CN115851927A

CN115851927A - Lung squamous carcinoma molecular typing and survival risk gene group, diagnosis product and application

Info

Publication number: CN115851927A
Application number: CN202210949735.3A
Authority: CN
Inventors: 周彤; 胡志元; 周伟庆; 马琳琳; 陆俊欢
Original assignee: Shanghai Shanzhun Medical Technology Co ltd
Current assignee: Shanghai Shanzhun Medical Technology Co ltd
Priority date: 2022-08-09
Filing date: 2022-08-09
Publication date: 2023-03-28

Abstract

The invention belongs to the technical field of biology, and discloses a group of genes for molecular typing of lung squamous carcinoma and survival risk assessment. The invention also discloses application of a reagent for detecting the gene expression level of the gene group in preparation of products, and the products are used for determining the molecular subtype of the squamous cell lung carcinoma and evaluating the survival risk of patients with the squamous cell lung carcinoma. The product comprises a second generation sequencing (NGS) detection kit, a fluorescent quantitative PCR detection kit, a gene chip and a protein chip. The invention also discloses a method for carrying out lung squamous carcinoma molecular typing and survival risk assessment by using the detection kit.

Description

Lung squamous carcinoma molecular typing and survival risk gene group, diagnosis product and application

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a gene group for carrying out molecular typing on squamous cell lung carcinoma and evaluating survival risk of squamous cell lung carcinoma patients, and an in-vitro diagnosis product and application thereof.

Background

Lung cancer is one of the most common malignant tumors worldwide. According to the statistical data published by the cancer center of the country in 2019, lung cancer is the first place of the morbidity and mortality of malignant tumors in China. 78.7 ten thousand cases of new lung cancer in China in 2015, 57.26/10 ten thousand of cases, 63.1 ten thousand of deaths of lung cancer and 45.87/10 ten thousand of deaths of lung cancer. Non-small cell lung cancer (NSCLC) is the most common pathological subtype of lung cancer, accounting for about 85% of the total lung cancer, and can be further classified into adenocarcinoma, squamous cell carcinoma, large cell carcinoma, etc. For early stage non-small cell lung cancer (IA-IIA stage), surgical treatment is mainly used, and most of the treatment is not assisted by chemotherapy. However, the 5-year survival rate after these early non-small cell lung cancers is only 49-71%. How to identify the high risk group among these early stage lung cancer patients and to perform postoperative adjuvant therapy early to improve the 5-year survival rate of this part of patients is the current new research direction for non-small cell lung cancer. For NSCLC patients who lose the chance of surgical treatment in the late clinical period, the traditional palliative chemotherapy has limited curative effect and obvious toxic and side effects, which leads to poor prognosis of the patients. The discovery of NSCLC driving genes and the research and development of corresponding molecular targeted drugs, and the clinical application of immune checkpoint inhibitors are promoted in recent years, so that the lung cancer diagnosis and treatment are greatly promoted to enter the times of 'precise medicine' and 'individualized diagnosis and treatment'. However, it should be noted that not all patients, whether targeted or immunotherapy, may benefit. Squamous cell carcinoma of Lung (LSC) in NSCLC occurs mainly in older men and is closely associated with smoking, accounting for about 30% of NSCLC. Squamous cell lung carcinoma is generally relatively insensitive to conventional chemotherapy and radiation therapy and, because of the small number of drivers, less effective targeted therapies than lung adenocarcinoma in NSCLC.

In recent years, the development of high-throughput detection technology and analysis method brings new opportunities for tumor research. The molecular biology typing and distant metastasis or survival risk assessment of the tumor, which are provided based on gene expression profiles and molecular biology characteristics, can better reflect the biological behavior of the tumor, and have potential guiding significance for clinical treatment. Although belonging to the large class of non-small cell lung cancer, our earlier studies found that the gene expression profile of squamous cell lung cancer and the changes in gene expression that are closely related to prognosis were very different from lung adenocarcinoma. There are many reports on the clinical studies on molecular typing and survival risk assessment of lung adenocarcinoma, but the studies focusing on squamous cell lung carcinoma are not common.

Disclosure of Invention

In one aspect, the invention provides a set of genes for determining the molecular subtype of squamous cell lung carcinoma and/or assessing the risk of survival of patients with squamous cell lung carcinoma, which includes genes associated with molecular typing and risk of survival assessment. In one embodiment, the population of genes further comprises a reference gene. Molecular subtypes of squamous cell lung carcinoma include the LSC1 subtype, LSC2 subtype, LSC3 subtype, LSC4 subtype, LSC5 subtype, and mixed subtypes.

In one aspect, the invention also provides reagents for detecting the expression level of a gene in the gene population of the invention. In a preferred embodiment, the reagent is a reagent for detecting the amount of RNA, particularly mRNA, transcribed from the gene of the invention; or it is a reagent that detects the amount of cDNA complementary to mRNA. In a specific embodiment, the agent is a primer, a probe, or a combination thereof.

In another aspect, the invention also provides a product for molecular typing and/or risk of survival assessment of squamous cell lung carcinoma comprising an agent of the invention. The invention also provides the application of the gene group or the reagent in preparing products. The product is used for determining lung squamous carcinoma molecular subtype and/or evaluating the survival risk of lung squamous carcinoma patients. In one embodiment, the product is a next generation sequencing kit, a real-time fluorescent quantitative PCR detection kit, a gene chip, a protein chip, an ELISA diagnostic kit, or an Immunohistochemical (IHC) kit. In a preferred embodiment, the product is a secondary sequencing kit or a real-time fluorescent quantitative PCR detection kit.

In one aspect, the invention also provides a method for determining the subtype and/or risk of survival of a lung squamous carcinoma molecule in a subject, the method comprising: (1) providing a sample of a subject; (2) Determining the expression level of a gene in the gene population of the invention in the sample; (3) Determining the subject's molecular subtype and/or risk of survival of squamous cell lung carcinoma.

Drawings

FIG. 1 shows the differential expression of genes involved in molecular typing and survival risk assessment of squamous cell lung carcinoma among different genes.

FIG. 2 shows the results of Kaplan-Meier survival analysis of different molecular subtypes of squamous cell lung carcinoma. The survival risk of each subtype is different, the 5-year relapse-free survival rate of the LSC2 subtype and the LSC4 subtype is better, and the 5-year relapse-free survival rate of the LSC1 subtype, the LSC3 subtype and the LSC5 subtype is relatively poorer.

FIG. 3 shows the results of Kaplan-Meier survival analysis in different risk groups for squamous cell lung carcinoma cases. The 5-year relapse-free survival rate was higher in the low-risk group (relapse risk index of 0-35) and significantly higher than in the high-risk group (relapse risk index of 36-100).

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.

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. In case of conflict, the present application will control. Experimental procedures not specifically identified herein may generally be carried out, for example, under the conventional conditions described in Sambrook et al, molecular Cloning: A Laboratory Manual,4th, cold Spring harbor, N.Y.,2012, or under the conditions recommended by the manufacturer.

When an amount, concentration, or other value or parameter is expressed in terms of a range, preferred range, or upper preferable numerical value and lower preferable numerical value, it is understood that any range defined by any pair of upper range limits or preferred numerical values in combination with any lower range limits or preferred numerical values is specifically disclosed, regardless of whether the range is specifically disclosed. Unless otherwise indicated, numerical ranges set forth herein are intended to include the endpoints of the ranges and all integers and fractions (decimal) within the range.

The term "about" or "approximately" when used in conjunction with a numerical variable generally means that the value of the variable and all values of the variable are within experimental error (e.g., within 95% confidence interval for the mean) or within ± 10% of the specified value, or more.

The terms "optional" or "optionally present" mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

The expressions "comprising" or similar expressions "including", "containing" and "having" and the like which are synonymous are open-ended and do not exclude additional, unrecited elements, steps or components. The expression "consisting of …" excludes any element, step or ingredient not specified. The expression "consisting essentially of …" means that the scope is limited to the specified elements, steps or components, plus optional elements, steps or components that do not materially affect the basic and novel characteristics of the claimed subject matter. It is to be understood that the expression "comprising" covers the expressions "consisting essentially of …" and "consisting of …".

The expression "at least one" or "one or more" may denote 1, 2, 3, 4, 5, 6,7 or more, and up to the total number of the modified set thereof.

Detection of gene expression levels as described herein can be accomplished, for example, by detecting a nucleic acid of interest (e.g., an RNA transcript), or by detecting the amount of a polypeptide of interest (e.g., an encoded protein), e.g., by detecting protein expression levels using proteomic methods. The amount of a polypeptide of interest, e.g., a polypeptide, protein, or protein fragment encoded by a gene of interest, can be normalized to the amount of total protein in the sample or the amount of polypeptide encoded by a reference gene. The amount of target nucleic acid, e.g., DNA of the target gene, RNA transcript thereof, or cDNA complementary to the RNA transcript, can be normalized to the amount of total DNA, total RNA, or total cDNA in the sample, or to the amount of DNA, RNA transcript, or cDNA complementary to the RNA transcript of a set of reference genes.

As used herein, the term "polypeptide" refers to a compound consisting of amino acids joined together by peptide bonds, including full-length or amino acid fragments of a polypeptide. Herein, "polypeptide" and "protein" are used interchangeably.

The term "nucleotide" includes deoxyribonucleotides and ribonucleotides. The term "nucleic acid" refers to a polymer composed of two or more nucleotides, and encompasses deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and nucleic acid analogs.

The term "RNA transcript" refers to total RNA, i.e., coding or non-coding RNA, including RNA derived directly from tissue or peripheral blood samples, and also including RNA derived indirectly from tissue or blood samples after cell lysis. Total RNA includes tRNA, mRNA, and rRNA, where mRNA includes mRNA transcribed from a target gene, as well as mRNA from other non-target genes. The term "mRNA" may include both precursor and mature mrnas, either full length or fragments thereof. In this context, the RNA which can be used for detection is preferably mRNA, more preferably mature mRNA. The term "cDNA" refers to DNA having a base sequence complementary to RNA. One skilled in the art can obtain from the DNA of a gene its RNA transcript and/or cDNA complementary to its RNA transcript using methods known in the art, for example, by chemical synthesis methods or molecular cloning methods.

In this context, a target nucleic acid (e.g., an RNA transcript) can be detected and quantified, for example, by hybridization, amplification, or sequencing methods. For example, the amount of the target nucleic acid can be obtained by hybridizing the RNA transcript to a probe or a primer to form a complex and detecting the amount of the complex. 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 (which may be DNA or RNA) comprising at least 5 nucleotides, e.g., comprising 5 to 100 nucleotides, which is capable of hybridizing under the specified conditions to a target nucleic acid (e.g., an RNA transcript of a target gene or an amplification product of an RNA transcript, or cDNA complementary to an RNA transcript) to form a complex. The hybridization probes may also include markers for detection. The term "TaqMan probe" is a probe based on the TaqMan technique, which carries a fluorescent group such as FAM, TET, HEX, NED, VIC, cy5, or the like at the 5 'end and a fluorescence quenching group such as TAMRA and BHQ groups or a non-fluorescence quenching group (TaqMan MGB probe) at the 3' end, has a nucleotide sequence capable of hybridizing with a target nucleic acid, and reports the amount of nucleic acid forming a complex therewith when applied to real-time fluorescent quantitative PCR (RT-PCR).

The term "reference gene" or "reference gene" as used herein refers to a gene that can be used as a reference to correct and normalize the expression level of a target gene, and reference genes that can be considered are included in the standard: (1) Stably expressed in a tissue, at a level that is unaffected or less affected by a pathological condition or drug treatment; (2) The expression level should not be too high to avoid making the expression data (such as obtained by second-generation sequencing) too high to be compared with the data obtained, and thus the accuracy of data detection and interpretation of other genes is not affected. Therefore, reagents useful for detecting the expression level of the reference gene of the present invention are also within the scope of the present invention. Reference genes that may be used in the present invention include, but are not limited to, "housekeeping genes". Herein, "reference gene" and "housekeeping gene" may be used interchangeably.

The term "housekeeping gene" refers to a class of genes whose products are essential for maintaining the basic vital activities of a cell, are continuously expressed in most or almost all tissues at various stages of an individual's growth, and are less affected by environmental factors.

In this context, the term "squamous cell lung carcinoma" refers to squamous cell lung carcinoma, a type of lung cancer, which is a non-small cell lung cancer, most of which originate in the larger bronchi, often central lung carcinoma, and which is closely associated with smoking. As used herein, squamous cell lung carcinoma includes, but is not limited to, primary squamous cell lung carcinoma and metastatic squamous cell lung carcinoma. In one embodiment of the invention, the molecular subtypes of squamous cell lung carcinoma include the LSC1 subtype, the LSC2 subtype, the LSC3 subtype, the LSC4 subtype, the LSC5 subtype, and mixed subtypes.

Herein, the term "molecular typing" or "subtype typing" of squamous cell lung carcinoma refers to a classification method of squamous cell lung carcinoma based on the gene expression profile of squamous cell lung carcinoma tumor tissue.

As used herein, the term "prognosis" refers to the prediction of the course and outcome of development of squamous cell lung cancer, including but not limited to the prediction of the risk of survival of squamous cell lung cancer. Lung cancer with a lower risk of survival has a better prognosis, whereas the prognosis is worse.

By "survival risk assessment" is meant herein the assessment of the likelihood of disease progression or death due to squamous cell lung carcinoma and its related causes in a patient with squamous cell lung carcinoma over a specified period of time starting at random. Herein, "disease progression" includes, but is not limited to, tumor cell proliferation, recurrence and metastasis. Herein, "survival risk assessment" and "relapse risk assessment" are used interchangeably. Herein, the terms "risk of relapse" and "risk of survival" may be used interchangeably. Herein, the survival risk assessment is performed by calculating a relapse risk score (also called a relapse risk index).

Gene group of the present invention

In one general aspect, the present invention provides a panel of gene groups, which includes genes related to molecular typing of squamous cell lung carcinoma and survival risk assessment.

The lung squamous carcinoma molecular typing and survival risk assessment related genes can comprise: (1) 18 coagulation-related genes; (2) 25 peptide cross-linking related genes; and (3) 7 nicotine metabolism-related genes.

(1) Blood coagulation related genes: c4BPA, FGA, HNF1B, SFTA, SFTA3, SFTPB, CAPN8, CHIA, CLDN18, CRTAC1, FGG, GKN2, HP, ORM1, PLA2G1B, RASGRF, ROS1, and SERPIND1;

(2) Peptide crosslinking-related genes: SPRR1A, SPRR1B, SPRR2A, SPRR2B, SPRR2D, SPRR2E, A ML1, CRCT1, IL36B, IL G, IL RN, IVL, LCE3E, PGLYRP, PI3, RAET1L, SBSN, SERPINB13, SPRR2C, SPRR2G, SPRR, SPRR4, TMPRSS11B, TMPRSS D and TMPRSS11F;

(3) Genes related to nicotine metabolism: DNAJB3, UGT1A1, UGT1A2P, UGT A6, ADH7, NTRK2, and UGT1A4.

In one embodiment, the present invention provides a set of genes comprising genes associated with molecular typing of squamous cell lung carcinoma and assessing risk of survival, as described above: (1) one or more of 18 coagulation-related genes; (2) one or more of the 25 peptide cross-linking related genes; and (3) one or more of 7 nicotine metabolism-related genes.

In one embodiment, the gene population includes 50 genes associated with molecular typing of squamous cell lung carcinoma and risk of survival assessment (see table 1), which includes 18 genes associated with coagulation, 25 genes associated with peptide cross-linking, and 7 genes associated with nicotine metabolism, as described above.

In another embodiment, the gene population comprises 16 lung squamous carcinoma molecular typing and survival risk assessment-associated genes (see table 2), which comprise (1) 6 coagulation-associated genes: c4BPA, FGA, HNF1B, SFTA, SFTA3 and SFTPB; (2) 6 peptide crosslinking-related genes: SPRR1A, SPRR1B, SPRR2A, SPRR2B, SPRR D and SPRR2E; and (3) 4 nicotine metabolism-related genes: DNAJB3, UGT1A1, UGT1A2P and UGT1A6.

In a preferred embodiment, the gene population may further comprise a reference gene. Preferably, the reference gene is a housekeeping gene. Preferably, the reference gene comprises at least 1 (e.g. 1, 2, 3, 4, 5, 6 or 7) of: ACTB, GAPDH, GUSB, TFRC, MRPL19, PSMC4, and SF3A1. In one embodiment, the gene cluster of the invention may further comprise at least one (e.g. 1, 2, 3, 4, 5 or 6), preferably at least 3, most preferably 6 reference genes: ACTB, GAPDH, GUSB, MRPL19, PSMC4, SF3A1 and TFRC. In a specific embodiment, the reference genes comprise GAPDH, GUSB, TFRC, MRPL19, PSMC4 and SF3A1. In a specific embodiment, the reference genes comprise three of ACTB, GAPDH, GUSB, TFRC, MRPL19, PSMC4, and SF3A1. In another specific embodiment, the reference genes comprise ACTB, GAPDH, and GUSB.

In a preferred embodiment, the gene group of the present invention comprises 50 molecular typing and survival risk assessment-associated genes as described above, and a reference gene. In a specific embodiment, the reference genes include GAPDH, GUSB, TFRC, MRPL19, PSMC4, and SF3A1, and the gene groups are shown in table 1.

In yet another preferred embodiment, the gene group of the present invention comprises 16 molecular typing and survival risk assessment-associated genes as described above, and a reference gene. In one embodiment, the reference genes comprise three of ACTB, GAPDH, GUSB, TFRC, MRPL19, PSMC4, and SF3A1. In a specific embodiment, the reference genes include ACTB, GAPDH, and GUSB, and the gene groups are shown in table 2.

TABLE 1

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TABLE 2

Serial number	Function(s)	Name of gene
			1	Blood coagulation related gene	C4BPA
2	Blood coagulation related gene	FGA
			3	Blood coagulation related gene	HNF1B
4	Blood coagulation related gene	SFTA2
			5	Blood coagulation related gene	SFTA3
6	Blood coagulation related gene	SFTPB
			7	Peptide cross-linking related genes	SPRR1A
8	Peptide cross-linking related genes	SPRR1B
			9	Peptide cross-linking related genes	SPRR2A
10	Peptide cross-linking related genes	SPRR2B
			11	Peptide cross-linking related genes	SPRR2D
12	Peptide cross-linking related genes	SPRR2E
			13	Genes associated with nicotine metabolism	DNAJB3
14	Genes associated with nicotine metabolism	UGT1A1
			15	Genes associated with nicotine metabolism	UGT1A2P
16	Genes associated with nicotine metabolism	UGT1A6
			17	Reference gene	ACTB
18	Reference gene	GAPDH
			19	Reference gene	GUSB

In a specific embodiment, the gene populations of the invention can be used to determine molecular subtypes of squamous cell lung carcinoma and/or to assess the risk of survival of patients with squamous cell lung carcinoma.

Molecular subtypes of squamous cell lung carcinoma may include the LSC1 subtype, the LSC2 subtype, the LSC3 subtype, the LSC4 subtype, the LSC5 subtype, and mixed subtypes. The survival risk may include a low risk and a high risk.

It will be appreciated by those skilled in the art that the gene populations of the present invention are not limited to the combinations listed above. In view of the present disclosure, those skilled in the art should be able to combine the genes related to molecular typing and survival risk assessment of the present invention with reference genes to obtain a gene group comprising combinations of different genes, and such gene groups are also within the scope of the present invention.

Diagnostic product of the invention

In still another aspect, the present invention relates to a reagent for detecting the expression level of genes in the gene group of the present invention and its use in the preparation of detection/diagnostic products. The gene groups are as described above.

The reagent or the detection/diagnosis product can be used for determining the molecular subtype of the lung squamous carcinoma and/or evaluating the survival risk of the lung squamous carcinoma patient. It will be appreciated by those skilled in the art that the selections in the reagent or product may each correspond to a gene in the gene population of the invention. By way of example, when a plurality of alternatives are listed, such as the primers of SEQ ID NO.113 to SEQ ID NO.150 or the probes of SEQ ID NO.151 to SEQ ID NO.169, it is not meant that the reagent or product of the invention necessarily comprises all of these primers or probes, but that the reagent or product will comprise those primers or probes corresponding to the genes encompassed therein. For example, the reagent or product may comprise at least one of the primers having the sequence shown in SEQ ID NO.113 to SEQ ID NO.150 and/or at least one of the probes having the sequence shown in SEQ ID NO.151 to SEQ ID NO. 169.

In a preferred embodiment, the reagent is used to detect the amount of a nucleic acid of interest (e.g., a DNA, RNA transcript or cDNA complementary to an RNA transcript of a gene in a gene population of the invention). More preferably, the reagents are used to detect the amount of RNA transcripts, in particular mRNA, of the genes in the gene population of the invention, or to detect the amount of cDNA complementary to mRNA. In one embodiment, the reagent is a reagent that detects the amount of RNA transcript, particularly mRNA, of a gene of interest (i.e., a gene in the gene population of the invention). In yet another embodiment, the reagent is a reagent that detects the amount of cDNA complementary to the mRNA.

In a preferred embodiment, the reagent is a probe or primer or a combination thereof, which is capable of hybridizing with a partial sequence of a target nucleic acid (e.g., a gene of the gene group of the present invention, an RNA transcript thereof, or a cDNA complementary to the RNA transcript) to form a complex. Preferably, the probes and primers are highly specific for the target nucleic acid. Probes and primers may be artificially synthesized.

In one embodiment, the reagent is a primer. In one embodiment, the primer has a sequence as shown in SEQ ID NO.1-SEQ ID NO. 100. In one embodiment, the primer has a sequence as shown in SEQ ID NO.1-SEQ ID NO.112 (see also Table 3). In another embodiment, the primer has a sequence as shown in SEQ ID NO.113-SEQ ID NO. 144. In another embodiment, the primer has a sequence as shown in SEQ ID NO.113-SEQ ID NO.150 (see also Table 4).

In a preferred embodiment, the primers are used for next-generation sequencing, preferably for targeted sequencing. In a specific embodiment, the primer is used for targeted sequencing and has a sequence shown as SEQ ID NO.1-SEQ ID NO. 100. In a specific embodiment, the primers are used for targeted sequencing and have the sequence shown as SEQ ID No.1-SEQ ID No.112 (Table 3).

In another preferred embodiment, the primers are used in quantitative PCR, preferably real-time fluorescent quantitative PCR (RT-PCR), such as SYBR Green RT-PCR based on SYBR Green dye and TaqMan RT-PCR based on TaqMan technology. TaqMan RT-PCR may be, for example, multiplex RT-PCR and singleplex RT-PCR. In one embodiment, the primers are used in SYBR Green RT-PCR and have the sequence shown in SEQ ID NO.113-SEQ ID NO. 144. In one embodiment, the primers are used in SYBR Green RT-PCR and have the sequence shown as SEQ ID NO.113-SEQ ID NO.150 (see also Table 4). In another embodiment, the primers are used in TaqMan RT-PCR and have the sequence shown as SEQ ID NO.113-SEQ ID NO. 144. In another embodiment, the primers are used in TaqMan RT-PCR and have the sequence shown as SEQ ID NO.113-SEQ ID NO.150 (Table 4). In a specific embodiment, the primers are used in single or multiplex RT-PCR and have the sequence shown in SEQ ID NO.113-SEQ ID NO. 144. In a specific embodiment, the primers are used in single or multiplex RT-PCR and have the sequence shown in SEQ ID NO.113-SEQ ID NO.150 (Table 4).

In one embodiment, the primers are used to make a detection/diagnostic product that is a targeted sequencing-based next-generation sequencing kit or a real-time fluorescent quantitative PCR kit.

In another embodiment, the reagents are probes, including but not limited to probes for detection by RT-PCR, in Situ Hybridization (ISH), DNA or RNA imprinting, and gene chip technology.

In one embodiment, the probe is a probe that can be used for in situ hybridization. The probe used for in situ hybridization may be, for example, a probe used for two-color silver-stained in situ hybridization (DISH), DNA fluorescence in situ hybridization (DNA-FISH), RNA fluorescence in situ hybridization (RNA-FISH), chromogenic In Situ Hybridization (CISH), or the like. The probe may carry a label. The label may be a fluorophore (e.g., alexa Fluor dye, FITC, texas Red, cy3, cy5, etc.), biotin, digoxigenin, and the like.

In another embodiment, the probes can be used in gene chip assays. The probe may also carry a label. The label may be a fluorophore. In one embodiment, the probes may be used to prepare detection/diagnostic products, which are gene chips.

In a preferred embodiment, the probes are used for RT-PCR. In one embodiment, the probe is used in TaqMan RT-PCR. In one embodiment, the probe is a TaqMan probe. In one embodiment, the probe has a sequence as shown in SEQ ID NO.151-SEQ ID NO. 166. In one embodiment, the probe has a sequence as shown in SEQ ID NO.151-SEQ ID NO.169 (see also Table 4). In a specific embodiment, the probe is a TaqMan probe having a sequence shown as SEQ ID NO.151-SEQ ID NO. 166. In a specific embodiment, the probe is a TaqMan probe having a sequence shown as SEQ ID NO.151-SEQ ID NO. 169.

In one embodiment, the probes may be used to prepare detection/diagnostic products that are real-time fluorescent quantitative PCR detection kits.

In yet another embodiment, the reagent is a combination of a primer and a probe. Preferably, the probe is a TaqMan probe. In one embodiment, the primer and probe combination is used in RT-PCR, such as single or multiplex RT-PCR. In one embodiment, the primer has a sequence as shown in SEQ ID NO.113-SEQ ID NO. 144. In one embodiment, the primer has a sequence as shown in SEQ ID NO.113-SEQ ID NO. 150. In one embodiment, the probe has a sequence as shown in SEQ ID NO.151-SEQ ID NO. 166. In one embodiment, the probe has a sequence as shown in SEQ ID NO.151-SEQ ID NO. 169. In a specific embodiment, the primer has a sequence shown as SEQ ID NO.113-SEQ ID NO.144, and the probe is a TaqMan probe having a sequence shown as SEQ ID NO.151-SEQ ID NO. 166. In a specific embodiment, the primer has a sequence shown as SEQ ID No.113-SEQ ID No.150, and the probe is a TaqMan probe having a sequence shown as SEQ ID No.151-SEQ ID No.169 (see also Table 4).

In one embodiment, the probes and primers can be used to prepare a diagnostic product that is a real-time fluorescent quantitative PCR detection kit, such as a multiplex or singleplex real-time fluorescent quantitative PCR detection kit.

In alternative embodiments, the reagents are used to detect the amount of polypeptide encoded by the gene of interest (a gene in the gene population of the invention). Preferably, the agent is an antibody, antibody fragment or affinity protein that is capable of specifically binding to a polypeptide encoded by a gene of interest. More preferably, the agent is an antibody or antibody fragment capable of specifically binding to a polypeptide encoded by the gene of interest. The antibody, antibody fragment, or affinity protein may also be labeled for detection, such as with an enzyme (e.g., horseradish peroxidase), a radioisotope, a fluorescent label (e.g., alexa Fluor dye, FITC, texas Red, cy3, cy5, etc.), a chemiluminescent substance (e.g., luminol), biotin, and quantum dot labeling (Qdot), among others. Thus, in a preferred embodiment, the agent is an antibody or antibody fragment capable of specifically binding to a polypeptide encoded by the gene of interest, and optionally carrying a label for detection. In one embodiment, the label is selected from the group consisting of an enzyme, a radioisotope, a fluorescent label, a chemiluminescent substance, biotin, and a quantum dot label. In one embodiment, the reagents are used to prepare a detection/diagnostic product that is a protein chip (e.g., a protein microarray), an ELISA diagnostic kit, or an Immunohistochemistry (IHC) kit.

Thus, in another aspect, the invention provides a product (preferably a kit) useful for determining the molecular subtype of squamous cell lung carcinoma and/or for assessing the risk of survival of patients with squamous cell lung carcinoma. The product (preferably a kit) comprises the agent of the invention. The product can be a second generation sequencing kit based on targeted sequencing, a real-time fluorescent quantitative PCR kit, a gene chip, a protein chip, an ELISA diagnostic kit or an Immunohistochemical (IHC) kit or a combination thereof.

In one embodiment, the product is a Next Generation Sequencing (NGS) -based diagnostic product. In a specific embodiment, the product comprises a reagent that detects the expression level of a gene of the gene population of the invention. In one embodiment, the gene population includes 56 genes, namely 50 molecular typing and survival risk assessment-associated genes as described above and 6 housekeeping genes (see also table 1). In one embodiment, the gene group of the present invention comprises 19 genes, i.e., 16 molecular typing and survival risk assessment-associated genes as described above, and 3 housekeeping genes, including three of ACTB, GAPDH, GUSB, TRFC, MRPL19, PSMC4, and SF3A1. In yet another embodiment, the gene population of the invention comprises 19 genes, namely 16 molecular typing and survival risk assessment-associated genes as described above and 3 housekeeping genes (see also table 2). In a specific embodiment, the next-generation sequencing (NGS) -based diagnostic product comprises primers having the sequence shown as SEQ ID No.1-SEQ ID No.112 (see also table 3).

In a further embodiment, the diagnostic product is a fluorescent quantitative PCR based diagnostic product, preferably a real-time fluorescent quantitative PCR (RT-PCR), such as SYBR Green RT-PCR and TaqMan RT-PCR. TaqMan RT-PCR may for example be multiplex RT-PCR and singleplex RT-PCR. In one embodiment, the diagnostic product comprises a reagent that detects the expression level of a gene of the gene population of the invention. In one embodiment, the gene population includes 56 genes, namely 50 molecular typing and survival risk assessment-associated genes as described above and 6 housekeeping genes (see also table 1). In one embodiment, the gene population includes 19 genes, 16 molecular typing and survival risk assessment-associated genes as described above and 3 housekeeping genes (see also table 2). In a specific embodiment, the diagnostic product based on fluorescent quantitative PCR comprises primers having the sequence shown as SEQ ID NO.113-SEQ ID NO. 150. In another specific embodiment, the fluorescent quantitative PCR based diagnostic product comprises a TaqMan probe having a sequence as shown in SEQ ID NO.151-SEQ ID NO. 169. In a preferred embodiment, the diagnostic product based on fluorescent quantitative PCR comprises primers having the sequence shown as SEQ ID NO.113-SEQ ID NO.150 and TaqMan probes having the sequence shown as SEQ ID NO.151-SEQ ID NO.169 (see also Table 4).

In one embodiment, the product is an in vitro diagnostic product. In a specific embodiment, the product is a diagnostic kit.

In one embodiment, the product is used to determine the molecular subtype of squamous cell lung carcinoma and/or to assess the risk of survival of patients with squamous cell lung carcinoma.

In a preferred embodiment, the product further comprises a total RNA extraction reagent, a reverse transcription reagent, a secondary sequencing reagent and/or a quantitative PCR 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, RNA storm CD201, qiagen 73504, invitrogen K156002, and ABI AM1975.

The reverse transcription reagent may be a reverse transcription reagent conventional in the art, and preferably comprises a dNTP solution and/or an RNA reverse transcriptase. Examples of reverse transcription reagents include, but are not limited to, NEB M0368L, thermo K1622, ABI4366596.

The second-generation sequencing reagent can be a reagent conventionally used in the field as long as the requirement of second-generation sequencing on the obtained sequence can be met. The second-generation sequencing reagent may be a commercially available product, examples of which include, but are not limited to, illumina Inc

Reagent Kit v3(150cycle)(MS-102-3001)、/>

Targeted RNA Index Kit A-96 indexes (384 Samples) (RT-402-1001). Secondary sequencing is a technique conventional in the art, such as targeted RNA-seq technology. Thus, the secondary sequencing reagents may also comprise reagents that can be tailored for constructing RNA-seq targeted libraries Illumina, e.g. < >>

Targeted RNA Custom Panel Kit(96Samples)(RT-102-1001)。

The quantitative PCR reagent is a reagent which is conventionally used in the field as long as the requirement of quantitative PCR on the obtained sequence can be met. The quantitative PCR reagent may be commercially available. The quantitative PCR technique is a quantitative PCR technique conventional in the art, preferably a real-time fluorescent quantitative PCR technique, such as SYBR Green RT-PCR and Taqman RT-PCR techniques. The PCR reagents preferably further comprise reagents for constructing a library for quantitative PCR. Preferably, the quantitative PCR reagents may also comprise real-time fluorescent quantitative PCR reagents, such as reagents for SYBR Green RT-PCR (e.g., SYBR Green premix, such as SYBR Green PCR Master Mix) and reagents for Taqman RT-PCR (e.g., taqman RT-PCR Master Mix). One skilled in the art will be able to select appropriate quantitative PCR reagents depending on the quantitative PCR technique used. The detection platform for quantitative PCR detection can be ABI7500 real-time fluorescence quantitative PCR instrument or Roche

480 II real-time fluorescence quantitative PCR instrument or all other PCR instruments capable of real-time fluorescence quantitative detection.

In a specific embodiment, the article of manufacture is a targeted RNA-seq based next-generation sequencing kit comprising primers having sequences as shown in table 3. Optionally, the product further comprises one or more selected from: total RNA extraction reagent, reverse transcription reagent and second generation sequencing reagent. Preferably, the second generation sequencing reagent is a reagent that can be customized for the construction of a library Illumina targeting RNA-seq.

In yet another specific embodiment, the product is a kit of SYBR Green RT-PCR comprising primers having sequences as shown in table 4 (SEQ ID No.151-SEQ ID No. 169), optionally further comprising one or more selected from: total RNA extraction reagent, reverse transcription reagent and reagent for SYBR Green RT-PCR.

In another specific embodiment, the product is a TaqMan RT-PCR assay kit comprising a primer (SEQ ID No.113-SEQ ID No. 150) having a sequence as shown in table 4 and a TaqMan probe (SEQ ID No.151-SEQ ID No. 169), optionally further comprising one or more of: total RNA extraction reagent, reverse transcription reagent and reagent for TaqMan RT-PCR.

The diagnostic product of the invention (preferably in the form of a kit) also preferably comprises means for taking a test sample from a subject; such as a device for extracting tissue or blood from a subject, preferably any blood sampling needle, syringe, etc. that can be used for blood sampling. The subject is a mammal, preferably a human, in particular a patient suffering from squamous cell lung carcinoma.

Methods and applications of the invention

In yet another aspect, the invention also relates to a method for determining the molecular subtype and/or risk of survival of squamous cell lung carcinoma in a subject, said method comprising

(1) Providing a sample of the subject and providing a sample of the subject,

(2) Determining the expression level of a gene in the gene group of the present invention in the sample, and

(3) Determining the subject's molecular subtype of squamous cell lung carcinoma and/or risk of survival.

The methods of the invention may be used for diagnostic or non-diagnostic purposes.

The subject for use in the method of the invention is a mammal, preferably a human, in particular a patient with squamous cell lung carcinoma.

The sample used in step (1) is not particularly limited as long as the expression level of the genes in the gene group can be obtained therefrom, and for example, total RNA, total protein, and the like, preferably total RNA, of a subject (e.g., a lung squamous carcinoma patient) can be extracted from the sample. The sample is preferably a sample of tissue, blood, plasma, body fluid or a combination thereof, preferably a tissue sample, in particular a paraffin tissue sample. In a preferred embodiment, the sample is a tumor tissue sample or a tissue sample comprising tumor cells. In a preferred embodiment, the sample is a tissue with a high content of tumor cells. In one embodiment, the sample is lung squamous carcinoma tumor tissue.

The step (2) may be performed by using a method for measuring the expression level of a gene known in the art. The skilled person can select the type and amount of sample in step (1) as required, and select the conventional techniques in the art to perform the determination in step (2). Preferably, the expression level of a gene of interest (e.g., a gene associated with the molecular typing and survival risk assessment of the present invention) is normalized based on the expression level of a reference gene. Methods for normalizing the expression level of a gene are well known to those skilled in the art.

In one embodiment, step (2) can be carried out by detecting the amount of the polypeptide encoded by the target gene (the gene in the gene group of the present invention). The detection can be accomplished by reagents as described above and techniques known in the art, including, but not limited to, enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay techniques (e.g., immunochemiluminometric assay, chemiluminescent enzyme immunoassay, electrochemiluminescent immunoassay), flow cytometry, immunohistochemical methods (IHC).

In a preferred embodiment, step (2) may be carried out by detecting the amount of the target nucleic acid. The detection may be accomplished by using the reagents described above and techniques known in the art, including but not limited to molecular hybridization techniques, quantitative PCR techniques, or nucleic acid sequencing techniques, among others. Molecular hybridization techniques include, but are not limited to, ISH techniques (e.g., DISH, DNA-FISH, RNA-FISH, CISH techniques, etc.), DNA or RNA imprinting techniques, gene chip techniques (e.g., microarray or microfluidic chip techniques), and the like, preferably in situ hybridization techniques. Quantitative PCR techniques include, but are not limited to, semi-quantitative PCR and RT-PCR techniques, preferably RT-PCR techniques such as SYBR Green RT-PCR technique, taqMan RT-PCR technique. Nucleic acid sequencing techniques include, but are not limited to, sanger sequencing, next Generation Sequencing (NGS), third generation sequencing, single cell sequencing techniques, and the like, with second generation sequencing being preferred, and targeted RNA-seq techniques being more preferred. More preferably, the detection is effected using the reagents of the invention.

In a preferred embodiment, in step (2), the expression level of the genes in the gene population of the invention is determined using a second generation sequencing technique. In one embodiment, the genes of the gene group are as shown in table 1 or table 2. In one embodiment, the gene population includes 50 molecular typing and risk of survival assessment-associated genes and 6 housekeeping genes as described above, and also see table 1. In yet another embodiment, the gene cluster comprises 16 molecular typing and survival risk assessment-associated genes and 3 housekeeping genes as described above, and also see table 2.

In a specific embodiment, step (2) may comprise:

(2 a-1) extracting total RNA in the sample;

(2 a-2) converting the total RNA, which is optionally purified, into cDNA, which is then prepared into a library that can be used for second-generation sequencing; and

(2 a-3) sequencing the library obtained in step (2 a-2), optionally normalizing the expression level of the gene associated with molecular typing and survival risk assessment according to the expression level of the housekeeping gene.

The extraction of step (2 a-1) can be performed by methods routine in the art, preferably using commercially available RNA extraction kits to extract total RNA from fresh frozen or paraffin-embedded tissues of a subject. In a more preferred embodiment, the extraction can be performed using RNA storm CD201 or Qiagen 73504.

In a preferred embodiment, the step (2 a-2) may comprise the steps of:

reverse transcribing the extracted total RNA to produce cDNA for the gene of interest; and

(ii) preparing the resulting cDNA into a library for sequencing.

In a preferred embodiment, the cDNA is amplified in step (2 a-2) using primers as shown in Table 3 to prepare a library for sequencing.

Step (2 a-3) may be accomplished by RNA sequencing. The sequencing method may be an RNA-seq sequencing method for determining the expression level of a gene, which is conventional in the art. Secondary sequencing is preferably performed using an Illumina NextSeq/MiSeq/MiniSeq/iSeq series sequencer. The gene in the gene group of the invention is amplified by using the primer in the kit, and the obtained gene sequence can be subjected to second-generation sequencing according to the difference of the libraries prepared in the step (2 a-2). Preferably, the second generation sequencing is a targeted RNA-seq technique, paired-end sequencing or single-end sequencing with Illumina NextSeq/MiSeq/MiniSeq/iSeq sequencer. Such a process may be automated by the instrument itself.

In step (2), the expression level of the gene in the gene group of the present invention can also be determined by a fluorescent quantitative PCR method. In one embodiment, the gene population includes 16 molecular typing and risk of survival assessment-associated genes and 3 housekeeping genes as described above, and also see table 2.

In a specific embodiment, step (2) may comprise:

(2 b-1) extracting total RNA in the sample;

(2 b-2) reverse transcribing the total RNA of (2-1) into cDNA; and

(2 b-3) subjecting the obtained cDNA to real-time fluorescent quantitative PCR (RT-PCR) detection, optionally normalizing the expression level of the gene associated with molecular typing and survival risk assessment according to the expression level of the housekeeping gene.

The extraction of step (2 b-1) can be performed by methods routine in the art, preferably using commercially available RNA extraction kits to extract total RNA from fresh frozen or paraffin-embedded tissues of a subject. In a more preferred embodiment, the extraction can be performed using RNA storm CD201 or Qiagen 73504. The reverse transcription of step (2 b-2) can be performed using a commercially available reverse transcription kit. In a preferred embodiment, the RT-PCR method of step (2 b-3) is TaqMan RT-PCR. Preferably, the genes shown in Table 2 can be separately detected by RT-PCR using primers and probes, which are TaqMan probes. Preferably, the sequences of the primers and probes are shown in table 4. In one embodiment, single or multiplex RT-PCR assays are performed using primers and probes as shown in Table 4.

In an alternative embodiment, the RT-PCR method described in step (2 b-3) is SYBR Green RT-PCR, and the genes shown in Table 2 can be detected separately or simultaneously using primers and a commercially available SYBR Green premix. Preferably, the sequence of the primer is shown as SEQ ID NO.113-SEQ ID NO.150 (see also Table 4).

The RT-PCR assay can be performed using ABI7500 real-time fluorescence quantitative PCR instrument (Applied Biosystems) or Roche

480 II) is carried out. After the reaction was completed, the Ct value of each gene was recorded and represents the expression level of each gene.

In one embodiment of the present invention, the step (3) may be performed by statistically analyzing the expression level of the gene in the gene group of the present invention in the sample obtained in the step (2). Lung squamous carcinoma molecular typing and recurrence risk prediction can optionally be performed according to the Single Sample prediction SSP (Single Sample Predictor) pioneered by Hu et al (see Hu Z, et al, BMC genetics.2006, 7 96) and Parker et al optimized methods (see Parker JS, et al, journal of Clinical oncology: of the American Society of Clinical oncology.2009,27 (8): 1160-7). Analyzing the gene expression data obtained in step (2) to obtain subtype classification of a single sample, and calculating the recurrence risk.

In one embodiment, step (3) comprises molecular typing of squamous cell lung carcinoma, which comprises determining the molecular subtype of squamous cell lung carcinoma in the subject based on the expression level of each gene in the sample of the subject obtained in step (2).

The present inventors obtained an expression profile of the gene of the present invention by using 495 cases of the lung squamous carcinoma gene expression amounts with complete clinical information in the TCGA database by an epiig gene expression profile analysis program (see Zhou T, et al,2006.Environ Health perspective 114 (4), 553-559, chou jw, et al,2007.Bmc Bioinformatics 8, 427). Furthermore, according to the expression profile of the genes, a hierarchical clustering method is adopted to compare the similarity among all the detected genes and group the genes; comparing the similarity of expression profiles among lung squamous carcinoma samples, grouping the lung squamous carcinomas, and dividing the lung squamous carcinomas into LSC1 subtypes, LSC2 subtypes, LSC3 subtypes, LSC4 subtypes, LSC5 subtypes and mixed subtypes; and taking the gene expression profile of the lung squamous carcinoma molecule subtype as standard test data for carrying out molecular typing and survival risk assessment on the sample.

The lung squamous carcinoma molecular subtypes may include LSC1 subtype, LSC2 subtype, LSC3 subtype, LSC4 subtype, LSC5 subtype, and mixed subtypes:

the LSC1 subtype is mainly characterized by high expression of related coagulation genes, low expression of related peptide crosslinking genes, low expression of related nicotine metabolism genes and low recurrence-free survival rate in 5 years;

the LSC2 subtype is mainly characterized by low expression of related coagulation genes, high expression of related peptide crosslinking genes, low expression of related nicotine metabolism genes, no relapse in 5 years and moderate survival rate;

the LSC3 subtype is mainly characterized by high expression of related coagulation genes, high expression of related peptide crosslinking genes, low expression of related nicotine metabolism genes and low recurrence-free survival rate in 5 years;

the LSC4 subtype is mainly characterized by low expression of related coagulation genes, high expression of related peptide crosslinking genes, high expression of related nicotine metabolism genes and high survival rate without relapse in 5 years;

the LSC5 subtype is mainly characterized by expression in related coagulation genes, low expression of related peptide cross-linking genes, high expression of related nicotine metabolism genes and low recurrence-survival rate in 5 years;

the mixed subtype is lung squamous carcinoma not belonging to LSC1 subtype, LSC2 subtype, LSC3 subtype, LSC4 subtype and LSC5 subtype.

In a particular aspect, the invention provides a method for determining the molecular subtype of squamous cell lung carcinoma in a test sample, said method comprising

(a) Obtaining a gene expression profile of the test sample, the gene expression profile being based on the expression levels of genes in the gene population of the invention in the test sample;

(b) Obtaining a gene expression profile of a squamous cell lung carcinoma sample which has been molecularly classified into an LSC1 subtype, an LSC2 subtype, an LSC3 subtype, an LSC4 subtype and an LSC5 subtype in a training set of the squamous cell lung carcinoma sample, wherein the gene expression profile is based on the expression level of genes in the gene group of the invention in the squamous cell lung carcinoma sample;

(c) Calculating a Pearson correlation coefficient between the gene expression profile of the test sample obtained in step (a) and the gene expression profile of the lung squamous carcinoma sample of LSC1 subtype, LSC2 subtype, LSC3 subtype, LSC4 subtype or LSC5 subtype obtained in step (b); and

(d) Determining that the gene expression profile of the test sample has the highest correlation coefficient and the highest confidence limit with the gene expression profile of a subtype X lung squamous carcinoma sample, wherein X is selected from the group consisting of LSC1 subtype, LSC2 subtype, LSC3 subtype, LSC4 subtype, and LSC5 subtype, and wherein,

when the Pearson correlation coefficient of the gene expression profile of the test sample and the gene expression profile of the X subtype lung squamous carcinoma sample is the highest and the confidence limit is more than or equal to 0.8, judging the test sample as an X subtype, wherein the X subtype is an LSC1 subtype, an LSC2 subtype, an LSC3 subtype, an LSC4 subtype or an LSC5 subtype;

and when the reliability is lower than 0.8, judging the test sample as the mixed type.

In some embodiments, the test sample is from a squamous cell lung carcinoma patient.

In one embodiment, in step (a), the expression profile of the gene group of the invention in LSC1 subtype, LSC2 subtype, LSC3 subtype, LSC4 subtype, LSC5 subtype is established as standard test data based on the expression data of the gene group of the invention in a statistically significant number of lung squamous carcinoma specimens (training set). In one embodiment, the standard test data is a weighted average of the expression levels of the genes in the gene populations of the invention in each subtype.

After obtaining data on the expression levels of the genes in the gene populations of the invention, one skilled in the art can apply techniques known in the art to obtain a weighted average of the expression levels of each set of genes. In yet another particular aspect, the invention provides a method for determining the risk of survival of a patient with squamous cell lung carcinoma, the method comprising

(a') obtaining a gene expression profile from a test sample from said patient with squamous cell lung carcinoma, said gene expression profile being based on the expression levels of genes in the gene population of the invention in said test sample;

(b') obtaining a gene expression profile of a lung squamous carcinoma specimen that has been molecularly classified into an LSC1 subtype, an LSC2 subtype, an LSC3 subtype, an LSC4 subtype and an LSC5 subtype in a training set of lung squamous carcinoma specimens, said gene expression profile being based on the expression levels of genes in the gene cluster of the invention in said lung squamous carcinoma specimen;

(c ') calculating a Pearson correlation coefficient between the gene expression profile of said test sample obtained in step (a ') and the gene expression profile of the squamous cell lung carcinoma sample of LSC1 subtype, LSC2 subtype, LSC3 subtype, LSC4 subtype or LSC5 subtype obtained in step (b ');

(d ') calculating a relapse Risk score (Risk of Recurrence, ROR) of the patient with squamous cell lung cancer based on the Pearson correlation coefficient obtained in step (c'); and

(e ') determining the risk of survival of said squamous cell lung carcinoma patient based on the risk of recurrence score calculated in step (d'), wherein

A recurrence risk score of 0-35, defining the risk of survival of said squamous cell lung carcinoma patient as low risk;

the recurrence risk score is 36-100, and the risk of survival of the lung squamous carcinoma patient is defined as high risk.

The molecular subtype and/or risk of survival of squamous cell lung carcinoma patients determined according to some embodiments of the present invention may be used to guide the treatment of squamous cell lung carcinoma patients with different risks of survival.

Thus, in a particular aspect, the invention provides a method of treating squamous cell lung carcinoma in a patient suffering therefrom, the method comprising

(a') obtaining a gene expression profile from a test sample from said patient with lung squamous carcinoma, said gene expression profile being based on the expression levels of genes in the gene population of the invention in said test sample;

(c ') calculating a Pearson correlation coefficient between the gene expression profile of the test sample obtained in step (a ') and the gene expression profile of the lung squamous carcinoma sample of the LSC1 subtype, LSC2 subtype, LSC3 subtype, LSC4 subtype or LSC5 subtype obtained in step (b ');

(d ') calculating a relapse Risk score (Risk of Recurrence, ROR) of the patient with squamous cell lung cancer based on the Pearson correlation coefficient obtained in step (c');

a recurrence risk score of 36-100, defining the risk of survival of said patient with squamous cell lung carcinoma as high risk; and

(f ') treating by administering at least one of radiation therapy, chemotherapy and biological therapy to the squamous cell lung carcinoma patient whose risk of survival is defined as high risk in step (e').

In one embodiment, the gene expression profile of the test sample is obtained based on the expression levels of the 50 genes associated with molecular typing of lung squamous carcinoma and risk of survival assessment (see also Table 1) as described above. Preferably, the expression levels of the 50 genes associated with molecular typing of squamous cell lung carcinoma and risk of survival assessment are normalized using a set of reference genes. In a preferred embodiment, the reference gene is selected from GAPDH, GUSB, TFRC, MRPL19, PSMC4 and SF3A1.

In yet another embodiment, the gene expression profile of the test sample is obtained based on the expression levels of the 16 lung squamous carcinoma molecular typing and survival risk assessment associated genes as described above (see also table 2). Preferably, the expression levels of the 16 genes associated with molecular typing of lung squamous carcinoma and survival risk assessment are normalized by using a group of reference genes. In a preferred embodiment, the reference gene is selected from ACTB, GAPDH and GUSB.

In one embodiment, a recurrence risk score is calculated using the 50 genes associated with molecular typing of squamous cell lung carcinoma and risk of survival assessment as described above (see also Table 1),

ROR = (0.19 × lsc 1) + (0.03 × lsc 2) + (0.12 × lsc 3) + (-0.22 × lsc 4) + (0.10 × lsc 5); wherein the content of the first and second substances,

"LSC1" represents the Pearson correlation coefficient of the test sample with the LSC1 subtype lung squamous carcinoma sample; "LSC2" represents the Pearson correlation coefficient of the test sample with the LSC2 subtype squamous cell carcinoma sample; "LSC3" represents the Pearson correlation coefficient of the test sample with the LSC3 subtype squamous cell carcinoma sample; "LSC4" represents the Pearson correlation coefficient of the test sample with the LSC4 subtype squamous cell carcinoma sample; "LSC5" represents the Pearson correlation coefficient of the test sample with the LSC5 subtype squamous cell carcinoma specimen.

In another embodiment, a relapse risk score is calculated using the 16 molecular typing of squamous cell lung carcinoma and genes associated with risk assessment for survival (see also Table 2) as described above,

ROR = (0.20 × lsc 1) + (-0.03 × lsc 2) + (0.14 × lsc 3) + (-0.11 × lsc 4) + (0.10 × lsc 5); wherein the content of the first and second substances,

"LSC1", "LSC2", "LSC3", "LSC4" and "LSC5" are as defined above.

Correspondingly, the invention also provides application of the gene group in molecular typing of the squamous cell lung carcinoma and/or assessing the survival risk of patients with the squamous cell lung carcinoma. The invention also provides application of the gene group or the reagent for detecting the expression level of the genes in the gene group in preparation of products for carrying out molecular typing on the squamous cell lung carcinoma and/or evaluating the survival risk of patients with the squamous cell lung carcinoma. In a preferred embodiment, the product is a detection/diagnostic kit. In one embodiment, the product is an in vitro diagnostic product. The reagents are as described above. The product is as described above. According to the methods or uses of the present invention, squamous cell lung carcinoma may be divided into different molecular subtypes, which may include the LSC1 subtype, the LSC2 subtype, the LSC3 subtype, the LSC4 subtype, the LSC5 subtype, and mixed subtypes. According to the methods or uses of the present invention, the risk of survival of a patient with squamous cell lung cancer can be assessed, which can include low risk and high risk.

Advantageous effects

According to the invention, by analyzing the gene expression profile and clinical data of the squamous cell lung carcinoma patient, a model for molecular typing and survival risk assessment of squamous cell lung carcinoma based on multi-gene expression and a detection method based on a second-generation sequencing and PCR platform are established, so that help is provided for improving the clinical treatment efficiency of squamous cell lung carcinoma.

According to the expression level of the genes in the gene group in the squamous cell lung carcinoma sample, a molecular typing system of the squamous cell lung carcinoma is established, the squamous cell lung carcinoma can be divided into different subtypes, and more targeted individualized treatment is provided for patients with the squamous cell lung carcinoma belonging to different subtypes. On the other hand, according to the method and the application of the invention, the recurrence risk of the lung squamous carcinoma patient can be well predicted, and the method has important guiding significance for clinical treatment. The binding molecule subtype and risk score can be used to make a prognosis for patients with squamous cell lung carcinoma. The molecular typing and risk evaluation of the squamous cell lung carcinoma are carried out on patients with the squamous cell lung carcinoma, so that the dominant population with different treatment schemes can be screened out, and a potential treatment approach is provided. For patients with low recurrence risk, radiotherapy and chemotherapy can be omitted, so that adverse reactions and economic burden of treatment are reduced; for patients with high recurrence risk, chemotherapy, radiotherapy or biological treatment should be given as an adjuvant in time in order to obtain the greatest clinical benefit. For patients in the advanced stage who cannot be operated, molecular diagnosis based on expression profiles can help identify populations where one treatment regimen can benefit, improve treatment efficiency, and avoid ineffective treatment.

Compared with the current molecular typing method of the squamous cell lung carcinoma, the molecular typing method has the advantages that the subtype typing of the squamous cell lung carcinoma is carried out, the survival risk of patients with the squamous cell lung carcinoma is also evaluated, the prognosis of the patients with the squamous cell lung carcinoma is comprehensively evaluated, and the possible benefit of treatment is also obtained. Another advantage of the present invention is that a plurality of selectable genes or combinations of genes are provided as supplementary embodiments, which can be used to supplement the present invention when applied to cancer patients if the detection of the expression level of a gene or genes is not effective or functional due to the pathological condition of the patient or other reasons (e.g., abnormal expression of a gene or genes), so that the detection results based on the present invention are more stable and reliable.

Examples

The invention is further illustrated by the following examples, which are not intended to limit the invention thereto. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions. Reagents and instruments used in the examples herein are all commercially available.

Example 1: screening of gene groups related to lung squamous carcinoma molecular typing and survival risk assessment

The method comprises the following steps: 495 clinically informative squamous cell carcinoma gene expression levels in the TCGA database were analyzed by an EPIG gene expression profiling program (see Zhou, chou et al,2006.Environ Health perspective 114 (4), 553-559 Chou, zhou et al,2007.Bmc Bioinformatics 8, 427), genes closely related to the risk of recurrence of squamous cell carcinoma were screened out, they were grouped into coagulation-related genes, peptide cross-linking-related genes, and nicotine metabolism-related genes according to their functions, and genes having a large contribution rate to typing and recurrence risk were calculated and preferred in each group of genes.

As a result: the co-screening obtains 50 genes and 6 housekeeping genes related to the molecular typing and survival risk of the lung squamous carcinoma, namely 56 gene test combinations. The gene list is shown in Table 1.

Using the 56 gene test combinations screened, lung squamous carcinomas can be classified as LSC1 subtype, LSC2 subtype, LSC3 subtype, LSC4 subtype, LSC5 subtype or mixed subtypes:

the LSC2 subtype is mainly characterized by low expression of related coagulation genes, high expression of related peptide crosslinking genes, low expression of related nicotine metabolism genes and high survival rate without relapse in 5 years;

the LSC3 subtype is mainly characterized by moderate expression of related coagulation genes, high expression of related peptide crosslinking genes, low expression of related nicotine metabolism genes and low recurrence-free survival rate in 5 years;

the LSC4 subtype is mainly characterized by low expression of related coagulation genes, medium expression of related peptide cross-linking genes, high expression of related nicotine metabolism genes and high survival rate without relapse in 5 years;

the LSC5 subtype is mainly characterized by moderate expression of related coagulation genes, low expression of related peptide crosslinking genes, high expression of related nicotine metabolism genes and low survival rate without relapse within 5 years;

the mixed subtype is squamous cell carcinoma of lung not belonging to LSC1 subtype, LSC2 subtype, LSC3 subtype, LSC4 subtype and LSC5 subtype.

Example 2: gene test combination for lung squamous carcinoma molecular typing and survival risk assessment

Test combinations of 56 genes screened according to example 1 were used for molecular typing of squamous cell lung carcinoma and survival risk assessment.

56 Gene test combination:

the experimental method comprises the following steps: a56 gene test combination (see Table 1) was used, wherein 50 lung squamous carcinoma molecules were typed and survival risk assessment related gene groups (coagulation related genes: C4BPA, FGA, HNF1B, SFTA, SFTA3, SFTPB, CAPN8, CHIA, CLDN18, CRTAC1, FGG, GKN2, HP, ORM1, PLA2G1B, RASGRF1, ROS1 and SERPIND1; peptide crosslink related genes SPRR1A, SPRR1B, SPRR2A, SPRR2B, SPRR2D, SPRR2E, A ML1, CRCT1, IL36B, IL G, IL RN, IVL, LCE3 3524 zxft 353, PI3, RAET1L, SBSN, SERPINB13, SPRR2C, SPRR2G, SPRR3, SPRR4, TMPRSS11B, TMPRSS D and TMPRSS11F, and nicotine metabolism related genes DNAJB3, UGT1A1, UGT1A2P, UGT A6, ADH7, NTRK2 and GAUGT 1A4 are used to determine and assess the risk of lung squamous carcinoma patients, including the risk of normalized expression of the relevant genes for lung squamous carcinoma (MRSF 6, PSD 19, PSD, 19 and the risk of lung carcinoma) as internal control molecules. The 50 genes related to molecular typing and survival risk assessment of squamous cell lung carcinoma in Table 1 were used in calculating the recurrence risk index (i.e., recurrence risk score).

The experimental results are as follows:

according to the standard test data obtained in example 1, using the molecular typing method for squamous cell lung carcinoma as described above (see "method and application of the present invention"), 50 cases of squamous cell lung carcinoma were molecularly typed using the expression levels of genes related to molecular typing and survival risk assessment of lung squamous carcinoma (normalized by the expression levels of GAPDH, GUSB, TFRC, MRPL19, PSMC4 and SF3 A1) shown in table 1, and squamous cell lung carcinoma tumors were classified into LSC1 subtype, LSC2 subtype, LSC3 subtype, LSC4 subtype, LSC5 subtype or mixed subtype.

By calculating the number and time of survival of different subtypes and taking the tumor development progress or death observed in 5 years of lung squamous carcinoma cases as an observation event, a Kaplan-Meier survival curve can be drawn to obtain the 5-year relapse-free survival rate and indicate the relapse risk of each subtype. The different recurrence risks of the subtypes indicate that the recurrence risk of each subtype of the squamous cell lung cancer is different.

the LSC5 subtype is mainly characterized by moderate expression of related coagulation genes, low expression of related peptide crosslinking genes, high expression of related nicotine metabolism genes and low recurrence-free survival rate in 5 years;

2. Relapse risk assessment

Calculating the recurrence risk of the tumor by adopting a Cox model, taking the tumor development progress or death as an observation terminal point, determining a corresponding coefficient according to the relative risk of the Pearson correlation coefficient between the tested tumor and each subtype on the influence of survival, and calculating the recurrence risk score by the following calculation method:

calculation of relapse Risk score (Risk of Recurrence, ROR): ROR ranges from 0 to 100, where: 0-35, low risk; 36-100, high risk;

"LSC1" represents the Pearson correlation coefficient of the test tumor with LSC1 subtype squamous cell lung carcinoma tumors; "LSC2" represents the Pearson correlation coefficient of the test tumor with LSC2 subtype squamous cell lung carcinoma tumor; "LSC3" represents the Pearson correlation coefficient of the test tumor with LSC3 subtype squamous cell lung carcinoma tumor; "LSC4" represents the Pearson correlation coefficient of the test tumor with LSC4 subtype squamous cell lung carcinoma tumor; "LSC5" represents the Pearson correlation coefficient of the test tumor with the LSC5 subtype squamous cell lung carcinoma tumor.

Based on the calculated risk of recurrence score, the risk of tumor recurrence can be divided into two groups, low risk (0-35) and high risk (36-100). The results show that the recurrence risk index may indicate the risk of survival for patients with squamous cell lung carcinoma: the survival rate for the low-risk group without recurrence in 5 years is higher, and the survival rate for the high-risk group without recurrence in 5 years is lower.

19 Gene test combination:

the molecular typing method and the survival risk score calculation method of the 19 gene test combination are similar to those of the 56 gene test combination. The 19 gene test combination (see table 2) included: 16 lung squamous carcinoma molecular typing and survival risk assessment related gene groups (blood coagulation related genes: C4BPA, FGA, HNF1B, SFTA, SFTA3 and SFTPB; peptide cross-linking related genes: SPRR1A, SPRR1B, SPRR A, SPRR2B, SPRR2D and SPRR2E; nicotine metabolism related genes: DNAR 3, UGT1A1, UGT1A2P and UGT1A 6) for determining lung squamous carcinoma molecular subtypes and assessing survival risk of lung squamous carcinoma patients; and 3 internal reference genes (including ACTB, GAPDH, and GUSB) as internal standards, which were used to normalize the expression levels of the molecular typing and survival risk-associated genes. When calculating the recurrence risk index, 16 lung squamous carcinoma molecular typing and survival risk assessment related genes in the table 2 are adopted.

The experimental results are as follows:

1. molecular typing of squamous cell lung carcinoma

The expression levels of 16 genes related to molecular typing and survival risk assessment of squamous cell lung carcinoma (normalized by the expression levels of ACTB, GAPDH, and GUSB) shown in table 2 were used to molecularly type 495 cases of squamous cell lung carcinoma, and squamous cell lung carcinoma tumors were classified into LSC1 subtype, LSC2 subtype, LSC3 subtype, LSC4 subtype, LSC5 subtype, or mixed subtypes (fig. 1 and 2). The results were similar to the 56 gene test combination.

2. Relapse risk assessment

"LSC1", "LSC2", "LSC3", "LSC4" and "LSC5" are as defined above.

Based on the calculated risk of recurrence scores, the risk of tumor recurrence can be divided into two groups, low risk (0-35) and high risk (36-100) (fig. 3). The results were similar to the 56 gene test combination.

Example 3: second-generation sequencing detection kit for determining lung squamous carcinoma molecular subtype and evaluating survival risk of lung squamous carcinoma patient

According to the 56 gene test combination in example 2, a second generation sequencing detection kit was designed, which contains primers for specifically amplifying the 56 gene cDNA, and the sequences of the primers are shown in Table 3. Methods for determining molecular subtypes of squamous cell lung carcinoma and assessing the risk of survival of patients with squamous cell lung carcinoma using the second generation sequencing assay kit are described below.

Step 1: taking the tumor or paraffin-embedded tissue of a subject, and acquiring the region containing the tumor cells in the subject as an original material by using a method in the detection kit.

Step 2: total RNA was extracted from the tissue. RNA storm CD201RNA or Qiagen RNease FFPE kit RNA extraction kit can be used for extraction.

And step 3: the resulting RNA is made into a library for sequencing. Making the RNA of the obtained tissue into a library for targeted RNA-seq technology next generation sequencing, wherein the preparation method of the library comprises the following steps:

(3-1): use of

II reverse transcriptase (New England Biolabs, # M0368L) reverse transcribes the RNA extracted in step (2) into cDNA.

(3-2): using Illumina

The target RNA library building kit (# 15034457) processes the obtained cDNA to prepare a library for sequencing, and the specific steps are as follows: (i) hybridization: adding TOP (specific composition shown in Table 3) 4.5 μ l, mixing, adding OB1 21 μ l, heating to 70 deg.C, and slowly gradient cooling to 30 deg.C; (ii) extension and connection: adsorbing the product in the step (i) by using a magnetic frame, then discarding the supernatant, washing the supernatant twice by using AM1 and UB1 in the kit, then discarding the supernatant, adding 36 mu l of ELM4, and incubating the supernatant for 45 minutes at 37 ℃ in a PCR instrument or a metal bath; (iii) ligating the sequencing tag (Index) to the product obtained in (ii), followed by PCR: adsorbing the product obtained in the step (ii) by using a magnetic frame, then discarding the supernatant, adding 18 mu l of HP3 diluted by 40 times, adsorbing by using the magnetic frame, then sucking 16 mu l, adding 17.3 mu l of TDP1, 0.3 mu l of PMM2 and 6.4 mu l of Index, mixing uniformly, and carrying out PCR amplification for 32 cycles;(iv) purifying the DNA by using a Gnome DNA (QuestGenomics, nanjing) purification kit to obtain a library.

And 4, step 4: the resulting DNA library was subjected to secondary sequencing with NextSeq/MiSeq/MiniSeq/iSeq. Paired-end sequencing or single-end sequencing was performed with an Illumina NextSeq/MiSeq/MiniSeq/iSeq sequencer. This process is automated by the instrument itself (Illumina).

And 5: and (5) carrying out statistical analysis on results. And (4) carrying out statistical analysis on the obtained sequencing result. Subjects were then molecularly typed for squamous cell lung carcinoma using the method described in example 2, and a recurrence risk score was calculated and the subject's risk of survival was predicted.

TABLE 3

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Example 4: quantitative PCR detection kit for determining lung squamous carcinoma molecular subtype and evaluating survival risk of lung squamous carcinoma patient

According to the 19 gene test combination in example 2, a quantitative PCR detection kit was designed, which comprises primers for PCR amplification of the 19 gene, and TaqMan probes for quantification of the amplified product, the sequences of the primers and probes are shown in Table 4. The kit can be used for single or multiple RT-PCR detection. The method for molecular typing and recurrence risk assessment of lung squamous cell carcinoma by single RT-PCR detection using the kit is as follows.

The experimental method comprises the following steps: taking lung squamous carcinoma tumor tissues, extracting RNA in tumor cells, and respectively detecting the expression levels of genes by adopting TaqMan RT-PCR technology and using primers and probes shown in Table 4. The method comprises the following steps:

Step 2: total RNA was extracted from the tissue. The RNA extraction kit can be used for extraction using RNAscope CD201RNA or Qiagen RNease FFPE kit.

And step 3: and (3) detecting by RT-PCR. The RT-PCR detection method is Taqman RT-PCR, and the genes shown in the table 4 are respectively subjected to RT-PCR detection. The method comprises the following steps:

(3-1): extracting total RNA from the subject;

(3-2): carrying out reverse transcription on the RNA obtained in the step (3-1), and specifically comprising the following steps: taking sample RNA with the total amount of about 2 mu g (for example, taking 11 mu l of sample RNA with the total amount of about 200 ng/. Mu.l), and carrying out reverse transcription together with 11 mu l of reference RNA (Thermo K1622 reverse transcription kit) to obtain sample cDNA and reference cDNA; adding 80. Mu.l of RNase-free water to the sample cDNA to dilute it 5-fold, and adding 180. Mu.l of RNase-free water to the reference cDNA to dilute it 10-fold;

(3-3): and (3) carrying out TaqMan RT-PCR detection on the cDNA sample corresponding to each gene obtained in the step (3-2) to respectively detect 16 lung squamous carcinoma molecule typing and survival risk related genes and 3 reference genes (see table 2). The method comprises the following steps: preparing a reaction system per hole: (3-2) 2. Mu.l (total amount 100-400 ng) of the obtained cDNA sample, 1.4. Mu.l of the forward and reverse specific primers and TaqMan fluorescent probe (10. Mu.M) shown in Table 4, 10. Mu.l of the reaction premix, 6.6. Mu.l of DEPC water; (ii) inactivating the reverse transcriptase at 95 ℃ for 2 minutes; (iii) amplification and detection: denaturation at 95 ℃ for 25 seconds, annealing at 60 ℃, extension and fluorescence detection for 60 seconds, and performing 45 cycles with a 60 ℃ delay period; after the amplification reaction was completed, the Ct value of each gene was recorded and represents the expression level of each gene.

And 4, step 4: and (5) carrying out statistical analysis on results. And (4) carrying out statistical analysis on the obtained sequencing result. Subjects were then molecularly typed for squamous cell lung carcinoma using the method described in example 2 and the risk of survival was predicted.

TABLE 4

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Claims

1. A set of genes for determining molecular subtype of squamous cell lung carcinoma and/or assessing survival risk of patients with squamous cell lung carcinoma, which comprises genes related to molecular typing and survival risk assessment, wherein the genes related to molecular typing and survival risk assessment comprise:

(1) One or more of the following coagulation-related genes: c4BPA, FGA, HNF1B, SFTA2, SFTA3, SFTPB, CAPN8, CHIA, CLDN18, CRTAC1, FGG, GKN2, HP, ORM1, PLA2G1B, RASGRF, ROS1, and SERPIND1;

(2) One or more of the following peptide cross-linking related genes: SPRR1A, SPRR1B, SPRR2A, SPRR2B, SPRR2D, SPRR2E, A ML1, CRCT1, IL36B, IL G, IL RN, IVL, LCE3E, PGLYRP, PI3, RAET1L, SBSN, SERPINB13, SPRR2C, SPRR2G, SPRR, SPRR4, TMPRSS11B, TMPRSS D and TMPRSS11F;

(3) One or more of the following nicotine metabolism-related genes: DNAJB3, UGT1A1, UGT1A2P, UGT A6, ADH7, NTRK2, and UGT1A4.

2. The gene group of claim 1, comprising 16 molecular typing and survival risk assessment-associated genes, comprising:

(1) Blood coagulation related genes: c4BPA, FGA, HNF1B, SFTA, SFTA3 and SFTPB;

(2) Peptide crosslinking-related genes: SPRR1A, SPRR1B, SPRR2A, SPRR2B, SPRR D and SPRR2E;

(3) Genes related to nicotine metabolism: DNAJB3, UGT1A1, UGT1A2P, and UGT1A6.

3. The gene group of claim 1, comprising 50 molecular typing and survival risk assessment-associated genes, comprising:

(1) Blood coagulation related genes: c4BPA, FGA, HNF1B, SFTA2, SFTA3, SFTPB, CAPN8, CHIA, CLDN18, CRTAC1, FGG, GKN2, HP, ORM1, PLA2G1B, RASGRF, ROS1, and SERPIND1;

(2) Peptide crosslinking-related genes: SPRR1A, SPRR1B, SPRR2A, SPRR B, SPRR2D, SPRR2E, A ML1, CRCT1, IL36B, IL G, IL RN, IVL, LCE3E, PGLYRP, PI3, RAET1L, SBSN, SERPINB13, SPRR2C, SPRR2G, SPRR, SPRR4, TMPRSS11B, TMPRSS D and TMPRSS11F;

4. The gene population of any one of claims 1-3, further comprising a reference gene;

preferably, the reference gene comprises 1, more preferably 3, most preferably 6 of: ACTB, GAPDH, GUSB, TFRC, MRPL19, PSMC4 and SF3A1.

5. The gene population of claim 2, further comprising a reference gene; preferably, the reference gene comprises three of ACTB, GAPDH, GUSB, TFRC, MRPL19, PSMC4 and SF3 A1; more preferably, the reference genes comprise ACTB, GAPDH and GUSB.

6. The gene population of claim 3, further comprising a reference gene; preferably, the reference genes include GAPDH, GUSB, TFRC, MRPL19, PSMC4 and SF3A1.

7. A reagent for detecting the expression level of a gene in the gene group according to any one of claims 1 to 6.

8. The reagent according to claim 7, which is a reagent for detecting the amount of RNA, particularly mRNA, transcribed from the gene; alternatively, it is a reagent for detecting the amount of cDNA complementary to mRNA.

9. The reagent of claim 7 or 8, which is a primer, a probe, or a combination thereof.

10. The reagent of claim 9, which is a primer;

preferably, the primer has a sequence shown as SEQ ID NO.1-SEQ ID NO.100 or SEQ ID NO.1-SEQ ID NO.112, or has a sequence shown as SEQ ID NO.113-SEQ ID NO.144 or SEQ ID NO.113-SEQ ID NO. 150.

11. The reagent of claim 9, which is a probe;

preferably, the probe is a TaqMan probe;

more preferably, the probe is a TaqMan probe having a sequence as shown in SEQ ID NO.151-SEQ ID NO.166 or SEQ ID NO.151-SEQ ID NO. 169.

12. The reagent according to claim 9, which is a combination of a primer and a probe,

preferably, the primer has a sequence shown as SEQ ID NO.113-SEQ ID NO.144 or SEQ ID NO.113-SEQ ID NO.150, and the probe is a TaqMan probe having a sequence shown as SEQ ID NO.151-SEQ ID NO.166 or SEQ ID NO.151-SEQ ID NO. 169;

more preferably, the primer has a sequence shown in SEQ ID NO.113-SEQ ID NO.150, and the probe is a TaqMan probe having a sequence shown in SEQ ID NO.151-SEQ ID NO. 169.

13. The reagent of claim 7, which is a reagent for detecting the amount of a polypeptide encoded by said gene, preferably said reagent is an antibody, an antibody fragment or an affinity protein.

14. A product for molecular typing and/or risk of survival assessment of squamous cell lung carcinoma comprising an agent of any one of claims 7-13.

15. Use of the gene population of any one of claims 1-6, the agent of any one of claims 7-13, or the product of claim 14 for determining the molecular subtype of squamous cell lung carcinoma and/or for assessing the risk of survival of a patient with squamous cell lung carcinoma.

16. Use of the gene cluster of any one of claims 1-6 or the agent of any one of claims 7-13 in the manufacture of a product for determining the molecular subtype of squamous cell lung carcinoma and/or assessing the risk of survival of patients with squamous cell lung carcinoma.

17. The product of claim 14 or the use of claim 16, wherein the product is in the form of an in vitro diagnostic product, preferably in the form of a diagnostic kit.

18. The product of claim 14 or the use of claim 16, wherein the product is a secondary sequencing kit, a real-time fluorescent quantitative PCR detection kit, a gene chip, a protein chip, an ELISA diagnostic kit, or an Immunohistochemical (IHC) kit, or a combination thereof.

19. The product or use of claim 18, wherein the product is a secondary sequencing kit comprising primers having a sequence as set forth in SEQ ID No.1-SEQ ID No.112, and optionally comprising one or more of: total RNA extraction reagent, reverse transcription reagent and second generation sequencing reagent.

20. The product or use of claim 18, wherein the product is a real-time fluorescent quantitative PCR assay kit comprising primers having the sequence shown as SEQ ID No.113-SEQ ID No. 150.

21. The product or use of claim 20, wherein the real-time fluorescent quantitative PCR detection kit further comprises a TaqMan probe, and optionally comprises one or more selected from the group consisting of: a total RNA extraction reagent, a reverse transcription reagent and a reagent for TaqMan RT-PCR.

22. The product or use of claim 21, wherein the real-time fluorescent quantitative PCR detection kit comprises a primer having a sequence as shown in SEQ ID No.113-SEQ ID No.150 and a TaqMan probe having a sequence as shown in SEQ ID No.151-SEQ ID No. 169.

23. The product or use of claim 20, wherein the real-time fluorescent quantitative PCR detection kit further comprises one or more selected from the group consisting of: total RNA extraction reagent, reverse transcription reagent and reagent for SYBR Green RT-PCR.

24. The gene population of any one of claims 1 to 6, the agent of any one of claims 7 to 13, the product of any one of claims 14 and 17 to 23, the use of any one of claims 15 to 23, wherein the lung squamous carcinoma comprises the LSC1 subtype, the LSC2 subtype, the LSC3 subtype, the LSC4 subtype, the LSC5 subtype and mixed subtypes.