JP7410480B2 - Fusion genes in cancer - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act Published on the website of the 78th Academic Meeting of the Japanese Cancer Society.

The present invention relates to a method for detecting whether gene fusion associated with cancer has occurred; a kit for detecting whether gene fusion associated with cancer has occurred; a preparation for treating cancer; The present invention relates to antibodies against proteins; polynucleotides targeting mRNA encoding fusion proteins; fusion proteins or fragments thereof, or cDNA encoding them; methods for synthesizing cDNA of fusion genes related to cancer; and the like.

A fusion gene is generated by the fusion of two different genes due to chromosomal translocation. Since fusion genes are one of the causes of cancer, individualized cancer treatment is being carried out by identifying fusion genes and using molecular targeting drugs for the identified fusion genes.

The RET (rearranging transfection) gene is a gene that exists near the centromere of the long arm of chromosome 10 and consists of 21 exons. The protein encoded by the RET gene is a receptor tyrosine kinase and has a cell membrane-spanning region in the center, a tyrosine kinase region at the carboxyl terminal side, and an extracellular region at the amino terminal side. It is known that there are three types of proteins, each with 1072 amino acids (RET9), 1106 amino acids (RET43), and 1114 amino acids (RET51), due to differences in splicing at the carboxyl terminal. RET phosphorylates its own tyrosine and activates it by forming a dimer through the ligand/GFR complex.

In papillary thyroid cancer, the RET gene fuses with other genes (H4, ELE1, etc.) due to intrachromosomal inversion or interchromosomal translocation, resulting in the expression of the fusion tyrosine kinase RET-PTC, which has the ability to cause cancer. It has been reported that (Non-Patent Document 1). Since many of RET's fusion partner molecules have a complex-forming domain, it is thought that the fusion protein with RET itself also forms a complex, and this complex formation is caused by RET's tyrosine kinase. It is thought that it causes constant autophosphorylation of activity, abnormally activates intracellular signals, and causes canceration and cell proliferation (Non-Patent Document 1). In fact, when the RET-PTC fusion gene was introduced into mouse normal NIH3T3 cells, they were transformed into cancer cell-like cells, and furthermore, when a RET kinase inhibitory compound was introduced into thyroid cancer cell line cells expressing the RET-PTC fusion gene, Since RET-PTC fusion protein can suppress proliferation, it has been shown that it can be a therapeutic target molecule for thyroid cancer (Non-patent Documents 2 to 4). Furthermore, it has been reported that the KIF5B-RET fusion gene is expressed in patients with non-small cell lung cancer (Patent Document 1), and that the PIBF1-RET fusion gene is expressed in patients with bronchiolopulmonary cell carcinoma. There is a report (Patent Document 2).

On the other hand, MGEA5 (Meningioma Expressed Antigen 5) is an enzyme that removes O-linked N-acetylglucosamine (O-GlcNAc) modification of serine/threonine residues in proteins, and is known to be particularly highly expressed in the brain and pancreas. ing. MGEA5 resides mainly in the cytoplasm and also contains histone acetyl transferase. However, it has not been reported that the MGEA5 gene is fused with a gene such as the RET gene or that it is associated with cancer.

International Publication No. 2012/014795 pamphlet Japanese Patent Application Publication No. 2015-109806

Eur J Endocrinol.2006 Nov;155(5):645-53. CancerRes. 2002 Dec 15;62(24):7284-90. J Clin Endocrinol Metab. 2006 Oct;91(10):4070-6. J BiolChem. 2007 Oct 5;282(40):29230-40.

An object of the present invention is to provide a biomarker that can serve as a target for cancer therapy.

In order to solve the above problem, the present inventors prepared total RNA from 4000 cases of cancer tissues, synthesized cDNA, and then detected the fusion gene using the method described in Example 1 described below. Ta. As a result, it was confirmed that a fusion gene between the MGEA5 gene and the RET gene (ie, MGEA5-RET fusion gene) was present in the colon cancer tissue sample. Furthermore, the cDNA of the MGEA5-RET fusion gene was identified, and breakpoints in the MGEA5-RET fusion gene were also identified. Furthermore, as a result of analyzing various cancer tissue samples using the same method, we identified 37 new fusion genes in addition to the MGEA5-RET fusion gene. As mentioned above, a fusion gene with RET and a protein encoded by the fusion gene are known to induce cancer and serve as therapeutic target molecules for cancer. From this, the fusion gene of the present invention and the proteins encoded by the fusion gene of the present invention (for example, MGEA5-RET fusion protein and SLC12A2-RET fusion protein) are fully expected to become therapeutic target molecules for cancer. . The present invention was completed based on these findings.

That is, the present invention is as follows.
[1] A method for detecting a fusion gene in cancer, comprising the following step (a).
(a) One or more fusion proteins selected from the following [Fusion Protein Group] (hereinafter sometimes referred to as the "Fusion Protein"), or the fusion protein, in a biological sample collected from a subject. Detecting the encoding genomic DNA, mRNA, or cDNA;
[Fusion protein group]
MGEA5-RET fusion protein TBC1 D5-LPP fusion protein ACER1-MLLT1 fusion protein NAB2-STAT6 fusion protein POU2F1-MAML2 fusion protein TMEM41B-FUS fusion protein ZDHHC21-FOXO1 fusion protein LACAT1-SEPT9 fusion protein EIF3K-ACTN4 fusion protein SLC1 2A2-RET fusion protein GSK3B-GPHN fusion protein IL6ST-FOXO1 fusion protein PPP1CB-ALK fusion protein RNF40-CUTA fusion protein (SEPT5-GP1BB)-SEpt5 fusion protein PRIM2-ACTB fusion protein AZIN1-MLLT3 fusion protein RAP1B-HMGA2 fusion protein EZR-MLLT4 fusion protein OGT - AFF1 fusion protein RPSAP52 - HMGA2 fusion protein BCL11B - LOC105370659 fusion protein LOC101928865 - GAB2 fusion protein AQP10 - ATP8B2 fusion protein ARMC2 - FOXO3 fusion protein CBL - MAML2 fusion protein PRKCQ - PICALM fusion protein FGF1 - PRKA R2A fusion protein SLC44A1-BRAF fusion protein SMURF1 -FOXO1 fusion protein TCAF1-BRAF fusion protein CCNC-MLLT10 fusion protein PPP1R21-ALK fusion protein LOC107986457 -ARHGAP26 fusion protein SPTBN1-ALK fusion protein MKRN2-PPARG fusion protein PHLDB2-JAK2 fusion protein SMARCA2-NFIB fusion protein [2] The fusion protein , MGEA5-RET fusion protein, the detection method according to [1] above,
(i) the MGEA5-RET fusion protein comprises an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1;
(ii) the genomic DNA encoding the MGEA5-RET fusion protein comprises a nucleotide sequence having 80% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 38;
(iii) the mRNA encoding the MGEA5-RET fusion protein comprises a nucleotide sequence having 80% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 2;
(iv) The detection method, wherein the cDNA encoding the MGEA5-RET fusion protein contains a nucleotide sequence having 80% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 3.
[3] A kit for detecting a fusion gene in cancer, which includes an antibody that specifically binds to one or more fusion proteins selected from the following [fusion protein group] in a biological sample.
[Fusion protein group]
MGEA5-RET fusion protein TBC1 D5-LPP fusion protein ACER1-MLLT1 fusion protein NAB2-STAT6 fusion protein POU2F1-MAML2 fusion protein TMEM41B-FUS fusion protein ZDHHC21-FOXO1 fusion protein LACAT1-SEPT9 fusion protein EIF3K-ACTN4 fusion protein SLC1 2A2-RET fusion protein GSK3B-GPHN fusion protein IL6ST-FOXO1 fusion protein PPP1CB-ALK fusion protein RNF40-CUTA fusion protein (SEPT5-GP1BB)-SEpt5 fusion protein PRIM2-ACTB fusion protein AZIN1-MLLT3 fusion protein RAP1B-HMGA2 fusion protein EZR-MLLT4 fusion protein OGT - AFF1 fusion protein RPSAP52 - HMGA2 fusion protein BCL11B - LOC105370659 fusion protein LOC101928865 - GAB2 fusion protein AQP10 - ATP8B2 fusion protein ARMC2 - FOXO3 fusion protein CBL - MAML2 fusion protein PRKCQ - PICALM fusion protein FGF1 - PRKA R2A fusion protein SLC44A1-BRAF fusion protein SMURF1 -FOXO1 fusion protein TCAF1-BRAF fusion protein CCNC-MLLT10 fusion protein PPP1R21-ALK fusion protein LOC107986457 -ARHGAP26 fusion protein SPTBN1-ALK fusion protein MKRN2-PPARG fusion protein PHLDB2-JAK2 fusion protein SMARCA2-NFIB fusion protein [4] In biological samples , a primer set of a forward primer and a reverse primer that anneals to genomic DNA encoding one or more fusion proteins selected from the following [fusion protein group], or a probe that hybridizes to the genomic DNA. Kit for detection of fusion genes in.
[Fusion protein group]
MGEA5-RET fusion protein TBC1 D5-LPP fusion protein ACER1-MLLT1 fusion protein NAB2-STAT6 fusion protein POU2F1-MAML2 fusion protein TMEM41B-FUS fusion protein ZDHHC21-FOXO1 fusion protein LACAT1-SEPT9 fusion protein EIF3K-ACTN4 fusion protein SLC1 2A2-RET fusion protein GSK3B-GPHN fusion protein IL6ST-FOXO1 fusion protein PPP1CB-ALK fusion protein RNF40-CUTA fusion protein (SEPT5-GP1BB)-SEpt5 fusion protein PRIM2-ACTB fusion protein AZIN1-MLLT3 fusion protein RAP1B-HMGA2 fusion protein EZR-MLLT4 fusion protein OGT - AFF1 fusion protein RPSAP52 - HMGA2 fusion protein BCL11B - LOC105370659 fusion protein LOC101928865 - GAB2 fusion protein AQP10 - ATP8B2 fusion protein ARMC2 - FOXO3 fusion protein CBL - MAML2 fusion protein PRKCQ - PICALM fusion protein FGF1 - PRKA R2A fusion protein SLC44A1-BRAF fusion protein SMURF1 -FOXO1 fusion protein TCAF1-BRAF fusion protein CCNC-MLLT10 fusion protein PPP1R21-ALK fusion protein LOC107986457 -ARHGAP26 fusion protein SPTBN1-ALK fusion protein MKRN2-PPARG fusion protein PHLDB2-JAK2 fusion protein SMARCA2-NFIB fusion protein [5] In biological samples , a kit for detecting a fusion gene in cancer, comprising a reverse primer or a probe that hybridizes to mRNA encoding one or more fusion proteins selected from the following [fusion protein group].
[Fusion protein group]
MGEA5-RET fusion protein TBC1 D5-LPP fusion protein ACER1-MLLT1 fusion protein NAB2-STAT6 fusion protein POU2F1-MAML2 fusion protein TMEM41B-FUS fusion protein ZDHHC21-FOXO1 fusion protein LACAT1-SEPT9 fusion protein EIF3K-ACTN4 fusion protein SLC1 2A2-RET fusion protein GSK3B-GPHN fusion protein IL6ST-FOXO1 fusion protein PPP1CB-ALK fusion protein RNF40-CUTA fusion protein (SEPT5-GP1BB)-SEpt5 fusion protein PRIM2-ACTB fusion protein AZIN1-MLLT3 fusion protein RAP1B-HMGA2 fusion protein EZR-MLLT4 fusion protein OGT - AFF1 fusion protein RPSAP52 - HMGA2 fusion protein BCL11B - LOC105370659 fusion protein LOC101928865 - GAB2 fusion protein AQP10 - ATP8B2 fusion protein ARMC2 - FOXO3 fusion protein CBL - MAML2 fusion protein PRKCQ - PICALM fusion protein FGF1 - PRKA R2A fusion protein SLC44A1-BRAF fusion protein SMURF1 -FOXO1 fusion protein TCAF1-BRAF fusion protein CCNC-MLLT10 fusion protein PPP1R21-ALK fusion protein LOC107986457 -ARHGAP26 fusion protein SPTBN1-ALK fusion protein MKRN2-PPARG fusion protein PHLDB2-JAK2 fusion protein SMARCA2-NFIB fusion protein [6] The following [ A primer set of a forward primer and a reverse primer that anneals to cDNA encoding one or more fusion proteins selected from the group of fusion proteins, or a probe that hybridizes to the cDNA, for detection of fusion genes in cancer. kit.
[Fusion protein group]
MGEA5-RET fusion protein TBC1 D5-LPP fusion protein ACER1-MLLT1 fusion protein NAB2-STAT6 fusion protein POU2F1-MAML2 fusion protein TMEM41B-FUS fusion protein ZDHHC21-FOXO1 fusion protein LACAT1-SEPT9 fusion protein EIF3K-ACTN4 fusion protein SLC1 2A2-RET fusion protein GSK3B-GPHN fusion protein IL6ST-FOXO1 fusion protein PPP1CB-ALK fusion protein RNF40-CUTA fusion protein (SEPT5-GP1BB)-SEpt5 fusion protein PRIM2-ACTB fusion protein AZIN1-MLLT3 fusion protein RAP1B-HMGA2 fusion protein EZR-MLLT4 fusion protein OGT - AFF1 fusion protein RPSAP52 - HMGA2 fusion protein BCL11B - LOC105370659 fusion protein LOC101928865 - GAB2 fusion protein AQP10 - ATP8B2 fusion protein ARMC2 - FOXO3 fusion protein CBL - MAML2 fusion protein PRKCQ - PICALM fusion protein FGF1 - PRKA R2A fusion protein SLC44A1-BRAF fusion protein SMURF1 -FOXO1 fusion protein TCAF1-BRAF fusion protein CCNC-MLLT10 fusion protein PPP1R21-ALK fusion protein LOC107986457 -ARHGAP26 fusion protein SPTBN1-ALK fusion protein MKRN2-PPARG fusion protein PHLDB2-JAK2 fusion protein SMARCA2-NFIB fusion protein [7] The fusion protein , MGEA5-RET fusion protein, the detection kit according to any one of [3] to [6] above,
(i) the MGEA5-RET fusion protein comprises an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1;
(ii) A nucleotide sequence in which a primer set of a forward primer and a reverse primer that anneals to the genomic DNA encoding the MGEA5-RET fusion protein has 80% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 38 and its complement; A primer set that amplifies nucleotide residues corresponding to positions 852 to 854 of SEQ ID NO: 38 in the genomic DNA containing the sequence,
(iii) The probe that hybridizes to the genomic DNA encoding the MGEA5-RET fusion protein contains a nucleotide sequence having 80% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 38 and its complementary sequence. is a probe that hybridizes to nucleotide residues corresponding to positions 852 to 854 of SEQ ID NO: 38,
(iv) the reverse primer that hybridizes to the mRNA encoding the MGEA5-RET fusion protein comprises a nucleotide sequence having 80% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 2; A reverse primer that reversely transcribes nucleotide residues corresponding to positions 2618 to 2619 of
(v) The probe that hybridizes to the mRNA encoding the MGEA5-RET fusion protein comprises a nucleotide sequence having 80% or more sequence identity with the nucleotide sequence shown in SEQ ID No. 2, A probe that hybridizes to nucleotide residues corresponding to positions 2618 and 2619,
(vi) A nucleotide sequence in which the primer set of a forward primer and a reverse primer that anneals to the cDNA encoding the MGEA5-RET fusion protein has 80% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 3, and its complementary sequence. A primer set that amplifies nucleotide residues corresponding to positions 2175 to 2176 of SEQ ID NO: 3 in the cDNA containing
(vii) the probe that hybridizes to the cDNA encoding the MGEA5-RET fusion protein comprises a nucleotide sequence having 80% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 3 and a complementary sequence thereof; The detection kit described above is a probe that hybridizes to nucleotide residues corresponding to positions 2175 and 2176 of SEQ ID NO: 3.
[8] A therapeutic agent for cancer, comprising a substance that suppresses the expression or physiological activity of one or more fusion proteins selected from the following [fusion protein group], which suppresses the expression or physiological activity of the fusion protein: The therapeutic agent is administered to a subject in whom is detected.
[Fusion protein group]
MGEA5-RET fusion protein TBC1 D5-LPP fusion protein ACER1-MLLT1 fusion protein NAB2-STAT6 fusion protein POU2F1-MAML2 fusion protein TMEM41B-FUS fusion protein ZDHHC21-FOXO1 fusion protein LACAT1-SEPT9 fusion protein EIF3K-ACTN4 fusion protein SLC1 2A2-RET fusion protein GSK3B-GPHN fusion protein IL6ST-FOXO1 fusion protein PPP1CB-ALK fusion protein RNF40-CUTA fusion protein (SEPT5-GP1BB)-SEpt5 fusion protein PRIM2-ACTB fusion protein AZIN1-MLLT3 fusion protein RAP1B-HMGA2 fusion protein EZR-MLLT4 fusion protein OGT - AFF1 fusion protein RPSAP52 - HMGA2 fusion protein BCL11B - LOC105370659 fusion protein LOC101928865 - GAB2 fusion protein AQP10 - ATP8B2 fusion protein ARMC2 - FOXO3 fusion protein CBL - MAML2 fusion protein PRKCQ - PICALM fusion protein FGF1 - PRKA R2A fusion protein SLC44A1-BRAF fusion protein SMURF1 -FOXO1 fusion protein TCAF1-BRAF fusion protein CCNC-MLLT10 fusion protein PPP1R21-ALK fusion protein LOC107986457 -ARHGAP26 fusion protein SPTBN1-ALK fusion protein MKRN2-PPARG fusion protein PHLDB2-JAK2 fusion protein SMARCA2-NFIB fusion protein [9] Fusion protein , MGEA5-RET fusion protein, the therapeutic agent according to [8] above,
The therapeutic agent, wherein the MGEA5-RET fusion protein contains an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1.
[10] An antibody that specifically binds to one or more fusion proteins selected from the following [fusion protein group] in a biological sample.
[Fusion protein group]
MGEA5-RET fusion protein TBC1 D5-LPP fusion protein ACER1-MLLT1 fusion protein NAB2-STAT6 fusion protein POU2F1-MAML2 fusion protein TMEM41B-FUS fusion protein ZDHHC21-FOXO1 fusion protein LACAT1-SEPT9 fusion protein EIF3K-ACTN4 fusion protein SLC1 2A2-RET fusion protein GSK3B-GPHN fusion protein IL6ST-FOXO1 fusion protein PPP1CB-ALK fusion protein RNF40-CUTA fusion protein (SEPT5-GP1BB)-SEpt5 fusion protein PRIM2-ACTB fusion protein AZIN1-MLLT3 fusion protein RAP1B-HMGA2 fusion protein EZR-MLLT4 fusion protein OGT - AFF1 fusion protein RPSAP52 - HMGA2 fusion protein BCL11B - LOC105370659 fusion protein LOC101928865 - GAB2 fusion protein AQP10 - ATP8B2 fusion protein ARMC2 - FOXO3 fusion protein CBL - MAML2 fusion protein PRKCQ - PICALM fusion protein FGF1 - PRKA R2A fusion protein SLC44A1-BRAF fusion protein SMURF1 -FOXO1 fusion protein TCAF1-BRAF fusion protein CCNC-MLLT10 fusion protein PPP1R21-ALK fusion protein LOC107986457 -ARHGAP26 fusion protein SPTBN1-ALK fusion protein MKRN2-PPARG fusion protein PHLDB2-JAK2 fusion protein SMARCA2-NFIB fusion protein [11] The fusion protein , the antibody according to [10] above, which is an MGEA5-RET fusion protein,
The antibody, wherein the MGEA5-RET fusion protein comprises an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1.
[12] Has RNA interference (RNAi) ability, and
A polynucleotide that specifically hybridizes to mRNA encoding one or more fusion proteins selected from the following [fusion protein group].
[Fusion protein group]
MGEA5-RET fusion protein TBC1 D5-LPP fusion protein ACER1-MLLT1 fusion protein NAB2-STAT6 fusion protein POU2F1-MAML2 fusion protein TMEM41B-FUS fusion protein ZDHHC21-FOXO1 fusion protein LACAT1-SEPT9 fusion protein EIF3K-ACTN4 fusion protein SLC1 2A2-RET fusion protein GSK3B-GPHN fusion protein IL6ST-FOXO1 fusion protein PPP1CB-ALK fusion protein RNF40-CUTA fusion protein (SEPT5-GP1BB)-SEpt5 fusion protein PRIM2-ACTB fusion protein AZIN1-MLLT3 fusion protein RAP1B-HMGA2 fusion protein EZR-MLLT4 fusion protein OGT - AFF1 fusion protein RPSAP52 - HMGA2 fusion protein BCL11B - LOC105370659 fusion protein LOC101928865 - GAB2 fusion protein AQP10 - ATP8B2 fusion protein ARMC2 - FOXO3 fusion protein CBL - MAML2 fusion protein PRKCQ - PICALM fusion protein FGF1 - PRKA R2A fusion protein SLC44A1-BRAF fusion protein SMURF1 -FOXO1 fusion protein TCAF1-BRAF fusion protein CCNC-MLLT10 fusion protein PPP1R21-ALK fusion protein LOC107986457 -ARHGAP26 fusion protein SPTBN1-ALK fusion protein MKRN2-PPARG fusion protein PHLDB2-JAK2 fusion protein SMARCA2-NFIB fusion protein [13] The fusion protein , the polynucleotide according to [12] above, which is an MGEA5-RET fusion protein,
The polynucleotide, wherein the mRNA encoding the MGEA5-RET fusion protein contains a nucleotide sequence having 80% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 2.
[14] One or more fusion proteins selected from the following [fusion protein group] or fragments thereof, or cDNA encoding them.
[Fusion protein group]
MGEA5-RET fusion protein TBC1 D5-LPP fusion protein ACER1-MLLT1 fusion protein NAB2-STAT6 fusion protein POU2F1-MAML2 fusion protein TMEM41B-FUS fusion protein ZDHHC21-FOXO1 fusion protein LACAT1-SEPT9 fusion protein EIF3K-ACTN4 fusion protein SLC1 2A2-RET fusion protein GSK3B-GPHN fusion protein IL6ST-FOXO1 fusion protein PPP1CB-ALK fusion protein RNF40-CUTA fusion protein (SEPT5-GP1BB)-SEpt5 fusion protein PRIM2-ACTB fusion protein AZIN1-MLLT3 fusion protein RAP1B-HMGA2 fusion protein EZR-MLLT4 fusion protein OGT - AFF1 fusion protein RPSAP52 - HMGA2 fusion protein BCL11B - LOC105370659 fusion protein LOC101928865 - GAB2 fusion protein AQP10 - ATP8B2 fusion protein ARMC2 - FOXO3 fusion protein CBL - MAML2 fusion protein PRKCQ - PICALM fusion protein FGF1 - PRKA R2A fusion protein SLC44A1-BRAF fusion protein SMURF1 -FOXO1 fusion protein TCAF1-BRAF fusion protein CCNC-MLLT10 fusion protein PPP1R21-ALK fusion protein LOC107986457 -ARHGAP26 fusion protein SPTBN1-ALK fusion protein MKRN2-PPARG fusion protein PHLDB2-JAK2 fusion protein SMARCA2-NFIB fusion protein [15] The fusion protein , the fusion protein or a fragment thereof according to [14] above, which is the MGEA5-RET fusion protein, or a cDNA encoding the same,
(i) the MGEA5-RET fusion protein is an isolated protein or a recombinant protein comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1; The fragment of the RET fusion protein contains amino acid residues corresponding to positions 725 to 726 of SEQ ID NO: 1 as a fusion site,
(ii) the cDNA encoding the MGEA5-RET fusion protein is a cDNA containing a nucleotide sequence having 80% or more sequence identity with the nucleotide sequence of SEQ ID NO: 3, and encodes a fragment of the MGEA5-RET fusion protein; cDNA is characterized in that it contains nucleotide residues corresponding to positions 2175 to 2176 of SEQ ID NO: 3 as a fusion site,
The fusion protein or a fragment thereof, or cDNA encoding the same.
[16] A method for synthesizing cDNA of a cancer-related fusion gene, which comprises the following steps (A) to (C).
(A) Selecting a fusion gene that satisfies the following conditions (1) to (5) from a database of cancer-related fusion genes;
(1) DNA sequence information of the fusion gene has been revealed;
(2) It is not a fusion gene known to exist in cultured cells;
(3) A fusion gene in which two different gene fragments are fused;
(4) The fusion site is a single fusion gene;
(5) It is a fusion gene whose location on the genomic DNA has been specified;
(B) Synthesizing cDNA with an adapter added to the upstream end and/or downstream end based on mRNA in a biological sample collected from a cancer patient;
(C) A forward primer that anneals to the adapter added to the upstream end of the cDNA synthesized in step (B), and a reverse primer that anneals to the cDNA of the downstream gene fragment of the fusion gene selected in step (A). Perform a PCR (Polymerase Chain Reaction) reaction using a primer, or perform a PCR (Polymerase Chain Reaction) reaction using a forward primer that anneals to the cDNA of the upstream gene fragment of the fusion gene selected in step (A), and synthesize in step (B). a step of performing PCR using a reverse primer that anneals to an adapter added to the downstream end of the cDNA;
[17] The method according to [16] above, wherein in step (A), at least 700 types of fusion genes are selected.
[18] In step (C), a forward primer that anneals to the adapter added to the upstream end of the cDNA synthesized in step (B) and a downstream gene fragment of the fusion gene selected in step (A) are added. The method described in [16] or [17] above, characterized in that PCR is performed using a reverse primer that anneals to the cDNA of.

Further, as another embodiment of the present invention,
A method for determining cancer (in other words, a method for collecting data for determining [diagnosing] cancer) includes the following steps (a) and (b).
(a) Detecting the subject fusion protein, or genomic DNA, mRNA, or cDNA encoding the subject fusion protein in a biological sample collected from a subject;
(b) In step (a), if the subject fusion protein, or genomic DNA, mRNA, or cDNA encoding the subject fusion protein, is detected, the subject may detect the subject fusion protein (i.e., the subject fusion protein) ) is a process of evaluating whether there is a high possibility that the patient is a cancer patient;

Further, as another embodiment of the present invention, a primer set of a forward primer and a reverse primer that anneals to genomic DNA encoding the subject fusion protein in a biological sample, for use in a method for detecting a fusion gene in cancer, and Probes that hybridize to genomic DNA can be mentioned.

Other embodiments of the present invention include reverse primers and probes that hybridize to mRNA encoding the fusion protein in a biological sample for use in a method for detecting a fusion gene in cancer.

In addition, as another embodiment of the present invention, a primer set of a forward primer and a reverse primer that anneals to the cDNA encoding the fusion protein for use in a method for detecting a fusion gene in cancer, and a primer set that hybridizes to the cDNA is provided. Probes can be mentioned.

Further, as another embodiment of the present invention,
A substance that suppresses the expression or physiological activity of the subject fusion protein for use in the treatment of cancer, said substance being administered to a subject in which the expression or physiological activity of the subject fusion protein is detected;
The Substance for use as a therapeutic agent for cancer;
Use of the Substance in the manufacture of cancer therapeutics;
A method for treating cancer, comprising the step (p) of administering the subject substance to a subject in which expression or physiological activity of the subject fusion protein is detected;
can be mentioned.

According to the present invention, it is not only possible to detect whether or not a person is suffering from cancer, but also to identify cancer patients in which the present fusion gene is present among cancer patients. As mentioned above, a fusion gene with RET and a protein encoded by the fusion gene are known to induce cancer and serve as therapeutic target molecules for cancer. From this, the fusion gene of the present invention and the proteins encoded by the fusion gene of the present invention (for example, MGEA5-RET fusion protein and SLC12A2-RET fusion protein) are fully expected to become therapeutic target molecules for cancer. . Therefore, cancer patients in which the MGEA5-RET fusion gene or SLC12A2-RET fusion gene is present may be treated using substances that suppress the expression or physiological activity of the MGEA5-RET fusion protein or SLC12A2-RET fusion protein. It becomes possible.

FIG. 3 is a diagram showing the results of PCR detection of mRNA of the MGEA5-RET fusion gene present in the colon cancer tissue of case #1. Lane M shows DNA size markers. "*" in lane 1 indicates a PCR product (estimated 4367 nucleotide pairs [bp]) obtained by PCR using cDNA prepared from colon cancer tissue as a template and the MGRET_F and MGRET_R primer sets. "#" in lane 2 indicates a PCR product (estimated 3390 bp) obtained by PCR using the PCR product obtained in lane 1 as a template and the MGRET-NEST_F and MGRET-NEST_R primer sets. FIG. 3 is a diagram showing the results of detecting the MGEA5-RET fusion gene present in the colon cancer tissue of case #1 using PCR. Lane M shows DNA size markers. Lane 1 shows the results of PCR using the primer sets of MG-RET_1F and MG-RET_1R, Lane 2 shows the results of PCR using the primer sets of MG-RET_CF and MG-RET_2R, and Lane 3 shows the results of PCR using the primer sets of MG-RET_1F and MG-RET_1R. Lane 4 shows the results of PCR using the primer sets of MG-RET_CF and MG-RET_3R, lane 5 shows the results of PCR using the primer sets of MG-RET_CF and MG-RET_4R, and lane 5 shows the results of PCR using the primer sets of MG-RET_CF and MG-RET_3R. The results of PCR using the MG-RET_5R primer set are shown, and lane 6 shows the results of PCR using the MG-RET_CF and MG-RET_6R primer sets.

The method for detecting fusion genes in cancer of the present invention includes:
One or more fusion proteins selected from the following [fusion protein group] (i.e., the subject fusion protein) in a biological sample collected from a subject (also referred to as "subject"); encoding the subject fusion protein; (hereinafter sometimes referred to as "the fusion gene"); mRNA (messenger RNA) encoding the fusion protein (hereinafter sometimes referred to as "mRNA of the fusion gene"); and/or the fusion protein. cDNA (complementary DNA) encoding (hereinafter sometimes referred to as "cDNA of the subject fusion gene"); The present detection method is a method to assist a doctor in diagnosing whether or not a subject is a cancer patient in which the subject fusion gene is present, and the detection method Does not include.
[Fusion protein group]
MGEA5-RET fusion protein TBC1 D5-LPP fusion protein ACER1-MLLT1 fusion protein NAB2-STAT6 fusion protein POU2F1-MAML2 fusion protein TMEM41B-FUS fusion protein ZDHHC21-FOXO1 fusion protein LACAT1-SEPT9 fusion protein EIF3K-ACTN4 fusion protein SLC1 2A2-RET fusion protein GSK3B-GPHN fusion protein IL6ST-FOXO1 fusion protein PPP1CB-ALK fusion protein RNF40-CUTA fusion protein (SEPT5-GP1BB)-SEpt5 fusion protein PRIM2-ACTB fusion protein AZIN1-MLLT3 fusion protein RAP1B-HMGA2 fusion protein EZR-MLLT4 fusion protein OGT - AFF1 fusion protein RPSAP52 - HMGA2 fusion protein BCL11B - LOC105370659 fusion protein LOC101928865 - GAB2 fusion protein AQP10 - ATP8B2 fusion protein ARMC2 - FOXO3 fusion protein CBL - MAML2 fusion protein PRKCQ - PICALM fusion protein FGF1 - PRKA R2A fusion protein SLC44A1-BRAF fusion protein SMURF1 -FOXO1 fusion protein TCAF1-BRAF fusion protein CCNC-MLLT10 fusion protein PPP1R21-ALK fusion protein LOC107986457 -ARHGAP26 fusion protein SPTBN1-ALK fusion protein MKRN2-PPARG fusion protein PHLDB2-JAK2 fusion protein SMARCA2-NFIB fusion protein

The kit for detecting a fusion gene in cancer of the present invention includes an antibody that specifically binds to the fusion protein in a biological sample (hereinafter sometimes referred to as "the fusion protein target antibody"). Kits for use in gene detection;
A primer set of a forward primer and a reverse primer that anneals to the genomic DNA encoding the subject fusion protein in a biological sample (hereinafter sometimes referred to as "the subject genomic DNA detection primer set") and/or a probe that hybridizes to the genomic DNA. (hereinafter sometimes referred to as "the subject genomic DNA detection probe"), a kit for use in detecting fusion genes in cancer;
A reverse primer (hereinafter sometimes referred to as "reverse primer for detecting mRNA") and/or a probe (hereinafter sometimes referred to as "probe for detecting mRNA") that hybridizes to mRNA encoding the subject fusion protein in a biological sample. ), a kit for use in detecting fusion genes in cancer;
A primer set of a forward primer and a reverse primer that anneals to the cDNA encoding the subject fusion protein (hereinafter sometimes referred to as "this subject cDNA detection primer set"); and/or a probe that hybridizes to the cDNA (hereinafter referred to as "this subject cDNA detection primer set"); A kit for use in detecting fusion genes in cancer, including a cDNA detection probe (sometimes referred to as "cDNA detection probe");
(These kits may be collectively referred to as "this kit" hereinafter). The present kit is an application invention relating to a kit for detecting a fusion gene in cancer, and these kits include components that are generally used in this type of detection kit, such as a carrier, a pH buffer, a stabilizer, and handling materials. Package inserts such as instructions and instructions for detecting fusion genes in cancer are usually included.

The therapeutic agent for cancer of the present invention contains a substance that suppresses the expression or physiological activity of the fusion protein (hereinafter sometimes referred to as the "substance"), which is specified for the purpose of "treating cancer." The therapeutic agent may be used alone as a pharmaceutical (preparation), or may be further mixed with other ingredients to form a composition (hereinafter sometimes referred to as "the therapeutic agent"). It may also be used as a pharmaceutical composition.

An antibody that targets the fusion protein of the present invention is an antibody that specifically binds to the fusion protein in a biological sample (i.e., an antibody targeting the fusion protein), and specifically includes a fusion site in the fusion protein. An antibody whose epitope is a polypeptide. The fusion protein-targeted antibody is useful for cancer treatment as well as cancer diagnosis.

A polynucleotide that targets the mRNA of the fusion gene of the present invention has RNA interference (RNAi) ability and hybridizes specifically to the mRNA encoding the fusion protein (i.e., the mRNA of the fusion gene). A polynucleotide (hereinafter sometimes referred to as "mRNA target polynucleotide of the fusion gene"), and polynucleotides with RNAi ability include siRNA (small interfering RNA), shRNA (short hairpin RNA), miRNA (micro RNA), antisense nucleic acids, and the like. The mRNA target polynucleotide of the subject fusion gene is useful for cancer treatment.

The "XY fusion protein" in the above [fusion protein group] means a protein in which an X protein fragment and a Y protein fragment are fused in order from the amino (N) terminus to the carboxyl (C) terminus. When the fusion protein is an MGEA5-RET fusion protein, it means a protein in which an MGEA5 protein fragment and a RET protein fragment are fused in order from the N-terminus to the C-terminus.

Specifically, the mRNA of the subject fusion gene is a nucleotide having 80% or more sequence identity with the mRNA of the subject fusion gene (hereinafter sometimes referred to as "mRNA of Table 1") shown in Table 1 below. Specifically, the cDNA of the fusion gene includes a nucleotide sequence having 80% or more sequence identity with the cDNA synthesized based on the mRNA shown in Table 1. Examples of the fusion protein include fusion proteins that include an amino acid sequence having 80% or more sequence identity with the amino acid sequence encoded by the mRNA in Table 1. Further, specific examples of the present fusion gene include genomic DNA containing a nucleotide sequence having 80% or more sequence identity with the nucleotide sequence encoding the mRNA shown in Table 1.

The nucleotide sequence of the mRNA in Table 1 can be understood from the content described in the item "mRNA of the subject fusion gene" in Table 1. For example, the nucleotide sequence of "MGEA5{NM_012215" which is the mRNA encoding the MGEA5-RET fusion protein. .3}:r.1_2618-RET{NM_020975.4}:r.2327_5617'' is the MGEA5 gene registered with the NCBI with the number NM_012215.3. nucleotide residues 1 to 2618 of the mRNA [specifically, the mRNA consisting of the nucleotide sequence of SEQ ID NO: 22]) and "RET{NM_020975.4}:r.2327_5617" (i.e., NM_020975.4 in NCBI). The mRNA of the fusion gene (specifically, the nucleotide residues 2327 to 5617 of the mRNA consisting of the nucleotide sequence of SEQ ID NO: 25) of the RET gene registered with the number MGEA5-RET gene mRNA consisting of the nucleotide sequence number 2). Furthermore, considering that the 444th to 446th nucleotide residues of the mRNA are translation initiation codons, and the 3825th to 3827th nucleotide residues are termination codons, cDNA synthesized based on the mRNA (i.e. It can be understood that the cDNA of the MGEA5-RET gene is a cDNA consisting of the nucleotide sequence of SEQ ID NO: 3. Furthermore, based on the nucleotide sequence of the cDNA, it can be understood that the protein encoded by the cDNA (ie, MGEA5-RET fusion protein) is a protein consisting of the amino acid sequence of SEQ ID NO: 1. Similarly, regarding the mRNA encoding the subject fusion protein other than the mRNA encoding the MGEA5-RET fusion protein, from the contents of Table 1, the nucleotide sequence of the mRNA, the nucleotide sequence of the cDNA synthesized based on the mRNA, The sequence, the amino acid sequence encoded by the mRNA, the fusion site, etc. can be understood.

A suitable example of the present fusion protein is the MGEA5-RET fusion protein. In that case, the MGEA5-RET fusion protein specifically includes the amino acid sequence shown in SEQ ID NO: 1 and a sequence of 80% or more. Examples of genomic DNA encoding the MGEA5-RET fusion protein include those containing an identical amino acid sequence, and specifically, genomic DNA that has 80% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 38. Examples of mRNA encoding the MGEA5-RET fusion protein include those containing a nucleotide sequence having a sequence identity of 80% or more with the nucleotide sequence shown in SEQ ID NO: 2. Specifically, examples of the cDNA encoding the MGEA5-RET fusion protein include a nucleotide sequence having 80% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 3. Examples include those containing arrays.

As used herein, "a polynucleotide that has RNAi ability and specifically hybridizes to the mRNA of the subject fusion gene" means, specifically, when the subject fusion protein is an MGEA5-RET fusion protein, Specifically hybridizes to nucleotide residues corresponding to positions 2618 to 2619 of SEQ ID NO: 2 in the mRNA of the MGEA5-RET fusion gene, which contains a nucleotide sequence having 80% or more sequence identity with the nucleotide sequence of SEQ ID NO: 2. , preferably an mRNA of the MGEA5 gene comprising a nucleotide sequence having 80% or more sequence identity with the nucleotide sequence of SEQ ID NO: 22, and/or having 80% or more sequence identity with the nucleotide sequence of SEQ ID NO: 25. It refers to a polynucleotide (preferably a single-stranded polynucleotide) containing a nucleotide sequence that does not specifically hybridize to the mRNA of the RET gene. The length of the mRNA target polynucleotide of the fusion gene is, for example, 10 nucleotides or more (preferably 11, 12, 13, 14, 15, 16, 17, or 18 nucleotides or more) and 50 nucleotides or less (preferably 46, 43, 40, 39, 36, or 30 nucleotides or less).

The fusion protein may be an isolated protein (hereinafter sometimes referred to as the "isolated fusion protein") or a recombinant protein (hereinafter sometimes referred to as the "recombinant fusion protein"); In addition, as a fragment of the fusion protein, the isolated fusion protein or Any fragment of the recombinant fusion protein of the present invention may be used, and the cDNA encoding the fusion protein of the present invention may be any cDNA encoding the fusion protein of the present invention (i.e., cDNA of the fusion gene of the present invention); The cDNA encoding the fragment of the fusion protein of the present invention includes the present isolated protein containing the fusion site (for example, when the present fusion protein is the MGEA5-RET fusion protein, nucleotide residues corresponding to positions 2175 to 2176 of SEQ ID NO: 3). Any cDNA encoding the fusion protein or a fragment of the recombinant fusion protein (in other words, a cDNA fragment of the fusion gene) may be used. The length of the fragment of the isolated fusion protein or the recombinant fusion protein of the present invention is not particularly limited, and for example, when the fusion protein of the present invention is a fragment of the MGEA5-RET fusion protein, it is within the range of 3 to 1126 amino acids (e.g. , 6-1100 amino acids, 10-1000 amino acids, 10-600 amino acids, 10-300 amino acids, 10-100 amino acids). Furthermore, the length of the cDNA fragment of the fusion gene is not particularly limited; for example, when the fusion protein is an MGEA5-RET fusion protein, it is within the range of 9 to 3381 nucleotides (for example, 18 to 3303 nucleotides). , 30-3003 nucleotides, 30-1803 nucleotides, 30-903 nucleotides, 30-303 nucleotides).

The method of synthesizing the cDNA of the cancer-related fusion gene of the present invention includes: (1) DNA sequence information of the fusion gene has been revealed from a cancer-related fusion gene database; (2) culture. It is not a fusion gene that is known to exist in cells; (3) it is a fusion gene in which two different gene fragments are fused; (4) the fusion site is a single fusion gene; and (5) the genome A step (A) of selecting a fusion gene that satisfies conditions (1) to (5); the fusion gene is a fusion gene whose location on the DNA has been specified; Step (B) of synthesizing cDNA with an adapter added to the upstream end and/or downstream end; a forward primer (hereinafter referred to as , sometimes referred to as "forward primer for adapter") and a reverse primer that anneals to the cDNA of the downstream gene fragment of the fusion gene selected in step (A) (hereinafter referred to as "reverse primer for fusion gene"). PCR (Polymerase Chain Reaction) reaction is performed using a forward primer (hereinafter referred to as a fusion gene) that anneals to the cDNA of the upstream gene fragment of the fusion gene selected in step (A). A reverse primer that anneals to the adapter added to the downstream end of the cDNA synthesized in step (B) (hereinafter sometimes referred to as a "reverse primer for adapters") is used. The method is not particularly limited as long as it includes steps (A) to (C) (hereinafter sometimes referred to as "synthesis method") of step (C) of performing PCR using the method. Using the present synthesis method, cDNA of the MGEA5-RET fusion gene of the present invention can be synthesized.

As used herein, "genomic DNA" generally refers to a double-stranded polynucleotide, specifically, an antisense strand (i.e., an mRNA template) and a sense strand that is its complementary sequence, unless otherwise specified. It is a double-stranded polynucleotide consisting of. In addition, in this specification, "mRNA" generally refers to a single-stranded polynucleotide, unless otherwise specified, and specifically, a single-stranded polynucleotide synthesized using the antisense strand of genomic DNA as a template. It is a nucleotide. Furthermore, in this specification, "cDNA" generally refers to a double-stranded polynucleotide, specifically a double-stranded polynucleotide synthesized using mRNA as a template, unless otherwise specified.

As used herein, the "upstream end" and "downstream end" of cDNA refer to the "near end" of the cDNA when viewed from the promoter when the cDNA is operably linked to the promoter. and "distal end". In addition, in this specification, "upstream gene fragment" and "downstream gene fragment" of the fusion gene refer to the "upstream gene fragment" and "downstream gene fragment" of the fusion gene when the fusion gene is operably linked to the promoter. , respectively, mean a "near gene fragment" and a "distant gene fragment" from the promoter.

In the present invention, cancers include, for example, colorectal cancer (colon cancer or rectal cancer); stomach cancer; liver cancer; brain tumor; lung cancer (specifically, adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, epithelial cancer, undifferentiated cancer, large cell cancer, small cell cancer); esophageal cancer; duodenal cancer; small intestine cancer; skin cancer; breast cancer; prostate cancer; bladder cancer; vaginal cancer; Cervical cancer; Endometrial cancer; Kidney cancer; Pancreatic cancer; Spleen cancer; Tracheal cancer; Bronchial cancer; Head and neck cancer; Gallbladder cancer; Bile duct cancer; Testicular cancer; leukemia; mediastinal tumors (e.g., thymoma, thymic carcinoma, germ cell tumor, malignant lymphoma, goiter), etc. Among these, the effect was demonstrated in this example. Preferred examples include colon cancer, lung cancer, stomach cancer, mediastinal tumor (eg, thymus cancer), pancreatic cancer, and head and neck cancer.

In the present invention, biological samples include, for example, tears, blood (e.g. plasma, serum), urine, cells (preferably cells in which cancer may exist), tissues (preferably cells in which cancer may exist). (tissues in which cancer may exist), organs (preferably organs in which cancer may exist), and tissues are preferred.

The above-mentioned subject is not particularly limited as long as it is a human, and for example, a subject whose presence or absence of cancer is unknown, genomic DNA or mRNA encoding the subject fusion protein or the subject MGEA5-RET fusion protein, Alternatively, cancer patients whose presence or absence of cDNA is unknown can be mentioned. Such subjects include subjects who have had cancer in the past and have since been completely cured of cancer, but at the time of the study, it is unknown whether or not they have cancer, and subjects who have not received the fusion protein or the MGEA5- This also includes subjects in which it is unclear whether genomic DNA, mRNA, or cDNA encoding the RET fusion protein exists.

As specifically shown in this example below, the amino acid sequence of SEQ ID NO: 1 is based on the MGEA5-RET fusion gene (i.e., the upstream MGEA5 gene fragment and the downstream This is the amino acid sequence encoded by the fusion gene consisting of the RET gene fragment on the side. More specifically, the amino acid sequence of SEQ ID NO: 1 is the amino acid sequence of the amino (N) terminal region of MGEA5 (specifically, in the MGEA5 protein containing the amino acid sequence of SEQ ID NO: 24, 1 to 725 of SEQ ID NO: 24). amino acid residue) and the amino acid sequence of the carboxyl (C) terminal region of RET (specifically, in the RET protein containing the amino acid sequence of SEQ ID NO: 27, amino acid residues 713 to 1114 of SEQ ID NO: 27) The fusion site is located between the 725th glutamic acid (E) and the 726th glutamic acid in the amino acid sequence of SEQ ID NO: 1. Since there are many single nucleotide polymorphisms (SNPs) in the MGEA5-RET fusion gene in humans, there are amino acid sequences in the human population that have 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 1. There may be MGEA5-RET fusion proteins that include. Similarly, fusion proteins may exist that include amino acid sequences that have 80% or more sequence identity to the amino acid sequences encoded by the mRNAs in Table 1.

In step (a) of the present detection method, the method for detecting the present fusion protein may include, for example, an immunoassay using an antibody targeting the present fusion protein. Suitable examples of such immunological measurement methods include immunohistochemical staining, ELISA, EIA, RIA, Western blotting, dot blotting, flow cytometry, and the like. Here, the flow cytometry method uses fluorescent substances (e.g., allophycocyanin [APC], phycoerythrin [PE], FITC [fluorescein isothiocyanate], Alexa Fluor 488, Alexa Fluor 647, Alexa Fluor 700, PE-Texas Red, PE-Cy5, PE -Cy7) using a fluorescence-activated cell sorter (FACS) using an antibody targeting the fusion protein of the present invention.

The nucleotide sequence of SEQ ID NO: 38 is an MGEA5-RET fusion gene detected in a sample derived from human colon cancer tissue, as specifically shown in this example below. The nucleotide sequence of SEQ ID NO: 38 more specifically includes the 5' region of the MGEA5 gene (i.e., the nucleotide sequence from the 5' end of the MGEA5 gene to the breakpoint present in intron 12) and the RET The 3' region of the gene (that is, the nucleotide sequence from the breakpoint in intron 11 of the RET gene to the 3' end) is a nucleotide sequence fused via thymidine, and the breakpoint is SEQ ID NO: 38. It is located between adenosine (A) at position 852 and thymidine (T) at position 854 in the nucleotide sequence. Since there are many SNPs of the MGEA5-RET fusion gene in humans, there may be an MGEA5-RET fusion gene in the human population that includes a nucleotide sequence having 80% or more sequence identity with the nucleotide sequence of SEQ ID NO: 38. .

In step (a) of the present detection method, methods for detecting the present fusion gene include, for example, the LAMP (Loop-Mediated Isothermal Amplification) method using the present genomic DNA detection primer set, the PCR (polymerase chain reaction) method ( For example, real-time PCR method [intercalator method, 5'-nuclease method, cycling probe method, etc.], droplet digital PCR [ddPCR] method); ligase chain reaction (LCR) method; next-generation sequencer Sequencing methods; Southern hybridization methods using the present genomic DNA detection probes, microarray methods, ISH (in situ hybridization) methods, etc. can be mentioned.

The nucleotide sequence of SEQ ID NO: 2 is the mRNA of the MGEA5-RET fusion gene detected in a sample derived from human colon cancer tissue, as specifically shown in this example below. More specifically, the nucleotide sequence of SEQ ID NO: 2 is the 5' region of the mRNA of the MGEA5 gene (specifically, 1 to 2618 of SEQ ID NO: 22 in the mRNA of the MGEA5 gene containing the nucleotide sequence of SEQ ID NO: 22). nucleotide residue) and the 3' region of the RET gene mRNA (specifically, nucleotide residues 2327 to 5617 of SEQ ID NO: 25 in the RET gene mRNA containing the nucleotide sequence of SEQ ID NO: 25) The fusion site is located between the 2618th guanosine (G) and the 2619th guanosine in the nucleotide sequence of SEQ ID NO: 2. Since there are many SNPs of the MGEA5-RET fusion gene in humans, there is mRNA of the MGEA5-RET fusion gene that contains a nucleotide sequence with 80% or more sequence identity to the nucleotide sequence of SEQ ID NO: 2 in the human population. It is possible. Similarly, there may be mRNAs that contain nucleotide sequences that have 80% or more sequence identity to the mRNAs in Table 1.

In step (a) of the present detection method, methods for detecting the mRNA of the present fusion gene include, for example, RT (Reverse Transcription)-PCR using the present mRNA detection reverse primer; Examples include Northern blotting method, microarray method, and ISH method.

The nucleotide sequence of SEQ ID NO: 3 is the cDNA of the MGEA5-RET fusion gene detected in a sample derived from human colon cancer tissue, as specifically shown in this example below. More specifically, the nucleotide sequence of SEQ ID NO: 3 is the 5' region of the cDNA of the MGEA5 gene (specifically, in the cDNA of the MGEA5 gene containing the nucleotide sequence of SEQ ID NO: 23, 1 to 2175 of SEQ ID NO: 23). nucleotide residue) and the 3' region of the cDNA of the RET gene (specifically, nucleotide residues 2137 to 3345 of SEQ ID NO: 26 in the cDNA of the RET gene, which includes the nucleotide sequence of SEQ ID NO: 26) The fusion site is located between the 2175th guanosine (G) and the 2176th guanosine in the nucleotide sequence of SEQ ID NO: 3. Since there are many SNPs of the MGEA5-RET fusion gene in humans, there is a cDNA of the MGEA5-RET fusion gene in the human population that contains a nucleotide sequence that has 80% or more sequence identity with the nucleotide sequence of SEQ ID NO: 3. It is possible. Similarly, there may be cDNAs containing nucleotide sequences that have 80% or more sequence identity with cDNAs synthesized based on the mRNAs in Table 1.

In step (a) of the present detection method, methods for detecting the cDNA of the present fusion gene include, for example, the LAMP method using the present cDNA detection primer set, the PCR method (for example, real-time PCR method [intercalator method, '-nuclease method, cycling probe method, etc.], ddPCR method); LCR method; sequencing method using a next-generation sequencer; Southern hybridization method using the cDNA detection probe, microarray method, ISH method; be able to. The cDNA of the fusion gene can be synthesized from the mRNA of the fusion gene by PCR using reverse transcriptase.

As used herein, the term "next generation sequencer" is also referred to as a second generation sequencer, and refers to a nucleotide sequence determination device that can simultaneously determine the nucleotide sequences of tens of millions of DNA fragments. Sequencing principles in next-generation sequencers include, for example, the bridge PCR method and the sequencing-by-synthesis method, in which DNA to be detected is amplified on a flow cell and sequencing is performed while it is synthesized, and the emulsion PCR method and DNA An example of this principle is the pyrosequencing method, which determines the sequence by measuring the amount of pyrophosphate released during synthesis. More specifically, next-generation sequencers include the MiniSeq, MiSeq, NextSeq, HiSeq, and HiSeqX series manufactured by Illumina, and the Roche 454 GS FLX sequencer manufactured by Roche.

Among the fusion protein target antibodies, the antibody that specifically binds to the MGEA5-RET fusion protein (hereinafter referred to as the "MGEA5-RET target antibody") has a sequence identity of 80% or more with the amino acid sequence of SEQ ID NO: 1. specifically binds to an MGEA5-RET fusion protein comprising an amino acid sequence having an amino acid sequence of SEQ ID NO: 24, preferably an MGEA5 protein comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 24, and/or SEQ ID NO: 27. This antibody does not specifically bind to the RET protein that contains an amino acid sequence that has 80% or more sequence identity with the amino acid sequence of . Specifically, the present MGEA5-RET target antibody has a polypeptide containing the fusion site in the MGEA5-RET fusion protein containing an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 as an epitope. Examples include antibodies. The length of the epitope of the fusion protein target antibody is, for example, 3 or more amino acids (preferably 4 or 5 amino acids or more) and 40 amino acids or less (preferably 30, 20, 10, or 8 amino acids or less).

The origin, type, class, form, etc. of the fusion protein target antibody are not particularly limited. For example, the fusion protein target antibody includes antibodies derived from humans; antibodies derived from non-human animals such as mice and rats; polyclonal antibodies, and Clonal antibodies (mixtures of several to dozens of types of antibodies), monoclonal antibodies; chimeric or humanized antibodies in which a part of the antibody region (e.g., constant region) is replaced with a region derived from a different species; F(ab') ₂ antibody fragment obtained by digestion with F(ab')2 antibody fragment, Fab' antibody fragment obtained by reducing F(ab') ₂ antibody fragment, Fab etc. obtained by digesting a monoclonal antibody with papain, Included are recombinant antibodies such as scFv in which an antibody heavy (H) chain variable region and an antibody light (H) chain variable region are linked by amino acid bridges; and the like.

Preferably, the fusion protein target antibody is isolated. Further, the antibody targeting the fusion protein of the present invention is preferably an antibody produced by artificial manipulation (eg, the above-mentioned recombinant antibody). Such "antibodies derived from or produced from cells produced by artificial manipulation" do not include antibodies that have not been subjected to artificial manipulation, such as antibodies produced from naturally occurring B cells. do not have.

As used herein, "separated" and "separated" mean that something (i.e., an antibody or protein) exists in a state different from its original state due to human manipulation or the like. means that In other words, "isolated antibodies or proteins" include antibodies or proteins derived from a certain individual that have not been subjected to external manipulation (artificial manipulation), and that have not been subjected to external manipulation (artificial manipulation). Antibodies and proteins contained in body fluids (blood, plasma, serum, etc.) are not included.

The fusion protein-targeting antibody of the present invention can be obtained by a known method. For example, a non-human animal is immunized with the recombinant fusion protein or a fragment thereof as an antigen, and a hybridoma that produces an antibody is prepared from the immunized non-human animal using cell fusion technology, and the antigen is immobilized. A culture supernatant containing the antibody targeting the fusion protein of the present invention can also be obtained by screening by ELISA using a plate. The fusion protein target antibody can be separated and purified from the culture supernatant using known antibody purification techniques.

The subject fusion protein targeting antibody, the subject isolated/recombinant fusion protein, or a fragment thereof is a cDNA encoding the subject fusion protein targeting antibody (hereinafter sometimes referred to as "this subject fusion protein targeting antibody cDNA") or a subject fusion protein target antibody. An expression vector is prepared by operably linking cDNA or a fragment thereof downstream of a promoter in a vector containing a promoter, and expression can be achieved by introducing the vector into a host cell or host organism. The type of promoter and vector in the expression vector can be appropriately selected depending on the type of host cell or host organism to be introduced.

When yeast (e.g., Saccharomyces Cerevisiae, Schizosaccharomyces Pombe, etc.) is used as a host cell, expression vectors include, for example, vectors such as YEP13 (ATCC 37115), YEp24 (ATCC 37051), YCp50 (ATCC 37419), or those derived from such vectors. Examples of promoters include promoters of glycolytic genes such as hexose kinase, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal1 promoter, gal10 promoter, heat shock protein promoter, MFα1 promoter, CUP1 promoter. etc. can be mentioned.

In addition, when mammalian cells (e.g., human-derived Namalwa cells, monkey-derived COS cells, CHO cells derived from Chinese hamster ovary, T cells derived from humans, mice, etc.) are used as host cells, the expression vector Examples include pcDNAI, pcDM8 (manufactured by Funakoshi), pAGE107 (Japanese Unexamined Patent Publication No. 3-22979; Cytotechnology, 3, 133, (1990)), pAS3-3 (Japanese Unexamined Patent Publication No. 2-227075), pCDM8 (Nature, 329, 840, (1987)), pcDNAI/Amp (manufactured by Invitrogen), pREP4 (manufactured by Invitrogen), pAGE103 (J.Biochemistry, 101, 1307 (1987)), pAGE210, pMSGV vector (Tamada k et al., Clin Cancer Examples include retrovirus vectors such as Res 18:6436-6445 (2002)) and pMSCV vector (manufactured by Takara Bio Inc.), or those derived from such vectors.

Examples of the promoter in the above vector include the cytomegalovirus (CMV) IE (immediate early) gene promoter, SV40 early promoter, retrovirus promoter, metallothionein promoter, heat shock promoter, SRα promoter, NFAT promoter, and HIF promoter. etc. can be mentioned.

In addition, when insect cells (for example, Sf9 cells, Sf21 cells, which are ovary cells of Spodoptera arugiperda, High5 cells, which are ovarian cells of Trichoplusiani, etc.) are used as host cells, the expression vector can be used, for example, in the recombinant baculovirus production method. Transfer vectors used include, specifically, vectors such as pVL1392, pVL1393, and pBlueBacIII (all manufactured by Invitrogen), or vectors derived from such vectors, and promoters include, for example, polyhedrin promoter, p10 promoter, etc. can be mentioned.

Furthermore, when using plant cells (e.g., tobacco, potato, tomato, carrot, soybean, canola, alfalfa, rice, wheat, barley, etc.) as host cells, expression vectors include, for example, Ti plasmids, tobacco mosaic virus vectors, etc. Examples of promoters include cauliflower mosaic virus (CaMV) 35S promoter, rice actin 1 promoter, and the like.

The above-mentioned expression vectors may further include enhancer regions or ribosome binding site (RBS) base sequences to further increase gene expression efficiency, or may contain vectors that are specific to the type of host cells for host cell screening. drug resistance genes (e.g., spectinomycin resistance gene, chloramphenicol resistance gene, tetracycline resistance gene, kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, hygromycin resistance gene, blasticidin resistance gene, geneticin resistance gene) It is preferable to further include a resistance gene, etc.). The enhancer region is usually placed upstream of the promoter, and the RBS is usually placed between the promoter and the subject fusion protein target antibody cDNA, the subject fusion gene cDNA, or a fragment thereof. The nucleotide sequence of the fusion protein target antibody cDNA, cDNA of the fusion gene, or a fragment thereof, which is incorporated into an expression vector, may have a codon sequence optimized according to the host cell in which it is expressed. Expression vectors can be produced by known methods using genetic recombination techniques.

A host cell can be obtained by introducing (transfecting) an expression vector into a host cell by a method depending on the type of host cell.

When using the above-mentioned yeast as a host cell, the method for introducing the expression vector into the yeast may be any method of introducing DNA into the yeast, such as electroporation method (Methods. Enzymol., 194, 182 (1990)), Methods such as the spheroplast method (Proc. Natl. Acad. Sci. U.S.A., 84, 1929 (1978)) and the lithium acetate method (J. Bacteriology, 153, 163 (1983)) can be mentioned.

In addition, when using the above-mentioned mammalian cells as host cells, the method for introducing the expression vector into the mammalian cells may be any method of introducing DNA into the mammalian cells, such as the electroporation method as described above. (Cytotechnology, 3, 133 (1990)), calcium phosphate method (JP-A-2-227075), lipofection method (Proc. Natl. Acad. Sci. U.S.A., 84, 7413 (1987)), virus infection method, etc. can be mentioned. As described above, such a virus infection method involves injecting the CAR expression vector (International Publication No. 2016/056228 pamphlet) and packaging plasmid into GP2-293 cells (manufactured by Takara Bio Inc.) and Plat-GP cells (manufactured by Cosmo Bio Inc.). A method of producing a recombinant virus by transfecting packaging cells such as PG13 cells (ATCC CRL-10686), PA317 cells (ATCC CRL-9078), etc.), and infecting T cells with such recombinant virus. be able to.

In addition, when using the above insect cells as host cells, methods for introducing expression vectors into insect cells include, for example, "Current Protocols in Molecular Biology", "Baculovirus Expression Vectors, A Laboratory Manual, W.H. Freeman and Company, New York". The expression vector (transfer vector) and baculovirus-derived genomic DNA were co-transfected into the above insect cells according to the method described in "Bio/Technology, 6, 47 (1988)", etc. Examples include methods for producing recombinant baculoviruses. Examples of such co-transfection methods include methods such as the calcium phosphate method (JP-A-2-227075) and the lipofection method (Proc. Natl. Acad. Sci. U.S.A., 84, 7413 (1987)). .

In addition, when using the above-mentioned plant cells as host cells, methods for introducing the expression vector into the plant cells include, for example, a method using Agrobacterium (JP-A No. 59-140885, JP-A No. 60-1989). 70080), electroporation method (Japanese Patent Application Laid-Open No. 60-251887), method using a particle gun (gene gun) (Japanese Patent No. 2606856, Japanese Patent No. 2517813). I can do it.

The fusion protein-targeting antibody of the present invention can be obtained by culturing the host cells obtained by the method described above in a culture medium appropriate for the host cells.

Furthermore, using transgenic animal production technology, transgenic animals such as mice, cows, goats, sheep, chickens, pigs, etc., into which a vector containing the subject fusion protein target antibody cDNA has been introduced, are produced, and the blood of such transgenic animals is It is also possible to produce a large amount of antibodies targeting the fusion protein from milk or the like.

Among the primer sets for detecting genomic DNA in this case, the primer set that anneals to the genomic DNA encoding the MGEA5-RET fusion protein (hereinafter sometimes referred to as "primer set for detecting MGEA5-RET genomic DNA") has SEQ ID NO: 38. In an MGEA5-RET fusion gene containing a nucleotide sequence having 80% or more sequence identity with the nucleotide sequence and its complementary sequence, nucleotide residues corresponding to positions 852 to 854 of SEQ ID NO: 38 are specifically amplified, preferably , MGEA5 gene comprising a nucleotide sequence having 80% or more sequence identity with the nucleotide sequence of SEQ ID NO: 36 and its complementary sequence, and/or a nucleotide sequence having 80% or more sequence identity with the nucleotide sequence of SEQ ID NO: 37 and a primer set (ie, a pair of polynucleotides, preferably a pair of single-stranded polynucleotides) that does not specifically amplify the RET gene containing the complementary sequence thereof. Specifically, the forward primer of the MGEA5-RET genomic DNA detection primer set is a nucleotide sequence on the 5' end side from the breakpoint (for example, nucleotide residues corresponding to positions 1 to 852 of SEQ ID NO: 38). A primer that anneals to a complementary sequence. In addition, in the MGEA5-RET genomic DNA detection primer set, the reverse primer specifically includes the nucleotide sequence on the 3' end side from the breakpoint (for example, nucleotide residues corresponding to positions 854 to 2163 of SEQ ID NO: 38). ) is a primer that anneals to

Among the genomic DNA detection probes, the probe that hybridizes to the genomic DNA encoding the MGEA5-RET fusion protein contains a nucleotide sequence having 80% or more sequence identity with the nucleotide sequence of SEQ ID NO: 38 and its complementary sequence. In the MGEA5-RET fusion gene, it specifically hybridizes to the nucleotide residues corresponding to positions 852 to 854 of SEQ ID NO: 38 (i.e., the breakpoint), and preferably has a sequence of 80% or more with the nucleotide sequence of SEQ ID NO: 36. Specifically for the MGEA5 gene containing a nucleotide sequence with identity and its complementary sequence, and/or the RET gene containing a nucleotide sequence with 80% or more sequence identity with the nucleotide sequence of SEQ ID NO: 37 and its complementary sequence. It is a non-hybridizing probe (preferably a single-stranded polynucleotide). The length of the genomic DNA detection probe is, for example, 10 nucleotides or more (preferably 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides or more) and 1000 nucleotides or less (preferably 900, 800, 700, 600, 500, 400, 300, or 200 nucleotides or less).

Among the reverse primers for detecting mRNA in this case, the reverse primer that hybridizes to the mRNA encoding the MGEA5-RET fusion protein (hereinafter sometimes referred to as "reverse primer for detecting MGEA5-RET mRNA") has the nucleotide sequence of SEQ ID NO: 2. In the mRNA of the MGEA5-RET fusion gene containing a nucleotide sequence with 80% or more sequence identity, nucleotide residues corresponding to positions 2618 to 2619 of SEQ ID NO: 2 are specifically reverse transcribed, preferably SEQ ID NO: 22. A reverse primer (preferably a single-stranded polynucleotide) that does not specifically reverse transcribe the mRNA of the MGEA5 gene, which includes the nucleotide sequence of SEQ ID NO: 25, and/or the mRNA of the RET gene, which includes the nucleotide sequence of SEQ ID NO: 25. Specifically, the reverse primer for detecting MGEA5-RET mRNA is a primer that anneals to a nucleotide sequence on the 3' end side from the fusion site (for example, nucleotide residues corresponding to positions 2619 to 5921 of SEQ ID NO: 2).

Among the present mRNA detection probes, the probe that hybridizes to the MGEA5-RET fusion protein contains a nucleotide sequence that has 80% or more sequence identity with the nucleotide sequence of SEQ ID NO: 2, and in the mRNA of the MGEA5-RET fusion gene, A nucleotide sequence that specifically hybridizes to the nucleotide residue corresponding to positions 2618 to 2619 of SEQ ID NO: 2 (i.e., the fusion site) and preferably has 80% or more sequence identity with the nucleotide sequence of SEQ ID NO: 22. A probe that does not specifically hybridize to the RET gene mRNA (preferably 1 (full-stranded polynucleotide). The length of the present mRNA detection probe is, for example, 10 nucleotides or more (preferably 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides or more) and 1000 nucleotides or less (preferably 900 nucleotides or more). , 800, 700, 600, 500, 400, 300, or 200 nucleotides or less).

Among the primer sets for cDNA detection in this case, the primer set of the forward primer and reverse primer that anneals to the cDNA encoding the MGEA5-RET fusion protein (hereinafter sometimes referred to as "MGEA5-RET cDNA detection primer set") has the sequence number In the cDNA of the MGEA5-RET fusion gene, which contains a nucleotide sequence having 80% or more sequence identity with the nucleotide sequence of SEQ ID NO: 3 and its complementary sequence, the nucleotide residues corresponding to positions 2175 to 2176 of SEQ ID NO: 3 are specifically cDNA of the MGEA5 gene, preferably comprising a nucleotide sequence having a sequence identity of 80% or more with the nucleotide sequence of SEQ ID NO: 23 and a complementary sequence thereof, and/or a nucleotide sequence of 80% or more with the nucleotide sequence of SEQ ID NO: 26. A primer set (i.e., a pair of polynucleotides, preferably a pair of single-stranded polynucleotides) that does not specifically amplify the cDNA of the RET gene, and includes a nucleotide sequence having sequence identity of 1 and its complementary sequence. Specifically, the forward primer of the MGEA5-RET cDNA detection primer set is a complementary sequence of the nucleotide sequence on the 5' end side from the breakpoint (for example, nucleotide residues corresponding to positions 1 to 2175 of SEQ ID NO: 3). This is a primer that anneals to. In addition, the reverse primer of the MGEA5-RET cDNA detection primer set specifically targets the nucleotide sequence on the 3' end side from the breakpoint (for example, nucleotide residues corresponding to positions 2176 to 3384 of SEQ ID NO: 3). Primer for annealing.

Among the cDNA detection probes of the present invention, the probe that hybridizes to the cDNA encoding the MGEA5-RET fusion protein is an MGEA5 probe that includes a nucleotide sequence having 80% or more sequence identity with the nucleotide sequence of SEQ ID NO: 3 and its complementary sequence. - In the cDNA of the RET fusion gene, it specifically hybridizes to the nucleotide residues corresponding to positions 2175 to 2176 of SEQ ID NO: 3 (i.e., the fusion site), and preferably has at least 80% of the nucleotide sequence of SEQ ID NO: 23. It specifically hybridizes to the cDNA of the MGEA5 gene, which includes a nucleotide sequence with sequence identity, and/or the cDNA of the RET gene, which includes a nucleotide sequence with 80% or more sequence identity to the nucleotide sequence of SEQ ID NO: 26. The probe is a non-soybean probe (preferably a single-stranded polynucleotide). The length of the cDNA detection probe is, for example, 10 nucleotides or more (preferably 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides or more) and 1000 nucleotides or less (preferably 900 nucleotides or more). , 800, 700, 600, 500, 400, 300, or 200 nucleotides or less).

From the viewpoint of detection sensitivity and specificity of the fusion gene, the forward primer and reverse primer in the primer set for detecting the genomic DNA of the subject matter should be used so that the length of the amplified product is 5,000 nucleotide pairs or less (for example, 4,000, 3,000, 2,000, or 1,000 nucleotide pairs). It is preferable to design it so that the number of nucleotide pairs or less). Furthermore, from the viewpoint of detection sensitivity and specificity of mRNA of the present fusion gene, the reverse primer for detecting the subject mRNA is preferably designed such that the length of the amplified product is 5000 nucleotide pairs or less as described above. In addition, the forward primer and reverse primer in the cDNA detection primer set are designed so that the length of the amplified product is 5000 nucleotide pairs or less as described above, from the viewpoint of detection sensitivity and specificity of the cDNA of the fusion gene. It is preferable. These primers can be appropriately designed using known techniques, for example, Primer Express (registered trademark) software (manufactured by Applied Biosystems) can be used. The length of these primers is, for example, 10 nucleotides or more (preferably 13, 16, 20, 23, 26, or 30 nucleotides or more) and 100 nucleotides or less (preferably 90, 80, 70, 60, 50, or less than 40 nucleotides).

In the present invention, the primers, probes, and adapters may be DNA, RNA, or a DNA/RNA chimera, but are preferably DNA. Also, in some or all of them, PNA (polyamide nucleic acid, peptide nucleic acid), LNA (registered trademark, locked nucleic acid, Bridged Nucleic Acid, crosslinked nucleic acid), ENA (registered trademark, 2'-O,4'-C- The nucleotides may be substituted with artificial nucleic acids such as Ethylene-bridged nucleic acids), GNA (Glycerol nucleic acid), and TNA (Threosenucleic acid).

In the present invention, the antibodies, primers, and probes are labeled in order to visualize and/or quantify the subject fusion protein, subject fusion gene, mRNA of the subject fusion gene, or cDNA of the subject fusion gene to be detected. objects can be used. Labeling substances in such labeled substances include peroxidase (for example, horseradishperoxidase), alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, penicillinase, Enzymes such as catalase, apoglucose oxidase, urease, luciferase or acetylcholinesterase; Fluorescent substances mentioned above; Green Fluorescence Protein (GFP), Cyan Fluorescence Protein (CFP), Blue Fluorescence Protein (BFP) ), fluorescent proteins such as Yellow Fluorescence Protein (YFP), Red Fluorescence Protein (RFP), and luciferase; radioactive isotopes such as ³ H, ¹⁴ C, ¹²⁵ I, or ¹³¹ I; biotin, avidin, or chemiluminescent substances; combinations of reporter and quencher fluorescent substances or structures (e.g., combinations of 5-FAM [5-Carboxyfluorescein] and 5-TAMRA [5-Carboxytetramethylrhodamine], VIC and MGB [ Minor Groove Binder].

In the present invention, "sequence identity of 80% or more" means that the sequence identity is at least 80%, preferably 85% or more, more preferably 88% or more, even more preferably 90% or more, Still more preferably 92% or more, particularly preferably 94% or more, particularly more preferably 96% or more, even more preferably 98% or more, even more preferably 99% or more, and most preferably 100% sequence identity. do. Amino acid sequence identity is determined using the algorithm BLAST by Carlin and Arthur (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990, Proc. Natl. Acad. Sci. USA 90: 5873, 1993) called BLASTX or BLASTP. It can be determined using the program (Altschul SF, et al: J Mol Biol 215: 403, 1990). When analyzing an amino acid sequence using BLASTX, the parameters are, for example, score=50 and wordlength=3. When using the BLAST and Gapped BLAST programs, use the default parameters for each program. Specific techniques for these analysis methods are well known to those skilled in the art (eg, http://www.ncbi.nlm.nih.gov/). Furthermore, the identity of nucleotide sequences can be determined using a program called BLASTN (Altschul SF, et al: J Mol Biol 215: 403, 1990) based on the algorithm BLAST. When analyzing a nucleotide sequence using BLASTN, the parameters are, for example, score=100 and wordlength=12.

The substance is not particularly limited as long as it directly or indirectly suppresses the expression and/or physiological activity of the MGEA5-RET fusion protein. Such substances include, for example, the mRNA target polynucleotide of the present fusion gene; an expression vector capable of expressing the mRNA target polynucleotide of the present fusion gene; an antibody targeting the present fusion protein that has the effect of suppressing the expression of the MGEA5-RET fusion protein; Examples include low molecular weight compounds;

In addition, when the present fusion protein is an MGEA5-RET fusion protein or an SLC12A2-RET fusion protein, the substance that suppresses the physiological activity of the present fusion protein is, for example, the kinase domain in RET (specifically, SEQ ID NO: 1 Substances that inhibit tyrosine kinase activity (for example, Sorafenib, Sunitinib, Cabozantinib, XL184, Vandetanib, Ponatinib, Cabozantinib, Alectinib, the compound described in Patent No. 5759568) can be mentioned.

The therapeutic agent preferably contains the substance as an active ingredient, and if necessary, it may contain conventional pharmaceutically acceptable carriers, binders, stabilizers, excipients, diluents, and pH buffers. Ingredients such as agents, disintegrants, isotonic agents, additives, coating agents, solubilizers, lubricants, solubilizers, lubricants, flavoring agents, sweeteners, solvents, gelling agents, nutrients, etc. It may be added. Specifically, such ingredients include water, physiological saline, animal fat and oil, vegetable oil, lactose, starch, gelatin, crystalline cellulose, gum, talc, magnesium stearate, hydroxypropyl cellulose, polyalkylene glycol. , polyvinyl alcohol, and glycerin.

The therapeutic agent may be administered orally in the form of powder, granules, tablets, capsules, syrups, suspensions, etc., or by injection (e.g. Mention may be made of parenteral administration, such as intranasal administration (e.g., subcutaneous injection, intravenous injection, intramuscular injection), or intranasal administration in the form of a spray, with oral administration being preferred.

The dosage of the substance in the therapeutic agent is appropriately determined depending on age, body weight, sex, symptoms, sensitivity to drugs, etc., and is, for example, in the range of 0.1 μg to 200 mg/kg (body weight)/day. It is.

The above-mentioned "cancer-related fusion gene database" is a collection of information on fusion genes that have been reported to be associated with cancer, using computer-based information processing for convenient storage, searching, and updating. Examples of such databases include COSMIC (Catalogue of Somatic Mutations in Cancer), Mitelman DataBase, TCGA (The Cancer Genome Atlas), and FusionGDB. Furthermore, the "database of cancer-related fusion genes" preferably includes information for determining whether conditions (1) to (5) above are satisfied. Note that when the database is updated, the latest version can be used.

The number of fusion genes selected in the above step (A) depends on the number of fusion genes stored in the database, and cannot be unconditionally specified. For example, when using COSMIC, the number of fusion genes selected is at least about 700 ( 699) types of fusion genes can be selected.

In step (B) above, the method for synthesizing cDNA with adapters added to the upstream end and/or downstream end involves first isolating and purifying total RNA from a biological sample collected from a cancer patient. , then, for example, using mRNA as a template, it contains a reverse primer that anneals to mRNA, such as an oligo dT (deoxythymidine) primer or a random primer, and an adapter (SMART) consisting of a single-stranded polynucleotide, and is synthesized. SMART (Switching Mechanism At 5'End of RNA Template) method using forward primer that anneals to cDNA consisting of single-stranded polynucleotide and reverse transcriptase derived from Moloney Murine Leukemia Virus; using mRNA as a template, oligo dT primer and After synthesizing cDNA by performing PCR using a reverse primer that anneals to mRNA such as a random primer and reverse transcriptase, dA (deoxyadenine) is added to the blunted cDNA end, and adapter ligation is performed using an adapter. law; etc.

The nucleotide sequence of the adapter is not particularly limited, but preferably a nucleotide sequence that does not exist in humans. Whether or not a nucleotide sequence exists in humans can be confirmed using, for example, a homology search using NCBI (National Center for Biotechnology Information) BLAST. In addition, the adapter is IL-N7 adapter DNA (manufactured by Qiagen)

In step (C) above, in order to amplify and synthesize cDNA with an adapter added to the upstream end and/or downstream end, PCR is performed using a forward primer for adapters and a group of reverse primers for fusion genes. Alternatively, PCR is performed using a group of forward primers for the fusion gene and a reverse primer for the adapter, but the effect has been demonstrated in this example, which will be described later. A preferred example is performing PCR using a group of reverse primers. Such a group of reverse primers for a fusion gene can be appropriately set based on a transcription product database such as Ensembl, and specifically, the reverse primer group shown in Table 3 of Examples described below can be mentioned.

The forward primer for the adapter may be any single-stranded polynucleotide that anneals with part or all of the antisense strand of the adapter, and the reverse primer for the fusion gene may be the fusion gene selected in step (A). Of these, a single-stranded polynucleotide that anneals to a part of the sense strand of the cDNA of the downstream gene fragment (specifically, in this example described later, shown in Table 1) may be used; The forward primer for the fusion gene may be a single-stranded polynucleotide that anneals to a part of the antisense strand of the cDNA of the upstream gene fragment of the fusion gene selected in step (A); The reverse primer for the adapter may be a single-stranded polynucleotide that anneals to part or all of the sense strand of the adapter. The length of these primers is, for example, 10 nucleotides or more (preferably 13, 16, 20, 23, 26, or 30 nucleotides or more) and 100 nucleotides or less (preferably 90, 80, 70, 60, 50, or less than 40 nucleotides).

The length of the adapter is, for example, within the range of 10 to 50 nucleotides (for example, 10 to 50 nucleotides, 20 to 50 nucleotides, 30 to 50 nucleotides, 40 to 50 nucleotides, 10 to 40 nucleotides, 10 to 30 nucleotides, 10-20 nucleotides, 20-40 nucleotides, 20-30 nucleotides). In addition, in this specification, the nucleotide sequence of mRNA is applied as "t (thymine residue)" in the nucleotide sequences of SEQ ID NO: 2, SEQ ID NO: 22, and SEQ ID NO: 25 as "u (uracil residue)". .

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the technical scope of the present invention is not limited to these examples.

1. Identification of the MGEA5-RET fusion gene in cancer patients Total RNA was prepared from 4000 cancer tissues surgically removed at the Shizuoka Prefectural Cancer Center, and cDNA was synthesized using a next-generation sequencer (IonProton [Thermo Fisher Scientific The fusion gene was detected using a custom panel, HOPE fusion panel ver. This panel was independently developed by the inventors. Specifically, it targets cDNA obtained from the mRNA of 409 reported fusion genes, and 5' partner genes (i.e., upstream A primer set that can amplify a region approximately 100 to 300 bases in length between the breakpoint of the 3' partner gene (gene) fragment and the 3' partner gene (i.e. downstream gene) fragment, and the 203 types that make up the 409 types of fusion genes mentioned above. It includes a primer set for confirming that the gene is expressed. Furthermore, the above panel includes a primer set for a 3'/5' imbalance assay regarding the RET gene, which is a 3' partner gene of a fusion gene reported in lung cancer. Since the 3' partner gene of the fusion gene is missing its 5' region (i.e., the upstream gene region), the number of reads in the 3' region (i.e., the downstream gene region) is lower than that in the 5' region. increases. The above-mentioned 3'/5' imbalance assay is an assay that estimates a new fusion gene by utilizing the difference in the number of reads between the 5' region and 3' region of one of the genes constituting the fusion gene (Reference “J Exp Clin Cancer Res. 2018 Mar 27; 37(1):68. doi: 10.1186/s13046-018-0735-1.)

The 3'/5' imbalance score (= [number of reads in 3' region] - [number of reads in 5' region]) ÷ total number of reads) of the RET gene was calculated for 4000 cancer tissue cases, and the value was 0. As a result of screening using those exceeding the threshold of 0.045 as an index, 14 types of cases were identified. Among these 14 types of cases, existing fusion genes KIF5B-RET and CCDC6-RET were detected in 5 cases. On the other hand, in the remaining nine cases, the fusion gene was not detected using the HOPE fusion panel ver.1 panel, so fusion was performed using the HOPE fusion panel ver.3 panel and QIAseqTargeted RNAscan Panel Kit (manufactured by Qiagen). After amplifying the cDNA of the gene and detecting the fusion gene using a next-generation sequencer (MiSeq [manufactured by Illumina]) and analysis software (Biomedical Genomics Workbench; manufactured by Qiagen), a case of a colorectal cancer patient was detected. Regarding #1, a novel fusion gene, MGEA5-RET, was identified. Specifically, the above-mentioned HOPE fusion panel ver.3 panel was produced according to the following procedure. First, we selected one of the data files containing information on 818,568 types of fusion genes in the data of COSMIC (Catalogue of Somatic Mutations in Cancer) version 81, a public database that collects somatic mutations in cancer. 2) fusion genes that exist in cultured cells, and 3) complex fusion genes (specific There are 174 types of genes, excluding fusion genes 1) to 3) (e.g., fusion genes in which three or more genes are fused, fusion genes with two or more fusion sites, fusion genes with unclear chromosomal location, etc.). Approximately 700 (699) types of fusion gene groups were selected. Table 2 shows information regarding the 3' partner gene (ie, downstream gene) fragment for each of the selected fusion gene groups. For example, number 1 in Table 2, "ABL1{ENST00000318560}:r.461_5766", indicates that the 461st to 5766th nucleotide residues of the ABL1 gene cDNA are the downstream gene fragment in the fusion gene, and the 461st nucleotide This means that the residue is fused with the upstream gene fragment. A group of reverse primers (manufactured by Qiagen) (see Table 3) that can anneal to the 699 types of downstream gene fragments (gene cDNA fragments) shown in Table 2 and amplify the fusion site, and A forward primer that anneals to the added IL-N7 adapter DNA (manufactured by Qiagen) was mixed to form a panel of HOPEfusion panel ver.3. By using the HOPE fusion panel ver.3 panel, it is possible to detect novel fusion genes for upstream genes. The fusion gene in 2) above was excluded because it was thought that the fusion gene may have been generated due to in vitro passage of cultured cells (possibility of artifact).

2. Identification of cDNA of MGEA5-RET fusion gene In order to generate cDNA of MGEA5-RET fusion gene, first, using the total RNA of case #1 as a template, PrimeScrip II 1st strand cDNA Synthesis Kit (manufactured by Takara Bio Inc.) was used. A reverse transcription reaction was performed to produce single-stranded cDNA. Specifically, 1 μL of total RNA (0.01 ng/μL) obtained from the colorectal cancer tissue of case #1 by a standard method, 1 μL of oligo (dT) primer, 1 μL of dNTPMixture, and 7 μL of RNase-free water. Mix and heat at 65℃ for 5 minutes, cool on ice, add 4μL of 5x PrimeScript Buffer, 0.5μL of RNase inhibitor, 1.0μL of PrimeScriptIIRtase, and 4.5μL of RNase-free water, and heat at 42℃. After incubating for 60 minutes, the mixture was heated at 95° C. for 5 minutes and cooled on ice.

Next, using the prepared single-stranded cDNA as a template, PCR was performed using PrimeSTAR GXL DNA Polymerase (manufactured by Takara Bio Inc.), and a double-stranded cDNA of the MGEA5-RET gene was prepared and amplified. Specifically, 0.5 μL of the prepared single-stranded cDNA, 10 μL of 5× PrimeSTAR GXL Buffer, 4 μL of dNTP Mixture, 0.5 μL of MGRET_F primer (single-stranded DNA consisting of the nucleotide sequence of SEQ ID NO: 4), After mixing 0.5 μL of MGRET_R primer (single-stranded DNA consisting of the nucleotide sequence of SEQ ID NO: 5), 1 μL of PrimeSTAR GXL DNA Polymerase, and 33.5 μL of RNase-free water, the mixture was incubated at 98°C for 10 seconds and at 60°C. PCR was performed under the following conditions: 35 cycles of 15 seconds and 4 minutes at 68°C; followed by 1 cycle of 10 minutes at 68°C; and the PCR products were separated by 1.0% agarose gel electrophoresis.

As a result, a DNA band was detected at the position of the size marker of 4 kbp to 5 kbp (see lane 1 in FIG. 1). The above MGRET_F primer and MGRET_R primer respectively target the 5' region of the MGEA5 gene mRNA (specifically, the complementary strand of the 84th to 109th nucleotide residues of SEQ ID NO: 22) and the 3' region of the RET gene mRNA. (specifically, nucleotide residues 4131 to 4158 of SEQ ID NO: 25), and the above results showing that DNA was amplified with these primer sets indicate that the 5' region of the MGEA5 gene and , indicating fusion with the 3' region of the RET gene.

Next, a second PCR was performed using the PCR products amplified by the MGRET_F primer and MGRET_R primer as templates. Specifically, 0.5 μL of PCR product, 10 μL of 5× PrimeSTAR GXL Buffer, 4 μL of dNTP Mixture, 0.5 μL of MGRET-NEST_F primer (single-stranded DNA consisting of the nucleotide sequence of SEQ ID NO: 6), 0. After mixing 5 μL of MGRET-NEST_R primer (single-stranded DNA consisting of the nucleotide sequence of SEQ ID NO: 7), 1 μL of PrimeSTAR GXL DNA Polymerase, and 33.5 μL of RNase-free water, the mixture was incubated at 98°C for 10 seconds and at 60°C. PCR was performed under the following conditions: 35 cycles of 15 seconds and 3 minutes at 68°C; followed by 1 cycle of 10 minutes at 68°C; and the PCR products were separated by 1.0% agarose gel electrophoresis. After confirming the target DNA band at the position of the 3 kbp to 4 kbp size marker (see lane 2 in Figure 1), cut out the band and purify the DNA using QIAEx II Gel Extraction kit (manufactured by Qiagen). , collected in 20 μL of sterile aqueous solution.

The recovered PCR product was incorporated into an E. coli cloning vector using Mighty TA-cloning Reagent Set for PrimeSTAR (Takara Bio Inc.). Specifically, 1 μL of the collected PCR product solution, 1 μL of 10× Buffer, 0.5 μL of dATP solution, and 0.5 μL of A-overhanging enzyme were mixed and incubated at 65°C for 10 minutes to remove the PCR product. Adenosine (dA) was added to the 3' end. Next, 1 μL of dA-added PCR product, 1 μL of cloning vector (pMD20-T-vector), 3 μL of sterile water, and 5 μL of ligation Mighty Mix were mixed and incubated at 16°C for 30 minutes to complete the ligation reaction. went. Thereafter, the entire amount was added to 100 μL of E. coli competent cells (Competent Quick DH5a, manufactured by Toyobo), incubated on ice for 1 hour, kept warm at 42° C. for 40 seconds, and then transferred to ice to perform transformation. Thereafter, it was applied to an LB agar plate containing the selective drug ampicillin and incubated at 37°C for 16 hours. Colonies that appeared on the agar plate were picked up and cultured in an LB liquid medium containing ampicillin, followed by miniprep using the alkaline method to recover plasmid DNA from E. coli. The E. coli strain for which the presence of a plasmid containing the desired PCR product (gene) was confirmed using restriction enzymes was cultured overnight at 37°C in 50 mL of LB medium containing ampicillin, and after harvesting, HiSpeed Plasmid Midi Plasmid DNA was purified using Kit (manufactured by Qiagen). Thereafter, Sanger sequencing was performed using the 14 types of primers shown in Table 4, and it was found that the 5' region of the cDNA of the MGEA5 gene (specifically, the nucleotide residues 1 to 2175 of SEQ ID NO: 23) ) and the 3' region of the cDNA of the RET gene (specifically, nucleotide residues 2137 to 3345 of SEQ ID NO: 26) of the MGEA5-RET fusion gene (specifically, SEQ ID NO: 3). A cDNA consisting of the nucleotide sequence was identified. The cDNA of the MGEA5-RET gene is generated from the mRNA of the MGEA5-RET gene, which consists of the nucleotide sequence of SEQ ID NO: 2, and contains the N-terminal region of MGEA5 (specifically, amino acid residues 1 to 725 of SEQ ID NO: 24). MGEA5-RET fusion protein (specifically, from the amino acid sequence of SEQ ID NO: 1) and the C-terminal region of RET (specifically, amino acid residues 713 to 1114 of SEQ ID NO: 27) It was confirmed that it encodes a protein.

なお、Ｍ１３ ｐｒｉｍｅｒ ＲＶ、及びＭ１３ ｐｒｉｍｅｒ Ｍ４は、クローニングベクター（pMD20-T-vector）のクローニングサイトの両端にアニーリングするプライマーである。

Note that M13 primer RV and M13 primer M4 are primers that anneal to both ends of the cloning site of the cloning vector (pMD20-T-vector).

3. Identification of breakpoints in the MGEA5-RET fusion gene Based on the nucleotide sequence information of the cDNA of the MGEA5-RET fusion gene, it was determined that the 3' end of exon 12 of the MGEA5 gene and the 5' end of exon 12 of the RET gene are fused. became clear. Therefore, it was speculated that the DNA in intron 12 of the MGEA5 gene and the DNA in intron 11 of the RET gene were cleaved, and as a result of fusion of both DNA fragments, an MGEA5-RET fusion gene was generated. To confirm this, PCR was performed using AmpliTaq 360 DNA Polymerase (manufactured by Thermo Fisher Scientific) using the genomic DNA of case #1 as a template. Specifically, 1 μL of genomic DNA obtained from the colorectal cancer tissue of case #1 by a standard method, 25 μL of AmpliTaq Gold 360 Master Mix, 1 μL of 360 GC Enhancer, and 22 μL of sterile water were mixed, and the mixture was prepared as shown in Table 5. PCR was performed using the primer set shown in 95°C for 2 minutes for 1 cycle; then 35 cycles of 95°C for 1 minute, 58°C for 1 minute, and 72°C for 3 minutes; then 72°C for 6 minutes. was carried out under the following conditions: 1 cycle. PCR products were separated by 0.8% agarose gel electrophoresis.

As a result, in lanes 1 to 3, PCR products containing one clear band were not confirmed, whereas in lanes 4 to 6, PCR products containing one clear band were confirmed ( (See Figure 2). Among lanes 4 to 6, the PCR product in lane 4, which has the shortest base length (i.e., the PCR product obtained with the MG-RET_CF primer and the MG-RET_4R primer), is said to be relatively easy to sequence. Based on this idea, this PCR product was subjected to Sanger sequencing using the primers shown in Table 5. As a result, breakpoints were found in intron 12 of the MGEA5 gene and intron 11 of the RET gene (see Tables 6 and 7), and as a result, the MGEA5 gene in the 5' region and the RET gene in the 3' region , it was revealed that they were fused via thymidine (see Table 8).

表中の四角で囲ったヌクレオチド配列は、ＭＧＥＡ５遺伝子のエクソン１２を示し、それ以外のヌクレオチド配列は、ＭＧＥＡ５遺伝子のイントロン１２を示す。また、２つの下線で示したヌクレオチド配列の相補配列には、５’末端から順に、それぞれＭＧ－ＲＥＴ＿１Ｆプライマー及びＭＧ－ＲＥＴ＿ＣＦプライマーがアニーリングする。また、矢印（↓）はブレイクポイントを示す。

The nucleotide sequence enclosed in a box in the table represents exon 12 of the MGEA5 gene, and the other nucleotide sequences represent intron 12 of the MGEA5 gene. Furthermore, the MG-RET_1F primer and the MG-RET_CF primer are annealed to the complementary sequences of the two underlined nucleotide sequences in order from the 5' end, respectively. Also, an arrow (↓) indicates a breakpoint.

表中の四角で囲ったヌクレオチド配列は、ＲＥＴ遺伝子のエクソン１２を示し、それ以外のヌクレオチド配列は、ＲＥＴ遺伝子のイントロン１１を示す。また、６つの下線で示したヌクレオチド配列には、５’末端から順に、それぞれＭＧ－ＲＥＴ＿１Ｒプライマー、ＭＧ－ＲＥＴ＿２Ｒプライマー、ＭＧ－ＲＥＴ＿３Ｒプライマー、ＭＧ－ＲＥＴ＿４Ｒプライマー、ＭＧ－ＲＥＴ＿５Ｒプライマー、及びＭＧ－ＲＥＴ＿６Ｒプライマーがアニーリングする。また、矢印（↓）はブレイクポイントを示す。

The nucleotide sequence enclosed in a box in the table indicates exon 12 of the RET gene, and the other nucleotide sequences indicate intron 11 of the RET gene. In addition, the six underlined nucleotide sequences include, in order from the 5' end, the MG-RET_1R primer, the MG-RET_2R primer, the MG-RET_3R primer, the MG-RET_4R primer, the MG-RET_5R primer, and the MG-RET_6R primer. is annealed. Also, an arrow (↓) indicates a breakpoint.

表中の１重下線で示したヌクレオチド配列は、５’領域のＭＧＥＡ５遺伝子のヌクレオチド配列（具体的には、配列番号３６の１～８５２番目のヌクレオチド残基）を示す。また表中の２重下線で示したヌクレオチド配列は、３’領域のＲＥＴ遺伝子のヌクレオチド配列（具体的には、配列番号３７の６８５～１９９５番目のヌクレオチド残基）を示す。小文字のＴ（ｔ）は、５’領域のＭＧＥＡ５遺伝子と３’領域のＲＥＴ遺伝子の間に挿入されたチミジンを示す。

The single underlined nucleotide sequence in the table indicates the nucleotide sequence of the MGEA5 gene in the 5' region (specifically, nucleotide residues 1 to 852 of SEQ ID NO: 36). Furthermore, the double underlined nucleotide sequence in the table indicates the nucleotide sequence of the RET gene in the 3' region (specifically, nucleotide residues 685 to 1995 of SEQ ID NO: 37). The lowercase letter T (t) indicates thymidine inserted between the MGEA5 gene in the 5' region and the RET gene in the 3' region.

4. Identification of fusion genes other than the MGEA5-RET fusion gene in cancer patients. Cancer tissue samples obtained from patients with MGEA5-RET fusion gene were analyzed using the HOPE fusion panelver.3 panel in the same manner as in Example 1. Novel fusion proteins (TBC1D5-LPP fusion protein, ACER1-MLLT1 fusion protein, NAB2-STAT6 fusion protein, POU2F1-MAML2 fusion protein, TMEM41B-FUS fusion protein, ZDHHC21-FOXO1 fusion protein, LACAT1-SEPT9 fusion protein, EIF3K-ACTN4 fusion protein Protein, SLC12A2-RET fusion protein, GSK3B-GPHN fusion protein, IL6ST-FOXO1 fusion protein, PPP1CB-ALK fusion protein, RNF40-CUTA fusion protein, (SEPT5-GP1BB)-SEpt5 fusion protein, PRIM2-ACTB fusion protein, AZIN1- MLLT3 fusion protein, RAP1B-HMGA2 fusion protein, EZR-MLLT4 fusion protein, OGT-AFF1 fusion protein, RPSAP52-HMGA2 fusion protein, BCL11B-LOC105370659 fusion protein, LOC101928865-GAB2 fusion protein, AQP10-ATP8B2 fusion protein, ARM C2-FOXO3 fusion protein, CBL-MAML2 fusion protein, PRKCQ-PICALM fusion protein, FGF1-PRKAR2A fusion protein, SLC44A1-BRAF fusion protein, SMURF1-FOXO1 fusion protein, TCAF1-BRAF fusion protein, CCNC-MLLT10 fusion protein, PPP1R21-ALK fusion protein, Novel fusion genes encoding LOC107986457-ARHGAP26 fusion protein, SPTBN1-ALK fusion protein, MKRN2-PPARG fusion protein, PHLDB2-JAK2 fusion protein, and SMARCA2-NFIB fusion protein were identified (see Table 9). Two types of fusion genes have been identified in the NAB2-STAT6 fusion gene that differ in the length of the STAT6 gene fragment in the 3' region, and in the AQP10-ATP8B2 fusion gene, the length of the AQP10 gene fragment in the 5' region differs. Two different types of fusion genes were identified, and three types of fusion genes were identified among the TCAF1-BRAF fusion genes, with different combinations of the length of the TCAF1 gene fragment in the 5' region and the length of the BRAF gene fragment in the 3' region. (See Table 9).

The present invention contributes to personalized medicine for cancer.

Claims (8)

A method for detecting a fusion gene in cancer, comprising the following step (a).
(a) Detecting the MGEA5-RET fusion protein, or genomic DNA, mRNA, or cDNA encoding the fusion protein in a biological sample collected from a subject ; ( i ) the M GEA5-RET fusion protein comprises an amino acid sequence having 90 % or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1;
(ii ) the genomic DNA encoding the M GEA5-RET fusion protein comprises a nucleotide sequence having 90 % or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 38;
(iii ) the mRNA encoding the M GEA5-RET fusion protein comprises a nucleotide sequence having 90 % or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 2;
(iv ) Detection according to claim 1 , characterized in that the cDNA encoding the M GEA5-RET fusion protein comprises a nucleotide sequence having 90 % or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 3. Method. A kit for detecting a fusion gene in cancer, comprising a primer set of a forward primer and a reverse primer that anneals to genomic DNA encoding the MGEA5-RET fusion protein in a biological sample, or a probe that hybridizes to the genomic DNA . A kit for detecting a fusion gene in cancer, comprising a reverse primer or probe that hybridizes to mRNA encoding an MGEA5-RET fusion protein in a biological sample . A kit for detecting a fusion gene in cancer, comprising a primer set of a forward primer and a reverse primer that anneals to cDNA encoding the MGEA5-RET fusion protein, or a probe that hybridizes to the cDNA . ( i ) the M GEA5-RET fusion protein comprises an amino acid sequence having 90 % or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1;
(ii ) The primer set of a forward primer and a reverse primer that anneals to the genomic DNA encoding the M GEA5-RET fusion protein has a nucleotide sequence that has 90 % or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 38 and its complement. A primer set that amplifies nucleotide residues corresponding to positions 852 to 854 of SEQ ID NO: 38 in the genomic DNA containing the sequence,
(iii ) The probe that hybridizes to the genomic DNA encoding the M GEA5-RET fusion protein contains a nucleotide sequence having 90 % or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 38 and its complementary sequence. is a probe that hybridizes to nucleotide residues corresponding to positions 852 to 854 of SEQ ID NO: 38,
(iv ) When the reverse primer that hybridizes to the mRNA encoding the M GEA5-RET fusion protein contains the nucleotide sequence having 90 % or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 2, A reverse primer that reversely transcribes nucleotide residues corresponding to positions 2618 to 2619 of
(v ) The probe that hybridizes to the mRNA encoding the M GEA5-RET fusion protein contains a nucleotide sequence having 90 % or more sequence identity with the nucleotide sequence shown in SEQ ID No. 2, A probe that hybridizes to nucleotide residues corresponding to positions 2618 to 2619,
(vi ) A nucleotide sequence in which the primer set of a forward primer and a reverse primer that anneals to cDNA encoding the M GEA5-RET fusion protein has 90 % or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 3, and its complementary sequence. A primer set that amplifies nucleotide residues corresponding to positions 2175 to 2176 of SEQ ID NO: 3 in the cDNA containing
(vii ) The probe that hybridizes to the cDNA encoding the M GEA5-RET fusion protein contains a nucleotide sequence having 90 % or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 3 and its complementary sequence, The detection kit according to any one of claims 3 to 5 , which is a probe that hybridizes to nucleotide residues corresponding to positions 2175 to 2176 of SEQ ID NO: 3. MGEA5-RET fusion protein, a fragment containing the fusion site thereof, or cDNA encoding the same . ( i ) the M GEA5-RET fusion protein is an isolated protein or a recombinant protein comprising an amino acid sequence having 90 % or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 ; The fragment containing the fusion site of the RET fusion protein contains amino acid residues corresponding to positions 725 to 726 of SEQ ID NO: 1 as the fusion site,
(ii ) The cDNA encoding the M GEA5-RET fusion protein is a cDNA containing a nucleotide sequence having 90 % or more sequence identity with the nucleotide sequence of SEQ ID NO: 3, and contains the fusion site of the M GEA5-RET fusion protein. characterized in that the cDNA encoding the fragment contains nucleotide residues corresponding to positions 2175 to 2176 of SEQ ID NO: 3 as a fusion site;
The fusion protein according to claim 7 , a fragment containing the fusion site thereof, or a cDNA encoding the same.