in therapeutic/recording trial and nuclear in clinical trial: of clinical trial Group. Recent findings by domestic scholars are: the survival rate of the oral squamous cell carcinoma stage III patient can be obviously improved by combining the recombinant adenovirus p53 gene therapy with conventional chemotherapy, but the survival rate of the oral squamous cell carcinoma stage IV patient is not obviously promoted, and the establishment of molecular typing of HNSCC and individualized treatment thereof is imperative.

Therefore, the establishment of molecular typing based on the guidance of molecular markers and effective personalized clinical treatment schemes from the genetic level has important significance for clinical personalized treatment and accurate head and neck treatment of HNSCC patients.

Disclosure of Invention

The invention provides a group of genes for HNSCC molecular typing and a detection kit thereof, aiming at the problems that the existing HNSCC typing method is relatively limited, especially the research on HNSCC molecular typing gene groups of Chinese people is very limited, the accuracy of the existing assessment method is not high, and the clinical personalized treatment of HNSCC cannot be guided.

The invention provides a group of genes for molecular typing of head and neck squamous cell carcinoma, which specifically comprises the following 18 genes: TP53 gene, CDKN2A gene, FAT1 gene, CASP8 gene, AJUBA gene, PIK3CA gene, NOTCH1 gene, KMT2D gene, NSD1 gene, HLA-A gene, HRAS gene, FBXW7 gene, RB1 gene, PIK3R1 gene, TRAF3 gene, NFE2L2 gene, CUL3 gene and PTEN gene.

The invention also provides application of the genes for molecular typing of the head and neck squamous cell carcinoma in preparing a kit for molecular typing of the head and neck squamous cell carcinoma. Therefore, the invention provides a group of PCR primers for molecular typing of head and neck squamous cell carcinoma and a group of probes for molecular typing of head and neck squamous cell carcinoma. The nucleotide sequences of the PCR primers are shown as SEQ ID No.1 to SEQ ID No.36, and the nucleotide sequences of the probes are shown as SEQ ID No.37 to SEQ ID No. 54. The invention also provides the application of the PCR primer and the probe for the molecular typing of the head and neck squamous cell carcinoma in the preparation of a kit and a gene chip for the molecular typing of the head and neck squamous cell carcinoma.

Another aspect of the present invention also provides a kit for molecular typing of head and neck squamous cell carcinoma, which comprises PCR primers capable of amplifying a set of genes for molecular typing of head and neck squamous cell carcinoma according to claim 1 and/or probes capable of being complementary to a set of gene sequences for molecular typing of head and neck squamous cell carcinoma according to claim 1. The nucleotide sequence of the PCR primer can be shown as SEQ ID No.1 to SEQ ID No. 36. The nucleotide sequences of the probes can be shown as SEQ ID No.37 to SEQ ID No. 54.

In addition, the invention also provides a gene chip for molecular typing of the head and neck squamous cell carcinoma, which comprises a solid phase carrier and a probe, wherein the nucleotide sequence of the probe can be shown as SEQ ID No.37 to SEQ ID No. 54.

The invention introduces the gene-based molecular typing into the molecular typing of the HNSCC for the first time, further enhances the rationality and accuracy of the HNSCC typing, and improves the capability of guiding the clinical treatment of the HNSCC. Meanwhile, the method is verified and optimized in the population of the Chinese HNSCC, and a molecular typing scheme suitable for the population of the Chinese HNSCC patients is created, so that a key technical support is provided for realizing early diagnosis, effective blocking and individualized treatment of the HNSCC, and the method has important clinical significance in the accurate treatment of the HNSCC.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

The invention establishes a gene molecular marker combination model through the combined application of gene detection, molecular marker combination and a data mining algorithm, utilizes polygene to evaluate HNSCC molecular typing, and mainly comprises the following steps:

(1) collecting clinical diagnosis data and gene expression profile data of HNSCC, and constructing an HNSCC gene expression profile database containing known 2 thousands of genes and 789 samples;

(2) statistical analysis is carried out on the gene expression pattern, 18 genes which are closely related to HNSCC typing are screened out, and the genes are respectively as follows: TP53 gene, CDKN2A gene, FAT1 gene, CASP8 gene, AJUBA gene, PIK3CA gene, NOTCH1 gene, KMT2D gene, NSD1 gene, HLA-A gene, HRAS gene, FBXW7 gene, RB1 gene, PIK3R1 gene, TRAF3 gene, NFE2L2 gene, CUL3 gene and PTEN gene.

(3) The 18 gene expression patterns in the HNSCC clinical sample are detected by methods such as PCR, gene expression chip and high-throughput sequencing, and typing evaluation is carried out on the HNSCC clinical sample.

The detection method for evaluating HNSCC molecular typing provided by the invention mainly comprises the following steps:

(1) RNA was extracted from clinical specimens. The clinical sample is tumor tissue taken from the subject, may be a fresh sample or a formalin-fixed paraffin embedded (FFPE) sample, or may be peripheral blood or saliva of the subject;

(2) the expression pattern and expression level of the above 18 genes in clinical samples of the subjects were examined by gene expression detection method and molecular typing of HNSCC was evaluated by statistical method. The gene expression detection method comprises digital PCR, quantitative RT-PCR, a gene chip, a high-throughput sequencing technology and the like, and preferably selects the digital PCR and the gene chip.

The kit for HNSCC molecular typing of the invention comprises primers and/or probes, wherein the primers are preferably synthetic oligonucleotide fragments with high gene specificity, and only need to be complementary with partial sequences of genes in the gene group for evaluating HNSCC molecular typing of the invention and amplify the genes of the gene group for evaluating HNSCC molecular typing of the invention. Preferably, the nucleotide sequence of the primer is shown as SEQ ID NO. 1-SEQ ID NO.36 in the sequence table. The probe is preferably a synthetic oligonucleotide fragment with high gene specificity, as long as it is complementary to a partial sequence of a gene in the HNSCC molecular typing genome to be evaluated according to the present invention. Preferably, the nucleotide sequence of the probe is shown as SEQ ID NO. 37-SEQ ID NO.54 in the sequence table.

Example 1Screening to assess HNSCC molecular typing genomes

789 HNSCC tumor gene whole gene sequencing, exon sequencing, RNA sequencing, gene expression chip and clinical variable related data published or stored in TCGA, COSMIC and other databases are analyzed by an EPIG gene expression profiling program, and HNSCC molecular typing gene groups are screened and evaluated. The analysis comprises the steps of carrying out unsupervised clustering analysis to identify an expression profile and related genes; and (5) analyzing the stability of the candidate gene. Candidate genes (r > 0.7, P < 0.001) were selected by first calculating the correlation coefficient of each gene to each other in 789 samples by clustering analysis and forming gene-specific expression profiles (r > 0.7, P < 0.001) by clustering, and then calculating the correlation of each gene to the specific expression profiles. The experimental results are as follows: the 18 genes that gave the highest score for assessing molecular typing of HNSCC were screened and are listed in table 1 below.

Table 1: 18 genes that can be used to assess molecular typing of HNSCC

Serial number	Name of gene	Gene identity number ID
			1	TP53	NM_001126118.1
2	CDKN2A	XM_011517679.1
			3	FAT1	NM_005245.3
4	CASP8	XM_006712793.2
			5	AJUBA	NM_198086.2
6	PIK3CA	NM_006218.2
			7	NOTCH1	NM_017617.4
8	KMT2D	NM_003482.3
			9	NSD1	NM_022455.4
10	HLA-A	NM_002116.7
			11	HRAS	NM_001130442.2
12	FBXW7	NM_033632.3
			13	RB1	NM_000321.2
14	PIK3R1	NM_001242466.1
			15	TRAF3	NM_145726.2
16	NFE2L2	NM_001145412.2
			17	CUL3	NM_001257198.1
18	PTEN	NM_000314.6

Example 2Molecular typing for HNSCC assessment by digital PCR method and Gene expression chip

In this example, the expression levels of the 18 genes screened in example 1 in paraffin-embedded tumor tissues and matched paracarcinoma tissues of 100 groups of patients with HNSCC in China were detected by using digital PCR and gene expression chip method. Primers and probes used for detection by the digital PCR method were synthesized by Gene Synthesis (Shanghai Biotech, Inc.) using Primer Premier5.0 design based on the human whole gene sequence published by NCBI (national center for Biotechnology information). The nucleotide sequences of the primers are shown in the following table 2 (SEQ ID Nos. 1 to 36 in the sequence table), and the nucleotide sequences of the probes are shown in the following table 3 (SEQ ID Nos. 37 to 54 in the sequence table).

Table 2: 18 gene PCR primer sequences for evaluating HNSCC molecular typing

Table 3: 18 genetic probe sequences useful for assessing molecular typing of HNSCC

Digital PCR (digital PCR) is a newly developed third-generation PCR technology, and compared with a quantitative real-time PCR (qPCR), the digital PCR technology can greatly improve the sensitivity, accuracy and repeatability of a detection method and realize absolute quantification in a real sense. At present, the digital PCR technology mainly includes two types, namely, a large-scale integrated microfluidic chip digital PCR (cdpcr) and a droplet digital PCR (ddPCR), in which a DNA sample to be detected is randomly distributed into a micro-reactor or a droplet of a chip before PCR amplification, the micro-reactor or the droplet contains or does not contain a nucleic acid target molecule to be detected, or at least contains a nucleic acid target molecule to be detected, and each micro-reactor or droplet is an independent PCR reactor. After PCR amplification, the detector detects each micro-reactor or micro-droplet, if a fluorescence signal exists, the micro-reactor or micro-droplet is judged to be '1', if no fluorescence signal exists, the micro-reactor or micro-droplet is judged to be '0', and finally the copy number or the concentration of the target sequence in the sample to be detected is calculated according to the Poisson distribution principle and the proportion of the positive micro-droplets.

In this example, total RNA was extracted from paraffin-embedded samples confirmed to be HNSCC by pathological diagnosis using Trizol RNA extraction kit; treatment with DNase to ensure complete decontamination of genomic DNA; obtaining cDNA after reverse transcription. Detection of expression of 18 genes was performed using digital PCR. The digital PCR is completed on a QX200 micro-drop digital PCR system of Bio-Rad company, after the PCR reaction is finished, the molecular typing is carried out according to the expression modes and the expression levels of 18 genes in the 100 groups of samples through statistical analysis and by combining with corresponding clinical data, the molecular typing is totally classified into types 4 (I-IV), and the specific results are shown in the following table 4.

Table 4: molecular typing results of HNSCC by digital PCR method

Example 3Molecular typing to assess HNSCC by Gene expression chips

In this example, a gene expression chip method was used to detect the expression levels of 18 genes selected in example 1 in paraffin-embedded tumor tissues and matched paracarcinoma tissues of 100 groups of patients with HNSCC in China, which are the same as those in example 2. The probes used for the gene expression chip were synthesized by gene synthesis company (Shanghai Biotech, Ltd.) using Primer premier5.0 designed based on the human whole gene sequence published by NCBI (national center for Biotechnology information). The nucleotide sequence of the probe is shown in sequence tables SEQ ID No. 37-SEQ ID No. 54.

The gene expression chip is a detection method which is characterized in that a large number of DNA segments or oligonucleotide segments with specific sequences are fixed on carriers such as glass, silicon chips and the like at high density according to a matrix by a microelectronic technology and a micromachining technology, a sample to be detected is labeled by fluorescent molecules, then is hybridized with the large DNA or oligonucleotide segments on the chip, and gene information is obtained by fluorescent scanning and computer analysis. The method is characterized in that the method can carry out rapid, accurate and high-flux detection and analysis on nucleic acid sequence information in a trace sample, and can simultaneously obtain expression abundance and expression pattern correlation maps of thousands of genes.

In this example, total RNA was extracted from paraffin-embedded samples confirmed to be HNSCC by pathological diagnosis using Trizol RNA extraction kit; treatment with DNase to ensure complete decontamination of genomic DNA; obtaining cDNA after reverse transcription. Mixing cDNA with gene chip, and hybridizing; the expression abundance of the gene was detected by scanning the fluorescent signal. After the reaction of the gene chip is finished, according to the expression patterns and expression levels of 18 genes in the 100 groups of samples, the molecular typing is carried out according to the example 2 by statistical analysis and combination of corresponding clinical data, and the specific results are shown in the table 5.

Table 5: gene expression chip HNSCC molecular typing results

Comparing the embodiment 2 with the embodiment 3, 18 typing related gene expression detections are carried out on the same group of 100 groups of Chinese HNSCC by adopting a digital PCR and a gene expression chip, and the results of the two detection methods on molecular typing of the HNSCC are basically consistent. The results show that 18 HNSCC typing related genes obtained by combining HNSCC whole genome sequencing, exon sequencing and RNA sequencing with clinical data can be used for evaluating HNSCC molecular typing.

Example 4Molecular typing for assessment of HNSCC by high throughput sequencing

In order to further verify the molecular typing method of the same group of 100 Chinese HNSCC samples in example 2 and example 3, the invention further adopts a high-throughput sequencing technology to randomly sample the typed (I-IV) samples in example 2 and example 3 (each subtype is 10 groups of samples, namely 10 HNSCC cancer tissues and 10 corresponding paired paracarcinoma tissues) for carrying out transcriptome sequencing verification. Transcriptome sequencing based on high throughput sequencing technology enables analysis of the structure and expression levels of transcripts in a sample, providing the most comprehensive transcriptome information. The method comprises the following specific steps:

step 1: total RNA was extracted from 100 groups of Chinese HNSCC sample tissues (RNA extraction kit from tissues was used from Roche).

Step 2: the resulting RNA is made into a library for sequencing. Preparing the RNA of the obtained tissue into a library for second-generation sequencing, wherein the preparation method of the library comprises the following steps:

the RNA of the extracted tissue is used for generating cDNA of the screened 18 molecular typing genes by reverse transcriptase under the direction of a specific primer. The ends were filled in and 5' phosphorylated, and 30. mu.l of DNA, 45. mu.l of pure water, 10. mu.l of T4DNA ligase buffer with 10mM ATP, and 4. mu.l of buffer containing 10mM dNTP Mix, 5. mu.l of T4DNA polymerase, l. mu.l of Klenow enzyme, and 5. mu.l of T4 ligase were mixed and incubated at 20 ℃ for 30 minutes (reagents from Illumina sample preparation kit PE-102-. End suspension A: the product of the above step was dissolved in 32. mu.l buffer, 5. mu.l of Klenow buffer, 1mM dATP 10. mu.l, Klenow E χ o-3. mu.l were added, and the mixture was held at 37 ℃ for 30 minutes (reagents from Illumina sample preparation kit), and the product was ligated by QIAGEN MinElute PCR purification kit: DNA was dissolved in 10. mu.l of buffer, and 2. mu.l of DNA ligase buffer, 25. mu.l of PE Adapter Oligo Mix 10. mu.l and 5. mu.l of DNA ligase were added, and the mixture was kept at 20 ℃ for 15 minutes (reagent: Illumina sample preparation kit), and after incubation, DNA was purified using QIAGEN QIAquick PCR purification kit to obtain a library.

And step 3: the nucleotide sequence of the primer is shown in sequence tables SEQ ID No. 1-SEQ ID No.36, and the obtained DNA library is subjected to second generation sequencing by MiSeq. Paired-end sequencing was performed with Illumina MiSeq sequencer. This process is done automatically by the instrument itself (Illumina).

And 4, step 4: and (6) analyzing results. Analyzing the obtained sequencing result, wherein the sequencing result shows that: there were significant differences in the expression of the molecular typing genes in each HNSCC subtype compared to paired paraneoplastic tissues.

Example 5HNSCC molecular typing PCR kit

The PCR technology utilizes specific primers and probes to realize the in vitro amplification and quantification of target genes. The kit provided by the invention can be used for evaluating HNSCC molecular typing by adopting a real-time quantitative PCR technology and a digital PCR technology, and preferably adopts the digital PCR technology. The invention provides a HNSCC molecular typing PCR kit, which comprises a specific primer and/or a probe, wherein the nucleotide sequence of the primer is shown as SEQ ID No. 1-SEQ ID No.36 in a sequence table, and the nucleotide sequence of the probe is shown as SEQ ID No. 37-SEQ ID No.54 in the sequence table.

The technology of preparing detection kits by using probes and primers with known nucleotide sequences is well known to those skilled in the art, and therefore, the specific steps of the kit for HNSCC molecular typing provided by the present invention are not described herein.

Example 6HNSCC molecular typing gene chip

The gene chip is a gene expression profile chip, mainly using oligonucleotide fragment as probe and solidified on the chip. The mRNA of a sample to be detected (a processing group) and the mRNA of a control sample are marked by two different fluorescent molecules, then the samples are hybridized with a chip at the same time, and the expression difference of related genes of HNSCC molecular typing is detected by analyzing the ratio of the fluorescent intensity of the hybridization of the two samples and the probe, so that the molecular typing of the HNSCC is evaluated. The invention provides a HNSCC molecular typing gene chip, which comprises a specific probe, wherein the nucleotide sequence of the probe is shown as SEQ ID No. 37-SEQ ID No.54 in a sequence table.

The technology of preparing gene chips by using probes with known nucleotide sequences is well known to those skilled in the art, and therefore, the specific steps of the gene chip for HNSCC molecular typing provided by the present invention are not repeated herein.

The foregoing is merely an exemplary embodiment of the invention. The scope of the present invention is not limited thereto, and any person skilled in the art can conceive of changes or substitutions within the technical scope of the present invention without any inventive work, and shall be covered thereby.

Claims

1. Use of a set of genes for molecular typing of head and neck squamous cell carcinoma for the preparation of a kit for molecular typing of head and neck squamous cell carcinoma, characterized in that the set of genes for molecular typing of head and neck squamous cell carcinoma consists of 18 genes as follows: TP53 gene, CDKN2A gene, FAT1 gene, CASP8 gene, AJUBA gene, PIK3CA gene, NOTCH1 gene, KMT2D gene, NSD1 gene, HLA-A gene, HRAS gene, FBXW7 gene, RB1 gene, PIK3R1 gene, TRAF3 gene, NFE2L2 gene, CUL3 gene and PTEN gene.

2. The application of a group of PCR primers for molecular typing of the head and neck squamous cell carcinoma in preparing a kit for molecular typing of the head and neck squamous cell carcinoma is characterized in that the nucleotide sequences of the PCR primers are shown as SEQ ID No.1 to SEQ ID No. 36.

3. The application of a group of probes for molecular typing of head and neck squamous cell carcinoma in preparing a kit and a gene chip for molecular typing of head and neck squamous cell carcinoma is characterized in that the nucleotide sequences of the probes are shown as SEQ ID No.37 to SEQ ID No. 54.

4. A kit for molecular typing of head and neck squamous cell carcinoma comprising PCR primers capable of amplifying a set of genes for molecular typing of head and neck squamous cell carcinoma and/or probes complementary to a set of gene sequences for molecular typing of head and neck squamous cell carcinoma, the set of genes for molecular typing of head and neck squamous cell carcinoma consisting of 18 genes: TP53 gene, CDKN2A gene, FAT1 gene, CASP8 gene, AJUBA gene, PIK3CA gene, NOTCH1 gene, KMT2D gene, NSD1 gene, HLA-A gene, HRAS gene, FBXW7 gene, RB1 gene, PIK3R1 gene, TRAF3 gene, NFE2L2 gene, CUL3 gene and PTEN gene.

5. The kit for molecular typing of head and neck squamous cell carcinoma according to claim 4, wherein the nucleotide sequence of said PCR primer is shown in SEQ ID No.1 to SEQ ID No. 36.

6. The kit for molecular typing of head and neck squamous cell carcinoma according to claim 4, wherein the nucleotide sequence of said probe is as shown in SEQ ID No.37 to SEQ ID No. 54.

7. A gene chip for molecular typing of head and neck squamous cell carcinoma is characterized by comprising a solid phase carrier and a probe, wherein the nucleotide sequence of the probe is shown as SEQ ID No.37 to SEQ ID No. 54.