CN104762399A - Tumor circulating DNA KRAS mutation detection method - Google Patents

Tumor circulating DNA KRAS mutation detection method Download PDF

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Publication number
CN104762399A
CN104762399A CN201510185937.5A CN201510185937A CN104762399A CN 104762399 A CN104762399 A CN 104762399A CN 201510185937 A CN201510185937 A CN 201510185937A CN 104762399 A CN104762399 A CN 104762399A
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test kit
gt
ggt
treatment
sequence according
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CN201510185937.5A
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Chinese (zh)
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王志敏
徐烨
黄薇
蔡三军
李聪
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上海产业技术研究院
上海人类基因组研究中心
复旦大学附属肿瘤医院
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Priority to CN201510185937.5A priority Critical patent/CN104762399A/en
Publication of CN104762399A publication Critical patent/CN104762399A/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/156Polymorphic or mutational markers

Abstract

The invention relates to a tumor circulating DNA KRAS mutation detection method. The invention specifically relates to primer sequences (SEQ ID NO: 3 and 4) used for the detection, a kit and compositions comprising the primer sequences, an application of the primer sequences, and the like. According to the invention, with a detection method for detecting KRAS second exon gene mutation through second-generation high-throughput sequencing aiming at the tumor circulating DNA, defects such as inadequate sensitivity of traditional sequencing methods can be overcome.

Description

Cycling tumor DNA KRAS detection method

Technical field

The present invention relates to cycling tumor DNA KRAS detection method.

Background technology

Mutation detection specific based on the high-flux sequence of Peripheral Circulation DNA is strong, highly sensitive.By the abrupt climatic change of Peripheral Circulation DNA KRAS, the impact of potential side effect over-treatment can avoided and bring, and other patients can carry out more positive treatment.Construction force evolution mathematical model can determine personalized cancer therapeutic regimen further.

K-ras is a kind of proto-oncogene, is about 35kb, is positioned at No. 12 karyomit(e)s, is one of RAS gene family member, encoded K-ras albumen, all has relation with the generation of tumour, propagation, migration, diffusion and vasculogenesis.The gene that K-ras gene (K-ras, p21) family is relevant to human tumor has three kinds---and H-ras, K-ras and N-ras, be positioned on 11,12 and No. 1 karyomit(e)s respectively.K-ras gene has another name called p21 gene because of the ras albumen of 21kD of encoding.In ras gene, KRAS has the greatest impact to human cancer, and it is as molecular switch: the path that can control regulating cell growth when normal; When occurring abnormal, then cause cell continued propagation, and stop cell self-destructive.It participates in intracellular signal transmission, and when KRAS gene mutation, this gene forever activates, and can not produce normal ras albumen, makes Cellular Signaling Transduction Mediated disorderly, uncontrolled cellular proliferation and canceration.Research shows that the human malignancies of about 30% is relevant with RAS transgenation, and the product after ras sudden change can be in active state always.In leukemia, lung cancer, the rectum cancer and carcinoma of the pancreas, KRAS is all very common, and wherein in the rectum cancer, 30%-35% patient has sudden change.The existence of itself and tumour cell, propagation, migration, diffusion and vasculogenesis all have relation.KRAS gene is divided into saltant type and wild-type, common mutations site is, on No. 12 codons of KRAS gene 2 exon and No. 13 codons, wherein have 7 mutantional hotspot: G12C (GGT>TGT), G12R (GGT>AGT), G12S (GGT>CGT), G12V (GGT>GTT), G12D (GGT>GAT), G12A (GGT>GCT), G13D (GGC>GAC).These 7 kinds of sudden changes have accounted for more than 90%.

Detect KRAS gene mutation be understand in depth oncogene situation, understand the development prognosis of various cancer, the important indicator of chemicotherapy curative effect.On July 20th, 2009, U.S. food Drug Administration (FDA) discloses the revision to EGF-R ELISA (EGFR) targeted drug working instructions, wherein clearly set forth the EGFR acceptor inhibitor drug effect for associated cancer treatment and KRAS gene Close relation, and carry out KRAS gene mutation detection before requiring to use such medicine to help doctor to judge applicable crowd.The dimension gram that FDA this time have updated the production of Amgen company is replaced than (Vectibix, Victibix Panitumumab) and ImClone company produce Erbitux (Erbitux, Cetuximab Cetuximab) package insert in " instruction manual " and " clinical study " part.If 12 or 13 codons indicating KRAS gene in patient tumors cell are undergone mutation, so this patient would not benefit from the treatment of EGFR inhibitor class medicine, so do not recommend the patient that KRAS gene mutation occurs to use this type of medicine.Meanwhile, Ministry of Health of the People's Republic of China has delivered " colorectal cancer diagnosis and treatment specification (2010 editions) " (defend do doctor political affairs send out (2010) No. 165) on October 14th, 2010.Specification is pointed out, carries out the diagnosis and treatment of early stage disease medication gene screening and occur for prevention colorectal cancer, prevent from playing an important role to advanced stages, treatment and reduction colorectal cancer mortality ratio in crowd.This specification explicitly points out when patient is defined as recurrence or metastatic colorectal carcinoma accepts Erbitux, Victibix, must detect the K-ras gene appearance of tumor tissues.Now in developed countries such as America and Europes, the routine inspection that K-RAS gene test must have been done before having become PATIENTS WITH LARGE BOWEL treatment, be current doctor understand colorectal cancer patients gene status the most directly, most effective means, and become the foundation can submitting an expense account relevant anti-EGFR medical expense.

Carry out generation order-checking for patient tissue sample, carrying out medication guide based on this result is current general scheme.But in actual clinical therapeutic process, observe after adopting Cetuximab to treat for some time to the patient of KRAS gene wild-type and occur resistance.How does this resistance produce? now there are some researches show that cancer may come from somatic mutation, cancer gene is constantly accumulated in mutant cells, and the latter also completes self evolution gradually.The genomics research of single cell is the emphasis of cancer Study on Evolution to have investigator to think, it can crack order and the time of cancer sudden change, and for people understand, cancer is suddenlyd change, the mechanism of resistance provides good thinking.But we think the complex system that unicellular order-checking can not help us to understand and the specific characteristic of resolving individual patient is formed.The qualification mode of conventional sequencing and typing cannot reach high-sensitivity detection to the low ratio sudden change mixing mutational site generation in sample, more accurate quantification cannot be reached, and under " natural pressure " of chemicotherapy, in this " adaptability situation ", some extremely low sudden change temporarily increases along with tumour progression, and this " convergent evolution " result in the generation of resistance just.There is investigator to think and determine personalized cancer therapeutic regimen by power mathematical model of evolving.Doctor repeatedly can not obtain tissue samples for same patient, therefore by peripheral blood carry out nothing wound or Wicresoft become inevitable choice.And the present invention will provide effective guidance program for this reason.

Summary of the invention

The invention discloses a kind of detection method by the two generations high-flux sequence detection KRAS Second Exon transgenation for cycling tumor DNA, to overcome traditional sequencing methods under-sensitive shortcoming.

Specifically, the present invention uses the primer shown in SEQ ID NO:3 and/or 4 to implement described detection method.

Therefore, the invention provides the primer sequence shown in SEQ ID NO:3 and 4.

The present invention also provides a kind of combined sequence, and this combination comprises SEQ ID NO:3 and 4.

The present invention also provides a kind of method detecting KRAS gene mutation in Peripheral Circulation DNA, the method comprises use SEQ ID NO:3 and/or 4 and increases, and check order to amplified production, thus whether 12 and/or 13 codons detecting this KRAS gene exist sudden change.

The present invention also provides a kind of and judges tumour patient, especially large bowel cancer (such as colorectal cancer) the patient method that can benefit from the treatment of EGFR inhibitor class medicine, the method comprises the KRAS gene used in SEQ ID NO:3 and/or 4 amplification peripheral blood in patients Circulating DNA, and check order to amplified production, thus whether 12 and/or 13 codons detecting this KRAS gene exist sudden change; Wherein, if 12 and/or 13 codons of KRAS gene exist sudden change in patient tumors cell, so this patient can not benefit from the treatment of EGFR inhibitor class medicine.

In a specific embodiment, described sudden change is selected from:

(1)G12C(GGT>TGT);

(2)G12R(GGT>AGT);

(3)G12S(GGT>CGT);

(4)G12V(GGT>GTT);

(5)G12D(GGT>GAT);

(6) G12A (GGT>GCT); With

(7)G13D(GGC>GAC)。

The present invention also provides a kind of test kit, and this test kit contains the primer sequence shown in SEQ ID NO:3 and/or 4.

Test kit of the present invention can be used for implementing aforesaid method.

Also can containing being applicable to the reagent carrying out PCR in test kit, and primer described in guidance technology librarian use implements the specification sheets of aforesaid method.

The present invention also provides SEQ ID NO:3 and/or 4 preparing the purposes in test kit.Described test kit can be used for:

(1) whether 12 and/or 13 codons detecting KRAS gene in Peripheral Circulation DNA exist sudden change;

(2) judge tumour patient, especially can rectal cancer patient benefit from the treatment of EGFR inhibitor class medicine;

(3) avoid the over-treatment of tumour patient and the impact of potential side effect that brings thereof;

(4) for auxiliary diagnosis leukemia, lung cancer, large bowel cancer (such as colorectal cancer) and carcinoma of the pancreas; With

(5) for detecting the existence of tumour cell, propagation, migration, diffusion and vasculogenesis.

Embodiment

The present invention is based on the detection of KRAS gene mutation in Peripheral Circulation DNA, can judge tumour patient, especially can PATIENTS WITH LARGE BOWEL (as colorectal cancer patients) benefit from the treatment of EGFR inhibitor class medicine.

The present invention uses the primer shown in SEQ ID NO:3 and/or 4 to carry out the detection of KRAS gene mutation in described Peripheral Circulation DNA.Preferably, the present invention uses the primer shown in SEQ ID NO:3 and 4 to carry out described detection.

Detection generally includes and uses SEQ ID NO:3 and/or 4 to increase, and checks order to amplified production, thus whether 12 and/or 13 codons detecting this KRAS gene exist sudden change.

If 12 and/or 13 codons of KRAS gene exist sudden change in detected result display patient tumors cell, so this patient can not benefit from the treatment of EGFR inhibitor class medicine.

Described sudden change includes but not limited to following sudden change:

(1)G12C(GGT>TGT);

(2)G12R(GGT>AGT);

(3)G12S(GGT>CGT);

(4)G12V(GGT>GTT);

(5)G12D(GGT>GAT);

(6) G12A (GGT>GCT); With

(7)G13D(GGC>GAC)。

EGFR inhibitor class medicine is that this area is known, includes but not limited to that people are in treatment tumour, especially conventional in colorectal cancer various EGFR inhibitor class medicines.This kind of medicine includes but not limited to that the dimension gram that Amgen company produces replaces the Erbitux (Erbitux, Cetuximab Cetuximab) produced than (Vectibix, Victibix Panitumumab) and ImClone company.

Except above-mentioned application, detected result also contributes to people and avoids the over-treatment of tumour patient and the impact of potential side effect that brings thereof; Auxiliary diagnosis leukemia, lung cancer, large bowel cancer and carcinoma of the pancreas; With detect the existence of tumour cell, propagation, migration, diffusion and vasculogenesis.

Sequence of the present invention can provide in various suitable mode, such as composition or test kit.Can containing the various carriers (such as water) for dissolving and/or stablize described sequence in composition.In some cases, sequence also can directly be present in the reaction mixture for carrying out PCR.

Usually, sequence of the present invention is provided in the mode of test kit.Primer sequence in test kit also can provide in the form of compositions.Also can comprise all ingredients carried out needed for PCR in test kit, include but not limited to various required enzyme, damping fluid, dNTP mixture etc.The specification sheets that primer described in guidance technology librarian use implements aforesaid method also can be comprised in test kit.

In the present invention, term " contains " or " comprising " represents that various composition can be applied in mixture of the present invention or composition together.Therefore, term " primarily of ... composition " and " by ... form " be included in term and " contain " or in " comprising ".

As no specific instructions, various raw material of the present invention all can be obtained by commercially available; Or prepare according to the ordinary method of this area.Unless otherwise defined or described herein, all specialties used herein and scientific words and those skilled in the art the same meaning be familiar with.In addition any method similar or impartial to described content and material all can be applicable in the inventive method.

Other aspects of the present invention, due to disclosure herein, are apparent to those skilled in the art.

Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, measures according to national standard usually.If there is no corresponding national standard, then according to general international standard, normal condition or carry out according to the condition that manufacturer advises.

Embodiment 1

SEQ ID NO:1-4 of the present invention is synthesized by Sangon Biotech (Shanghai) Co., Ltd..

Embodiment 2

The KRAS Second Exon of 18 routine Colonic Carcinoma tissues and pairing Circulating DNA checks order on ABI 3730 sequenator.We collect the colorectal cancer frozen tissue of 18 example operation in 2014, and tissue DNA is adopted in extracting; Extracting patient peripheral blood, separated plasma, and take out tumour and carry Circulating DNA.Obtain SEQ ID NO:5 by primer 1,2 and detect product (PCR reaction conditions 95 DEG C of 15min; 94 DEG C of 30s, 62 DEG C of 30s, 72 DEG C of 30s, 12 take turns, and often take turns-0.5 DEG C; 94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 30s, 28 take turns; 72 DEG C of 7min, 4 DEG C of ∞).Purified pcr product, ABI 3730 sequenator checks order, and carries out sequencing result and compare.

We find in tumor tissues, have 8 routine samples to undergo mutation, and find 3 example sudden changes in Circulating DNA.The two overall concordance rate reaches 83.3%, positive sample concordance rate 37.5%, and relation conefficient is 0.5 (table 1).

Table 1: based on generation order-checking tumor tissues and Circulating DNA results contrast

* relation conefficient remarkable (two tail) in 0.05 level.R is Pearson correlation coefficient.Boldface type is designated sudden change.

Embodiment 3

Adopt with the identical sample of case study on implementation 2, obtain SEQ ID NO:5 by primer 3,4, electrophoresis detection product, adopt AMPure XP beads (magnetic bead) purifying, carry out DNA accurate quantification with PicoGreen fluorescence dye; Add index joint: index Kit-PCR primers (FC-121-1012, FC-121-1011), carries out secondary PCR reaction, again adopt AMPure XP beads (magnetic bead) purifying, carry out DNA accurate quantification with PicoGreen fluorescence dye, be diluted to 0.015nM, 1ul sample loading.MiSeq sequenator carries out high depth order-checking to PCR primer; And carry out data results.

We find in tumor tissues, have 8 routine samples to undergo mutation, and in Circulating DNA, find 7 example sudden changes, the two overall concordance rate reaches 94.4%, positive sample concordance rate 87.5%, and relation conefficient is 0.892 (table 2).

Table 2: based on two generations order-checking tumor tissues and Circulating DNA results contrast

* relation conefficient is remarkable (two tail) in 0.01 level.R is Pearson correlation coefficient.Boldface type is designated sudden change.

Embodiment 4

18 routine Colonic Carcinomas organize KRAS Second Exon to compare at MiSeq sequencing result (experimental technique is with embodiment 3) with 18 routine Colonic Carcinoma tissues and Peripheral Circulation DNA at ABI 3730 sequencing result (experimental technique is with embodiment 2).

18 routine Colonic Carcinomas are organized in ABI 3730 sequencing result and MiSeq sequencing result concordance rate reaches 100%, 8 routine samples are had to undergo mutation in 18 routine Colonic Carcinoma tissues, 7 example sudden changes are found in Circulating DNA, the two overall concordance rate reaches 94.4%, positive sample concordance rate 87.5%, relation conefficient is 0.892 (table 3).

Table 3: tumor tissues generation sequencing result compares with Circulating DNA two generation sequencing result

* relation conefficient is remarkable (two tail) in 0.01 level.R is Pearson correlation coefficient.Boldface type is designated sudden change.

The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.

Claims (10)

1. be selected from following sequence:
(1) tcgtcggcagcgtcagatgtgtataagagacaggcctgctgaaaatgactgaa (SEQ IDNO:3); With
(2)gtctcgtgggctcggagatgtgtataagagacagagaatggtcctgcaccagtaa(SEQID NO:4)。
2. one group of sequence, it comprises:
(1) tcgtcggcagcgtcagatgtgtataagagacaggcctgctgaaaatgactgaa (SEQ IDNO:3); With
(2)gtctcgtgggctcggagatgtgtataagagacagagaatggtcctgcaccagtaa(SEQID NO:4)。
3. a test kit, is characterized in that, described test kit comprises sequence according to claim 1 or one group of sequence according to claim 2.
4. test kit as claimed in claim 3, is characterized in that, described test kit is also containing the reagent carried out needed for PCR, and primer described in guidance technology librarian use implements the specification sheets of aforesaid method.
5. the test kit as described in claim 3 or 4, is characterized in that, described test kit is used for:
(1) whether 12 and/or 13 codons detecting KRAS gene in Peripheral Circulation DNA exist sudden change;
(2) judge tumour patient, especially can PATIENTS WITH LARGE BOWEL benefit from the treatment of EGFR inhibitor class medicine;
(3) avoid the over-treatment of tumour patient and the impact of potential side effect that brings thereof;
(4) for auxiliary diagnosis leukemia, lung cancer, large bowel cancer and carcinoma of the pancreas; With
(5) for detecting the existence of tumour cell, propagation, migration, diffusion and vasculogenesis.
6. sequence according to claim 1 or one group of sequence according to claim 2 are preparing the purposes in test kit, and wherein, described test kit is used for:
(1) whether 12 and/or 13 codons detecting KRAS gene in Peripheral Circulation DNA exist sudden change;
(2) judge tumour patient, especially can PATIENTS WITH LARGE BOWEL benefit from the treatment of EGFR inhibitor class medicine;
(3) avoid the over-treatment of tumour patient and the impact of potential side effect that brings thereof;
(4) for auxiliary diagnosis leukemia, lung cancer, large bowel cancer and carcinoma of the pancreas; With
(5) for detecting the existence of tumour cell, propagation, migration, diffusion and vasculogenesis.
7. purposes as claimed in claim 6, is characterized in that, described test kit is also containing the reagent carried out needed for PCR, and primer described in guidance technology librarian use implements the specification sheets of aforesaid method.
8. sequence according to claim 1 or the purposes of one group of sequence according to claim 2 in the reagent for the preparation of following application, wherein, described application comprises:
(1) whether 12 and/or 13 codons detecting KRAS gene in Peripheral Circulation DNA exist sudden change;
(2) judge tumour patient, especially can PATIENTS WITH LARGE BOWEL benefit from the treatment of EGFR inhibitor class medicine;
(3) avoid the over-treatment of tumour patient and the impact of potential side effect that brings thereof;
(4) auxiliary diagnosis leukemia, lung cancer, large bowel cancer and carcinoma of the pancreas; With
(5) existence of tumour cell, propagation, migration, diffusion and vasculogenesis is detected.
9. test kit as claimed in claim 5 or the purposes according to any one of claim 6-8, it is characterized in that, described sudden change is selected from:
(1)G12C(GGT>TGT);
(2)G12R(GGT>AGT);
(3)G12S(GGT>CGT);
(4)G12V(GGT>GTT);
(5)G12D(GGT>GAT);
(6) G12A (GGT>GCT); With
(7)G13D(GGC>GAC)。
10. the composition detected, is characterized in that, said composition contains sequence according to claim 1, or one group of sequence according to claim 2.
CN201510185937.5A 2015-04-17 2015-04-17 Tumor circulating DNA KRAS mutation detection method CN104762399A (en)

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Cited By (4)

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CN107151706A (en) * 2017-06-27 2017-09-12 郴州市第人民医院 The kit and method of KRAS gene mutation in a kind of detection plasma DNA
CN107164543A (en) * 2017-06-16 2017-09-15 上海长海医院 KRAS as biomarker in cancer of pancreas purposes
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