KR101800366B1 - Composition for detecting mutations of epidermal growth factor receptor gene and kit comprising the same using cfDNA in plasma - Google Patents

Abstract

The present invention relates to a composition for detecting epithelial cell growth factor receptor gene mutation and a kit comprising the same, and more particularly, to a primer and a probe set composition for detecting epithelial cell growth factor receptor gene mutation, an EGFR gene mutation detection And a kit for detecting an EGFR gene mutation comprising a primer / probe set of a part of the composition of the present invention. The primer / probe set of the present invention can predict the prognosis of the cancer patient's response to the therapeutic agent, not only the diagnosis but also the metastasis or recurrence of the cancer, so it is possible to judge the necessity of the administration of the chemotherapeutic agent, And can be usefully used for purposes of monitoring cancer metastasis or recurrence.

Description

TECHNICAL FIELD The present invention relates to a composition for detecting an epithelial cell growth factor receptor gene mutation using plasma free cucumber DNA (cfDNA), and a kit comprising the same. (Composition for detecting mutations of epidermal growth factor receptor and cfDNA in plasma)

The present invention relates to a composition for detecting epithelial cell growth factor receptor gene mutation and a kit comprising the same, and more particularly, to a primer and a probe set composition for detecting epithelial cell growth factor receptor gene mutation, an EGFR gene mutation detection And a kit for detecting an EGFR gene mutation comprising a primer / probe set of a part of the composition of the present invention.

Cancer refers to a group of abnormal cells caused by continuous division and proliferation by destroying the balance between cell division and death by various causes, and is also called a tumor or neoplasm. It generally develops in more than 100 parts of the body, including organs, white blood cells, bones, lymph nodes, etc., and develops into serious symptoms through the phenomenon of invasion into surrounding tissues and metastasis to other organs.

Cancer therapy is being developed continuously, and dozens of therapies for various cancers currently used in clinical trials are available. However, until now, clinicians are suffering from two difficulties. First, it takes several weeks for the therapeutic effect to take effect. In other words, the therapeutic effects of anticancer drugs can not be judged within a few days, but they gradually appear over several weeks, so it takes a long time to judge the prescription drug to be ineffective. If the treatment is not effective enough, the patient will be considered for other types of treatment. If the clinician can not select the appropriate treatment for the patient at the time of initiation of treatment, the time will be delayed And the prognosis of the disease.

The second difficulty is that there are a number of patients who do not respond to the treatment. For example, lapatinib, a breast cancer treatment, has been shown to have therapeutic effects when high levels of HER2 protein (HER2 positive) and low levels of EGRF protein are present. However, metastatic HER2 negative breast cancer did not respond to lapatinib, indicating that lapatinib was ineffective. These results suggest that patients with breast cancer need to know precisely whether they are HER2 negative or positive before treatment, so that appropriate treatment can be selected.

Therefore, if the therapeutic response to the therapeutic agent and its side effects can be predicted in advance, it will be possible to lower the dropout rate of the drug and improve the compliance of the drug due to the wrong selection of the drug. 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.

EGFR, on the other hand, is a type of protein tyrosine kinase of the erbB receptor family. Upon binding of a growth factor ligand, such as an epidermal growth factor (EGF), the receptor may form a homodimer with another EGFR molecule or another class member such as erbB2 (HER2), erbB3 (HER3 ), Or erbB4 (HER4).

The formation of homologous and / or heterodimeric erbB receptors results in the phosphorylation of key tyrosine residues in the intracellular domain and induces the stimulation of many intracellular signaling pathways involved in cell proliferation and survival. Deregulation of erbB class signaling promotes proliferation, invasion, metastasis, angiogenesis and tumor cell survival and is described in many human cancers, including lung, head and neck cancer and breast cancer.

Therefore, a number of agents that currently represent a reasonable target for the development of anti-cancer drugs and that target EGFR, including zetitib (IRESSA (TM), TARCEVA (TM), are currently clinically applicable. In 2004, activation mutations in EGFR were reported to be correlated with responses to zetti-nip therapy in non-small-cell lung cancer (NSCLC) (Science [2004] Vol. 304, 1497-500 And New England Journal of Medicine [2004] Vol. 350, 2129-39). It is known that the most common EGFR activating mutations, L858R and delE746_A750, are associated with responsiveness to small molecule tyrosine kinase inhibitors such as zetytipine and erotinib as compared to wild-type (WT) wild-type EGFR. Ultimately, acquisition tolerance for therapies using zetti nip or elotinib is caused, for example, by mutation of the gatekeeper residue T790M, which is detected in 50% of clinically resistant patients It has been reported.

Thus, the presence of the EGFR mutation acts as a strong predictor of drug sensitivity in response to EGFR kinase inhibitors such as zetitibe or elotinib, and therefore an effective and rapid detection method of the EGFR mutation is required for an optimal therapeutic approach .

On the other hand, most cancers, although different in timing and frequency, can penetrate into neighboring tissues and migrate through lymphatic vessels or blood vessels, resulting in metastatic spread of tumor cells elsewhere. Metastasis accounts for 90% of cancer deaths. Penetration and metastasis are very complex processes, and their genetic-biochemical factors are not yet well-known. Cancer metastasis is largely divided into lymphatic metastasis and hematogenous metastasis. It is known that most metastasis to the distant organs from the origin of the tumor is due to hematogenous metastasis. When a metastasis occurs in a cancer patient, it is difficult to diagnose which part of the cancer cell is colonized and proliferates, and the prognosis of the disease is not good because the cancer cell can spread rapidly through the blood. Therefore, continuous monitoring is necessary to prevent cancer metastasis or recurrence in cancer patients or patients whose primary treatment of cancer has been completed, and this monitoring should be performed as simple and quick as possible without infringing the quality of life of the patient There is a need to be.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

Therefore, there is a need to detect EGFR mutation easily and rapidly, to select a drug therapy strategy for EGFR-related cancer patients, or to use as a monitoring means to prevent cancer metastasis or recurrence. The present invention has been completed based on the finding of a primer / probe set suitable for application to cfDNA in a patient's blood, and a kit suitable for the primer / probe set.

Accordingly, an object of the present invention is to provide a primer and a probe set composition for detecting an epidermal growth factor receptor (EGFR) gene mutation.

Another object of the present invention is to provide a kit for detecting mutation of EGFR gene comprising a composition of the present invention and a kit for detecting mutation of EGFR gene comprising a partial primer / probe set of the composition of the present invention.

In order to accomplish the above object, the present invention provides primer and probe set composition for detecting epidermal growth factor receptor (EGFR) gene mutation.

In order to accomplish another object of the present invention, there is provided a kit for detecting mutation of EGFR gene comprising a composition of the present invention and a primer / probe set of a part of the composition of the present invention.

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

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

The present invention relates to a primer and a probe set composition for detecting an epidermal growth factor receptor (EGFR) mutation, comprising a forward primer of SEQ ID NO: 3, a reverse primer of SEQ ID NO: 4, a forward primer of SEQ ID NO: The reverse primer of SEQ ID NO: 10, the reverse primer of SEQ ID NO: 10, the forward primer of SEQ ID NO: 13, the reverse primer of SEQ ID NO: 14 and the reverse primer of SEQ ID NO: 1, the reverse primer of SEQ ID NO: 2, the forward primer of SEQ ID NO: 5, the reverse primer of SEQ ID NO: 6, and the reverse primer of SEQ ID NO: 15 to SEQ ID NO: 17, 24 to 27, The reverse primer of SEQ ID NO: 5, the reverse primer of SEQ ID NO: 6, and the reverse primer of SEQ ID NO: 6 and the polyline of the probe selected from the group consisting of SEQ ID NOS: 28, 29, A polynucleotide comprising at least one polynucleotide selected from the group consisting of nucleotide sets.

The composition of the present invention may preferably be for predicting the responsiveness of an EGFR inhibitor to treatment. In 2004, a mutation in EGFR was reported to correlate with the response to zepitibem therapy in non-small-cell lung cancer (NSCLC) (Science [2004] Vol. 304, 1497-500 New England Journal of Medicine [2004] Vol. 350, 2129-39), many relevant EGFR mutations have been identified. The EGFR inhibitor may be, but is not limited to, erlotinib, gefitinib or Afatinib.

In the present invention, the term " primer " means an oligonucleotide in which the synthesis of a primer extension product complementary to a nucleic acid chain (template) is induced, that is, the presence of a polymerizing agent such as a nucleotide and a DNA polymerase, It can act as a starting point for synthesis under pH conditions. Preferably, the primer is a deoxyribonucleotide and is a single strand. The primers used in the present invention may include naturally occurring dNMPs (i.e., dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural nucleotides. In addition, the primers may also include ribonucleotides.

The primer should be long enough to be able to prime the synthesis of the extension product in the presence of the polymerizing agent. The suitable length of the primer is determined by a number of factors, such as the temperature, the application, and the source of the primer, but is typically 15-30 nucleotides. Short primer molecules generally require lower temperatures to form a sufficiently stable hybrid complex with the template. The term " annealing " or " priming " means that the oligodeoxynucleotide or nucleic acid is apposited to the template nucleic acid, which polymerizes the nucleotide to form a complementary nucleic acid molecule to the template nucleic acid or a portion thereof .

In the present invention, " probe " is designed as a kind of taqman probe used for quantitative PCR. Preferably, a fluorescent material (HEX, VIC, FAM dye) is attached to the probe, and BHQ1 can be used as a quencher on the 3 'side of all the probes. The TaqMan probe is an oligonucleotide tagged with the 5 'end as a fluorescent substance and the 3' end as a quencher. The TaqMan probe specifically hybridizes to the template DNA in the annealing step, but there is a quencher at the 3 'end of the probe However, if the TaqMan probe hybridizes to the template due to the 5 '→ 3' exonuclease activity of the Taq DNA polymerase in the next step of extension, the fluorescent material is separated from the probe and is inhibited by the quencher And fluorescence is emitted quantitatively by the principle of the fluorescence emitted by the PCR reaction.

The primers and probes specific for the genomic DNA gene mutation in the present invention may be one for detecting mutations in the epidermal growth factor receptor (EGFR) gene. Preferably, 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, and each independently have the forward primer of SEQ ID NO: 3, the reverse primer of SEQ ID NO: 4, The reverse primer of SEQ ID NO: 8, the polynucleotide set of the probe selected from the group consisting of SEQ ID NOs: 18 to 23, 30, the forward primer of SEQ ID NO: 9, the reverse primer of SEQ ID NO: 10, the forward primer of SEQ ID NO: A reverse primer of SEQ ID NO: 14 and a polynucleotide set of a probe selected from the group consisting of SEQ ID NO: 31, 36 to 38, a forward primer of SEQ ID NO: 1, a reverse primer of SEQ ID NO: 2, a forward primer of SEQ ID NO: Reverse primers and SEQ ID NOs: 15 to 17, 24 to 27 A forward primer of SEQ ID NO: 12, a reverse primer of SEQ ID NO: 12, a forward primer of SEQ ID NO: 5, a reverse primer of SEQ ID NO: 6, and a group consisting of SEQ ID NOs: 28, 29 and 32 to 35 And a set of polynucleotides of the probe selected in SEQ ID NO.

The measurement of the PCR reaction can be performed according to a method 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, Preferably by measuring the fluorescence value for each PCR reaction of each undifferentiated droplet.

Specifically, since the probe is combined with FAM, HEX, VIC fluorescent dye (fluorescent substance) or EvaGreen fluorescent dye, it can be performed by measuring fluorescence thereon. This process can be performed by a commercially available detector (for example, Droplet Reader from biorad), which detects the droplet fluorescence signal of each sample in the apparatus, counts the number of positive and negative droplets, and automatically Analysis can be completed.

In this case, probes to be added to the PCR reaction solution and probes to be added to the standard PCR reaction solution for detection may be respectively associated with different fluorescent materials.

Meanwhile, the present invention provides a kit for detecting EGFR gene mutation comprising the primer / probe set of the present invention.

The kit of the present invention may further include an oligomer (blocker) designed to prevent non-specific binding of a mutation detection probe to a wild-type sequence using a wild-type sequence corresponding to a mutation position to reduce background noise have.

The kit of the present invention can be preferably used for the detection of mutations of the EGFR gene by PCR reaction using the primer / probe set of the present invention. The kit of the present invention may further comprise tools and / or reagents known in the art used for PCR or detection thereof. The kit of the present invention may further comprise a tube, a well plate, an instructional material describing a method of use and the like to be used for mixing the components as necessary.

In addition, the kit of the present invention may be a research use only (RUO) or an investigation use only (IVD) kit. IVD kits also include IVD-CDx kits.

The reverse primer of SEQ ID NO: 7, the reverse primer of SEQ ID NO: 8, and the poly primer of the probe selected from the group consisting of SEQ ID NOS: 18 to 21, 23 and 30, A kit for detecting EGFR gene mutation comprising a set of nucleotides as an active ingredient.

The present invention also provides a polynucleotide set of a probe selected from the group consisting of the forward primer of SEQ ID NO: 9, the reverse primer of SEQ ID NO: 10, the forward primer of SEQ ID NO: 13, the reverse primer of SEQ ID NO: 14, As an active ingredient, an EGFR gene mutation detecting kit.

The present invention also provides a polynucleotide set of the forward primer of SEQ ID NO: 1, the reverse primer of SEQ ID NO: 2, the forward primer of SEQ ID NO: 5, the reverse primer of SEQ ID NO: 6, and the probes selected from the group consisting of SEQ ID NOs: 15 to 17 and 40 A kit for detecting EGFR gene mutation which is contained as an active ingredient.

The present invention also provides a polynucleotide set of a probe selected from the group consisting of the forward primer of SEQ ID NO: 11, the reverse primer of SEQ ID NO: 12, the forward primer of SEQ ID NO: 13, the reverse primer of SEQ ID NO: 14, As an active ingredient, an EGFR gene mutation detecting kit.

The kit of the present invention is used in qPCR (quantitative PCR) or digital PCR method.

The template applicable to the kit of the present invention can be used without restriction as long as mutation detection of EGFR is necessary and PCR reaction is possible. Preferably, the kit of the present invention comprises DNA isolated from FFPE (formalin fixed paraffin embedded) A cDNA synthesized from a tissue-derived RNA by a reverse transcription reaction, or a cfDNA (cell-free DNA) isolated from blood as a template.

CfDNA circulating in human plasma has been studied in a variety of physiological and pathological conditions such as inflammatory disorders, oxidative stress and malignant tumors. The precise mechanism involved in the release of cfDNA into the bloodstream is unclear, but seems likely to be a synergistic effect of apoptosis, cellular necrosis and active release from cells. CfDNA circulating through blood vessels is a potentially useful biomarker. DNA levels and fragmentation patterns present interesting potential for diagnostic and prognostic prediction purposes. In particular, the biomarker can be detected easily and rapidly without deteriorating the quality of life because the biomarker can be easily detected from the patient's blood.

The tissue obtained from the patient after biopsy is usually fixed with formalin (formaldehyde) or the like. The immobilized biological sample is generally dehydrated and embedded in a solid support such as paraffin, and the sample thus prepared is called an FFPE sample. Nucleic acids in the FFPE sample, especially DNA, are present in immobilized cells and are either fragmented or cross-linked by formalin, so it is necessary to remove the paraffin and dissolve the fixed cells to elute the DNA and other nucleic acids in the cells.

In the present invention, the term " paraffin " comprehensively refers to a foraging medium of a biological sample used in all analyzes including morphological, immunohistochemical and enzymatic histochemical analysis. That is, the paraffin in the present invention may be a petroleum-based paraffin wax alone, and may be any one selected from the group consisting of all kinds of petroleum-based paraffin waxes, May be included. Herein, the petroleum paraffin wax refers to a mixture of hydrocarbons which are solid at room temperature derived from petroleum.

In general, a specimen of a cancer patient treated with FFPE is cut into a thickness of 5 to 10 μm using a rotary microtome, and then a nucleic acid containing DNA is isolated through a commercially available nucleic acid separation kit for FFPE or a device utilizing the same . Kits / devices for separating nucleic acids from FFPE include, for example, the Tissue Preparation System from Siemens and VERSANT tissue preparation reagents.

In addition, the kit of the present invention can be used as a template for DNA isolated from blood circulating CTC (circulating tumor cell) or cDNA synthesized by reverse transcription from the CTC-derived RNA. CTC is a tumor cell found in the peripheral blood of a malignant tumor patient. Because CTC plays an important role in the process of metastasis, CTC is considered to be crucial in the study and diagnosis of cancer, but the number of circulating tumor cells in the peripheral blood is very rare, and there are fewer than a dozen There is a need for a detection system that requires a degree of sensitivity to detect tumor cells.

Therapeutic reactivity in the present invention can be defined as "responsive" for a therapeutic agent if the growth rate is inhibited as a result of contact with the therapeutic agent compared to its growth in the absence of contact with the therapeutic agent. The growth of cancer can be measured in various ways, for example, the expression of a tumor marker suitable for the size of the tumor or its tumor type can be measured. In addition, the above " reactivity " may indicate an increase in survival time on a significant survival curve.

Cancer is "non-responsive" to the therapeutic agent unless the growth rate is suppressed or suppressed to a very low extent as a result of contact with the therapeutic agent as compared to its growth not in contact with the therapeutic agent. As noted above, cancer growth can be measured in a variety of ways, for example, the expression of tumor markers suitable for the size of the tumor or its tumor type can be measured. Non-responsive measures can be assessed using additional criteria beyond the growth size of the tumor, including the patient's quality of life, metastasis, and the like.

The therapeutic response to cancer therapy may be therapeutic response to an inhibitor of epidermal growth factor receptor (EGFR), and thus the cancer of the present invention may be lung cancer, breast cancer, bladder cancer, stomach cancer.

EGFR is a protein product of the oncogene erbB or ErbB1. ErbB or ErbB1 is one of the ERBB family of protooncogenes, which is known to be an important factor in the development of many cancers. It has been observed that the expression of EGFR is increased in breast cancer, bladder cancer, stomach cancer and the like, including lung cancer.

A variety of EGFR target drugs have been developed to treat epithelial cancers such as lung cancer, breast cancer, bladder cancer and stomach cancer, and in particular, have been developed for the treatment of various EGFR target drugs such as Gefitinib (AstraZeneca UK Ltd., trade name "IRESSA"), Erlotinib , Inc. and OSI Pharmaceuticals, Inc., trade name "TARCEVA") and Afatinib (Boehringer Ingelheim GmbH corp., Trade name "GIOTRIF"). Zetytipine and elotinib are quinazoline compounds that inhibit tyrosine kinase activity of EGFR and inhibit phosphorylation thereby inhibiting cell growth.

The nucleic acid isolated from the sample of the present invention is preferably a genomic DNA, more preferably a genomic DNA presumed to have a mutation.

The compositions or kits of the invention can preferably be used for EGFR mutation detection in automated or semi-automated methods. In the above, automation can be achieved by injecting a sample (sample); Relocation or movement of a substrate (e.g., tube, plate) that has been extracted, separated, or reacted; Reagent, buffer stock, replenishment; Means that all or most of the process, except equipment maintenance, takes place through means other than human (for example, a robot).

The composition or kit of the present invention showed excellent mutation detection ability in detecting each mutation in the EGFR gene mutation, which was superior to conventional comparative products.

For reference, the above-mentioned nucleotide and protein work can be referred to the following references (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982); Sambrook et al Inc., San Diego, Calif. (1990), < RTI ID = 0.0 > Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press De Andres et al., BioTechniques 18: 42044 (1995); Held et al., ≪ Diagnostic 2: 84-91 (2000); K. Specht et al., Am. J. Pathol., 158: 419 -29 (2001)).

The primer / probe set of the present invention can predict the prognosis of the cancer patient's response to the therapeutic agent, not only the diagnosis but also the metastasis or recurrence of the cancer, so it is possible to judge the necessity of the administration of the chemotherapeutic agent, And can be usefully used for purposes of monitoring cancer metastasis or recurrence.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the constitutions (MMX 1 to 4) of an EGFR mutation detection kit comprising a probe and a primer of the present invention, and shows probes contained in each MMX and the kinds of detectable EGFR mutations.
Figure 2 shows the result of linearity of the kit sensitivity by diluting the mutation rate in order to measure the analytical sensitivity of the EGFR mutation detection kit comprising the probe and the primer of the present invention (A: MMX 1, B: MMX 2) .
FIG. 3 shows the results of linearity of the kit sensitivity by diluting the mutation rate in order to measure the analytical sensitivity of the EGFR mutation detection kit comprising the probe and the primer of the present invention (A: MMX 3, B: MMX 4) .
FIG. 4 shows the results of confirming whether the EGFR mutation detection kit containing the probe and the primer of the present invention can detect 42 kinds of EGFR mutation sites and detecting only EGFR mutation sites contained in each MMX A: MMX 1, B: MMX 2).
FIG. 5 shows the results of confirming whether the EGFR mutation detection kit containing the probe and primer of the present invention can detect 42 kinds of EGFR mutation sites and detecting only EGFR mutation sites contained in each MMX A: MMX 3, B: MMX 4).
6 is a result of detecting mutation in a reference FFPE DNA using the EGFR mutation detection kit comprising the probe and the primer of the present invention (A: exon 19 E746-A750 del detection, B: L858R detection, C: T790M detection, D: G719S detection).
FIG. 7 shows the results of detection of EGFR mutation from cfDNA in one of the clinical patients (patient 5, patient # 5) using the EGFR mutation detection kit comprising the probe and the primer of the present invention.
FIG. 8 is a table summarizing the results of detection of EGFR mutation from cfDNA of 9 clinical patients and detection of EGFR mutation from FFPE using an EGFR mutation detection kit containing the probe and primer of the present invention (A: Result comparison table, B: ddPCR result data)
Fig. 9 shows the results of evaluating the equivalence of the EGFR mutation detection kit containing the probe and primer of the present invention and the licensed product Cobas (R) EGFR kit (Roche Molecular Diagnostics) using the FFPE sample as a clinical sample (d: MMX1, B: MMX2).
10 shows the results of evaluating the equivalence of the EGFR mutation detection kit containing the probe and the primer of the present invention and the licensed product Cobas® EGFR kit (Roche Molecular Diagnostics) using the FFPE sample as a clinical sample through ddPCR (A: MMX3, B: MMX4).

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

≪ Example 1 >

Isolation of cell-free DNA (cfDNA) from plasma

Plasma cfDNA was isolated using a circulating nucleic acid production kit (QIAGEN) according to the manufacturer's recommendations. The blood of the patient was centrifuged at 3000 rpm for 10 minutes, and 1 ml of plasma was transferred to a 15 ml tube while taking care of the cell debris. To this sample, 800 μl of the lysis buffer and 100 μl of the reconstituted protease K were mixed. After brief vortexing, the tubes were incubated at 60 DEG C for 30 minutes. 1.8 ml of binding buffer is mixed into the sample, vortexed briefly, and reacted on ice for 5 minutes. After attaching the vacuum connection tube provided in the circulating nucleic acid kit to the vacuum pump, the column and the passing fraction tube were connected to the upper part of the vacuum connection tube. The sample was put into a passing fraction tube and a vacuum pump was run to pass the sample in the column. To wash the column, 700 μl of washing solution was put in the passing fraction tube, and the vacuum pump was operated again to wash the column. The passing fraction tube and the vacuum connector tube were discarded and the filter was attached to a new collection tube. And centrifuged at 14000 rpm for 3 minutes to completely remove the washing solution in the column. After centrifugation, the column was coupled to a new collection tube and the column was dried at 56 < 0 > C for 10 minutes. The column was transferred to a new 1.5 ml microtube, and 50 용 of elution buffer was added to the center of the column, followed by incubation at room temperature for 1 minute. Thereafter, the DNA sample was eluted by centrifugation at 14000 rpm for 1 minute at room temperature. It was stored at 4 ° C until use or at -70 ° C for long-term storage.

≪ Example 2 >

Production of standard material vector

Standard material vectors (named mini-clones) were constructed to validate designed primers and probes, and to make the reference material required for ddPCR performance. The mini-clone was first synthesized in about 300 bp of the EGFR mutation region, ie, the probe site in the middle of each exon. The synthesized DNA fragment was inserted between the universal link sequence of pIDTSmart Amp vector and the clone was transformed into E. coli DH5α cells.

In order to maximize the efficiency of ddPCR (droplet digital PCR) by linearizing a standard material vector in a circular form or a super-coiled form, the restriction enzyme was treated with a reference material vector. Standard vector (Miniclone DNA) ClaI restriction enzyme was reacted at 37 ° C for 30 min. After the reaction product was quantified, it was stored at -20 ° C until use.

In the present invention, the level after PCR amplification of an object to be detected may be entirely different depending on the sample to be tested, so a criterion for discriminating whether amplification by a primer / probe specific for a mutation is necessary. For this purpose, the reference material vector can be obtained by transforming a polynucleotide of 100 to 350 bp covering a genomic DNA gene mutation into an ordinary vector. Preferably, the reference material vector of the present invention can be used by inserting about 300 bp in the pIDTSmart Amp vector in the region where the mutation occurred in each exon of EGFR, i.e., the probe position as the center.

Preferably, the reference material vector of the present invention may be a vector containing a DNA fragment of about 300 bp in which a mutation has occurred in each exon of EGFR, that is, a probe position in the middle, which is transformed into a host cell such as Escherichia coli Amplification, and extraction. More preferably, the reference material vector of the present invention is a polynucleotide of 100 to 350 bp in position 159701 to 159701 of the EGFR gene in the case of exon 18, in the case of exon 19, 160501 of EGFR gene 100 to 350 bp polynucleotide at 160900 base, exon 20, 100 to 350 bp polynucleotide at 167101 to 167500 base, exon 21 of EGFR gene, 100 to 350 bp at 177551 to 177930 base of EGFR gene, The polynucleotide DNA fragment of 350 bp may be inserted into the pIDTSmart Amp vector.

≪ Example 3 >

Primer / probe design and selection

In order to develop a biomarker for EGFR, a lung cancer-related gene, the mutation position was confirmed based on the cosmic number (http://cancer.sanger.ac.uk). Primer 3 primers for each gene were designed using a primer 3 program, and the rest except for EGFR-21 was designed so that the forward region overlap with the intron region. At this time, the Tm value of the primer was 58 ° C to 60 ° C and the GC% was designed to be 40% to 60%.

The probes were designed with the taqman probe to select those that met the criteria. HEX / FAM reporter fluorescence was attached to the 5 'wild type prolove, and FAM dye was attached to the 5' mutant probe to detect the amplification. BHQ1 was used as a quencher on the 3 'side of all probes. 3, 12, 6, and 3 probes were designed and synthesized on EGFR exons 18, 19, 20, and 21, respectively. The probes designed by the present inventors have allele specificity and most probes have cosmic numbers.

In order to reduce the background noise, a blocker oligomer was constructed so that the probe for mutation detection could not be non-specifically bound to the wild type sequence by using a wild type sequence corresponding to the mutation position.

The information of the designed primer (Table 1), probe (Table 2), and blocker oligomer (Table 3) are as follows.

<Example 4>

Mutation detection ability of primer and probe

The QX200 ™ Droplet digital PCR system (Bio-RAD, USA) was used as a ddPCR instrument. For the preparation of the samples, 20 μl of each was added to 8-strip PCR tubes containing MMX1, MMX2, MMX3 and MMX4 mixture. Ultrapure water (NTC), positive control (PC) and template DNA . Information on the MMX1, MMX2, MMX3 and MMX4 mixtures is shown in Table 4 below. Specific information on primers and probes contained in each MMX is shown in Tables 5 and 6 below.

8-Strip PCR tubes were capped, vortexed, spin down and incubated at room temperature for 10 min. The prepared samples were loaded into a sample well of a cartridge by taking 20ul of 8-Strip PCR tube using an 8-channel Electronic Pipette as a droplet generation step. Droplet Generation Oil was loaded into the oil loading well of the cartridge by 70ul each, and the droplet generator gasket was mounted on the top and bottom of the gasket, and the QX200 ™ Droplet Generator was put into operation. Droplet generated in the process of droplet generation was transferred to a 96-well plate using an 8-channel multi-pipet, and the plate was covered with a red line of Pierceable Foil Heat Seal (BIO-RAD, 181-4040) ™ PCR Plate Sealer (180 ° C, 5 sec. Sealing) and sealed. PCR reaction Verity 96-Well Thermal Cycler was used. After PCR reaction, amplified PCR product was separated into FAM and HEX. The resulting product was performed with QuantaSoft® sofware supplied by Bio-RAD.

On the other hand, the EGFR mutation sites which can be detected by the MMXs 1 to 4 shown in Table 4 are shown in Fig.

As shown in Fig. 1, the EGFR mutation detection kit (hereinafter referred to as &quot; EGFR IVD kit &quot;) of the present invention includes all of the MMXs 1 to 4, and each construct can detect 42 mutation sites of EGFR Respectively. They can also distinguish mutations with FAM and HEX. For example, we used a wild-type HEX probe to measure the quality of a sample with FAM at 19 del in MMX1 of FIG. Therefore, mutation types were distinguished by FAM and HEX, respectively.

In addition, based on the data published in the previous paper, mutation sites related to the efficacy of EGFR tyrosine kinase inhibitor medicines (elotinib, zetitib and apatipin) were shown, and the red color was EGFR mutant And yellow indicates the EGFR mutation that is effective for the drug.

<4-2> EGFR mutation detection ability

Human genomic DNA (Promega corp, USA) was used as a non-template control (NTC) and positive control (PC) as a negative control (NC) to verify the performance of the EGFR IVD kit described in Table 4 above. 40 miniclone were used. The reason for using 40 reference materials is that the EGFR kit can detect 42 mutation sites, but three sites contain the same sequencing.

To identify the sensitivity of the EGFR mutation detection kit, each miniclone was spiked with gDNA used as PC to dilute to 1, 0.5, 0.1 and 0.02%.

The results are shown in Fig.

As shown in FIG. 2, the sensitivity of the PCR mixture mixed with MMX 1 to 4 was as high as 0.02% in the 24 mutation sites, and the remaining 16 mutation sites were detected in 0.1% .

That is, the minimum detection concentration of the EGFR IVD kit was 0.1 to 0.02%.

&Lt; Example 5 >

Mutation detection test of EGFR IVD kit

40 standard reference materials were used to determine whether the EGFR IVD kit of the present invention could detect 42 mutation sites of EGFR exons 18 to 21. In addition, considering the heterozygote, one of the features of cancer, miniclone was substituted with wildtype gDNA %. &Lt; / RTI &gt; The amount of DNA used in this test was 12 ng / sample, and all 40 samples were reacted with MMX1-4 respectively to perform ddPCR.

The results are shown in Fig.

As shown in FIG. 3, only the EGFR mutations detectable by MMX 1 to 4 were detected, and no cross-reaction was observed between the mixturer containing probes. In FIG. 3, a cut-off is set based on NTC and gDNA. When blue or green droplets are detected, a mutation is detected. In FIG. 3, the type of mutation indicated by each blue or green droplet Lt; RTI ID = 0.0 &gt; 1 &lt; / RTI &gt;

&Lt; Example 6 >

Mutation detection in reference FFPE DNA using EGFR IVD kit

The EGFR IVD kit of the present invention is applicable to ddPCR. On the other hand, the advantage of ddPCR is that absolute quantification is possible, unlike qPCR. In other words, we can calculate the number of DNA copies by converting the amount of inserted DNA into genome equivalence (GE). Therefore, ddPCR was performed to demonstrate the performance of the EGFR IVD kit using FFPET (Horizon, UK) with an accurate mutation frequency.

We used Horizon reference FFPET, which contains exon 19del, L858R, T790M and G719S as a multi-plex, and diluted to 1/2 as well as the existing mutant frequency for more accurate quantitative analysis.

The results are shown in Fig.

As shown in FIG. 6, the results of the experiments showed that the ddPCR-based EGFR IVD kit was highly accurate. That is, the X axis in FIG. 6 represents the tumor content of the FFPE sample, and the Y axis of the graph represents the number of mutations detected by the EGFR IVD kit of the present invention. 1000GE backgroud means 1000 copies. Therefore, it can be seen that the EGFR IVD kit of the present invention detects a copy number almost identical to the tumor% contained in each FFPE sample.

Based on the above results, it can be confirmed that the EGFR IVD kit is a kit capable of EGFR mutation analysis by semi-quantitation according to each mutation frequency with a reference FFPE that can quantitatively determine the number of copies.

&Lt; Example 7 >

Comparison of results using cfDNA or FFPE samples

We compared the results obtained with the PNA technique and the sanger sequencing method using FFPE samples obtained from patients such as ddPCR analysis of cfDNA isolated from blood. That is, blood samples were taken from a patient who had metastasis (stage 4) or recurrence (no stage) in a local hospital that was contracted with IRB to see if cfDNA extracted from Plasma could be detected during the performance test of the EGFR IVD kit. In the hospital, the patient's tissues (FFPE) were used for PNA or sequencing, but the blind test was performed to compare the results.

The collected blood was centrifuged at 3000 rpm for 10 minutes, and cfDNA was isolated using a QIAamp Circulating Nucleic acid kit in 1 ml of plasma. The isolated cfDNA had an average concentration of 0.7 ng / ul and used double 3 ul for detection with the EGFR IVD kit according to the present invention. This shows that the EGFR IVD kit according to the present invention is capable of detecting EGFR mutation even at a sample amount of about 2 ng.

The results are shown in Fig.

As shown in Figure 8, a sample of nine clinical patients was tested with the EGFR IVD kit and the EGFR mutation detected in cfDNA was consistent with the EGFR mutation detected in the FFPE sample. That is, the EGFR IVD kit according to the present invention can detect EGFR mutations not only in the FFPE sample but also in the cfDNA isolated from the patient's blood.

In addition, although conventional EGFR-licensed kits require about 25-50 ng per reaction, the EGFR IVD kit of the present invention can detect EGFR mutations in samples of 3 ng or less than 1.5 ng, Lt; RTI ID = 0.0 &gt; EGFR &lt; / RTI &gt;

&Lt; Example 8 >

Assessment of equality with licensed products

To obtain clinical samples from the hospitals to which the IRB was accorded and to demonstrate the performance of the EGFR IVD kit, ddPCR was performed for the equality test with the licensed product Cobas® EGFR kit (Roche Molecular Diagnostics).

The results are shown in Fig. 9 and Table 7 below.

With reference to FIG. 9, the X-axis is NTC, gDNA, PC, and patient samples as a sample, and cut-off based on gDNA (negative control) can be regarded as more positive than fluorescence seen above.

The green color in MMX1 indicates the presence of the PCR reaction by measuring the quality of the sample. In the MMX1 sample, the FAM-labeled sample indicates the exon 19del mutant and the MMX2 HEX indicates the T790M-detected patient.

As shown in FIG. 9 and Table 7, patient samples detected with exon 19 del mutation when detected with Cobas® EGFR kit were used, and EGFR IVD kit was used to detect all samples with Cobas® EGFR kit And the exon 19 del mutation was detected in the same manner as the result. Thus, it was confirmed that the accuracy of the EGFR IVD kit was excellent.

As described above, the primer / probe set of the present invention is capable of predicting the reactivity of a cancer patient to a therapeutic agent, not only diagnosis but also cancer metastasis or recurrence, For the purpose of presenting clues and for monitoring the transfer or recurrence of cancer.

<110> Gencurix Inc.          Seoul National University R & DB Foundation <120> Composition for detecting mutations of epidermal growth factor          receptor gene and kit comprising the same using cfDNA in plasma <130> NP15-0029 <160> 44 <170> Kopatentin 2.0 <210> 1 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Primer (C1-F1) <400> 1 tgaggatctt atgcgaa 17 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> Primer (C1-R1) <400> 2 ctgtgcatgc cagggacctt 20 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer (C5-F15) <400> 3 gatcccaatg cgaaggtgag aaa 23 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer (C5-R18) <400> 4 cagcatgcaa agcagaaact ca 22 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer (C29-F1) <400> 5 aaaattcccg tcgcaatatc aa 22 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer (C29-R1) <400> 6 aggttcagag caacatggac 20 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer (C36-F1) <400> 7 ccctccagat gcgaagcc 18 <210> 8 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Primer (C36-R1) <400> 8 cagccgatgc aaggg 15 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer (C36-F18) <400> 9 atctgcctca catgcctc 18 <210> 10 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer (C36-R16) <400> 10 tttgtgttcc atgccata 18 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer (C36-F17) <400> 11 ccacactaat tgacgtgcc 19 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer (C36-R14) <400> 12 ggtggaaatt ggtgaggc 18 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer (C44-F1) <400> 13 acaccgcagc aattatgtca 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer (C44-R1) <400> 14 tgcctcatgc cttctgcatg 20 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe (EP2-2) <400> 15 tcaaagtgct aatttccggt gc 22 <210> 16 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Probe (EP3-3) <400> 16 aaaagatcaa agtatgcagc tccg 24 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Probe (EP4-3) <400> 17 aaaagatcaa agtgctgaat tccg 24 <210> 18 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Probe (EP22-1) <400> 18 catgcctatc aaggaatcga aagcca 26 <210> 19 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Probe (EP31-1) <400> 19 ccgtcatgct caagtatctc cgaaagcca 29 <210> 20 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Probe (EP33-1) <400> 20 ccgtcgctaa tgcaattccg aaagccaaca 30 <210> 21 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Probe (EP20) <400> 21 cgctatcaag gaaatgcaag ccaaca 26 <210> 22 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Probe (EP26) <400> 22 ttccaattgc tatcaaggtt gctt 24 <210> 23 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Probe (EP28) <400> 23 tcccgtcatg ctcgcaacat ctccga 26 <210> 24 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Probe (EP15-) <400> 24 ttcccgtcgc taaatggagc aat 23 <210> 25 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Probe (EP19) <400> 25 aggaaccaac atctaattaa gccaa 25 <210> 26 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Probe (EP21) <400> 26 cgctatatgc gaatcatctc cgaaagc 27 <210> 27 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Probe (EP32) <400> 27 cgctatcaaa atgccatctc cgaaagc 27 <210> 28 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Probe (EP30) <400> 28 cgctaatgca attccaacat ctccgaa 27 <210> 29 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Probe (EP35) <400> 29 caaggaacat gcgaaagcca acaagga 27 <210> 30 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe (EP36) <400> 30 gcatctgcat gccctccacc gt 22 <210> 31 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Probe (EP37-Y) <400> 31 catgagatgc atgatgagct gca 23 <210> 32 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe (EP38-2) <400> 32 tacgtgaatg ccatcgtgga ca 22 <210> 33 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe (EP40-2) <400> 33 tggacaaccc atgccacgtg t 21 <210> 34 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Probe (EP41) <400> 34 cgtggacggt aacatggacg tgt 23 <210> 35 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Probe (EP42-2) <400> 35 caattgtgga cagcgtgg 18 <210> 36 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe (EP45) <400> 36 cacaatgctt gggcgggcca a 21 <210> 37 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe (EP46) <400> 37 ccaaacagat gctgcggaag a 21 <210> 38 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Probe (EP47) <400> 38 gattttggat gcgccaaact gctg 24 <210> 39 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe (EP47-3) <400> 39 ttttgggcgt gccccaaact g 21 <210> 40 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Probe (EP29) <400> 40 agagaagcaa cccactcgat gtgagttt 28 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe (EP46-3) <400> 41 tggccaacca cagctgggtg 20 <210> 42 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Probe (E-ex19-B1) <400> 42 cgtcgctatc aaccggaatt aagagaagca 30 <210> 43 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe (E-ex21-B1) <400> 43 gattttgggc tgccgccaaa ct 22 <210> 44 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe (E-ex18-B1) <400> 44 aagtgctggg ccctccggtg 20

Claims (18)

i) the forward primer of SEQ ID NO: 9, ii) the reverse primer of SEQ ID NO: 10, iii) the forward primer of SEQ ID NO: 13, iv) the reverse primer of SEQ ID NO: 14 and v) (EGFR), which is used for qPCR (quantitative PCR) or digital PCR method, is used as a template and a cell-free DNA (cfDNA) separated from blood is contained as an active ingredient in a set of polynucleotides of probes. ) &Lt; / RTI &gt; primer and probe set composition for detecting gene mutation.
The primer and probe set composition according to claim 1, wherein the EGFR mutation detection is for predicting the responsiveness to an EGFR inhibitor.
3. The primer and probe set composition of claim 2, wherein the EGFR inhibitor is selected from the group consisting of erlotinib, gefitinib, and Afatinib.
2. The primer and probe set composition according to claim 1, wherein the probe is bound to a fluorescent material.
5. The primer and probe set composition according to claim 4, wherein the fluorescent material is at least one selected from the group consisting of HEX (hexachlorofluorescein), FAM (fluorescein amidite) and EverGreen dye.
A research use only (RUO) kit for detecting EGFR gene mutation comprising the primer and probe set composition of claim 1 as an active ingredient.
An IVD (investigation use only) kit for detecting EGFR gene mutation comprising the primer and probe set composition of claim 1 as an active ingredient.
A reverse primer of SEQ ID NO: 13, a reverse primer of SEQ ID NO: 14, a reverse primer of SEQ ID NO: 14, and a polynucleotide set of a probe selected from the group consisting of SEQ ID NOs: 31, 36 and 39 A kit for detecting mutations in EGFR gene using qPCR (quantitative PCR) or digital PCR using a cell-free DNA (cfDNA) isolated from blood as a template.
9. The kit of claim 8, wherein the kit further comprises a blocking oligomer of SEQ ID NO: 43.
The composition of claim 1, wherein the composition comprises a forward primer of SEQ ID NO: 3, a reverse primer of SEQ ID NO: 4, a forward primer of SEQ ID NO: 7, an inverted primer of SEQ ID NO: 8 and a group of SEQ ID NOs: 18 to 21, 23 and 30 A polynucleotide set of selected probes;
A polynucleotide set of a probe selected from the group consisting of the forward primer of SEQ ID NO: 1, the reverse primer of SEQ ID NO: 2, the forward primer of SEQ ID NO: 5, the reverse primer of SEQ ID NO: 6, and SEQ ID NOs: 15 to 17 and 40; And
A forward primer of SEQ ID NO: 11, a reverse primer of SEQ ID NO: 12, a forward primer of SEQ ID NO: 13, a reverse primer of SEQ ID NO: 14, and a set of polynucleotides of probes selected from the group consisting of SEQ ID NOS: 32 to 35 and 41 A primer and probe set composition for detecting an epidermal growth factor receptor (EGFR) mutation in a sample, wherein the composition further comprises at least one set of polynucleotides.
A research use only (RUO) kit for detecting EGFR gene mutation comprising the primer and probe set composition of claim 10 as an active ingredient.
An IVD (investigation use only) kit for detecting EGFR gene mutation comprising the primer and probe set composition of claim 10 as an active ingredient.
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