CN110923329A

CN110923329A - Application of FGFR4 point mutation in prediction of sensitivity of non-small cell lung cancer patient to immune checkpoint inhibitor therapy

Info

Publication number: CN110923329A
Application number: CN202010081244.2A
Authority: CN
Inventors: 张琳; 张史钺
Original assignee: Zhiben Medical Science And Technology (shanghai) Co Ltd
Current assignee: Zhiben Medical Science And Technology (shanghai) Co Ltd
Priority date: 2020-02-06
Filing date: 2020-02-06
Publication date: 2020-03-27
Anticipated expiration: 2040-02-06
Also published as: CN110923329B

Abstract

The invention relates to the field of clinical molecular diagnostics, in particular to application of FGFR4 point mutation in predicting the sensitivity of non-small cell lung cancer patients to immune checkpoint inhibitor therapy and predicting the tumor mutation load degree of the non-small cell lung cancer patients. The method is beneficial to simplifying detection content, reducing the detection cost of patients, quickening the issuing time of detection reports, and the detection of the gene mutation state is more reliable.

Description

Application of FGFR4 point mutation in prediction of sensitivity of non-small cell lung cancer patient to immune checkpoint inhibitor therapy

Technical Field

The invention relates to the field of clinical molecular diagnostics, in particular to application of FGFR4 point mutation in predicting the sensitivity of non-small cell lung cancer patients to immune checkpoint inhibitor therapy.

Background

Tumor immunotherapy has been developed to be very popular, and among them, Immune Checkpoint Inhibitors (ICI) are more "star" drugs in the field of tumor therapy in recent years, and have entered first-line treatment of non-small cell lung cancer. Although the effect of the immune checkpoint inhibitor is good, the overall Objective Remission Rate (ORR) is still only about 20%, so how to accurately screen the population with benefit becomes a problem to be urgently solved by clinicians.

PD-L1, TMB (tumor mutational burden) and MSI (microsatellite instability) are three immunotherapeutic biomarkers (biomarker) that have been approved by FDA or recommended by NCCN guidelines, but each of these three biomarkers has advantages and disadvantages. PD-L1 is most widely used as an immunotherapy biomarker, and PD-L1 IHC detection is also approved by the FDA as a concomitant diagnosis of Pembrolizumab first-line drug administration. However, the results of multiple clinical trials show that the prediction ability of the expression of PD-L1 on the curative effect of immunotherapy is inconsistent, part of PD-L1 negative patients still can benefit from the immunotherapy, and the sustained remission time is not inferior to that of PD-L1 positive patients; TMB is also the immunotherapeutic biomarker recommended by the non-small cell lung cancer NCCN guidelines, but TMB thresholds are difficult to establish consensus given the differences in TMB algorithms by different companies or laboratories; MSI has been used as a key biomarker for tumors to allow FDA to agree to administer medication based on MSI status, rather than histopathological type, but the tumor MSI-H ratio is too low, and clinical popularization has certain limitations. The most important point is that the overlapping rate of PD-L1 positive, TMB high expression and MSI-H is only 0.6% in the existing research (including 11348 cases of solid tumors), which suggests that many potential immunotherapy benefit groups are missed by any biomar alone. Further exploration of immunotherapeutic biorarkers is required.

With the development of the second generation sequencing in the precise treatment of tumors, somatic mutations of specific genes are found to possibly influence the immune function of the tumors or the response to immunotherapy, namely, the specific somatic mutations are suggested to be potential predictors of immunotherapy. EGFR mutations and ALK rearrangements are potential predictors of poor prognosis for ICI immunotherapy. A retrospective analysis found that only 3.6% of these patients responded to ICI immunotherapy, while the response rate for EGFR wild-type and ALK-negative or unknown patients was 23.3%. Meta-analysis of 5 trials involving 3025 patients with advanced non-small cell lung cancer (NSCLC) treated with PD- (L)1 inhibitors revealed no improvement in overall survival compared to docetaxel in EGFR-mutated patients. EGFR-mutated or ALK-rearranged lung cancers show lower PD-L1 and CD8+ T cell infiltration, which may be responsible for poor ICI immunotherapy response. In addition to the synergistic mutations of EGFR and ALK that alter TP53 and KRAS and those in the DNA Damage Response (DDR) pathway of homologous recombination repair and mismatch repair (HRR-MMR) or HRR and base excision repair (HRR-BER) are considered to be positive predictors of better efficacy of ICI immunotherapy, whereas the synergistic mutations of KRAS and STK11 are associated with poor prognosis of ICI immunotherapy. However, these gene mutations still do not cover all potential immunotherapeutic benefit groups as biorarers, and there is still a need in the art for methods and tools for more efficient and accurate identification of non-small cell lung cancer patients for treatment with immune checkpoint inhibitors.

PD-L1 and TMB have been considered as the 2 most common predictive biomarkers for the selection of NSCLC patients susceptible to treatment with immune checkpoint inhibitors. However, the PD-L1 test lacks a uniform standard due to the different anti-PD- (L)1 drugs having their own corresponding PD- (L)1 detection antibodies and platforms; in addition, the expression of PD-L1 has the characteristic of dynamic change, so that the relationship between the expression of PD-L1 and the effect of immunotherapy is still controversial; on the other hand, although a large number of random control studies and large-sample real-world studies have confirmed the correlation between TMB and the immune efficacy, TMB still can only reflect the tumor mutation number, but cannot prompt the state of the tumor microenvironment, and TMB detection has high requirements on a technical platform, a long working period and high cost, which restricts clinical application thereof.

Disclosure of Invention

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

the invention relates to application of a detection agent of FGFR4 (Fibroblast growth factor receptor 4) point mutation in preparation of a kit for predicting the sensitivity of a non-small cell lung cancer patient to an immune checkpoint inhibitor therapy, wherein the existence of the FGFR4 point mutation is an indication that the non-small cell lung cancer patient is sensitive to the immune checkpoint inhibitor therapy.

The invention also relates to application of the FGFR4 point mutation detection agent in preparing a kit for predicting the degree of tumor mutation load of a patient with non-small cell lung cancer, wherein the existence of the FGFR4 point mutation is an indication of high tumor mutation load.

Currently, Immune Checkpoint Inhibitor (ICI) treatment of NSCLC patients uses only PD-L1 and TMB as biomarkers for suitable treatment. However, the objective response rate is still only around 20% in NSCLC patients selected by PD-L1 or TMB, while some NSCLC patients with PD-L1 negative expression have also been reported to respond to ICI. In the invention, by considering FGFR4 point mutation, the TMB degree in NSCLC patients can be accurately predicted, so that ICI-sensitive populations can be predicted, blind medication is avoided, and the economic performance of ICI treatment is improved.

Compared with the co-mutation of other gene combinations, the FGFR4 point mutation screened out in the invention is used as a biomarker for predicting a population sensitive to ICI in NSCLC patients, and the prediction result is more accurate; and the FGFR4 point mutation adopted in the invention can be used as an independent prediction risk factor in practical application, thereby improving the detection efficiency. The method is beneficial to simplifying detection content, reducing the detection cost of a patient and shortening the time for issuing a detection report, and compared with the PD-L1 immunohistochemical method which needs manual interpretation of immunohistochemical fragments and the TMB which needs manual determination of a threshold value, the detection of the gene mutation state is more reliable.

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.

FIG. 1 is a comparison of the Tumor Mutation Burden (TMB) of a FGFR4 mutant patient (MUT) with a wild type patient (WT) in one embodiment of the invention;

FIG. 2 is a comparison of Tumor Mutation Burden (TMB) between a wild-type patient (WT) of FGFR4 and different variant variants (MUT) [ point mutation patient (mutation), amplification mutation patient (amp), and Fusion/Rearrangement mutation (Fusion/reconstruction) patient ] according to one embodiment of the present invention;

FIG. 3 is a diagram showing the analysis of mutation sites of the FGFR4 gene in one embodiment of the invention;

figure 4 is a comparison of the efficacy of FGFR4 mutant patients (MUT) in one embodiment of the invention compared to wild type patients (WT) on immunotherapy with immune checkpoint inhibitors;

figure 5 is a comparison of the efficacy of FGFR1 mutant patients (MUT) in one embodiment of the invention compared to wild type patients (WT) on immunotherapy with immune checkpoint inhibitors;

figure 6 is a comparison of the efficacy of FGFR2 mutant patients (MUT) in one embodiment of the invention compared to wild type patients (WT) on immunotherapy with immune checkpoint inhibitors;

figure 7 is a comparison of the efficacy of FGFR3 mutant patients (MUT) in one embodiment of the invention compared to wild type patients (WT) on immunotherapy with immune checkpoint inhibitors;

FIG. 8 is a comparison of the proportion of patients with FGFR4 Mutations (MUT) compared to wild type patients (WT) who received a sustained benefit from administration of an immune checkpoint inhibitor in one embodiment of the invention;

figure 9 is a graph of the independent risk factors associated with the efficacy of immunotherapy using immune checkpoint inhibitors in a Cox multifactorial regression analysis in one embodiment of the invention.

Detailed Description

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. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in 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 present invention relates to the use of a detector of a FGFR4 point mutation in the manufacture of a kit for predicting the sensitivity of a non-small cell lung cancer patient to an immune checkpoint inhibitor therapy, wherein the presence of the FGFR4 point mutation is indicative of the sensitivity of the non-small cell lung cancer patient to the immune checkpoint inhibitor therapy.

In some embodiments, the FGFR4 gene species is a mammal;

in some embodiments, the FGFR4 gene species is a primate;

in some embodiments, the FGFR4 gene species is human; gene ID 2264 NM-002011.5.

From the above, it is to be understood that the present invention provides a novel marker for predicting the sensitivity of non-small cell lung cancer patients to immune checkpoint inhibitor therapy: FGFR4 point mutation. Clinical studies have shown that the degree of TMB in patients with non-small cell lung cancer is statistically different from that in patients without point mutation. And the prognosis of patients with FGFR4 mutation after receiving immunotherapy is obviously better than that of patients with FGFR4 wild type.

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.

Immune checkpoints according to the invention include, but are not limited to, programmed death receptor 1 (PD 1), PD-L1, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4); also included are some newly discovered immune checkpoints such as lymphocyte activation gene 3 (LAG 3), CD160, T cell immunoglobulin and mucin-3 (TIM-3), T cell activated V domain immunoglobulin inhibitor (VISTA), adenosine A2a receptor (A2 aR), and the like.

Preferred immune checkpoint inhibitors are PD1 inhibitors and/or PD-L1 inhibitors.

The PD1 inhibitor may further be selected from one or more of Nivolumab (OPDIVO; BMS-936558), Pembrolizumab (MK-3475), Jembrolizumab, lambrolizumab, Pidilizumab (CT-011) Terepril mab (JS 001), and Iplilimumab.

The PD-L1 inhibitor may further be selected from one or more of Atezolizumab (MPDL 3280A), JS003, Durvalumab, Avelumab, BMS-936559, MEDI4736 and MSB001071 0010718C.

The terms "mutation load", "mutation load (mutation load)" and "mutation load (mutation load)" 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, a point mutation may refer to a mutation which may be a Single Nucleotide Polymorphism (SNP), a base substitution, a single base insertion or base deletion, or a silent mutation (e.g., a synonymous mutation).

Assessing FGFR4 gene mutations includes determining whether there is a mutation, such as a frameshift mutation, in its coding region. In some embodiments, the mutation is at nucleotide 173-2581 of the FGFR4 gene. In some preferred embodiments, assessing FGFR4 gene mutations comprises determining whether there is a mutation in its coding region that truncates the FGFR4 protein.

In some embodiments, FGFR4 gene expression, e.g., protein expression levels of the FGFR4 gene, are assessed following determination of the presence of a mutation in the coding region of the FGFR4 gene that truncates the FGFR4 protein.

In some embodiments, the non-small cell lung cancer patient is 40-80 years of age, e.g., 50, 60, or 70 years of age.

In some embodiments, the pathological type of the non-small cell lung cancer patient comprises squamous cell lung carcinoma and/or adenocarcinoma of the lung.

In some embodiments, the kit further comprises a detection agent for TP53 gene mutation and/or KMT2C gene mutation.

The relation between TP53 gene mutation and/or KMT2C gene mutation and the application is described in Chinese patent CN110305965A, published 2019, 10 months and 08 days.

Since the FGFR4 gene is a gene capable of encoding protein, and thus the mutation of the gene is usually expressed at the transcription level and the response level, the skilled person can detect the mutation from the RNA and protein level to indirectly reflect whether the point 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 flight mass spectrometer detection.

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"). Second generation sequencing produces thousands to millions of sequences simultaneously in a parallel sequencing process. NGS is distinguished from "Sanger sequencing" (one generation sequencing), which is based on electrophoretic separation of chain termination products in a single sequencing reaction. 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 some embodiments, the detection agent is detected at the protein level.

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, cerebrospinal fluid, tissue or tissue lysate, cell culture supernatant, semen, and saliva sample of the non-small cell lung cancer patient.

In some embodiments, the tissue is lung cancer tissue or a tissue adjacent to a cancer.

Among them, preferred test samples are blood, serum, plasma, and more preferred are those derived from peripheral blood.

According to yet another aspect of the present invention, there is also provided a method for predicting the sensitivity of a non-small cell lung cancer patient to immune checkpoint inhibitor therapy, the method comprising:

a) measuring the presence or absence of a FGFR4 point mutation using the detection agent as described above; and b) optionally, assessing TP53 gene mutation and/or KMT2C gene mutation and/or expression in tumor tissue of the patient.

An ideal scenario for diagnosis is a situation where a single event or process may cause various diseases, e.g. in infectious diseases. In all other cases, correct diagnosis can be very difficult, especially when the etiology of the disease is not fully understood, as in the case of many cancer types. As the skilled artisan will appreciate, diagnosis without biochemical markers is 100% specific and with the same 100% sensitivity for a given multifactorial disease. Conversely, biochemical markers (e.g., FGFR4 point mutations, TP53 gene mutations, and/or KMT2C gene mutations) can be used to assess, for example, the presence, absence, or severity of a disease with some likelihood or predictive value. Thus, in routine clinical diagnosis, a combination of various clinical symptoms and biological markers is often considered to diagnose, treat and control underlying diseases.

In some embodiments, the methods are used for prognostic evaluation of non-small cell lung cancer patients following immune checkpoint inhibitor therapy.

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

Examples

The research method adopted by the embodiment of the invention is as follows:

comprehensive genomic analysis

FFPE tumor samples from chinese non-small cell lung cancer (NSCLC) patients and paired peripheral whole blood control samples were studied. All patients provided written informed consent. Next generation sequencing for targeted capture (NGS) at OrigiMed involves a combination comprising 450 cancer-associated genes. DNA was extracted from all unstained FFPE sections and whole blood containing not less than 20% of tumor content by DNA FFPE Tissue Kit and DNA Mini Kit (QIAamp), respectively, and then quantified by dsDNA HS determination Kit (Qubit). The 250bp sonicated DNA was fragmented using KAPA Hyper Prep Kit (KAPA Biosystems) to construct a library, which was then PCR amplified and quantified. Hybrid capture was performed using custom combinations, this group and the human genome covering 2.6Mb, targeting 450 cancer-associated genes and some frequently rearranged introns. The captured libraries were mixed, denatured and diluted to 1.5-1.8 pM, followed by paired-end sequencing on Illumina NextSeq 500 according to the manufacturer's protocol.

Wherein the samples were subjected to quality detection using the following three primer pairs for amplifying the ACTIN gene:

i) 5'-CACACTGTGCCCATCTATGAGG-3' and 5'-CACGCTCGGTGAGGATCTTC-3' of the group consisting of,

ii) 5'-CACACTGTGCCCATCTATGAGG-3' and 5'-TCGAAGTCCAGGGCAACATAGC-3', and

iii) 5'-CACACTGTGCCCATCTATGAGG-3' and 5'-AAGGCTGGAAGAGCGCCTCGGG-3', which amplify fragments of 100bp, 200bp and 300bp, respectively. And when the three groups of primers are amplified to the target fragment, judging that the tissue sample is qualified.

Genome alteration analysis

Genomic alterations, including single base Substitutions (SNVs), short and long insertion deletions, Copy Number Variations (CNVs), and gene rearrangements and fusions were evaluated. Alignment of the original reads to the human genome reference sequence (hg19) was performed using a Burrows-Wheeler Aligner, followed by PCR deduplication using Picard's MarkDuplicates algorithm. Variants with read depths less than 30x, strand bias greater than 10% or VAF < 0.5% were removed. Common Single Nucleotide Polymorphisms (SNPs) defined as from the dbSNP database (version 147) or with frequencies exceeding 1.5% of exome sequencing project 6500 (ESP6500) or exceeding 1.5% of the 1000 genome project were also excluded.

Whether the identified mutation is true is judged by the following criteria:

(1) for point mutations:

the sequencing coverage depth of the position of the point mutation is more than 500 times; a quality value for each read comprising the point mutation of >40, and a base quality value corresponding to the point mutation on each read comprising the point mutation of > 21; the number of the reads containing the point mutation is more than or equal to 5; a ratio of reads in forward to reads in reverse of all reads comprising the point mutation < 1/6; and the frequency of the variant allele of the tumor tissue/the frequency of the variant allele of the control tissue is more than or equal to 20;

(2) for indels (indels):

if the consecutive identical bases in the indel are <5, the sequencing coverage depth of the position of the indel is >600 times; the quality value of each read containing the indels is > 40; (ii) a base quality value corresponding to the indel mutation on each read comprising the indel of > 21; the number of reads containing the insertion deletion is more than or equal to 5; the ratio of forward read length to reverse read length in all reads containing the indel is < 1/6; the frequency of the variant allele of the tumor tissue/the frequency of the variant allele of the control tissue is more than or equal to 20;

if the continuous identical basic groups in the insertion deletion are more than or equal to 5 and less than 7, the sequencing coverage depth of the position of the insertion deletion is more than 60 times; the quality value of each read containing the indels is > 40; (ii) a base quality value corresponding to the indel mutation on each read comprising the indel of > 21; the number of reads containing the insertion deletion is more than or equal to 5; the ratio of forward read length to reverse read length in all reads containing the indel is < 1/6; (ii) the variant allele frequency of the tumor tissue/variant allele frequency of a control tissue > 20; and the frequency of the variant allele of the tumor tissue is more than or equal to 10 percent;

if the continuous same basic groups in the insertion deletion are more than or equal to 7, the sequencing coverage depth of the position of the insertion deletion is more than 60 times; the quality value of each read containing the indels is > 40; (ii) a base quality value corresponding to the indel mutation on each read comprising the indel of > 21; the number of reads containing the insertion deletion is more than or equal to 5; the ratio of forward read length to reverse read length in all reads containing the indel is < 1/6; (ii) the variant allele frequency of the tumor tissue/variant allele frequency of a control tissue > 20; and the frequency of the variant allele of the tumor tissue is more than or equal to 20 percent.

(3) Amplifying mutations

Refers to the type of variation in copy number variation of a gene. Amplification is an increased copy number of CNV. CNVs, i.e. copy number variations, generally refer to copy number duplications, deletions of large genomic fragments ranging from 1kb to several Mb in length.

TMB calculation

In addition to routine detection of genomic changes, TMB is also determined by NGS-based algorithms. TMB was estimated by counting somatic mutations including SNVs and indels per megabase of coding region sequence examined. The driver mutations and known germline changes in dbSNP were excluded.

Immunohistochemistry

Immunohistochemical (IHC) staining procedures were performed as previously described. Briefly, deparaffinization, rehydration and target recovery were performed, followed by incubation with monoclonal antibodies against PD-L1 (DAKO, clones 22C3 and 28-8). The slides were incubated with a ready-to-use chromogenic reagent consisting of a secondary antibody molecule and a horseradish peroxidase (HRP) molecule coupled to a dextran polymer backbone. Subsequent enzymatic conversion with the addition of chromophores and enhancers results in the precipitation of visible reaction products at the antigenic site. The samples were then counterstained with hematoxylin.

Public database queue data acquisition

To further validate the clinical predictive role of FGFR4 variants on immune checkpoint inhibitor therapy, the present invention downloaded a cohort of 240 non-small cell lung cancer cohorts including patient clinical baseline data, immune checkpoint inhibitor therapy efficacy assessment data, and patient genomic data in the tumor genomics database, cbioport website (http:// www.cbioportal.org /).

Example 1

A total of 3433 non-small cell lung cancer (NSCLC) patients were enrolled in the study. The characteristics of the patients are shown in table 1. Most patients are male (1901/3433, 55.4%), with a median age at diagnosis of 60 years. The most common histological type is lung adenocarcinoma(N =2859, 83.3%). 1024 cases (29.8%) in stage I, 311 cases (9.1%) in stage II, 574 cases (16.7%) in stage III, and 1233 cases (35.9%) in stage IV. TMB measurements were performed on 3433 patients. The median TMB of the whole population was 4.6 muts/Mb (IQR, 2.3-9.2). Tumors with TMB of more than or equal to 10muts/Mb account for 24.3%. This example found that age was positively correlated with TMB (p <0.001). In addition, male patient group (p <0.001) and squamous cell carcinoma patient group: (p <0.001) is higher.

TABLE 1 characterization of NSCLC patients

Example 2 frequency of occurrence of pathogenic FGFR4 mutations in the chinese NSCLC population and correlation with the immunotherapeutic biomarker TMB

34 of 3433 NSCLC patients carried FGFR4 gene mutation, accounting for 1%. The FGFR4 mutation was not significantly different from the wild type in the age, sex, disease stage and pathological type ratio of the two groups of patients (table 2). The TMB of FGFR4 mutant patients was significantly higher than that of FGFR4 wild-type patients (median TMB: 6.55 vs.4.6,p<0.001) (fig. 1).

FGFR4 variant (MUT) types include 24 point mutations (mutation), 9 amplifications (amp), and 1 Fusion/Rearrangement (Fusion/Rearrangement). The TMB of FGFR4 point-mutant patients was significantly higher than that of FGFR wild-type (median TMB: 12 vs.4.6,p<0.001) and FGFR amplified variant patients (median TMB: 12 vs, 4.6 of the total weight of the steel,p= 0.006) (fig. 2).

The FGFR4 mutation sites were relatively scattered, and there were no distinct hot spot mutation regions (fig. 3).

TABLE 2 correlation between FGFR4 mutation and clinicopathological characteristics in NSCLC patients

Example 3

To further validate the FGFR4 mutation against immune checkpoint inhibitionPredictive value of agent (ICIs) treatment, the inventors performed external validation by downloading public database cohort information. The inventors downloaded the cohort data uploaded by Rizvi et al at the cBioPortal website (http:// www.cbioportal.org /), which included 240 non-small cell Lung Cancer patients receiving anti-PD- (L)1 monotherapy or anti-PD- (L)1+ anti-CTLA-4 combination regimens, and specific patient baseline data were referenced (Liu S-y, Dong Z-y, Wu S-p et al. Clinical release of PD-L1expression and CD8+ T cells injection in tissues with EGFR-mutated and dALK-rearrarged Cancer. Lung Cancer 2018; 125: 86-92.). Of the 240 patients, 6 patients with FGFR4 mutation (2.5%), patients with FGFR4 mutation had longer median PFS after immunotherapy than patients with FGFR4 wild type (median PFS: 13.17 vs. 3.17 months,p= 0.025) (fig. 4), however, the same sample was analyzed, and it could be found that none of the FGFR1/2/3 mutations in the same family as FGFR4 could predict the therapeutic effect of the immunotherapy for non-small cell lung cancer patients, and the survival analysis results of the threepValues were all greater than 0.05 (fig. 5-7), indicating that FGFR4 is specific for the prediction of immunotherapy efficacy. Further analysis of the proportion of patients with FGFR4 mutations who received immunotherapy that continued benefit was significantly higher in patients with FGFR4 wild-type (80% vs. 29.3%,p= 0.03) (fig. 8). The Cox multifactorial analysis results further indicated that FGFR4 gene variation is an independent predictive risk factor for prognosis of immunotherapy (FGFR 4-MUT vsFGFR4-WT, HR: 0.22, 95% CI:0.055-0.92, p = 0.037) (fig. 9).

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

Use of a detector of a FGFR4 point mutation in the manufacture of a kit for predicting the sensitivity of a non-small cell lung cancer patient to an immune checkpoint inhibitor therapy, wherein the presence of the FGFR4 point mutation is indicative of the sensitivity of the non-small cell lung cancer patient to the immune checkpoint inhibitor therapy.
2. The use of claim 1, wherein the kit further comprises a detection agent for TP53 gene mutation and/or KMT2C gene mutation.
Use of a detector of a FGFR4 point mutation in the manufacture of a kit for predicting the degree of tumor mutation burden in a non-small cell lung cancer patient, wherein the presence of a FGFR4 point mutation is indicative of a high tumor mutation burden.
4. The use according to claim 3, wherein the kit further comprises a detection agent for the mutation of TP53 gene and/or the mutation of KMT2C gene.
5. The use of any one of claims 1 to 4, wherein the immune checkpoint inhibitor is a PD1 inhibitor and/or a PD-L1 inhibitor.
6. The use of any one of claims 1 to 4, wherein the detection agent is detected at the nucleic acid level.
7. The use of claim 6, wherein 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.
8. The use of any one of claims 1 to 4, wherein the detection agent is detected at the protein level.
9. The use of claim 8, 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.
10. The use of any one of claims 1 to 4, wherein 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.
11. The use according to claim 10, wherein 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 non-small cell lung cancer patient.