MULTIPLEX PCR METHODS FOR DETECTING GENE FUSIONS, KITS AND COMPOSITIONS
원문보기
IPC분류정보
국가/구분
United States(US) Patent
공개
국제특허분류(IPC7판)
C12Q-001/686
C12Q-001/6883
출원번호
16225118
(2018-12-19)
공개번호
20190185910
(2019-06-20)
발명자
/ 주소
Li, Jingfeng
Guo, Xiaomin
출원인 / 주소
Li, Jingfeng
인용정보
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0인용 특허 :
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초록▼
Methods for detecting presence or absence of at least two known gene fusions in isolated genomic DNA comprise subjecting isolated genomic DNA to multiplex PCR. For each known gene fusion, the multiplex PCR employs one or a plurality of forward primers which hybridize to a first gene adjacent its fus
Methods for detecting presence or absence of at least two known gene fusions in isolated genomic DNA comprise subjecting isolated genomic DNA to multiplex PCR. For each known gene fusion, the multiplex PCR employs one or a plurality of forward primers which hybridize to a first gene adjacent its fusion breakpoint location, and one or a plurality of reverse primers which hybridize to a second gene adjacent its fusion breakpoint location. The primers hybridize to the respective gene at consecutive respective positions separated from one another by a plurality of base pairs. Amplified products are detected and respectively represent the presence of a gene fusion. Amplified products may be Sanger sequenced to determine the fusion breakpoints. The identified specific fusion is monitored by a designed fusion PCR. Drug-resistant mutations are detected using multiplex mutation real-time PCR in patient plasma cell-free DNA during targeted therapy, for example, tyrosine kinase inhibitor-targeted therapy.
대표청구항▼
1. A method for detecting the presence or absence of at least two known gene fusions in a tissue sample, wherein each gene fusion is formed between a respective first gene at a first fusion breakpoint location and a respective second gene at a second fusion breakpoint location, the method comprising
1. A method for detecting the presence or absence of at least two known gene fusions in a tissue sample, wherein each gene fusion is formed between a respective first gene at a first fusion breakpoint location and a respective second gene at a second fusion breakpoint location, the method comprising (a) providing genomic DNA isolated from the tissue sample,(b) subjecting the isolated genomic DNA to multiplex PCR, wherein, for each known gene fusion, the multiplex PCR employs one or a plurality of forward primers which hybridize to the respective first gene adjacent the first fusion breakpoint location, wherein the plurality of forward primers hybridize to the respective first gene at consecutive respective positions along the first gene and are separated from one another by a first plurality of base pairs, and one or a plurality of reverse primers which hybridize to the respective second gene adjacent the second fusion breakpoint location, wherein the plurality of reverse primers hybridize to the respective second gene at consecutive respective positions along the second gene and are separated from one another by a second plurality of base pairs, and(c) detecting if one or more amplified products are formed, each amplified product respectively representing the presence of a gene fusion. 2. The method of claim 1, wherein the first plurality of base pairs and the second plurality of base pairs are each from about 0.1 to about 4 kb, from about 0.25 to about 3 kb, or from about 0.5 to about 2 kb. 3. The method of claim 1, wherein the first plurality of base pairs and the second plurality of base pairs are each about 0.5 to about 1 kb. 4. The method of claim 1, for detecting the presence or absence of three or more known gene fusions in a tissue sample. 5. The method of claim 1, wherein the first gene of the respective gene fusions are selected from the group consisting of EML4, KIF5B, KLC1, TGF, SEC31A, TPR, SQSTM1, DCTN1, STRN, PPFIBP1 and HIP1 and the second gene of the respective gene fusions are ALK. 6. The method of claim 5, wherein the first fusion breakpoint location of the respective gene fusions are selected from the group consisting of EML4 intron 2, EML4 intron 6, EML4 intron 13, EML4 intron 14, EML4 intron 15, EML4 intron 18, EML4 intron 17, EML4 intron 20, KIF5B intron 24, KIF5B intron 17, KIF5B intron 15, KLC1 intron 9, TFG intron 3, SEC31A intron 21, TPR intron 15, SQSTM1 intron 5, DCTN1 intron 26, STRN intron 3, PPFIBP1 intron 8, PPFIBP1 intron 12, HIP1 intron 21, HIP1 intron 28, and HIP1 intron 30, and the second fusion breakpoint location of the respective gene fusions are ALK intron 19. 7. The method of claim 1, wherein the first gene of the respective gene fusions are selected from the group consisting of slc34a2, SDC4, CD74, EZR, LRIG3, TPM3, GOPC, and CCDC6, and the second gene of the respective gene fusions are ROS1. 8. The method of claim 7, wherein the first fusion breakpoint location of the respective gene fusions are selected from the group consisting of slc34a2 intron 4, slc34a2 intron 12, SDC4 intron 2, SDC4 intron 4, CD74 intron 6, EZR intron 10, LRIG3 intron 16, TPM3 intron 2, TPM3 intron 8, GOPC intron 4, GOPC intron 8, and CCDC6 intron 6, and the second fusion breakpoint location of the respective gene fusions are selected from the group consisting of ROS1 intron 31, ROS1 intron 33, and ROS1 intron 34. 9. The method of claim 1, wherein the first gene of the respective gene fusions are selected from the group consisting of KIF5B, TRIM33, NCOA4 and CUX1, and the second gene of the respective gene fusions are RET. 10. The method of claim 9, wherein the first fusion breakpoint location of the respective gene fusions are selected from the group consisting of KIF5B intron 15, KIF5B intron 16, KIF5B intron 23, KIF5B intron 24, TRIM33 intron 14, NCOA4 intron 6, and CUX1 intron 19, and the second fusion breakpoint location of the respective gene fusions are selected from the group consisting of RET intron 11, RET intron 10, and RET intron 7. 11. The method of claim 1, for detecting the presence or absence of at least three or more gene fusions, wherein two or more multiplex PCR according to step (c) are performed and wherein the primers for the respective known gene fusions are divided among the multiplex PCRs to minimize dimer formation between primers. 12. The method of claim 1, wherein at least one amplified product is detected in step (c), the method further comprising (d) subjecting genomic DNA isolated from the tissue sample to an individual PCR for each gene fusion for which forward primer(s) and reverse primer(s) were employed in step (b), the respective individual PCRs employing the forward primer(s) and the reverse primer(s) employed in the multiplex PCR for the respective gene fusion; and(e) in each individual PCR, detecting if an amplified product is formed, the amplified product representing the presence of the respective gene fusion. 13. The method of claim 12, further comprising sequencing an amplified product detected in step (e), and designing a forward primer and a reverse primer around the gene fusion in the amplified product, operable to amplify a fragment encompassing the gene fusion. 14. The method of claim 13, wherein the fragment comprises from about 50 to about 150 base pairs. 15. The method of claim 13, wherein sequencing an amplified product detected in step (e) is conducted by Sanger sequencing. 16. A method for assessing cancer progression or cancer regression during targeted treatment, comprising determining the presence of a gene fusion in a patient's tissue sample and designing a forward primer and a reverse primer around the gene fusion operable to amplify a fragment encompassing the gene fusion, according to claim 13, and monitoring the amount of fusion product in patient plasma cell-free DNA (cfDNA) using the designed primers in a PCR during the targeted treatment. 17. A method for monitoring a patient undergoing targeted therapy with a drug to which resistance may develop, comprising detecting the presence or absence of one or more acquired functional mutations associated with the drug resistance in a tissue sample from the patient according to the method of claim 1. 18. The method of claim 17, wherein the step of detecting the presence or absence of one or more acquired functional mutations associated with the drug resistance is repeated at least once during the targeted therapy. 19. The method of claim 17, wherein the drug is crizotinib and the step of detecting the presence or absence of one or more acquired functional mutations associated with the drug resistance detects the presence or absence of crizotinib-resistant ALK mutations and crizotinib-resistant ROS1 mutations. 20. The method of claim 16, wherein the genomic DNA is plasma cell-free DNA. 21. The method of claim 17, wherein (a) the crizotinib-resistant ALK mutations are selected from 1511Tins, L1152R, C1156Y, 11171T, 11171S, F1174V, F1174C, L1196M, L1198F, G1202R, S1206Y and G1269A, and combinations thereof; or(b) the crizotinib-resistant ROS1 mutations are selected from L2026M, G2032R, D2032N, L2155S, S1986Y and S1986F, and combinations thereof. 22. The method of claim 1, wherein at least one of the known gene fusions is BCR-ABL1, RBN15-MKL1, NPM1-ALK, IGH-MYC, RUNX1-RUNX1T1, ETV6-RUNX1, IGH-MAF, PML-RARA, FGFR2-KIAA1967, FGFR3-TACC3, CD74-NTRK1, MPRIP-NTRK1, TPM3-NTRK1, LMNA-NTRK1, SQSTM1-NTRK1, TPR-NTRK1, PEAR1-NTRK1, IRF2BP2-NTRK1, RFWD2-NTRK1, TP53-NTRK1, TFG-NTRK1, NFASC-NTRK1, BCAN-NTRK1, MDM4-NTRK1, RABGAP1L-NTRK1, PPL-NTRK1, CHTOP-NTRK1, ARHGEF2-NTRK1, TAF-NTRK1, CEL-NTRK1, SSBP2-NTRK1, GRIPAP1-NTRK1, LRRC71-NTRK1, MRPL24-NTRK1, QKI-NTRK2, NACC2-NTRK2, VCL-NTRK2, AGBL4-NTRK2, PAN3-NTRK2, AFAP1-NTRK2, DAB21P-NTRK2, TRIM24-NTRK2, SQSTM1-NTRK2, ETV6-NTRK3, BTBD1-NTRK3, EML4-NTRK3, TFG-NTRK3, RBPMS-NTRK3, or LYN-NTRK3. 23. A method for detecting the presence or absence of a known gene fusion in a tissue sample, wherein the gene fusion is formed between a first gene at a first fusion breakpoint location and a second gene at a second fusion breakpoint location, the method comprising (a) providing genomic DNA isolated from the tissue sample,(b) subjecting the isolated genomic DNA to PCR amplification, the PCR amplification employing one or a plurality of forward primers which hybridize to the respective first gene adjacent the first fusion breakpoint location, wherein the plurality of forward primers hybridize to the respective first gene at consecutive respective positions along the first gene and are separated from one another by a first plurality of base pairs, and one or a plurality of reverse primers which hybridize to the respective second gene adjacent the second fusion breakpoint location, wherein the plurality of reverse primers hybridize to the respective second gene at consecutive respective positions along the second gene and are separated from one another by a second plurality of base pairs, and(c) detecting if an amplified product is formed, the amplified product representing the presence of the gene fusion. 24. The method of claim 23, wherein the first plurality of base pairs and the second plurality of base pairs are each from about 0.1 to about 4 kb, from about 0.25 to about 3 kb, or from about 0.5 to about 2 kb. 25. The method of claim 24, wherein the first plurality of base pairs and the second plurality of base pairs are each from about 0.5 to about 1 kb. 26. The method of claim 23, wherein (a) the first gene is EML4, KIF5B, TGF, SEC31A, TPR, SQSTM1, DCTN1, STRM, or PPFIBP1, and the second gene is ALK,(b) the first gene is slc34a2, SDC4, CD74, EZR, LRIG3, TPM3, GOPC OR CCDC6, and the second gene is ROS1;(c) the first gene is KIF5B, TRIM33, NCOA4 or CUX1, and the second gene is RET; or(d) the gene fusion is BCR-ABL1, RBN15-MKL1, NPM1-ALK, IGH-MYC, RUNX1-RUNX1T1, ETV6-RUNX1, IGH-MAF, PML-RARA, FGFR2-KIAA1967, FGFR3-TACC3, CD74-NTRK1, MPRIP-NTRK1, TPM3-NTRK1, LMNA-NTRK1, SQSTM1-NTRK1, TPR-NTRK1, PEAR1-NTRK1, IRF2BP2-NTRK1, RFWD2-NTRK1, TP53-NTRK1, TFG-NTRK1, NFASC-NTRK1, BCAN-NTRK1, MDM4-NTRK1, RABGAP1L-NTRK1, PPL-NTRK1, CHTOP-NTRK1, ARHGEF2-NTRK1, TAF-NTRK1, CEL-NTRK1, SSBP2-NTRK1, GRIPAP1-NTRK1, LRRC71-NTRK1, MRPL24-NTRK1, QKI-NTRK2, NACC2-NTRK2, VCL-NTRK2, AGBL4-NTRK2, PAN3-NTRK2, AFAP1-NTRK2, DAB21P-NTRK2, TRIM24-NTRK2, SQSTM1-NTRK2, ETV6-NTRK3, BTBD1-NTRK3, EML4-NTRK3, TFG-NTRK3, RBPMS-NTRK3, or LYN-NTRK3. 27. The method of claim 23, further comprising sequencing the amplified product detected in step (c) by Sanger sequencing, and designing a forward primer and a reverse primer around the gene fusion operable to amplify a fragment encompassing the gene fusion. 28. The method of claim 27, wherein the fragment comprises from about 50 to about 150 base pairs. 29. A method for assessing cancer progression or cancer regression during targeted treatment, comprising determining the presence of a gene fusion in a patient's tissue sample and designing a forward primer and a reverse primer around the gene fusion operable to amplify a fragment encompassing the gene fusion, according to claim 27, and monitoring the amount of fusion product in patient plasma cell-free DNA (cfDNA) using the designed primers in a PCR during the targeted treatment. 30. A method for monitoring a patient undergoing targeted therapy with a drug to which resistance may develop, comprising detecting the presence or absence of one or more acquired functional mutations associated with the drug resistance in a tissue sample from the patient according to the method of claim 23. 31. The method of claim 30, wherein the step of detecting the presence or absence of the one or more acquired functional mutations associated with the drug resistance is repeated at least once during the targeted therapy. 32. The method of claim 30, wherein the drug is crizotinib and the step of detecting the presence or absence of the one or more acquired functional mutations associated with the drug resistance detects the presence or absence of a crizotinib-resistant ALK mutation or a crizotinib-resistant ROS1 mutation. 33. The method of claim 29, wherein the genomic DNA is plasma cell-free DNA. 34. A kit for detecting the presence or absence of a gene fusion in a tissue sample, wherein (a) the gene fusion is at least one ALK gene fusion, the kit comprising reverse primers of SEQ ID NO: 1-5 and forward primers of SEQ ID NO: 6-141;(b) the gene fusion is at least one ROS1 gene fusion, the kit comprising reverse primers of SEQ ID NO: 142-165 and forward primers of SEQ ID NO:166-208; or(c) the gene fusion is at least one RET gene fusion, the kit comprising reverse primers of SEQ ID NO: 209-216 and forward primers of SEQ ID NO: 217-251. 35. A kit according to claim 34, further comprising a negative control isolated DNA sample which is free of a positive fusion DNA. 36. A composition comprising an isolated DNA sample from a patient non-small cell lung cancer tumor, and (a) reverse primers of SEQ ID NO: 1-5, and forward primers of SEQ ID NO: 6-141;(b) reverse primers of SEQ ID NO: 142-165 and forward primers of SEQ ID NO:166-208; or(c) reverse primers of SEQ ID NO: 209-216 and forward primers of SEQ ID NO: 217-251. 37. A composition according to claim 36, further comprising a polymerase.
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