CN112921091B - Use of FLT3 gene mutation in predicting sensitivity of non-small cell lung cancer patient to immune checkpoint inhibitor therapy - Google Patents

Abstract

The invention provides application of FLT3 gene mutation in predicting the sensitivity of non-small cell lung cancer patients to ICI therapy of immune checkpoint inhibitor and predicting the tumor mutation load degree of non-small cell lung cancer patients.

Description

Use of FLT3 gene mutation in predicting 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 FLT3 gene mutation in prediction of ICI therapy sensitivity.

Background

In recent years, a series of breakthrough achievements are achieved in tumor immunotherapy, which becomes a revolutionary therapeutic means beyond surgical treatment, radiotherapy, chemotherapy and targeted therapy, and particularly, a therapeutic scheme based on emerging Immune Checkpoint Inhibitors (ICIs) such as PD-1/PD-L1 and CTLA-4 is widely studied in various solid tumors and has entered first-line treatment of Non-small cell lung cancer (NSCLC). 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 immune therapy benefit groups are key problems to be solved urgently in the development of future tumor immune therapy.

Currently, various biomarkers of immunotherapy response are being explored (Biomarker). 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 non-small cell lung cancer, expression of PD-L1 can be used as a predictive marker for anti-PD-1/PD-L1 therapeutic response. The KEYNOTE-024 research result shows that NSCLC patients with high expression of PD-L1 benefit most from the treatment of the palivizumab, particularly late-stage patients with negative driving genes with expression of PD-L1 being more than or equal to 50 percent have better first-line treatment effect than chemotherapy. The KEYNOTE-042 study showed that the Pabollizumab could significantly improve the Overall Survival (OS) of NSCLC patients with PD-L1 expression more than or equal to 1%. 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. Moreover, the detection criteria of PD-L1 remain controversial, and PD-L1 changes in different anatomies of tumors and as treatment progresses.

Tumor Mutational Burden (TMB), which refers to the total number of somatic mutations per million bases in a specific region of the tumor genome, has been shown to correlate with therapeutic efficacy of immune checkpoint inhibitors for a variety of tumor types, including melanoma, non-small cell lung cancer and bladder cancer. In 2019, the NCCN guidelines listed TMB as the recommended detection method for metastatic non-small cell lung cancer receiving immunotherapy. Based on the KEYNOTE-158 trial, in 6 months of 2020, FDA accelerated approval of Pabollizumab for the monotherapy of patients with unresectable or metastatic solid tumors with high tumor mutation burden (TMB-H) and disease progression after previous treatment, regardless of cancer type. Nevertheless, the use of TMB as a predictor still faces some problems. First, the CheckMate-227 and CheckMate-528 studies suggest that patients with high TMB benefit from immunotherapy, but the results of exploratory analysis of the KEYNOTE series of studies show that, regardless of whether TMB is high or low, Pabollizumab in combination with chemotherapy shows survival benefit in first line therapy in patients with squamous and non-squamous NSCLC. Secondly, the detection of TMB has no standard method due to different detection products and different algorithms of different detection organizations in China. Furthermore, there is currently a question of what TMB threshold can distinguish valid or invalid patients.

DNA Mismatch repair deficiency (dMMR) or Microsatellite instability (MSI-H) also show good predictive effects in digestive system tumors such as colorectal and gastric cancers. The FDA is used for treating MSI-H or dMMR advanced stage treated solid tumor patients through anti-tumor drug Pabollizumab trans-tumor indication according to the accelerated approval of molecular markers for the first time. However, the ratio of MSI-H in the tumor is too low, the clinical popularization has certain limitation, and particularly, the prediction value of the dMMR/MSI-H on the curative effect of the lung cancer immunotherapy needs more research and data to verify. The instability of PD-L1 expression, TMB and MSI is an important index for predicting the curative effect of the immunity, and many potential immune therapy benefit groups can be missed by using any biomarker singly. In the clinical research of other novel biomarkers for predicting the curative effect of immunotherapy, researchers find that a T cell inflammation Gene Expression Profile (GEP) comprising IFN-gamma response genes related to antigen presentation, chemokine expression, cytotoxicity activity and adaptive immunity can predict the clinical effect of the PD-1 inhibitor by analyzing a tumor tissue specific Gene expression profile of a patient treated by the Pabolizumab, and verify the clinical effect in melanoma, lung cancer, head and neck squamous cell carcinoma and the like. In addition, in future research, correlation research between biomarkers and combined use thereof are also main directions of future tumor immunotherapy, and the combination of multiple biomarkers may realize more accurate treatment and bring more clinical benefits to patients.

With the development of the second-generation sequencing in the precise treatment of tumors, more and more researches show that the somatic mutation of a specific gene can influence the tumor immune function or the response to immunotherapy, namely, the specific somatic mutation can be a potential immunotherapy predictor. The POLE gene coding DNA polymerase is involved in DNA nucleotide and base excision repair pathways, the present research has proved the POLE gene mutation as the biological marker of the survival benefit of multiple cancer immunotherapy, the researchers find that the POLE/POLD1 mutant patients have a median OS significantly better than that of non-carriers (34 months vs.18 months) by analyzing MSK-IMPACT patients receiving immunotherapy solid tumor. 26% of patients with mutation in the POLE/POLD1 gene incorporated MSI-H (microsatellite highly unstable), and the mutant group OS still benefited after removal of this portion of patients (28 months vs.16 months). Even though it is generally considered that a Microsatellite stabilized MSS (MSS) patient cannot benefit from immunotherapy, whether or not it can benefit from immunotherapy can be judged by the POLE/POLD1 gene mutation. Finally, multifactorial analysis demonstrated that the POLE/POLD1 mutation could be a completely new independent indicator for predicting the benefit of immunotherapy. In addition, related researches also show that gene mutations such as ARID1, ALRP1B, MUC16, PRKDC, NFE2L2 and the like are forward prediction factors with better curative effect on ICI immunotherapy; whereas EGFR, ALK, CDKN2A, KEAP1, and other genetic mutations are associated with poor prognosis for ICI immunotherapy. However, these above gene mutations as biomarkers still do not cover all potential immunotherapeutic benefit populations and there remains a need in the art for more efficient and accurate methods and tools for identifying suitable immune checkpoint inhibitors for treating lung cancer patients.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to find a method for effectively predicting the sensitivity of NSCLC patients to immune checkpoint inhibitor therapy, and the following technical scheme is specially proposed in the application for achieving the aim:

the invention provides an application of a detection agent of FLT3 gene mutation in preparation of a kit for predicting or screening the sensitivity of NSCLC patients to immune checkpoint inhibitor therapy; preferably, the presence of the FLT3 mutation is indicative of the NSCLC patient being susceptible to immune checkpoint inhibitor therapy.

The invention also provides application of the FLT3 mutation detection agent in preparing a kit for predicting or screening the tumor mutation load degree of NSCLC patients; preferably, the presence of the FLT3 mutation is indicative of a high tumor mutation load.

In some aspects, the kit further comprises a detection agent for other gene mutations.

In some aspects, the immune checkpoint inhibitor is a PD-1 inhibitor and/or a PD-L1 inhibitor.

In some aspects, 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.

In some aspects, the detection agent detects 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.

In some aspects, 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.

In some aspects, the kit further comprises sample treatment reagents comprising at least one of sample lysis reagents, sample purification reagents, and sample nucleic acid extraction reagents.

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

The present invention also provides a method of predicting or screening a NSCLC patient for susceptibility to immune checkpoint inhibitor therapy, comprising detecting the presence or absence of a mutation in FLT3 gene using a detection agent.

The invention also provides a kit for predicting or screening the sensitivity of NSCLC patients to immune checkpoint inhibitor therapy, which is characterized by comprising reagents for detecting FLT3 mutation; preferably, other reagents for detecting mutations in genes including, but not limited to: one or more of FGFR4, POLE, POLD1, ARID1A, ARID1B, FGFR4, MUC16, or NOTCH 1-4.

A kit for predicting, assessing or screening the degree of tumor mutation burden in a non-small cell lung cancer patient, comprising reagents for the detection of FLT3 mutation; preferably, other reagents for detecting mutations in genes including, but not limited to: one or more of FGFR4, POLE, POLD1, ARID1A, ARID1B, FGFR4, MUC16, or NOTCH 1-4.

The beneficial technological effect of this application:

1. immune checkpoint inhibitor treatment of patients uses only PD-L1 expression, TMB, etc. as biomarkers suitable for treatment. However, not all patients with high PD-L1 expression or TMB-H responded to immunotherapy, the present invention screens FLT3 gene mutations as biomarkers to predict populations susceptible to ICI in NSCLC patients; according to the invention, through FLT3 gene mutation, the TMB level 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.

2. The FLT3 gene mutation adopted in the invention can be used as a risk prediction factor in practical application, so that the detection efficiency is improved, and the result is more reliable.

3. The method of the invention is beneficial to simplifying the detection content, reducing the detection cost of the patient, accelerating the detection report issuing time and being suitable for popularization and application.

Drawings

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

FIG. 1 is a comparison of Tumor Mutation Burden (TMB) in patients with FLT3 gene Mutation (MT) and wild-type patients (WT) according to one embodiment of the present invention;

FIG. 2 is a graph showing the comparison of the expression of PD-L1 in a patient (MT) with a mutant FLT3 gene and in a wild-type patient (WT) in accordance with one embodiment of the present invention;

FIG. 3 is a diagram showing the analysis of mutation sites of FLT3 gene in one embodiment of the present invention;

FIG. 4 is a graph comparing the efficacy of NSCLC FLT3 gene mutant patients (MT) and wild-type patients (WT) receiving immunotherapy with immune checkpoint inhibitors in one embodiment of the present invention;

FIG. 5 is a graph showing the comparison of the proportion of persons who continue to benefit from an immune checkpoint inhibitor in patients with a mutant FLT3 gene (MUT) versus wild type patients (WT) in one embodiment of the present invention;

FIG. 6 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 present invention.

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 reagents or instruments used are not indicated by manufacturers, and are all conventional products available on the market.

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 present invention relates to the use of a detection agent for a mutation in the FLT3 gene in the manufacture of a kit for predicting or screening a non-small cell lung cancer patient for sensitivity to an immune checkpoint inhibitor therapy, preferably, the presence of a mutation in the FLT3 gene is indicative of said non-small cell lung cancer patient being sensitive to an immune checkpoint inhibitor therapy.

The invention also relates to the application of the detection agent of FLT3 gene mutation in the preparation of a kit for predicting or screening the degree of tumor mutation load of a patient with non-small cell lung cancer, wherein the existence of FLT3 gene mutation is an indicator of high tumor mutation load.

FLT3(Fms-like tyrosine kinase 3) of the present invention belongs to the class III Receptor tyrosine kinase (RTK III) family members, and in recent years, many large sample studies have demonstrated that the activating mutation of FLT3 plays a very important pathological role in the development of AML and the progression of disease. The gene mutation of FLT3 can cause FLT3 to generate autophosphorylation, further cause FLT3 to generate ligand-independent constitutive activation, and further activate downstream abnormal signal transduction, thereby playing roles in promoting proliferation and inhibiting apoptosis, and leading the clinical prognosis of leukemia patients with the mutant phenotype to be poor.

In some embodiments, the FLT3 gene species is a mammal; preferably, it is a primate. In some preferred embodiments, the FLT3 gene species is human; gene ID:2322 NM-004119.3.

It will be appreciated that the present invention provides a novel marker for predicting the sensitivity of non-small cell lung cancer patients to immune checkpoint inhibitor therapy: mutation of FLT3 gene. 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 gene mutation. And the prognosis of patients with FLT3 mutation after immunotherapy is obviously better than that of FLT3 wild-type patients.

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 newly discovered immune checkpoints such as lymphocyte activation gene 3(LAG3), 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 (A2aR), 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 (JS001), Cedilizumab (IBI308), Carrilizumab (Eleka) and Terrilizumab (Baizelan). The PD-L1 inhibitor may further be selected from one or more of Atezolizumab (Tecnriq; MPDL3280A), JS003, Durvalumab (Imfinzi), Avelumab (Bavencio), BMS-936559, MEDI4736 and MSB 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, 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).

Evaluating FLT3 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 nucleotides 67-3048 of the FLT3 gene. In some preferred embodiments, assessing FLT3 gene mutations comprises determining whether there is a mutation in its coding region that truncates the FLT3 protein.

In some embodiments, FLT3 gene expression, e.g., protein expression level of FLT3 gene, is assessed following determination of the presence of a mutation in the coding region of FLT3 gene that truncates the FLT3 protein.

In some embodiments, the pathological types of the non-small cell lung cancer 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: one or more of FGFR4, POLE, POLD1, ARID1A, ARID1B, FGFR4, MUC16, or NOTCH 1-4. .

Since FLT3 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, those skilled in the art can detect the mutation from the RNA and protein level to indirectly reflect whether 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/454FLX, 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 non-small cell lung cancer patient.

In some embodiments, the tissue is non-small cell lung cancer tissue or tissue adjacent to cancer. Wherein, the preferable detection samples are blood, serum and plasma; more preferably, from peripheral blood.

According to yet another aspect of the present invention, there is also provided a method for predicting or screening non-small cell lung cancer patient sensitivity to immune checkpoint inhibitor therapy, the method comprising: the presence or absence of a mutation in FLT3 gene was detected using the detection agent described above. In some embodiments, the methods are used for prognostic evaluation of non-small cell lung cancer patients following immune checkpoint inhibitor therapy.

According to a further aspect of the invention, the invention also provides a kit for predicting, assessing or screening non-small cell lung cancer patient susceptibility to immune checkpoint inhibitor therapy, comprising reagents for mutation detection of FLT3 gene; 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.

According to still another aspect of the present invention, the present invention also provides a kit for predicting, evaluating or screening the degree of tumor mutation load of a patient with non-small cell lung cancer, wherein the kit comprises reagents for detecting mutation of FLT3 gene; 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

The present invention was studied using the following methodology in particular

Sample material: test samples FFPE tumor samples from chinese non-small cell lung cancer patients and paired peripheral whole blood control samples (all patients provided written informed consent). Studies have been performed by targeted capture NGS sequencing analysis, specifically involving a combination of 551 cancer-associated genes.

The test method comprises the following steps:

1) the invention uses a finished product commercial kit to extract the DNA of the FFPE slices and the whole blood samples with the tumor cell ratio of more than 20 percent, and the extracted nucleic acid enters the library construction after being qualified by the quantit quantification and the agent 2100 analysis.

Specifically, the method comprises the following steps: the library construction of the invention uses a probe hybridization capture method, the library construction and the hybridization capture reagent are commercialized reagents, and the probe is customized. Extracting nucleic acid with qualified quality control, breaking the nucleic acid to about 200-300 bp by an enzyme cutting method, then carrying out terminal repair and joint connection, purifying joint connection products by using AMPure Beads, carrying out PCR amplification on the purified products to construct a pre-library for hybridization, hybridizing the pre-library with the qualified quality control by using a customized probe, and capturing target fragments to form a final library. The final library is first quantified by using the qubit4.0, and then the insert size of the library is detected by using Agilent 4200TapeStation, and the concentration and the fragment of the library meet expectations and then the on-machine sequencing is carried out according to the requirements.

After the library was qualified, the different libraries were posing according to the requirements of the target off-machine data volume and then sequenced using Illumina Novaseq 6000 for PE150 bp. Adding four kinds of fluorescence-labeled dNTPs, DNA polymerase and a joint primer into a sequenced flow cell for amplification, releasing corresponding fluorescence every time one fluorescently-labeled dNTP is added when each sequencing cluster extends a complementary chain, and acquiring sequence information of a fragment to be detected by a sequencer through capturing a fluorescence signal and converting an optical signal into a sequencing peak through computer software.

The invention aims to sequence 551 genes, and the detection interval exceeds 2.25 Mbp. Of these 530 genes covered the entire exon (coding region 1.41 Mb). The kit can detect SNV, InDel, CNV, Fusion, MSI and TMB, and can provide relevant detection results for patients such as targeting, chemotherapy, immunotherapy, genetic risk and the like.

2) Genome alteration analysis

The present invention detects the content of genomic alterations, including single base Substitutions (SNV), short fragment insertion deletions (Indel), gene Copy Number Variations (CNV), and gene rearrangements and fusions. The original sequenced sequence was aligned to the human genome reference sequence (hg19) using bwa-mem (version 0.7.17). And (3) taking paired leukocyte DNA of the sequencing sample as a control, and removing the embryonic line variation to obtain the sample somatic variation.

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

for point mutations and short fragment indels: the effective sequencing coverage depth of the position of the point mutation is more than 200; each sequenced sequence comprising the mutation has a quality value of >40, and each base supporting the mutation on the sequence has a base quality value of > 20; the point mutation is judged as somatic mutation by Fisher's exact test; the number of all sequences supporting the mutation is more than or equal to 6; the average value of the shortest distance between each base distance supporting the mutation and the tail segment of the sequence fragment is less than or equal to 8; this mutation can be significantly distinguished from the background mutation at that site in the background set of mutations constructed from healthy human specimens by Fisher's exact test or Z test.

TMB calculation: in the invention, TMB is defined as the number of SNV and Indel somatic mutations contained in each million bases in the range of a detected coding region, wherein 1) the mutation is synonymous, 2) AF is less than 2 percent, 3) the frequency recorded by a Cosmic public database is more than 100,4) NMPA/FDA clearly approves a medication target point, and the medication site is recommended by NCCN guidelines; the above 4 classes of sites were not included in the TMB calculation

Immunohistochemistry: detection of PD-L1 expression was performed using immunohistochemical methods. And (3) performing immunohistochemical detection on wax blocks or sections with more than 100 tumor cells observed under a mirror, performing pretreatment on the white sections by baking and cleaning, then dyeing the sections by using Dako Link 48 (monoclonal antibodies 22C3 and 28-8) stainers respectively, and after dyeing, cleaning and sealing the sections, and observing the result under the mirror.

Acquiring public database queue data: in order to further verify the clinical prediction effect of FLT3 gene mutation on immune checkpoint inhibitor treatment and the potential mechanism of influencing immune treatment, the invention downloads the number of non-small cell lung cancer patients in the receiving immune treatment queue of MSKCC 350 cases in a tumor genomics database cBioPortal website (http:// www.cbioportal.org /), including clinical baseline data of patients, curative effect evaluation data of immune checkpoint inhibitor treatment and patient genome data.

Example 1 characterization of non-Small cell Lung cancer patients

The present invention included a total of 2300 non-small cell lung cancer patients into the study. The characteristics of the patients are shown in table 1. The median age at diagnosis was 63 years. 2066 patients had clear pathological type information, and 1816 (79.0%) and 250 (10.9%) patients had lung adenocarcinoma and lung squamous carcinoma, respectively. TMB testing was performed on 2300 patients. The median TMB of the whole population was 2.94mut/Mb (IQR, 0.74-5.88). Tumors with TMB of more than or equal to 10muts/Mb account for 12.3%. This example found a positive correlation of age with TMB (p < 0.001). In addition, the TMB values were higher in the patient group of squamous cell lung carcinoma (p < 0.001).

TABLE 1 non-Small cell Lung cancer patient characteristics

Example 2 frequency of occurrence of FLT3 Gene mutations in the Chinese NSCLC population and correlation with immunotherapeutic biomarkers PD-L1, TMB

When the statistical analysis of FLT3 gene mutation of 2300 non-small cell lung cancer patients shows that 45 patients in 2300 non-small cell lung cancer patients carry FLT3 gene mutation, which accounts for 1.96%. The FLT3 mutation was not significantly different in age from the wild type two groups of patients (table 2). The TMB of FLT3 mutant patients was significantly higher than that of FLT3 wild-type patients (median TMB: 10.3vs.2.84mut/Mb, p ═ 0.001 (fig. 1).) among 2300 NSCLC patients, 927 patients had tumor tissues tested by PD-L1 immunohistochemistry, with PD-L1 negative (PD-L1TPS score ≦ 1% 20.8% in patients, PD-L1 weakly positive (PD-L1TPS score ≦ 1% -25%) and strongly positive PD-L1 expression (PD-L1TPS score ≧ 25%) 14.2% and 5.26%, respectively, FLT3 mutant patients had PD-L1 positive expression rate (PD-L1TPS ≧ 1%) that was not significantly correlated with FLT3 wild-type patients (30.3% vs.41.1%, p ═ 0.1905) (fig. 2).

TABLE 2 correlation between FLT3 mutation and clinical pathological characteristics in NSCLC patients

Example 3 analysis of mutation site of FLT3 Gene

Further analysis of mutation sites of FLT3 gene revealed that the FLT3 gene variant forms were mainly missense mutation and nonsense mutation, and the main sites were Y166, E204 and S883; the site of frameshift mutation is R849Vfs 4. However, the gene variation sites were relatively scattered and no hot spot mutation region was evident overall (FIG. 3).

Example 4 validation of clinical data on predictive value of FLT3 mutation for ICI treatment

External validation is performed by downloading the public database queue information. The 1662 pan cancer patient cohort data uploaded by Rizvi et al was downloaded at the cBioPortal website (http:// www.cbioportal.org /), and the Rizvi cohort included 350 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 (Samstein RM, Lee CH, Shoushtari AN, et al. Tumor Multiparticulate load prediction summary cancer multiple cancer types. nat Genet 2019; 51: 202-6.). In the cohort of non-small cell lung cancers receiving immunotherapy, 9 patients with FLT3 mutations (2.8%) had longer median PFS after immunotherapy than FLT3 wild-type patients (median OS: not reached vs.10.2 months, P ═ 0.034) (fig. 4). Further analysis of the sustained benefit population (sustained benefit over 12 months) of FLT3 mutant patients receiving immunotherapy was higher than that of FLT3 wild-type patients (77.8% vs. 44.2%, p ═ 0.04286) (fig. 5), which indicates that FLT3 mutation can predict the therapeutic efficacy of immunotherapy in non-small cell lung cancer patients.

The Cox multifactorial analysis results further indicated that FLT3 gene mutation is an independent predictive risk factor for prognosis of immunotherapy (FLT3 MT vs. FLT3 WT, HR: 024, 95% CI:0.06-0.96, p ═ 0.0432) (fig. 6).

Thus, the mutation of FLT3 gene can be used as a prediction factor for the degree of tumor mutation load of non-small cell lung cancer patients and the potential prediction factor of the non-small cell lung cancer patients on immune checkpoint inhibitor therapy.

Finally, 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; although the present application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions 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.

Claims (4)

1. Use of a FLT3 mutation detection agent in the preparation of a kit for predicting or screening susceptibility of chinese non-small cell lung cancer patients to immune checkpoint inhibitor therapy; the independent presence of the FLT3 mutation is indicative that the non-small cell lung cancer patient is susceptible to immune checkpoint inhibitor therapy; the mutation is a point mutation; the immune checkpoint inhibitor is a PD-1 inhibitor and/or a PD-L1 inhibitor.
2. The application of the FLT3 mutation detection agent in preparing a kit for predicting or screening the tumor mutation load degree TMB of a Chinese non-small cell lung cancer patient; the independent presence of the FLT3 mutation is indicative of a high tumor mutation burden; the mutation is a point mutation.
3. 3. The use of any one of claims 1-2, 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, nucleic acid sequencing, denaturing gradient gel electrophoresis, nucleic acid typing chip detection, denaturing high performance liquid chromatography, in situ hybridization, biological mass spectrometry and HRM method.
4. 4. The use of any one of claims 1-2, wherein the detection agent is detected at the protein level; the detection agent is used for executing any one of the following methods: biological mass spectrometry, amino acid sequencing, electrophoresis, and detection using antibodies specifically designed for the mutation site.

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