WO2016018115A1 - Method for diagnosing responsiveness of therapeutic agent for second line treatment of cancer patient using circulating tumor cells - Google Patents

Abstract

The present invention relates to a method for diagnosing the responsiveness of a therapeutic agent for a second line treatment of a cancer patient using circulating tumor cells. More specifically, the present invention relates to a method for calculating a predicted value of treatment responsiveness which comprises several steps of separating and detecting CTC from a blood sample of a patient to provide information for determination of the second line treatment through detection of gene mutation information in the cancer patient, the method being suitable for automation. The method of the present invention can be useful for the purpose of guiding future treatment, including determination of the need for administering an anti-cancer therapeutic agent, since it is possible to predict and diagnose the responsiveness of a patient to the therapeutic agent by calculating the predicted value of treatment responsiveness using the blood of a patient who is subject to the second line treatment and from whom obtaining an FFPE tissue sample is difficult.

Description

 【Specification】

 [Name of invention]

 Therapeutic Reactivity Diagnosis Method for Secondary Treatment of Cancer Patients Using Circulating Tumor Cells

 <ι> This application claims the priority of Korean Patent Application No. 10-2014-0099187 filed on August 1, 2014, the entirety of which is a reference of the present application.

 <2>

 The present invention relates to a method for diagnosing reactivity of a therapeutic agent for secondary treatment of cancer patients using circulating tumor cells, and more specifically, by detecting genetic variation information of cancer patients.

In order to provide information for judging a second ine treatment, the present invention relates to a method suitable for automation as a method of calculating a treatment response predictive including multiple steps of detecting and detecting CTC from a patient's blood sample. .

 <4>

 Background Art

 Cancer is a group of abnormal cells caused by continuous division and proliferation due to the disruption of the balance between cell division and death by various causes, also called tumors or neoplasms. It usually occurs in more than 100 different parts of the body, including organs, white blood cells, bones, lymph nodes, etc., and develops into severe symptoms through infiltration into surrounding tissues and metastases to other organs.

 <6>

 <7> Therapeutics for cancer have been continuously developed, and there are dozens of treatments for various cancers currently used in clinical practice. However, to date, clinicians have been experiencing two difficulties. The first is that it takes several weeks for the treatment to be effective, and it is impossible to know in advance which treatment is effective for individual patients. In other words, the therapeutic effect of anticancer drugs is not able to be determined within a few days, but gradually appears over several weeks, so it takes a long time to determine that the prescribed drug is ineffective. If the therapeutic effect is inappropriate then consider changing to another type of treatment. If the clinician fails to select the appropriate treatment for the patient at the start of the treatment, the effective treatment will be delayed. It has a fatal effect on disease progression, recurrence and prognosis.

<8> <9> ¥ ^처 ^ young treatment 처 ¥ Invisible patients ᅳ Many zones; It is a point. For example, lapatinib, a breast cancer drug, has been shown to be effective in the treatment of high levels of HER2 protein (HER2-positive) and low levels of EGRF protein. However, metastatic HER2-negative breast cancers do not respond to lapatinib, indicating that lapatinib is ineffective. These findings suggest that breast cancer patients can select the appropriate treatment by confirming whether they are HER2 negative or black positive before the treatment.

 <10>

 Therefore, if the treatment response and the side effects of the therapeutic agent can be predicted in advance, the drug suitable for the patient can be selected in advance, thereby reducing the dropout rate caused by the wrong selection of the drug and increasing the compliance of the drug. There will be. It will also avoid the time it takes for the drug to take effect and the risk of side effects that the patient may experience.

 <12>

 Under this concept, a search for a marker related to various drug reactions or a commercial in vitro diagnostic or companion diagnostic kit using the same has been developed, and some of them have already been commercialized and used in clinical practice. However, these methods are mainly applicable to first line treatment, which is difficult to apply after the second treatment.

 <14>

 In addition, the diagnostic methods of response to the therapeutic agents are generally less reliable than the highly experienced experimenter. As a result, in some cases, the patient's sample should be provided to the service provider according to a predetermined process in a central lab to obtain relatively meaningful results.

(inter-laboratory variation), inter-observer variation, and day-to-day variation may raise questions about the reliability of the overall system.

 <16>

 As described above, the conventional method may cause an error in the diagnosis result according to the place, time, and experimenter. Thus, in order to obtain a stable result, a method or an automation process that requires the involvement of the experimenter is preferably excluded.

<18> Throughout this specification, a number of papers and patent documents are referred to and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

 <20>

 [Detailed Description of the Invention]

 [Technical problem]

 <2i> As a result of our intensive research to develop a method for calculating treatment responsiveness using the blood of a patient who is difficult to obtain FFPE tissue samples, the present inventors have isolated CTC from blood samples of patients. The present invention has been completed by developing a method for calculating a therapeutic response, including several steps of detection, in particular a method suitable for automation.

 <22>

 Accordingly, an object of the present invention is to provide a method for judging secondary treatment by detecting genetic mutation information of lung cancer patients. It is to provide a method for calculating a therapeutic response response comprising a.

 <24>

 Technical Solution

 In order to achieve the above object, the present invention provides the following steps from a blood sample of a patient in order to provide information for judging a second treatment in the genetic mutation information detection of lung cancer patients It provides a method of calculating the therapeutic response included.

 (A) isolating CTC (circulat ing tumor cel l) from the blood of the patient;

 (B) isolating genomic DNA from CTC cells;

 (C) preparing a PCR reaction solution comprising the genomic DNA, primers and probes specific for genomic DNA gene mutations;

(D) Standards comprising standard DNA, primers and probes specific for genomic DNA gene mutations, wherein the standard vector is processed to be linear DNA by restriction enzymes.

Preparing a PCR reaction solution;

(E) micronizing the PCR reaction solution of step (d) and the standard PCR reaction solution of step (f) into a plurality of droplets, respectively; (F) performing a PCR reaction such that PCR reaction is performed on each of the plurality of small droplets;

 (G) measuring PCR reaction in all or part of each micronized droplet;

 (H) calculating a mutation rate (% mutat ion) from the measurement of PCR reaction in the PCR reaction solution and the standard PCR reaction solution;

(I) calculating a predictive value predicting that the higher the mutation rate, the higher the therapeutic responsiveness.

 <35>

 Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The following reference provides one of the descriptions (ski l l) having a general definition of several terms used in the specification of the present invention: Singleton et al. , DICTIONARY OF MICROBIOLOGY AND MOLECULAR BI0L0TY (2th ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walkered., 1988); and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY.

 <37>

 Hereinafter, the content of the present invention will be described in more detail.

 <39>

 In order to provide information for judging secondary treatment by detecting genetic variation information of a lung cancer patient, the present invention calculates a therapeutic response predictive value including the following step from a blood sample of a patient. Provide a method.

 (A) isolating CTC (circulat ing tumor cel l) from the blood of the patient;

 (B) isolating genomic DNA from CTC cells;

 (C) preparing a PCR reaction solution comprising the genomic DNA and primers and probes specific for genomic DNA gene mutations;

(D) Standards comprising standard DNA, primers and probes specific for genomic DNA gene mutations, wherein the vector of reference material has been subjected to restriction enzymes to form linear DNA.

Preparing a PCR reaction solution;

(E) micronizing the PCR reaction solution of step (d) and the standard PCR reaction solution of step (f) into a plurality of droplets, respectively;

(F) PCR reactions are performed such that the PCR reaction is performed in each of the plurality of small rooms. Performing;

 (G) measuring PCR reaction in all or part of each micronized droplet;

 (H) calculating a mutation rate (% mutat ion) from a measurement of PCR reaction in a PCR reaction solution and a standard PCR reaction solution;

(I) calculating a predictive value predicting that the higher the mutation rate, the higher the therapeutic responsiveness.

 <50>

 The method of the present invention is preferably an automated or semi-automated method. Automation in the above is the introduction of a sample (sample); Rearrangement or transfer with extraction, separation, reaction complete substrates (eg tubes, plates); Injection of reagents, buffers into stock, complement; This means that all or most of the process, except for the maintenance of equipment, is done by means other than humans (eg, robots).

 <52>

 Each step will be described in detail below.

 <54>

 Step (a) is the step of separating CTCs from the blood of the patient.

 CTCs are tumor cells found in the peripheral blood of malignant tumor patients. Since CTC plays an important role in the cancer metastasis process, CTC is considered to be very important in cancer research and diagnosis.However, the number of circulating tumor cells in peripheral blood is very rare, and dozens or less mixed with millions of normal blood cells There is a need for a detection system that requires a sensitivity that is capable of detecting tumor cells.

 <57>

 Step (b) is the step of separating genomic DNA from CTC cells.

 Isolation of DNA in CTCs can be performed according to conventional methods known in the art. The nucleic acid to be isolated from the CTC of the present invention is preferably genomic DNA, more preferably genomic DNA assumed to carry mutations.

 <60>

 Step (c) is a step of preparing a PCR reaction solution containing primers and probes specific for the DNA and genomic DNA gene mutations.

The isolated DNA was then subjected to primer / probe sets and PCR reactions for PCR reactions. Or a combination such as a buffer (eg, commercial PCR premixes). The primer set can specifically amplify the mutation (mutation) of the genomic DNA gene of interest. PCR premixes may include DNA polymerase for PCR reactions (eg Tag polymerase), dyes for quantitative detection of PCR reactions (eg fluorescent dyes), buffers suitable for PCR reactions, dNTPs, and the like. .

 <63>

 (D) step (d) is to prepare a standard PCR reaction solution containing primers and probes specific for genomic DNA gene mutations, such as standard DNA genomic DNA gene mutation to the standard DNA vector (restriction enzyme) to be a linear DNA Step.

 In step (d), the step (c) is used except that a standard material vector is used instead of genomic DNA in step (c) as a template for PCR amplification. Restriction enzymes to be treated in the standard vector can be selected from any of the restriction enzymes present in the vector if the vector can be linearized, Clal was used in the embodiment of the present invention.

 <66>

 Since the level after PCR amplification of the detection target in the present invention may vary widely depending on the target sample, a criterion for determining whether to amplify by primer / probe specific to the mutation is necessary. The standard vector is for this purpose

100-350 bp of polynucleotides covering DNA gene mutations can be used transformed into a conventional vector. Preferably, the standard vector of the present invention may be used by inserting about 300bp into the pIDTSmart Amp vector by mutating the exon of the EGFR, that is, the probe position in the center.

 <68>

Preferably, the standard vector of the present invention may be a vector containing a DNA fragment of about 300 bp with each mutation in the exon of the EGFR, that is, the probe position in the center, which is applied to a host cell such as E. coli. After transformation, amplification and extraction can be used. More preferably, the standard vector of the present invention is 1597 of the EGFR gene (genbank accession no. NG_007726) for axon ₁₈ to 100 to 350 bp of polynucleotide at base 100100, for the axon 19, EGFR gene Of polynucleotides 100 to 350 bp at 160501 base in 160501, 100 to 350 bp polynucleotide at 167101 to 167500 base in EGFR gene for axon 20, and 177551 to 177930 bases for EGFR gene in axon 21 for axon 21 100 to 350 bp poly The nucleotide DNA fragment may be inserted into the pIDTSmart Amp vector.

 <70>

 In the present invention, "primer" refers to an oligonucleotide, a nucleic acid chain

 It can serve as a starting point for the synthesis under conditions in which the synthesis of primer extension products complementary to (template) is induced, i.e., the presence of polymerizers such as nucleotides and DNA polymerases, and conditions of suitable temperature and pH. Preferably, the primer is deoxyribonucleotide and single chain. Primers used in the present invention may include natural ly occur ing dNMP (ie, dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural nucleotides. In addition, the primer may also include ribonucleotides.

 <72>

 The primer should be long enough to prime the synthesis of the extension product in the presence of the polymerizer. Suitable lengths of primers are typically 15-30 nucleotides, depending on a number of factors, such as silver, application, and source of the primer. Short primer molecules generally require lower temperatures to form a more complex stable complex with the template. The term "annealing" or "priming" refers to the juxtaposition of oligodioxynucleotides or nucleic acids to a template nucleic acid, where the polymerase polymerizes the nucleotides to complement the template nucleic acid or portion thereof. To form nucleic acid molecules.

<74> ^■

 In the present invention, "probe" is designed as a kind of taqman probe used for quantitative PCR, Preferably, the probe is attached with fluorescent material (HEX, VIC, F 颜 dye), TAMRA can be used as a quencher on the 3 'side. TaqMan probes are generally ol igonuc leotides tagged with the 5' end as the fluorescent material and the 3 'end as the quencher material, and the TaqMan probe is the anneal ing step. Although hybr idi zat ion is specific to template DNA at 3 ', the quencher at the 3' end of the probe does not fluoresce when light is applied, but in the next step, the extension step 5 '→ 3' exonuc 1 possessed by Taq DNA polymerase. When the TaqMan probe with hybr idi zat ion is decomposed by the ease activity, the fluorescence is quantitatively determined by PCR reaction based on the principle that the fluorescent material is separated from the probe, released by the quencher, and fluoresced. Will be released.

<76> <77> Primers and probes specific for genomic DNA gene mutations in the present invention is EGFR

(epidermal growth factor receptor) may be to detect mutations in the gene. Preferably, primers and probes specific for genomic DNA gene mutations are used in the same sequence for the PCR reaction solution and the standard PCR reaction solution, each independently a forward primer of SEQ ID NO: 1, a reverse primer of SEQ ID NO: 2, and SEQ ID NO: 9 Polynucleotide set of probes selected from the group consisting of 13 to 13, forward primer of SEQ ID NO: 3, reverse primer of SEQ ID NO: 4 and polynucleotide set of probes selected from the group consisting of SEQ ID NOs: 14 to 42, forward primer of SEQ ID NO: 5 ， A polynucleotide set of probes selected from the group consisting of reverse primer of SEQ ID NO: 6 and SEQ ID NOs: 43-50, a forward primer of SEQ ID NO: 7, a reverse primer of SEQ ID NO: 8, and a probe selected from the group consisting of SEQ ID NOs: 51 to 54 Robe's Polynucle O lactide may be at least one selected from the group consisting of the set.

<78>

 Step (e) is a step of micronizing the PCR reaction solution of step (e) and the standard PCR reaction solution of step (f) into a plurality of droplets, respectively.

 Before each PCR reaction, each PCR reaction solution is divided into a plurality of small droplets. This micronization process allows each small chamber to be subjected to a subsequent PCR reaction to target each droplet. Depending on whether the DNA is amplified, positive or negative, and the number of copies of the target DNA can be calculated through the Poisson distribution, there is an advantage that does not require a standard curve unlike conventional methods. The micronized microdroplets of the present invention may be about lnl in size, and may be micronized to 10, 000 to 25, 000 for convenience of measurement of PCR reaction and reaction.

 <81>

 Step (f) is a step of performing a PCR reaction so that PCR reaction is performed on each of the plurality of small drops.

PCR reactions are performed using the sample genomic DNA or the DNA of the standard material vector as a template. PCR reaction can be carried out according to methods known in the art, generally should be carried out under conditions that do not cross the primer / probe cross-linking, according to the method of the present invention based on a standard vector (vector) Because the value can be set, the PCR reaction can be performed even under some conditions where crosslinking is allowed. PCR reaction conditions, for example at 95 ^° C for 10 minutes at the enzyme activation reactions and, 94 ^° C 30 seconds, and the cool ing process of one minutes to 40 cycles, 98 ^° at C 10 seconds and 4 ^° C at 60 ^° C PCR can be performed via. <84>

 (G) is a step of measuring the PCR reaction in all or part of each micronized droplet.

 The determination of the PCR reaction can be performed according to methods known in the art, but can be measured by an optical quantitative analysis system using a probe labeled with a reporter fluorescent dye and / or a quencher fluorescent dye. And preferably, by measuring the fluorescence value for the PCR reaction of each micronized droplet.

Specifically, since the FAM, HEX, VIC fluorescent dye (fluorescent material) or EvaGreen fluorescent dye is used in combination with the probe, it may be performed by measuring the fluorescence thereof. This process can be performed by a commercially available detection device (e.g., Biorad's Droplet Reader), which detects the droplet fluorescence signal of each sample and the number of posi- tive and negative drop let, respectively. The count can be automatically completed until the analysis.

 At this time, the probe added to the PCR reaction solution and the probe added to the standard PCR reaction solution for detection may be associated with different fluorescent materials. .

<89>

 Step (h) is a step of calculating the mutation rate (% mutat ion) from the measurement of the PCR reaction in the PCR reaction solution and the standard PCR reaction solution.

 <9i> The mutation rate (% mutat ion) is calculated by comparing the measured values of PCR reactions in the PCR reaction solution and the standard PCR reaction solution by using a mutation rate at a ratio equal to or higher than the threshold value corresponding to the measurement value in the standard PCR reaction solution. (% mutat ion) can be calculated

 <92>

 In step (i), the predictive value predicts that the higher the mutation rate, the higher the therapeutic responsiveness.

 <94>

 The therapeutic responsiveness in the present invention can be defined as "banung" for a therapeutic agent if lung cancer growth rate is inhibited as a result of contact with the therapeutic agent as compared to its growth not in contact with the therapeutic agent. The growth of lung cancer can be measured in various ways, for example, the expression of tumor markers appropriate to the size of the tumor or its tumor type can be measured. In addition, the "banungseong" may indicate a significant increase in the survival time on the survival curve.

Lung cancer is associated with the treatment compared to its growth rate where the growth rate is not in contact with the treatment. As a moist result, it is "non-fungling" for the therapeutic agent if it is inhibited or not to a very low degree. As mentioned above, the growth of lung cancer can be measured in a variety of ways, eg, the expression of tumor markers appropriate to the size of the tumor or its tumor type can be measured. Non-ungular measures may be assessed using additional criteria beyond the growth size of the tumor, including patient quality of life and metastasis.

 <97>

 The treatment response to the lung cancer treatment agent may be treatment responsiveness to an inhibitor of epidermal growth factor receptor (EGFR).

 <99>

 <ioo> EGFR is a protein product of the oncogene erbB or ErbBl. erbB or ErbBl is part of the ERBB family of protooncogenes known to be important factors in numerous cancer developments.

 <101>

 <102> A variety of EGFR target drugs have been developed for the treatment of epithelial cell carcinoma such as lung cancer. OSI Pharmaceuticals, Inc., trade name "TARCEVA") is a representative drug. Zephytinib and erlotinib are quinazoline compounds that inhibit cell growth by inhibiting tyrosine kinase activity of EGFR to inhibit phosphorylation.

 <103>

 For reference, reference may be made to the above-mentioned nucleotide and protein operations (Maniat is et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982)). Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press (1989); Deutscher, M., Guide to Protein Pur ifi cat i Methods Enzymology, vol. 182. Academic Press. San Diego, CA (1990); Ausubel et al., Current Protocols of Molecular Biology, John Wiley and Sons (1997); Rupp and Locker, Lab Invest. 56: A67 (1987); De Andres et al., BioTechniques 18: 42044 (1995); Held et al., Genome Research 6: 986-994 (1996); TE Godfrey et al. J. Molec. Diagnost ics 2: 84-91 (2000); K. Specht et al. , Am. J. Pathol. 158: 419-29 (2001)).

 <105>

Advantageous Effects Therefore, the method of the present invention calculates the therapeutic responsiveness using the blood of the patient who is difficult to obtain the FFPE tissue sample, and can predict and diagnose the patient's therapeutic agent. It can be useful for purposes such as determining the necessity of administration and providing clues about the direction of future treatment.

<107> ^*

 [Brief Description of Drawings]

 1 shows a vector map of a pIDTSmart Amp back jump.

 2 is a conceptual diagram of a CTC separation method of the method of the present invention.

 3 is an example of PCR reaction results in CTC cells according to the method of the present invention.

<111> '

 [Form for implementation of invention]

 Hereinafter, the present invention will be described in detail by way of examples.

 However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

 <114>

 <115> <Example 1>

 <i i6> CTC Separation

 <i i7> CTCs were obtained by CTC separation apparatus based on magnetophores i s blood samples obtained from cancer patients. The conceptual diagram of the CTC separation method by magnetophoresis is shown in FIG. The separation method of the CTC will be described in more detail as follows.

<118>

 <i i9> A microfluidic chip (mi crof luidics chip) prepared for CTC separation was installed in a blood and buffer-injectable CTC separation device. Blood was flowed through the inlets at both ends while flowing the buffer into the center channel. In the blood, an antibody (EpCam ant ibody) attached with a magnetic substance was added to the CTC to specifically bind to the CTC so that the CTC was specifically magnetized. The ferromagnetic material pre-installed on the microfluidics chip is magnetized by the permanent magnet installed in the CTC separation device, and the CTC cells are moved to the central CTC separation channel by the magnetic force generated from the magnetized ferromagnetic material. After the flow of blood, CTC collected on the side of the CTC separation channel was used for subsequent experiments.

 <120>

<121><Example2> <122> Construction of Standard Material Vectors

 A standard vector (named mini-clone) was constructed to validate the designed primers and probes, and to make the standards needed to perform ddPCR. In the process of mini-clone, about 300bp was synthesized by mutating the exon of each EGFR. The synthesized DNA fragment was inserted between the universal link sequence of the pIDTSmart Amp vector (see FIG. 1), and the produced clone was transformed into E. coli DH5a cells.

 <124>

 In order to maximize efficiency in ddPCR (droplet digital PCR) by limiting the standard vector existing in the circular form or the super-coiled form, the restriction enzyme was treated in the standard vector. Standard Vector (Miniclone DNA) Clal restriction enzyme was reacted at 37 ° C for 30 minutes, and the reaction product was quantified and stored at -20 ° C until use.

<126>

 <127> <Example 3>

 <128> Primer / Probe Design and Selection

 <129> Cosmic number to develop a biomarker of EGFR, a lung cancer gene

Mutation sites were identified based on (http://cancer.sanger.ac.uk). The exon primers of the genes were designed so that forward could overlap with the intron part except for EGFR-21 using primer3 program. At this time, the Tm value of the primer was 58 ~ 60 ^° C and GC% was designed as 40 ~ 60%.

 <130>

 Probes were designed as taqman probes by screening those that met conditions.

 HEX / VIC reporter fluorescence was attached to 5 'wild type probe, and FAM dye was attached to 5' mutant probe to detect amplification afterwards. TAMRA was used as quencher on the 3 'side of all probes. 4, 31, 8, and 4 probes were designed and synthesized at EGFR exon 18, 19, 20, and 21, respectively. Probes designed by the inventors had allele specifics and almost all probes had cosmic numbers.

 <132>

The information of the designed primers (Table 1) and probes (Tables 2 and 3) is as follows. <135> [Table 1]

 <136>

<137>

 <138> [Table 2]

WT: wildtype; mt: mutant (hereinafter ^ᅵ same as Table 3)

<139> <140>

 <141> [Table 3]

 <143>

 <Example 4>

 <145> Comparative experiment with licensed product

 <146>

<147> A comparative assay was compared to the sensitivity for the correlation test between the method by the Cobas EGFR mutat i on test kit approved by the KFDA and the method in the present invention. <148>

 Mutation frequencies of 5%, 1%, 0.5%, 0.1%, 0.05%,

 Samples adjusted by dilution in 7 steps up to 0.02% and 0.01% were simultaneously measured by the method according to the Covas EGFR gene mutation test kit and the method in the present invention. Covas EGFR gene mutation testing was performed according to the manufacturer's instructions.

 <150>

 <i5i> samples were tested in three cases. Firstly, Horizon

 DNA extracted from FFPE tissue (exon 19, delE746— A750 (l)) and mutant genomic DNA from Horizon were used.

 <152>

 First, as a result of measuring the minimum mutation frequency using a template spiking gDNA to Miniclone, the minimum measurement result of the Cobas EGFR mutation test was 0.5% to 5%, whereas the method of the present invention In this case, it was confirmed that the test can be performed from 0.02% to 0.1%, and the results of particular note are that the mutation position of 2239_2257> GT cannot be measured by the COVAS EGFR gene mutation test 0.05% according to the method of the present invention. Sensitivity of the result was confirmed to have more than equivalent results compared to the approved method of COVA EGFR gene mutation test.

 <154>

 <155> [Table 4]

<156> <i57> * ddPCR uses 33ng gDNA (10 copies) as template

 4

 <i 58> ** Cobas EGFR mutat ion kit uses 50ng (1.5x10 copies) as template

<159>

<i60> Secondly, the sensitivity analysis was performed for the correlation test using DNA extracted from Hor izon's FFPE tissue and Horizon's mutant genomic DNA. Although measured by the method of the present invention according to the position of the minimum measured value was determined to 0.02% ~ 0.5% value. This indicates that at least ₁₀ times the sensitivity is shown in the licensed product comparison analysis.

<161>

 Table 5 fi. ddPC -bated RUO kit Jj- Cobas Ox U j seat

 R »d (/ ^: Not AyalteUe, X ： Mo test

 <163>

<i64> * ddPCR uses 33ng gDNA (10 ⁴ copies) as template

 4

 ** Cobas EGFR mutat ion kit uses 50ng (1.5xl0 copies) as template

<166>

 <i67> Third, EGFR gene mutations were measured in DNA isolated from human CTC cells. DNA was isolated from the CTC isolated in Example 4, and the mutation was confirmed according to the method of the present invention using the primer / probe of the present invention.

<168>

As a result, as shown in FIG. 3, as in the case of P5 and P44 probes, a small room in which PCR products were amplified due to the presence of mutations was detected, and through this, the CTC also detected EGFR mutations in CTC. It was found that the prediction is possible. <170>

 Industrial Applicability

 <i7i> As described above, the method of the present invention calculates the anticoagulant predictive value using the blood of the patient who is difficult to obtain the FFPE tissue sample, and thus predicts the responsiveness and diagnosis of the patient's therapeutic agent. It can be useful for the purpose of presenting clues about the direction of future treatment, including the necessity of administering anticancer drugs.

Claims

[Range of request]  [Claim 1]  In order to provide information for judging second line treatment by detecting genetic mutation information of a lung cancer patient, a method of calculating a therapeutic response predictive value from a blood sample of a patient includes the following steps:  (a) isolating circulating tumor cells (CTC) from the blood of the patient;  (b) isolating genomic DNA from CTC cells;  (c) preparing a PCR reaction solution comprising a primer and a probe specific for the genomic DNA and genomic DNA gene mutations;  (d) preparing a standard PCR reaction solution containing standard DNA, primers and probes specific for genomic DNA gene mutations, wherein the standard vector is subjected to restriction enzymes to form linear DNA;  (e) micronizing the PCR reaction solution of step (d) and the standard PCR reaction solution of step (f) into a plurality of droplets, respectively;  (f) performing a PCR reaction to perform a PCR reaction on each of the plurality of small droplets;  (g) determining the PCR reaction in all or part of each micronized droplet;  (h) calculating a mutation rate ᅳ mutation) from the measurement of PCR reaction in the PCR reaction solution and the standard PCR reaction solution;  (i) calculating predictions that predict that the higher the mutation rate, the higher the therapeutic responsiveness. [Claim 2]  The method of claim 1, wherein the therapeutic response is a therapeutic response to an inhibitor of EGFR (epidermal growth factor receptor). [Claim 3]  The method of claim 12, wherein the EGFR inhibitor is erlotinib or gefitinib. [Claim 4] The method of claim 1, wherein the genomic DNA gene is an epidermal growth factor receptor (EGFR) gene. [Claim 5]  The method of claim 1, wherein the primers and probes specific for the genomic DNA gene mutation are used in the same sequence for the PCR reaction solution and the standard PCR reaction solution, each independently a forward primer of SEQ ID NO: 1, a reverse primer of SEQ ID NO: 2 And a polynucleotide set of probes selected from the group consisting of SEQ ID NOs: 9 to 13, a forward primer of SEQ ID NO: 3, a reverse primer of SEQ ID NO: 4, and a polynucleotide set of probes selected from the group consisting of SEQ ID NOs: 14 to 42, SEQ ID NO: Polynucleotide set of probes selected from the group consisting of forward primer of SEQ ID NO: 5, reverse primer of SEQ ID NO: 6 and SEQ ID NOs: 43-50, forward primer of SEQ ID NO: 7, reverse primer of SEQ ID NO: 8, and SEQ ID NOs: 51-54 Poly of probe selected from the group consisting of At least one selected from the group consisting of nucleotide sets. [Claim 6]  The method of claim 5, wherein the probe is associated with a fluorescent material. [Claim 7]  The method of claim 5, wherein the probe added to the PCR reaction solution and the probe added to the standard PCR reaction solution are each associated with different fluorescent materials. [Claim 8]  The method of claim 1, wherein the restriction enzyme is Clal. [Claim 9] The method of claim 1, wherein the small droplets are micronized to 10,000 to 25,000. [Claim 10]  The method of claim 1, wherein the measurement of PCR reaction is performed by measuring the fluorescence value for each PCR reaction of each micronized droplet. [Claim 111]  The method of claim 1, wherein the mutation rate (% mutat ion) is calculated by comparing the measured value of the PCR reaction in the PCR reaction reaction solution and the standard PCR reaction reaction solution, the ratio of the threshold value or more corresponding to the measurement value in the standard PCR reaction reaction solution To calculate a ¾ mutat ion. [Claim 12]  The method of claim 1, wherein the method is performed by automation or semi-automation.