CN113322325A - Application of gene group as detection index in oral squamous cell carcinoma diagnosis - Google Patents

Abstract

The invention discloses an application of a gene group as a detection index in diagnosis of oral squamous cell carcinoma, wherein the gene group comprises CKMT2, GABRP and/or KRT23, CKMT2, GABRP and KRT23 are expressed in oral squamous cell carcinoma patients in a down-regulation manner, and whether a subject has oral squamous cell carcinoma or whether the risk of the subject has the oral squamous cell carcinoma can be judged by detecting the expression level of CKMT2, GABRP and/or KRT 23. The invention also discloses application of the CKMT2, the GABRP and/or the KRT23 in constructing a calculation model for predicting oral squamous cell carcinoma.

Description

Application of gene group as detection index in oral squamous cell carcinoma diagnosis

Technical Field

The invention belongs to the field of biological medicine, and relates to application of a gene group as a detection index in oral squamous cell carcinoma diagnosis.

Background

As one of the most common malignant tumors of the head and the neck, oral cancer has the characteristics of fast growth, strong infiltration, easy occurrence of lymph node metastasis and the like. Oral cancer is highly metastatic due to the abundance of vascular lymphatic vessels in the human head and face, with common sites of metastasis including the gums, tongue, soft and hard palate, jaw bone, floor of mouth, oropharynx, salivary glands, upper and lower lips, upper frontal sinus, and facial skin mucosa (Ribeiro I P, Barroso L, Marques F, et al. Early detection and personified treatment in oral cancer: the anatomical of microorganisms associated with J. Molecular Cytogenetics, 2016, 9 (1): 85). Recent studies have shown that there are about 30 million new cases of oral Cancer worldwide each year (Gao Wei, Li John-Zeng-Hong, Chen Si-Qi. Desrigerated breast-expressed X-linked 4 (BEX 4) expression models growth of oral squamous cell carcinosa [ J ]. J. exp. Clin. Cancer Res., 2016,35 (1): 92). Despite clinical surgical intervention and concomitant drug therapy in patients with oral cancer, patient prognosis remains unattractive (Kim B, Kim D. The role of dental care providers and orders and maxima of clinical outcomes in The clinical and clinical management of orders in The United States [ J ]. General department, 2013,61 (7): 47.), especially in The case of distant metastases. It is statistically estimated that about 50% of patients have primary diagnosis with cervical lymph node metastasis, and that the survival rate of the patients is only 50% (Martha a arellano-garcia, Roger li, Xiaojun liu. Identification of tetranectin as a potential biological marker for a metastatic organic cancer [ J ]. Int J Mol Sci, 2010, 11 (9): 21-3106.). Therefore, the research of mechanism for oral cancer is still necessary.

With the advance of modern medical technology, omics research brings unprecedented help to the advance of medicine, and genome research reveals molecular mechanisms of many complex human diseases. The development of the second generation sequencing technology provides a research means with high accuracy, high throughput, high sensitivity and low running cost for the research of genomics, and the technology is widely applied to the search of candidate genes of diseases (Shridhar K, Walia GK, Agrarwal A, et al. DNA methylation markers for Oral pre-cancer progression: A clinical review [ J ]. Oral Oncol, 2016, 53: 1-9.). The gene plays an important regulating role in the cell physiological process, and the regulating mechanism of oral cancer, especially oral squamous cell carcinoma, caused by gene induction cannot be ignored. Therefore, the search for the gene related to oral squamous cell carcinoma is of great significance for realizing the diagnosis and treatment of oral squamous cell carcinoma.

Disclosure of Invention

In order to remedy the deficiencies of the prior art, it is an object of the present invention to provide a biomarker associated with oral squamous cell carcinoma, which marker can be used to diagnose whether a subject has or is at risk of developing oral squamous cell carcinoma.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides application of an agent for detecting biomarkers in a sample in preparing a product for diagnosing oral squamous cell carcinoma, wherein the biomarkers comprise CKMT2, GABRP and/or KRT 23.

Further, the reagent comprises a reagent for detecting the level of the biomarker by a sequencing technology, a nucleic acid hybridization technology, a nucleic acid amplification technology and a protein immunity technology.

Further, the agent is selected from:

a probe that specifically recognizes the biomarker;

primers that specifically amplify the biomarkers; or

An antibody that specifically binds to the biomarker.

Further, the sample is selected from the group consisting of tissue, blood.

The invention provides a product for diagnosing oral squamous cell carcinoma, which comprises an agent for detecting the level of CKMT2, GABRP and/or KRT23 in a sample.

Further, the level of CKMT2, GABRP and/or KRT23 in the sample is determined by measuring the protein level or mRNA level of CKMT2, GABRP and/or KRT23 in the sample.

Further, the protein levels of CKMT2, GABRP and/or KRT23 in the sample are measured by using immunostaining, immunofluorescence, western blot or ELISA.

Further, mRNA levels of CKMT2, GABRP and/or KRT23 in the sample are measured by using microarray, RNA-seq, in situ hybridization, RNA-scope, and conventional semi-quantitative or quantitative RT-PCR.

Further, the product also includes reagents for processing the sample.

The invention provides the use of biomarkers comprising CKMT2, GABRP and/or KRT23 in the construction of a computational model or a system incorporating the computational model for predicting oral squamous carcinoma.

Further, the calculation model is operated by a bioinformatics method with the level of the biomarker as an input variable.

The invention has the advantages and beneficial effects that:

according to the invention, CKMT2, GABRP and/or KRT23 are selected as biomarkers, so that whether a subject suffers from oral squamous cell carcinoma or not can be diagnosed, or the risk of the subject suffering from oral squamous cell carcinoma can be judged, and doctors are guided to take treatment strategies, means and measures timely.

Drawings

Fig. 1 shows the differential mRNA expression profile of CKMT2 gene, wherein panel a: TCGA; and B: GEO;

FIG. 2 shows the differential mRNA expression profile of the GABRP gene, where panel A: TCGA; and B: GEO;

figure 3 shows KRT23 gene mRNA differential expression profiles, wherein panel a: TCGA; and B: GEO;

figure 4 shows ROC plots of CKMT2 gene diagnosis of oral squamous cell carcinoma, where panel a: TCGA; and B: GEO;

fig. 5 shows ROC plots of GABRP gene diagnosis of oral squamous carcinoma, in which panel a: TCGA; and B: GEO;

figure 6 shows ROC plots of KRT23 gene for diagnosing oral squamous carcinoma, where panel a: TCGA; and B: GEO;

figure 7 shows ROC plots of CKMT2+ GABRP gene diagnosis of oral squamous cell carcinoma, where panel a: TCGA; and B: GEO;

figure 8 shows ROC plots of CKMT2+ KRT23 gene for diagnosing oral squamous cell carcinoma, where panel a: TCGA; and B: GEO;

fig. 9 shows ROC plots of GABRP + KRT23 gene for diagnosing oral squamous carcinoma, where panel a: TCGA; and B: GEO;

figure 10 shows ROC plots for the combination diagnosis of oral squamous cell carcinoma by CKMT2+ GABRP + KRT23, wherein panel a: TCGA; and B: GEO.

Detailed Description

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 invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

The terms "abundance," "level," and "content" are used interchangeably herein to refer to a quantitative content (e.g., weight or mole), a semi-quantitative content, a relative content (e.g., weight% or mole% within a grade), a concentration, and the like. Thus, these terms encompass the absolute or relative amounts or concentrations of cancer treatment biomarkers in a sample.

The term "and/or" means and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

In the present invention, the term "including" is used to mean, and is used interchangeably with, the phrase "including but not limited to".

In the present invention, the term "biomarker" means a compound, preferably a gene, which is differentially present (i.e. increased or decreased) in a biological sample from a subject or a group of subjects having a first phenotype (e.g. having a disease) compared to a biological sample from a subject or a group of subjects having a second phenotype (e.g. no disease). The term "biomarker" generally refers to the presence/concentration/amount of one gene or the presence/concentration/amount of two or more genes.

The term "biomarker value" or "biomarker level" refers to a value measured or derived for at least one corresponding biomarker in a subject, and which is typically at least partially indicative of the abundance or concentration of the biomarker in a sample taken from the subject. Thus, a biomarker value may be a measured biomarker value, which is a biomarker value measured for a subject, or alternatively may be a derived biomarker value, which is a value derived from one or more measured biomarker values, for example, by applying a function to one or more measured biomarker values. The biomarker values may be in any suitable form, depending on the manner in which the values are determined. For example, biomarker values may be determined using high throughput techniques such as sequencing platforms, array and hybridization platforms, mass spectrometry, immunoassays, immunofluorescence, flow cytometry, or any combination of these techniques. In a preferred example, biomarker values relate to the abundance or activity level of an expression product or other measurable molecule, quantified using techniques such as quantitative RT-PCR, sequencing, and the like. In this case, the biomarker values may be in the form of amplification levels or cycle numbers, which are logarithmic representations of biomarker concentrations within the sample, as known to those skilled in the art. In other preferred examples, immunofluorescence of cells containing the expression product is used to quantify biomarker values.

The "level", "abundance" or "amount" of a biomarker is the detectable level or amount in a sample. These can be measured by methods known to those skilled in the art and also disclosed herein. These terms include quantitative content or level (e.g., weight or mole), semi-quantitative content or level, relative content or level (e.g., weight% or mole% within a grade), concentration, and the like. Thus, these terms include the absolute or relative amounts or levels of the biomarkers in the sample. The level or amount of expression of the biomarker assessed can be used to determine whether the subject is diseased or whether there is a risk of disease. In particular embodiments in which the level of a biomarker is "reduced" relative to a reference or control, the reduced level can refer to an overall reduction in the level of the biomarker (e.g., protein or nucleic acid (e.g., mRNA)) of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more, as compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue, as detected by standard art-known methods, such as those described herein. In certain embodiments, a reduced level refers to a reduction in the level/amount of a biomarker in a sample, wherein the reduction is at least about 0.9 x, 0.8 x, 0.7 x, 0.6 x, 0.5 x, 0.4 x, 0.3 x, 0.2 x, 0.1 x, 0.05 x, or 0.01 x the level/amount of the corresponding biomarker in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In certain embodiments, wherein the level of the biomarker is "about the same" as a reference or control, the level of the biomarker differs by less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or even less as compared to the level of the biomarker (e.g., protein or nucleic acid (e.g., mRNA or cDNA)) in the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue, as detected by standard art-known methods such as those described herein.

The term "nucleic acid" or "polynucleotide" as used herein includes RNA, mRNA, miRNA, cRNA, cDNA, mtDNA, or DNA. The term generally refers to a polymeric form of nucleotides, ribonucleotides or deoxyribonucleotides or a modified form of any type of nucleotide that is at least 10 bases long. The term includes both single-stranded and double-stranded forms of DNA or RNA.

In a specific embodiment of the invention, the biomarker comprises CKMT2, GABRP, KRT 23.

In the present invention, CKMT2 (gene ID: 76722) includes CKMT2 gene and homologs, mutations, and isoforms thereof. The term encompasses full-length, unprocessed CKMT2, as well as any form of CKMT2 that results from processing in a cell. The term encompasses naturally occurring variants (e.g., splice variants or allelic variants) of CKMT 2.

GABRP (gene ID: 2568) includes the GABRP gene and homologs, mutations, and isoforms thereof. The term encompasses full-length, unprocessed GABRP, as well as any form of GABRP that results from processing in a cell. The term encompasses naturally occurring variants (e.g., splice variants or allelic variants) of GABRP.

KRT23 (gene ID: 25984) includes the KRT23 gene and homologs, mutations, and isoforms thereof. The term encompasses full-length, unprocessed KRT23, as well as any form of KRT23 that results from processing in a cell. The term encompasses naturally occurring variants (e.g., splice variants or allelic variants) of KRT 23.

The gene ID of the above gene is available as https:// www.ncbi.nlm.nih.gov/gene/.

The term "primer" refers to an oligonucleotide that, when paired with a DNA strand, is capable of priming the synthesis of a primer extension product in the presence of a suitable polymerizing agent. The primer is preferably single-stranded for maximum amplification efficiency, but may also be double-stranded. The primer must be long enough to prime the synthesis of extension products in the presence of the polymerization agent. The length of the primer depends on many factors, including the application, the temperature to be used, the template reaction conditions, other reagents, and the source of the primer. For example, depending on the complexity of the target sequence, the primer can be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, 500 to the 3 ' end of the primer a length of one base shorter than the template sequence to allow extension of the nucleic acid strand, although the 5 ' end of the primer can extend in length beyond the 3 ' end of the template sequence. In certain embodiments, the primer may be a large polynucleotide, such as about 35 nucleotides to several kilobases or more. The primer may be selected to be "substantially complementary" to a sequence on the template that is designed to hybridize thereto and serve as a synthesis initiation site. By "substantially complementary" is meant that the primers are sufficiently complementary to hybridize to the target polynucleotide. Ideally, a primer does not contain a mismatch to the template to which it is designed to hybridize, but this is not required. For example, a non-complementary nucleotide residue can be attached to the 5' end of the primer, while the remainder of the primer sequence is complementary to the template. Alternatively, a non-complementary nucleotide residue or a stretch of non-complementary nucleotide residues may be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the template sequence to hybridize therewith, thereby forming a template for synthesizing a primer extension product.

The term "probe" refers to a molecule that binds to a particular sequence or subsequence or other portion of another molecule. Unless otherwise indicated, the term "probe" generally refers to a nucleic acid probe that binds to another nucleic acid (also referred to herein as a "target polynucleotide") by complementary base pairing. Probes can bind target polynucleotides that lack complementarity to the entire sequence of the probe, depending on the stringency of the hybridization conditions. Probes may be directly or indirectly labeled and include primers within their scope.

The term "sample" includes any biological sample that may be extracted, untreated, treated, diluted or concentrated from a subject. The sample may include, without limitation, biological fluids such as whole blood, serum, red blood cells, white blood cells, plasma, saliva, urine, feces (i.e., feces), tears, sweat, cortex, nipple aspirates, catheter lavage, tumor exudate, synovial fluid, ascites, peritoneal fluid, amniotic fluid, cerebrospinal fluid, lymph fluid, fine needle aspirate, amniotic fluid, any other bodily fluid, cell lysate, cell secretion products, inflammatory fluid, semen, and vaginal secretion. Samples may include tissue samples and biopsies, tissue homogenates, and the like. Advantageous samples may include samples comprising detectable amounts of any one or more of the biomarkers as taught herein. In certain embodiments, the sample contains blood, particularly peripheral blood, or a fraction or extract thereof. In particular embodiments, the sample comprises cancer cells or tumor cells.

The term "gene" refers to a nucleic acid strand that encodes a functional polypeptide or RNA strand. Although the exon regions of the gene are transcribed to form mRNA, the term "gene" also includes regulatory regions, such as promoters and enhancers, that control the expression of the exon regions.

The term "immobilized" means that the molecular species of interest is immobilized on a solid support, suitably by covalent bonds. The covalent bond may be achieved in different ways depending on the molecular nature of the molecular species. Furthermore, molecular species may also be immobilized on a solid support by electrostatic forces, hydrophobic or hydrophilic interactions, or van der waals forces. The above-mentioned physicochemical interactions generally occur in the interaction between molecules. In particular embodiments, all that is required is that the molecule (e.g., nucleic acid or polypeptide) remain immobilized or attached to the support under the conditions in which the support is intended to be used, e.g., in applications requiring nucleic acid amplification and/or sequencing or in antibody binding assays. For example, oligonucleotides or primers are immobilized such that the 3' end is available for enzymatic extension and/or at least a portion of the sequence is capable of hybridizing to a complementary sequence. In some embodiments, immobilization may occur by hybridization to a surface-attached primer, in which case the immobilized primer or oligonucleotide may be in the 3 '-5' orientation. In other embodiments, immobilization may occur by means other than base-pairing hybridization, such as covalent attachment.

The term "solid support" refers to a solid inert surface or body to which molecular species (e.g., nucleic acids and polypeptides) can be immobilized. Non-limiting examples of solid supports include glass surfaces, plastic surfaces, latex, dextran, polystyrene surfaces, polypropylene surfaces, polyacrylamide gels, gold surfaces, and silicon wafers. In some embodiments, the solid support is in the form of a membrane, chip, or particle. In some embodiments, the solid support may be a glass surface (e.g., the plane of a flow cell channel). In some embodiments, the solid support may comprise an inert surface or substrate that has been "functionalized", such as by applying a layer or coating of an intermediate material comprising reactive groups that allow covalent attachment to a molecule (e.g., a polynucleotide). For example, such a support may comprise a polyacrylamide hydrogel supported on an inert surface (e.g., glass). Molecules (e.g., polynucleotides) can be directly covalently attached to an intermediate material (e.g., a hydrogel), but the intermediate material can itself be a non-covalently attached substrate or matrix (e.g., a glass matrix). The support may comprise a plurality of particles or beads, each having a different attached molecular species.

Detection of biomarker nucleic acids

In some embodiments, the biomarker is assessed by determining biomarker nucleic acid transcript levels. In an illustrative nucleic acid-based assay, nucleic acids are isolated from cells contained in a biological sample according to standard methods (Sambrook et al, 1989, supra; and Ausubel et al, 1994, supra). The nucleic acid is typically fractionated or whole cell RNA. In some embodiments, the nucleic acid is amplified by a template-dependent nucleic acid amplification technique. A variety of template-dependent methods can be used to amplify the cancer treatment biomarker sequences present in a given template sample. An exemplary nucleic acid amplification technique is the polymerase chain reaction (referred to as PCR).

In certain advantageous embodiments, the template-dependent amplification involves transcript quantification in real time. For example, real-time PCR techniques can be used to quantify RNA or DNA. By determining the concentration of the target DNA amplification product in a PCR reaction that completes the same number of cycles and is within its linear range, the relative concentration of a particular target sequence in the original DNA mixture can be determined. If the DNA mixture is cDNA synthesized from RNA isolated from different tissues or cells, the relative abundance of the particular mRNA from which the target sequence is derived can be determined for each tissue or cell. Real-time PCR is typically performed using any PCR instrument available in the art. In general, instruments for real-time PCR data collection and analysis include a thermal cycler, optics for fluorescence excitation and emission collection, and optionally a computer and data acquisition and analysis software.

In certain embodiments, the target nucleic acid is quantified using blotting techniques, which are well known to those skilled in the art. Southern blotting involves the use of DNA as a target, whereas Northern blotting involves the use of RNA as a target. Each providing different types of information, although in many respects, cDNA blots are similar to blots or RNA material. Briefly, probes are used to target DNA or RNA species that have been immobilized on a suitable substrate, often a nitrocellulose filter. The different substances should be spatially separated to facilitate analysis. This is usually done by gel electrophoresis of the nucleic acid material, followed by "blotting" onto the filter. Subsequently, the blotted target is incubated with a probe (usually labeled) under conditions that promote denaturation and rehybridization. Removing the unbound probe to complete the detection. After detection/quantification, the results observed in a given subject can be compared to a reference group or control subject population that is statistically significant herein. In this way, the amount of cancer biomarker nucleic acid detected can be correlated with the progression or severity of the disease.

Chip hybridization utilizes biomarker-specific oligonucleotides attached to a solid substrate, which may consist of a particulate solid phase designed as a microarray, such as a nylon filter, glass slide, or silicon chip. Microarrays are known in the art and consist of a surface on which probes with sequences corresponding to gene products (e.g., cdnas) can be specifically hybridized or bound at known locations to detect biomarker gene expression.

Quantification of hybridization complexes is well known in the art and can be accomplished by any of several methods. These methods are typically based on the detection of labels or markers, such as any radioactive, fluorescent, biological or enzymatic labels or tags used as standard in the art. Labels may be applied to the oligonucleotide probes or to RNA derived from the biological sample.

In general, mRNA quantification can be suitably performed together with a calibration curve to achieve accurate mRNA determination. Furthermore, it is preferred to quantify the transcripts originating from the biological sample by comparison with a control sample, said sample being characterized by a known expression pattern of the transcripts examined.

Detection of biomarker proteins

In some embodiments, cancer therapy biomarkers are assessed at the protein expression level by demonstrating the presence of the protein (isolated or one or in the cell), or by one or more known functional properties of the biomarker. For example, anti-CKMT 2, GABRP, KRT23 antibodies for use in protein assays specific for CKMT2 or specific for GABRP or specific for KRT23 are known in the art, are commercially available, and can also be readily produced by one of skill in the art. Antibodies and antigen-antibody complexes can be detected by several assays well known in the art, including immunofluorescence assays, immunohistochemistry, Fluorescence Activated Cell Sorting (FACS) analysis, enzyme-linked immunosorbent assays (ELISA), Radioimmunoassays (RIA), light emission immunoassays, and western blot analysis.

In particular embodiments, immunofluorescence or immunocytochemistry is performed to detect the protein. Cells, such as tumor cells, can be isolated or enriched by methods known in the art. Isolation or enrichment of cells refers to a process in which the percentage of specific cells (e.g., tumor cells) is increased relative to the percentage in the sample prior to the enrichment procedure. Purification is an example of enrichment. In other embodiments, antibodies directed to surface markers on tumor cells can be attached to a solid support for isolation. Procedures for separation may include the use of antibody magnetic beads (e.g., Miltenyi)^TMBeads), affinity chromatography, "panning" using antibodies attached to a solid matrix, or any other convenient technique, such as Laser Capture Microdissection. Other techniques that provide particularly accurate separation include FACS. Once deposited on the slide, the cells can be fixed and probed with labeled antibodies to detect cancer diagnostic biomarkers.

Antibodies specific for cancer biomarkers can be directly conjugated to fluorescent markers including fluorescein, FITC, rhodamine, Texas Red, Cy3, Cy5, Cy7, and other fluorescent markers and the filters observed under a fluorescent microscope equipped with appropriate filters. The antibody may also be conjugated to an enzyme that initiates the reaction upon addition of an appropriate substrate, thereby providing a colored precipitate on the cells with the biomarker protein detected. The slide can then be viewed by a standard optical microscope. Alternatively, the primary antibody specific for the cancer diagnostic biomarker may be further bound to a secondary antibody conjugated to a detectable moiety.

Immunohistochemistry is in principle very similar to immunofluorescence or immunocytochemistry, however, for example, in contrast to cell suspensions, tissue specimens are probed with antibodies specific for cancer treatment biomarkers. The biopsy specimen is fixed and processed and optionally embedded in paraffin and, if necessary, sectioned to provide a cell or tissue slide for subsequent detection with heparanase-specific antibodies. Alternatively, frozen tissue cryostats can be sectioned and then antibody probed to avoid fixation-induced antigen masking. Antibodies, as in immunofluorescence or immunocytochemistry, are coupled to a fluorescent or enzyme-linked detectable moiety and used to probe tissue sections by methods described for immunofluorescence, and then viewed by fluorescence or confocal microscopy depending on the detection method used. After the reaction product is formed, visualization of the reaction product precipitate can be observed by standard optical microscopy if an enzymatically detectable moiety is utilized.

In other embodiments, assays such as ELISA and RIA are used, which follow similar principles for detecting specific antigens. As an illustrative example, CKMT2, GABRP or KRT23 may be measured by using RIA with CKMT2, GABRP or KRT23 specific antibodies, typically radiolabeled with 125I. In an ELISA assay, CKMT2, GABRP or KRT23 specific antibodies are chemically linked to the enzyme. Capture antibodies specific for CKMT2, GABRP or KRT23 are immobilized on a solid support. Unlabeled samples, such as protein extracts from biological samples, are then incubated with the immobilized antibodies under conditions in which non-specific binding is blocked, and unbound antibodies and/or proteins are removed by washing. Bound CKMT2, GABRP or KRT23 is detected by a second CKMT2, GABRP or KRT 23-specific labeled antibody. In RIA, antibody binding is measured directly by measuring radioactivity, whereas in ELISA binding is detected by the reaction of a colorless substrate to a colored reaction product as a function of the activity of the linked enzyme. Thus, the change can be easily detected by spectrophotometry.

Protein biomarker expression can also be detected by luminescence immunoassay. Much like ELISA and RIA, in a luminescent immunoassay, the biological sample/protein extract to be tested is immobilized on a solid support and probed with a specific label (labeled antibody). The label is luminescent again and upon binding emits light as an indication of specific recognition. Luminescent labels include substances that emit light when activated by electromagnetic radiation, electrochemical excitation, or chemical activation, and may include fluorescent and phosphorescent substances, scintillators, and chemiluminescent substances. The label may be part of a catalytic reaction system, such as an enzyme, enzyme fragment, enzyme substrate, enzyme inhibitor, coenzyme, or catalyst; a part of a chromogen system, such as a fluorophore, dye, chemiluminescent, luminescent or sensitizing agent; dispersible particles (which may be non-magnetic or magnetic), solid supports, liposomes, ligands, receptors, hapten radioisotopes, and the like.

Western blot analysis is another method for assessing the amount of cancer biomarker polypeptides in a biological sample. Protein extracts from biological samples of cells (e.g., tumor cells) are lysed in a denaturing ionization environment and aliquots are applied to a polyacrylamide gel matrix. As it migrates toward the anode, the proteins will separate based on molecular size characteristics. The antigen is then transferred to a nitrocellulose, PVDF or nylon membrane, and then membrane blocking is performed to minimize non-specific binding. The membrane is probed with an antibody directly coupled to the detectable moiety or subsequently probed with a secondary antibody containing the detectable moiety. Typically, horseradish peroxidase or alkaline phosphatase is conjugated to an antibody and the activity is visualized using a chromogenic or luminescent substrate.

In particular embodiments, protein capture arrays are used that allow for the simultaneous detection and/or quantification of large numbers of proteins. For example, low density protein arrays on filter membranes, it is now possible to use protein arrays to analyze protein expression in body fluids, such as serum of healthy or diseased subjects and in subjects before and after drug treatment. Exemplary protein capture arrays include arrays comprising spatially addressed antigen binding molecules, commonly referred to as antibody arrays, which can facilitate extensive parallel analysis of a variety of proteins defining a proteome or a sub-proteome. Antibody arrays have been shown to have desirable specificity and acceptable background characteristics.

Diagnostic product

The invention provides a product for diagnosing oral squamous carcinoma, which comprises a reagent for detecting the biomarker in a sample; and instructions for using the product to assess whether the subject is suffering from or susceptible to oral squamous carcinoma.

The diagnostic product may also optionally include suitable reagents for detecting the marker, positive and negative controls, wash solutions, blotting membranes, microtiter plates, dilution buffers, and the like. For example, a protein-based assay diagnostic product can include (i) at least one cancer biomarker polypeptide; and (ii) an antibody that specifically binds to a cancer therapy biomarker polypeptide. Alternatively, the nucleic acid-based test kit can comprise (i) a cancer treatment biomarker polynucleotide; and (ii) a primer or probe that specifically hybridizes to the cancer treatment biomarker polynucleotide. Enzymes suitable for amplifying nucleic acids may also be included, including various polymerases (reverse transcriptase, Taq, Sequenase^TMDNA ligase, etc.), deoxyribonucleotides and buffers to provide the reaction mixture required for amplification. Such kits will also typically contain a different container for each individual reagent and enzyme, and each primer or probe, in a suitable manner.

Any form of sample assay capable of detecting a sample biomarker described herein may be used. Typically, the assay will quantify the biomarkers in the sample to an extent, for example whether their concentration or amount is above or below a predetermined threshold. Such kits may take the form of test strips, dipsticks, cartridges, chip-based or bead-based arrays, multi-well plates, or a series of containers, and the like. One or more reagents are provided to detect the presence and/or concentration and/or amount of a selected sample biomarker. The sample from the subject may be dispensed directly into the assay or indirectly from a stored or previously obtained sample.

Calculation model

In the present invention, biomarkers may be determined individually, or in one embodiment of the invention, they may be determined simultaneously, for example using a chip or bead-based array technology. The concentration of the biomarkers is then interpreted independently, for example using individual retention of each marker, or a combination thereof.

As the skilled artisan will appreciate, the step of associating a marker level with a certain likelihood or risk may be implemented and realized in different ways.

The logarithmic function used to correlate marker combinations with disease preferably employs algorithms developed and obtained by applying statistical methods. For example, suitable statistical methods are Discriminant Analysis (DA) (i.e., linear, quadratic, regular DA), Kernel methods (i.e., SVM), nonparametric methods (i.e., k-nearest neighbor classifiers), PLS (partial least squares), tree-based methods (i.e., logistic regression, CART, random forest methods, boosting/bagging methods), generalized linear models (i.e., logistic regression), principal component-based methods (i.e., SIMCA), generalized additive models, fuzzy logic-based methods, neural network-and genetic algorithm-based methods. The skilled person will not have problems in selecting a suitable statistical method to evaluate the marker combinations of the invention and thereby obtain a suitable mathematical algorithm. In one embodiment, the statistical method used to obtain the mathematical algorithm used in assessing oral squamous carcinoma is selected from DA (i.e., linear, quadratic, regular discriminant analysis), Kernel method (i.e., SVM), non-parametric method (i.e., k-nearest neighbor classifier), PLS (partial least squares), tree-based method (i.e., logistic regression, CART, random forest method, boosting method), or generalized linear model (i.e., logarithmic regression).

The area under the receiver operating curve (= AUC) is an indicator of the performance or accuracy of the diagnostic procedure. The accuracy of a diagnostic method is best described by its Receiver Operating Characteristics (ROC). ROC plots are line graphs of all sensitivity/specificity pairs derived from continuously varying decision thresholds across the entire data range observed.

The clinical performance of a laboratory test depends on its diagnostic accuracy, or the ability to correctly classify a subject into a clinically relevant subgroup. Diagnostic accuracy measures the ability to correctly discriminate between two different conditions of the subject under investigation. Such conditions are, for example, health and disease or disease progression versus no disease progression.

One convenient goal to quantify the diagnostic accuracy of a laboratory test is to express its performance by a single numerical value. The most common global metric is the area under the ROC curve (AUC). Conventionally, this area is always ≧ 0.5 (if not, the decision rule can be reversed to do so). The range of values was between 1.0 (test values that perfectly separated the two groups) and 0.5 (no significant distribution difference between the test values of the two groups). The area depends not only on a particular part of the line graph, such as the point closest to the diagonal or the sensitivity at 90% specificity, but also on the entire line graph. This is a quantitative, descriptive representation of how the ROC plot is close to perfect (area = 1.0).

Overall assay sensitivity will depend on the specificity required to carry out the methods disclosed herein. In certain preferred settings, a specificity of 75% may be sufficient, and statistical methods and resulting algorithms may be based on this specificity requirement. In a preferred embodiment, the method for assessing an individual at risk for oral squamous cell carcinoma is based on specificity of 80%, 85%, or also preferably 90% or 95%.

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.

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 results of the analysis showed that CKMT2, GABRP, KRT23 all exhibited differential expression in the TCGA and GEO databases, as shown in table 1 and figures 1-3, wherein CKMT2, GABRP, KRT23 were down-regulated in oral squamous cell carcinoma compared to paracancerous 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 CKMT2, GABRP, KRT23 and their combination have high accuracy in diagnosing oral squamous cell carcinoma, especially their combination has high accuracy, sensitivity and specificity, and AUC values, sensitivity and specificity in the training set are 0.915, 0.909 and 0.802 respectively; the AUC values, sensitivity, specificity in the validation set were 0.949, 0.933, 0.868, 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 an agent for detecting a biomarker in a sample for the manufacture of a product for diagnosing oral squamous cell carcinoma, wherein the biomarker comprises CKMT2, GABRP and/or KRT 23.

2. The use of claim 1, wherein the reagents comprise reagents for detecting biomarker levels by sequencing techniques, nucleic acid hybridization techniques, nucleic acid amplification techniques, protein immunization techniques.

3. Use according to claim 2, wherein said agent is selected from:

a probe that specifically recognizes the biomarker;

primers that specifically amplify the biomarkers; or

An antibody that specifically binds to the biomarker.

4. The use according to any one of claims 1 to 3, wherein the sample is selected from the group consisting of tissue, blood.

5. The use of claim 1, wherein the level of CKMT2, GABRP and/or KRT23 in the sample is determined by measuring the protein level or mRNA level of CKMT2, GABRP and/or KRT23 in the sample.

6. The use of claim 5, wherein the protein level of CKMT2, GABRP and/or KRT23 in the sample is measured by using immunostaining, immunofluorescence, Western blotting or ELISA.

7. The use of claim 5, wherein the mRNA level of CKMT2, GABRP and/or KRT23 in the sample is measured using microarray, RNA-seq, in situ hybridization, RNA-scope and conventional semi-quantitative or quantitative RT-PCR.

8. The use of claim 1, wherein the product further comprises a reagent for processing the sample.

9. Use of a biomarker in the construction of a computational model or a system incorporating the computational model for predicting oral squamous carcinoma, wherein the biomarker comprises CKMT2, GABRP and/or KRT 23.

10. Use according to claim 9, wherein the computational model is operated by bioinformatics methods with the level of a biomarker as an input variable.

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