KR101864331B1 - Predicting kit for survival of lung cancer patients and the method of providing the information for predicting survival of lung cancer patients - Google Patents

Abstract

The present invention relates to a kit for predicting the survival time of a lung cancer patient using a combined epithelial growth factor receptor (EGFR) mutation and a method for providing information for predicting the survival period.
The present invention enables relatively accurate and easy detection of EGFR complex mutations deeply associated with the survival period of lung cancer patients. Accordingly, the present invention more accurately predicts the prognosis of lung cancer patients, which is the main cause of cancer death, and enables therapeutic contrast accordingly.

Description

Predicting kit for survival of lung cancer patients and the method of providing the information for predicting survival of lung cancer patients

The present invention relates to a kit for predicting the survival period of lung cancer patients and a method of providing information for predicting the survival period, and more particularly, to a lung cancer patient using a complex mutation of the epidermal growth factor receptor (EGFR). It relates to a kit for predicting survival time and a method of providing information for predicting survival time.

Lung cancer is one of the leading causes of cancer deaths, accounting for 30% of cancer deaths worldwide. According to the WHO year-end report, as of 2012, there were 1,824,701 new cases of lung cancer worldwide, of which approximately 1,590,000 patients were reported to have died from the disease. During the same period, 21,753 new lung cancer patients occurred in Korea, and 16,654 died. Lung cancer is a terrifying, multi-frequency disease that ranks first in the domestic and international cancer mortality rate (Jung KW et al., Prediction of cancer incidence and mortality in Korea, 2014. Cancer Res Treat 2014; 46: 124-130).

Cancer-causing mutations represent various types of genetic changes, which can act as an important factor in maintaining the development and progression of cancer. Selecting an appropriate targeted therapeutic agent to identify activated cancer-causing mutations can improve the medical outcome of many lung cancer patients. The application of targeted therapeutics based on cancer-causing mutations has made tumor genotyping assays available for treatment decision making (Kris MG et al., Using multiplexed assays of oncogenic drivers in lung cancers to select targeted drugs. Jama 2014; 311: 1998 -2006).

On the other hand, lung cancer is a type of epithelial cell cancer characterized by abnormally accelerated growth of epithelial cells, and epidermal growth factor receptors (hereinafter "EGFR") expressed on the surface of epithelial cells are present. It is known to play an important role in the process of cancer. Therefore, EGFR has been recognized as a target for lung cancer treatment, and a representative example of cancer treatment targeting EGFR is the development of an EGFR tyrosine kinase inhibitor (EGFR-TKI).

The EGFR mutation is more frequently found in lung adenocarcinoma patients in East Asia than in other regions, and advances in tumor genotyping analysis have made it possible to confirm the EGFR mutation in a small amount of samples.

Among the EGFR mutations, exon 19 deletions around the LREA motif (amino acid residues 747 to 750) account for 45% of the EGFR mutations, followed by the L858R point mutation of exon 21 accounts for 40% (Shigematsu H et al. ., Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers.J Natl Cancer Inst 2005; 97: 339-346; Sequist LV et al, Molecular predictors of response to epidermal growth factor receptor antagonists in non-small- cell lung cancer.J Clin Oncol 2007; 25: 587-595; Tokumo M et al., The relationship between epidermal growth factor receptor mutations and clinicopathologic features in non-small cell lung cancers.Clin Cancer Res 2005; 11: 1167-1173 .). In addition, as EGFR protein mutations, exon 18 point mutations at the G719 position account for 3%, exon 21 L861Q mutations account for 2%, and these activated mutations are EGFR-tyrosine kinase inhibitors (TKIs). (Mitsudomi T et al., Epidermal growth factor receptor in relation to tumor development: EGFR gene and cancer. Febs j 2010; 277: 301-308; Mitsudomi T et al., Mutations of the epidermal growth factor receptor) gene and related genes as determinants of epidermal growth factor receptor tyrosine kinase inhibitors sensitivity in lung cancer.Cancer Sci 2007; 98: 1817-1824; Yeh P et al., DNA-Mutation Inventory to Refine and Enhance Cancer Treatment (DIRECT): a catalog of clinically relevant cancer mutations to enable genome-directed anticancer therapy.Clin Cancer Res 2013; 19: 1894-1901).

On the other hand, the in-frame insertion mutation of EGFR exon 20, which accounts for 4-10% of the EGFR protein mutations, and other rare mutations, e.g., L747S, D761Y, T790M, T854A, have resistance to EGFR-TKIs ( Mitsudomi T et al., Mutations of the epidermal growth factor receptor gene and related genes as determinants of epidermal growth factor receptor tyrosine kinase inhibitors sensitivity in lung cancer.Cancer Sci 2007; 98: 1817-1824; Pao W et al., Rational, biologically based treatment of EGFR-mutant non-small-cell lung cancer.Nat Rev Cancer 2010; 10: 760-774; Yasuda H et al., EGFR exon 20 insertion mutations in non-small-cell lung cancer: preclinical data and clinical implications.Lancet Oncol 2012; 13: e23-31; Yasuda H et al., Structural, biochemical, and clinical characterization of epidermal growth factor receptor (EGFR) exon 20 insertion mutations in lung cancer.Sci Transl Med 2013; 5: 216ra177. ).

For the medical application of a more precise genotyping system, the biological and medical importance of rare EGFR mutations is increasing. Even if there is a mutation at the residue of the approximate position of genomic DNA, each may exhibit different reactivity to EGFR-TKI. For example, in the case of in-frame insertion of EGFR exon 20, it is considered an EGFR-TKI resistance mutant due to a low response rate (<5%) to EGFR-TKI and a short interval for disease control, but A763_Y746 ins FQEA is currently It has been reported as a sensitive mutation for EGFR-TKI. Therefore, there is a need for continuous research on the biological and medical significance of these mutations.

An object of the present invention is to provide a kit for predicting the survival time of lung cancer patients using the EGFR complex mutation.

Another object of the present invention is to provide a method of providing information for predicting the survival time of lung cancer patients using EGFR complex mutations.

In general, simple mutations are commonly known as EGFR (Epidermal Growth Factor Receptor) mutations related to lung cancer, but some multiple mutations are unknown, and furthermore, the biological and medical Nothing has been revealed about the meaning.

The inventors of the present invention came to the present invention by discovering a complex mutation related to the survival time of lung cancer patients among EGFR mutations.

According to an embodiment of the present invention, it includes a probe or a set of primers capable of hybridizing with a gene encoding an epidermal growth factor receptor (EGFR) mutation,

The mutations are two or more complex mutations selected from the group consisting of L858R mutation, G719A mutation, G719S mutation, exon 19 deletion, V689L mutation, L833V mutation, H870R mutation, A871G mutation, R776H mutation, I706T mutation, E709K mutation, and R776H mutation. It provides a kit for predicting the survival period of a lung cancer patient, characterized in that.

According to another embodiment of the present invention, it includes a protein containing an epidermal growth factor receptor (EGFR) mutation, or an antibody capable of binding thereto,

The mutations are two or more complex mutations selected from the group consisting of L858R mutation, G719A mutation, G719S mutation, exon 19 deletion, V689L mutation, L833V mutation, H870R mutation, A871G mutation, R776H mutation, I706T mutation, E709K mutation, and R776H mutation. It provides a kit for predicting the survival period of a lung cancer patient, characterized in that.

In the above embodiment, the protein containing the EGFR mutation may provide a kit for predicting the survival period of a lung cancer patient, characterized in that it specifically binds to an antibody in the serum of a lung cancer patient, or the antibody is a lung cancer patient It is possible to provide a kit for predicting the survival time of lung cancer patients, characterized in that it specifically binds to the EGFR mutation of.

In addition, the gene encoding EGFR may have a nucleotide sequence represented by SEQ ID NO: 1, and the EGFR may have an amino acid sequence represented by SEQ ID NO: 2.

Preferably, the complex mutation is an EGFR-TKI sensitive mutation, for example, at least one mutation selected from the group consisting of L858R mutation, G719A mutation, G719S mutation and exon 19 deletion,

With atypical mutations of exon 18, the V689L mutation, the I706T mutation and the E709K mutation; With an atypical mutation of exon 20, the R776H mutation; And atypical mutations of exon 21, which may include one or more mutations selected from the group consisting of L833V mutation, H870R mutation, and A871G mutation.

In addition, preferably, the complex mutation may include an L858R mutation and an exon 19 deletion.

According to another embodiment of the present invention, comprising the step of detecting an EGFR (Epidermal Growth Factor Receptor) mutation or a gene encoding the same,

The mutations are two or more complex mutations selected from the group consisting of L858R mutation, G719A mutation, G719S mutation, exon 19 deletion, V689L mutation, L833V mutation, H870R mutation, A871G mutation, R776H mutation, I706T mutation, E709K mutation, and R776H mutation. It provides a method of providing information for predicting the survival period of a lung cancer patient, characterized in that.

Here, the gene encoding the EGFR may have a nucleotide sequence represented by SEQ ID NO: 1.

In addition, the EGFR may have an amino acid sequence represented by SEQ ID NO: 2.

Preferably, the complex mutation is an EGFR-TKI sensitive mutation, for example, at least one mutation selected from the group consisting of L858R mutation, G719A mutation, G719S mutation and exon 19 deletion,

With atypical mutations of exon 18, the V689L mutation, the I706T mutation and the E709K mutation; With an atypical mutation of exon 20, the R776H mutation; And atypical mutations of exon 21, which may include one or more mutations selected from the group consisting of L833V mutation, H870R mutation, and A871G mutation.

In addition, preferably, the complex mutation may include an L858R mutation and an exon 19 deletion.

However, in the present invention, the detection of the complex mutation is performed by a next-generation sequencing method, so that it is possible to more easily and accurately detect the EGFR complex mutation related to the survival period of a lung cancer patient.

In one embodiment of the present invention, "multiple mutation" refers to a combination of two or more independently isolated mutations in the EGFR tyrosine kinase domain (EGFR-TKD).

In one embodiment of the present invention, "EGFR-TKI sensitizing mutation" refers to a mutation having a high response rate of approximately 62-82% to EGFR tyrosine kinase inhibitor (EGFR-TKI). will be.

In one embodiment of the present invention, the "prognosis" includes overall survival in the patient, disease-free survival, progression-free survival, event-free survival, prediction of the likelihood of recurrence of cancer, and the likelihood of metastasis of the tumor. It is not intended to mean the prediction of the likelihood of recovery of the disease or the onset or outcome of the disease.

In one embodiment of the present invention, "Disease-free survival" is a term that is well known to those skilled in the art, and refers to survival without a disease to be monitored.

In one embodiment of the present invention, the "primer" can serve as an initiation point for template-directed DNA synthesis under suitable conditions (ie, four different nucleoside triphosphates and polymerases) in a suitable buffer at a suitable temperature. Refers to a single-stranded oligonucleotide that is present. The suitable length of the primer varies depending on various factors such as temperature and the application of the primer, but is typically 15-30 nucleotides. Short primer molecules generally require lower temperatures to form sufficiently stable hybrid complexes with the template.

The sequence of the primer does not need to have a sequence that is completely complementary to some of the sequences of the template, and it is sufficient to have sufficient complementarity within a range capable of hybridizing with the template to perform a unique function of the primer. Therefore, the primer in the present invention does not need to have a sequence that is completely complementary to the above-described nucleotide sequence as a template, and it is sufficient if it has sufficient complementarity within a range capable of hybridizing to this gene sequence to function as a primer. The design of such a primer can be easily carried out by a person skilled in the art by referring to the nucleotide sequence described above, for example, it can be done using a primer design program (eg, PRIMER 3 program).

In one embodiment of the present invention, the "probe" refers to a linear oligomer of natural or modified monomers or linkages, includes deoxyribonucleotides and ribonucleotides, and can specifically hybridize to a target nucleotide sequence, , Either naturally occurring or artificially synthesized. The probe of the present invention is preferably single-chain and is an oligodeoxyribonucleotide.

The measurement of the change in the expression level of the gene can be carried out through various methods known in the art. For example, RT-PCR (Sambrook et al., Molecular Cloning.A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)), Northern Blotting (Peter B. Kaufma et al., Molecular and Cellular Methods in Biology and Medicine) , 102-108, CRC press), hybridization reaction using cDNA microarray (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)) or in situ hybridization reaction (Sambrook et al. , Molecular Cloning.A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)).

When performing according to the RT-PCR protocol, first, total RNA is isolated from the cells treated with the sample, and then single-stranded cDNA is prepared using oligo dT primers and reverse transcriptase. Then, a single-stranded cDNA is used as a template, and a PCR reaction is performed using a gene-specific primer set. Gene-specific primer sets are listed in Table 2 below. Then, the PCR amplification product is subjected to electrophoresis, and the formed band is analyzed to measure the change in the expression level of the gene.

In one embodiment of the present invention, the "next-generation sequencing method" means a new concept of nucleotide sequence capable of reading a large amount of nucleotide sequence and generating a large amount of nucleotide sequence data for a sample to be analyzed within a short time. Examples of the analysis technology include sequencing techniques using equipment such as Roche/454, Illumina/Solexa, and SOLiD (Michael L. Metzker, Aapplications of next-generation sequencing; Sequencing technologies the) next generation, Nature Reviews Genetics, Vol. 11, pp 31-46, January 2010). The next-generation sequencing process can be divided into the following three steps.

(1) capture of exome

In order to find a disease-causing gene, the whole-genome can be sequenced using a next-generation sequencing method, or it can be sequenced by targeting only the exome region (Targeted sequencing). When sequencing only exome regions, it is advantageous in terms of cost and efficiency. In addition, since changes in genes often appear as direct diseases such as cancer, it can be said that viewing the change in the base sequence in the exome region is effective in finding the causative gene. In order to sequence only exomes, a library capable of capturing only exomes is required. SureSelect Human All Exon Kits (http://www.genomics.agilent.com) are the most used, but are not limited thereto. SureSelect Human All Exon Kits are designed based on exons (gene set of the human genome defined by the participation of Consensus CDS, NCBI, EBI, UCSC, Wellcome Trust Sanger Institute) exons, and include a region corresponding to 1.22% of the human genome. have.

(2) Massive parallel DNA sequencing

Next Generation Sequencing (NGS) is faster than the existing capillary sequencing and can perform a larger amount of sequencing at once, and uses the vector used in the existing capillary sequencing method. Since the amplification process of the sample is omitted, there is an advantage of avoiding experimental errors occurring in this process. Currently, NGS systems manufactured by three companies are mainly used. Roche's 454 GS FLX, launched in 2004, is the first NGS device to be introduced. This device performs sequence verification using a pyrosequencing method and an emulsifier-polymerase chain reaction. And, according to the intensity of light emitted in the final stage of the experiment, a specific base can be identified. When operated for 7 hours, a sequence of about 100 Mb can be confirmed, and it shows a significantly higher performance than the existing ABI 3730 device capable of confirming a sequence of 440 kb at the same time. Illumina's Illimina Genome Analyzer introduces the concept of sequencing by synthesis. After attaching DNA fragments consisting of only one strand on a glass plate, the fragments are polymerized and clustered. To achieve. When going through this process, the sequence confirmation method is performed while confirming the type of base attached to the DNA fragment to be tested. In about 4 days, 40 to 50 million fragments with a length of 32-40 bases are produced. Life Technologies' SOLiD (Sequencing by Oligo Ligation) device attaches a piece of DNA to be tested to a magnetic bead of 1 μm in size, and then performs sequence confirmation using an emulsifier-polymerase chain reaction. When confirming the sequence, 8-mer fragments are repeatedly attached. At the 4th and 5th positions of this 8-mer, the bases to be used for actual sequence identification are located. A fluorescent substance is connected to the remaining part attached to it, indicating which base complementarily binds to the DNA fragment to be tested. If all 8-mers are attached 5 times per binding cycle and the same operation is performed 5 times, the sequence of a DNA fragment consisting of a total of 25 bases can be confirmed. The feature of the SOLiD device is sequence identification using two-base encoding. This method identifies the same site through two sequence identification when determining the sequence of one base. Sequence confirmation is performed by shifting the sequence by one base for each binding cycle toward the adhesive attached to the magnetic beads (adaptor). This process has the advantage of eliminating errors that occur in sequence confirmation experiments.

(3) Analysis of nucleotide sequence data

In order to find the cause of the disease, it is necessary to investigate what changes have occurred from the existing gene sequence, so the work of comparing the sequence reads of the individual (patient) with the reference genome is performed. This task is called mapping. After finding the difference between an individual and a standard sequence through mapping, appropriate selection criteria are determined, and only reliable sequence variation information is extracted (Variant Calling). This variation information is either a single nucleotide sequence variation (SNV) or a short indel. Then, the base sequence mutation information is compared with the existing database (dbSNP) to determine whether it is an already discovered mutation or a newly discovered mutation. And it predicts whether the mutation will lead to changes in amino acids or not, and what effect it will have on protein structure. This process is called Annotation. Information on extracted single nucleotide sequence mutations and short insertions/deletions are listed in a database to further improve the quality of information, or research to find the cause of disease through integrated research with Genome Wide Association Study (GWAS) is conducted. You can also do it.

However, unless there is a specific definition in the specification of the present invention, all scientific and technical terms used in the present specification have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

The present invention makes it possible to relatively accurately and easily detect complex mutations of the EGFR protein that are deeply related to the survival time of lung cancer patients.

Accordingly, the present invention more accurately predicts the prognosis of lung cancer patients, which is the main cause of cancer death at present, and enables therapeutic preparation accordingly.

1 is a graph showing the analysis of disease-free survival of 61 cases of lung adenocarcinoma with EGFR mutations with EGFR single mutations and EGFR complex mutations.
FIG. 2 is a graph showing the total survival time of 61 cases of lung adenocarcinoma with EGFR mutations with EGFR single mutations and EGFR complex mutations.
3 shows the missense mutations of major genes related to lung cancer in a case with a single mutation of EGFR.
4 shows the missense mutations of major genes related to lung cancer in a case with an EGFR complex mutation.

Hereinafter, the present invention is described in detail by the following examples. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Patient characterization and preparation of tumor DNA samples

Samples of a total of 143 patients who were diagnosed with p-stage IB-IIA lung adenocarcinoma from a hospital affiliated with Yonsei Medical Center and received postoperative adjuvant chemotherapy based on platinum formulations and surgical treatment for the purpose of cure after being diagnosed with lung adenocarcinoma of stage IB to IIA are provided. received. Among them, the following experiments were performed on 61 cases showing EGFR mutations and not treated with EGFR-TKI before tumor genotyping analysis.

All samples were fixed with paraffin and then loaded onto silanized slides at a thickness of 4 μM. Each slide of all blocks was H&E stained, and after reconfirming the presence of cancer cells, a dense area was marked. Thereafter, the cancer cell dense area was microdissected, and then DNA (gDNA) was extracted using a QIAamp DNA extraction kit (Qiagen, Valencia, CA, USA).

Generation of libraries for next-generation sequencing (NGS)

10 μg of gDNA was amplified using Ion AmpliSeqTM Custom Panel (Life Technologies). Genetic data pool (Multiplex pool) was purified using Agencourt AmPure XP beads (Beckman Coulter Incorporated), and combined with Iron Xpress barcode adapters (Life Technologies). The size and amount of pieces of each library were analyzed by a BioAnalyzer using a high sensitivity chip (High Sensitivity Chip, Agilent, Santa Clara, CA). The library was diluted and emulsions PCR reactions were performed using the Onetouch™ reaction kit (Life Technologies); Thereafter, the amount of the emulsion PCR product was increased using DynaBeadsR MyOneTM Streptavidin C1 Beads (Life Technologies). Finally, a sequencing primer and a polymerase were mixed in the ion spheres of which the amount was increased, and 5 318 v2 chips were loaded. The library includes Ion One Touch 200 Template Kit v2 DL (Life Technologies) and Iron PGM Sequencing 200 kit v2 (Ion PGM Sequencing 200 kit v2, Life Technologies) in deep coverage (1,000X objective) and 318 v2 chip kit ( Life Technologies) to perform sequencing with an Iron Torrent PGM sequencer. Sequence confirmation was performed by collating with the human GRCh37 genome as a reference sequence, and base calling was performed by Iron Torrent Suite V3.4.2 (Ion Torrent Suite V3.4.2) using tmap-f3 in the Ion Torrent server. Was done. Iron Torrent Variant Caller (ITVC) v3.4 was used to detect mutations and increase the mutation frequency to 5% or more. Bam (Binary sequence Alignment/Map format) and FASTQ files (alignment) were performed based on the result of extracting nucleotide sequence information, and mutations in nucleotide sequences such as Single Nucleotide Polymorphisms (SNPs) or INDELs It was used to extract information.

Statistical analysis

Based on the library prepared as described above, 61 EGFR mutations were analyzed and the results are shown in Table 1. Variables are expressed in percent and compared by ^{χ 2 -test.} The difference in distribution of continuous variables between two independent samples was performed using the Mann-Whitney U test. The analysis was performed using IBM SPSS Statistical Version 20 (IBM Corp), the statistical test was performed with a two-sided test, and P-value <0.05 means statistical significance.

Types of EGFR mutations Number of cases frequency(%) Single mutation Exon 19 defect 24 39.3 Exon 19 insertion V738_K739insKIPVAI One 1.6 Exon 20 insertion M766_A767insASV One 1.6 D770_N771insG+N771T One 1.6 Exon 20 mutation N771F One 1.6 Exon 21 mutation L858R 17 27.9 L861R One 1.6 Complex mutation L858R+V689L One 1.6 L858R+L833V One 1.6 L858R+H870R One 1.6 L858R+A871G One 1.6 L858R+R776H One 1.6 L858R+exon 19 defect One 1.6 G719A+I706T One 1.6 G719S+E709K One 1.6 G719S+R776H One 1.6 Exon 19 defect+I706T 2 3.3 D770_N771insNPY+H773Y One 1.6 L688F+G824S One 1.6 E749Q+A750P One 1.6 T785I+Y813H+V845M+V851I+G857R One 1.6 total 61 100

('Frequency' in Table 1 represents the number of cases in% relative to the total number of cases.)

As shown in Table 1, a single mutation accounted for 46 cases (75.4%) as the most common mutation among EGFR mutations. More specifically, as the single mutation, 24 exon 19 deletions (39.3%), L858R point mutations 17 cases (27.9%), and EGFR exon 20 insertion mutations accounted for 2 cases (3.2%). Exon 20 and 19 insertions or point mutations associated with L861R showed very little frequency.

On the other hand, complex mutations accounted for 15 cases (24.6%) out of a total of 61 cases, and many of the complex mutations of the EGFR protein (10 cases, 66.7%) were G719X ( n = 3), L858R ( n = 6), and exons. It can be seen that it is associated with EGFR TKI-sensitive mutations, such as the 19 deletion ( n = 1). In addition, one case was found to contain L858R mutation and exon 19 deletion, exon 20 insertion + H773Y mutation, or other rare mutations such as E749Q+A750P and L688F+G824S double mutations were observed, exon 20 and exon Multiple mutations distributed in 21 (T785I+Y813H+V845M+V851I+G857R) were also observed.

In the present invention, in detecting EGFR protein mutations, the entire sequence of exons 18 to 21 constituting EGFR-TKD can be identified by using a next-generation sequencing method, whereby in addition to a single mutation known as EGFR protein mutation related to lung adenocarcinoma. EGFR complex mutations can be quickly and easily identified.

Clinical and pathological characterization of cases with EGFR complex mutations

Among 61 lung cancer patients with EGFR protein mutations, the clinical and pathological characteristics of a single mutation and a complex mutation were compared and analyzed, and the results are shown in Table 2 below.

characteristic Single mutation (n=46) Complex mutation (n=15) p-value Age (mean±SD); year 59.6±10.52 58.9±7.93 0.778* gender male 10 7 0.061** female 36 8 Smoking status Non-smoker 39 11 0.488** Current smoker 4 2 Past smokers 3 2 stage IB 4 One 0.970** IIA 16 5 IIB 2 One IIIA 24 8 Maximum tumor diameter (cm) 2.9±0.96 3.4±1.01 0.075** Histological subtype Scale dominant type 3 0 0.732** Reward advantage type 31 9 Nipple/fine nipple dominant type 7 4 Solid dominant type that forms mucins 3 One Etc 2 One

As shown in Table 2, the age distribution of 61 lung adenocarcinoma patients with mutations in EGFR-TKD was 59 ± 9.9 years (range 34 to 78 years old), 17 cases (2%) for men, and 44 cases for women. (72.1%), and there was no significant difference in age at the time of diagnosis of lung cancer in male and female patients.

In addition, in many cases (50 cases, 82%), there was no smoking experience, and there were only 5 cases (9.8%) of current smokers and past smokers.

From these clinical analysis results, it can be seen that there is no significant difference in the age or sex distribution of lung adenocarcinoma in the case of EGFR single mutation and EGFR complex mutation, and smoking experience or p-stage at the time of diagnosis affects the type of EGFR mutation. It can be seen that it does not.

In addition, in the distribution of histological subtypes, complex mutations were not observed in the lepidic predominant types, and the papillary/micropapillary predominant types and solid predominant types forming mucins. with mucin production type), there were 4 cases (26.7%) and 1 case (6.7%) of complex mutations, respectively, but this did not reach statistical significance.

In addition, in terms of the size of the lung mass, the mass value of the EGFR complex mutation was relatively larger than that of the EGFR single mutation, but this also did not reach statistical significance (2.9 ± 0.96 vs. 3.4). ± 1.01 cm).

Survival Analysis of Cases with EGFR Complex Mutations

In order to determine whether a case with an EGFR complex mutation adversely affects the clinical prognosis, survival analysis was performed using a Kaplan-Myer evaluator. Disease Free Survival (DFS) and Overall Survival (OS). And the results are shown in Figs. 1 and 2, respectively. However, the analysis was performed using IBM SPSS statistics version 20 (IBM Corp), the statistical test was performed by a two-sided test, and a P-value <0.05 means statistical significance.

The above survival analysis was performed for a total of 81.9 months (95% Confidence Interval: 65.7 to 98.1 months), and during the analysis, lung adenocarcinoma recurred in 33 cases (54.1%) of 61 cases, and 15 cases (24.6%) were analyzed. During this period, he died of the same disease.

As shown in Figs. 1 and 2, there was no significant difference in both single mutations and complex mutations in disease-free survival. However, in the case of the total survival period, it was found that the complex mutation has a significantly shorter period than that of the single mutation (p=0.007, Log Rank test), and through this, the complex mutation has very low reactivity to EGFR-TKI. It could be easily predicted.

Analysis of reasons related to survival of EGFR complex mutations

In order to determine the reason for the short survival period in lung adenocarcinoma patients with complex mutations, the AKT1, BRAF , DDR2 , ERBB2 , FGFR1 , KRAS , MAPK2K1 , prototypes of the prototype with break apart of the ALK gene identified by FISH. MET , NRAS , PI3CA , PTEN , RET , ROS1 mutations and co-occurrence of the intron 2 deletion of the BIM gene, such as co-mutation (Co-mutation) was confirmed, and the results are shown in FIGS. 3 and 4.

3 and 4 each shows a missense mutation of a major lung cancer-related gene in a case with an EGFR single mutation or a complex mutation, and the frequency of co-occurrence-mutation is a case with an EGFR single mutation and an EGFR complex. There were no significant differences between cases with mutations. However, multiple missense mutations were found in various parts of the genes to be tested in 4 cases with complex mutations, which can be seen to have significant differences from cases showing a single mutation (P=0.003, χ ² -test). .

In the above, the present invention has been described centering on the above embodiments, but these are only examples, and the present invention describes various modifications and other equivalent embodiments that are obvious to those of ordinary skill in the art. It is to be understood that it may be carried out within the scope of the appended claims.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> Predicting kit for survival of lung cancer patients and the method of providing the information for predicting survival of lung cancer patients <130> DPB150014 <160> 2 <170> KopatentIn 2.0 <210> 1 <211> 9821 <212> DNA <213> Homo sapiens <400> 1 gccggagtcc cgagctagcc ccggcggccg ccgccgccca gaccggacga caggccacct 60 cgtcggcgtc cgcccgagtc cccgcctcgc cgccaacgcc acaaccaccg cgcacggccc 120 cctgactccg tccagtattg atcgggagag ccggagcgag ctcttcgggg agcagcgatg 180 cgaccctccg ggacggccgg ggcagcgctc ctggcgctgc tggctgcgct ctgcccggcg 240 agtcgggctc tggaggaaaa gaaagtttgc caaggcacga gtaacaagct cacgcagttg 300 ggcacttttg aagatcattt tctcagcctc cagaggatgt tcaataactg tgaggtggtc 360 cttgggaatt tggaaattac ctatgtgcag aggaattatg atctttcctt cttaaagacc 420 atccaggagg tggctggtta tgtcctcatt gccctcaaca cagtggagcg aattcctttg 480 gaaaacctgc agatcatcag aggaaatatg tactacgaaa attcctatgc cttagcagtc 540 ttatctaact atgatgcaaa taaaaccgga ctgaaggagc tgcccatgag aaatttacag 600 gaaatcctgc atggcgccgt gcggttcagc aacaaccctg ccctgtgcaa cgtggagagc 660 atccagtggc gggacatagt cagcagtgac tttctcagca acatgtcgat ggacttccag 720 aaccacctgg gcagctgcca aaagtgtgat ccaagctgtc ccaatgggag ctgctggggt 780 gcaggagagg agaactgcca gaaactgacc aaaatcatct gtgcccagca gtgctccggg 840 cgctgccgtg gcaagtcccc cagtgactgc tgccacaacc agtgtgctgc aggctgcaca 900 ggcccccggg agagcgactg cctggtctgc cgcaaattcc gagacgaagc cacgtgcaag 960 gacacctgcc ccccactcat gctctacaac cccaccacgt accagatgga tgtgaacccc 1020 gagggcaaat acagctttgg tgccacctgc gtgaagaagt gtccccgtaa ttatgtggtg 1080 acagatcacg gctcgtgcgt ccgagcctgt ggggccgaca gctatgagat ggaggaagac 1140 ggcgtccgca agtgtaagaa gtgcgaaggg ccttgccgca aagtgtgtaa cggaataggt 1200 attggtgaat ttaaagactc actctccata aatgctacga atattaaaca cttcaaaaac 1260 tgcacctcca tcagtggcga tctccacatc ctgccggtgg catttagggg tgactccttc 1320 acacatactc ctcctctgga tccacaggaa ctggatattc tgaaaaccgt aaaggaaatc 1380 acagggtttt tgctgattca ggcttggcct gaaaacagga cggacctcca tgcctttgag 1440 aacctagaaa tcatacgcgg caggaccaag caacatggtc agttttctct tgcagtcgtc 1500 agcctgaaca taacatcctt gggattacgc tccctcaagg agataagtga tggagatgtg 1560 ataatttcag gaaacaaaaa tttgtgctat gcaaatacaa taaactggaa aaaactgttt 1620 gggacctccg gtcagaaaac caaaattata agcaacagag gtgaaaacag ctgcaaggcc 1680 acaggccagg tctgccatgc cttgtgctcc cccgagggct gctggggccc ggagcccagg 1740 gactgcgtct cttgccggaa tgtcagccga ggcagggaat gcgtggacaa gtgcaacctt 1800 ctggagggtg agccaaggga gtttgtggag aactctgagt gcatacagtg ccacccagag 1860 tgcctgcctc aggccatgaa catcacctgc acaggacggg gaccagacaa ctgtatccag 1920 tgtgcccact acattgacgg cccccactgc gtcaagacct gcccggcagg agtcatggga 1980 gaaaacaaca ccctggtctg gaagtacgca gacgccggcc atgtgtgcca cctgtgccat 2040 ccaaactgca cctacggatg cactgggcca ggtcttgaag gctgtccaac gaatgggcct 2100 aagatcccgt ccatcgccac tgggatggtg ggggccctcc tcttgctgct ggtggtggcc 2160 ctggggatcg gcctcttcat gcgaaggcgc cacatcgttc ggaagcgcac gctgcggagg 2220 ctgctgcagg agagggagct tgtggagcct cttacaccca gtggagaagc tcccaaccaa 2280 gctctcttga ggatcttgaa ggaaactgaa ttcaaaaaga tcaaagtgct gggctccggt 2340 gcgttcggca cggtgtataa gggactctgg atcccagaag gtgagaaagt taaaattccc 2400 gtcgctatca aggaattaag agaagcaaca tctccgaaag ccaacaagga aatcctcgat 2460 gaagcctacg tgatggccag cgtggacaac ccccacgtgt gccgcctgct gggcatctgc 2520 ctcacctcca ccgtgcagct catcacgcag ctcatgccct tcggctgcct cctggactat 2580 gtccgggaac acaaagacaa tattggctcc cagtacctgc tcaactggtg tgtgcagatc 2640 gcaaagggca tgaactactt ggaggaccgt cgcttggtgc accgcgacct ggcagccagg 2700 aacgtactgg tgaaaacacc gcagcatgtc aagatcacag attttgggct ggccaaactg 2760 ctgggtgcgg aagagaaaga ataccatgca gaaggaggca aagtgcctat caagtggatg 2820 gcattggaat caattttaca cagaatctat acccaccaga gtgatgtctg gagctacggg 2880 gtgactgttt gggagttgat gacctttgga tccaagccat atgacggaat ccctgccagc 2940 gagatctcct ccatcctgga gaaaggagaa cgcctccctc agccacccat atgtaccatc 3000 gatgtctaca tgatcatggt caagtgctgg atgatagacg cagatagtcg cccaaagttc 3060 cgtgagttga tcatcgaatt ctccaaaatg gcccgagacc cccagcgcta ccttgtcatt 3120 cagggggatg aaagaatgca tttgccaagt cctacagact ccaacttcta ccgtgccctg 3180 atggatgaag aagacatgga cgacgtggtg gatgccgacg agtacctcat cccacagcag 3240 ggcttcttca gcagcccctc cacgtcacgg actcccctcc tgagctctct gagtgcaacc 3300 agcaacaatt ccaccgtggc ttgcattgat agaaatgggc tgcaaagctg tcccatcaag 3360 gaagacagct tcttgcagcg atacagctca gaccccacag gcgccttgac tgaggacagc 3420 atagacgaca ccttcctccc agtgcctgaa tacataaacc agtccgttcc caaaaggccc 3480 gctggctctg tgcagaatcc tgtctatcac aatcagcctc tgaaccccgc gcccagcaga 3540 gacccacact accaggaccc ccacagcact gcagtgggca accccgagta tctcaacact 3600 gtccagccca cctgtgtcaa cagcacattc gacagccctg cccactgggc ccagaaaggc 3660 agccaccaaa ttagcctgga caaccctgac taccagcagg acttctttcc caaggaagcc 3720 aagccaaatg gcatctttaa gggctccaca gctgaaaatg cagaatacct aagggtcgcg 3780 ccacaaagca gtgaatttat tggagcatga ccacggagga tagtatgagc cctaaaaatc 3840 cagactcttt cgatacccag gaccaagcca cagcaggtcc tccatcccaa cagccatgcc 3900 cgcattagct cttagaccca cagactggtt ttgcaacgtt tacaccgact agccaggaag 3960 tacttccacc tcgggcacat tttgggaagt tgcattcctt tgtcttcaaa ctgtgaagca 4020 tttacagaaa cgcatccagc aagaatattg tccctttgag cagaaattta tctttcaaag 4080 aggtatattt gaaaaaaaaa aaaagtatat gtgaggattt ttattgattg gggatcttgg 4140 agtttttcat tgtcgctatt gatttttact tcaatgggct cttccaacaa ggaagaagct 4200 tgctggtagc acttgctacc ctgagttcat ccaggcccaa ctgtgagcaa ggagcacaag 4260 ccacaagtct tccagaggat gcttgattcc agtggttctg cttcaaggct tccactgcaa 4320 aacactaaag atccaagaag gccttcatgg ccccagcagg ccggatcggt actgtatcaa 4380 gtcatggcag gtacagtagg ataagccact ctgtcccttc ctgggcaaag aagaaacgga 4440 ggggatggaa ttcttcctta gacttacttt tgtaaaaatg tccccacggt acttactccc 4500 cactgatgga ccagtggttt ccagtcatga gcgttagact gacttgtttg tcttccattc 4560 cattgttttg aaactcagta tgctgcccct gtcttgctgt catgaaatca gcaagagagg 4620 atgacacatc aaataataac tcggattcca gcccacattg gattcatcag catttggacc 4680 aatagcccac agctgagaat gtggaatacc taaggatagc accgcttttg ttctcgcaaa 4740 aacgtatctc ctaatttgag gctcagatga aatgcatcag gtcctttggg gcatagatca 4800 gaagactaca aaaatgaagc tgctctgaaa tctcctttag ccatcacccc aaccccccaa 4860 aattagtttg tgttacttat ggaagatagt tttctccttt tacttcactt caaaagcttt 4920 ttactcaaag agtatatgtt ccctccaggt cagctgcccc caaaccccct ccttacgctt 4980 tgtcacacaa aaagtgtctc tgccttgagt catctattca agcacttaca gctctggcca 5040 caacagggca ttttacaggt gcgaatgaca gtagcattat gagtagtgtg gaattcaggt 5100 agtaaatatg aaactagggt ttgaaattga taatgctttc acaacatttg cagatgtttt 5160 agaaggaaaa aagttccttc ctaaaataat ttctctacaa ttggaagatt ggaagattca 5220 gctagttagg agcccacctt ttttcctaat ctgtgtgtgc cctgtaacct gactggttaa 5280 cagcagtcct ttgtaaacag tgttttaaac tctcctagtc aatatccacc ccatccaatt 5340 tatcaaggaa gaaatggttc agaaaatatt ttcagcctac agttatgttc agtcacacac 5400 acatacaaaa tgttcctttt gcttttaaag taatttttga ctcccagatc agtcagagcc 5460 cctacagcat tgttaagaaa gtatttgatt tttgtctcaa tgaaaataaa actatattca 5520 tttccactct attatgctct caaatacccc taagcatcta tactagcctg gtatgggtat 5580 gaaagataca aagataaata aaacatagtc cctgattcta agaaattcac aatttagcaa 5640 aggaaatgga ctcatagatg ctaaccttaa aacaacgtga caaatgccag acaggaccca 5700 tcagccaggc actgtgagag cacagagcag ggaggttggg tcctgcctga ggagacctgg 5760 aagggaggcc tcacaggagg atgaccaggt ctcagtcagc ggggaggtgg aaagtgcagg 5820 tgcatcaggg gcaccctgac cgaggaaaca gctgccagag gcctccactg ctaaagtcca 5880 cataaggctg aggtcagtca ccctaaacaa cctgctccct ctaagccagg ggatgagctt 5940 ggagcatccc acaagttccc taaaagttgc agcccccagg gggattttga gctatcatct 6000 ctgcacatgc ttagtgagaa gactacacaa catttctaag aatctgagat tttatattgt 6060 cagttaacca ctttcattat tcattcacct caggacatgc agaaatattt cagtcagaac 6120 tgggaaacag aaggacctac attctgctgt cacttatgtg tcaagaagca gatgatcgat 6180 gaggcaggtc agttgtaagt gagtcacatt gtagcattaa attctagtat ttttgtagtt 6240 tgaaacagta acttaataaa agagcaaaag ctattctagc tttcttcttc atattttaat 6300 tttccaccat aaagtttagt tgctaaattc tattaatttt aagattgtgc ttcccaaaat 6360 agttctcact tcatctgtcc agggaggcac agttctgtct ggtagaagcc gcaaagccct 6420 tagcctcttc acggatctgg cgactgtgat gggcaggtca ggagaggagc tgcccaaagt 6480 cccatgattt tcacctaaca gccctgatca gtcagtactc aaagcttgga ctccatccct 6540 gaaggtcttc ctgattgata gcctggcctt aataccctac agaaagcctg tccattggct 6600 gtttcttcct cagtcagttc ctggaagacc ttaccccatg accccagctt cagatgtggt 6660 ctttggaaac agaggtcgaa ggaaagtaag gagctgagag ctcacattca taggtgccgc 6720 cagccttcgt gcatcttctt gcatcatctc taaggagctc ctctaattac accatgcccg 6780 tcaccccatg agggatcaga gaagggatga gtcttctaaa ctctatattc gctgtgagtc 6840 caggttgtaa gggggagcac tgtggatgca tcctattgca ctccagctga tgacaccaaa 6900 gcttaggtgt ttgctgaaag ttcttgatgt tgtgacttac cacccctgcc tcacaactgc 6960 agacataagg ggactatgga ttgcttagca ggaaaggcac tggttctcaa gggcggctgc 7020 ccttgggaat cttctggtcc caaccagaaa gactgtggct tgattttctc aggtgcagcc 7080 cagccgtagg gccttttcag agcaccccct ggttattgca acattcatca aagtttctag 7140 aacctctggc ctaaaggaag ggcctggtgg gatctacttg gcactcgctg gggggccacc 7200 ccccagtgcc actctcacta ggcctctgat tgcacttgtg taggatgaag ctggtgggtg 7260 atgggaactc agcacctccc ctcaggcaga aaagaatcat ctgtggagct tcaaaagaag 7320 gggcctggag tctctgcaga ccaattcaac ccaaatctcg ggggctcttt catgattcta 7380 atgggcaacc agggttgaaa cccttatttc tagggtcttc agttgtacaa gactgtgggt 7440 ctgtaccaga gcccccgtca gagtagaata aaaggctggg tagggtagag attcccatgt 7500 gcagtggaga gaacaatctg cagtcactga taagcctgag acttggctca tttcaaaagc 7560 gttcaattca tcctcaccag cagttcagct ggaaaggggc aaataccccc acctgagctt 7620 tgaaaacgcc ctgggaccct ctgcattctc taagtaagtt atagaaacca gtctcttccc 7680 tcctttgtga gtgagctgct attccacgta ggcaacacct gttgaaattg ccctcaatgt 7740 ctactctgca tttctttctt gtgataagca cacactttta ttgcaacata atgatctgct 7800 cacatttcct tgcctggggg ctgtaaaacc ttacagaaca gaaatccttg cctctttcac 7860 cagccacacc tgccatacca ggggtacagc tttgtactat tgaagacaca gacaggattt 7920 ttaaatgtaa atctattttt gtaactttgt tgcgggatat agttctcttt atgtagcact 7980 gaactttgta caatatattt ttagaaactc atttttctac taaaacaaac acagtttact 8040 ttagagagac tgcaatagaa tcaaaatttg aaactgaaat ctttgtttaa aagggttaag 8100 ttgaggcaag aggaaagccc tttctctctc ttataaaaag gcacaacctc attggggagc 8160 taagctaggt cattgtcatg gtgaagaaga gaagcatcgt ttttatattt aggaaatttt 8220 aaaagatgat ggaaagcaca tttagcttgg tctgaggcag gttctgttgg ggcagtgtta 8280 atggaaaggg ctcactgttg ttactactag aaaaatccag ttgcatgcca tactctcatc 8340 atctgccagt gtaaccctgt acatgtaaga aaagcaataa catagcactt tgttggttta 8400 tatatataat gtgacttcaa tgcaaatttt atttttatat ttacaattga tatgcattta 8460 ccagtataaa ctagacatgt ctggagagcc taataatgtt cagcacactt tggttagttc 8520 accaacagtc ttaccaagcc tgggcccagc caccctagag aagttattca gccctggctg 8580 cagtgacatc acctgaggag cttttaaaag cttgaagccc agctacacct cagaccgatt 8640 aaacgcaaat ctctggggct gaaacccaag cattcgtagt ttttaaagct cctgaggtca 8700 ttccaatgtg cggccaaagt tgagaactac tggcctaggg attagccaca aggacatgga 8760 cttggaggca aattctgcag gtgtatgtga ttctcaggcc tagagagcta agacacaaag 8820 acctccacat ctgtcgctga gagtcaagaa cctgaacaga gtttccatga aggttctcca 8880 agcactagaa gggagagtgt ctaaacaatg gttgaaaagc aaaggaaata taaaacagac 8940 acctctttcc atttcctaag gtttctctct ttattaaggg tggactagta ataaaatata 9000 atattcttgc tgcttatgca gctgacattg ttgccctccc taaagcaacc aagtagcctt 9060 tatttcccac agtgaaagaa aacgctggcc tatcagttac attacaaaag gcagatttca 9120 agaggattga gtaagtagtt ggatggcttt cataaaaaca agaattcaag aagaggattc 9180 atgctttaag aaacatttgt tatacattcc tcacaaatta tacctgggat aaaaactatg 9240 tagcaggcag tgtgttttcc ttccatgtct ctctgcacta cctgcagtgt gtcctctgag 9300 gctgcaagtc tgtcctatct gaattcccag cagaagcact aagaagctcc accctatcac 9360 ctagcagata aaactatggg gaaaacttaa atctgtgcat acatttctgg atgcatttac 9420 ttatctttaa aaaaaaagga atcctatgac ctgatttggc cacaaaaata atcttgctgt 9480 acaatacaat ctcttggaaa ttaagagatc ctatggattt gatgactggt attagaggtg 9540 acaatgtaac cgattaacaa cagacagcaa taacttcgtt ttagaaacat tcaagcaata 9600 gctttatagc ttcaacatat ggtacgtttt aaccttgaaa gttttgcaat gatgaaagca 9660 gtatttgtac aaatgaaaag cagaattctc ttttatatgg tttatactgt tgatcagaaa 9720 tgttgattgt gcattgagta ttaaaaaatt agatgtatat tattcattgt tctttactcc 9780 tgagtacctt ataataataa taatgtattc tttgttaaca a 9821 <210> 2 <211> 1210 <212> PRT <213> Homo sapiens <400> 2 Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala 1 5 10 15 Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln 20 25 30 Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe 35 40 45 Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn 50 55 60 Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys 65 70 75 80 Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val 85 90 95 Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr 100 105 110 Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn 115 120 125 Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu 130 135 140 His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu 145 150 155 160 Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met 165 170 175 Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro 180 185 190 Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln 195 200 205 Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg 210 215 220 Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys 225 230 235 240 Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp 245 250 255 Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro 260 265 270 Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly 275 280 285 Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His 290 295 300 Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu 305 310 315 320 Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val 325 330 335 Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn 340 345 350 Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp 355 360 365 Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr 370 375 380 Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu 385 390 395 400 Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp 405 410 415 Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln 420 425 430 His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu 435 440 445 Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser 450 455 460 Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu 465 470 475 480 Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu 485 490 495 Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro 500 505 510 Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn 515 520 525 Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly 530 535 540 Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro 545 550 555 560 Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro 565 570 575 Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val 580 585 590 Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp 595 600 605 Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys 610 615 620 Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly 625 630 635 640 Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu 645 650 655 Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg Arg His 660 665 670 Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu 675 680 685 Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu 690 695 700 Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly Ser 705 710 715 720 Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu Gly Glu 725 730 735 Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg Glu Ala Thr Ser 740 745 750 Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met Ala Ser 755 760 765 Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser 770 775 780 Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe Gly Cys Leu Leu Asp 785 790 795 800 Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn 805 810 815 Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp Arg Arg 820 825 830 Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro 835 840 845 Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Gly Ala 850 855 860 Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile Lys Trp 865 870 875 880 Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln Ser Asp 885 890 895 Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ser 900 905 910 Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser Ser Ile Leu Glu 915 920 925 Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr 930 935 940 Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg Pro Lys 945 950 955 960 Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Ala Arg Asp Pro Gln 965 970 975 Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His Leu Pro Ser Pro 980 985 990 Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp Met Asp 995 1000 1005 Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro Gln Gln Gly Phe Phe 1010 1015 1020 Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser Ser Leu Ser Ala 1025 1030 1035 1040 Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg Asn Gly Leu Gln 1045 1050 1055 Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg Tyr Ser Ser Asp 1060 1065 1070 Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp Asp Thr Phe Leu Pro 1075 1080 1085 Val Pro Glu Tyr Ile Asn Gln Ser Val Pro Lys Arg Pro Ala Gly Ser 1090 1095 1100 Val Gln Asn Pro Val Tyr His Asn Gln Pro Leu Asn Pro Ala Pro Ser 1105 1110 1115 1120 Arg Asp Pro His Tyr Gln Asp Pro His Ser Thr Ala Val Gly Asn Pro 1125 1130 1135 Glu Tyr Leu Asn Thr Val Gln Pro Thr Cys Val Asn Ser Thr Phe Asp 1140 1145 1150 Ser Pro Ala His Trp Ala Gln Lys Gly Ser His Gln Ile Ser Leu Asp 1155 1160 1165 Asn Pro Asp Tyr Gln Gln Asp Phe Phe Pro Lys Glu Ala Lys Pro Asn 1170 1175 1180 Gly Ile Phe Lys Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val 1185 1190 1195 1200 Ala Pro Gln Ser Ser Glu Phe Ile Gly Ala 1205 1210

Claims (16)

A probe or primer set that can hybridize with a gene encoding an EGFR (Epidermal Growth Factor Receptor) mutation,
Said mutation being a complex mutation comprising a G719S mutation and an E709K mutation; And
A complex mutation comprising a G719S mutation and a R776H mutation,
Wherein the EGFR is represented by SEQ ID NO: 2. delete delete delete delete A protein comprising an EGFR (Epidermal Growth Factor Receptor) mutation, or an antibody capable of binding thereto,
Said mutation being a complex mutation comprising a G719S mutation and an E709K mutation; And
A complex mutation comprising a G719S mutation and a R776H mutation,
Wherein the EGFR is represented by SEQ ID NO: 2. delete delete delete delete Detecting an EGFR (Epidermal Growth Factor Receptor) mutation or a gene encoding the EGFR mutation,
Said mutation being a complex mutation comprising a G719S mutation and an E709K mutation; And
A complex mutation comprising a G719S mutation and a R776H mutation,
Wherein said EGFR is represented by SEQ ID NO: 2. delete delete delete delete 12. The method of claim 11,
Wherein the detecting step is performed by a next-generation nucleotide sequence analysis method for predicting the survival period of lung cancer patients.

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