Method for predicting therapeutic response to EGFR tyrosine kinase inhibitors in EGFR-mutated non-small cell lung cancer

Abstract

The present invention provides a method for early prediction of the therapeutic success of non-small cell lung cancer with an EGFR inhibitor, which comprises a step of confirming the presence or absence of an EGFR gene mutation in DNA derived from a blood sample of a non-small cell lung cancer patient after administration of an EGFR inhibitor.The present invention also provides a kit for early prediction of the therapeutic success of non-small cell lung cancer with an EGFR inhibitor, which comprises a primer set for amplifying a DNA fragment containing an EGFR gene mutation.

Description

[Technical Field]

[0001] The present invention relates to a method for early prediction of therapeutic success of non-small cell lung cancer with an EGFR inhibitor, which comprises a step of confirming the presence or absence of an EGFR gene mutation in DNA derived from a blood sample of a non-small cell lung cancer patient after administration of the EGFR inhibitor.The present invention also relates to a kit for early prediction of therapeutic success of non-small cell lung cancer with an EGFR inhibitor, which comprises a primer set for amplifying a DNA fragment containing an EGFR gene mutation. [Background technology]

[0002] While tissue-based testing has traditionally been used to detect EGFR mutations in non-small cell lung cancer, a liquid-based detection method has recently been approved as an alternative (Non-Patent Document 1). However, test results are only used to determine whether or not treatment with an EGFR inhibitor is appropriate, and their diagnostic significance in predicting or assessing therapeutic efficacy remains unclear. Furthermore, it remains unclear whether early assessment of molecular remission in plasma can serve as an alternative means of assessing clinical remission and / or longer-term efficacy. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Karlovich C et al. Assessment of EGFR Mutation Status in Matched Plasma and Tumor Tissue of NSCLC Patients from a Phase I Study of Rociletinib (CO-1686). Clin Cancer Res. 2016 May 15;22(10):2386-95. Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide a method for early prediction of therapeutic success of non-small cell lung cancer with an EGFR inhibitor, which comprises a step of confirming the presence or absence of an EGFR gene mutation in DNA derived from a blood sample of a non-small cell lung cancer patient after administration of an EGFR inhibitor. Another object of the present invention is to provide a kit for early prediction of therapeutic success of non-small cell lung cancer with an EGFR inhibitor, which comprises a primer set for amplifying a DNA fragment containing an EGFR gene mutation. [Means for solving the problem]

[0005] The inventors administered afitinib to patients with non-small cell lung cancer who were positive for EGFR mutations in plasma, and found that patients who achieved molecular complete response (CMR) for EGFR mutations in plasma DNA had a longer progression-free period than patients who did not.

[0006] Based on the above findings, the present invention has been completed. That is, the present invention is as follows. [1] A method for early prediction of the therapeutic success of non-small cell lung cancer with an EGFR inhibitor, comprising a step of confirming whether or not an EGFR gene mutation is present in DNA derived from a blood sample of a non-small cell lung cancer patient after administration of an EGFR inhibitor. [2] The early prediction method described in [1], wherein DNA derived from a blood sample of a non-small cell lung cancer patient before administration of an EGFR inhibitor contains a mutation in the EGFR gene. [3] The early prediction method according to [1] or [2], wherein the EGFR gene mutation includes at least one of c.2573T>G of the EGFR gene or a deletion of exon 19 of the EGFR gene. [4] The early prediction method described in [3], wherein the confirmation step includes amplifying a DNA fragment containing c.2573T>G of the EGFR gene using a primer set consisting of a primer represented by sequence number 1 and a primer represented by sequence number 2. [5] The early prediction method described in [4], wherein the confirmation step further comprises detecting a DNA fragment containing c.2573T>G of the amplified EGFR gene using a labeled probe represented by sequence number 8. [6] An early prediction method according to any one of [3] to [5], wherein the confirmation step includes amplifying a DNA fragment in which the 19th exon of the EGFR gene is deleted using a primer set consisting of a primer represented by sequence number 3 and a primer represented by sequence number 4. [7] The early prediction method described in [6], wherein the confirmation step further comprises detecting a DNA fragment in which the 19th exon of the amplified EGFR gene is deleted using a labeled probe represented by sequence number 10. [8] The early prediction method according to any one of [1] to [7], wherein the checking step is carried out at least two weeks after administration of the EGFR inhibitor. [9] An early prediction method according to any one of [1] to [8], further comprising a step of determining that the non-small cell lung cancer is likely to be treated with an EGFR inhibitor if the presence of an EGFR gene mutation is not confirmed in DNA derived from a blood sample of the non-small cell lung cancer patient after administration of an EGFR inhibitor.

[10] The early prediction method according to any one of [1] to [9], wherein the EGFR inhibitor is afatinib.

[11] A kit for early prediction of the therapeutic response of non-small cell lung cancer to EGFR inhibitors, which includes a primer set that amplifies DNA fragments containing EGFR gene mutations.

[12] An early prediction kit described in

[11] for non-small cell lung cancer patients in whom DNA derived from a blood sample before administration of an EGFR inhibitor contains a mutation in the EGFR gene.

[13] The kit for early prediction according to

[11] or

[12] , wherein the mutation in the EGFR gene is at least one of c.2573T>G of the EGFR gene or a deletion of exon 19 of the EGFR gene.

[14] An early prediction kit according to

[13] , wherein the primer set for amplifying a DNA fragment containing c.2573T>G of the EGFR gene is a primer set consisting of a primer represented by sequence number 1 and a primer represented by sequence number 2.

[15] The kit for early prediction according to

[14] , further comprising a labeled probe represented by sequence number 8.

[16] An early prediction kit according to any one of

[13] to

[15] , wherein the primer set for amplifying a DNA fragment in which the 19th exon of the EGFR gene is deleted is a primer set consisting of a primer represented by sequence number 3 and a primer represented by sequence number 4.

[17] The kit for early prediction according to

[16] , further comprising a labeled probe represented by sequence number 10.

[18] The kit for early prediction according to any one of

[11] to

[17] , wherein the EGFR inhibitor is afatinib. [Effects of the Invention]

[0007] By confirming whether or not EGFR gene mutations are present in DNA derived from blood samples of non-small cell lung cancer patients after administration of an EGFR inhibitor, the present invention makes it possible to predict the effectiveness of EGFR inhibitor treatment for non-small cell lung cancer at an early stage, thereby enabling the selection of a treatment method at an earlier stage than conventional methods. [Brief explanation of the drawings]

[0008] [Figure 1] Figure 1 provides an overview of afatinib monotherapy for chemotherapy-naive patients with advanced NSCLC harboring EGFR-sensitive mutations. [Figure 2] Figure 2 shows the steps of the digital PCR method. (A) A sample containing the target nucleic acid was mixed with assay reagents to a volume of 40 μL. (B) Using a microfluidic chip, the sample containing the assay reagents was divided into 8 million individual 5 pL droplets, with only one target molecule present in any given droplet. (C) PCR amplification caused droplets containing specific sequences to emit fluorescence. (D) The fluorescent signal intensity was measured by passing each droplet through a laser spot located in a microfluidic channel on a reading chip. [Figure 3] Figure 3 is a table showing the characteristics of patients with advanced NSCLC harboring EGFR-sensitive mutations treated with afatinib monotherapy. [Figure 4]FIG. 4 shows the median progression-free period (mPFS) between patients who were positive for plasma EGFR mutation and patients who were negative for EGFR mutation during administration of afatinib. [Figure 5] Figure 5(A) shows that patients who achieved CMR within 2 weeks after afatinib administration, excluding patients who discontinued afatinib administration within 2 weeks, had a significantly longer PFS than patients who did not achieve CMR within 2 weeks. Figure 5(B) shows that patients who achieved CMR within 4 weeks after afatinib administration, excluding patients who discontinued afatinib administration within 4 weeks, also had a significantly longer PFS than patients who did not achieve CMR within 4 weeks. DETAILED DESCRIPTION OF THE INVENTION

[0009] The present invention provides a method for early prediction of the therapeutic success of non-small cell lung cancer with an EGFR inhibitor, which comprises a step of confirming the presence or absence of an EGFR gene mutation in DNA derived from a blood sample of a non-small cell lung cancer patient after administration of an EGFR inhibitor (hereinafter sometimes abbreviated as "the method of the present invention").

[0010] The method of the present invention includes a step of confirming whether or not an EGFR gene mutation is present in DNA derived from a blood sample of a non-small cell lung cancer patient after administration of an EGFR inhibitor (hereinafter sometimes abbreviated as the "confirmation step of the present invention").

[0011] Non-small cell lung cancer in the method of the present invention includes adenocarcinoma, squamous cell carcinoma, large cell carcinoma, and the like, and may be any of them.

[0012] The non-small cell lung cancer patient in the method of the present invention is not particularly limited as long as the patient has been pathologically diagnosed as suffering from non-small cell lung cancer. The stage of non-small cell lung cancer may be any stage, but the method of the present invention is more effective in stage III and stage IV. The non-small cell lung cancer patient in the method of the present invention may be a patient who has recurred after surgery. In addition, the non-small cell lung cancer patient in the method of the present invention is preferably a patient who has not been treated with an EGFR inhibitor. Examples of EGFR inhibitors include, but are not limited to, gefitinib, erlotinib, osimertinib, and afatinib.

[0013] The blood sample used in the method of the present invention is not particularly limited as long as it is a blood sample containing DNA collected from a patient with non-small cell lung cancer, and examples include blood, serum, and plasma. Because blood or serum may be contaminated with fragmented genomic DNA derived from leukocytes, it is preferable to use plasma. DNA can be isolated from blood, serum, and plasma by known methods, for example, using a commercially available product such as the QIAmp Circulating Nucleic Acid Kit.

[0014] In the method of the present invention, DNA derived from a blood sample refers to DNA released into the blood from cells. In cancer patients, this DNA contains a large amount of circulating tumor DNA (ctDNA) released from tumor cells. The concentration of circulating DNA is known to be elevated in cancer patients. It is believed to be released into the blood due to apoptosis, necrosis, or secretion from cancer cells, and has been reported to reflect copy number variation, gene mutation, or methylation of the primary tumor. Therefore, it is preferable that DNA derived from a blood sample of a non-small cell lung cancer patient before administration of an EGFR inhibitor contains a mutation in the EGFR gene.

[0015] The EGFR gene mutation in the method of the present invention may be any mutation that activates the function of EGFR, and includes at least one mutation in the EGFR gene that results in a deletion of exon 19 of the EGFR gene (deletion of an amino acid listed in Table 1) or a substitution of amino acid Leu, the 858th amino acid of EGFR, with Arg. Examples of EGFR genes with a deletion of exon 19 include deletions of the nucleotide sequences listed in Table 1. Examples of EGFR gene mutations that result in a substitution of amino acid Leu, the 858th amino acid of EGFR, with Arg include c.2573T>G of the EGFR gene. Furthermore, the EGFR gene mutation in the method of the present invention may be a mutation in an EGFR gene other than those described above, and may include EGFR gene mutations that have been reported to date, as listed in Lindeman NI et al., Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology., Journal of Thoracic Oncology, Volume 8, Number 7, pages 823-859, July 2013.

[0016] [Table 1]

[0017] The confirmation step of the present invention is carried out after administering an EGFR inhibitor to a patient with non-small cell lung cancer. The EGFR inhibitor is not particularly limited as long as it can inhibit the function of EGFR, but examples include gefitinib, erlotinib, osimertinib, and afatinib. Among these, afatinib is preferred.

[0018] The dosage of an EGFR inhibitor varies depending on the type of EGFR inhibitor, the route of administration, the patient's age, weight, symptoms, etc., and cannot be generally defined. However, in the case of oral administration, the active ingredient amount per day for an adult is typically several mg to 2 g, preferably 5 mg to several tens of mg, administered once or in divided doses several times a day. In the case of injection, the active ingredient amount for an adult is approximately 0.1 mg to approximately 500 mg, and the daily dose can be administered once or in divided doses several times a day. In particular, afatinib can be administered orally at 40 mg once a day.

[0019] The confirmation step of the present invention is not particularly limited as long as it can detect EGFR gene mutations in DNA derived from blood samples of non-small cell lung cancer patients, and can be performed by, for example, RFLP, PCR-SSCP, ASO hybridization, direct sequencing, ARMS, denaturing gradient gel electrophoresis, RNase A cleavage, chemical cleavage, DOL, TaqMan PCR, Invader, MALDI-TOF / MS, TDI, molecular beacon, dynamic allele-specific hybridization, padlock probe, UCAN, nucleic acid hybridization using a DNA chip or DNA microarray, ECA, or digital PCR, but is preferably a method that includes a step of amplifying DNA fragments containing EGFR gene mutations. Examples of methods that include a step of amplifying DNA fragments containing EGFR gene mutations include PCR-based methods, and among PCR-based methods, digital PCR is preferred.

[0020] When the confirmation step of the present invention involves a method for amplifying a DNA fragment containing an EGFR gene mutation, a primer set for amplifying the DNA fragment containing an EGFR gene mutation is used. The primer set may be any primer set designed to amplify the DNA fragment containing the EGFR gene mutation to be detected in the confirmation step of the present invention. For example, the primer set may be a pair of nucleic acids that are partial nucleotide sequences of the EGFR gene, including a nucleic acid comprising a nucleotide sequence of about 15 to about 50 bases, preferably about 15 to about 30 bases, that hybridizes to a portion of the complementary strand sequence 5' from the mutation site to be detected, and a nucleic acid comprising a nucleotide sequence of about 15 to about 50 bases, preferably about 15 to about 30 bases, that hybridizes to a portion of the sequence 3' from the mutation site, and the length of the nucleic acid fragment amplified by the primer set is about 50 to about 1,000 bases, preferably about 50 to about 500 bases, and more preferably about 50 to about 200 bases.

[0021] When the EGFR gene mutation in the method of the present invention is c.2573T>G of the EGFR gene, a DNA fragment containing c.2573T>G of the EGFR gene can be amplified using a primer set consisting of the primer shown in SEQ ID NO: 1 and the primer shown in SEQ ID NO: 2. Furthermore, when the EGFR gene mutation in the method of the present invention is a deletion of exon 19 of the EGFR gene, a DNA fragment in which exon 19 of the EGFR gene has been deleted can be amplified using a primer set consisting of the primer shown in SEQ ID NO: 3 and the primer shown in SEQ ID NO: 4.

[0022] The DNA fragment containing the EGFR gene mutation amplified in the confirmation step of the present invention can be detected by known means, for example, using a labeled probe containing a base sequence complementary to the base sequence containing the EGFR gene mutation. The probe is labeled at its 5' end with a fluorescent dye such as FAM or TET and at its central or 3' end with a quencher (quenching substance) such as ZEN or IABkF. In its original state, the quencher absorbs the fluorescent energy, so no fluorescence is detected. Probes are prepared for both wild-type and mutant EGFR alleles, and preferably labeled with fluorescent dyes with different fluorescent wavelengths (e.g., one allele with FAM and the other with TET) for simultaneous detection. Furthermore, the 3' end is phosphorylated to prevent PCR extension from occurring from the probe. When PCR is performed with primers designed to amplify a partial sequence of genomic DNA containing the region that hybridizes with the probe and Taq DNA polymerase, the probe hybridizes to the template DNA and simultaneously an extension reaction from the PCR primer occurs. As the extension reaction progresses, the hybridized probe is cleaved by the 5' nuclease activity of Taq DNA polymerase, liberating the fluorescent dye, which is no longer affected by the quencher, and fluorescence can be detected. Fluorescence intensity increases exponentially with template amplification.

[0023] In the confirmation step of the present invention, an amplified DNA fragment containing c.2573T>G of the EGFR gene can be detected with a labeled probe represented by SEQ ID NO: 8. Furthermore, an amplified DNA fragment lacking exon 19 of the EGFR gene can be detected with a labeled probe represented by SEQ ID NO: 10.

[0024] The confirmation step of the present invention may be performed at any time after administration of an EGFR inhibitor, but is preferably performed two weeks or later after administration of an EGFR inhibitor.

[0025] The method of the present invention can determine whether a patient is likely to be successfully treated with an EGFR inhibitor if, after administration of an EGFR inhibitor, no mutations in the EGFR gene are detected in DNA from the patient's blood sample. Therefore, the method of the present invention further includes determining whether a patient is likely to be successfully treated with an EGFR inhibitor if no mutations in the EGFR gene are detected in DNA from the patient's blood sample after administration of an EGFR inhibitor. If the frequency of mutant EGFR genes detected in DNA from the patient's blood sample after administration of an EGFR inhibitor is lower than the frequency (cutoff value) of mutant EGFR genes detected as false positives in DNA from healthy individuals' blood samples, the absence of a mutation in the EGFR gene (or molecular complete response (CMR)) can be determined. Examples of mutant EGFR genes that can be detected include EGFR genes with a deletion of exon 19 and EGFR genes resulting in a substitution of the 858th amino acid of EGFR, Leu, with Arg. Examples of EGFR genes with a deletion of exon 19 include deletions of the nucleotide sequences listed in Table 1. An example of an EGFR gene mutation resulting in a substitution of Leu (amino acid 858) with Arg is c.2573T>G of the EGFR gene. Cutoff values include, but are not limited to, 4 events for a deletion of exon 19 and 2 events for c.2573T>G.

[0026] The present invention also provides a kit for early prediction of the therapeutic success of non-small cell lung cancer with an EGFR inhibitor, which kit includes a primer set for amplifying a DNA fragment containing a mutation in the EGFR gene (hereinafter sometimes abbreviated as "the kit of the present invention").

[0027] The primer set for amplifying a DNA fragment containing a mutation in the EGFR gene in the kit of the present invention may be the primer set used in the method of the present invention, more specifically, a primer set consisting of a primer represented by SEQ ID NO: 1 and a primer represented by SEQ ID NO: 2, or a primer set consisting of a primer represented by SEQ ID NO: 3 and a primer represented by SEQ ID NO: 4.

[0028] The kit of the present invention preferably further comprises a nucleic acid probe capable of detecting a DNA fragment containing an EGFR gene mutation amplified using the primer set with high sensitivity and quantitative detection, the nucleic acid probe having a nucleotide sequence complementary to a nucleotide sequence in the amplified fragment and labeled with a labeling substance to facilitate and enhance detection. For example, the kit of the present invention may further comprise a labeled probe represented by SEQ ID NO: 8 as a probe for detecting a DNA fragment containing c.2573T>G of the amplified EGFR gene. The kit of the present invention may further comprise a labeled probe represented by SEQ ID NO: 10 as a probe for detecting a DNA fragment in which exon 19 of the amplified EGFR gene is deleted.

[0029] The primer set and probe used in the kit of the present invention can be obtained by chemically synthesizing part or all of the base sequence and / or its complementary strand sequence based on known base sequence information using a commercially available automatic DNA / RNA synthesizer, etc. The kit containing the primer set and probe can be provided as a solid in a dried or alcohol-precipitated state, or can be provided in a dissolved state in water or an appropriate buffer solution (e.g., TE buffer, etc.).

[0030] In addition to the primer set and probe, the kit of the present invention may further contain other substances necessary for the reaction of detecting DNA fragments containing EGFR gene mutations, which do not adversely affect the reaction when stored in the presence of other substances. Alternatively, the kit of the present invention may be provided as a reagent kit containing the other substances as reagents separate from the reagents containing the primer set and probe. Examples of such other substances include a reaction buffer, dNTPs, and a thermostable DNA polymerase.

[0031] The present invention will be described in more detail with reference to the following examples, but these examples are merely illustrative of the present invention and do not limit the scope of the present invention in any way. [Example]

[0032] Patients with advanced NSCLC harboring EGFR-sensitive mutations who had not received chemotherapy (EGFR-TKI) received once-daily afatinib monotherapy (40 mg / body) until disease progression (PD) or toxicity (Figure 1). Plasma DNA was obtained from patients at the start of treatment (day 0), 2 weeks, 4 weeks, 8 weeks, 12 weeks, 24 weeks, 48 weeks, and at PD. Three clinically relevant EGFR mutations (exon 19 deletion, exon 20 T790M, and exon 21 L858R) were analyzed using plasma DNA using a multiplexed, pico-droplet digital PCR assay (RainDrop® system, RainDance Technologies, Billerica, MA) (Figure 2). A molecular complete response (CMR) was defined as "a condition in which the frequency of EGFR mutations detected in DNA derived from blood samples of non-small cell lung cancer patients after treatment with EGFR inhibitors is less than the frequency of EGFR mutations detected as false-positive in DNA derived from blood samples of healthy individuals (cutoff values)." The cutoff values used were 4 events for exon 19 deletion, 2 events for exon 21 L858R, and 3 events for exon 20 T790. The study described in this example has been registered with UMIN (ID: 000015847).

[0033] Materials and methods Sample collection Whole blood was collected from all subjects into BD Biosciences Vacutainer EDTA blood collection tubes. The blood was centrifuged at 1500 × g for 10 minutes at 4°C, and the plasma supernatant (2–3.5 mL) was transferred to a 50 mL conical tube (BD Falcon) and stored at -80°C until use. Plasma DNA was isolated using the QIAmp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Plasma DNA was dissolved in AVE buffer (45 μL). Approximately 20 μL of plasma DNA was concentrated to approximately 10 μL using a SpeedVac (Thermo Scientific).

[0034] EGFR mutation detection in a multiplex format A multiplex assay was developed to identify three common EGFR mutations and their corresponding wild-type sequences. Briefly, 20.0 μL of TaqMan Genotyping Master Mix (Life Technologies) was mixed with assay reagents, including 2.0 μL of 10 μM forward and reverse primers, 2.0 μL of 4 μM FAM- and TET-labeled probe, 4.0 μL of Droplet Stabilizer (RainDance Technologies, Billerica, MA), and 4.0 μL of DNase- and RNase-free sterile water. The final reaction volume was 40 μL, containing 8 μL of patient-derived plasma DNA sample. The primer, probe, and quencher sequences, along with their concentrations, are listed in Tables 2 and 3.

[0035] [Table 2]

[0036] [Table 3]

[0037] Following the manufacturer's instructions, a uniformly sized droplet population (emulsion) was generated from the assay solution containing the plasma DNA sample by hydrodynamic flow focusing in a droplet-generating microfluidic chip (Souse chip, RainDance Technologies). The emulsion was collected in a PCR tube strip (Axygen, Tewksbury, MA) containing eight 0.2 mL conical-bottom PCR tubes. The PCR tube strip, containing a total of 75 μL of emulsion and carrier oil, was tightly closed with an 8-Strip Dome Cap (Axygen) and placed in a thermal cycler equipped with a thermal lid (Proflex PCR system, Life Technologies). The emulsion was thermal cycled under the conditions listed in Table 4.

[0038] [Table 4]

[0039] The thermally cycled emulsion was transferred to a second microfluidic chip (Source chip, RainDance Technologies), and the endpoint fluorescent signal was measured according to the manufacturer's instructions.

[0040] Data analysis Droplet event data were analyzed using RainDrop Analyst software (RainDance Technologies) according to the manufacturer's instructions. Briefly, sample data was loaded with a droplet size gate template (RainDance Technologies). A correction matrix was created within RainDrop Analyst software using data from the positive control sample. The correction matrix was applied to data from each sample to remove crosstalk fluorescent signals from TET and FAM fluorescent molecules. The size and position of the wild-type and mutant gates were determined by manual selection of areas containing wild-type or mutant clusters in the positive control. For each unknown sample, the number of PCR-positive droplet events was counted within each gate. The number of events within each gate was converted to the number of events per assay using the total number of raw droplets. When analyzing clinical samples, the results of the EGFR mutation status in the tissue sample were concealed until the results of the EGFR mutation status in the plasma sample were revealed.

[0041] result In this study, 55 patients were studied (Figure 3). The efficacy of afatinib was comparable to previous reports (overall remission rate: 78.6%, median progression-free survival (mPFS): 14.2 months). At the start of treatment, 62.5% (35 / 56) of patients were EGFR mutation-positive in plasma. Patients with EGFR mutation-positive plasma had a slightly shorter PFS than patients without EGFR mutations, but this was not significant (p = 0.24, log-rank) (Figure 4). Of patients with EGFR mutation-positive plasma at the start of treatment, 60.6% achieved CMR within 2 weeks and 87.5% achieved CMR within 4 weeks (Figure 5). Excluding patients who discontinued afatinib treatment by week 2, patients who achieved CMR within 2 weeks had a significantly longer PFS than patients who did not achieve CMR within 2 weeks (13.6 versus 7.5 months, p = 0.0001). After excluding patients who discontinued afatinib treatment by week 4, patients who achieved a CMR at 4 weeks also had a significantly longer PFS compared with patients who did not achieve a CMR at 4 weeks (13.6 versus 5.1 months, p < 0.0001). Among patients who achieved a CMR by week 4, the time to achieving a CMR did not affect PFS (p = 0.59). [Industrial Applicability]

[0042] By confirming whether or not EGFR gene mutations are present in DNA derived from blood samples of non-small cell lung cancer patients after administration of an EGFR inhibitor, the present invention makes it possible to predict the effectiveness of EGFR inhibitor treatment for non-small cell lung cancer at an early stage, thereby enabling the selection of a treatment method at an earlier stage than conventional methods. This application is based on US Provisional Patent Application No. 62 / 507,010, the entire contents of which are incorporated herein by reference.

Claims

1. The method includes a step of confirming whether or not an EGFR gene mutation is present in DNA derived from a blood sample of a non-small cell lung cancer patient two weeks after starting administration of an EGFR inhibitor, DNA from blood samples of non-small cell lung cancer patients before administration of EGFR inhibitors contained mutations in the EGFR gene, The EGFR gene mutation includes at least one of c.2573T>G of the EGFR gene or a deletion of exon 19 of the EGFR gene; A test method for early prediction of progression-free survival in patients with non-small cell lung cancer treated with EGFR inhibitors.

2. The testing method of claim 1, wherein the confirmation step comprises amplifying a DNA fragment containing c.2573T>G of the EGFR gene using a primer set consisting of a primer represented by sequence number 1 and a primer represented by sequence number 2.

3. The testing method of claim 2, wherein the confirmation step further comprises detecting a DNA fragment containing c.2573T>G of the amplified EGFR gene with a labeled probe represented by sequence number 8.

4. The testing method according to any one of claims 1 to 3, wherein the confirming step comprises amplifying a DNA fragment in which exon 19 of the EGFR gene is deleted using a primer set consisting of a primer represented by SEQ ID NO: 3 and a primer represented by SEQ ID NO:

4.

5. The testing method according to claim 4, wherein the confirmation step further comprises detecting the amplified DNA fragment lacking exon 19 of the EGFR gene using a labeled probe represented by sequence number 10.

6. The method for testing according to any one of claims 1 to 5, wherein the EGFR inhibitor is afatinib.

7. A kit for predicting progression-free survival time of a patient with non-small cell lung cancer treated with an EGFR inhibitor, 2 weeks after starting administration of the EGFR inhibitor, for the patient in whom DNA derived from a blood sample before administration of the EGFR inhibitor contains a mutation in the EGFR gene, the kit comprising a primer set for amplifying a DNA fragment containing a mutation in the EGFR gene, A kit for predicting whether the EGFR gene mutation is at least one of c.2573T>G of the EGFR gene or a deletion of exon 19 of the EGFR gene.

8. The prediction kit according to claim 7, wherein the primer set for amplifying a DNA fragment containing c.2573T>G of the EGFR gene is a primer set consisting of a primer represented by sequence number 1 and a primer represented by sequence number 2.

9. The prediction kit of claim 8, further comprising a labeled probe represented by SEQ ID NO:

8.

10. The prediction kit according to any one of claims 7 to 9, wherein the primer set for amplifying a DNA fragment in which the 19th exon of the EGFR gene is deleted is a primer set consisting of a primer represented by SEQ ID NO: 3 and a primer represented by SEQ ID NO:

4.

11. The predictive kit of claim 10, further comprising a labeled probe represented by SEQ ID NO:

10.

12. The prediction kit according to any one of claims 7 to 11, wherein the EGFR inhibitor is afatinib.

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