WO2018212247A1 - Method for predicting therapeutic efficacy of egfr tyrosine kinase inhibitor for egfr-mutant non-small cell lung cancer - Google Patents

Abstract

Provided is a method for early predicting the therapeutic efficacy of an EGFR inhibitor drug for non-small cell lung cancer, said method comprising a step for confirming the presence or absence of mutation of EGFR gene in DNA derived from a blood sample of a non-small cell lung cancer patient after the administration of the EGFR inhibitor drug. Also provided is a kit for early predicting the therapeutic efficacy of an EGFR inhibitor drug for non-small cell lung cancer, said kit comprising a primer set for amplifying a DNA fragment containing mutation of EGFR gene.

Description

Prediction method of therapeutic efficacy of EGFR tyrosine kinase inhibitor in EGFR mutant non-small cell lung cancer

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 the administration of the EGFR inhibitor, the therapeutic efficacy of the non-small cell lung cancer with the EGFR inhibitor It relates to an early prediction method. The present invention also relates to a kit for early prediction of the efficacy of treatment of non-small cell lung cancer with an EGFR inhibitor, comprising a primer set for amplifying a DNA fragment containing a mutation in the EGFR gene.

In order to detect EGFR mutation in non-small cell lung cancer, examination using a tissue has been conventionally performed, but a detection method using a liquid sample has recently been approved as an alternative method (Non-patent Document 1). However, the test results were used only to determine whether the treatment with EGFR inhibitor is applicable, and the diagnostic significance in predicting the therapeutic effect and determining the effect was unknown. Furthermore, it was unclear whether early assessment of molecular genetic remission in plasma could be an alternative to clinical remission and / or longer-term efficacy assessment.

An object of the present invention is to treat non-small cell lung cancer with an EGFR inhibitor, comprising the 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 the EGFR inhibitor It is to provide an early prediction method of response. Another object of the present invention is to provide a kit for early prediction of the efficacy of treatment of non-small cell lung cancer with an EGFR inhibitor, comprising a primer set for amplifying a DNA fragment containing a mutation in the EGFR gene.

We administer afitinib to non-small cell lung cancer patients who are positive for EGFR mutations in plasma, and patients who have a complete molecular genetic response (CMR) for EGFR mutations in plasma DNA are not It has been found that it has a longer duration of hate than the patient.

Based on the above findings, the present invention has been completed.
That is, the present invention is as follows.
[1] Successful treatment of non-small cell lung cancer with an EGFR inhibitor, comprising the 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 the EGFR inhibitor Early prediction method.
[2] The early prediction method according to [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 of the EGFR gene.
[3] The early prediction method according to [1] or [2], wherein the mutation of the EGFR gene includes at least one of c.2573T> G of the EGFR gene or deletion of the 19th exon of the EGFR gene.
[4] The step of confirming includes amplifying a DNA fragment containing c.2573T> G of the EGFR gene using a primer set consisting of the primer represented by SEQ ID NO: 1 and the primer represented by SEQ ID NO: 2. The early prediction method according to [3].
[5] The early step according to [4], wherein the step of confirming further comprises detecting a DNA fragment containing c.2573T> G of the amplified EGFR gene with a labeled probe represented by SEQ ID NO: 8 Prediction method.
[6] The step of confirming includes amplifying a DNA fragment from which the 19th exon of the EGFR gene is deleted using a primer set comprising the primer represented by SEQ ID NO: 3 and the primer represented by SEQ ID NO: 4. The early prediction method according to any one of [3] to [5].
[7] The early step according to [6], wherein the confirming step further includes detecting a DNA fragment from which the 19th exon of the amplified EGFR gene is deleted with a labeled probe represented by SEQ ID NO: 10. Prediction method.
[8] The early prediction method according to any one of [1] to [7], wherein the step of confirming is performed after 2 weeks after administration of the EGFR inhibitor.
[9] When the presence of a mutation in the EGFR gene is not confirmed in DNA derived from a blood sample of a non-small cell lung cancer patient after administration of the EGFR inhibitor, the step of determining that there is a successful treatment of the non-small cell lung cancer with the EGFR inhibitor The early prediction method according to any one of [1] to [8], further including:
[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 efficacy of treatment of non-small cell lung cancer with an EGFR inhibitor, comprising a primer set for amplifying a DNA fragment containing a mutation in the EGFR gene.
[12] The kit for early prediction according to [11] for a non-small cell lung cancer patient in which DNA derived from a blood sample before administration of the EGFR inhibitor contains a mutation of the EGFR gene.
[13] The early prediction kit according to [11] or [12], wherein the mutation of the EGFR gene is at least one of c.2573T> G of the EGFR gene or deletion of the 19th exon of the EGFR gene.
[14] The primer set for amplifying a DNA fragment containing EGFR gene c.2573T> G is a primer set comprising the primer represented by SEQ ID NO: 1 and the primer represented by SEQ ID NO: 2, The kit for early prediction as described.
[15] The kit for early prediction according to [14], further comprising a labeled probe represented by SEQ ID NO: 8.
[16] The primer set for amplifying a DNA fragment from which the 19th exon of the EGFR gene is deleted is a primer set comprising the primer represented by SEQ ID NO: 3 and the primer represented by SEQ ID NO: 4. [13] to [13] [15] The kit for early prediction according to any one of [15].
[17] The kit for early prediction according to [16], further comprising a labeled probe represented by SEQ ID NO: 10.
[18] The kit for early prediction according to any one of [11] to [17], wherein the EGFR inhibitor is afatinib.

The present invention provides an early treatment of non-small cell lung cancer with an EGFR inhibitor by confirming whether or not there is a mutation in the EGFR gene in DNA derived from a blood sample of a non-small cell lung cancer patient after administration of the EGFR inhibitor It becomes possible to predict the response, and it is possible to select a treatment method at an earlier stage than the conventional method.

FIG. 1 is a diagram showing an overview of afatinib monotherapy for advanced NSCLC patients with EGFR-sensitive mutations who have not received chemotherapy. FIG. 2 is a diagram showing the procedure of the digital PCR method. (A) A sample containing the target nucleic acid was mixed with an assay reagent so as to be 40 μL. (B) Using a microfluidic chip, the sample containing the assay reagent was divided into 8 million individual 5 pL droplets so that there was only one target molecule in any droplet. (C) Fluorescence was emitted to a droplet containing a specific sequence by PCR amplification. (D) The fluorescence signal intensity was measured by passing droplets one by one through a laser spot located in the microfluidic channel on the reading chip. FIG. 3 is a table showing the characteristics of advanced NSCLC patients with EGFR-sensitive mutations who received afatinib monotherapy. FIG. 4 is a diagram showing the median progression-free period (mPFS) of patients with positive and negative EGFR mutations in plasma at the time of afatinib administration. Fig. 5 (A) shows that patients who had stopped afatinib administration by the second week after administration of afatinib were excluded from the subject of verification, and those who became CMR in 2 weeks did not become CMR in 2 weeks It is a figure which shows that PFS was significantly long compared with the patient. Figure 5 (B) shows that patients who discontinued afatinib by 4 weeks after administration of afatinib were excluded from the verification, and patients who became CMR in 4 weeks also did not become CMR in 4 weeks It is a figure which shows that PFS was significantly long compared with the other patient.

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 the administration of the EGFR inhibitor, the therapeutic efficacy of the non-small cell lung cancer with the EGFR inhibitor Is provided (hereinafter may be abbreviated as “method of the present invention”).

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 abbreviated as “the confirmation step of the present invention”). May be).

The non-small cell lung cancer in the method of the present invention includes adenocarcinoma, squamous cell carcinoma, large cell carcinoma and the like, and any of them may be used.

The non-small cell lung cancer patient in the method of the present invention is not particularly limited as long as it is a patient 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 when it is stage III or stage IV. The non-small cell lung cancer patient in the method of the present invention may be a patient who has relapsed 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. EGFR inhibitors include, but are not limited to, gefitinib, erlotinib, osmeltinib, and afatinib.

The blood sample in the method of the present invention is not particularly limited as long as it is a blood sample containing DNA collected from a non-small cell lung cancer patient, and examples thereof include blood, serum, or plasma. Since 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 a known means, for example, using a commercially available product such as QIAmp Circulating Nucleic Acid Kit.

The DNA derived from a blood sample in the method of the present invention refers to DNA released from cells into the blood, and cancer patients contain a large amount of circulating tumor DNA (ctDNA) released from tumor cells. Circulating DNA levels are known to be elevated in cancer patients, and are thought to be released into the blood by cancer cell apoptosis, necrosis, or secretion from cancer cells. , Reported to reflect genetic mutation, or methylation. Therefore, it is preferable that the DNA derived from the blood sample of a non-small cell lung cancer patient before administration of the EGFR inhibitor contains a mutation of the EGFR gene.

The mutation of the EGFR gene in the method of the present invention may be any mutation as long as the function of EGFR is activated. Among them, deletion of the 19th exon of the EGFR gene (described in Table 1) At least one mutation in the EGFR gene that results in the replacement of Leu, the 858th amino acid of EGFR, with Arg. Examples of the EGFR gene from which the 19th exon has been deleted include deletion of the base sequences described in Table 1. The EGFR gene mutation c.2573T> G is exemplified as a result of replacing Leu, which is the 858th amino acid of EGFR, with Arg. Further, the mutation of the EGFR gene in the method of the present invention may be a mutation of an EGFR gene other than the above, and 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, Page 823-859, EGFRJuly Are listed, and may be those.

The confirmation step of the present invention is performed after EGFR inhibitor administration to non-small cell lung cancer patients. The EGFR inhibitor is not particularly limited as long as it can inhibit the function of EGFR, and examples thereof include gefitinib, erlotinib, osimertinib, and afatinib. Of these, afatinib is preferred.

In addition, the dose of EGFR inhibitor varies depending on the type of EGFR inhibitor, administration route, patient age, weight, symptoms, etc., and cannot generally be specified. As a result, about several mg to 2 g, preferably about 5 mg to several tens mg can be administered once to several times a day. In the case of injection, it is sufficient to administer about 0.1 mg to about 500 mg as an active ingredient amount in an adult, and the daily dose can be administered once or divided into several times. In particular, in the case of afatinib, 40 mg can be administered once a day by oral administration.

The confirmation step of the present invention is not particularly limited as long as it is a method capable of detecting a mutation in the EGFR gene in DNA derived from a blood sample of a non-small cell lung cancer patient. For example, the RFLP method, PCR-SSCP method, ASO high level Hybridization, direct sequencing method, ARMS method, denaturing gradient gel electrophoresis method, RNaseA cleavage method, chemical cleavage method, DOL method, TaqMan PCR method, invader method, MALDI-TOF / MS method, TDI method, molecular beacon method , Dynamic allele specific hybridization method, padlock probe method, UCAN method, nucleic acid hybridization method using DNA chip or DNA microarray, ECA method, digital PCR method, etc. A method comprising a step of amplifying a DNA fragment comprising Examples of the method including the step of amplifying a DNA fragment containing a mutation of the EGFR gene include a method using PCR, and among the methods using PCR, a digital PCR method is preferable.

In the confirmation step of the present invention, when a method including a step of amplifying a DNA fragment containing a mutation in the EGFR gene is used, a primer set for amplifying the DNA fragment containing a mutation in the EGFR gene is used. The primer set may be any as long as it is designed to amplify a DNA fragment containing a mutation of the EGFR gene to be detected in the confirmation step of the present invention. For example, the primer set is a partial base sequence of the EGFR gene, which hybridizes to a part of the complementary strand sequence 5 ′ to the mutation site to be detected, preferably about 15 to about 50 bases, preferably about 15 to A nucleic acid containing a base sequence of about 30 bases and a base sequence of about 15 to about 50 bases, preferably about 15 to about 30 bases, which hybridizes to a part of the sequence 3 'to the base of the mutation site A pair of nucleic acids which is a combination of nucleic acids and whose nucleic acid fragments amplified by them have a fragment length of about 50 to about 1,000 bases, preferably about 50 to about 500 bases, more preferably about 50 to about 200 bases. It is done.

When the mutation of the EGFR gene in the method of the present invention is c.2573T> G of the EGFR gene, the DNA fragment containing c.2573T> G of the EGFR gene is represented by the primer represented by SEQ ID NO: 1 and SEQ ID NO: 2. Amplification can be performed using a primer set consisting of the primers to be prepared. In addition, when the mutation of the EGFR gene in the method of the present invention is the deletion of the 19th exon of the EGFR gene, the DNA fragment from which the 19th exon of the EGFR gene is deleted is the primer and sequence represented by SEQ ID NO: 3 Amplification can be performed using a primer set consisting of the primer represented by No. 4.

The DNA fragment containing the mutation of the EGFR gene amplified in the confirmation step of the present invention can be detected by known means, for example, labeled with a base sequence complementary to the base sequence containing the mutation of the EGFR gene It can be detected with a probe. The probe is labeled with a fluorescent dye such as FAM or TET at the 5 'end, and with a quencher (quenching substance) such as ZEN or IABkF near the center or at the 3' end. Fluorescence is not detected because it absorbs fluorescence energy. It is preferable to prepare probes for both wild-type EGFR and mutant EGFR alleles and label them with fluorescent dyes having different fluorescence wavelengths (for example, one allele is FAM and the other is TET) for collective detection. Further, the 3 'end is phosphorylated so that PCR extension reaction from the probe does not occur. When PCR is performed with a primer designed to amplify a partial sequence of genomic DNA containing a region that hybridizes with the probe and Taq DNA polymerase, the probe hybridizes with the template DNA, and at the same time, an extension reaction from the PCR primer occurs. However, when the extension reaction proceeds, the hybridized probe is cleaved by the 5 ′ nuclease activity of Taq DNA polymerase, the fluorescent dye is released and is not affected by the quencher, and fluorescence is detected. The fluorescence intensity increases exponentially by amplification of the template.

In the confirmation step of the present invention, the amplified DNA fragment containing c.2573T> G of the EGFR gene can be detected with the labeled probe represented by SEQ ID NO: 8. Further, a DNA fragment from which the 19th exon of the amplified EGFR gene is deleted can be detected with a labeled probe represented by SEQ ID NO: 10.

The confirmation step of the present invention may be performed at any time point after the administration of the EGFR inhibitor, but is preferably after the second week after the administration of the EGFR inhibitor.

In the method of the present invention, after the administration of the EGFR inhibitor, if the presence of a mutation in the EGFR gene is not confirmed in the DNA before the administration of the EGFR inhibitor, it is determined that the treatment of non-small cell lung cancer with the EGFR inhibitor is successful Can do. Therefore, the method of the present invention is effective in treating non-small cell lung cancer with an EGFR inhibitor when the presence of a mutation in the EGFR gene is not confirmed in DNA derived from a blood sample of a non-small cell lung cancer patient after administration of the EGFR inhibitor. The process of determining is further included. Here, the frequency of the mutant EGFR gene detected in the DNA derived from the blood sample of the non-small cell lung cancer patient after administration of the EGFR inhibitor is When the frequency is less than the cutoff value, it can be determined that the presence of a mutation in the EGFR gene is not confirmed (or complete molecular genetic remission (CMR)). Examples of the mutant EGFR gene to be detected include an EGFR gene in which the 19th exon is deleted, and an EGFR gene that results in the replacement of Leu, which is the 858th amino acid of EGFR, with Arg. Examples of the EGFR gene from which the 19th exon has been deleted include deletion of the base sequences described in Table 1. The EGFR gene mutation c.2573T> G is exemplified as a result of replacing Leu, which is the 858th amino acid of EGFR, with Arg. The cut-off values include, but are not limited to, 4 events for the 19th exon deletion and 2 events for c.2573T> G.

The present invention also provides a kit for early prediction of the efficacy of treatment of non-small cell lung cancer with an EGFR inhibitor, comprising a primer set for amplifying a DNA fragment containing a mutation in the EGFR gene (hereinafter referred to as “kit of the present invention”). May be omitted).

The primer set used in the method of the present invention may be a primer set for amplifying a DNA fragment containing an EGFR gene mutation in the kit 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, a primer set comprising a primer represented by SEQ ID NO: 3 and a primer represented by SEQ ID NO: 4 Can be mentioned.

The kit of the present invention is a nucleotide complementary to the nucleotide sequence in the amplified fragment, capable of quantitatively detecting a DNA fragment containing a mutation of the EGFR gene amplified using the above primer set. It preferably further comprises a nucleic acid probe having a sequence and labeled with a labeling substance for easy and sensitive detection. For example, the kit of the present invention can further include a labeled probe represented by SEQ ID NO: 8 as a probe for detecting a DNA fragment containing the amplified EGFR gene c.2573T> G. Moreover, the kit of the present invention can further include a labeled probe represented by SEQ ID NO: 10 as a probe for detecting a DNA fragment from which the 19th exon of the amplified EGFR gene is deleted.

The primer set and probe used in the kit of the present invention can be obtained by using a commercially available DNA / RNA automatic synthesizer or the like, based on known base sequence information, by using a commercially available DNA / RNA automatic synthesizer or the like to partially or entirely include the base sequence and / or its complementary strand sequence It can be obtained by chemical synthesis. The kit including the primer set and the probe can be provided as a solid in a dry state or in an alcohol precipitate state, or provided in a state dissolved in water or a suitable buffer (eg, TE buffer). You can also

In addition to the primer set and probe described above, the kit of the present invention is another substance necessary for the reaction for detecting a DNA fragment containing a mutation in the EGFR gene, and adversely affects the reaction by storing it in the coexisting state. The substance which does not affect can be further contained. Alternatively, the kit of the present invention may be provided as a reagent kit containing the other substance as a reagent separate from the reagent containing the primer set and the probe. Examples of such other substances include reaction buffers, dNTPs, and heat-resistant DNA polymerase.

The present invention will be described more specifically with reference to the following examples, but the examples are merely illustrative of the present invention and do not limit the scope of the present invention.

Patients with advanced NSCLC with EGFR-sensitive mutations who have not received chemotherapy (EGFR-TKI) received afatinib monotherapy (40 mg / body) once daily until disease progression (PD) or cessation of toxicity ( FIG. 1). Plasma DNA from patients was obtained at the start of administration (Day 0), 2 weeks, 4 weeks, 8 weeks, 12 weeks, 24 weeks, 48 weeks and PD. Three clinically correlated EGFR mutations (exon 19 deletion, exon 20 T790M and exon 21 L858R) were multiplexed, pico-droplet digital (PCR assay (RainDrop (R) system, RainDance Technologies, Billerica, MA) (FIG. 2). Molecular genetic complete remission (CMR): “The frequency of mutated EGFR gene detected in DNA from blood samples of non-small cell lung cancer patients after EGFR inhibitor administration is false positive in DNA from healthy human blood samples It is defined as “when the frequency (cutoff value) of the mutated EGFR gene detected is less than”. As cut-off values, 4 events were used for exon 19 deletion, 2 events were used for exon 21 L858R, and 3 events were used for exon 20 T790. The research described in this example is registered in UMIN (ID: 000015847).

Samples 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). About 20 μL of plasma DNA was concentrated to about 10 μL by SpeedVac (Thermo Scientific).

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

According to the manufacturer's instructions, a uniform population of water droplets (emulsion) was prepared from the assay solution containing the plasma DNA sample by hydrodynamic flow convergence with a droplet preparation microfluidic chip (Souse chip, RainDance Technologies). Emulsions were collected in PCR tube strips (Axygen, Tewksbury, MA) containing 8 0.2 mL conical bottom PCR tubes. PCR tube strips containing a total volume of 75 μL of emulsion and carrier oil were tightly closed with 8-Strip® Dome® Cap® (Axygen) and placed in a thermal cycler with a thermal lid (Proflex® PCR® system, Life Technologies Technologies). The emulsion was thermal cycled under the conditions listed in Table 4.

The thermally cycled emulsion was transferred to a second microfluidic chip (Souse chip, RainDance Technologies), and the endpoint fluorescence signal was measured according to the manufacturer's instructions.

Data analysis Droplet event data was analyzed with 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 in RainDrop Analyst software using data from positive control samples. A correction matrix was applied to the data from each sample to eliminate crosstalk fluorescence signals from TET and FAM fluorescent molecules. The size and location of 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 in each gate was converted to the number of events per assay using the total number of untreated droplets. When analyzing clinical samples, EGFR mutation status results in tissue samples were hidden until EGFR mutation status results in plasma samples appeared.

Results In this example, 55 patients were verified (FIG. 3). Afatinib was as effective as previously reported (total remission rate: 78.6%, median progression-free period (mPFS): 14.2 months). At the start of administration, plasma EGFR mutations were positive in 62.5% (35/56) of patients. EGFR mutation positive patients in plasma had slightly shorter PFS than negative patients, but were not significant (p = 0.24, log-rank) (FIG. 4). Of the patients who were positive for plasma EGFR mutation at the start of administration, 60.6% reached CMR in 2 weeks and 87.5% in 4 weeks, respectively (FIG. 5). Patients who discontinued afatinib by week 2 were excluded from the validation, and those who became CMR in 2 weeks had significantly longer PFS than those who did not become CMR in 2 weeks ( 13.6 versus 7.5 months, p = 0.0001). Patients who discontinued afatinib by week 4 were excluded from the validation, and those who became CMR at 4 weeks also had significantly longer PFS than those who did not become CMR at 4 weeks (13.6 versus 5.1 months, p <0.0001). Among patients who became CMR by 4 weeks, the time to reach CMR did not affect PFS (p = 0.59).

The present invention provides an early treatment of non-small cell lung cancer with an EGFR inhibitor by confirming whether or not there is a mutation in the EGFR gene in DNA derived from a blood sample of a non-small cell lung cancer patient after administration of the EGFR inhibitor It becomes possible to predict the response, and it is possible to select a treatment method at an earlier stage than the conventional method.
This application is based on US Provisional Patent Application No. 62 / 507,010, the entire contents of which are incorporated herein.

Claims (18)

1. A method for early prediction of the efficacy of treatment of non-small cell lung cancer with an EGFR inhibitor, comprising the step of confirming whether an EGFR gene mutation is present in DNA derived from a blood sample of a non-small cell lung cancer patient after the administration of the EGFR inhibitor .
2. The early prediction method according to claim 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 of the EGFR gene.
3. The method for early prediction according to claim 1 or 2, wherein the mutation of the EGFR gene includes at least one of c.2573T> G of the EGFR gene or 19th exon deletion of the EGFR gene.
4. The step of confirming comprises amplifying a DNA fragment containing c.2573T> G of the EGFR gene using a primer set consisting of the primer represented by SEQ ID NO: 1 and the primer represented by SEQ ID NO: 2. 3. The early prediction method according to 3.
5. The early prediction method according to claim 4, wherein the step of confirming further comprises detecting a DNA fragment containing the amplified EGFR gene c.2573T> G with the labeled probe represented by SEQ ID NO: 8.
6. The step of confirming comprises amplifying a DNA fragment from which the 19th exon of the EGFR gene is deleted using a primer set comprising the primer represented by SEQ ID NO: 3 and the primer represented by SEQ ID NO: 4. The early prediction method according to any one of 3 to 5.
7. The early prediction method according to claim 6, wherein the step of confirming further comprises detecting a DNA fragment from which the 19th exon of the amplified EGFR gene is deleted with a labeled probe represented by SEQ ID NO: 10.
8. The early prediction method according to any one of claims 1 to 7, wherein the step of confirming is performed after 2 weeks after administration of the EGFR inhibitor.
9. When the presence of a mutation in the EGFR gene is not confirmed in DNA derived from a blood sample of a non-small cell lung cancer patient after administration of the EGFR inhibitor, the method further comprises determining that the treatment of non-small cell lung cancer with an EGFR inhibitor is successful. The early prediction method according to any one of claims 1 to 9.
10. The early prediction method according to any one of claims 1 to 9, wherein the EGFR inhibitor is afatinib.
11. A kit for early prediction of successful treatment of non-small cell lung cancer with EGFR inhibitors, including a primer set that amplifies a DNA fragment containing a mutation in the EGFR gene.
12. The kit for early prediction according to claim 11, for a non-small cell lung cancer patient in which DNA derived from a blood sample before administration of an EGFR inhibitor contains a mutation of the EGFR gene.
13. The kit for early prediction according to claim 11 or 12, wherein the mutation of the EGFR gene is at least one of c.2573T> G of the EGFR gene or 19th exon deletion of the EGFR gene.
14. The primer set which amplifies a DNA fragment containing c.2573T> G of EGFR gene is a primer set consisting of the primer represented by SEQ ID NO: 1 and the primer represented by SEQ ID NO: 2, Prediction kit.
15. The kit for early prediction according to claim 14, further comprising a labeled probe represented by SEQ ID NO: 8.
16. The primer set for amplifying a DNA fragment from which the 19th exon of the EGFR gene is deleted is a primer set consisting of the primer represented by SEQ ID NO: 3 and the primer represented by SEQ ID NO: 4. An early prediction kit according to claim 1.
17. The kit for early prediction according to claim 16, further comprising a labeled probe represented by SEQ ID NO: 10.
18. The kit for early prediction according to any one of claims 11 to 17, wherein the EGFR inhibitor is afatinib.

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