JP5769952B2 - Highly sensitive detection method for EML4-ALK fusion gene - Google Patents

Description

  The present invention relates to a highly sensitive detection method, detection primer, and kit for an EML4-ALK fusion gene.

  The EML4-ALK fusion gene is a product in which the N-terminus of EML4 (echinoderm microtubule associated protein like 4) on the second chromosome and the C-terminus of ALK (anaplastic lymphoma receptor tyrosine kinase) are fused. ALK is a receptor tyrosine kinase, but becomes an active substance by fusion with EML4 and exhibits canceration ability (Patent Documents 1 and 2). Activation of ALK is observed in EML4-ALK fusion gene-expressing cells, and it has been clarified that an ALK inhibitor is effective in suppressing cell proliferation. That is, it can be said that the EML4-ALK fusion gene is a sensitivity defining factor of an ALK inhibitor. Expression of the EML4-ALK fusion gene has been observed in lung cancer tumors, and the expression frequency is said to be about 5%.

  At present, for the detection of EML4-ALK fusion gene from cancer tissue, a quantitative method by a reverse transcription polymerase chain reaction (RT-PCR) method for a frozen specimen has been reported (Non-patent Document 1). Since it is necessary to amplify about 200 bp to about 1300 bp including the fusion point of ALK and ALK, it cannot be applied to the measurement in a paraffin-embedded specimen in which nucleic acid is degraded or deteriorated. However, in lung cancer, it is difficult to collect a living tissue, and an analysis using a relatively available paraffin-embedded specimen is desirable. As a method for detecting an EML4-ALK fusion gene in a paraffin-embedded specimen, an immunohistochemical staining method or a fluorescence in situ hybridization (FISH) method is used. However, these methods have low detection sensitivity and are difficult to discriminate when detecting variants with similar fusion points. Therefore, EML4-ALK fusion using paraffin-embedded specimens is more sensitive and accurate. There is a demand for the construction of a gene detection method.

JP 2008-295444 A JP 2010-501175 A

Clin Cancer Res 2008; 14: 6618-24.

  An object of the present invention is to provide a method for detecting an EML4-ALK fusion gene with high sensitivity and high accuracy in a very small amount of sample such as a paraffin-embedded sample (nucleic acid, particularly mRNA). It is in providing the primer and kit which are used for.

  As a result of intensive research to solve the above problems, the present inventor used a primer capable of amplifying a region of about 100 bp or less including the fusion point of the EML4-ALK fusion gene listed below. It was found that the presence or absence of the EML4-ALK fusion gene can be detected by mass spectrometry using an extension reaction (MassARRAY method). That is, for each of Variant 1, Variant 2, Variant 3a, Variant 3b, Variant 4, Variant 5a, Variant 5b, Variant 6, and Variant 7 of the EML4-ALK fusion gene, about 100 bp including each fusion point of EML4 and ALK gene By amplifying the fusion point using an amplification primer capable of amplifying the following region, it was possible to detect all of the fusion gene in a paraffin-embedded section. Furthermore, the presence of all the fusion genes can be detected with high sensitivity and high accuracy by carrying out a single base extension reaction using the amplified product as a template and a complementary primer capable of hybridizing to the fusion point. Made possible.

The present invention
[1] (A) A step of preparing a template DNA from a sample,
(B) A combination of primers for amplification for amplifying a region including a fusion point with respect to the template DNA, a combination of SEQ ID NO: 11 and SEQ ID NO: 15, a combination of SEQ ID NO: 20 and SEQ ID NO: 18 or 21, and a sequence number 23 or 26 and SEQ ID NO: 24, SEQ ID NO: 32 and SEQ ID NO: 33, SEQ ID NO: 35 or 38 and SEQ ID NO: 36 or 39, SEQ ID NO: 41 and SEQ ID NO: 45, SEQ ID NO: 47 and SEQ ID NO: Amplification in the base sequence region that can be amplified by using at least one amplification primer combination selected from 48 combinations, combinations of SEQ ID NO: 50 and SEQ ID NO: 51, and combinations of SEQ ID NO: 56 and SEQ ID NO: 54 A method of detecting an EML4-ALK fusion gene comprising the step of:
[2] A method for detecting variant 1 of the EML4-ALK fusion gene, wherein the amplification primer is a combination of SEQ ID NO: 11 and SEQ ID NO: 15 in the method of [1] above;
[3] A method for detecting variant 2 of the EML4-ALK fusion gene, wherein the amplification primer is a combination of SEQ ID NO: 20 and SEQ ID NO: 18 or 21 in the method of [1] above;
[4] A method for detecting variant 3a of the EML4-ALK fusion gene, wherein the amplification primer is a combination of SEQ ID NO: 23 or 26 and SEQ ID NO: 24 in the method of [1] above;
[5] A method for detecting variant 3b of the EML4-ALK fusion gene, wherein the amplification primer is a combination of SEQ ID NO: 32 and SEQ ID NO: 33 in the method of [1] above;
[6] A method for detecting variant 4 of the EML4-ALK fusion gene, wherein the amplification primer is a combination of SEQ ID NO: 35 or 38 and SEQ ID NO: 36 or 39 in the method of [1] above;
[7] A method for detecting variant 5a of the EML4-ALK fusion gene, wherein the amplification primer is a combination of SEQ ID NO: 41 and SEQ ID NO: 45 in the method of [1] above;
[8] A method for detecting variant 5b of the EML4-ALK fusion gene, wherein the amplification primer is a combination of SEQ ID NO: 47 and SEQ ID NO: 48 in the method of [1] above;
[9] A method for detecting variant 6 of the EML4-ALK fusion gene, wherein the amplification primer is a combination of SEQ ID NO: 50 and SEQ ID NO: 51 in the method of [1] above;
[10] A method for detecting variant 7 of the EML4-ALK fusion gene, wherein the amplification primer is a combination of SEQ ID NO: 56 and SEQ ID NO: 54 in the method of [1] above;
[11] (a) A combination of amplification primers for amplifying a region containing a fusion point, which is a combination of SEQ ID NO: 11 and SEQ ID NO: 15, a combination of SEQ ID NO: 20 and SEQ ID NO: 18 or 21, and SEQ ID NO: 23 or 26 And SEQ ID NO: 24, SEQ ID NO: 32 and SEQ ID NO: 33, SEQ ID NO: 35 or 38 and SEQ ID NO: 36 or 39, SEQ ID NO: 41 and SEQ ID NO: 45, SEQ ID NO: 47 and SEQ ID NO: 48 Amplifying the amplifiable base sequence region by using at least one combination of primers for amplification selected from the combination of SEQ ID NO: 50 and SEQ ID NO: 51, the combination of SEQ ID NO: 56 and SEQ ID NO: 54;
(B) a method for detecting an EML4-ALK fusion gene comprising a step of performing a single base extension reaction using the amplification product as a template and a detection primer complementary to a region sandwiching the fusion point;
[12] Variant 1 of EML4-ALK fusion gene in which the amplification primer is a combination of SEQ ID NO: 11 and SEQ ID NO: 15 and the extension primer is SEQ ID NO: 13 and / or SEQ ID NO: 16 in the method of [11] above How to detect
[13] In the method of [11] above, an EML4-ALK fusion gene wherein the amplification primer is a combination of SEQ ID NO: 20 and SEQ ID NO: 18 or 21, and the extension primer is SEQ ID NO: 19 and / or SEQ ID NO: 22 A method for detecting variant 2;
[14] In the method of [11] above, an EML4-ALK fusion gene wherein the amplification primer is a combination of SEQ ID NO: 23 or 26 and SEQ ID NO: 24, and the extension primer is SEQ ID NO: 25 and / or SEQ ID NO: 28 A method for detecting variant 3a;
[15] EML4-ALK fusion gene variant 3b in which the amplification primer is a combination of SEQ ID NO: 32 and SEQ ID NO: 33 and the extension primer is SEQ ID NO: 31 and / or SEQ ID NO: 34 in the method of [11] above How to detect
[16] An EML4-ALK fusion in which the amplification primer is a combination of SEQ ID NO: 35 or 38 and SEQ ID NO: 36 or 39, and the extension primer is SEQ ID NO: 37 and / or SEQ ID NO: 40 in the method of [11] above A method for detecting variant 4 of a gene;
[17] Variant 5a of EML4-ALK fusion gene in which the amplification primer is a combination of SEQ ID NO: 41 and SEQ ID NO: 45 and the extension primer is SEQ ID NO: 43 and / or SEQ ID NO: 46 in the method of [11] How to detect
[18] A method for detecting variant 5b of the EML4-ALK fusion gene in which the amplification primer is a combination of SEQ ID NO: 47 and SEQ ID NO: 48 and the extension primer is SEQ ID NO: 49 in the method of [11] above;
[19] A method for detecting variant 6 of the EML4-ALK fusion gene in which the amplification primer is a combination of SEQ ID NO: 50 and SEQ ID NO: 51 and the extension primer is SEQ ID NO: 52 in the method of [11] above;
[20] In the method of [11], variant 7 of the EML4-ALK fusion gene, wherein the amplification primer is a combination of SEQ ID NO: 56 and SEQ ID NO: 54, and the extension primer is SEQ ID NO: 55 and / or SEQ ID NO: 58 How to detect
[21] Combination of SEQ ID NO: 11 and 12, Combination of SEQ ID NO: 14 and 15, Combination of SEQ ID NO: 17 and 18, Combination of SEQ ID NO: 20 and 21, Combination of SEQ ID NO: 23 and 24, Combination of SEQ ID NO: 26 and 27 , Combinations of SEQ ID NOs: 29 and 30, combinations of SEQ ID NOs: 32 and 33, combinations of SEQ ID NOs: 35 and 36, combinations of SEQ ID NOs: 38 and 39, combinations of SEQ ID NOs: 41 and 42, combinations of SEQ ID NOs: 44 and 45, sequences A primer set selected from a combination of Nos. 47 and 48, a combination of SEQ ID Nos. 50 and 51, a combination of SEQ ID Nos. 53 and 54, and a combination of SEQ ID Nos. 56 and 57;
About.

  According to the present invention, the presence of variant 1, variant 2, variant 3a, variant 3b, variant 4, variant 5a, variant 5b, variant 6 and variant 7 of the EML4-ALK fusion gene is detected with high sensitivity and high accuracy. Made possible.

It is a mass spectrum which shows the detection result of each EML4-ALK fusion gene variant using a plasmid. It is a mass spectrum which shows a result when ALK (1) is used (detection base is T) among the mass spectra shown in FIG. It is a mass spectrum which shows the result when Variant1 (1) is used (detection base is G) among the mass spectra shown in FIG. It is a mass spectrum which shows a result when Variant3a (1) is used among the mass spectra shown in Drawing 1 (detection base is C). It is a mass spectrum which shows a result when Variant1 (2) is used (detection base is A) among the mass spectra shown in FIG. It is a mass spectrum which shows an example [Variant3a (2); MKN1 (wild type ALK gene expression strain)] of a detection result of EML4-ALK fusion gene variant 3a using a cell line. In order to compare detection sensitivity (see FIG. 8), electrophoresis showing the detection results of EML4-ALK fusion gene variant 3a by PCR amplification in plasmids, cell lines (H2228), formalin-fixed paraffin-embedded sections (FFPE) It is a pattern. In order to compare with the detection sensitivity in the PCR amplification method implemented in FIG. 7, it is a mass spectrum which shows the detection result of the EML4-ALK fusion gene variant 3a by a MassARRAY system in a formalin fixed paraffin embedding section (FFPE). The detection result of EML4-ALK fusion gene variant 3a by PCR amplification in plasmid, cell line (H2228), formalin-fixed paraffin-embedded section (FFPE) using 1/10 of the sample used in FIG. It is an electrophoresis pattern shown. It is a mass spectrum which shows the detection result of the EML4-ALK fusion gene variant 3a by a MassARRAY system in a formalin fixed paraffin embedding section (FFPE) using 1/10 amount of the sample used in FIG. It is explanatory drawing which shows typically the structure of each EML4-ALK fusion gene variant. The sequence of the fusion point of EML4-ALK fusion gene variant 1 and the region in the vicinity thereof (SEQ ID NO: 1), and each primer (forward primer, reverse primer, and extension primer included in primer sets 1 and 2 shown in Table 4) It is explanatory drawing which shows the same target sequence. It is explanatory drawing which shows the target sequence of each primer contained in the primer set 1 and 2 shown in Table 4 and the arrangement | sequence (sequence number 2) of the fusion point of EML4-ALK fusion gene variant 2 and its vicinity region (sequence number 2). It is explanatory drawing which shows the fusion point of EML4-ALK fusion gene variant 3a and the sequence | arrangement (sequence number 3) of the vicinity region, and the target sequence of each primer contained in the primer sets 1 and 2 shown in Table 4. It is explanatory drawing which shows the fusion point of EML4-ALK fusion gene variant 3b, the sequence | arrangement (sequence number 4) of the vicinity region, and the target sequence of each primer contained in the primer sets 1 and 2 shown in Table 4. It is explanatory drawing which shows the fusion point of EML4-ALK fusion gene variant 4 and the sequence | arrangement (sequence number 5) of the vicinity region, and the target sequence of each primer contained in the primer sets 1 and 2 shown in Table 4. It is explanatory drawing which shows the fusion point of EML4-ALK fusion gene variant 5a and the sequence | arrangement (sequence number 6) of the vicinity region, and the target sequence of each primer contained in the primer sets 1 and 2 shown in Table 4. It is explanatory drawing which shows the target sequence of each primer contained in the primer set 1 shown in Table 4 and the sequence (sequence number 7) of the fusion point of EML4-ALK fusion gene variant 5b and its vicinity region (sequence number 7). It is explanatory drawing which shows the fusion point of EML4-ALK fusion gene variant 6 and the sequence (sequence number 8) of the vicinity region, and the target sequence of each primer contained in the primer set 1 shown in Table 4. It is explanatory drawing which shows the fusion point of EML4-ALK fusion gene variant 7 and the sequence | arrangement (sequence number 9) of the vicinity region, and the target sequence of each primer contained in the primer sets 1 and 2 shown in Table 4. FIG. 5 is an explanatory diagram showing a fusion point of a wild-type ALK gene and a sequence in the vicinity thereof (SEQ ID NO: 10) and a target sequence of each primer included in primer sets 1 and 2 shown in Table 4.

  In this specification, the definitions of terms such as DNA, RNA, nucleic acid, gene, gene expression, code, complementation, template, primer, hybridization, PCR, etc. are currently widely used in molecular biology, genetics, genetic engineering, etc. It has the same meaning as a commonly used term.

  Unless otherwise specified, gene manipulation techniques in this specification can be performed according to known techniques such as “Molecular Cloning” Sambrook, J. et al., Cold Spring Harbor Laboratory Press, 1989, and the like.

  In the present specification, the nucleic acid may be DNA or RNA, a natural one, or a synthesized one. Examples of natural nucleic acids include genomic DNA, mRNA, tRNA, rRNA, hnRNA and the like recovered from an organism. Further, as a synthesized nucleic acid, DNA synthesized by a known chemical synthesis method such as β-cyanoethyl phosphoramidite method, DNA solid phase synthesis method, a nucleic acid synthesized by a known nucleic acid amplification method such as PCR, Examples include cDNA synthesized by reverse transcription reaction.

  As used herein, “wild type gene” refers to a gene before fusion occurs. Specifically, they are the EML4 gene of NM — 09063.3 and the ALK gene of NM — 000434.3, which are represented by the access number of Gene Bank of NCBI.

In the present specification, the “EML4-ALK fusion gene” is a gene in which part of the EML4 gene and part of the ALK gene are fused to have tumorigenicity (FIG. 11). Specifically, it is indicated by NCBI Gene Bank accession number.
"EML4-ALK fusion gene variant 1" in which exon 1-13 of the EML4 gene and exon 20 of the ALK gene are fused in the positive direction (AB274722.1: Fig. 11),
“EML4-ALK fusion gene variant 2” (AB275788.1: FIG. 11) in which exon 1-20 of EML4 gene and exon 20 of ALK gene were fused in the positive direction
"EML4-ALK fusion gene variant 3a" in which exon 1-6 of EML4 gene and exon 20 of ALK gene were fused in the positive direction (AB3744361.1: Fig. 11),
-"EML4-ALK fusion gene variant 3b" in which exon 1-6 of EML4 gene, cryptic exon and exon 20 of ALK gene are fused in the positive direction (AB3744362.1: Fig. 11)
"EML4-ALK fusion gene variant 4" in which exon 1-14 of EML4 gene and unknown sequence and exon 20 of ALK gene were fused in the opposite orientation (AB3744363.1: Fig. 11)
"EML4-ALK fusion gene variant 5a" in which exon 1-2 of EML4 gene and exon 20 of ALK gene were fused in the positive direction (AB3744361: Fig. 11),
"EML4-ALK fusion gene variant 5b" in which exon 1-2 of EML4 gene and a part of intron 19 of ALK gene and exon 20 of ALK gene were fused in the positive direction (AB3744365.1: Fig. 11)
"EML4-ALK fusion gene variant 6" in which exon 1-13 of EML4 gene, unknown sequence and exon 20 of ALK gene were fused in the positive direction (AB46211.1: Fig. 11),
"EML4-ALK fusion gene variant 7" in which exon 1-14 of EML4 gene and exon 20 of ALK gene were fused in the positive direction (AB46212.1: Fig. 11)
Is included.

  The “fusion point” in the present specification indicates a site where the EML4 gene and the ALK gene are linked. The fusion point of EML4-ALK fusion gene variant 1 is the site where EML4 gene and ALK gene are in contact (FIG. 12), and the fusion point of EML4-ALK fusion gene variant 2 is the site where EML4 gene and ALK gene are in contact ( 13), the fusion point of EML4-ALK fusion gene variant 3a is the site where EML4 gene and ALK gene are in contact (FIG. 14), and the fusion point of EML4-ALK fusion gene variant 3b is between EML4 gene and ALK gene. Is a site where the EML4 gene and the cryptic exon are in contact, or a site where the ALK gene and the cryptic exon are in contact (FIG. 15). The fusion point of the EML4-ALK fusion gene variant 4 is EML4. Not between gene and ALK gene Since the sequence is inserted, it is the site where the EML4 gene and the unknown sequence are in contact, or the site where the ALK gene and the unknown sequence are in contact (FIG. 16). The fusion point of the EML4-ALK fusion gene variant 5a is the EML4 gene and the ALK gene. The fusion point of the EML4-ALK fusion gene variant 5b is a part of the EML4 gene and the ALK gene, since a part of the intron 19 of the ALK gene is inserted between the EML4 gene and the ALK gene. The site where intron 19 is in contact or the site where ALK gene and ALK gene are in contact with intron 19 (FIG. 18). The fusion point of EML4-ALK fusion gene variant 6 is an unknown sequence inserted between EML4 gene and ALK gene. Because there is a part where EML4 gene and unknown sequence are in contact Is a portion contacting the ALK gene and unknown sequence (FIG. 19), the fusion point of the EML4-ALK fusion gene variant 7 is a portion contacting the EML4 gene and ALK gene (Figure 20).

  The fusion gene can be prepared with reference to, for example, JP-A-2008-295444. MRNA is extracted from cells or tissues having the fusion gene, for example, lung tissue derived from human lung cancer patients. Subsequently, this mRNA can be subjected to a reverse transcriptase reaction in the presence of a random primer or oligo dT primer to synthesize a first strand cDNA. Using the obtained first strand cDNA, it can be subjected to PCR using two kinds of primers sandwiching a partial region of the target gene to obtain the fusion gene. Further, as shown in Example 1 of the present invention, a fusion gene can be prepared by amplifying and ligating a target region from an already cloned vector.

  The biological sample that can be used in the present invention is not limited as long as the EML4-ALK fusion gene can be detected. However, pathological specimens (biopsy, cytology, surgical specimen, pleural effusion), frozen tissue specimens in sputum, paraffin-embedded specimens Etc. can be used. Paraffin-embedded specimens include those fixed with alcohol and those fixed with formalin. In particular, the method of the present invention is preferable because the EML4-ALK fusion gene can be detected even in a small amount of paraffin-embedded specimen of tissue.

  The biological sample only needs to contain somatic cells. Tissue or fluid samples isolated from an individual can be used, such as blood, tumor biopsy, spinal fluid, intrathoracic fluid, respiratory organs, saliva, cells (including but not limited to blood cells) Including, but not limited to, samples of tumors, organs, and even in vitro cell culture components. Preferably, tissue from an invasive tumor biopsy can be used conveniently.

  Any number of procedures well known in the art can be used as a method for preparing nucleic acid molecules from the sample.

  The disease targeted in the present invention is not limited as long as it is a disease in which a correlation is observed in tumor formation with an “EML4-ALK fusion gene” produced by fusing a part of the EML4 gene and a part of the ALK gene. As a disease that correlates with Variant 1, Variant 2, Variant 3a, Variant 3b, Variant 4, Variant 5a, Variant 5b, Variant 6, and Variant 7 of the EML4-ALK fusion gene, lung cancer may be mentioned.

  For such diseases, inhibitors having ALK tyrosine kinase inhibitory activity (ALK inhibitors) can be used as therapeutic agents. Examples of the ALK inhibitor include PF-02341066, TAE684 and the like. The substance exhibiting medicinal effect may be a low molecular compound or a protein (particularly an antibody). By using the method for detecting an EML4-ALK fusion gene according to the present invention to determine the presence or absence of the gene, the usage policy of the ALK inhibitor can be determined.

  The term “probe” refers to a molecule that can detectably distinguish between target molecules of different structures. Detection can be accomplished in a variety of different ways, depending on the type of probe used and the type of target molecule. Thus, for example, detection may be based on identification of the activity level of the target molecule, but is preferably based on detection of specific binding. Examples of such specific binding include nucleic acid probe hybridization.

  As used herein, the term “primer” refers to polynucleotide synthesis along a complementary strand when placed under conditions that catalyze the synthesis of a primer extension product that is complementary to the polynucleotide. An oligonucleotide that can act as a starting position. Such conditions include four different nucleotide triphosphates or nucleoside analogs and one or more agents for polymerization, such as DNA polymerase and / or reverse transcriptase, in an appropriate buffer ("buffer" "Includes substitutions that are cofactors or that affect pH, ionic strength, etc.) and at the appropriate temperature. The primer must be long enough to prime the synthesis of the extension product even in the presence of an agent for the polymerase. A typical primer includes a sequence that is at least about 5 nucleotides in length and is substantially complementary to the target sequence, although somewhat longer primers are preferred. Typically, the primer contains about 15-26 nucleotides, but longer primers may be used.

  A primer always contains a sequence that is substantially complementary to a target sequence that is the specific sequence to be amplified and that the primer can anneal to. The primer may optionally contain additional sequences. As the additional sequence, an analysis sequence required for measurement by the MassARRAY system described later, and other sequences required for measurement can be appropriately added. The design of these primers can be appropriately performed according to the measurement method using known primer design software (for example, provided by Primer 3: NCBI).

  In the present invention, an amplification primer capable of amplifying a target gene region from a severely damaged biological sample is used. Further, in order to detect a target gene with high sensitivity, an extension primer can be used with the amplification product amplified with the amplification primer as a template. The extension primer of the present invention can also be used as a probe.

  Numerous known analysis procedures can be used to detect the presence or absence of the EML4-ALK fusion gene of the present invention. In general, detection of the presence or absence of a fusion gene requires an amplification reaction and optionally a signal generating system. Table 1 lists mutation detection techniques, some based on PCR. These can also be used in combination with the signal generation systems listed in Table 2. Table 3 lists other amplification techniques. For many current detection methods of fusion genes, see Nollau et al., Clin. Chem. 43, 1114-1120 (1997) and standard textbooks such as Laboratory Protocols for Mutation Detection, edited by U. Landegren, Oxford University. Press (1996) and PCR, 2nd edition by Newton & Graham, BIOS Scientific Publishers Limited (1997).

  Preferred mutation detection techniques include electrophoresis, MassARRAY method, oligonucleotide array (DNA chip) method, DNA sequencing method, ARMS (registered trademark), ALEX (registered trademark), COPS, Taqman, PNA-LNA PCR clamp method. There are PCR and FRET techniques based on molecular beacons, RFLP, and restriction sites. The MassARRAY method is a particularly preferable method.

  Hereinafter, several inspection methods will be exemplified.

  In the test method, detection of the fusion point generated in the EML4-ALK fusion gene can be performed, for example, by directly determining the base sequence of the EML4-ALK fusion gene of the subject. In this method, first, a DNA sample is prepared from a biological sample obtained from a subject. These samples can be prepared based on RNA extracted from tissues or cells, for example.

  In this method, the DNA containing the fusion point in the EML4-ALK fusion gene is then isolated using the DNA sample. Isolation of the DNA is performed using a primer that hybridizes to a nucleic acid sequence capable of amplifying a nucleic acid sequence containing a fusion point in the EML4-ALK fusion gene even in a sample having a damaged nucleic acid such as a formalin-fixed paraffin-embedded sample, It can also be performed by PCR or the like using the prepared DNA sample as a template. In this method, the size of the isolated DNA base sequence is then determined. The size of the base sequence of the isolated DNA can be determined by methods known to those skilled in the art.

  Primers that can be used in the present invention include a combination of SEQ ID NO: 11 and SEQ ID NO: 15 as primers for amplification of variant 1 of the EML4-ALK fusion gene (bases 1742 to 1779 in AB274722.1), EML4-ALK fusion gene A combination of SEQ ID NO: 20 and SEQ ID NO: 18 or 21 (base Nos. 2499 to 2532 in AB2758829.1) as an amplification primer for variant 2 of SEQ ID NO: 23 or 26 as an amplification primer for variant 3a of the EML4-ALK fusion gene And a combination of SEQ ID NO: 24 (bases 711 to 743 in AB3744361.1) and a combination of SEQ ID NO: 32 and SEQ ID NO: 33 (prime 712 to AB3744362.1 as primers for amplification of variant 3b of the EML4-ALK fusion gene) 87th base), a combination of SEQ ID NO: 35 or 38 and SEQ ID NO: 36 or 39 (bases 1695 to 1723 in AB3744363.1) as a primer for amplification of variant 4 of the EML4-ALK fusion gene, EML4-ALK fusion gene A combination of SEQ ID NO: 41 and SEQ ID NO: 45 as primers for amplification of variant 5a (bases 252 to 295 in AB3744364.1), SEQ ID NO: 47 and SEQ ID NO: 48 as amplification primers for variant 5b of the EML4-ALK fusion gene Combination (bases 252 to 279 in AB3744365.1), combination of SEQ ID NO: 50 and SEQ ID NO: 51 as primers for amplification of variant 6 of EML4-ALK fusion gene (bases 1540 to 1560 in AB4622411.1), EML -Amplification for amplifying the amplifiable base sequence region by using a combination of SEQ ID NO: 56 and SEQ ID NO: 54 (base Nos. 1655 to 1715 in AB4622412.1) as a primer for amplification of variant 7 of ALK fusion gene A primer is mentioned, More specifically, each primer set 1 or 2 used in the Example can be mentioned. In addition, the base sequence prescribed | regulated by the base number in the parenthesis described immediately after each combination is a base sequence (however, except the sequence corresponding to a primer sequence) amplified by each primer set.

  Further, as a control, an amplification primer for amplifying the ALK gene sequence (NM_004304.3) or EML4 gene (NM_019063.3) registered as a wild type in GeneBank can be used. For example, as a primer for amplification of the ALK gene, a combination of SEQ ID NO: 59 and SEQ ID NO: 60 (4072 to 4099 bases of NM_004304.3), or a combination of SEQ ID NO: 62 and SEQ ID NO: 63 (4023-23 of NM_004304.3) An amplification primer that amplifies the base sequence region that can be amplified by using the base No. 4062) (FIG. 21).

  In the present specification, “amplify the base sequence region that can be amplified by using a certain primer set” means that the base sequence amplified by the primer set in the base sequence of the gene to be amplified [primer Means that the entire length of the sequence (including the sequence corresponding to the overlapping or complementary sequence portion of the primer sequence and the gene to be amplified, but not including the additional random sequence portion) or a part thereof is amplified. To do.

  First, a DNA sample is prepared from a subject. Next, the prepared DNA sample is amplified with the same amplification primer as described above, and the DNA is isolated. The isolated DNA fragment is then separated according to its size. The size of the detected DNA fragment is then compared to a control. Since the ALK gene and the EML4 gene do not exist on the same RNA unless they are fused, they are not detected by the method of the present invention. Therefore, the presence or absence of the target fusion gene can be determined by whether or not a DNA fragment of the target size has been obtained.

  The test method of the present invention can be performed by various methods capable of detecting a fusion gene other than the method for directly determining the size of the base sequence of DNA derived from a subject as described above. For example, the following methods can be mentioned.

  In the above method, it is complicated and it is difficult to test a large number of specimens. Further, in the identification method based on the size of the DNA fragment obtained by the PCR method, when examining variants of a plurality of EML4-ALK fusion genes, it is sometimes difficult to distinguish each other because the length of the amplifiable region is narrow. is there. Therefore, a method that directly detects the fusion point is preferable.

  An example of this method is a single base extension reaction. In the single base extension reaction, an unlabeled oligonucleotide primer is annealed immediately before the base position to be examined, and its 3 'end is extended with one molecule of ddNTP. Since dNTP is not contained in the reaction solution, only the site complementary to the target base is extended. This DNA fragment is analyzed and detected by a gene analysis system. As a measuring method using this method, there is a MassARRAY system (Sekenom).

  The detection of the EML4-ALK fusion gene by the MassARRAY system will be described. First, a DNA sample is prepared from a subject, and then the prepared DNA sample is amplified with the same amplification primer as described above to isolate the DNA. As the amplification primer, the amplification primers mentioned above can be used. Next, using this amplification product as a template, a single base extension reaction is performed with an extension primer, and the extended base is determined based on the mass of the obtained substance. The extension primer is preferably designed in a region including the fusion point so that the fusion point of the EML4-ALK fusion gene can be detected.

  When the ALK gene and the EML4 gene are not fused, they do not exist on the same RNA, and therefore are not amplified with the amplification primer and are not extended with the extension primer. Therefore, they are not detected by the method of the present invention. When the EML4-ALK fusion gene is present, it can be amplified with the primer for amplification and extended with the extension primer including the fusion point, so that the presence or absence of the EML4-ALK fusion gene can be accurately determined. .

  In addition, by adding a random sequence of about 10 bases to the 5 ′ end of the sequence complementary to the EML4-ALK fusion gene, the PCR amplification product becomes out of the mass spectroscopic range, and the single base extension reaction product is specific. Can be detected. These amplification primers and extension primers can be designed by Assay Designer (Sequenom).

  In addition, analysis by multiplex PCR is possible by examining amplification primers and extension primers so that each variant can be specifically detected.

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

<< Example 1: Material and EML4-ALK fusion gene detection method >>
1. Construction of template DNA pDNR-dual donor vector / EML4-ALK variant 1 which is a vector containing the full length of variant 1 (SEQ ID NO: 1) was used as a template DNA for detecting EML4-ALK variant 1 (Kinki University School of Medicine tumor) Donated by internal medicine, pDNR-dual donor vector is Clontech). As a template DNA for detecting EML4-ALK variant 3b (SEQ ID NO: 4), pcDNA3.1 (+) / EML4-ALK variant 3b, which is a vector containing the full length of variant 3b, was used (provided by Kinki University School of Medicine) ). EML4-ALK variant 2 (SEQ ID NO: 2), EML4-ALK variant 3a (SEQ ID NO: 3), EML4-ALK variant 4 (SEQ ID NO: 5), EML4-ALK variant 5a (SEQ ID NO: 6), EML4-ALK variant 5b ( SEQ ID NO: 7), EML4-ALK variant 6 (SEQ ID NO: 8), and template DNA for detection of EML4-ALK variant 7 (SEQ ID NO: 9) were each fused with each other using an In fusion system (Clontech). A plasmid in which a DNA sequence of about 1,000 bp sandwiched was introduced into pcDNA3.1 (+) (Invitrogen) was prepared and used.

  As ALK, about 1 sandwiching a fusion point for detecting ALK, using as a template a cDNA obtained by reversing RNA extracted from MKN1 cells (obtained from JCRB cell bank) which is a human gastric cancer cell line expressing wild type ALK. A plasmid into which a DNA sequence (SEQ ID NO: 10) of 1,000 bp was introduced by TA cloning was prepared and used. The fusion point for ALK detection means a part where exon 19 and exon 20 are in contact with each other for convenience.

2. Extraction method of sample and template As a fusion gene expression strain and non-expression strain in cell lines, NCI-H3122 (EML4-ALK / variant 1 gene expression strain: obtained from Kinki University School of Medicine) and NCI- H2228 (EML4-ALK / variant 3a and 3b gene expression strain: obtained from Kinki University School of Medicine Oncology (or ATCC)), human gastric cancer cell line MKN1 (wild type ALK expression strain: obtained from JCRB cell bank) was used. . These cell lines were cultured according to a conventional method.

  As an example of a clinical specimen in this measurement system, a formalin-fixed paraffin-embedded section of a lung cancer patient was used. Formalin-fixed paraffin-embedded sections were prepared according to a conventional method.

  RNeasy Plus Mini Kit (Qiagen) was used for RNA extraction from cell lines, and RNeasy FFPE Kit (Qiagen) was used for RNA extraction from paraffin-embedded sections. The extracted RNA was quantified with NanoDrop2000 (Thermo Fisher Scientific) and subjected to reverse transcription reaction with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) to prepare template DNA. The prepared template DNA was stored at −80 ° C. until use.

3. Detection method of EML4-ALK fusion gene EML4-ALK fusion gene and wild type ALK gene are detected by MassARRAY system (Seakenom) using MALDI-TOF (Matrix Assisted Laser Desorption / Ionization-Time of Flight) mass spectrometer. went. The genes to be detected are nine types of fusion genes (variant 1, variant 2, variant 3a, variant 3b, variant 4, variant 5a, variant 6, and variant 7) and 10 types of ALK. After a specific region containing the fusion point of each variant was amplified by PCR, an extension primer was hybridized to the region containing the fusion point, and a single base extension reaction was performed. The DNA fragment produced by the single base extension reaction was applied to a matrix chip, analyzed from the mass number of the DNA fragment by a MALDI-TOF mass spectrometer, and the detection base was determined to identify the gene to be measured. For detection of ALK, a primer was also designed on an exon (exon 19) that does not contain a fusion point and does not have a fusion gene.

  The detection reaction was performed according to the protocol using iPLEX (TM) Gold (Sequenom). Table 4 summarizes the primers used for the PCR reaction and the primer for single base extension reaction. In the forward primer and the reverse primer shown in Table 4, except for SEQ ID NOS: 32 and 56, a random sequence consisting of 10 bases unrelated to the base sequence to be amplified is added to the 5 'end. Primers were synthesized by Sigma Genosys. For the primers listed in Table 4, first, according to Assay Designer (Sequenom), 150 candidates of primers capable of amplification including a fusion point and primers capable of single-base extension reaction were obtained. It was determined by examining whether amplification can be performed and whether even a small amount of sample can be detected.

  Table 5 shows the bases detected under the above measurement conditions.

<< Example 2: Confirmation of detection of each EML4-ALK fusion gene variant using a plasmid >>
Using 0.1 ng of the plasmid DNA prepared in Example 1, it was investigated whether the wild type ALK gene and the EML4-ALK fusion gene could be detected by the method shown in Example 1. The measured measurement conditions are as shown in Table 5.

  The detection results (each mass spectrum) are shown in FIG. In FIG. 1, the number in parentheses after the gene name indicates the set name (whether set 1 or set 2 was used). Moreover, although description of the horizontal axis and the vertical axis | shaft was abbreviate | omitted, a horizontal axis shows mass (mass) and a vertical axis | shaft shows intensity | strength (intensity). As an example when the detection base is T, G, C, or A, ALK (1), Variant1 (1), Variant3a (1), or Variant1 (2) shown in FIG. Show. In each mass spectrum (the higher the mass is toward the right side), the two dotted lines on the right side indicate the positions of peaks represented by the types of bases extended by one base. For example, in ALK (1), T (left side) ) And A (right side) are shown below. Similarly, in Variant1 (1), G (left side) and C (right side), and in Variant3a (1), G (left side) and C are shown. (Right), Variant1 (2) shows the position of each peak when T (left) and A (right). Note that the dotted line at the left end of each mass spectrum indicates the position of the peak that appears when no single-base extension occurs. As a result, the wild-type ALK gene and the EML4-ALK fusion gene could be identified under all measurement conditions using the prepared primer.

<< Example 3: Detection of EML4-ALK fusion gene variant 1 and EML4-ALK fusion gene variant 3a using cells >>
Using 1 ng of the template cDNA prepared in Example 1, it was examined whether the wild type ALK gene and the EML4-ALK fusion gene can be detected by the method shown in Example 1. The examined measurement conditions are NCI-H3122 (EML4-ALK / variant 1 gene expression strain), which is a human lung cancer cell line, and whether EML4-ALK / variant 1 gene can be identified, or NCI-H2228 (EML4-ALK / variant 3a gene). And 3b gene expression strain) can identify the EML4-ALK / variant 3a gene, and the human gastric cancer cell line MKN1 (wild-type ALK gene expression strain) can identify the wild-type ALK gene.

  As a result, using the prepared primer set Variant1 (2), that is, set 2 of the primer set for EML4-ALK variant 1 described in Tables 4 and 5, the EML4-ALK / variant 1 gene was expressed in NCI-H3122. Could be identified. Also, Variant3a (2), ie, set 2 of the primer set for EML4-ALK variant 3a described in Table 4 and Table 5, can be used to identify the EML4-ALK / variant 3a gene in NCI-H2228. It was. In addition, using ALK (2), that is, set 2 of the primer set for ALK described in Tables 4 and 5, the wild-type ALK gene could be identified in MKN1. On the other hand, in MKN1, it was confirmed that the gene could not be detected under EML4-ALK / variant 3a gene measurement conditions. Similar results were obtained when set 1 was used instead of set 2. As an example, the result of measuring MKN1 using Variant 3a (2) is shown in FIG.

<< Example 4: Detection of EML4-ALK fusion gene in clinical specimen >>
According to Example 1, mRNA was extracted from formalin-fixed paraffin-embedded sections (FFPE) of lung cancer patients to prepare template cDNA. Using 4 ng of the cDNA, it was examined whether the EML4-ALK fusion gene could be detected by the method shown in Example 1. The examined conditions were as follows so that EML4-ALK fusion gene variant 1, variant 2, variant 3a, variant 3b, variant 4, variant 5a, variant 5b, variant 6, and variant 7 could be detected.

  As a result, EML4-ALK fusion gene variant 3a was detected. From the detection of EML4-ALK fusion gene variant 3a, it was identified that this lung cancer patient possesses EML4-ALK fusion gene variant 3a.

<< Example 5: Sequence analysis of PCR amplification product of clinical specimen >>
The sequence analysis of the PCR amplification product obtained in the amplification reaction of the MassARRAY system obtained in Example 4 was performed. The PCR amplification product was subcloned with TOPO TA Cloning Kit (Invitrogen), and the insertion sequences of the obtained single colonies were analyzed by direct sequencing. As a result, it was the same as variant 3a of the EML4-ALK fusion gene. From this, it was confirmed that the clinical specimen used had variant 3a of the EML4-ALK fusion gene.

<< Example 6: Examination 1 of detection sensitivity of EML4-ALK fusion gene >>
The detection sensitivity of EML4-ALK fusion gene variant 3a by the MassARRAY system according to Example 1 was compared with the PCR method. As a result of PCR, the amplification product obtained in the amplification process of MassARRAY system was used. Confirmation of the amplified product by PCR was performed with a bioanalyzer (Agilent Technology).

  Using 1 ng of plasmid (pcDNA3.1 (+) / EML4-ALK variant 3a) DNA, 4 ng of NCI-H2228 DNA, 10 ng of formalin-fixed paraffin-embedded section (FFPE) DNA, EML4-ALK fusion in both PCR amplification method and MassARRAY system Gene variant 3a could be detected. FIG. 7 shows the results of the PCR amplification method, and FIG. 8 shows the results of the MassARRAY system. The PCR amplification method showed the result of the plasmid, the cell line (H2228), and the formalin fixed paraffin embedded section (FFPE), and the MassARRAY system showed the result of the formalin fixed paraffin embedded section (FFPE).

  Next, 0.1 ng of plasmid (pcDNA3.1 (+) / EML4-ALK variant 3a) DNA, 0.4 ng of NCI-H2228 DNA, and 1 ng of formalin-fixed paraffin-embedded section (FFPE) DNA were similarly examined. As a result, detection of EML4-ALK fusion gene variant 3a using formalin-fixed paraffin-embedded sections (FFPE) could not be confirmed by PCR amplification, but was detected by MassARRAY system. The results of the PCR amplification method are shown in FIG. 9, and the results of the MassARRAY system are shown in FIG. The PCR amplification method showed the results of plasmid, cell line (H2228), formalin-fixed paraffin-embedded section (FFPE), and MassARRAY system showed the results of formalin-fixed paraffin-embedded section (FFPE).

  Formalin-fixed paraffin-embedded sections (FFPE) are relatively easy to obtain clinical specimens, but the amount collected is small and valuable. From the above, even when a very small amount of sample is used, the EML4-ALK fusion gene can be detected by using the MassARRAY system.

<< Example 7: Examination 2 of detection sensitivity of EML4-ALK fusion gene >>
In clinical specimens, a small amount of abnormal cells are mixed with a large amount of normal cells. It was examined whether the target EML4-ALK fusion gene could be detected even when such contaminants were mixed.

  Similarly to Example 3, it was examined whether EML4-ALK fusion gene variant 3a in the cell line could be detected. Specifically, the template DNA of MKN1 and NCI-H2228 is 0: 1 (w / w), 1: 0 (w / w), 1: 1 (w / w), 9: 1 (w / w), The mixture was mixed at a ratio of 99: 1 (w / w), and a total of 4 ng was used to examine detection of EML4-ALK fusion gene variant 3a.

  As a result, EML4-ALK fusion gene variant 3a could be clearly detected under any conditions. From this, it was shown that the EML4-ALK fusion gene can be detected with high accuracy by this method.

  Variant 1, variant 2, variant 3a, variant 3b, variant 4, variant 5a, variant 5b, variant 6 of the EML4-ALK fusion gene in paraffin-embedded sections relatively available from lung cancer patients Thus, the presence of variant 7 can be detected, and the effectiveness of the ALK inhibitor can be diagnosed quickly and accurately.

  The base sequences represented by the sequences of SEQ ID NOs: 11 to 64 in the sequence listing are primer sequences.

Claims (10)

(A) A combination of amplification primers that amplify a region including a fusion point, which is a combination of SEQ ID NO: 11 and SEQ ID NO: 15, a combination of SEQ ID NO: 20 and SEQ ID NO: 18 or 21, and SEQ ID NO: 23 or 26 and SEQ ID NO: 24 combinations, SEQ ID NO: 32 and SEQ ID NO: 33, SEQ ID NO: 35 or 38 and SEQ ID NO: 36 or 39, SEQ ID NO: 41 and SEQ ID NO: 45, SEQ ID NO: 47 and SEQ ID NO: 48, SEQ ID NO: Amplifying the amplifiable base sequence region by using at least one combination of primer for amplification selected from the combination of 50 and SEQ ID NO: 51, the combination of SEQ ID NO: 56 and SEQ ID NO: 54;
(B) using the amplification product as a template and performing a single base extension reaction using a detection primer complementary to a region sandwiching the fusion point ;
(C) A method of detecting an EML4-ALK fusion gene comprising the step of determining the extended base by mass spectrometry of the DNA fragment generated by the single base extension reaction . The method according to claim 1 , wherein variant 1 of the EML4-ALK fusion gene is detected, wherein the amplification primer is a combination of SEQ ID NO: 11 and SEQ ID NO: 15, and the extension primer is SEQ ID NO: 13 and / or SEQ ID NO: 16. Method. The method according to claim 1 , wherein variant 2 of the EML4-ALK fusion gene, wherein the amplification primer is a combination of SEQ ID NO: 20 and SEQ ID NO: 18 or 21, and the extension primer is SEQ ID NO: 19 and / or SEQ ID NO: 22. How to detect. The method according to claim 1 , wherein the EML4-ALK fusion gene variant 3a is used, wherein the amplification primer is a combination of SEQ ID NO: 23 or 26 and SEQ ID NO: 24, and the extension primer is SEQ ID NO: 25 and / or SEQ ID NO: 28. How to detect. The method according to claim 1 , wherein variant 3b of the EML4-ALK fusion gene is detected, wherein the amplification primer is a combination of SEQ ID NO: 32 and SEQ ID NO: 33, and the extension primer is SEQ ID NO: 31 and / or SEQ ID NO: 34. Method. The variant of the EML4-ALK fusion gene according to claim 1 , wherein the amplification primer is a combination of SEQ ID NO: 35 or 38 and SEQ ID NO: 36 or 39, and the extension primer is SEQ ID NO: 37 and / or SEQ ID NO: 40 Method for detecting 4. The method according to claim 1 , wherein variant 5a of the EML4-ALK fusion gene, wherein the amplification primer is a combination of SEQ ID NO: 41 and SEQ ID NO: 45 and the extension primer is SEQ ID NO: 43 and / or SEQ ID NO: 46, is detected. Method. The method according to claim 1 , wherein variant 5b of the EML4-ALK fusion gene is detected, wherein the amplification primer is a combination of SEQ ID NO: 47 and SEQ ID NO: 48, and the extension primer is SEQ ID NO: 49. The method according to claim 1 , wherein variant 6 of the EML4-ALK fusion gene is detected, wherein the amplification primer is a combination of SEQ ID NO: 50 and SEQ ID NO: 51, and the extension primer is SEQ ID NO: 52. The method according to claim 1 , wherein variant 7 of the EML4-ALK fusion gene, wherein the amplification primer is a combination of SEQ ID NO: 56 and SEQ ID NO: 54 and the extension primer is SEQ ID NO: 55 and / or SEQ ID NO: 58, is detected. Method.