CN113801937A

CN113801937A - Product for diagnosing lung cancer

Info

Publication number: CN113801937A
Application number: CN202111237253.7A
Authority: CN
Inventors: 邓美; 许文清; 刘敏; 梁朝阳
Original assignee: China Japan Friendship Hospital
Current assignee: China Japan Friendship Hospital
Priority date: 2021-10-22
Filing date: 2021-10-22
Publication date: 2021-12-17

Abstract

A product for diagnosing lung cancer includes reagents for detecting biomarkers including PCDH18, CDKN2A and/or HOXA9 in a sample of a subject. The biomarker is used for detecting lung cancer, so that the method is quick and effective, and provides a treatment target and an important basis for clinical application of gene therapy, drug therapy and the like.

Description

Product for diagnosing lung cancer

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to a product for diagnosing lung cancer.

Background

Lung cancer is a common malignant tumor, and currently, methods clinically used for lung cancer diagnosis include imaging examination, sputum cytopathology examination, endobronchial examination, molecular pathology examination and the like, but these methods are not easy to operate and are prone to false positive. In order to improve the diagnosis and treatment of lung cancer, many tumor markers have been applied to the field of lung cancer in recent years.

(1) Carcinoembryonic antigen (CEA): CEA is a proteoglycan complex, mainly exists in epithelial tissues of the digestive tract, pancreas, liver, biliary tract, lung, mammary gland and other tissues, is a tumor marker which is most widely applied at present, and has abnormal expression in various cancer tissues. The positive rate of CEA of lung cancer patients is about 50%, the adenocarcinoma is higher than that of squamous carcinoma, and the CEA increases along with the increase of clinical stages and the serum level increases.

(2) Cytokeratin 19 fragment (CYFRA 21-1): cytokeratins belong to the intermediate filament family and are widely distributed on normal tissue surfaces, such as stratified or squamous epithelia. In malignant epithelial cells, activated proteases accelerate cellular degradation, allowing release of large numbers of cytokeratin fragments into the blood. In lung cancer, adenocarcinoma and squamous carcinoma both express CK19, especially squamous carcinoma. The determination of cytokeratin has important clinical value for judging the typing stage of lung cancer, especially for diagnosing squamous cell carcinoma in non-small cell lung cancer.

(3) Neuron-specific enolase (NSE): NSE is an enolase involved in the glycolytic pathway, and is present in neural and neuroendocrine tissues. It was found to be present in tumours associated with neuroendocrine tissue origin, in particular in small cell lung cancer, with excessive NSE expression, resulting in a significant increase in serum NSE. NSE has higher sensitivity to small cell lung cancer, so that NSE is superior to other tumor markers in the aspect of small cell lung cancer diagnosis and treatment.

(4) Squamous cell carcinoma antigen (SCC-Ag): the squamous cell carcinoma-associated antigen is a glycoprotein present in the cytoplasm of squamous cell carcinomas such as uterus, cervix, lung, head and neck, particularly in cells of non-keratinized carcinomas. Compared with CEA, SCC-Ag has low sensitivity but high specificity to lung squamous carcinoma, but has no sensitivity or specificity as CYFRA 21-l.

(5) Cancer antigen-125 (cancer antigen 125, CA 125): cancer antigen 125 is a glycoprotein tumor-associated antigen, and is present in epithelial cells of ovarian tumors. CA125 was first found to have a significant increase in ovarian cancer patients, and later was found to have a higher positive rate of CA125 in lung cancer patients.

The tumor marker can reflect the existence and change of the tumor, provides guidance for the diagnosis of the tumor, has simple examination method and good repeatability, and becomes a research hotspot of cancer diagnosis in recent years. More reliable biomarkers closely related to lung cancer diagnosis are screened out, and a new method is provided for lung cancer diagnosis.

Disclosure of Invention

Biomarkers useful for diagnosing lung cancer, including PCDH18, CDKN2A, and/or HOXA9, are disclosed.

In one aspect, the invention provides a product for diagnosing lung cancer, the product comprising reagents for detecting biomarkers in a sample from a subject, the biomarkers comprising PCDH18, CDKN2A and/or HOXA 9.

The term "and/or" as used herein in phrases such as "a and/or B" is intended to include both a and B; a or B; a (alone); and B (alone). Likewise, the term "and/or" as used in phrases such as "A, B and/or C" is intended to encompass each of the following embodiments: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; a (alone); b (alone); and C (alone).

The term "biomarker" refers to a biological molecule present in an individual at different concentrations that can be used to predict the cancer status of the individual. Biomarkers can include, but are not limited to, nucleic acids, proteins, and variants and fragments thereof. A biomarker may be DNA comprising all or part of a nucleic acid sequence encoding the biomarker, or the complement of such a sequence. Biomarker nucleic acids useful in the present invention are considered to include DNA and RNA comprising all or part of any nucleic acid sequence of interest.

In the present invention, biomarkers such as PCDH18 (gene ID: 54510), CDKN2A (gene ID: 1029), HOXA9 (gene ID: 3205), including gene and its encoded protein 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 term "subject" refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, dogs, cats, rodents, and the like. Further, the subject is a human subject. The terms "subject", "individual" and "patient" are used interchangeably herein. Thus, the terms "subject," "individual," and "patient" encompass individuals having cancer (e.g., lung cancer), including those individuals who have undergone or who are candidates for resection (surgery) to remove cancerous tissue.

"biological sample," "specimen," and "test sample" are used interchangeably herein to refer to any material, biological fluid, tissue, or cell obtained or otherwise obtained from an individual. Fluid samples include blood (including whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, and serum), sputum, tears, mucus, nasal washes, nasal aspirates, respiration, urine, semen, saliva, meningeal fluid, amniotic fluid, glandular fluid, lymph fluid, nipple aspirates, bronchial aspirates, synovial fluid, joint aspirates, ascites, cells, cell extracts, and cerebrospinal fluid. The sample may be a combination of samples from an individual, such as a combination of a tissue and a fluid sample, if desired. The term "biological sample" also encompasses materials containing homogenized solid material, such as for example material from a stool sample, a tissue sample or a tissue biopsy. The term "biological sample" also includes material derived from tissue culture or cell culture. Any suitable method for obtaining a biological sample may be employed; exemplary methods include, for example, phlebotomy, swab (e.g., buccal swab), and fine needle aspiration biopsy procedures. Samples can also be collected, for example, by microdissection (e.g., Laser Capture Microdissection (LCM) or Laser Microdissection (LMD)), bladder irrigation, smear (e.g., PAP smear), or ductal lavage. A "biological sample" obtained or derived from an individual includes any such sample that has been processed in any suitable manner after being obtained from the individual, e.g., freshly frozen or formalin-fixed and/or paraffin-embedded.

Further, the sample is a tissue.

Further, the reagent includes a reagent for determining the expression level of the biomarker in the sample of the subject by quantitative PCR, NGS, northern blot (northern blot), southern blot (southern blot), microarray, SAGE, immunoassay (ELISA, EIA, agglutination, nephelometry, turbidimetry, Western blot (Western blot), immunoprecipitation, immunocytochemistry, flow cytometry, Luminex assay) or mass spectrometry.

Further, the immunoassay includes ELISA, EIA, agglutination, nephelometry, turbidimetry, Western immunoblotting (Western blot), immunoprecipitation, immunocytochemistry, flow cytometry, Luminex assay.

Further, the reagent comprises a primer, a probe and an antibody.

"primer" refers to an oligonucleotide that hybridizes to a sequence in a target nucleic acid (a "primer binding site") and can serve as a point at which synthesis is initiated along a complementary strand of the nucleic acid under conditions suitable for such synthesis

"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 polynucleotide probe that is capable of binding to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing. Depending on the stringency of the hybridization conditions, a probe can bind to a target polynucleotide that lacks complete sequence complementarity to the probe. The probe may be directly or indirectly labeled. Hybridization modalities, including, but not limited to: solution phase, solid phase, mixed phase or in situ hybridization assays.

"antibody" refers to an immunoglobulin molecule that recognizes and specifically binds a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combination of the foregoing, through at least one antigen binding site. As used herein, the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, single chain antibodies, antibody fragments (such as Fab, Fab ', F (ab') 2, and Fv fragments), single chain Fv (scfv) antibodies, multispecific antibodies (such as bispecific antibodies), monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen binding site of an antibody, and any other modified immunoglobulin molecule comprising an antigen binding site, so long as the antibody exhibits the desired biological binding activity. The antibody can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2). The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies may be naked or conjugated to other molecules, including but not limited to toxins and radioisotopes.

Furthermore, the product comprises a kit, a nucleic acid membrane strip, a preparation and a chip.

Reagents, tools, and/or instructions for performing the methods described herein can be provided in a kit. For example, a kit may comprise reagents, tools, and instructions for determining an appropriate therapy for a cancer patient. Such kits may include reagents for collecting (e.g., by biopsy) a tissue sample from a patient, and reagents for processing the tissue. The kit may also include one or more reagents for performing gene or gene product expression analysis, such as reagents for determining the expression level of a biomarker in a subject sample by quantitative PCR, NGS, northern blot, southern blot, microarray, SAGE, immunoassay, or mass spectrometry. Suitable buffers for the assay may also be included. Detection reagents required for any of these assays may also be included. Suitable reagents and methods are described in more detail below.

Kits characterized herein may also include an instruction sheet that describes how to perform these assays to measure gene or gene product expression. The instructions sheet may also include instructions on how to determine the reference set, including how to determine the expression levels of the genes or gene product markers in the reference set and how to compile expression data to establish a reference for comparison to the test patient. The instructions may also include instructions for determining the expression of the gene or gene product in the test patient and for comparing the expression level to the expression in the reference set in order to subsequently determine the appropriate chemotherapy for the test patient. Methods for determining appropriate chemotherapy are described above and can be described in detail in the specification.

The informational material included in the kit may be descriptive, instructive, marketing, or other material related to the use of the methods described herein and/or reagents for the methods described herein. For example, the informational material of the kit may contain contact information, such as a physical address, an email address, a website, or a telephone number, where the user of the kit may obtain a wealth of information about performing gene expression analysis and interpreting the results, particularly when they are applied to a human that may have a positive response to a particular therapeutic agent.

Further, the lung cancer comprises lung adenocarcinoma, lung squamous carcinoma, large cell lung cancer and small cell lung cancer, and preferably, the lung cancer is lung squamous carcinoma.

In another aspect, the present invention provides a device suitable for diagnosing lung cancer, comprising:

a) an analyzer unit comprising one or more detection reagents that specifically bind to a biomarker, the unit being adapted to determine the amount of the one or more biomarkers in the sample; and

b) an analyzer unit for comparing the one or more measured quantities with one or more reference quantities, thereby diagnosing lung cancer, said unit comprising a database of one or more reference quantities and a computer-implemented algorithm for performing the comparison;

the biomarker comprises PCDH18, CDKN2A and/or HOXA 9.

As used herein, the term "comparing" encompasses comparing the amount (expression level) of a biomarker as referred to herein comprised by a sample to be analyzed with the amount of a suitable reference source specified elsewhere in the specification. It is to be understood that comparison as used herein refers to comparison of corresponding parameters or values, e.g., an absolute amount to an absolute reference amount, while a concentration is compared to a reference concentration, or an intensity signal derived from a test sample is compared to the same type of intensity signal of a reference sample. The comparison mentioned in step (b) of the method of the invention may be carried out manually or by computational means. For computer-assisted comparison, the value of the measured quantity may be compared with a value corresponding to a suitable reference, which is stored in a database by a computer program. The computer program may further evaluate the result of the comparison, i.e. provide the required evaluation automatically in a suitable output format. The results may, preferably, serve as an aid in assessing whether a subject should undergo imaging-based diagnostic assessment as described elsewhere herein. For example, the results of the comparison may be given in raw data (absolute or relative quantities), and in some cases in terms of words, phrases, symbols, or indicators in the form of numerical values, which may indicate a particular diagnosis. Thus, the reference amount should be selected such that differences or similarities in the compared amounts allow the diagnosis of the present invention to be carried out.

Furthermore, the lung cancer comprises lung adenocarcinoma, lung squamous carcinoma, large cell lung cancer and small cell lung cancer.

Further, the lung cancer is squamous cell lung cancer.

In another aspect, the invention provides the use of a biomarker comprising PCDH18, CDKN2A and/or HOXA9 in the manufacture of a product for diagnosing lung cancer.

Further, the lung cancer is squamous cell lung cancer.

In another aspect, the invention provides a pharmaceutical composition comprising an enhancer of PCDH18, an inhibitor of CDKN2A, and/or an inhibitor of HOXA 9.

In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable buffer, carrier, or excipient.

By "pharmaceutically acceptable" is meant a non-toxic material that does not detract from the active ingredient. Such pharmaceutically acceptable buffers, carriers or Excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18 th edition, A.R Gennaro, eds., Mack Publishing Company (1990) and handbook of Pharmaceutical Excipients, 3 rd edition, A.Kibbe eds., Pharmaceutical Press (2000)).

The term "buffer" is intended to mean an aqueous solution containing an acid-base mixture with the aim of stabilizing the pH. Examples of buffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPSO, imidazole lactate, PIPES, SSPE, POPSO, TAPS, TABS, TAPSO and TES.

The carriers of the present invention include antimicrobial agents, isotonic agents, antioxidants, local anesthetics, suspending agents, dispersing agents, emulsifying agents, chelating agents, thickening agents, or solubilizing agents.

The excipient may be one or more of the following: carbohydrates, polymers, lipids, and minerals. Examples of carbohydrates include lactose, sucrose, mannitol, and cyclodextrins, which are added to the composition, for example, to facilitate lyophilization. Examples of polymers are starch, cellulose ethers, cellulose, carboxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, ethyl cellulose, methyl cellulose, propyl cellulose, alginates (alginates), carrageenans (carageenans), hyaluronic acid and its derivatives, polyacrylic acid, polysulfonates (polysulfonates), polyethylene glycol/polyethylene oxide, polyethylene oxide/polypropylene oxide copolymers, polyvinyl alcohol/polyvinyl acetate, poly (lactic acid), poly (glycolic acid) or copolymers thereof with various compositions, and polyvinylpyrrolidone (all of different molecular weights) which are added to the composition, for example to control viscosity, to achieve bio-adhesion, or to protect the active ingredient from chemical and proteolytic degradation. Examples of lipids are fatty acids, phospholipids, mono-, di-and triglycerides, ceramides, sphingolipids and glycolipids (all with different acyl chain lengths and saturations), egg lecithin (egg lecithins), soy lecithin, hydrogenated lecithin and soy lecithin, which are added to the composition for similar reasons as polymers. Examples of minerals are talc, magnesium oxide, zinc oxide and titanium oxide, which are added to the composition to obtain benefits such as reduced liquid accumulation or favorable pigment properties.

In another aspect, the present invention provides the use of the pharmaceutical composition as described above in the preparation of a medicament for the prevention or treatment of lung cancer.

Further, the lung cancer is squamous cell lung cancer.

In another aspect, the present invention provides the use of biomarkers comprising PCDH18, CDKN2A, and/or HOXA9 in the screening of candidate drugs for the prevention or treatment of lung cancer.

Further, the lung cancer is squamous cell lung cancer.

Drawings

FIG. 1 is a box line plot of differential expression of PCDH 18;

fig. 2 is a boxplot of differential expression of CDKN 2A;

FIG. 3 is a boxplot of HOXA9 differential expression;

FIG. 4 is a ROC plot of PCDH18 diagnosis of squamous cell lung carcinoma;

FIG. 5 is a ROC plot of CDKN2A diagnosis of squamous cell lung carcinoma;

FIG. 6 is a ROC plot of HOXA9 diagnosis of squamous cell lung carcinoma;

FIG. 7 is a ROC plot of the combined diagnosis of squamous cell lung carcinoma by PCDH18 and CDKN 2A;

FIG. 8 is a ROC plot of the combined diagnosis of squamous cell lung carcinoma by CDKN2A and HOXA 9;

FIG. 9 is a ROC plot of the combination of PCDH18 and HOXA9 for diagnosis of squamous cell lung carcinoma;

FIG. 10 is a ROC plot of the PCDH18+ CDKN2A + HOXA9 combined diagnosis of squamous cell lung carcinoma.

Detailed Description

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.

Example 1 Gene differentially expressed in Lung squamous carcinoma

Raw data for lung squamous carcinoma were downloaded from a cancer genomic profiling database (TCGA), followed by log-taking in R for subsequent analysis. After removing the samples with incomplete clinical information, the number of samples contained in the TCGA cohort was paracancerous: carcinoma =49: 502.

Differential expression analysis was performed using the "limma" package in the R software, with screening criteria for differential genes being adj. pvalue <0.01, | log2FC | > 1. Under this criterion, 8571 differentially expressed genes, 5455 up-regulated differentially expressed genes, and 3116 down-regulated differentially expressed genes were present in TCGA. The expression of the gene of the present invention in squamous cell lung carcinoma is shown in FIG. 1-3 and Table 1.

TABLE 1 Gene differential expression data

Example 2 diagnostic Performance analysis

The invention uses R language pROC to carry out ROC diagnosis analysis on the gene so as to screen biomarkers with diagnostic value. The larger the area under the ROC curve, the higher the diagnostic value.

The diagnostic efficacy data of the biomarkers and biomarker combinations according to the present invention are shown in fig. 4-10, table 2, and the biomarker combinations have better diagnostic efficacy than the individual biomarkers. The results prove that the biomarker and the biomarker combination related to the invention can be used for diagnosing squamous cell lung carcinoma.

TABLE 2 diagnostic potency of biomarkers and biomarker combinations

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Claims

1. A product for use in diagnosing lung cancer, the product comprising reagents for detecting biomarkers in a sample from a subject, the biomarkers comprising a combination of any two or three of PCDH18, CDKN2A, HOXA 9.

2. The product of claim 1, wherein the reagents comprise reagents for determining the expression level of a biomarker in a sample from a subject by quantitative PCR, NGS, northern blot, southern blot, microarray, SAGE, immunoassay, or mass spectrometry.

3. The product of claim 2, wherein said immunoassay comprises ELISA, EIA, agglutination, nephelometry, turbidimetry, western blot, immunoprecipitation, immunocytochemistry, flow cytometry, Luminex assay.

4. The product of claim 1, wherein the reagents comprise primers, probes, antibodies.

5. The product of claim 1, wherein the product comprises a kit, a nucleic acid membrane strip, a formulation, a chip.

6. A product according to any one of claims 1 to 5 wherein the lung cancer is squamous cell lung cancer.

7. A device suitable for diagnosing lung cancer, comprising:

the biomarker comprises any two or three of PCDH18, CDKN2A and HOXA 9.

8. Use of a biomarker in the manufacture of a product for the diagnosis of lung cancer, wherein the biomarker comprises a combination of any two or three of PCDH18, CDKN2A, HOXA 9.

9. A pharmaceutical composition comprising any two or three of an enhancer of PCDH18, an inhibitor of CDKN2A, and an inhibitor of HOXA 9.

10. Use of the pharmaceutical composition of claim 9 for the preparation of a medicament for the prevention or treatment of lung cancer.