Use of a PCDHGB1 mutation detection agent in the manufacture of a kit for predicting the sensitivity of a patient with lung adenocarcinoma to an immune checkpoint inhibitor therapy, wherein the presence of a PCDHGB1 mutation is indicative of the sensitivity of said patient with lung adenocarcinoma to an immune checkpoint inhibitor therapy; the mutation is a point mutation.

2. The use according to claim 1, wherein the presence of the PCDHGB1 mutation is an independent presence of the PCDHGB1 mutation.

3. The use according to claim 1, wherein the immune checkpoint inhibitor is a PD1 inhibitor and/or a PD-L1 inhibitor.

4. The use according to any one of claims 1 to 3, wherein the point mutations include, but are not limited to, single nucleotide polymorphisms, base substitutions/insertions/deletions, or silent mutations.

5. The use of any one of claims 1-3, wherein the detection agent is detected at the nucleic acid level; the detection agent is used for executing any one of the following methods: polymerase chain reaction, denaturing gradient gel electrophoresis, nucleic acid sequencing, nucleic acid typing chip detection, denaturing high performance liquid chromatography, in situ hybridization, biological mass spectrometry and HRM method.

6. The use of any one of claims 1 to 3, wherein the detection agent is detected at the protein level, and wherein the detection agent is used to perform any one of the following methods: biological mass spectrometry, amino acid sequencing, electrophoresis, and detection using antibodies specifically designed for the mutation site.

7. The use of any one of claims 1 or 3, wherein the kit further comprises reagents for detecting mutations in other genes, including but not limited to, POLE, POLD1, ARID1A, ARID1B, PCDH11X or NOTCH 1-4.

8. The use of claim 7, wherein the kit further comprises a sample treatment reagent comprising at least one of a sample lysis reagent, a sample purification reagent, and a sample nucleic acid extraction reagent; the sample is selected from at least one of blood, serum, plasma, cerebrospinal fluid, tissue or tissue lysate, cell culture supernatant, semen and saliva sample of the lung adenocarcinoma patient.

9. A kit for predicting, assessing or screening susceptibility of a patient with lung adenocarcinoma to treatment with an immune checkpoint inhibitor, comprising reagents for detection of mutations in the PCDHGB1 gene.

10. The kit of claim 9, wherein the kit further comprises reagents for detecting mutations in other genes, including but not limited to, one or more of POLE, POLD1, ARID1A, ARID1B, PCDH11X, or NOTCH 1-4.

Background

In recent years, treatment regimens based on emerging Immune Checkpoint Inhibitors (ICIs) such as PD-1/PD-L1, CTLA-4, and the like have been extensively studied in a variety of solid tumors and have entered first-line treatment of non-small cell lung cancer (NSCLC). Of the non-small cell lung cancers, approximately 55% are lung adenocarcinomas. Although the effect of the immune checkpoint inhibitor is good, the overall Objective Remission Rate (ORR) is still only about 20%, so that the development of a proper biomarker and the accurate screening of more populations benefiting from immunotherapy are key problems to be solved urgently in the development of future tumor immunotherapy.

The detection of tumor PD-L1 expression based on immunohistochemical staining is the most widely used biomarker for immunotherapy at present. In certain tumor types, such as lung adenocarcinoma, the expression of PD-L1 can be used as a predictive marker for the response to anti-PD-1/PD-L1 therapy. However, the results of multiple clinical trials show that the prediction ability of PD-L1 expression on the curative effect of immunotherapy is inconsistent, and some PD-L1 negative patients still benefit from immunotherapy, and although the overall effective rate of PD-L1 negative patients is lower, the sustained remission time is no less than that of PD-L1 positive patients. Due to the heterogeneity of tumors, the expression level of PD-L1 varies at different sites of the same tumor, and the expression level of PD-L1 changes as the treatment progresses. Also, the detection criteria of PD-L1 are controversial.

Tumor Mutational Burden (TMB) has been shown to correlate with therapeutic efficacy of immune checkpoint inhibitors for a variety of tumor types including melanoma, lung adenocarcinoma and bladder cancer. However, the use of TMB as a predictor still faces problems such as patients with high TMB benefit from immunotherapy, but studies have shown that either high or low TMB, palbociclumab in combination with chemotherapy, shows survival benefit in first line therapy in both squamous and non-squamous NSCLC patients; secondly, the detection of TMB has no standard method due to different detection products and different algorithms of different detection organizations in China. Furthermore, the TMB threshold problem remains a difficulty in distinguishing between valid or invalid patients.

The research shows that the tumor antigen is effective on immune cells in a tumor microenvironment, particularly CD8⁺The infiltration degree of T cells can predict the possibility of the tumor responding to immunotherapy, but a simple and effective molecular marker is lacked to reflect the immune characteristics of lung adenocarcinoma at present. In addition, recent studies have shown that mutation of the DNA mismatch repair (DDR) pathway gene leads to increased DNA damage and decreased DNA repair capacity in cancer cells, which can improve the effectiveness of immunotherapy. However, the DDR pathway involves a large number of genes, and a simple detection method is currently lacking.

More and more researches in precise treatment of second-generation sequencing tumors show that the somatic mutation of a specific gene can influence the immune function of the tumor or the response to immunotherapy, namely, the specific somatic mutation can be a potential prediction factor of immunotherapy. Therefore, there is still a need in the art to more efficiently and accurately identify methods and tools suitable for immune checkpoint inhibitors to treat lung cancer patients, and in view of this, the present invention is specifically proposed.

Disclosure of Invention

In order to achieve the above object of the present invention, the following technical solutions are adopted.

The invention firstly provides application of a PCDHGB1 mutation detection agent in preparation of a kit for predicting sensitivity of a lung adenocarcinoma patient to an immune checkpoint inhibitor therapy.

In some embodiments, the PCDHGB1 mutation detector is used as the sole mutation detector in the preparation of a kit for predicting the sensitivity of a lung adenocarcinoma patient to immune checkpoint inhibitor therapy.

The invention also provides application of the PCDHGB1 mutation in predicting the sensitivity of a lung adenocarcinoma patient to an immune checkpoint inhibitor therapy.

Further, the presence of the above PCDHGB1 mutation is an indication that the lung adenocarcinoma patient is susceptible to immune checkpoint inhibitor therapy.

In some preferred forms, the presence of the PCDHGB1 mutation is an independent presence of the PCDHGB1 mutation.

The invention also provides application of the PCDHGB1 mutation detection agent in preparation of a kit for predicting tumor mutation load degree of a lung adenocarcinoma patient.

The invention also provides application of the PCDHGB1 in predicting the tumor mutation load degree of a lung adenocarcinoma patient.

Further, the presence of the PCDHGB1 point mutation is an indication of a high tumor mutation load.

The invention also provides application of the PCDHGB1 mutation detection agent in preparation of a kit for predicting the PD-L1 gene expression level of a patient with lung adenocarcinoma.

The invention also provides application of the PCDHGB1 in predicting the expression level of the PD-L1 gene of a lung adenocarcinoma patient.

Further, the existence of the PCDHGB1 point mutation is an indication of high expression of the PD-L1 gene.

The invention also provides application of the PCDHGB1 mutation detection agent in preparation of a kit for predicting DDR pathway gene mutation frequency of lung adenocarcinoma patients.

The invention also provides application of the PCDHGB1 in predicting the DDR pathway gene mutation frequency of a lung adenocarcinoma patient.

Further, the existence of the PCDHGB1 mutation is an indication of high DDR pathway gene mutation frequency of the lung adenocarcinoma patients.

The invention also provides application of the PCDHGB1 mutation detection agent in preparation of a kit for predicting the infiltration level of immune cells in tumors of patients with lung adenocarcinoma.

The invention also provides a method for predicting the CD8 of the PCDHGB1 in the tumor of a patient with lung adenocarcinoma⁺Use of the degree of T cell infiltration.

Further, the PCDHGB1 mutation exists in the lung adenocarcinoma patient intratumoral CD8⁺An indication of high degree of T cell infiltration.

Further, the immune checkpoint inhibitor is a PD1 inhibitor and/or a PD-L1 inhibitor.

Further, the mutation is a point mutation; preferably, the point mutation includes, but is not limited to, a single nucleotide polymorphism, a base substitution/insertion/deletion, or a silent mutation.

The invention also provides application of the PCDHGB1 in predicting the tumor mutation load degree of a lung adenocarcinoma patient.

Further, the detection agent is detected at the nucleic acid level.

Preferably, the detection agent is used to perform any one of the following methods: polymerase chain reaction, denaturing gradient gel electrophoresis, nucleic acid sequencing, nucleic acid typing chip detection, denaturing high performance liquid chromatography, in situ hybridization, biological mass spectrometry and HRM method.

Further, the detection agent is detected at the protein level.

Preferably, the detection agent is used to perform any one of the following methods: biological mass spectrometry, amino acid sequencing, electrophoresis, and detection using antibodies specifically designed for the mutation site.

Furthermore, the kit also comprises a reagent for detecting other gene mutations.

Preferably, the additional genes include, but are not limited to, one or more of POLE, POLD1, ARID1A, ARID1B, PCDH11X, or NOTCH 1-4.

Further, the kit further comprises a sample treatment reagent, wherein the sample treatment reagent comprises at least one of a sample lysis reagent, a sample purification reagent and a sample nucleic acid extraction reagent.

Further, the sample is selected from at least one of blood, serum, plasma, cerebrospinal fluid, tissue or tissue lysate, cell culture supernatant, semen and saliva sample of the lung adenocarcinoma patient.

The invention also provides a kit for predicting, evaluating or screening the sensitivity of a patient with lung adenocarcinoma to an immune checkpoint inhibitor therapy, wherein the kit comprises a reagent for detecting PCDHGB1 gene mutation.

In some embodiments, the kit further comprises mutation detection reagents for other lung adenocarcinoma immunotherapy-sensitive genes, including but not limited to: POLE, POLD1, ARID1A, ARID1B, PCDH11X or NOTCH 1-4.

In some embodiments, the kit does not contain mutation detection reagents of other lung adenocarcinoma immunotherapy-sensitive genes, and the PCDHGB1 gene can be used as the only independent sensitivity marker.

The invention also provides a kit for predicting, evaluating or screening the tumor mutation load degree of a lung adenocarcinoma patient, wherein the kit comprises a reagent for detecting the mutation of other genes of PCDHGB 1.

In some embodiments, the kit further comprises reagents for detecting mutations in other lung adenocarcinoma immunotherapy-sensitive genes, including but not limited to: POLE, POLD1, ARID1A, ARID1B, PCDH11X or NOTCH 1-4.

The invention also provides a method for predicting the sensitivity of a patient with lung adenocarcinoma to immune checkpoint inhibitor therapy, which comprises detecting the PCDHGB1 gene mutation condition of the patient for prediction, or predicting by using the kit.

The invention also provides a method for predicting, evaluating or screening the tumor mutation load degree of a patient with lung adenocarcinoma, which comprises the step of detecting the PCDHGB1 gene mutation condition of the patient for prediction, or the kit is used for prediction.

The invention also provides a method for predicting, evaluating or screening the DDR pathway gene mutation frequency of a patient with lung adenocarcinoma, which comprises the step of detecting the PCDHGB1 gene mutation condition of the patient for prediction, or the kit is used for prediction.

The invention also provides a method for predicting, evaluating or screening the infiltration degree of immune cells in tumors of patients with lung adenocarcinoma, which comprises the step of detecting the PCDHGB1 gene mutation condition of the patients for prediction, or the kit is used for prediction.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

Figure 1 the PCDHGB1 mutation in the Hellmann & Miao cohort was significantly different from the wild type patient PFS.

Figure 2 Hellmann & Miao cohort PCDHGB1 mutant group ORR was significantly higher than wild type.

Figure 3 the proportion of PCDHGB1 mutant DCB in the Hellmann & Miao cohort was significantly higher than the wildtype group.

Figure 4 PCDHGB1 in the Hellmann & Miao cohort is an independent predictor of immunotherapeutic PFS.

FIG. 5 shows that the expression level of the PD-L1 gene of the PCDHGB1 mutant group in the TCGA-LUAD queue is obviously higher than that of the wild type group.

Figure 6 the TMB of patients in the PCDHGB1 mutant group (MUT) in the Hellmann & Miao cohort was significantly higher than in the Wild Type (WT) group.

FIG. 7 the PCDHGB1 mutant group (MUT) TMB in the TCGA-LUAD cohort was significantly higher than the wild-type group (WT).

FIG. 8 shows that PCDHGB1 gene mutation in Hellmann & Miao queue is related to high mutation frequency of DDR access gene.

FIG. 9 mutation of PCDHGB1 gene in TCGA-LUAD cohort is related to high mutation frequency of DDR pathway gene.

FIG. 10 the degree of infiltration of PCDHGB1 mutant group (MUT) CD8+ T cells in the TCGA-LUAD cohort was significantly higher than in the wild-type group (WT).

Detailed Description

Embodiments of the present application will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present application and should not be construed as limiting the scope of the present application. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.

The following basic terms or definitions are provided only to aid in understanding the present invention. These definitions should not be construed to have a scope less than understood by those skilled in the art. Unless defined otherwise below, all technical and scientific terms used in the detailed description of the present invention are intended to have the same meaning as commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.

As used herein, the terms "comprising," "including," "having," "containing," or "involving" are inclusive or open-ended and do not exclude additional unrecited elements or method steps. The term "consisting of …" is considered to be a preferred embodiment of the term "comprising". If in the following a certain group is defined to comprise at least a certain number of embodiments, this should also be understood as disclosing a group which preferably only consists of these embodiments.

Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun.

The terms "about" and "substantially" in the present invention denote an interval of accuracy that can be understood by a person skilled in the art, which still guarantees the technical effect of the feature in question. The term generally denotes a deviation of ± 10%, preferably ± 5%, from the indicated value.

Furthermore, the terms first, second, third, (a), (b), (c), and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

The term "nucleic acid" or "nucleic acid sequence" in the present invention refers to any molecule, preferably polymeric molecule, comprising units of ribonucleic acid, deoxyribonucleic acid, or analogues thereof. The nucleic acid may be single-stranded or double-stranded. The single-stranded nucleic acid may be a nucleic acid that denatures one strand of a double-stranded DNA. Alternatively, the single-stranded nucleic acid may be a single-stranded nucleic acid not derived from any double-stranded DNA.

The term "complementary" as used herein relates to hydrogen bonding base pairing between nucleotide bases G, A, T, C and U, such that when two given polynucleotides or polynucleotide sequences anneal to each other, a pairs with T, G pairs with C in DNA, G pairs with C, and a pairs with U in RNA.

Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment. It is therefore intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention are disclosed in or are apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention.

The invention relates to application of a detection agent of PCDHGB1 gene mutation in preparation of a kit for predicting or screening sensitivity of a patient with lung adenocarcinoma to an immune checkpoint inhibitor therapy, preferably, the existence of the PCDHGB1 gene mutation is an indication that the patient with lung adenocarcinoma is sensitive to the immune checkpoint inhibitor therapy.

The invention also relates to the application of the detection agent of PCDHGB1 gene mutation in the preparation of a reagent for predicting or screening the tumor mutation load degree, the PD-L1 gene expression level, the DDR channel gene mutation frequency and the intratumoral CD8 of a patient with lung adenocarcinoma⁺The application of the kit for the T cell infiltration degree, wherein the existence of PCDHGB1 gene mutation is an indication of high tumor mutation load, or an indication of high expression of PD-L1 gene, or an indication of high DDR pathway gene mutation frequency, or an indication of high immune cell infiltration degree in tumors.

The PCDHGB1 is protocadherin family B1, and the PCDHGB1 gene species can be mammals; preferably, it is a primate.

It will be appreciated that the present invention provides a novel marker for predicting the sensitivity of a patient with lung adenocarcinoma to immune checkpoint inhibitor therapy: PCDHGB1 gene mutation. Patient inoculation of PCDHGB1 mutation by public data miningThe prognosis of the patient is obviously better than that of a PCDHGB1 wild-type patient after immunotherapy, and the analysis of the current reported immunotherapy prediction molecular marker finds that the degree of TMB of the patient with PCDH11X mutation and the patient without mutation, the expression level of PD-L1 gene, the mutation frequency of DDR gene, CD8 gene of the patient with lung adenocarcinoma⁺There were statistical differences in the degree of T cell infiltration.

As used herein, the term "immune checkpoint" refers to some inhibitory signaling pathway present in the immune system. Under normal conditions, the immune checkpoint can maintain immune tolerance by adjusting the strength of autoimmune reaction, however, when the organism is invaded by tumor, the activation of the immune checkpoint can inhibit autoimmunity, which is beneficial to the growth and escape of tumor cells. By using the immune checkpoint inhibitor, the normal anti-tumor immune response of the body can be restored, so that the tumor can be controlled and eliminated.

The "immune checkpoint" of the present invention includes, but is not limited to, programmed death receptor 1 (PD-1), PD-L1, cytotoxic T immune cell associated antigen 4 (CTLA-4); also included are some newly discovered immune checkpoints such as immune cell activation gene 3 (LAG 3), T-cell immunoglobulin and ITIM domain (TIGIT), T-cell immunoglobulin and mucin-3 (TIM-3), T-cell activated V domain immunoglobulin inhibitor (VISTA), adenosine A2a receptor (A2 aR), sialic acid binding immunoglobulin-like lectin 7/9, and the like. In some embodiments, the immune checkpoint inhibitor of the present invention is preferably a PD-1 inhibitor and/or a PD-L1 inhibitor. The PD-1 inhibitor may further be selected from one or more of Nivolumab (Opdivo; BMS-936558), Pembrolizumab (Keytruda; MK-3475), lambrolizumab (MK-3475), Pidilizumab (CT-011), Tereprinizumab (JS 001), Cedilizumab (IBI308), Carrilizumab (Eleka) and Terrilizumab (Baizelan). The PD-L1 inhibitor may further be selected from one or more of Atezolizumab (Tecnriq; MPDL 3280A), JS003, Durvalumab (Imfinzi), Avelumab (Bavencio), BMS-936559, MEDI4736 and MSB 0010718C.

The terms "Mutation load", "Mutation burden" and "Mutation load (mutant burden)" are used interchangeably herein. In the context of tumors, the mutational burden is also referred to herein as "tumor mutational burden" or "TMB".

In the present invention, the "DDR pathway gene mutation frequency" refers to the frequency of gene mutation in the DNA mismatch repair (DNA damage response) pathway. DDR pathways include 9 pathways of Base Excision Repair (BER), Homologous Recombination (HR), mismatch repair (MMR), Vanconi anemia (FA), non-homologous end joining (NHEJ), DNA Damage Repair (DDR), nucleic acid excision repair (NER), DNA Double Strand Breaks (DSB) and Single Strand Breaks (SSBs), each pathway comprising a gene list from the Broad Institute MSigDB database (https:// www.gsea-MSigDB. The gene mutation frequency refers to the ratio of the number of samples with mutation of any gene in the 9 channels to the total number of samples.

In the present invention, "degree of CD8+ T cell infiltration" refers to how much of the relative content of CD8+ T cells leave the bloodstream and migrate into the tumor. CD8+ T cells are T cells expressing CD8 molecules on the cell surface, which are important effector cells in the resistance to viral infection, acute allograft rejection and killing of tumor cells. It plays an important role in the tumor immunotherapy process, and a plurality of studies report that the immunotherapy prognosis of patients with high CD8+ T cell infiltration degree is better.

In the present invention, the point mutation may be a Single Nucleotide Polymorphism (SNP), a base substitution, a base insertion or a base deletion, or a silent mutation (e.g., a synonymous mutation).

Assessing mutations in the PCDHGB1 gene includes determining the presence or absence of mutations, such as frameshift mutations, in its coding region.

In some embodiments, the mutation is at nucleotide 869-4528 of the PCDHGB1 gene. In some preferred embodiments, assessing a mutation in the PCDHGB1 gene comprises determining whether there is a mutation in its coding region that truncates the PCDHGB1 protein.

In some embodiments, the PCDHGB1 gene expression, e.g., the protein expression level of the PCDHGB1 gene, is assessed following determination of the presence of a mutation in the coding region of the PCDHGB1 gene that truncates the PCDHGB1 protein.

In some embodiments, the pathological types of the lung adenocarcinoma patient include lung adenocarcinoma and lung squamous carcinoma.

In some embodiments, the kit further comprises a detection agent for other gene mutations; preferably, the genes include, but are not limited to: POLE, POLD1, ARID1A, ARID1B, PCDH11X or NOTCH 1-4.

Since the PCDHGB1 gene is a gene encoding a protein, and thus mutations in the gene are also usually expressed at the transcriptional level and the response level, those skilled in the art can detect mutations in RNA and protein levels to indirectly reflect whether or not the gene mutation occurs, and these can be applied to the present invention.

In some embodiments, the detection agent detects at the nucleic acid level.

As the detection agent for a nucleic acid level (DNA or RNA level), a known agent known to those skilled in the art can be used, for example, a nucleic acid (usually a probe or primer) which can hybridize to the DNA or RNA and is labeled with a fluorescent label, and the like. And one skilled in the art would also readily envision reverse transcribing mRNA into cDNA and detecting the cDNA, and routine replacement of such techniques would not be outside the scope of the present invention.

In some embodiments, the detection agent is used to perform any one of the following methods:

polymerase chain reaction, denaturing gradient gel electrophoresis, nucleic acid sequencing, nucleic acid typing chip detection, denaturing high performance liquid chromatography, in situ hybridization, biological mass spectrometry and HRM method. In some embodiments, the polymerase chain reaction is selected from the group consisting of restriction fragment length polymorphism, single strand conformation polymorphism, Taqman probe, competitive allele-specific PCR, and allele-specific PCR.

In some embodiments, the biomass spectrometry is selected from a flying mass spectrometer assay, such as a Massarray assay.

In some embodiments, the nucleic acid sequencing method is selected from the Snapshot method.

In some embodiments of the invention, the nucleic acid sequencing method may be transcriptome sequencing or genome sequencing. In some further embodiments of the invention, the nucleic acid sequencing method is high throughput sequencing, also known as next generation sequencing ("NGS"). NGS is distinguished from "Sanger sequencing" (one generation sequencing), which is based on electrophoretic separation of chain termination products in a single sequencing reaction. NGS is a revolutionary revolution in the traditional Sanger sequencing technology, and can sequence hundreds of thousands to millions of nucleic acid molecules at a time. Sequencing platforms that can be used with the NGS of the present invention are commercially available and include, but are not limited to, Roche/454 FLX, Illumina/Solexa genome Analyzer, and Applied Biosystems SOLID system, among others. Transcriptome sequencing can also rapidly and comprehensively obtain almost all transcripts and gene sequences of a specific cell or tissue of a certain species in a certain state through a second-generation sequencing platform, and can be used for researching gene expression quantity, gene function, structure, alternative splicing, prediction of new transcripts and the like. In other embodiments of the present invention, the nucleic acid sequencing method can be single-molecule real-time sequencing, and the single-molecule DNA sequencing technology is a new generation of sequencing technology developed in recent 10 years, also referred to as third generation sequencing technology, and includes single-molecule real-time sequencing, true single-molecule sequencing, single-molecule nanopore sequencing, and the like.

In some embodiments, the detection agent is detected at the protein level.

In some embodiments, the detection agent is used to perform any one of the following methods: biological mass spectrometry, amino acid sequencing, electrophoresis, and detection using antibodies specifically designed for the mutation site. The detection method using an antibody specifically designed for the mutation site may further be immunoprecipitation, co-immunoprecipitation, immunohistochemistry, ELISA, Western Blot, or the like.

In some embodiments, the kit further comprises a sample treatment reagent; further, the sample processing reagent includes at least one of a sample lysis reagent, a sample purification reagent, and a sample nucleic acid extraction reagent.

In some embodiments, the sample is selected from at least one of blood, serum, plasma, pleural fluid, cerebrospinal fluid, tissue or tissue lysate, cell culture supernatant, semen, and saliva samples of the lung adenocarcinoma patient.

In some embodiments, the tissue is lung adenocarcinoma cancerous tissue or para-cancerous tissue. Wherein, the preferable detection samples are blood, serum and plasma; more preferably, from peripheral blood.

According to a further aspect of the invention, there is also provided a method for predicting or screening a patient with lung adenocarcinoma for sensitivity to immune checkpoint inhibitor therapy, the method comprising: the presence or absence of a mutation in the PCDHGB1 gene was detected using the detection reagent described above. In some embodiments, the methods are used for prognostic evaluation of lung adenocarcinoma patients after undergoing immune checkpoint inhibitor therapy.

According to a further aspect of the invention, the invention also provides a kit for predicting, assessing or screening susceptibility of a patient with lung adenocarcinoma to immune checkpoint inhibitor therapy, which kit comprises reagents for detecting mutations in the PCDHGB1 gene; in some preferred embodiments, reagents for the detection of mutations in other genes including, but not limited to: POLE, POLD1, ARID1A, ARID1B, PCDH11X or NOTCH 1-4.

According to a further aspect of the invention, the invention also provides a kit for predicting, evaluating or screening the degree of tumor mutation load of a patient with lung adenocarcinoma, which is characterized in that the kit comprises a reagent for detecting the mutation of the PCDHGB1 gene; in some preferred embodiments, reagents for the detection of mutations in other genes including, but not limited to: POLE, POLD1, ARID1A, ARID1B, PCDH11X or NOTCH 1-4.

According to still another aspect of the invention, the invention also provides a kit for predicting, evaluating or screening the expression level of the PD-L1 gene of a patient with lung adenocarcinoma, which is characterized in that the kit comprises a reagent for detecting PCDHGB1 gene mutation; in some preferred embodiments, reagents for the detection of mutations in other genes including, but not limited to: POLE, POLD1, ARID1A, ARID1B, PCDH11X or NOTCH 1-4.

According to a further aspect of the invention, the invention also provides a kit for predicting, evaluating or screening DDR gene mutation frequency of lung adenocarcinoma patients, which is characterized in that the kit comprises a reagent for detecting PCDHGB1 gene mutation; in some preferred embodiments, reagents for the detection of mutations in other genes including, but not limited to: POLE, POLD1, ARID1A, ARID1B, PCDH11X or NOTCH 1-4.

According to a further aspect of the present invention, there is also provided a method for predicting, assessing or screening for intratumoral CD8 in a patient with lung adenocarcinoma⁺A kit for the T cell infiltration degree is characterized by comprising a reagent for detecting PCDHGB1 gene mutation; in some preferred embodiments, reagents for the detection of mutations in other genes including, but not limited to: one or more of FGFR4, POLE, POLD1, ARID1A, ARID1B, FGFR4, MUC16, or NOTCH 1-4.

Embodiments of the present invention will be described in detail with reference to examples.

Examples

1. Data set

The present invention used a total of 4 independent queues of data sets for analysis (table 1). (1) A Hellmann & Miao cohort consisting of a pool of data sets published by Hellmann and Miao in 2018, respectively, the Hellman data set containing 75 patients with lung adenocarcinoma treated for ICI and the Miao data set containing 56 patients with acellular lung carcinoma treated for ICI, 49 of which were lung adenocarcinoma patients, both data sets detecting somatic mutations in the patients by whole exome sequencing of paracancerous control tissue and cancerous tissue, so we merged the two data sets and named the Hellmann & Miao cohort for analysis; (2) the TCGA-LUAD cohort, patients not receiving immunotherapy, contained 567 patients with lung adenocarcinoma full exome sequencing data and 509 patients with RNA-seq data. The data sets were downloaded at the tumor genomics database, cBioPortal website (http:// www.cbioportal.org /), and included patient clinical baseline data, immune checkpoint inhibitor treatment efficacy assessment data, and patient somatic mutation and gene expression data.

Table 1 data set used by the invention

Clinical index

Clinical indicators to which the present invention relates include Objective Remission Rate (ORR), sustained benefit (DCB), Progression Free Survival (PFS) and Overall Survival (OS). ORR is defined in terms of recistv.1.1 as the percentage of patients confirmed to have a Complete Response (CR) or a Partial Response (PR). Benefits (complete remission (CR), Partial Remission (PR), or Stable Disease (SD)) according to recistv.1.1 definition were sustained benefits (DCB) for more than 6 months, otherwise non-sustained benefits (NDB). PFS is defined as the time from initiation of immunotherapy to the date of disease progression or death. For the cohort of ICI treatments, the Hellmann & Miao cohort, PFS was calculated starting from the treatment start date. PFS and OS were calculated from the date of first diagnosis for the non-immunotherapy cohorts, i.e., TCGA-LUAD and TCGA-LUSC.

3. Prognostic comparison of patients with mutant PCDHGB1 with wild-type patients

The invention divides patients into two groups according to whether the PCDHGB1 is mutated or not, namely a PCDHGB1 mutation group (PCDHGB 1-MUT) and a PCDHGB1 wild-type group (PCDHGB 1-WT), analyzes PFS of ICI treatment of the two groups of patients, namely the PCDHGB1-MUT and the PCDHGB1-WT, through Kaplan-Meier (KM), and compares the PFS of the two groups of patients by using a log-rank test (log-rank test). The ORR and DCB ratios of the two groups of PCDHGB1-MUT and PCDHGB1-WT are respectively counted and compared by a Fisher accurate test method. A Cox regression model was used for multifactorial analysis.

4. Correlation analysis of PCDHGB1 mutation and TMB and PD-L1 gene expression, degree of immune cell infiltration in tumor and DDR pathway gene mutation frequency

Tumor Mutational Burden (TMB) is defined as the number of mutations (SNVs or indels) per Million Bases (MB) of the genome per patient. Two groups of patients, PCDHGB1-MUT and PCDHGB1-WT, TMB, were compared by Mann-Whitney U test.

Patients were divided into two groups, PCDHGB1-MUT and PCDHGB1-WT, depending on whether the PCDHGB1 mutation occurred. Differential expression gene analysis was performed using R-package edgeR, and the definition criteria of differential expression genes were as follows: p-values <0.05 and log2 (fold change) >0.58 or < (-0.58). The PD-L1 gene meets the above criteria and is considered to have significant differences in the expression levels in the two groups of patients.

TIMER software was used to analyze the CD8 between the PCDHGB1-WT and PCDHGB1-MUT subgroups in the TCGA-LUAD and TCGA-LUSC cohorts⁺Infiltration abundance of T cells. Two groups of patients, CD8, were treated with Mann-Whitney U test on PCDHGB1-MUT and PCDHGB1-WT⁺The degree of T cell infiltration was compared.

The gene set associated with the DDR pathway is from the Broad Institute MSigDB database (https:// www.gsea-MSigDB. The number of mutations in the DDR pathway gene in the two groups of patients, PCDHGB1-MUT and PCDHGB1-WT, was compared by Mann-Whitney U test.

5. Statistical analysis

Continuous variables were compared by Mann-Whitney U test and categorical variables were compared by Fisher's exact test. Survival analysis was estimated from the Kaplan Meier curve and p-value was calculated using the log rank test. P is no more than 0.05 for significance, and all statistical analyses were performed using rv.4.0.3 software.

Example 1 analysis of the relationship between PCDHGB1 Gene mutations and the efficacy of immunotherapy in clinical cohorts

In the Hellmann & Miao cohort, 8 patients with the PCDHGB1 mutation (6.1%) had longer median PFS after immunotherapy than the PCDHGB1 wild-type patients (median OS: 16.74 vs. 4.7 months; log-rank test, p = 0.038) (FIG. 1), suggesting that PCDHGB1 may be a predictor of therapeutic efficacy of immunotherapy.

The present example also analyzed the relationship between PCDHGB1 gene mutations and ORR. The ORR of the PCDHGB1 mutant group was 87.5% (7/8), whereas in patients of the PCDHGB1 wild type this ratio was only 28.44% (33/116), the ORR of the PCDHGB1 mutant group was significantly higher than that of the PCDHGB1 wild type group (Fisher exact test, p = 0.0015; fig. 2).

Furthermore, the present example also analyzed the relationship between PCDHGB1 gene mutation and DCB. The DCB of the PCDHGB1 mutant group was 87.5% (7/8), whereas in patients of the PCDHGB1 wild type this ratio was 44.82% (44/116), the DCB ratio of the PCDHGB1 mutant group was significantly higher than that of the PCDHGB1 wild type group (Fisher exact test, p = 0.027; fig. 3).

In this example, the predicted efficacy of PCDHGB1 on the prognosis of lung adenocarcinoma immunotherapy was further analyzed by Cox multifactor regression analysis, taking into account the influence of age, gender, and smoking history. The results show that the PCDHGB1 mutation can be used as an independent predictor for the prognosis of lung adenocarcinoma immunotherapy (HR value =0.2345, 95% CI: 0.05626-0.9772, P = 0.0464).

Therefore, this example 1 demonstrates that the PCDHGB1 gene mutation can be used as a predictor of sensitivity of lung adenocarcinoma patients to immune checkpoint inhibitor therapy through analysis of clinical cohorts of immunotherapy, and that the presence of the PCDHGB1 mutation is indicative of sensitivity of said lung adenocarcinoma patients to immune checkpoint inhibitor therapy; furthermore, Cox multifactorial regression analysis indicates that the PCDHGB1 mutation can be an independent predictor of lung adenocarcinoma patient sensitivity to immune checkpoint inhibitor therapy.

Example 2 correlation of PCDHGB1 Gene mutations with the expression level of the immunotherapeutic biomarker PD-L1 Gene

The detection of tumor PD-L1 expression based on immunohistochemical staining is one of the most widely used biomarkers for immunotherapy at present. The tumor tissue mutation and gene expression of patients are detected simultaneously in the TGCA-LUAD cohort, and the correlation between the PCDHGB1 gene mutation and the PD-L1 gene expression level is explored in the TCGA-LUAD cohort. The results show that the expression level of the PD-L1 gene in the tumor tissue of the PCDHGB1 mutant patient is obviously higher than that of the PCDHGB1 wild-type patient (log 2 (fold change) = 0.89, and p = 0.043) (FIG. 5).

In the embodiment, the relation between the PCDHGB1 mutation and the PD-L1 gene expression level is analyzed in a TCGA-LUAD queue, so that the PCDHGB1 mutation can be used as an indication of high expression of PD-L1, and further, the PCDHGB1 mutation can be used for predicting the curative effect of lung adenocarcinoma immunotherapy.

Example 3 correlation of mutations in the PCDHGB1 Gene with the immunotherapeutic biomarker TMB

TMB is one of the widely used prediction markers for the therapeutic effect of immunotherapy, and this example explores the relationship between PCDHGB1 gene mutation and TMB. In the Hellmann & Miao dataset, the TMB of the PCDHGB1 mutant patients was significantly higher than that of the PCDHGB1 wild-type patients (median TMB: 55.30 vs.14.03 MUT/Mb, p = 0.0036 (FIG. 6). likewise, in TCGA-LUAD, the TMB of the PCDHGB1 mutant patients was significantly higher than that of the PCDHGB1 wild-type patients (FIG. 7).

Therefore, the relation between the PCDHGB1 mutation and TMB was verified in two cohorts in this example, suggesting that the PCDHGB1 mutation may be an indication of high TMB, and further suggesting that the PCDHGB1 mutation may be predictive of lung adenocarcinoma immunotherapy efficacy.

Example 4 correlation of PCDHGB1 Gene mutations with immunotherapy biomarker DDR pathway Gene mutations

This example further examined the association of PCD11X mutation status and DDR gene mutation, as mutations in DDR pathway genes were reportedly correlated with response to immunotherapy in tumors. A trend was observed for the high frequency of mutations in the DDR gene in the PCDHGB1 mutant group. In the Hellmann & Miao cohort, the 7 DDR-related pathways, Base Excision Repair (BER), Fanconi Anemia (FA), non-homologous end joining (NHEJ), DNA Damage Repair (DDR), nucleic acid excision repair (NER), DNA Double Strand Breaks (DSB), and Single Strand Breaks (SSBs), all gave rise to more mutations in patients with the PCDHGB1 mutation; in the TCGA-LUAD cohort, the 6 pathways associated with DDR, i.e., Base Excision Repair (BER), Homologous Recombination (HR), mismatch repair (MMR), Fanconi Anemia (FA), DNA Damage Repair (DDR), and Single Strand Break (SSB), were all enriched for more mutations in patients with PCDHGB1 mutation (fig. 8, fig. 9).

In the embodiment, the relation between the PCDHGB1 mutation and the DDR pathway mutation is verified in Hellmann & Miao and TCGA-LUAD 2 queues, so that the PCDHGB1 mutation can be used as an indication of high DDR pathway mutation frequency, and further, the PCDHGB1 mutation is possible to predict the curative effect of lung adenocarcinoma immunotherapy.

Example 5 mutation of the PCDHGB1 Gene and CD8⁺Correlation between the degree of T cell infiltration

CD8⁺T cells are the most prominent cell type in the course of immunotherapy to exert tumor killing, and multiple studies have revealed CD8⁺The prognosis of patients with high degree of T cell infiltration is better. In the TGCA-LUAD cohort, the tumor tissue mutation and gene expression of patients were simultaneously examined. This example therefore analyzed the correlation between the PCDHGB1 mutation and the degree of CD8+ T cell infiltration in the TGCA-LUAD cohort. FIG. 10 shows that PCDHGB1 mutations in tumor tissue of patients with CD8⁺The infiltration degree of T cells is obviously higher than that of the wild-type patient of PCDHGB 1.

This example demonstrates the PCDHGB1 mutation and intratumoral CD8 in the TCGA-LUAD cohort⁺The relation of the infiltration degree of T cells indicates that the PCDHGB1 mutation can be used as CD8 in tumors⁺High T cell infiltration degree, and further suggests that the PCDHGB1 mutation can predict the curative effect of lung adenocarcinoma immunotherapy.

It should be noted that the above embodiments are only used for illustrating the technical solutions of the present application, and not for limiting the same, and although the present application is described in detail with reference to the foregoing embodiments, a person skilled in the art should understand that the technical solutions described in the foregoing embodiments can be modified or some or all of the technical features can be equivalently replaced, and the modifications or the replacements do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present application.