WO2017084027A1 - Kit pour la classification pronostique de la leucémie myélocytaire aigüe et méthode d'essai correspondante - Google Patents

Kit pour la classification pronostique de la leucémie myélocytaire aigüe et méthode d'essai correspondante Download PDF

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WO2017084027A1
WO2017084027A1 PCT/CN2015/094788 CN2015094788W WO2017084027A1 WO 2017084027 A1 WO2017084027 A1 WO 2017084027A1 CN 2015094788 W CN2015094788 W CN 2015094788W WO 2017084027 A1 WO2017084027 A1 WO 2017084027A1
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primer
gene
fragment
seq
nucleotide sequence
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PCT/CN2015/094788
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English (en)
Chinese (zh)
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王秀莉
洪燕
玄兆伶
李大为
梁峻彬
陈重建
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安诺优达基因科技(北京)有限公司
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Priority to PCT/CN2015/094788 priority Critical patent/WO2017084027A1/fr
Publication of WO2017084027A1 publication Critical patent/WO2017084027A1/fr

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

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  • the invention relates to a kit and a detection method which can be used for prognosis stratification of acute myeloid leukemia, and belongs to the technical field of molecular detection.
  • AML Acute Myelocytic Leukemia
  • Cytogenetic changes have greatly affected the treatment and prognosis of patients. With the continuous advancement of science and technology, more and more prognostic factors have been identified: (1) gene mutation; (2) cytogenetic diagnosis; (3) Clinical manifestations, including age, WBC, and clinical manifestations; (4) rate of decline in minimal residual disease (MRD) after treatment. Among them, gene mutation is one of the key factors affecting prognosis.
  • Non-Patent Documents 1 to 4 The most common mutations in genes are CEBPA, FLT3-ITD, and NMP1 gene mutations, which occur at about 15%, 35%, and 20%, respectively [Non-Patent Documents 1 to 4]. Due to the prevalence of multiple gene multi-mutation coexistence in AML cases, systematic detection of multi-gene/multi-site mutations is more conducive to prognostic stratification of AML, and also helps to determine treatment options and predict patient response to treatment. .
  • kits and assays for prognostic stratification of acute myeloid leukemia that are capable of rapidly and accurately detecting polygenic/multiple site mutations.
  • the present invention includes:
  • a kit for prognosis stratification of acute myeloid leukemia comprising an ITD region for detecting a FLT3 gene a primer set of 1 or a fragment thereof, a primer set 2 for detecting an NPM1 gene or a fragment thereof, and a primer set 3 for detecting a CEBPA gene or a fragment thereof;
  • Each of the primer sets includes an upstream primer and a downstream primer, and at least one of the upstream primer and the downstream primer is labeled with a fluorescent molecule.
  • the fluorescent molecule is selected from the group consisting of FAM, HEX, TET, VIC, Cy3, Cy5, and ROX7.
  • each primer set for detecting a gene of a similar size or a fragment thereof is labeled with a different fluorescent molecule.
  • the primer set 1 is composed of an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 1 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 2;
  • the primer set 2 is composed of an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 3 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 4;
  • the primer set 3 is the following primer set 31 to 34:
  • a primer set 31 consisting of an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 5 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 6;
  • a primer set 32 consisting of an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 7 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 8;
  • a primer set 33 consisting of an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 9 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 10;
  • Primer set 34 consists of an upstream primer as shown in SEQ ID NO: 11 and a downstream primer as shown in SEQ ID NO: 12.
  • a method for simultaneously detecting a gene mutation of an ITD region, an NPM1 gene, and a CEBPA gene of a FLT3 gene of a subject which comprises the kit according to any one of items 1 to 4, comprising:
  • A using the primer set 1 as a primer, using the genomic DNA of the sample from the subject as a template, amplifying the ITD region of the FLT3 gene of the sample or a fragment thereof, to obtain an amplification product A;
  • the method according to item 5 further comprising: comparing the ITD region of the FLT3 gene or a fragment thereof, the NPM1 gene or a fragment thereof, and the CEBPA gene or a fragment thereof based on the result of the multiplex fluorescent capillary gel electrophoresis If the size of one or more amplification products is the same as the size of the corresponding wild-type gene or a fragment thereof, such as the two, it is determined that the subject's gene has not been mutated, and if the two are inconsistent, the test is determined to be a test. The gene has been mutated.
  • the multi-gene multi-mutation with significant clinical guiding significance for the prognosis stratification of AML can be simultaneously detected, and the entire detection process from sample preparation to report is completed within one working day, and the detection result is fast and accurate. It can effectively provide rapid guidance on the prognosis of AML.
  • One of the advantages of the present invention over the prior art is that it is fast. Traditionally, it takes at least 3 days to complete the prognostic stratification of AML by one-generation sequencing; it takes at least one week to complete the prognostic stratification of AML by second-generation sequencing; and the method of the present invention is used to complete the prognostic stratification of AML. It takes less than a day to complete, which is extremely important for clinical testing.
  • another advantage of the present invention is that it is accurate, and the specific prognosis results detected by the present invention are consistent with the results of actual clinician feedback.
  • Figure 1 is the result of testing a male AML patient using the method of the examples.
  • Figure 2 is the result of testing a female AML patient using the method of the examples.
  • the present invention provides a kit for prognosis stratification of acute myeloid leukemia (kit of the invention) comprising a primer set 1 for detecting an ITD region of a FLT3 gene or a fragment thereof, for use in a primer set 2 for detecting the NPM1 gene or a fragment thereof, and a primer set 3 for detecting the CEBPA gene or a fragment thereof;
  • Each of the primer sets includes an upstream primer and a downstream primer, and at least one of the upstream primer and the downstream primer is labeled with a fluorescent molecule.
  • for detection refers to the use of a designated gene or a fragment thereof from a subject comprising the gene or a fragment thereof.
  • the source sample is amplified by PCR and the amplification product is given a fluorescent label that can be used for multiplex capillary gel electrophoresis.
  • the "subject” is a person who has already suffered from acute myeloid leukemia, preferably a person who has developed acute myeloid leukemia and has received clinical treatment and needs a prognostic evaluation.
  • Fluorescent molecular labeling of the nucleotide sequence can be carried out by methods known to those skilled in the art, which are typically labeled at the 5' or 3' end of the upstream primer and/or downstream primer.
  • fluorescent molecule include FAM, HEX, TET, VIC, Cy3, Cy5, and ROX7, but are not limited thereto.
  • Capillary electrophoresis also known as high performance capillary electrophoresis (HPCE)
  • HPCE high performance capillary electrophoresis
  • capillary gel electrophoresis is a zone electrophoresis in which a gel is transferred to a capillary as a support for separation.
  • Gel is a solid dispersion system with porosity, similar to the role of molecular sieves. The separation is separated by the gel force in the capillary, according to the volume of each molecule. The larger the volume is first Isolated, suitable for the analysis of biological macromolecules.
  • Capillary gel electrophoresis can identify oligonucleotides with one base difference and can be used to isolate DNA fragments. Fluorescence capillary gel electrophoresis distinguishes nucleotide sequences of different sizes by detecting fluorescent signals from fluorescent molecules labeled on nucleotide sequences. In the present specification, multiplex capillary capillary gel electrophoresis refers to simultaneous detection of fluorescent capillary gel electrophoresis from a plurality of fluorescent molecules.
  • each primer set for detecting genes of similar sizes or fragments thereof.
  • each gene and its fragment can be more significantly distinguished by fluorescent capillary gel electrophoresis.
  • the size is similar means that the difference is within ⁇ 30 bp.
  • nucleotide sequences of the ITD region, the NPM1 gene and the CEBPA gene of the human FLT3 gene are known, and amplification of primers for genes of known nucleotide sequences is well known to those skilled in the art, for example, according to, for example, "Molecule The Guide to Cloning Experiments (J. Sambrook. J., Huang Peitang et al., 3rd edition, 2005) is designed to be designed, or using computer software (such as Primer Premier 6.0 developed by Premier). Design.
  • PCR polymerase chain reaction
  • the PCR reaction procedure is generally a step of denaturation, annealing, extension, and the like.
  • the primer set 1 consists of an upstream primer having a nucleotide sequence such as SEQ ID NO: 1 and a downstream primer having a nucleotide sequence such as SEQ ID NO: 2;
  • Group 2 consists of an upstream primer as shown in SEQ ID NO: 3 and a downstream primer as shown in SEQ ID NO: 4;
  • the primer set 3 is the following primer set 31 to 34:
  • a primer set 31 consisting of an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 5 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 6;
  • a primer set 32 consisting of an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 7 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 8;
  • a primer set 33 consisting of an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 9 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 10;
  • Primer set 34 consists of an upstream primer as shown in SEQ ID NO: 11 and a downstream primer as shown in SEQ ID NO: 12.
  • each of the obtained amplification products shows a "high quality" peak in the multiplex fluorescent capillary gel electrophoresis measurement.
  • “high quality” means that the amplification peaks for each purpose are single, and no peaks appear.
  • the kit of the present invention may further comprise, in addition to the above primer sets 1 to 3, a method for extracting genomic DNA for use in a PCR amplification reaction for extracting total RNA for reverse transcription. And one or more of reagents or devices for multiplex capillary gel electrophoresis.
  • the kit of the present invention can simultaneously detect a gene mutation of the ITD region, the NPM1 gene and the CEBPA gene of the FLT3 gene in a subject.
  • the mutation of the ITD region, the NPM1 gene and the CEBPA gene of the FLT3 gene of the subject is simultaneously detected using the kit of the present invention, it can be carried out as follows:
  • Step A Using the primer set 1 as a primer and using the genomic DNA of the sample from the subject as a template, the ITD region of the FLT3 gene of the sample or a fragment thereof is amplified to obtain an amplification product A.
  • Step B Using the primer set 2 as a primer, the genomic DNA of the sample from the subject is used as a template to amplify the NPM1 gene of the sample or a fragment thereof to obtain an amplification product B.
  • Step C Using the primer set 3 as a primer, the cDNA obtained by reverse transcription of total RNA from a sample of the subject is used as a template to amplify the CEBPA gene of the sample or a fragment thereof to obtain an amplification product C.
  • the steps A, B, and C can be performed in any order or simultaneously.
  • sample from the subject may be, for example, any tissue or cells from the subject, preferably whole blood or bone marrow.
  • Obtaining genomic DNA from a sample from a subject can be carried out by methods well known to those skilled in the art, for example, using a commercially available kit. Furthermore, obtaining total RNA from a sample from a subject, and reverse transcription of the total RNA to obtain cDNA can be carried out by a method known to those skilled in the art, for example, using a commercially available kit.
  • step D is carried out: the above amplification product A, amplification product B and amplification product C are mixed to obtain a mixed sample, and the mixed sample is subjected to multiplex fluorescent capillary gel electrophoresis.
  • the size of the amplification product of one or more (preferably all) of the gene or fragment thereof and the CEBPA gene or a fragment thereof is identical to the size of the corresponding wild-type gene or a fragment thereof, such as both, and is determined to be the subject's
  • the gene did not mutate, and if the two were inconsistent, it was determined that the subject had a mutation in the gene.
  • the size of the ITD region of the wild-type FLT3 gene is 365 bp
  • the size of the wild-type NPM1 gene fragment is 283 bp
  • the fragment of the wild-type CEBPA gene is four, and the sizes thereof are 309 bp, 345 bp, 444 bp, and 416 bp.
  • the kit of the present invention can be used for prognosis stratification of acute myeloid leukemia, that is, in the case where the result is that the FLT3-ITD is not mutated and at least one of NMP1 or CEBPA is mutated, the prognosis of the patient can be determined.
  • the prognosis of the patient can be determined to be moderate; in the judgment result, the mutation of FLT3-ITD and the mutation of NMP1 and CEBPA are not mutated. In the case of this, it can be determined that the patient's prognosis is poor.
  • F or FS represents an upstream primer
  • R or RS represents a downstream primer
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, (denaturation at 95 ° C for 1 minute, annealing at 55 ° C for 30 seconds, extension at 72 ° C for 30 seconds) 30 cycles, 72 ° C extension for 5 minutes.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, (denaturation at 95 ° C for 1 minute, annealing at 55 ° C for 30 seconds, extension at 72 ° C for 30 seconds) 30 cycles, 72 ° C final extension for 5 minutes.
  • wild-type NPM1 gene fragment size is 283 bp
  • wild-type FLT3 gene ITD region fragment size is 365 bp
  • wild-type CEPBA gene fragment has four, its size is 309 bp, 345 bp, 444 bp And 415 bp. Whether or not an insertion or deletion mutation occurs in the gene to be detected is determined based on the size of the fragment corresponding to the peak.
  • the test results are shown in Figure 1. It can be seen that the fragment of the amplification product of the NPM1 gene is large. The small size was 283 bp, indicating that the detection gene was not mutated; the size of the four amplified fragments of CEBPA gene were 309 bp, 345 bp, 444 bp and 416 bp, both of which were wild type, indicating that the CEBPA detection gene was not mutated; FLT3 gene amplification product The size is 383 bp, indicating that the gene has an 18 bp insertion mutation. In this sample, only the insertion mutation occurred in FLT3, and the CEBPA and NPM1 genes did not mutate, indicating that the male AML sample had a poor prognosis.
  • the test results are shown in Figure 2. It can be seen that the size of the amplified product of NPM1 gene is 287 bp, indicating that the detection gene has a 4 bp insertion mutation; the four amplified fragments of CEBPA gene are 309 bp, 345 bp, 444 bp and 416 bp, respectively, all of which are wild-type fragments. , indicating that the CEBPA detection gene did not mutate; the size of the amplification product of the ITT region of the FLT3 gene was 365 bp, which was wild-type, indicating that the FLT3 of the sample did not change. In this sample, only the insertion mutation occurred in NPM1, and the CEBPA and FLT3 genes did not mutate, indicating that the prognosis of the female AML sample is better.
  • any technical feature or combination of technical features described in the specification as a component of a certain technical solution may also be applied to the embodiments that can be implemented without obscuring the gist of the present invention.
  • Other technical solutions; and, while being able to implement and not clearly deviating from the gist of the present invention, the technical features described as the constituent parts of the different technical solutions may be combined in any manner to constitute other technical solutions.
  • the present invention also encompasses the technical solutions obtained by the combination in the above case, and these technical solutions are equivalent to those described in the present specification.
  • kits and a detection method for prognostic stratification of acute myeloid leukemia capable of rapidly detecting polygenic/multiple site mutations.

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Abstract

La présente invention concerne un kit pour classification pronostique de la leucémie myélocytaire aigüe (LMA) et une méthode d'essai correspondante capable de détecter rapidement une mutation multigénique/multisite. Le kit permet de tester complètement des sites mutés avec une sensibilité d'essai élevée, il peut effectuer en une journée l'ensemble du processus d'expérimentation, depuis la préparation de l'échantillon jusqu'à la production de rapport, il permet d'obtenir rapidement un résultat précis et peut offrir un degré de couverture élevé. Ledit kit peut en outre fournir une ligne directrice efficace et rapide pour la classification pronostique de la LMA.
PCT/CN2015/094788 2015-11-17 2015-11-17 Kit pour la classification pronostique de la leucémie myélocytaire aigüe et méthode d'essai correspondante WO2017084027A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108624687A (zh) * 2018-05-11 2018-10-09 南京先声医学检验有限公司 引物、引物组合、试剂盒及其应用
CN110241200A (zh) * 2019-06-18 2019-09-17 苏州大学附属第一医院 Flt3-itd突变高灵敏度检测方法及试剂盒
CN112553306A (zh) * 2020-12-28 2021-03-26 北京思尔成生物技术有限公司 基于毛细管电泳片段分析和一代测序结合的融合基因核酸检测方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101948829A (zh) * 2010-05-18 2011-01-19 北京大学人民医院 一种检测cebpa基因突变的试剂盒
CN104508143A (zh) * 2012-03-12 2015-04-08 纪念斯隆-凯特琳癌症中心 用于急性髓细胞白血病的诊断、预后和治疗的方法和组合物

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101948829A (zh) * 2010-05-18 2011-01-19 北京大学人民医院 一种检测cebpa基因突变的试剂盒
CN104508143A (zh) * 2012-03-12 2015-04-08 纪念斯隆-凯特琳癌症中心 用于急性髓细胞白血病的诊断、预后和治疗的方法和组合物

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BENTHAUS, T.: "Rapid and sensitive screening for CEBPA mutations in acute myeloid leukaemia", BRITISH JOURNAL OF HAEMATOLOGY, vol. 143, no. 2, October 2008 (2008-10-01), pages 230 - 239, XP055597186 *
NOGUERA, NI: "Simultaneous detection of NPM1 and FLT3-ITD mutations by capillary electrophoresis in acute myeloid leukemia", LEUKEMIA, vol. 19, no. 8, August 2005 (2005-08-01), pages 1479 - 1482, XP055597196 *
TAYYAB, M.: "Distinct Gene Mutations, their Prognostic Relevance and Molecularly Targeted Therapies in Acute Myeloid Leukemia (AML", J CANCER SCI THER, vol. 6, no. 9, 5 September 2014 (2014-09-05), pages 337 - 349, XP055597207, DOI: 10.4172/1948-5956.1000292 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108624687A (zh) * 2018-05-11 2018-10-09 南京先声医学检验有限公司 引物、引物组合、试剂盒及其应用
CN110241200A (zh) * 2019-06-18 2019-09-17 苏州大学附属第一医院 Flt3-itd突变高灵敏度检测方法及试剂盒
CN110241200B (zh) * 2019-06-18 2024-01-12 苏州大学附属第一医院 Flt3-itd突变高灵敏度检测方法及试剂盒
CN112553306A (zh) * 2020-12-28 2021-03-26 北京思尔成生物技术有限公司 基于毛细管电泳片段分析和一代测序结合的融合基因核酸检测方法

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