KR20160001304A - Method for Detecting EML-ALK Positive Based on RT-PCR - Google Patents

Method for Detecting EML-ALK Positive Based on RT-PCR Download PDF

Info

Publication number
KR20160001304A
KR20160001304A KR1020140079584A KR20140079584A KR20160001304A KR 20160001304 A KR20160001304 A KR 20160001304A KR 1020140079584 A KR1020140079584 A KR 1020140079584A KR 20140079584 A KR20140079584 A KR 20140079584A KR 20160001304 A KR20160001304 A KR 20160001304A
Authority
KR
South Korea
Prior art keywords
seq
eml4
alk
mutation
pcr
Prior art date
Application number
KR1020140079584A
Other languages
Korean (ko)
Inventor
전병희
Original Assignee
주식회사 싸이토젠
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 주식회사 싸이토젠 filed Critical 주식회사 싸이토젠
Priority to KR1020140079584A priority Critical patent/KR20160001304A/en
Publication of KR20160001304A publication Critical patent/KR20160001304A/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Abstract

The present invention relates to a method for detecting EML4-ALK mutation, more specifically, to a PCR primer pair for detection of EML4-ALK mutation, a detection method of EML4-ALK mutation using nested PCR, and a method for detecting EML4-ALK mutation using a blood- The present invention relates to a method for determining an increased availability probability of a jotunit.
According to the present invention, it is possible to detect EML4-ALK mutation using blood lung cancer cells even in patients with lung cancer whose ALK-FISH test could not be performed due to difficulty in collecting lung cancer tissues. Thus, the EML4-ALK mutation- It can be confirmed more easily.

Description

Method for Detecting EML-ALK Positive Based RT-PCR Based on RT-PCR [

The present invention relates to a method for detecting EML4-ALK mutation, and more particularly, to a method for detecting EML4-ALK mutation, more specifically, a PCR primer pair for detection of EML4-ALK mutation, a method for detecting EML4-ALK mutation using nested PCR, The present invention relates to a method for determining an increased availability probability of a jotunit.

Lung cancer is the fourth most common cancer among Koreans in 2010 (10.3%) among cancer patients in Korea (202,053). However, the 5 - year survival rate is 19.7%, which is worse than other cancers. Lung cancer is classified into non - small cell lung cancer and small cell lung cancer according to the size and shape of cancer cells microscopically. Thus, distinguishing between non - small cell lung cancer and small cell lung cancer is different from clinical course and treatment.

According to the data released by the Central Cancer Registry in 2011, there were 192,561 cases of cancer in Korea in 2009, of which 14,300 cases of non-small cell lung cancer occurred in men and women, and 19,685 cases of non-small cell lung cancer occurred in 72,672 cases Respectively. The male to female sex ratio was 2.5: 1, which was higher in males (published by the Ministry of Health and Welfare's Central Cancer Registration Office on December 29, 2011).

ALK-positive non-small cell lung cancer is a lung cancer caused by the fusion of two genes, ALK and EML4, with a mutation in the EML4-ALK gene corresponding to 4 to 7% (7% of Asians) of all lung cancer patients and 167 Ten of the non-small cell lung cancer patients in Korea have been reported to have the EML4-ALK mutation ( J. Korean Med. Sci. , 27: 228-230, 2012).

The target anticancer drug for non-small cell carcinoma by EML4-ALK mutation is XALKORI (crizotinib) developed by Pfizer. Since the approval of Abbott's companion diagnostics in August 2011 by the US Food and Drug Administration (FDA) in collaboration with XALKORI, it has become a widely used, clinically validated and standardized method of genetic testing in the American Oncology community. It has been approved for CE-IVD testing in Europe and has been used in medical practice since September 2011 and is mainly used to support academic research and evaluation of new therapies. After the emergence of XALKORI, the treatment of non-small cell lung cancer (EGFR, EML4-ALK) was followed by the Iressa or conventional cytotoxic chemotherapy (with low therapeutic efficacy and high side effects) with or without mutation after EGFR mutation test. The paradigm shifts in the way of choosing the best cure / conventional cytotoxic chemotherapy.

The ALK gene mutation shows various types of gene fusion. Immunohistochemistry, RT-PCR, and FISH can be used for clinical tests. Currently, the standard diagnostic test for crizotinib is FDA Abbott's Vysis ALK FISH method has been used, but this method is difficult and complicated, which is costly and time-consuming to test many patients.

In addition, the anatomical nature of cancer patients who have difficulty in collecting lung cancer tissues and can not be tested for ALK-FISH is 8-18% ( Lung Cancer 84: 39-44, 2014, Cancer Cytopathol. Epub ahead of print 2014.4.10) It is known that there is a desperate need for finding a solution to this problem.

On the other hand, CTC (circulating tumor cell) is a small number of tumor cells that move away from the primary tumor tissue and circulate in the bloodstream. There are one to several thousand blood cancer cells per 1 mL of blood depending on the cancer type. Blood cancer cell-based testing is a powerful alternative to conventional biopsy. It is a cost-effective alternative to conventional biopsy. It reduces the patient's pain and risk due to biopsy, have. The present inventors conducted a basic study and feasibility evaluation on the production of a microfluidic module chip for separating cancer cells from blood in the previous study, and confirmed the capturing performance of cells and filed several patents (Korean Patent No. 1,254,679 Korean Patent Publication No. 2011-0115478, Korean Patent No. 1,254,680, Korean Patent No. 1,226,515)

Accordingly, the present inventors have made intensive efforts to develop a genetic test method for enabling crizotinib administration in patients who have difficulty in collecting lung cancer tissue, and as a result, they have succeeded in capturing lung cancer cells present in blood cancer cells, A method of confirming the EML4-ALK mutation with high accuracy was developed, and the present invention was completed.

An object of the present invention is to provide a pair of PCR primers for detection of EML4-ALK mutation for confirming EML4-ALK mutation of pancreatic cells in blood with high accuracy.

It is another object of the present invention to provide a method for detecting V1 type mutation of EML4-ALK with high accuracy.

It is still another object of the present invention to provide a method for detecting V3 type mutation of EML4-ALK with high accuracy.

It is yet another object of the present invention to provide a method for determining the increased availability of crizotinib using blood-derived cancer cells in patients with non-small-cell lung cancer.

In order to achieve the above object, the present invention provides a PCR primer pair for detection of EML4-ALK mutation comprising a forward primer selected from the group consisting of primers represented by SEQ ID NOS: 1 to 3 and a reverse primer represented by SEQ ID NO: 4 to provide.

The present invention also relates to

(a) isolating RNA from a patient's lung cancer cells;

(b) carrying out RT PCR using the separated RNA as a template and using primer pairs shown in SEQ ID NO: 1 and SEQ ID NO: 9;

(c) performing nested PCR using the PCR product of step (b) as a template and using a nested primer pair for the primer pair used in step (b); And

(d) detecting the V1 type mutation of EML4-ALK by the presence or absence of the nested PCR product of step (c);

Lt; RTI ID = 0.0 > EML4-ALK. ≪ / RTI >

The present invention also relates to

(a) isolating RNA from a patient's lung cancer cells;

(b) carrying out RT PCR using the separated RNA as a template and using primer pairs shown in SEQ ID NO: 2 and SEQ ID NO: 4;

(c) performing nested PCR using the PCR product of step (b) as a template and using a nested primer pair for the primer pair used in step (b); And

(d) detecting the V3 type mutation of EML4-ALK by the presence or absence of the nested PCR product of step (c);

Lt; RTI ID = 0.0 > EML4-ALK. ≪ / RTI >

 The present invention also provides a kit for detecting EML4-ALK mutation comprising a forward primer selected from the group consisting of primers represented by SEQ ID NO: 1 to SEQ ID NO: 3 and a reverse primer represented by SEQ ID NO: 4, A mutation detection kit is provided.

The present invention also includes a method of detecting the presence or absence of an EML4-ALK fusion mutation in blood-derived cancer cells of a non-small cell lung cancer patient,

The EML4-ALK fusion mutation

(i) exons 1-13 of the EML4 gene and exons 20-29 of the ALK gene;

(ii) exons 1-20 of the EML4 gene; exons 20-29 of the ALK gene;

(iii) exons 1-6a of the EML4 gene and exons 20-29 of the ALK gene;

(iv) exons 20-29 of the exon 1-6b + ALK gene of the EML4 gene;

(v) exon 15 of the EML4 gene and exon 20-29 of the ALK gene;

(vi) exon 14 of EML4 gene + exon 20-29 of linker of 11bp + ALK gene;

(vii) exon 2 of the EML4 gene + exon 20-29 of the ALK gene; And

(viii) a fusion mutation selected from the group consisting of exon 2 of EML4 gene, intron 19 of ALK gene, and exon 20-29 of ALK gene,

The presence of said fusion variant provides a method for determining increased potency of crytotinib in a patient suffering from non-small cell lung cancer, wherein the presence of the cryotozib is indicative of efficacy.

According to the present invention, it is possible to detect EML4-ALK mutation using blood lung cancer cells even in patients with lung cancer whose ALK-FISH test could not be performed due to difficulty in collecting lung cancer tissues. Thus, the EML4-ALK mutation- It can be confirmed more easily.

Figure 1 shows the EML4-ALK fusion mutation type.
Figure 2 shows the differences in V3a and V3b, the isoforms of type V3 among the EML4-ALK fusion mutations.
Fig. 3 shows the detection result of the V1 type mutation by the known VL type mutation detection method of EML4-ALK.
4 shows the detection results of the V1 type mutation using the primer for detecting V1 type mutation of EML4-ALK of the present invention.
FIG. 5 shows the detection results of the V1 type mutation by nested-PCR using the primer for detection of V1 type mutation of EML4-ALK of the present invention.
Fig. 6 shows the detection result of the V3 type mutation by the known method of detecting V3 mutation of EML4-ALK.
Fig. 7 shows the detection results of the V3 type mutation using the primer for detection of V3 type mutation of EML4-ALK of the present invention.
FIG. 8 shows the detection results of the V3 type mutation by nested-PCR using the primer for detection of V3 type mutation of EML4-ALK of the present invention.
FIG. 9 shows the sequencing results of V1 type mutation and V3 type mutation of EML4-ALK detected by the method of the present invention.
FIG. 10 shows the results of application of the EML4-ALK of the present invention to actual patient samples using a primer for detection of V1 type mutation.

ALK-positive non-small cell lung cancer is lung cancer caused by EML4-ALK mutation caused by fusion of two genes, ALK and EML4, and EML4-ALK mutation is associated with exons and / or intron regions of EML4 gene fused. And has various mutation types as shown below.

Variant 1: exon 1-13 (EML4) + exon 20-29 (ALK)

Variant 2: exon 1-20 (EML4) + exon 20-29 (ALK)

Variant 3a: exon 1-6a (EML4) + exon 20-29 (ALK)

Variant 3b: exon 1-6b (EML4) + exon 20-29 (ALK)

Variant 4a: exon 15 (EML4) + exon 20-29 (ALK)

Variant 4b: exon 14 (EML4) + linker of 11bp + exon 20-29 (ALK)

Variant 5a: exon 2 (EML4) + exon 20-29 (ALK)

Variant 5b: exon 2 (EML4) + intron 19 (ALK) + exon 20-29 (ALK)

Among these mutations, 16 cases of non-small cell carcinoma were confirmed in Korea. In 10 cases of EML4-ALK fusion mutation, 4 cases were V1 type mutation and 2 cases were V3b ( J. Korean Med. Sci. , 27: 228-230, 2012).

There are two isoforms in the V3 type, and 33bp in the EML4 intron 6 in the V3b type (Figure 2). Therefore, two types of PCR products can be generated when detecting V3 type PCR.

In one aspect, the present invention relates to a pair of PCR primers for detection of EML4-ALK mutation comprising a forward primer selected from the group consisting of primers represented by SEQ ID NOS: 1 to 3 and a reverse primer represented by SEQ ID NO: 4.

The detection limit of the known primers that have been used for the detection of EML4-ALK mutation is 0.1 to 1%. When the sample is in cancer tissue, there is no problem in detecting the mutation. However, in order to use a method of isolating and detecting cancerous cells in blood 0.1% or less. Therefore, a primer having a low detection limit is required.

In the present invention, in order to design a primer having high accuracy and detection efficiency, a primer was designed by the following method. The design of the primer is determined by using the primer3 ( http://bioinfo.ut.ee/primer3-0.4.0/ ) primer design program, and then the CBI homepage ( http://www.ncbi.nlm.nih.gov / ), The possibility of primer binding to other parts of the EML4-ALK region and the possibility of hairpin structure were avoided through a blast search. Direct comparison of the homology between the primer pairs prevented the formation of dimers, and GC The content was checked to be no more than 60%. The Tm values of the four primers were designed to be similar to each other so that PCR could be performed with any combination of primers. EML4 (E1E2) - EML4 (E1E2) - was designed between possible exon junctions to avoid the occurrence of mutiple bands due to primer binding to genomic DNA because of avoiding repeated or sequential sequences and using cDNA as a template FP primer (SEQ ID NO: 2)).

In the present invention, the primer pair includes a pair of primers represented by SEQ ID NO: 1 and SEQ ID NO: 4; A pair of primers represented by SEQ ID NO: 2 and SEQ ID NO: 4; And a pair of primers represented by SEQ ID NO: 3 and SEQ ID NO: 4 is preferably used.

The pair of primers represented by SEQ ID NO: 1 and SEQ ID NO: 4 is used for detecting the V1 type mutation of EML4-ALK, and the pair of primers represented by SEQ ID NO: 2 and SEQ ID NO: 4; And the primer pair represented by SEQ ID NO: 3 and SEQ ID NO: 4 can be used to detect the V3 type mutation of EML4-ALK.

In another aspect,

(a) isolating RNA from a patient's lung cancer cells;

(b) carrying out RT PCR using the separated RNA as a template and using primer pairs shown in SEQ ID NO: 1 and SEQ ID NO: 9;

(c) performing nested PCR using the PCR product of step (b) as a template and using a nested primer pair for the primer pair used in step (b); And

(d) detecting the V1 type mutation of EML4-ALK by the presence or absence of the nested PCR product of step (c);

Lt; RTI ID = 0.0 > of EML4-ALK. ≪ / RTI >

Herein, the nested primer pair may use the primer pairs shown in SEQ ID NO: 10 and SEQ ID NO: 4.

The RNA extraction in step (a) may be performed according to a method commonly used in the art, and may be performed using a commercially available RNA extraction kit. In the step (b), the complementary DNA strands are synthesized by reverse transcription using reverse transcriptase using reverse primer of the primer pair of the present invention as a template, Reaction can be carried out. The PCR reaction can be carried out using a PCR reaction mixture containing various components known in the art necessary for the PCR reaction. The PCR reaction mixture includes an appropriate amount of DNA polymerase, dNTP, PCR buffer solution and distilled water (dH 2 O) in addition to the complementary DNA synthesized by the reverse transcription reaction and the PCR primer pair provided in the present invention. The PCR buffer comprises Tris -HCl (Tris-HCl), MgCl 2, KCl and the like. At this time, the MgCl2 concentration greatly affects the specificity and yield of amplification. In general, when Mg 2+ is excessive, nonspecific PCR amplification products are increased, and when Mg 2+ is insufficient, the yield of PCR products is decreased. The PCR buffer solution may further contain an appropriate amount of Triton X-100 (Triton X-100). Also, PCR was performed by denaturing the template DNA at 94-95 ° C, followed by denaturation; Annealing; Followed by a cycle of amplification and extension followed by final elongation at 72 ° C. The denaturation and amplification can be performed at 94-95 ° C and 72 ° C, respectively, and the temperature at the time of binding can be varied depending on the type of the primer, and in the case of the primer of the present invention, it can be performed at 55-64 ° C . The time and number of cycles in each step can be determined according to the conditions commonly practiced in the art.

In the step (c), the DNA of the PCR product can be isolated by size according to a method well known in the art. Preferably by agarose gel or polyacrylamide gel electrophoresis or an ABI prism 3100 genetic analyzer-electropherogram. In this case, in order to use the fluorescence analyzer, PCR is performed in step (b) using a pair of primers having a fluorescent dye well known in the art.

The addition of the nested PCR step minimizes errors due to nonspecific reactions when detecting PCV, which can be useful when needed.

The PCR amplification result can preferably be confirmed by agarose gel electrophoresis. After electrophoresis, electrophoresis results can be analyzed by ethidium bromide staining. Methods for performing general reverse transcription, PCR and analysis of the results are well known in the art.

The standard recombinant DNA and molecular cloning techniques used in the present invention are well known in the art and are described in the following references (Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989); by Silhavy, TJ, Bennan, ML and Enquist, LW, Experiments with Gene Fusions, Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1984); and Ausubel, FM et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-lnterscience (1987)).

In the present invention, the lung cancer cell may be a blood circulating tumor cell (CTC) isolated from a blood sample of a patient.

A CTC (circulating tumor cell) is a small number of tumor cells that move away from the primary tumor tissue and circulate in the bloodstream. There are from one to several thousand blood cancer cells per mL of blood, depending on the cancer type. Blood cancer cell-based testing is a powerful alternative to conventional biopsy. It is a cost-effective alternative to conventional biopsy. It reduces the patient's pain and risk due to biopsy, have.

Methods for separating blood cancer cells from blood samples can be performed using the apparatuses and methods described in Korean Patent No. 1,254,679, Korean Patent Publication No. 2011-0115478, Korean Patent No. 1,254,680, and Korean Patent No. 1,226,515 It is not.

In yet another aspect,

(a) isolating RNA from a patient's lung cancer cells;

(b) carrying out RT PCR using the separated RNA as a template and using primer pairs shown in SEQ ID NO: 2 and SEQ ID NO: 4;

(c) performing nested PCR using the PCR product of step (b) as a template and using a nested primer pair for the primer pair used in step (b); And

(d) detecting the V3 type mutation of EML4-ALK by the presence or absence of the nested PCR product of step (c);

Lt; RTI ID = 0.0 > of EML4-ALK. ≪ / RTI >

Herein, the nested primer pair may use the primer pairs shown in SEQ ID NO: 3 and SEQ ID NO: 4.

In the present invention, the lung cancer cell may be a blood circulating tumor cell (CTC) isolated from a blood sample of a patient.

In another aspect, the present invention includes a PCR primer pair for detection of EML4-ALK mutation comprising a forward primer selected from the group consisting of primers represented by SEQ ID NO: 1 to SEQ ID NO: 3 and a reverse primer represented by SEQ ID NO: 4 And an EML4-ALK mutation detection kit.

The kit may further include a primer represented by SEQ ID NO: 9 and SEQ ID NO: 10.

The kit may further include a DNA polymerase and a PCR reaction buffer solution having the composition described above in order to facilitate the PCR reaction in addition to the PCR primer pair. In addition, electrophoresis The components necessary for the performance may be further included in the kit of the present invention.

In another aspect, the present invention relates to a method for detecting the presence or absence of EML4-ALK fusion mutation of blood-derived cancer cells in a patient suffering from non-small cell lung cancer,

The EML4-ALK fusion mutation

(i) exons 1-13 of the EML4 gene and exons 20-29 of the ALK gene;

(ii) exons 1-20 of the EML4 gene; exons 20-29 of the ALK gene;

(iii) exons 1-6a of the EML4 gene and exons 20-29 of the ALK gene;

(iv) exons 20-29 of the exon 1-6b + ALK gene of the EML4 gene;

(v) exon 15 of the EML4 gene and exon 20-29 of the ALK gene;

(vi) exon 14 of EML4 gene + exon 20-29 of linker of 11bp + ALK gene;

(vii) exon 2 of the EML4 gene + exon 20-29 of the ALK gene; And

(viii) a fusion mutation selected from the group consisting of exon 2 of EML4 gene, intron 19 of ALK gene, and exon 20-29 of ALK gene,

The presence of said fusion mutation relates to a method for determining the increased efficacy of crytotinib in a patient suffering from non-small cell lung cancer, wherein the crytotubium exhibits an efficacy.

The target anticancer drug for non-small cell lung cancer caused by EML4-ALK mutation is cryotorib (XALKORI, Pfizer). Currently, the treatment of non-small cell lung cancer is the Iressa / / It is proceeding in a way to select the treatment modality among conventional cytotoxic anticancer drugs.

However, in patients unable to invasive biopsy of lung cancer tissue, genetic testing for the EML4-ALK fusion mutation could not be performed and the efficacy of administration of clozotanib could not be confirmed. There was no.

The method of the present invention can determine the efficacy of clitorotinib by isolating blood circulating tumor cells (CTC) from the blood of non-small cell lung cancer patients and confirming the presence of EML4-ALK fusion mutations in the blood cancer cells.

In the present invention, the step of detecting the presence or absence of the EML4-ALK fusion mutation of the blood-derived cancer cell comprises the step of detecting the presence or absence of the forward primer selected from the group consisting of the primers represented by SEQ ID NO: 1 to SEQ ID NO: 3 and the reverse primer PCR using the PCR primer pair for detection of EML4-ALK mutation.

In one aspect of the invention, a method for detecting the presence or absence of an EML4-ALK fusion mutation in a method for determining the increased availability of crizotinib can be carried out comprising the following steps:

 (a) isolating RNA from blood-derived cancer cells of a patient suffering from non-small cell lung cancer;

(b) carrying out RT PCR using the separated RNA as a template and using primer pairs shown in SEQ ID NO: 1 and SEQ ID NO: 9;

(c) performing nested PCR using the PCR product of step (b) as a template and using a nested primer pair for the primer pair used in step (b); And

(d) detecting the fusion mutation of EML4-ALK with or without the nested PCR product of step (c).

Here, the nested primer pair may use the primer pairs shown in SEQ ID NO: 10 and SEQ ID NO: 4, but any pair of primers capable of amplifying the RT PCR product can be used without limitation.

In another aspect of the present invention, a method of detecting the presence or absence of an EML4-ALK fusion mutation in a method for determining increased potency of said crizotinib can be carried out comprising the following steps:

  (a) isolating RNA from blood-derived cancer cells of a patient suffering from non-small cell lung cancer;

(b) carrying out RT PCR using the separated RNA as a template and using primer pairs shown in SEQ ID NO: 2 and SEQ ID NO: 4;

(c) performing nested PCR using the PCR product of step (b) as a template and using a nested primer pair for the primer pair used in step (b); And

(d) detecting the fusion mutation of EML4-ALK with or without the nested PCR product of step (c).

Here, the nested primer pair may use the primer pairs shown in SEQ ID NO: 3 and SEQ ID NO: 4, but any pair of primers capable of amplifying the RT PCR product can be used without limitation.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Example 1: Detection of V1 type mutation of EML4-ALK by PCR

The H3122 cell line (the Matthew Meyerson - Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02115), a lung cancer cell line with a variant of V1 type of EML4-ALK, was cultured and RNA was extracted. CDNA was synthesized from the extracted RNA, and the synthesized cDNA was sequentially diluted and used as a template for PCR for V1 type detection of EML4-ALK.

PBMC cells and A549 cells (ATCC, CCL-185) were used as negative controls.

The composition of the mixture for the PCR reaction is shown in Table 1.

Mixture composition for PCR reaction matter content 10x LA PCR buffer II (Mg2 + plus) 2μl FP (10 pmol / ul) 2μl RP (10 pmol / ul) 2μl 10 mM dNTP 2μl TaKaRa LA-Taq. HS polymerase 0.2 [mu] l Template 1 μl Distilled water 10.8 μl Total volume 20μl

The PCR cycle was performed at 95 ° C for 10 minutes, followed by 40 cycles of 95 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 40 seconds as one cycle, and elongation was performed at 72 ° C for 10 minutes

The PCR primers used are as follows.

EML4 (E12) -FP: 5'-TTCACCCAACAGATGCAAATA-3 '(V1 forward primer) (SEQ ID NO: 1)

ALK (E20) -RP: 5'-AGGTCTTGCCAGCAAAGCAGTAGT-3 (reverse primer) (SEQ ID NO: 4)

Control primer sequence (FP): 5'-GTGCAGTGTTTAGCATTCTTGGGG-3 '(forward primer) (SEQ ID NO: 5)

Control primer sequence (RP): 5'-ATCCAGTTCGTCCTGTTCAGAGC-3 '(reverse primer) (SEQ ID NO: 6)

The amplified PCR product was electrophoresed on agarose gel to identify bands, and the identified bands were separated and sequenced.

As a result, the V1 mutation could be detected with a detection limit of 0.1% to 0.1% in both the PCR method using a known primer (FIG. 3) and the PCR method using the primer of the present invention (FIG. 4) I could see a cleaner band in the way.

Example 2: Detection of V3 type mutation of EML4-ALK by PCR

The H2228 cell line (ATCC, CRL-5935), a lung cancer cell line having the V3 type mutation of EML4-ALK, was cultured and RNA was extracted. CDNA was synthesized from the extracted RNA, and the synthesized cDNA was sequentially diluted and used as a template for PCR for V3 type detection of EML4-ALK.

PCR was carried out in the same manner as in Example 1 except for the primers used.

When performing PCR using EML4 (E1E2) -FP primer (SEQ ID NO: 2) and ALK (E20) -RP primer (SEQ ID NO: 4), PCR products of 789 bp (V3a type) or 822 bp (V3b type) (V3a type) or 420bp (V3b type) PCR when EML4 (E4) -FP primer (SEQ ID NO: 3) and ALK (E20) -RP primer The product is amplified.

The PCR primers used are as follows.

EML4 (E1E2) -FP: 5'-CCGGCAGTCTCGATGATAGTATTT-3 '(V3 forward primer) (SEQ ID NO: 2)

EML4 (E4) -FP: 5'-CACAAATTCGAGCATCACCTTCTC-3 '(V3 forward primer) (SEQ ID NO: 3)

ALK (E20) -RP: 5'-AGGTCTTGCCAGCAAAGCAGTAGT-3 '(reverse primer) (SEQ ID NO: 4)

Control primer sequence (FP): 5'-GTCAGCTCTTGAGTCACGAGTT-3 '(foward primer) (SEQ ID NO: 7)

Control primer sequence (RP): 5'-ATCCAGTTCGTCCTGTTCAGAGC-3 '(reverse primer) (SEQ ID NO: 8)

The amplified PCR product was electrophoresed on agarose gel to identify bands, and the identified bands were separated and sequenced.

As a result, the V3 mutation could be detected at a detection limit of 0.1% to 0.1% in the PCR method using known primers (FIG. 6), and the PCR method using the primer of the present invention (FIG. 7) E1E2) -FP primer showed a detection limit of 1 to 10%, whereas the detection limit of EML4 (E4) -FP primer was 0.1 to 1%.

Example 3: Detection of V1 and V3 type mutations using nested PCR

The first PCR used EML4 (E12) -FP (SEQ ID NO: 1) and the 3078RR primer (SEQ ID NO: 9), the 2nd PCR used the Fusion RT-S primer ALK (E20) -RP (SEQ ID NO: 2) was used.

3078RR primer sequence: 5'-ATCCAGTTCGTCCTGTTCAGAGC -3 '(SEQ ID NO: 9)

Fusion RT-S primer sequence: 5'-GTGCAGTGTTTAGCATTCTTGGGG-3 '(SEQ ID NO: 10)

As a result, as shown in FIG. 5, it was confirmed that in the case of the V1 type mutation, it was possible to detect the mutation type at the detection limit of 0.001%, which is 1000 times more improved than the known technology,

(E1) -FP (SEQ ID NO: 2) and ALK (E20) -RP (SEQ ID NO: 2) were used for the first PCR and EML4 SEQ ID NO: 3) and ALK (E20) -RP (SEQ ID NO: 2) were used.

As a result, as shown in FIG. 8, it was confirmed that in the case of the V3 type mutation, the detection of the mutation type was possible with the detection limit of 0.1%, and the sensitivity was improved by 10 times. Further, the PCR product was sequenced and it was confirmed that the mutation site was correctly detected (Fig. 9).

Example 4 Detection of EML4-ALK Mutation in Clinical Samples Using Nested PCR

Using the PCR and Nested PCR methods of Example 2 and Example 3, the EML4-ALK mutation in the clinical sample was detected.

Among the histologically proven non-small cell lung cancer patients, RNA from blood cancer cells isolated from the blood of patients with EML4-ALK positive (IRB 1209-029-424, September 12, 2012, Seoul National University Hospital) Respectively. CDNA was synthesized from the extracted RNA, and nested PCR was performed by the method of Example 3 using the synthesized cDNA as a template.

As a result, as shown in FIG. 10, the detection of the V1 type mutation was confirmed, and it was confirmed that EML4-ALK mutation could be detected in blood cancer cells of a patient having an actual EML4-ALK mutation.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> cytogenlab <120> Method for Detecting EML-ALK Positive Based on RT-PCR <130> P14-B136 <160> 10 <170> Kopatentin 2.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer-EML4 (E12) -FP <400> 1 ttcacccaac agatgcaaat a 21 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer-EML4 (E1E2) -FP <400> 2 ccggcagtct cgatgatagt attt 24 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer-EML4 (E4) -FP <400> 3 cacaaattcg agcatcacct tctc 24 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer-ALK (E20) -RP <400> 4 aggtcttgcc agcaaagcag tagt 24 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer-control FP <400> 5 gtgcagtgtt tagcattctt gggg 24 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer-control RP <400> 6 atccagttcg tcctgttcag agc 23 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer-control2 FP <400> 7 gtcagctctt gagtcacgag tt 22 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer-control2 RP <400> 8 atccagttcg tcctgttcag agc 23 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer-3078RR <400> 9 atccagttcg tcctgttcag agc 23 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer-Fusion RT-S <400> 10 gtgcagtgtt tagcattctt gggg 24

Claims (18)

A pair of PCR primers for detection of EML4-ALK mutation comprising a forward primer selected from the group consisting of primers represented by SEQ ID NO: 1 to SEQ ID NO: 3 and a reverse primer represented by SEQ ID NO:
4. The primer set of claim 1, wherein the pair of primers represented by SEQ ID NO: 1 and SEQ ID NO: 4; A pair of primers represented by SEQ ID NO: 2 and SEQ ID NO: 4; And a pair of primers represented by SEQ ID NO: 3 and SEQ ID NO: 4.
3. The pair of PCR primers for detecting EML4-ALK mutation according to claim 2, wherein the pair of primers represented by SEQ ID NO: 1 and SEQ ID NO: 4 detects the V1 type mutation of EML4-ALK.
3. The method according to claim 2, wherein the pair of primers represented by SEQ ID NO: 2 and SEQ ID NO: 4; And the pair of primers represented by SEQ ID NO: 3 and SEQ ID NO: 4 detects the V3 type mutation of EML4-ALK.
A method for detecting a V1 type mutation of EML4-ALK comprising the steps of:
(a) isolating RNA from a patient's lung cancer cells;
(b) carrying out RT PCR using the separated RNA as a template and using primer pairs shown in SEQ ID NO: 1 and SEQ ID NO: 9;
(c) performing nested PCR using the PCR product of step (b) as a template and using a nested primer pair for the primer pair used in step (b); And
(d) detecting the V1 type mutation of EML4-ALK by the presence or absence of the nested PCR product of step (c).
6. The method according to claim 5, wherein the nested primer pair is a pair of primers represented by SEQ ID NO: 10 and SEQ ID NO:
6. The method of claim 5, wherein the lung cancer cell is a circulating tumor cell (CTC) isolated from a blood sample of a patient.
A method for detecting a V3 type mutation of EML4-ALK comprising the steps of:
(a) isolating RNA from a patient's lung cancer cells;
(b) carrying out RT PCR using the separated RNA as a template and using primer pairs shown in SEQ ID NO: 2 and SEQ ID NO: 4;
(c) performing nested PCR using the PCR product of step (b) as a template and using a nested primer pair for the primer pair used in step (b); And
(d) detecting the V3 type mutation of EML4-ALK by the presence or absence of the nested PCR product of step (c).
9. The method according to claim 8, wherein the nested primer pair is a pair of primers represented by SEQ ID NO: 3 and SEQ ID NO:
6. The method of claim 5, wherein the lung cancer cell is a circulating tumor cell (CTC) isolated from a blood sample of a patient.
A kit for detecting EML4-ALK mutation comprising the primer pair of claim 1.
The kit for detecting EML4-ALK mutation according to claim 11, further comprising a primer represented by SEQ ID NO: 9 and SEQ ID NO: 10.
Detecting the presence or absence of an EML4-ALK fusion mutation in blood-derived cancer cells of a non-small cell lung cancer patient,
The EML4-ALK fusion mutation
(i) exons 1-13 of the EML4 gene and exons 20-29 of the ALK gene;
(ii) exons 1-20 of the EML4 gene; exons 20-29 of the ALK gene;
(iii) exons 1-6a of the EML4 gene and exons 20-29 of the ALK gene;
(iv) exons 20-29 of the exon 1-6b + ALK gene of the EML4 gene;
(v) exon 15 of the EML4 gene and exon 20-29 of the ALK gene;
(vi) exon 14 of EML4 gene + exon 20-29 of linker of 11bp + ALK gene;
(vii) exon 2 of the EML4 gene + exon 20-29 of the ALK gene; And
(viii) a fusion mutation selected from the group consisting of exon 2 of EML4 gene, intron 19 of ALK gene, and exon 20-29 of ALK gene,
Wherein the presence of said fusion mutation indicates that crizotinib is potentially useful in the treatment of non-small cell lung cancer.
14. The method according to claim 13, wherein the step of detecting the presence or absence of the EML4-ALK fusion mutation is carried out by an RT-PCR method using the primer pair of claim 1.
14. The method according to claim 13, wherein the step of detecting the presence or absence of the EML4-ALK fusion mutation is carried out in the following manner:
(a) isolating RNA from blood-derived cancer cells of a patient suffering from non-small cell lung cancer;
(b) carrying out RT PCR using the separated RNA as a template and using primer pairs shown in SEQ ID NO: 1 and SEQ ID NO: 9;
(c) performing nested PCR using the PCR product of step (b) as a template and using a nested primer pair for the primer pair used in step (b); And
(d) detecting the fusion mutation of EML4-ALK with or without the nested PCR product of step (c).
16. The method according to claim 15, wherein the nested primer pair is a pair of primers represented by SEQ ID NO: 10 and SEQ ID NO:
14. The method according to claim 13, wherein the step of detecting the presence or absence of the EML4-ALK fusion mutation is carried out in the following manner:
(a) isolating RNA from blood-derived cancer cells of a patient suffering from non-small cell lung cancer;
(b) carrying out RT PCR using the separated RNA as a template and using primer pairs shown in SEQ ID NO: 2 and SEQ ID NO: 4;
(c) performing nested PCR using the PCR product of step (b) as a template and using a nested primer pair for the primer pair used in step (b); And
(d) detecting the fusion mutation of EML4-ALK with or without the nested PCR product of step (c).
18. The method according to claim 17, wherein the nested primer pair is a pair of primers represented by SEQ ID NO: 3 and SEQ ID NO:
KR1020140079584A 2014-06-27 2014-06-27 Method for Detecting EML-ALK Positive Based on RT-PCR KR20160001304A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
KR1020140079584A KR20160001304A (en) 2014-06-27 2014-06-27 Method for Detecting EML-ALK Positive Based on RT-PCR

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
KR1020140079584A KR20160001304A (en) 2014-06-27 2014-06-27 Method for Detecting EML-ALK Positive Based on RT-PCR

Publications (1)

Publication Number Publication Date
KR20160001304A true KR20160001304A (en) 2016-01-06

Family

ID=55165206

Family Applications (1)

Application Number Title Priority Date Filing Date
KR1020140079584A KR20160001304A (en) 2014-06-27 2014-06-27 Method for Detecting EML-ALK Positive Based on RT-PCR

Country Status (1)

Country Link
KR (1) KR20160001304A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20190015869A (en) * 2017-08-07 2019-02-15 주식회사 싸이토젠 A method for analyzing eml4-alk gene variance

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20190015869A (en) * 2017-08-07 2019-02-15 주식회사 싸이토젠 A method for analyzing eml4-alk gene variance
WO2019031722A3 (en) * 2017-08-07 2019-04-11 주식회사 싸이토젠 Eml4-alk gene mutation analysis method
CN110997942A (en) * 2017-08-07 2020-04-10 西托根有限公司 EML4-ALK gene mutation analysis method
JP2020528755A (en) * 2017-08-07 2020-10-01 サイトジェン インコーポレーテッドＣｙｔｏｇｅｎ， Ｉｎｃ． EML4-ALK gene mutation analysis method
EP3666907A4 (en) * 2017-08-07 2021-05-05 Cytogen, Inc. Eml4-alk gene mutation analysis method
US11718882B2 (en) 2017-08-07 2023-08-08 Cytogen, Inc. EML4-ALK gene mutation analysis method

Similar Documents

Publication Publication Date Title
Vanni et al. The current state of molecular testing in the BRAF-mutated melanoma landscape
Park et al. Discordance of molecular biomarkers associated with epidermal growth factor receptor pathway between primary tumors and lymph node metastasis in non-small cell lung cancer
JP7191140B2 (en) TERT promoter mutations in a subset of gliomas and tumors
Kwon et al. Frequency of KRAS, BRAF, and PIK3CA mutations in advanced colorectal cancers: Comparison of peptide nucleic acid-mediated PCR clamping and direct sequencing in formalin-fixed, paraffin-embedded tissue
Patel et al. The translational potential of circulating tumour DNA in oncology
Konopka et al. PIK3CA mutations and amplification in endometrioid endometrial carcinomas: relation to other genetic defects and clinicopathologic status of the tumors
Furuki et al. Evaluation of liquid biopsies for detection of emerging mutated genes in metastatic colorectal cancer
Kimura et al. EGFR mutation of tumor and serum in gefitinib-treated patients with chemotherapy-naive non–small cell lung cancer
Kamal et al. The tumor microenvironment of colorectal cancer metastases: opportunities in cancer immunotherapy
Mariani et al. Concordant analysis of KRAS status in primary colon carcinoma and matched metastasis
WO2016109939A1 (en) Method and reagent for enrichment of circulating tumor dna
JP2015522277A (en) Prostate cancer diagnosis method and diagnostic substance
US20170002425A1 (en) Method for predicting long-term efficacy of vegf inhibitor
WO2014028222A1 (en) Biomarkers for identifying esophageal cancer patients for treatment with an anti-egfr drug
Wu et al. Opportunities of circulating tumor DNA in lung cancer
CN109161543B (en) DNA probe for enriching low-frequency DNA mutation and application thereof
CN109913482A (en) PIK3CA-I874R mutated gene and its application in Computer-aided Diagnosis of Breast Cancer
KR20160012949A (en) Use of method for the detection of multiple target nucleic acids using clamping probes and detection probes
KR20160001304A (en) Method for Detecting EML-ALK Positive Based on RT-PCR
US20220119893A1 (en) Process for classification of glioma
CN111154883B (en) Breast cancer related gene PIK3CA site g.179220986A &amp; gtT mutant and application thereof
Nicoś et al. PIK3CA mutations detected in patients with central nervous system metastases of non-small cell lung cancer
JP6792823B2 (en) Gene polymorphisms useful for predicting the therapeutic effect of cancer immunotherapy
Pinsolle et al. A Rare Fusion of CLIP1 and ALK in a Case of Non–Small-Cell Lung Cancer With Neuroendocrine Features
JP6346493B2 (en) Method for predicting the effect of peptide vaccine therapy

Legal Events

Date Code Title Description
A201 Request for examination
E902 Notification of reason for refusal
E601 Decision to refuse application