CN117106774A - Reagent for detecting methylation level and application of reagent in preparation of HPV negative head and neck squamous cell carcinoma diagnostic product - Google Patents

Abstract

The invention provides a reagent for detecting methylation level and application thereof in preparing HPV negative head and neck squamous cell carcinoma diagnostic products; the target region is selected from the full length or partial region of the negative strand of Chr2:118849988 ～ 118850355 using GRch38.p14 as the reference genome. According to the research of the invention, the methylation level of the full length or partial region of the Chr2:118849988 ～ 118850355 negative strand DNA is detected, so that diagnosis can be carried out on different pathological stages and different types of HPV negative head and neck squamous cell carcinoma patients, the detection sensitivity and specificity are high, and a new idea can be provided for screening and diagnosing the HPV negative head and neck squamous cell carcinoma patients.

Description

Reagent for detecting methylation level and application of reagent in preparation of HPV negative head and neck squamous cell carcinoma diagnostic product

Technical Field

The invention relates to the technical field of biomedicine, in particular to a reagent for detecting methylation level and application thereof in preparing HPV negative head and neck squamous cell carcinoma diagnostic products.

Background

Head and neck tumors originate mainly from mucosal epithelial cells of the oral cavity, pharynx or larynx, and more than 90% of head and neck tumors are squamous cell carcinomas, so these partial tumors are collectively referred to as head and neck squamous cell carcinomas (Head and Neck Squamous Cell Carcinoma, HNSCC).

Oral and laryngeal cancers are often associated with smoking, alcoholism, whereas pharyngeal cancers are due in part to infection by human papillomavirus (Human Papilloma Virus, HPV), principally HPV-16 type. Patients with HNSCC can be classified into two categories based on whether they are infected with HPV or not: HPV positive (HPV+HNSCC) and HPV negative (HPV-HNSCC); in general, HPV-positive HNSCC patients have a better prognosis. Survival in HNSCC patients has increased over the last 30 years, mainly due to increased survival in HPV-positive HNSCC patients; whereas the survival of HPV-negative HNSCC patients is not improved, i.e. the survival of HPV-negative HNSCC patients is not improved by current diagnostic and therapeutic means.

HPV-negative HNSCC is developed from mucosal epithelial cells undergoing hyperproliferative to dysplastic recurrence to invasive cancer, but most patients do not clinically develop significant pre-cancerous lesions, often have advanced cancer and developed lymph node metastasis upon diagnosis, and therefore the prognosis for HPV-negative HNSCC is poor.

Furthermore, there is currently no effective protocol for screening head and neck tumors, the diagnosis of which relies mainly on tissue biopsies of the lesion and pathological staging of the patient using imaging detection methods. Thus, there is an urgent need for improvements in screening and diagnostic methods for HPV-negative HNSCC patients.

Disclosure of Invention

Based on the detection, the invention provides the application of the reagent for detecting the methylation level with simplicity, convenience, high sensitivity and high specificity in preparing the diagnosis product of HPV negative head and neck squamous cell carcinoma.

The specific technical scheme is as follows:

according to one aspect of the invention there is provided an agent for detecting the methylation level of a target region comprising the full length or a partial region of the Chr2:118849988 ～ 118850355 negative strand, using GRch38.p14 as a reference genome.

In one embodiment, the partial region of the target region includes one or more of regions 1-2:

region 1 is the Chr2:118849988 ～ 118850180 negative strand, and

region 2 is the Chr2:118850154 ～ 118850355 negative strand.

In one embodiment, the reagent enables detection of the target region by one or more of the following methods:

methylation-specific PCR, bisulfite sequencing, methylation-specific microarray, whole genome methylation sequencing, pyrosequencing, methylation-specific high performance liquid chromatography, methylation-specific high resolution dissolution profile, methylation-sensitive restriction endonuclease, and fluorescent quantitative PCR.

In one embodiment, the reagent comprises a nucleic acid product that detects the methylation level of the target region;

optionally, the nucleic acid product comprises one or more of a primer pair and a detection probe.

In one embodiment, the nucleic acid product comprises one or more of the following primer pairs:

the methylation primer pair of the detection region 1 has a nucleotide sequence shown in SEQ ID NO. 7-8;

the nucleotide sequence of the unmethylated primer pair of the detection region 1 is shown as SEQ ID NO. 9-10; and

the methylation primer pair of the detection region 2 has a nucleotide sequence shown in SEQ ID NO. 11-12.

In one embodiment, the nucleic acid product comprises one or more of the following primer pairs in combination with a detection probe:

(1) Primer pairs with nucleotide sequences shown as SEQ ID NO. 13-14; the nucleotide sequence of the detection probe is shown as SEQ ID NO. 15;

(2) Primer pairs with nucleotide sequences shown as SEQ ID NO. 16-17, and the nucleotide sequence of the detection probe is shown as SEQ ID NO. 18;

(3) The nucleotide sequence of the primer pair is shown as SEQ ID NO. 19-20, and the nucleotide sequence of the detection probe is shown as SEQ ID NO. 21;

(4) The nucleotide sequence of the primer pair is shown as SEQ ID NO. 22-23, and the nucleotide sequence of the detection probe is shown as SEQ ID NO. 24;

(5) The nucleotide sequence of the primer pair is shown as SEQ ID NO. 25-26, and the nucleotide sequence of the detection probe is shown as SEQ ID NO. 27; and

(6) The nucleotide sequence of the primer pair is shown as SEQ ID NO. 28-29, and the nucleotide sequence of the detection probe is shown as SEQ ID NO. 30.

In one embodiment, the sample tested comprises a blood sample, an oral exfoliated cell sample, or a saliva sample.

According to another aspect of the present invention there is provided a kit for detecting HPV-negative head and neck squamous cell carcinoma comprising an agent as described in any of the preceding claims.

In one embodiment, the kit further comprises one or more of a nucleic acid extraction reagent, a methylation conversion reagent, a PCR reaction reagent, a sequencing reagent, a negative control and a positive control;

alternatively, the PCR reaction reagent comprises an amplification buffer, dNTPs, a DNA polymerase and Mg ²⁺ One or more of the following.

According to a further aspect of the present invention there is provided the use of an agent as defined in any one of the preceding claims or a kit as defined in any one of the preceding claims in the manufacture of a diagnostic product for HPV negative squamous cell carcinoma of the head and neck.

Compared with the prior art, the invention has the following beneficial effects:

The invention can diagnose different pathological stages and different types of HPV negative head and neck squamous cell carcinoma patients by detecting the methylation level of the full length or partial region of the Chr2:118849988 ～ 118850355 negative strand DNA, has high detection sensitivity and specificity, and can provide a new thought for screening and diagnosing HPV negative head and neck squamous cell carcinoma patients.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a statistical plot of methylation levels of cg09385093 in HNSCC cancer tissues and paracancerous normal tissues.

Detailed Description

The detailed description of the present invention will be provided to make the above objects, features and advantages of the present invention more obvious and understandable. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. The present invention may be embodied in many other forms than described herein and similarly modified by those skilled in the art without departing from the spirit of the invention, whereby the invention is not limited to the specific embodiments disclosed below.

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 to which this application belongs. The terminology used herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present application are commercially available or may be prepared by existing methods.

Terminology

Unless otherwise indicated or contradicted, terms or phrases used herein have the following meanings:

the term "and/or," "and/or," as used herein, includes any one of two or more of the listed items in relation to each other, as well as any and all combinations of the listed items in relation to each other, including any two of the listed items in relation to each other, any more of the listed items in relation to each other, or all combinations of the listed items in relation to each other. It should be noted that, when at least three items are connected by a combination of at least two conjunctions selected from the group consisting of "and/or", "and/or", it should be understood that, in the present application, the technical solutions include technical solutions that all use "logical and" connection, and also include technical solutions that all use "logical or" connection. For example, "a and/or B" includes three parallel schemes A, B and a+b. For another example, the technical schemes of "a, and/or B, and/or C, and/or D" include any one of A, B, C, D (i.e., the technical scheme of "logical or" connection), and also include any and all combinations of A, B, C, D, i.e., any two or three of A, B, C, D, and also include four combinations of A, B, C, D (i.e., the technical scheme of "logical and" connection).

The terms "plurality", "plural", "multiple", and the like in the present invention refer to, unless otherwise specified, an index of 2 or more in number. For example, "one or more" means one kind or two or more kinds.

As used herein, "a combination thereof," "any combination thereof," and the like include all suitable combinations of any two or more of the listed items.

The "suitable" in the "suitable combination manner", "suitable manner", "any suitable manner" and the like herein refers to the fact that the technical scheme of the present invention can be implemented, the technical problem of the present invention is solved, and the technical effect expected by the present invention is achieved.

Herein, "preferred", "better", "preferred" are merely to describe better embodiments or examples, and it should be understood that they do not limit the scope of the invention.

In the present invention, "further", "still further", "particularly" and the like are used for descriptive purposes to indicate differences in content but should not be construed as limiting the scope of the invention.

In the present invention, "optional" means optional or not, that is, means any one selected from two parallel schemes of "with" or "without". If multiple "alternatives" occur in a technical solution, if no particular description exists and there is no contradiction or mutual constraint, then each "alternative" is independent.

In the invention, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.

In the present invention, "diagnosis" includes auxiliary diagnosis, recurrence risk evaluation, evaluation of risk of canceration and degree of canceration, prognosis judgment, and the like. In the present invention, a diagnostic product is any product comprising any compound that can be tested on a biological sample from a patient to make a diagnostic assay or test as to whether the patient has certain diseases. In some cases, the test results of the diagnostic product may be used as an aid or reference for clinical diagnosis or to indicate the patient's risk of suffering from certain diseases. In the present invention, a diagnostic product is any product comprising the agents herein; in some embodiments, the diagnostic product is a kit of the invention, or the diagnostic product is a kit comprising a kit of the invention and other elements, e.g., the other elements may be sampling devices and/or detection elements based on other methods.

In the present invention, "methylation" is a form of chemical modification of DNA that can alter genetic manifestations without altering the DNA sequence. DNA methylation refers to covalent binding of a methyl group at the 5 th carbon position of cytosine of a genomic CpG dinucleotide under the action of a DNA methyltransferase. DNA methylation can cause alterations in chromatin structure, DNA conformation, DNA stability, and the manner in which DNA interacts with proteins, thereby controlling gene expression.

As used herein, "methylation level" refers to whether or not a cytosine in one or more CpG dinucleotides in a DNA sequence is methylated, or the frequency/proportion/percentage of methylation, and represents both qualitative and quantitative concepts. In practical application, different detection indexes can be adopted to compare the DNA methylation level according to practical conditions. As in some cases, the comparison may be made based on Ct values detected by the sample; in some cases, the ratio of gene methylation in the sample, i.e., number of methylated molecules/(number of methylated molecules+number of unmethylated molecules). Times.100, can be calculated and then compared; in some cases, statistical analysis and integration are also performed on each index to obtain a final judgment index.

In the present invention, the term "primer" refers to an oligonucleotide that can be used in an amplification method (e.g., polymerase chain reaction PCR) to amplify a target sequence based on a polynucleotide sequence corresponding to a target gene or a part of a region thereof. Typically, at least one of the PCR primers used to amplify a polynucleotide sequence is sequence specific for that polynucleotide sequence. The exact length of the primer will depend on many factors, including temperature, source of primer, and method used. For example, for diagnostic and prognostic applications, the oligonucleotide primers will typically contain at least 10, 15, 20, 25 or more nucleotides, but may also contain fewer nucleotides, depending on the complexity of the target sequence.

In the present invention, the "primer pair" refers to a pair of primers capable of hybridizing to a double strand of a target DNA molecule or capable of hybridizing to regions of the target DNA molecule located at both sides of a nucleotide sequence to be amplified.

In the present invention, the term "TaqMan probe" refers to a segment of an oligonucleotide sequence comprising a 5 '-end fluorescent reporter group and a 3' -end fluorescent quencher group. When the probe binds to the corresponding site on the DNA, the probe does not fluoresce because of the presence of a quenching group near the fluorescent group. During amplification, if the probe binds to the amplified strand, the 5'-3' exonuclease activity of the DNA polymerase (e.g., taq enzyme) digests the probe and the fluorescent group is far from the quenching group, its energy is not absorbed, i.e., a fluorescent signal is generated. The fluorescence signal is also identical to the target fragment with a synchronous exponential increase per PCR cycle.

Some embodiments of the invention provide a method for detecting the methylation level of a target region selected from the group consisting of the full length or a partial region of the negative strand of Chr2:118849988 ～ 118850355 using GRch38.p14 as a reference genome. In some of these embodiments, the partial region of the target region is one or more of region 1 and region 2;

Region 1 is the negative strand DNA of Chr2:118849988 ～ 118850180, and

region 2 is the negative strand DNA of Chr2:118850154 ～ 118850355.

It will be appreciated that the reagents described above may detect the full length or partial regions of regions 1 and 2 simultaneously, or may detect the full length or partial regions of regions 1 or 2 separately. The partial region comprises at least one CpG dinucleotide.

In some embodiments, the above-described reagents enable detection of the target region by, but not limited to, one or more of the following methods:

methylation-specific PCR, bisulfite sequencing, methylation-specific microarray, whole genome methylation sequencing, pyrosequencing, methylation-specific high performance liquid chromatography, methylation-specific high resolution dissolution profile, methylation-sensitive restriction endonuclease, and fluorescent quantitative PCR.

In some embodiments, the reagent comprises a nucleic acid product that detects the methylation level of the above-described target region.

Optionally, the nucleic acid product includes a primer pair, and may further include a detection probe corresponding to the primer pair.

In some examples, the nucleic acid product comprises a methylated primer pair of detection region 1 having the nucleotide sequence set forth in SEQ ID NOS.7-8.

In some examples, the nucleic acid product comprises a non-methylated primer pair of detection region 1 having the nucleotide sequences shown in SEQ ID NOS.9-10.

In some examples, the nucleic acid product comprises a methylated primer pair of detection region 2 having the nucleotide sequence set forth in SEQ ID NOS.11-12.

In some examples, the nucleic acid product comprises a combination of a primer pair and a detection probe that detects the full length or a partial region of the target region.

In one specific example, the nucleic acid product comprises a primer pair and a detection probe that detect the negative strand region of Chr2:118850012 ～ 118850119; the nucleotide sequence of the primer pair is shown as SEQ ID NO. 13-14, and the nucleotide sequence of the detection probe is shown as SEQ ID NO. 15.

In one specific example, the nucleic acid product comprises a primer pair and a detection probe that detect the negative strand region of Chr2:118850012 ～ 118850119; the nucleotide sequence of the primer pair is shown as SEQ ID NO. 16-17, and the nucleotide sequence of the detection probe is shown as SEQ ID NO. 18.

In one specific example, the nucleic acid product comprises a primer pair and a detection probe that detect the negative strand region of Chr2:118850214 ～ 118850307; the nucleotide sequence of the primer pair is shown as SEQ ID NO. 19-20, and the nucleotide sequence of the detection probe is shown as SEQ ID NO. 21.

In one specific example, the nucleic acid product comprises a primer pair and a detection probe that detect the negative strand region of Chr2:118850214 ～ 118850307; the nucleotide sequence of the primer pair is shown as SEQ ID NO. 22-23, and the nucleotide sequence of the detection probe is shown as SEQ ID NO. 24.

In one specific example, the nucleic acid product comprises a primer pair and a detection probe that detect the negative strand region of Chr2:118850279 ～ 118850346; the nucleotide sequence of the primer pair is shown as SEQ ID NO. 25-26, and the nucleotide sequence of the detection probe is shown as SEQ ID NO. 27.

In one specific example, the nucleic acid product comprises a primer pair and a detection probe that detect the negative strand region of Chr2:118850279 ～ 118850346; the nucleotide sequence of the primer pair is shown as SEQ ID NO. 28-29, and the nucleotide sequence of the detection probe is shown as SEQ ID NO. 30.

Further, the detection probe of any of the above examples is a TaqMan probe; fluorescent groups and quenching groups are respectively marked at the 5 'end and the 3' end of the detection probe.

Alternatively, the fluorophore is FAM, ROX, VIC or CY5; the quenching group is TAMRA, BHQ or MGB. When a plurality of different detection probes are included in the reaction system, each probe contains a different fluorescent group.

In some examples, the nucleic acid product further comprises a primer pair for detecting a reference gene and a corresponding detection probe.

In a specific example, the reference gene is ACTB gene, the nucleotide sequence of the primer pair for detecting the reference gene ACTB is shown as SEQ ID NO. 31-32, and the nucleotide sequence of the corresponding detection probe is shown as SEQ ID NO. 33.

Understandably, the detection probes of the reference gene are labeled with a fluorescent group and a quenching group different from those of the target region.

In some embodiments, the sample detected comprises a blood sample, an oral exfoliated cell sample, or a saliva sample.

According to the invention, by detecting the full length or partial region of the Chr2:118849988 ～ 118850355 negative chain, the sample of the HPV negative head and neck squamous cell carcinoma patient can be rapidly detected with high sensitivity and high specificity; provides a new idea for diagnosis and screening of HPV negative head and neck squamous cell carcinoma patients.

Further embodiments of the present invention provide a kit for detecting squamous cell carcinoma of head and neck, comprising a reagent as defined in any of the above embodiments.

In some embodiments, the kit further comprises one or more of a nucleic acid extraction reagent, a methylation conversion reagent, a PCR reaction reagent, a sequencing reagent, a negative control, and a positive control.

It will be appreciated that in other specific examples, the above kit may not include any of the nucleic acid extraction reagent, the methylation conversion reagent, the PCR reaction reagent, the sequencing reagent, the negative control, and the positive control, and the reagent not included may be reasonably obtained from the outside.

Alternatively, the PCR reaction reagents include an amplification buffer, dNTPs, a DNA polymerase, and Mg ²⁺ One or more of the following.

The kit is prepared from the reagent for detecting the full length or partial region of the Chr2:118849988 ～ 118850355 negative chain, and the head and neck squamous cell carcinoma can be rapidly, conveniently and accurately detected by adopting the kit.

Further embodiments of the invention also provide the use of the agent of any of the embodiments or the kit of any of the embodiments described above for the preparation of a HPV-negative head and neck squamous cell carcinoma diagnostic product.

The reagent for detecting the methylation level of the target region is proved to have higher detection sensitivity and specificity when being used for detecting HPV negative head and neck squamous cell carcinoma, and can effectively distinguish head and neck squamous cell carcinoma patients from healthy people.

The present invention will be further described with reference to specific examples and comparative examples, which should not be construed as limiting the scope of the invention.

Example 1:

the methylation level of the target region was analyzed using Sang Geer sequencing and the performance of HPV negative HNSCC patient tissue samples was examined. The method comprises the following specific steps:

1.1 sample collection

A total of 106 HNSCC patients diagnosed by pathological tissue biopsy (all patients are HPV-16 and HPV-18 seronegative, i.e. have not been infected with human papillomavirus type 16 and 18) were recruited, and their biopsy confirmed cancer tissue samples and corresponding paracancerous normal tissue samples were obtained, respectively. All samples were formalin-soaked and paraffin-embedded tissue samples, all samples were approved by the ethical committee for collection, and all volunteers signed informed consent prior to sample collection. The pathology information of the above 106 HNSCC patients is shown in table 1.

TABLE 1 pathological information of HPV negative HNSCC patients

1.2 extraction of sample DNA

Genomic DNA from paraffin-embedded tissue samples was extracted using QIAamp DNAFFPE Tissue Kit (56404) and the specific procedure was performed according to the kit instructions.

1.3 bisulfite conversion

The DNA extracted in step 1.2 was bisulphite converted using the DNA converting reagent of the Living technologies of Wuhan Ai Misen, huhan Chemicals 20200843, and the converted DNA was purified for subsequent experiments, with specific procedures being described in the kit instructions.

1.4 PCR amplification and Sang Geer sequencing

GRCh38 is used as a reference genome, target region is Chr2:118849988 ～ 118850355 negative strand DNA, a PCR method is used for amplifying the target region in tissue sample genome which is subjected to bisulfite conversion and purification, and PCR amplified products are sent to a sequencing company for Morgol sequencing.

In order to amplify the target region efficiently, it is divided into two regions to amplify separately: the untransformed, fully methylated and bisulfite-converted, fully unmethylated and bisulfite-converted DNA sequences corresponding to region 1 (Chr 2:118849988 ～ 118850180, minus strand) and region 2 (Chr 2:118850154 ～ 118850355, minus strand) are shown in Table 2.

TABLE 2 DNA sequence information for region 1 and region 2

Respectively taking the DNA samples which are extracted in the step 1.3 and are converted by sulfite as templates; region 1 was amplified using methylated primer pairs (SEQ ID NOS.7-8) and unmethylated primer pairs (SEQ ID NOS.9-10), region 2 was amplified using degenerate primers (SEQ ID NOS.11-12), and the sequence information for the primer pairs is shown in Table 3.

TABLE 3 primer information for PCR amplification and Sang Geer sequencing

All primer pairs shown in Table 3 were synthesized manually, and PCR amplification was performed according to the reaction procedure provided in Table 5, using the bisulfite-converted and purified tissue sample DNA as a template, and following the formulation of Table 4, a PCR reaction system was prepared. For each sample to be detected, when amplifying the target area 1, a 2-tube reaction system is required to be configured, and a methylation primer pair (SEQ ID NO. 7-8) and a non-methylation primer pair (SEQ ID NO. 9-10) are respectively used for amplification; when amplifying the target region 2, only a 1-tube reaction system is required to be configured, and degenerate primer pairs (SEQ ID NOS.11-12) are used for amplification. After the end of the PCR amplification, the amplified product was sequenced Sang Geer using the same primers as the PCR amplification, with sequencing from the 5 'and 3' ends.

TABLE 4 PCR reaction System

Component (A)	Volume of
		5×PrimeSTAR Buff(Mg 2+ Plus)	10μL
dNTP Mixture(2.5mM each)	4μL
		Upstream primer (10. Mu.M)	1μL
Downstream primer (10. Mu.M)	1μL
		Template DNA	5μL
PrimeSTAR HS DNA Polymerase(2.5U/μL)	0.5μL
		Sterilizing water	Make up to 50 mu L

TABLE 5 PCR amplification procedure

1.5 analysis of results

For bisulfite converted DNA sequences, if the sequencing result of the CpG dinucleotide site on the original DNA sequence before conversion is still CG, then the site is considered to be fully methylated; if the sequencing result of the CpG dinucleotide site on the initial DNA sequence is bimodal, namely CG and TG are simultaneously present, the site is considered to be partially methylated; when the sequencing result of one CpG dinucleotide site on the original DNA sequence is TG, the site is considered unmethylated.

1.5.1 sensitivity and specificity analysis for diagnosing HNSCC by methylation level of region 1

The total of 17 CpG sites in the region 1, and if 16 or more CpG sites in the region 1 are methylated (including complete methylation and partial methylation) in the sample to be detected, the sample is considered to be positive in methylation in the region 1; if the sample to be tested is unmethylated at all 17 CpG sites within region 1, then the sample is considered methylation negative within region 1.

Methylation analysis was performed on 212 cancer and paracancerous tissue samples collected in this example according to the criteria described above, and statistical analysis was performed to detect target region 1 using Sang Geer sequencing to diagnose sensitivity and specificity of HPV-negative HNSCC patients. Wherein 209 samples were successfully sequenced and 3 samples were failed to sequence; the sensitivity and specificity of Sang Geer sequencing to detect methylation level in region 1 to diagnose HNSCC tissue samples are shown in tables 6 and 7.

Sensitivity is the proportion of methylation positive samples in samples with the pathological detection result of positive cancers, and specificity is the proportion of methylation negative samples in normal samples beside cancers.

TABLE 6 methylation level diagnosis of HNSCC different tissue samples for region 1

As can be seen from Table 6, the methylation level of region 1 was analyzed by Sang Geer sequencing and the effect of diagnosing HNSCC patients negative for HPV was good. The sensitivity of diagnosing HNSCC by detecting the methylation level of the region is very high, the sensitivity of diagnosing oral cancer, pharyngeal cancer and laryngeal cancer is more than 96%, and the detection specificity is more than 75%.

TABLE 7 methylation level of region 1 diagnosis of HNSCC tissue samples of different pathological stages

Although the number of samples collected for pathological stage I was small, it is clear from Table 7 that the sensitivity of diagnosing stage I oral cancer tissue samples by detecting the methylation level of the region 1 was 100%. Furthermore, diagnosis of other HNSCC of different stages by detecting the methylation level of region 1 also has a higher sensitivity. Indicating that the HNSCC cancer tissue and the paracancerous normal tissue can be effectively distinguished according to the methylation level of the region 1.

1.5.2 sensitivity and specificity analysis for diagnosing HNSCC by methylation level of region 2

The total of 19 CpG sites in the region 2, and if 17 or more CpG sites in the region 2 are methylated (including complete methylation and partial methylation) in the sample to be detected, the sample is considered to be positive in methylation in the region 2; if the 19 CpG sites of the sample to be tested are all unmethylated in region 2, the sample is considered methylation negative in region 2.

According to the above criteria, the statistical analysis detects target region 2 using Sang Geer sequencing to diagnose sensitivity and specificity of HPV negative HNSCC patients, and the results are shown in tables 8 and 9.

TABLE 8 methylation level diagnosis of region 2 HNSCC different tissue samples

As can be seen from table 8, the effect of analyzing the methylation level of region 2 in the target region by using the Sang Geer sequencing method, and thus diagnosing HPV-negative HNSCC patients, was also very excellent: the sensitivity of diagnosing HNSCC cancer tissue samples according to the methylation level of the region 2 is not lower than 93.94 percent, and the specificity is higher than 82 percent.

From tables 6 and 8, it can be seen that the specificity of diagnosing HNSCC based on the methylation level of region 2 is relatively higher compared to region 1.

TABLE 9 methylation level diagnosis of region 2 HNSCC tissue samples at different pathological stages

As can be seen from Table 9, the sensitivity of diagnosing HNSCC cancer tissues of different stages by sequencing the methylation level of the analysis region 2 was also high, the sensitivity was not lower than 83.33% for the stage I and stage II cancer tissue samples, and the detection sensitivity was higher than 91% for the stage III and stage IV cancer tissue samples.

In conclusion, the Chr2:118849988 ～ 118850180 negative strand DNA and the Chr2:118850154 ～ 118850355 negative strand DNA are highly methylated in more than 100 HPV negative oral cancers, pharyngeal cancers and laryngeal cancers, and the HNSCC cancer tissue sample and the paracancerous normal tissue sample can be efficiently distinguished by analyzing the methylation level of the target region by taking the Chr2:118849988 ～ 118850180 negative strand DNA and the Chr2:118850154 ～ 118850355 negative strand DNA as target regions.

Furthermore, the inventors screened cg09385093 probes located in region 2, which have significant methylation level differences in HNSCC cancer tissue and paracancerous normal tissue, by analyzing methylation data of IIlumina Human Methylation K chips in the TCGA database. As shown in fig. 1, cg09385093 had significantly higher methylation levels in cancerous tissue than in paracancerous normal tissue. This result is consistent with the test result of the tissue sample in example 1.

Example 2:

from the results of example 1, it was found that it is possible to diagnose HNSCC by detecting the methylation level of the target region by liquid biopsy. Thus, this example detects the methylation level of the target region by fluorescent quantitative methylation-specific PCR (quantitative methylation specific PCR, qMSP) and diagnoses HNSCC patient body fluid samples based on the methylation level.

2.1 sample collection

258 volunteers (all volunteers were HPV-16 and HPV-18 seronegative) were enrolled, of which 78 were patients diagnosed with oral squamous cell carcinoma by pathological tissue biopsy, 77 were patients diagnosed with pharyngeal squamous cell carcinoma by pathological tissue biopsy, 53 were patients with laryngeal squamous cell carcinoma by pathological tissue biopsy, and the remaining 50 were healthy persons. Blood samples and buccal exfoliated cell samples were obtained separately for each volunteer (some patients who were reluctant to take buccal exfoliated cell samples were changed to saliva samples). All samples were collected by ethical committee approval and all volunteers signed informed consent prior to sample collection. The pathological stage information of the above 208 cancer patients is shown in Table 10.

TABLE 10 pathological staging of HNSCC patients

Type of cancer	Number of examples	Phase I	Stage II	Stage III	Stage IV
						Oral cancer	78	21	25	18	14
Pharyngeal cancer	77	6	15	27	29
						Laryngeal carcinoma (laryngeal carcinoma)	53	8	12	18	15
Totalizing	208	35	52	63	58

The collection method of the blood plasma sample, the oral cavity exfoliated cell sample and the saliva sample of the volunteer is specifically as follows:

collection of anti-plasma samples: greater than 8mL venous blood was withdrawn from each volunteer, and the collected anticoagulated blood samples were allowed to stand at room temperature and centrifuged at 3000rpm for 10 minutes to obtain plasma samples.

Collecting an oral cavity exfoliated cell sample: the volunteer needs to brush teeth or rinse mouth before taking the oral cavity cell sample, then uses the cotton swab to scrape at least 20 times on the two sides of the oral cavity, the oropharynx, the lingual face, the gum and other parts of the subject, then puts the cotton swab into the cell preservation liquid for rinsing, and takes the cell sediment after centrifuging the cell preservation liquid for standby.

Collection of saliva samples: volunteers were either brushing their teeth or rinsing their mouth with clear water before taking a saliva sample, then required to sit upright and low in a comfortable position, allow saliva to accumulate in the mouth for about 10 minutes, and then collect the saliva into a sterile 50mL centrifuge tube. The collected saliva sample is centrifuged for 10 minutes at 4 ℃ and 500g, and the cell sediment is taken after the supernatant is discarded, namely the oral cavity exfoliated cell sample is frozen for standby.

2.2 extraction of sample DNA

The cfDNA of the plasma samples was extracted using a magnetic bead serum/plasma free DNA (cfDNA) extraction kit (DP 709) from the company of the biochemical science and technology of the root of the heaven, beijing, limited, the specific procedure was performed according to the kit instructions.

Genomic DNA of the oral cast cell samples was extracted using the blood/cell/tissue genomic DNA extraction kit (DP 304) of the tiangen biochemical technology (beijing) limited, and the specific procedure was as per the specification.

2.3 transformation of sample DNA

All sample DNA was transformed with nucleic acid transforming reagent (Ehan apparatus 20200843) from the GmbH of the life technologies of Wuhan Ai Misen, and the transformed DNA was purified, the specific procedure being described in the kit instructions.

2.4 qMSP reaction

Primer pairs and TaqMan detection probes for methylation-specific fluorescent quantitative PCR were designed for partial regions of region 1 and region 2, and the combinations and sequence information of the primer pairs and detection probes are shown in Table 11. The primer pair has good amplification specificity, only the methylated DNA template is amplified, the unmethylated DNA template is not amplified, and other nonspecific amplification is not caused; the primer pair has good amplification efficiency, namely the amplification efficiency of the primer pair on each target region is between 90 and 110 percent.

TABLE 11 methylation detection primer pairs and detection probes

The qMSP reaction based on the TaqMan probe can realize simultaneous detection of a plurality of target genes in one reaction system, and only the fluorescent groups carried by the 5' end of each target gene specific detection probe are different. In this example, 2 target genes were detected per qMSP reaction system: target region and reference gene ACTB. The fluorescent group at the 5 'end of the TaqMan probe for amplifying the target region is FAM, and the 3' end of the TaqMan probe is a non-fluorescence quenching group NFQ; the fluorescent group at the 5 'end of the TaqMan probe for amplifying the reference gene ACTB is ROX, and the fluorescent quenching group at the 3' end is BHQ.

The qMSP reaction was performed using the bisulfite-converted DNA of step 2.3 as a template, using the primer pair and probe shown in Table 11, the amplification system was shown in Table 12, and the reaction procedure was shown in Table 13.

TABLE 12 qMSP reaction System

TABLE 13 qMSP reaction procedure

When each sample is subjected to qMSP reaction, the quantity of the reference gene ACTB needs to be detected in advance for monitoring the sample quality and judging the result. Within each PCR reaction plate, positive control wells and negative control wells also need to be provided simultaneously. The DNA template of the positive control well was 10 ³ copies/. Mu.L of plasmid containing the sequence ACTB after transformation and 10 ³ The copies/. Mu.L of plasmid mixture containing the DNA sequence of the target region after transformation (equal volume of the two are mixed), and other components are the same as those of the experimental tube; the template of the negative control well was TE buffer and the other components were the same as the experimental tube.

After the qMSP reaction is finished, a baseline is adjusted (a signal value corresponding to the 3 rd to 15 th cycles is generally set as a baseline level), a threshold value is set (the threshold value is required to be positioned in an exponential amplification period), a straight line crossing the threshold value and parallel to the X axis is called a threshold line, and the cycle number corresponding to the intersection point of the threshold line and the amplification curve is the Ct value.

The result of the qMSP reaction meets the following conditions, the detection result is considered to be reliable, otherwise, the current experiment is invalid: (1) negative control tubes were not amplified (i.e., no line lifting); (2) the positive control PCR tube has obvious index increasing period, and the Ct value of the target gene in the positive control PCR tube is between 26 and 30; (3) the Ct value of the reference gene ACTB of the sample to be detected is less than or equal to 33.

2.5 analysis of results

2.5.1 Sensitivity and specificity of detecting methylation level of region 1 or region 2 by qMSP method for diagnosing HNSCC plasma sample

For a plasma sample, the positive judgment value is 45, and if the Ct value of amplification by using one group of primer combination is less than or equal to 45, the sample is considered to be methylation positive in the amplification region; if the Ct value >45 for amplification using one of the primer sets, the sample is considered methylation negative in this amplified region.

According to the above criteria, methylation levels of region 1 and region 2 were obtained and the methylation levels of region 1 or region 2 were analyzed by qMSP method to diagnose sensitivity and specificity of HNSCC plasma samples, and the results are shown in table 14.

Table 14 qMSP method for detecting methylation level diagnostic HNSCC plasma samples in region 1 or region 2

From the above table, it can be seen that the methylation level of detection region 1 or region 2 can effectively distinguish between plasma samples from HNSCC patients and from plasma samples from healthy persons by performing a qMSP reaction using primer combinations 1 to 6.

The detection effect of the primer combination 1 and the primer combination 2 of the methylation level of the detection region 1 is equivalent: the sensitivity of the primer combination 1 and the primer combination 2 for detecting the oral cancer plasma sample is 78.21% and 79.49%, respectively; the sensitivity of detecting the pharyngeal cancer plasma sample is 67.53% and 66.23%, respectively; the sensitivity of detecting laryngeal cancer plasma samples is 66.04%; the detection specificity of both primer combinations was 96%.

The detection effect of primer combinations 3 to 6 of methylation level of detection region 2 is not significantly different either: the sensitivity of the primer combination 3-6 for detecting the oral cancer plasma sample is between 71.79 and 73.08 percent, the sensitivity of the primer combination for detecting the pharyngeal cancer plasma sample is between 71.43 and 74.03 percent, the sensitivity of the primer combination for detecting the laryngeal cancer plasma sample is between 71.70 and 75.47 percent, and the specificity of the primer combination for detecting HNSCC is between 96 and 98 percent.

In summary, the sensitivity of diagnosing oral cancer plasma samples by detecting the methylation level of region 1 is higher than region 2; whereas the methylation level of detection zone 2 is more sensitive to diagnose pharyngeal and laryngeal cancers than zone 1.

2.5.2 Sensitivity and specificity of detecting methylation level of region 1 or region 2 by qMSP method for diagnosing HNSCC oral cavity exfoliated cell sample

For an oral cavity exfoliated cell sample, the positive judgment value is 37, and if the Ct value of amplification by using one group of primer combination is less than or equal to 37, the sample is considered to be methylation positive in the amplification region; if the Ct value of one of the primer combinations for amplification is >37, the sample is considered methylation negative in this amplified region.

The sensitivity and specificity of HNSCC oral shed cell samples were diagnosed by detecting the methylation level of either region 1 or region 2 by the qMSP method, and the results are shown in table 15.

TABLE 15 qMSP method detection of methylation level of region 1 or region 2 diagnosis of HNSCC oral shed cell samples

From the above table, it can be seen that the methylation level of detection region 1 or region 2 can effectively distinguish between oral shed cell samples and oral shed cell samples of HNSCC patients by performing qMSP reactions using primer combinations 1 to 6.

Similar to the detection results of the plasma samples, the detection effect of the primer combination 1 and the primer combination 2 of the methylation level of the detection region 1 is equivalent: the sensitivity of the primer combination 1 and the primer combination 2 for detecting the oral cavity exfoliated cell sample is 91.03%; the sensitivity of detecting the oral cavity exfoliated cell samples is 75.32%; the sensitivity of detecting the laryngeal cancer oral cavity exfoliated cell sample is 73.58% and 75.47% respectively; the detection specificity of both primer combinations was 92% and 94%, respectively.

The detection effect of primer combinations 3 to 6 of methylation level of detection region 2 is not obviously different: the sensitivity of the primer combination 3-6 for detecting the oral cancer shedding sample is between 83.33 and 85.90 percent, the sensitivity of the primer combination for detecting the pharyngeal cancer shedding sample is between 79.22 and 81.82 percent, the sensitivity of the primer combination for detecting the laryngeal cancer shedding sample is between 79.25 and 83.02 percent, and the specificity of the primer combination for detecting HNSCC is between 94 and 98 percent.

The detection result of the oral cavity shedding cell sample is consistent with the detection result of the plasma sample, and the sensitivity of the oral cavity cancer shedding sample is higher than that of the area 2 through the methylation level of the detection area 1; whereas the methylation level of detection zone 2 is more sensitive to diagnose pharyngeal and laryngeal carcinoma shedding samples than zone 2.

Example 3:

the performance of the body fluid samples of HPV negative HNSCC patients was tested by a fluorescent quantitative methylation specific PCR method by combining analysis of region 1 and region 2.

As can be seen from example 2, the detection rate of the oral cancer sample in the region 1 is better than that in the region 2, and the detection rate of the pharyngeal cancer and laryngeal cancer samples in the region 2 is better than that in the region 1, and the detection performance of the two regions may have a synergistic effect. Thus, analysis was performed based on the qMSP detection results of 516 samples (258 plasma samples and 258 exfoliated cell samples) in example 2; when the detection of the region 1 and the region 2 are combined, the judgment standard of the methylation positive sample is different from the judgment standard of the single region, and the specific judgment standard is as follows:

for plasma samples, if the Ct value of qMSP method amplified region 1 is less than or equal to 45, region 1 is considered methylation positive, if the Ct value of qMSP method amplified region 1 is > 45, region 1 is considered methylation negative, and region 2 is judged as above. If at least one of the area 1 or the area 2 of a certain sample to be detected is methylation positive, the sample is considered as HNSCC positive sample; otherwise, HNSCC negative samples are obtained.

For the exfoliated cell sample, if the Ct value of the qMSP method amplified region 1 is less than or equal to 37, the region 1 is considered to be methylation positive, and if the Ct value of the qMSP method amplified region 1 is more than 37, the region 1 is considered to be methylation negative, and the determination of the region 2 is the same as above. If at least one of the area 1 or the area 2 of a certain sample to be detected is methylation positive, the sample is considered as HNSCC positive sample; otherwise, HNSCC negative samples are obtained.

3.1 Combined detection of Performance of region 1 and region 2 in plasma samples from HPV negative HNSCC patients

The primer combination 2 of the amplified region 1 and the primer combination 6 of the amplified region 2 were selected for qMSP reaction, thereby analyzing the effect of the combined detection of HNSCC plasma samples in both regions. The sensitivity and specificity of the plasma samples of HNSCC patients with different pathological stages detected by the region 1 and the region 2 are shown in tables 16-17.

TABLE 16 sensitivity of region 1, region 2 detection of HNSCC plasma samples of different pathological stages, singly or in combination

As can be seen from Table 16, the sensitivity of detecting HNSCC patient plasma samples alone in either region 1 or region 2 was better, but the sensitivity of detecting HNSCC patient plasma samples in both stage I and stage II was relatively low, which may be related to the lower amount of free DNA released into the blood by tumor cells at the early stage of cancer.

The sensitivity of the combined detection of the phase I oral cancer, pharyngeal cancer and laryngeal cancer plasma samples in the areas 1 and 2 is 66.67%, 66.67% and 62.50% respectively; the sensitivity of detecting the HNSCC plasma sample at the II stage is 84.00%, 73.33% and 83.33% respectively; the sensitivity of detecting the 3 HNSCC plasma samples in the III phase and the IV phase is higher than 80 percent. The combined detection effect of the areas 1 and 2 is obviously better than that of a single area.

TABLE 17 sensitivity and specificity of detection of HNSCC plasma samples in region 1, region 2, alone or in combination

As can be seen from table 17, the sensitivity of the combined detection of HNSCC patient plasma samples in region 1 and region 2 was significantly improved compared to the single-region diagnosis. Specifically, the sensitivity of the combined detection of the plasma samples of patients with oral cancer, pharyngeal cancer and laryngeal cancer in the areas 1 and 2 can be respectively improved to 84.62%, 79.22% and 81.13%, and the specificity of the detection of the plasma samples of healthy human blood is 94%.

3.2 Combined detection of Performance of region 1 and region 2 on shed cell samples from HPV negative HNSCC patients

Similar to the detection method of the plasma sample, the primer combination 2 of the amplified region 1 and the primer combination 6 of the amplified region 2 are selected to perform qMSP reaction, so that the effect of the combined detection of HNSCC exfoliated cell samples by the two regions is analyzed. The results are shown in tables 18 to 19.

TABLE 18 sensitivity of detection of HNSCC shed cell samples of different pathological stages, region 1, region 2, singly or in combination

As can be seen from Table 18, the sensitivity of diagnosing the samples of shed cells from HNSCC patients at stage III and stage IV by methylation levels in either region 1 or region 2 was better than the sensitivity of detecting the samples at stage I and stage II, respectively. Furthermore, it can be seen from tables 16 and 18 that HNSCC patients were diagnosed by detecting exfoliated cell samples, and that the detection sensitivity was higher for different cancer types and different pathological stages than for plasma samples.

Meanwhile, compared with single detection, the sensitivity of detecting the cell samples of the patients with the phase I HNSCC and the phase II HNSCC in the combined mode is obviously improved, the sensitivity of detecting the cell samples of the patients with the phase I oral cancer, the pharyngeal cancer and the laryngeal cancer is respectively 80.95%, 83.33% and 75%, and the sensitivity of detecting the cell samples of the patients with the phase II oral cancer, the pharyngeal cancer and the laryngeal cancer is respectively 100%, 80% and 83.33%. In addition, the sensitivity of the combined detection of HNSCC shedding cell samples of different pathological stages of III stage and IV stage in the regions 1 and 2 is not lower than 88.89%.

TABLE 19 sensitivity and specificity of detection of HNSCC-shed cell samples in region 1, region 2, either alone or in combination

As can be seen from table 19, the sensitivity of detecting the sample of the exfoliated cells of HNSCC patient in combination with the region 1 and the region 2 was better than that of the single-region diagnosis. Specifically, the sensitivity of the combined detection of the exfoliated cell samples of the patients with oral cancer, pharyngeal cancer and laryngeal cancer in the areas 1 and 2 can be respectively improved to 94.87%, 88.31% and 88.68%, and the specificity of the detection of the exfoliated cell samples of the healthy people is 92%.

In sum, by detecting the methylation level of the region 1 and the region 2 in a combined way, the body fluid sample for diagnosing the HNSCC patient has the advantages of high sensitivity, high specificity and the like; moreover, by detecting the methylation level of the above region, an early lesion sample can be specifically detected.

The embodiment of the invention provides a reagent for diagnosing or assisting in diagnosing head and neck squamous cell carcinoma based on a plasma sample or an exfoliated cell sample, and can diagnose whether a patient has the head and neck squamous cell carcinoma or not by detecting methylation levels of one or more of a Chr2:118849988 ～ 118850180 negative strand DNA region and a Chr2:118850154 ～ 118850355 negative strand DNA region in a subject sample, has the advantages of high sensitivity, high specificity and the like, and can provide a new idea for diagnosing and screening HPV negative HNSCC patients.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (10)

1. An agent for detecting the methylation level of a target region comprising the full length or a partial region of the negative strand of Chr2:118849988 ～ 118850355, using grch38.p14 as a reference genome.

2. The reagent of claim 1, wherein the partial region of the target region comprises one or more of regions 1-2:

Region 1 is the Chr2:118849988 ～ 118850180 negative strand, and

region 2 is the Chr2:118850154 ～ 118850355 negative strand.

3. The reagent according to any one of claims 1-2, wherein the reagent enables detection of the target region by one or several of the following methods:

methylation-specific PCR, bisulfite sequencing, methylation-specific microarray, whole genome methylation sequencing, pyrosequencing, methylation-specific high performance liquid chromatography, methylation-specific high resolution dissolution profile, methylation-sensitive restriction endonuclease, and fluorescent quantitative PCR.

4. A reagent according to claim 3, wherein the reagent comprises a nucleic acid product that detects the methylation level of the target region;

optionally, the nucleic acid product comprises one or more of a primer pair and a detection probe.

5. The reagent of claim 4, wherein the nucleic acid product comprises one or more of the following primer pairs:

the methylation primer pair of the detection region 1 has a nucleotide sequence shown in SEQ ID NO. 7-8;

the nucleotide sequence of the unmethylated primer pair of the detection region 1 is shown as SEQ ID NO. 9-10; and

The methylation primer pair of the detection region 2 has a nucleotide sequence shown in SEQ ID NO. 11-12.

6. The reagent of claim 4, wherein the nucleic acid product comprises one or more of the following primer pairs in combination with a detection probe:

(1) Primer pairs with nucleotide sequences shown as SEQ ID NO. 13-14; the nucleotide sequence of the detection probe is shown as SEQ ID NO. 15;

(2) Primer pairs with nucleotide sequences shown as SEQ ID NO. 16-17, and the nucleotide sequence of the detection probe is shown as SEQ ID NO. 18;

(3) The nucleotide sequence of the primer pair is shown as SEQ ID NO. 19-20, and the nucleotide sequence of the detection probe is shown as SEQ ID NO. 21;

(4) The nucleotide sequence of the primer pair is shown as SEQ ID NO. 22-23, and the nucleotide sequence of the detection probe is shown as SEQ ID NO. 24;

(5) The nucleotide sequence of the primer pair is shown as SEQ ID NO. 25-26, and the nucleotide sequence of the detection probe is shown as SEQ ID NO. 27; and

(6) The nucleotide sequence of the primer pair is shown as SEQ ID NO. 28-29, and the nucleotide sequence of the detection probe is shown as SEQ ID NO. 30.

7. The reagent according to any one of claims 1 to 2 and 4 to 6, wherein the sample to be tested comprises a blood sample, an oral exfoliated cell sample or a saliva sample.

8. A kit for detecting HPV negative head and neck squamous cell carcinoma comprising the agent of any one of claims 1-7.

9. The kit for detecting HPV negative head and neck squamous cell carcinoma of claim 8, further comprising one or more of a nucleic acid extraction reagent, a methylation conversion reagent, a PCR reaction reagent, a sequencing reagent, a negative control, and a positive control;

alternatively, the PCR reaction reagent comprises an amplification buffer, dNTPs, a DNA polymerase and Mg ²⁺ One or more of the following.

10. Use of the agent of any one of claims 1 to 7 or the kit of any one of claims 8 to 9 in the preparation of an HPV-negative head and neck squamous cell carcinoma diagnostic product.