CN113278704A - Marker and product for diagnosing oral squamous cell carcinoma - Google Patents

Abstract

The invention discloses a marker and a product for diagnosing oral squamous cell carcinoma, wherein the marker comprises CA3, CDKN2A and GABRP, wherein the expression of CA3 and GABRP is down-regulated in an oral squamous cell carcinoma patient; CDKN2A was up-regulated in oral squamous carcinoma patients. The invention provides an effective means for diagnosing oral squamous cell carcinoma.

Description

Marker and product for diagnosing oral squamous cell carcinoma

Technical Field

The invention relates to the field of disease diagnosis, in particular to a marker and a product for diagnosing oral squamous cell carcinoma.

Background

Oral Squamous Cell Carcinoma (OSCC) is one of the ten most common cancers worldwide, with about 300,000 New cases worldwide each year and about 50% five-year survival (Siegel R L, Miller K D, Jemal A. Cancer statistics, 201$ [ J ]. CA: A Cancer Journal for clinicians. 2018, 68(1): 7-30; Chow L Q M. Head and New Cancer [ J ]. New England and Journal of medicine.2020,382(1): 60-72.). Oral Cancer is a broad term malignancy, including those of the buccal, lingual, gingival, floor of mouth, hard palate, and the like, with about 95% of Oral malignancies being squamous cell carcinomas (Montero P H, Patel S G. Cancer of the Oral Cavity [ J ]. Surgical Oncology Clinics of North America. 2015, 24(3): 491 508). Smoking, drinking and viral infection are all the causes of oral squamous carcinoma, and smoking and drinking are two main causes of high oral squamous carcinoma. Although smoking is a risk factor for various cancers, the degree of pathogenesis is not clearly defined, and the overall risk caused by or increased by smoking cannot be accurately calculated in cases with cancer confirmed at present. Studies of the underlying mechanisms of Oral squamous cell carcinoma have shown that free radicals (e.g., reactive oxygen species generated by smoking contribute to the development and progression of Oral squamous cell carcinoma), and that an excess of iron and copper ions in saliva can enhance the action of free radicals (Nagler R, Weizman A, Gavish A. Cigarette serum, saliva, the translocator protein 18 kDa (TSPO), and Oral cancer [ J ]. Oral diseases. 2019, 25(8): 1843 and 1849.). Cigarette smoke has an inhibitory effect on immune cell production, can cause Inflammation, suppress mucosal immunity and cause a decrease in autoimmunity, resulting in oral cancer (Lee J, Taneja V, Vassallo R. Cigarette cooking and Inflammation [ J ]. Journal of Dental research 2012, 91(2): 142-. Smoking and drinking can inhibit metabolic enzymes related to 5-fluorouracil (5-FU) by inducing the activity of dihydropyrimidine dehydrogenase (DPD; the only catabolic enzyme of 5-FU), which in turn leads to oral cancer (Yamashita T, Kato K, Long N K, et al. Effects of eating and alcohol regulation on 5-tluoouracil-related metabolic enzymes in oral cavity cell cancer [ J ]. Molecular and Clinical Oncology 2014, 2(3): 429 and 434.).

Squamous cell carcinoma is characterized by a late diagnosis, a poor prognosis, a low overall survival rate and a tendency to local recurrence (Wang B, Zhang S, Yue K, et al, The recurrence and subvalue of oral square cell carcinosa: a report of 275 cases [ J ]. Chinese Journal of cancer. 2013, 32(11): 614-618.). The reasons for the high mortality of oral squamous carcinoma can be summarized as difficulty in early diagnosis or lack of specific indicators, difficulty in predicting tumor development and prognosis of the patient. Early identification and diagnosis of oral cancer can improve survival and reduce the incidence of complications resulting from treatment. In diagnosing oral cancer, many examination and testing techniques have been developed, such as cell vital staining, oral cytology, oral spectroscopy, and blood and saliva analysis. The sensitivity and specificity of the advanced non-invasive technology detection technologies provide a research direction for developing more effective oral cancer diagnosis methods. However, in system evaluation, there is a lack of sufficient evidence or key details to validate and eliminate the risk that these techniques may be biased.

With the rapid development of sequencing technology and the deep mining of disease genes, the molecular mechanism of tumor formation has become a research focus by using data mining technology to screen genes driving tumorigenesis and development from mass data (Zou J, Huss M, Abid A, et al A primer on deletion learning in genetics [ J ]. Nature genetics. 2019, 51(1): 12-18.). The method is characterized in that key molecules in the oral squamous cell carcinoma development process are explored, the effect of the key molecules in the oral squamous cell carcinoma development process is clarified, and personalized molecular markers are necessary for diagnosing the oral squamous cell carcinoma, and meanwhile, the markers have important significance for helping patients with the oral squamous cell carcinoma to find therapeutic molecular targets, carrying out prognosis judgment and customizing a therapeutic scheme in a personalized manner.

Disclosure of Invention

In order to make up for the defects of the prior art, the invention provides the following technical scheme:

in one aspect, the invention provides the use of an agent for determining biomarkers comprising any two or three of CA3, CDKN2A, GABRP in a sample in the manufacture of a diagnostic product for oral squamous carcinoma.

In a particular aspect, the diagnostic product comprises an agent for detecting the level of CA3, CDKN2A, and/or GABRP expression in a sample.

In another aspect, the invention provides a diagnostic product for oral squamous carcinoma comprising reagents for detecting the biomarkers CA3, CDKN2A and/or GABRP.

In a particular aspect, the reagent is a reagent that detects the amount of RNA transcribed from the biomarker.

In another specific aspect, the agent is an agent that detects the amount of a polypeptide encoded by the biomarker.

In yet another aspect, the present invention provides an apparatus for diagnosing/predicting oral squamous carcinoma, the apparatus comprising:

a processor;

an input module for inputting a level of a biomarker in a biological sample;

a computer-readable medium containing instructions that, when executed by the processor, perform an algorithm on the input levels of the biomarkers; and

an output module that indicates whether the subject has or is at risk of developing oral squamous cell carcinoma;

in a particular aspect, the biomarker is selected from CA3, CDKN2A, and/or GABRP.

In one aspect, the invention provides a computer-readable storage medium having stored thereon a computer program which, when executed by a processor, implements the apparatus as described above.

In another aspect, the present invention provides an electronic device comprising:

a client component, wherein the client component comprises a user interface;

a server component, wherein the server component comprises at least one memory unit configured to receive data input comprising sequencing data for biomarkers generated from a sample, the biomarkers comprising CA3, CDKN2A, and/or GABRP;

the user interface operatively coupled with the server component; and

a computer processor operatively coupled to the at least one memory unit, wherein the computer processor is programmed with an executable program for running an oral squamous carcinoma diagnostic model constructed from the biomarkers.

Has the advantages that:

according to the invention, by detecting the expression levels of CA3, CDKN2A and/or GABRP, the diagnosis of oral squamous cell carcinoma can be realized, the detection sensitivity is increased, the detection capability and efficiency are improved, and the capability of guiding the clinical treatment of the oral squamous cell carcinoma is improved.

Drawings

Fig. 1 shows the CA3 gene mRNA differential expression profile, in which panel a: TCGA; and B: GEO;

fig. 2 shows CDKN2A gene mRNA differential expression profiles, in which panel a: TCGA; and B: GEO;

FIG. 3 shows the differential mRNA expression profile of the GABRP gene, where panel A: TCGA; and B: GEO;

fig. 4 shows ROC plots of CA3 gene diagnosis of oral squamous carcinoma, in which panel a: TCGA; and B: GEO;

figure 5 shows ROC plots of CDKN2A gene diagnosis of oral squamous carcinoma, where panel a: TCGA; and B: GEO;

fig. 6 shows ROC plots of GABRP gene diagnosis of oral squamous carcinoma, in which panel a: TCGA; and B: GEO;

fig. 7 shows ROC plots of CA3+ CDKN2A gene diagnosis of oral squamous carcinoma, where panel a: TCGA; and B: GEO;

fig. 8 shows ROC plots of CA3+ GABRP gene diagnosis of oral squamous carcinoma, in which panel a: TCGA; and B: GEO;

fig. 9 shows ROC plots of CDKN2A + GABRP gene diagnosis of oral squamous carcinoma, where panel a: TCGA; and B: GEO;

figure 10 shows ROC plots for the combined diagnosis of oral squamous carcinoma by CA3+ CDKN2A + GABRP, where panel a: TCGA; and B: GEO.

Detailed Description

General definitions and terms

The invention will be described in further detail below with the understanding that the terminology is intended to be in the nature of words of description rather than of limitation.

The term "sample" or "test sample" refers to a composition obtained or derived from an individual of interest that comprises cellular entities and/or other molecular entities to be characterized and/or identified, e.g., based on physical, biochemical, chemical, and/or physiological characteristics. For example, the phrase "disease sample" and variations thereof refers to any sample obtained from an individual of interest that is expected or known to contain cellular and/or molecular entities to be characterized. The sample may be obtained from a tissue of the subject of interest or from peripheral blood of the subject. For example, the sample may be obtained from blood and other fluid samples of biological origin and tissue samples, such as biopsy tissue samples or tissue cultures or cells derived therefrom. The source of the tissue sample may be solid tissue, such as from a fresh, frozen and/or preserved organ or tissue sample, biopsy tissue or aspirate; blood or any blood component; a body fluid; cells from any time of pregnancy or development of the individual; or plasma. The term "sample" or "test sample" includes a biological sample that has been manipulated in any manner after it has been obtained, such as by treatment with a reagent, stabilization, or enrichment for certain components (e.g., proteins or polynucleotides), or embedding in a semi-solid or solid matrix for sectioning purposes. For purposes herein, a "slice" of a tissue sample means a single portion or piece of the tissue sample, e.g., a thin slice of tissue or cells cut from the tissue sample. Samples include, but are not limited to, whole blood, blood-derived cells, serum, plasma, lymph, synovial fluid, cell extracts, and combinations thereof.

The term "biomarker" refers to an indicator of a patient's phenotype (e.g., a pathological state or possible reactivity to a therapeutic agent) that can be detected in a biological sample from the patient. Biomarkers include, but are not limited to, DNA, RNA, proteins, carbohydrates, or glycolipid-based molecular markers.

In the present invention, the biomarkers include CA3, CDKN2A, and/or GABRP. Biomarkers such as CA3 (carbonic anhydrase 3, gene ID: 761), CDKN2A (cyclic dependent kinase inhibitor 2A, gene ID: 1029), GABRP (gamma-aminobutyric acid type A receptor subunit pi, gene ID: 2568); including genes and their encoded proteins and homologs, mutations, and isoforms. The term encompasses full-length, unprocessed biomarkers, as well as any form of biomarker that results from processing in a cell. The term encompasses naturally occurring variants (e.g., splice variants or allelic variants) of the biomarkers. The gene ID is available at https:// www.ncbi.nlm.nih.gov/gene/.

The term "hybridization" refers to the process by which two nucleic acid fragments bind by stable and specific hydrogen bonds under appropriate conditions to form a duplex complex.

The term "amplification primer" or "primer" refers to a nucleic acid fragment comprising 5 to 100 nucleotides, preferably 15 to 30 nucleotides, capable of initiating an enzymatic reaction (e.g., an enzymatic amplification reaction).

The term "(hybridization) probe" refers to a nucleic acid sequence comprising at least 5 nucleotides, e.g., 5 to 100 nucleotides, that hybridizes under specified conditions to an expression product of a target gene or an amplification product of the expression product to form a complex. The hybridization probes may also include labels for detection. Such labels include, but are not limited to, labels for fluorescent quantitative PCR or fluorescent in situ hybridization. In a preferred embodiment, the label may be FAM, HEX, VIC, Cy5, or the like. In another preferred embodiment, the marker may be biotin, digoxigenin, or the like.

The terms "level of expression" or "expression level" are generally used interchangeably and generally refer to the amount of a polynucleotide or amino acid product or protein in a biological sample. "expression" generally refers to the process by which information encoded by a gene is converted into structures that are present and operational in a cell. Thus, "expression" of a gene as used herein refers to transcription into a polynucleotide, translation into a protein, or even post-translational modification of a protein. Transcribed polynucleotides, translated proteins, or fragments of post-translationally modified proteins are also considered to be expressed, whether they are derived from transcripts produced or degraded by alternative splicing, or from post-translational processing of proteins (e.g., by proteolysis). "expressed genes" include those that are transcribed into a polynucleotide (e.g., mRNA) and then translated into a protein, as well as those that are transcribed into RNA but not translated into a protein (e.g., transfer RNA and ribosomal RNA).

The term "diagnosis" is used herein to refer to the identification or classification of a molecular or pathological state, disease or disorder. For example, "diagnosing" can refer to identifying a risk of developing oral squamous carcinoma, either by the involved tissue/organ (e.g., oral squamous carcinoma), or by a molecular characteristic (e.g., expression characterized by a particular gene or one or a combination of the proteins encoded by that gene).

"aiding diagnosis" refers to a method of aiding in making a clinical decision as to the presence or nature of a particular type of symptom or condition. For example, a method of aiding diagnosis of oral squamous cell carcinoma may comprise measuring the expression of certain genes in a biological sample from an individual.

The term "antibody" is used herein in the broadest sense and includes monoclonal antibodies (e.g., full length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity), and may also include certain antibody fragments (as described in more detail herein). The antibody may be a human, humanized and/or affinity matured antibody.

An "antibody fragment" comprises only a portion of an intact antibody, wherein the portion preferably retains at least one, preferably most or all, of the functions normally associated with the portion when present in an intact antibody. In one embodiment, the antibody fragment comprises the antigen-binding site of an intact antibody, thereby retaining the ability to bind antigen. In another embodiment, an antibody fragment (e.g., an antibody fragment comprising an Fc region) retains at least one of the biological functions normally associated with the Fc region when present in an intact antibody, such as FcRn binding, ADCC function, and complement binding. In one embodiment, the antibody fragment is a monovalent antibody having an in vivo half-life substantially similar to that of an intact antibody. For example, such an antibody fragment may comprise an antigen-binding arm linked to an Fc sequence capable of conferring in vivo stability to the fragment.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, unlike polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of one or more chains is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

The term "diabodies" refers to small antibody fragments having two antigen-binding sites, which fragments comprise a variable heavy domain (VH) linked to a variable light domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on the other chain and two antigen binding sites are created.

An "affinity matured" antibody is one that has one or more alterations in one or more hypervariable regions thereof which result in an improvement in the affinity of the antibody for an antigen compared to a parent antibody that does not have those alterations. In certain embodiments, an affinity matured antibody will have nanomolar or even picomolar affinity for the target antigen.

The detection of the gene expression level described herein can employ assay methods known in the art, including, but not limited to, methods that detect the amount of an RNA transcript of the biomarker or the amount of a polypeptide encoded by the biomarker.

The RNA transcript of the biomarker may be converted to cDNA complementary thereto by methods known in the art, and the amount of the RNA transcript may be obtained by determining the amount of complementary cDNA. The amount of RNA transcript of a biomarker, or cDNA complementary thereto, can be normalized to the amount of total RNA or total cDNA in a sample, or to the amount of RNA transcript of a panel of housekeeping genes, or cDNA complementary thereto.

In this context, RNA transcripts can be detected and quantified by methods such as hybridization, amplification, sequencing, including, but not limited to, methods of hybridizing RNA transcripts to probes or primers, methods of detecting the amount of RNA transcripts or their corresponding cDNA products by various quantitative PCR techniques or sequencing techniques based on the Polymerase Chain Reaction (PCR). The quantitative PCR techniques include, but are not limited to, fluorescent quantitative PCR, real-time PCR, or semi-quantitative PCR techniques. Such sequencing techniques include, but are not limited to, Sanger sequencing, second-generation sequencing, third-generation sequencing, single cell sequencing, and the like. Preferably, the sequencing technique is next generation sequencing, more preferably a targeted RNA-seq technique.

Herein, the amount of polypeptide can be detected by, for example, proteomics or reagents. Preferably, the agent is an antibody, an antibody fragment or an affinity protein.

Applications of

The invention relates to the use of reagents for determining biomarkers in a sample, including any two or three of CA3, CDKN2A, GABRP, in the manufacture of a diagnostic product for oral squamous carcinoma.

Preferably, the diagnostic product comprises reagents for detecting the level of CA3, CDKN2A and/or GABRP expression in a sample.

In one embodiment, the reagents comprise reagents for detecting the level of biomarker expression in a sample by digital imaging techniques, protein immunization techniques, dye techniques, nucleic acid sequencing techniques, nucleic acid hybridization techniques, chromatography techniques, mass spectrometry techniques.

As an alternative embodiment, the sample is a tissue sample; preferably, the tissue sample comprises plasma, serum or blood extract, brush, biopsy or surgically excised tissue or fluid sample from the subject.

In a preferred embodiment, the sample is a tumor tissue sample or a tissue sample comprising tumor cells.

Diagnostic product

The invention relates to a diagnostic product for oral squamous carcinoma, comprising reagents for detecting the biomarkers CA3, CDKN2A and/or GABRP.

In one embodiment, the agent is an agent that detects the level of expression of the biomarker.

In one embodiment, the diagnostic product is in the form of an in vitro diagnostic product.

In a specific embodiment, the diagnostic product is a diagnostic kit.

In one embodiment, the reagent is a reagent that detects the amount of RNA, particularly mRNA, transcribed from the biomarker. In yet another embodiment, the reagent is a reagent that detects the amount of cDNA complementary to the mRNA.

In a preferred embodiment, the diagnostic product further comprises a total RNA extraction reagent, a reverse transcription reagent and/or a secondary sequencing reagent.

The total RNA extraction reagent can be a total RNA extraction reagent which is conventional in the field. Examples include, but are not limited to Qiagen 73504, RNA storm CD201, Invitrogen and ABI AM 1975.

The reverse transcription reagent may be a reverse transcription reagent conventional in the art, and preferably includes a dNTP solution and/or an RNA reverse transcriptase. Examples of reverse transcription reagents include, but are not limited to, NEB M0368L, Thermo K1622, ABI 4366596.

The second-generation sequencing reagent may be a reagent conventionally used in the art as long as it can satisfy the requirement of second-generation sequencing of the resulting sequence. The second generation sequencing reagents may be commercially available products, examples of which include, but are not limited to, Illumina MIseq. Reagent Kit v3(150 cycles) (MS-102. sup. 3001), TruSeq. Targeted RNA Index KitA-96 Inds (384Samples) (RT-402. sup. 1001). Secondary sequencing is a technique conventional in the art, such as targeted RNA-seq technology. Thus, the second generation sequencing reagents may also include reagents that can be tailored for constructing a library Illumina targeting RNA-seq, such as the Targeted RNA Custom Panel Kit (96Samples) (RT-102-1001).

In a preferred embodiment, the agent is a probe or primer.

In alternative embodiments, the reagent is a reagent that detects the amount of the polypeptide encoded by the biomarker.

In particular embodiments, the agent is an antibody, antibody fragment, or affinity protein.

A "kit" is an article of manufacture (e.g., a package or container) containing probes for specifically detecting the biomarker genes or proteins of the present invention. In certain embodiments, the article of manufacture is marketed, distributed, or sold as a unit for performing the methods of the present invention.

Such kits may comprise carrier means compartmentalized to receive, in close confinement, one or more container means (e.g., vials, tubes, etc.), each container means comprising one of the separate components to be used in the method. For example, one of the container means may comprise a probe that carries or can carry a detectable label. Such probes may be polynucleotides specific for polynucleotides of one or more genes comprising gene expression characteristics. Where the kit utilizes nucleic acid hybridization to detect a target nucleic acid, the kit can also have a container containing one or more nucleic acids for amplifying the target nucleic acid sequence and/or a container containing a reporter means, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, fluorescent, or radioisotope label.

A kit will generally comprise the above-described container and one or more additional containers containing commercially and user-desired materials, including buffers, diluents, filters, needles, syringes, and package inserts containing instructions for use. A label may be present on the container to indicate that the composition is for a particular therapeutic or non-therapeutic application, and may also indicate the direction of in vivo or in vitro use, such as those described above. Other optional components of the kit include one or more buffers (e.g., blocking buffer, wash buffer, substrate buffer, etc.), other reagents (e.g., substrate chemically altered by enzymatic labeling), epitope retrieval solutions, control samples (positive and/or negative controls), control sections, and the like.

Device for measuring the position of a moving object

The present invention relates to a device for diagnosing/predicting oral squamous carcinoma, the device comprising:

a processor;

an input module for inputting a level of a biomarker in a biological sample;

a computer-readable medium containing instructions that, when executed by the processor, perform an algorithm on the input levels of the biomarkers; and

an output module that indicates whether the subject has or is at risk of developing oral squamous cell carcinoma;

in one embodiment, the biomarker is selected from CA3, CDKN2A, and/or GABRP.

An apparatus as applied herein shall at least comprise the above-mentioned modules. The modules of the device are operatively connected to each other. How the modules are operatively linked will depend on the type of module contained in the device.

The processor may execute a series of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as a memory. Instructions may be directed to a processor that may then program or otherwise configure the processor to implement the present disclosure. Examples of operations performed by a processor may include read, decode, execute, and write-back.

Readable storage medium

The invention relates to a computer-readable storage medium, characterized in that a computer program is stored thereon, which computer program, when being executed by a processor, realizes the aforementioned apparatus.

A readable storage medium, such as computer executable code, may take many forms, including but not limited to tangible storage media, carrier wave media, or physical transmission media. Non-volatile storage media include, for example, optical or magnetic disks, any storage device such as in any computer or the like, volatile storage media include dynamic memory, such as the main memory of such computer platforms. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media can take the form of electrical or electromagnetic signals, or acoustic or light waves, such as those generated during radio frequency and infrared data communications. Thus, common forms of computer-readable media include, for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards, paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM, and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer can read programming code and/or data. Many of these computer readable media may take the form of one or more sequences of one or more instructions that are executable by a processor to perform operations.

Electronic device

The invention relates to an electronic device comprising:

a client component, wherein the client component comprises a user interface;

a server component, wherein the server component comprises at least one memory unit configured to receive data input comprising sequencing data for biomarkers generated from a sample, the biomarkers comprising CA3, CDKN2A, and/or GABRP;

the user interface operatively coupled with the server component; and

a computer processor operatively coupled to the at least one memory unit, wherein the computer processor is programmed with an executable program for running an oral squamous carcinoma diagnostic model constructed from the biomarkers.

The following detailed description of embodiments of the present application will be made with reference to the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the present application, are given by way of illustration and explanation only, and are not intended to limit the present application.

Examples genetic markers associated with diagnosis of oral squamous cell carcinoma

1. Data and preprocessing

Public gene expression data and complete clinical annotations were searched in a gene expression integration database (GEO) and a cancer genomic profile database (TCGA). According to clinical information, oral clinical specimens (alveolar ridge, buccal mucosa, oral floor, tongue, lip, oral cavity and hard palate) are selected, specimens of anatomical parts such as hypopharynx, larynx, oropharynx, tonsil and the like are excluded, and samples with incomplete clinical information are removed.

For the data set in TCGA, RNA sequencing data (FPKM values) and clinical information for gene expression were downloaded from UCSC Xena (https:// gdc. Genes were annotated using the tideverse package, genes were de-duplicated and averaged, and FPKM was power-transformed by 2, converting FPKM values to million per kilobase (TPM) valued transcripts.

The gene expression data of the GSE30784 is downloaded from a GEO database (http:// www.ncbi.nlm.nih.gov/GEO /), and is annotated by using an annotation file, and the average value of a plurality of probes corresponding to the same gene is taken as the expression quantity of the gene, and then a gene expression matrix file is obtained.

Wherein, the TCGA data set is used as a training set, and the GEO data set is used as a verification set. After removing the sample with incomplete clinical information, the number of samples contained in the TCGA cohort was paracancerous: carcinoma =44:258, the amount of samples in the GEO cohort is paracarcinoma: carcinoma =45: 167.

2. Differential expression analysis

Differential expression analysis was performed using the "limma" package in the R software, with the screening criteria for differential genes being adj. Pvalue<0.01，|log₂FC|>1.5。

The analysis results showed that CA3, CDKN2A, GABRP all exhibited differential expression in the TCGA and GEO databases, as shown in table 1 and fig. 1-3, wherein CA3, GABRP were down-regulated in oral squamous cell carcinoma and CDKN2A was up-regulated in oral squamous cell carcinoma, compared to the paracarcinoma tissues.

TABLE 1 expression of the genes

3. Diagnostic efficacy analysis

The Receiver Operating Curve (ROC) is drawn by using the R package 'pROC', the AUC value, the sensitivity and the specificity of the differential expression gene serving as a detection variable are analyzed, and the diagnosis efficiency of the indicators alone or in combination is judged.

When the diagnostic efficacy of the individual index is judged, the expression level of the gene is directly used for analysis. When the diagnosis efficiency of the index combination is judged, firstly, glmnet is used for conducting Logistic regression on genes, the established Logistic regression model is utilized to calculate the prediction probability, and an ROC curve of the prediction result is drawn.

The results are shown in table 2 and fig. 4-10, and it can be seen from the table that CA3, CDKN2A, GABRP and their combination have high accuracy in diagnosing oral squamous cell carcinoma, especially the combination of the three has high accuracy, sensitivity and specificity, and the AUC value, sensitivity and specificity in the training set are 0.945, 0.864 and 0.911 respectively; the AUC values, sensitivity, specificity in the validation set were 0.921, 0.911, 0.772, respectively.

TABLE 2 Gene diagnostic potency analysis

The preferred embodiments of the present application have been described in detail with reference to the accompanying drawings, however, the present application is not limited to the details of the above embodiments, and various simple modifications can be made to the technical solution of the present application within the technical idea of the present application, and these simple modifications are all within the protection scope of the present application.

It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described in the present application.

In addition, any combination of the various embodiments of the present application is also possible, and the same should be considered as disclosed in the present application as long as it does not depart from the idea of the present application.

Claims (10)

1. Use of a reagent for determining a biomarker in a sample, wherein the biomarker comprises any two or three of CA3, CDKN2A, GABRP, in the manufacture of a diagnostic product for oral squamous carcinoma.

2. Use according to claim 1, wherein the diagnostic product comprises a reagent for detecting the level of CA3, CDKN2A and/or GABRP expression in a sample.

3. The use of claim 2, wherein the reagents comprise reagents for detecting the level of expression of a biomarker in a sample by digital imaging techniques, protein immunization techniques, dye techniques, nucleic acid sequencing techniques, nucleic acid hybridization techniques, chromatography techniques, mass spectrometry techniques.

4. The use of any one of claims 1-3, wherein the sample is a tissue sample.

5. The use of claim 2, wherein the agent is an agent that detects the amount of RNA transcribed from the biomarker.

6. The use of claim 5, wherein the agent is a probe or primer.

7. The use according to claim 5, wherein the diagnostic product further comprises a total RNA extraction reagent, a reverse transcription reagent and/or a secondary sequencing reagent.

8. The use of claim 2, wherein the agent is an agent that detects the amount of a polypeptide encoded by the biomarker.

9. An apparatus for diagnosing/predicting oral squamous carcinoma, the apparatus comprising:

a processor;

an input module for inputting the level of a biomarker in a biological sample, the biomarker selected from CA3, CDKN2A, and/or GABRP;

a computer-readable medium containing instructions that, when executed by the processor, perform an algorithm on the input levels of the biomarkers; and

an output module that indicates whether the subject has or is at risk of developing oral squamous cell carcinoma.

10. An electronic device, comprising:

a client component, wherein the client component comprises a user interface;

a server component, wherein the server component comprises at least one memory unit configured to receive data input comprising sequencing data for biomarkers generated from a sample, the biomarkers comprising CA3, CDKN2A, and/or GABRP;

the user interface operatively coupled with the server component; and

a computer processor operatively coupled to the at least one memory unit, wherein the computer processor is programmed with an executable program for running an oral squamous carcinoma diagnostic model constructed from the biomarkers.

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