CN104017874A - Molecular marker and kit for predicting platinum-containing chemotherapy curative effects and lifetime of later-period non-small cell lung cancer - Google Patents
Molecular marker and kit for predicting platinum-containing chemotherapy curative effects and lifetime of later-period non-small cell lung cancer Download PDFInfo
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Abstract
The invention belongs to the fields of medical molecular genetics and clinical medicine, and particularly relates to a molecular marker and a kit for predicting the clinical benefit rate and the progression-free survival of platinum-containing chemotherapy for later-period non-small cell lung cancer. The molecular marker comprises an RICTOR gene related to the clinical benefit rate and the progression-free survival of the platinum-containing chemotherapy for the later-period non-small cell lung cancer (NSCLC), and comprises a single-nucleotide polymorphism site rs6878291 on the gene. As an application of the molecular marker, the invention also provides the RICTOR gene polymorphism detection kit for an NSCLC patient prognostic the platinum-containing chemotherapy. The medical molecular and the kit can be used for predicting the clinical benefit rate and the progression-free survival of the platinum-containing chemotherapy for the later-period non-small cell lung cancer, and provides bases for individualized treatment of patients with the disease.
Description
Technical field
The invention belongs to medical molecular genetics and clinical medicine technical field, be specifically related to a kind ofly predict that advanced Non-small cell lung is accepted after platiniferous chemotherapy clinical Benefit rate and without progression of disease molecule marker and the test kit of lifetime.
Background technology
Malignant tumour is one of killer of serious threat human life health, and lung cancer is that sickness rate is the highest, the malignant tumour that mortality ratio is the highest, modal Lung Cancer Types is nonsmall-cell lung cancer (Non-small cell lung cancer, NSCLC), account for 80% left and right of whole patients with lung cancer, most of Patients with Non-small-cell Lung in the time making a definite diagnosis in late period, no longer be applicable to operative treatment, therefore become the standard care of advanced Non-small cell lung taking chemotherapy as main complex therapy, and platiniferous chemotherapy is widely used in clinical treatment advanced Non-small cell lung because having compared with strong antitumous effect.At present, platiniferous chemotherapy has obtained unusual effect in clinical treatment advanced Non-small cell lung: efficiently reach 30%, median survival interval 8 ~ 10 months, 2 years survival rates approximately 10% ~ 15%.But Different Individual shows significantly different susceptibility to platiniferous chemotherapy in clinical treatment.Therefore, further investigation affects Non-small Cell Iung Cancer After Chemotherapy correlative factors, improves clinical treatment efficiency, has important clinical meaning.As theoretical foundation, for lung cancer Personalized medicine provides basis, be focus and the difficult point of current lung cancer personalized medicine research field.
Along with the development of Protocols in Molecular Biology and genomics, lung cancer Personalized medicine is studied by traditional external susceptibility experiment research, develop into the research method of the high flux screening chemotherapy of tumors molecular marker based on genomics and proteomics fundamental research, comprise expression of tumor tissue spectrum, DNA methylation, miRNA, single nucleotide polymorphism (Single nucleotide polymorphism, SNP) etc.Along with the development of high-throughput techniques, existing and have specific SNP based on ubiquity progressively becomes in Personalized medicine the biological heredity molecular marked compound of widespread use the most.From chemotherapy of tumors curative effect evaluation, although chemotherapeutic efficacy and tumor tissues genome, the variation of transcribing group and expressing quantity are the most relevant, but most of patients with advanced NSCLC have been not suitable for operation, obtain tumor tissues very difficult, therefore conventional dependency of carrying out peripheral blood genome SNP polymorphic detection and explore chemotherapeutic efficacy and inherited genetic factors than the peripheral blood that is easier to acquisition.Therefore, detect SNP polymorphism by peripheral blood genomic dna and become the most frequently used research method of clinical assessment lung cancer chemotherapy curative effect.
MTOR(mammalian target of rapamycin, mTOR) signal path plays crucial regulating and controlling effect in cells survival, Growth of Cells and propagation, has close relationship with apoptosis and autophagy, protein synthesis, immunity, cell movement and metabolism etc.Research shows, mTOR signal path is abnormal to be developed relevantly with the generation of Several Kinds of Malignancy, and has inseparable relation with drug susceptibility.
rICTORthe RICTOR albumen of genes encoding is one of composition of mTORC2 complex body in mTOR path, research shows, mTORC2 complex body participates in cytoskeleton and forms, RICTOR as one of principal constituent of mTORC2 tumor development with shift in play a part very crucial, and thereby the activation completely that RICTOR participates in Akt affect the function of Akt passage downstream regulon, and then the function of adjusting Akt path in apoptosis.
The present invention has studied
rICTORthe pleomorphism site of gene and patients with advanced NSCLC are accepted clinical Benefit rate after platiniferous chemotherapy and without the progression of disease dependency of lifetime, find single nucleotide polymorphism of this gene and platiniferous chemotherapy clinical Benefit rate and without progression of disease significant correlation lifetime, this molecule marker can be applicable to predict patients with advanced NSCLC platiniferous chemotherapy prognosis situation, for this type of patient's Personalized medicine provides foundation.
Summary of the invention
The object of the present invention is to provide one to can be applicable to molecule marker and the test kit of dlinial prediction patients with advanced NSCLC platiniferous chemotherapy prognosis (after chemotherapy clinical Benefit rate and without progression of disease lifetime).
The molecule marker of prediction patients with advanced NSCLC platiniferous chemotherapy provided by the invention prognosis, this forecast function can be realized by the detection of the molecule marker to peripheral blood in patients genomic dna.Above-mentioned molecule marker comprises and advanced Non-small cell lung platiniferous chemotherapy clinical Benefit rate and relevant lifetime without progression of disease
rICTORmononucleotide polymorphism site rs6878291 on gene and this gene.
As the application of above-mentioned molecule marker, the present invention also provides to predict the prognosis of patients with advanced NSCLC platiniferous chemotherapy
rICTORgene pleiomorphism detection kit.
This test kit comprises:
(1) detect
rICTORthe genotypic Auele Specific Primer pair of rs6878291 pleomorphism site on gene, described Auele Specific Primer to refer to for
rICTORgene rs6878291 pleomorphism site and designing, can specific amplification goes out to comprise the primer pair of the DNA fragmentation in this SNP site, and Auele Specific Primer synthesizes the synthetic technology of available routine.Described primer pair can be used following sequence: forward primer: 5 '-CCCATCACTCTTACCTAAACTCTC-3 ' (SEQ ID NO.1); Reverse primer: 5 '-GACTGTGGTAGGCATTTGAAGATA-3 ' (SEQ ID NO.1).
(2) polymerase chain reaction (polymerase chain reaction, and sequencing reaction assembly PCR), comprise: 1U Taq enzyme 100 μ l and 10 × Taq damping fluid 1mL, 2mM dNTP mixed solution 1ml, 1U shrimp alkaline phosphotase 100 μ l, 1U exonuclease 100 μ l, BigDye v3.1 reagent and supporting damping fluid 1ml(Applied Biosystems thereof), 120mM ethylenediamine tetraacetic acid (EDTA) 1ml, 95% methane amide 1ml, dehydrated alcohol and ultrapure water.
Use mentioned reagent box and to predict lifetime without progression of disease patients with advanced NSCLC platiniferous chemotherapy clinical Benefit rate, concrete steps are as follows:
1) collection detected person's peripheral blood 2ml, is stored in-20 DEG C of refrigerators;
2) adopt saturated sodium-chloride method extracting genomic dna from peripheral blood sample;
3) use this detection kit, specific amplification comprises
rICTORthe DNA fragmentation of gene rs6878291 pleomorphism site, and obtained DNA fragmentation is checked order, the genotype data in rs6878291 site obtained;
4) according to obtained rs6878291 loci gene type data, detected person is carried out to platiniferous chemotherapeutic efficacy and prognosis evaluation.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, or the condition of advising according to manufacturer is carried out.
embodiment 1. predicts patients with advanced NSCLC platiniferous chemotherapy clinical Benefit rate and without the progression of disease molecule marker of lifetime
1, clinical sample collection
Research object is the Patients with Non-small-cell Lung of 1004 first visits.Inclusive criteria is: the new history of pathological diagnosis, do not accept chemotherapy, operation and radiotherapy for nonsmall-cell lung cancer in the past; Accept a line platiniferous chemotherapy; Health general status scoring PS 0-2; Without the dysfunction of main organs, routine blood test, hepatic and renal function and heart function normal; Can obtain the complete information of following up a case by regular visits to.All research objects are the Chinese Han Population of consanguinity-less relation, and geographically mainly from Shanghai and surrounding area thereof, all patients are all that to accept first platinum class be basic first-line treatment scheme.
Include patient in to above-mentioned, compile its essential information, clinical information and treatment information, and according to Principles in Informed Consent signature Informed Consent Form.Patient is entering after group acceptance two cycle therapy, carry out therapeutic evaluation (for details, see the appendix 1) according to solid tumor the standard of curative effect evaluation RECIST 1.0 versions, criterion is as follows: alleviate (CR) completely, partial rcsponse (PR), stable disease (SD) and progression of disease (PD).Clinical Benefit is CR+PR+SD.Be defined as by first chemotherapy to the time that progression of disease or any former cause death occur for the first time without progression of disease lifetime (PFS).
2, the extracting of genomic dna
The peripheric venous blood 2ml that uses the heparin tube collection research object of EDTA anti-freezing before Chemotherapy in Patients, is stored in-20 DEG C of refrigerators, adopts saturated sodium-chloride method extracting genomic dna.
3, SNPs somatotype
To comprising
rICTORthe DNA fragmentation of gene rs6878291 pleomorphism site carries out specific amplification, and obtained DNA fragmentation is checked order, and obtains the genotype data in rs6878291 site
4, statistical study
Calculate odds ratio (Odds Ratio taking non-Conditional Logistic Regression Model as rs6878291 pleomorphism site, and 95% fiducial interval (95% confidence interval OR), 95% CI), assess the impact of this loci polymorphism on lung cancer platiniferous chemotherapeutic efficacy with this.
Calculate risk than (Hazard Ratio taking Cox proportional hazards regression models as rs6878291 pleomorphism site, and 95% fiducial interval (95% confidence interval HR), 95% CI), assess the impact without the progression of disease time on lung cancer platiniferous chemotherapy of this loci polymorphism with this.
5, interpretation of result
rICTORwhen gene rs6878291 loci gene type is A/A or A/G, its clinical Benefit rate is higher, can reach 82.1%, and longer lifetime without progression of disease, and median survival interval is 9.4 months; When genotype is G/G, clinical Benefit rate significantly reduces, and is only 69.6%, and drops to 6.5 months without progression of disease median survival interval lifetime.Visible, the clinical Benefit rate of the genotypic NSCLC patient's platiniferous of this site G/G chemotherapy and without progression of disease significantly decline lifetime (seeing annex 2 and annex 3)
the use of embodiment 2. detection kit
1, the extraction of genomic dna
Adopt saturated sodium-chloride method extracting genomic dna
2, molecule marker somatotype
Use detection kit, the DNA fragmentation that first specific amplification comprises RICTOR gene rs6878291 pleomorphism site, primer sequence is as follows:
Forward primer: 5 '-CCCATCACTCTTACCTAAACTCTC-3 ' (SEQ ID NO.1)
Reverse primer: 5 '-GACTGTGGTAGGCATTTGAAGATA-3 ' (SEQ ID NO.1)
Reaction system (20ul) comprises: 10 × Taq damping fluid, 2 μ l, 2mM dNTP mixed solution 2 μ l, 1U Taq enzyme 1 μ l, sample DNA (about 50ng) 1 μ l and 5 μ M PCR primer 1 μ l.This amplified reaction carries out on pcr amplification instrument, and reaction conditions is: 95 DEG C 5 minutes; Carry out 30 circulations 95 DEG C 30 seconds, 55 DEG C 30 seconds, 72 DEG C 30 seconds; 72 DEG C 7 minutes; 4 DEG C of preservations.
Then the DNA fragmentation obtaining is checked order.Concrete steps are as follows:
1) PCR product purification.In 10 μ lPCR products, add 37 DEG C of 1U shrimp alkaline phosphotase and 1U exonucleases temperature to bathe 1 hour, then 75 DEG C of deactivations 15 minutes.
2) sequencing reaction.(10 μ l) comprise reaction system: BigDye v3.1 sequencing reagent and supporting damping fluid thereof (Applied Biosystems), 1 μ l PCR forward primer (3.2 μ M) and 3 μ l purified products.This sequencing reaction carries out on pcr amplification instrument, and reaction conditions is: 96 DEG C 10 seconds; Operation 25 circulations 96 DEG C 10 seconds, 50 DEG C 5 seconds, 60 DEG C 4 minutes; 60 DEG C 4 minutes; 4 DEG C of preservations.
3) sex change of sequencing reaction product, precipitation.Add 1 μ l 120mM ethylenediamine tetraacetic acid (EDTA), then collect product with dehydrated alcohol, with 75% ethanol wash products 2 times, finally add 10 μ l 95% formamide solns, to be detected.
4) ABI3130XL sequenator sample detection.
3, gene type assay
For the original gene type data of above-mentioned acquisition, use Bioedit software to carry out interpretation and analysis.
embodiment 3. carries out the service of predication of chemotherapy effect to advanced NSCLC patient
1, the extraction of genomic dna
Collect detected person's peripheral blood sample, adopt saturated sodium-chloride method extracting genomic dna.
2, genotype tests
Use test kit provided by the invention, somatotype is carried out in the RICTOR gene rs6878291 site of detected person's genomic dna.
3, the forecast analysis of individual platiniferous chemotherapeutic efficacy
By the analysis to detected person rs6878291 loci gene type, provide the analysis and assessment report of individual platinum-based chemotherapy curative effect: if genotype is A/A or A/G, clinical Benefit rate is 82.1%, is 9.4 months without progression of disease median survival interval; If genotype is G/G, clinical Benefit rate is 69.6%, is 6.5 months without progression of disease median survival interval.This result can be for doctor with reference to adjusting application method and dosage.
annex 1: curative effect evaluation standard (RESICT):
Measure the major diameter of all target focuses, and calculate the major diameter sum of all target focuses, compared with the major diameter sum of baseline state, tumour Objective evaluation standard is as follows: alleviate (CR)---all target foci disappearances completely; The major diameter sum of partial rcsponse (PR)---target focus and baseline epidemic situation comparison, at least reduce 30%; The major diameter sum comparison of minimum target focus that the major diameter sum of lesion growth (PD)---target focus is recorded to after starting with treatment, increases by 20%, or occurs one or more new focus; Pathology is stablized (SD)---between surplus partial rcsponse and progression of disease.
annex 2: non-Conditional Logistic Regression Model is analyzed the dependency of rs6878291 loci gene type and NSCLC patient's platinum-based chemotherapy clinical Benefit rate
| Gene | SNP | Genotype | Efficiently individual quantity/total number of persons | % | OR | 95%CI | P |
| RICTOR | rs6878291 | AA+AG | 689/839 | 82.1 | 1 | ||
| GG | 94/135 | 69.6 | 2.037 | 1.35-3.08 | 0.001 |
annex 3:Cox proportional hazards regression models analysis rs6878291 loci gene type and NSCLC patient's platinum-based chemotherapy are without the progression of disease dependency of lifetime
。
SEQUENCE LISTING
<110> Fudan University
<120> prediction advanced Non-small cell lung platiniferous chemotherapeutic efficacy and the molecule marker of lifetime and test kit
<130> 001
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 24
<212> DNA
<213>
<400> 1
cccatcactc ttacctaaac tctc 24
<210> 2
<211> 24
<212> DNA
<213>
<400> 2
gactgtggta ggcatttgaa gata 24
Claims (3)
1. the molecule marker that can predict advanced Non-small cell lung platiniferous chemotherapeutic efficacy and lifetime, is characterized in that comprising relevant to curative effect after advanced Non-small cell lung platiniferous chemotherapy and lifetime
rICTORgene, and mononucleotide polymorphism site rs6878291 on this gene.
2. predict advanced Non-small cell lung platiniferous chemotherapy clinical Benefit rate and without the progression of disease test kit of lifetime, it is characterized in that this test kit comprises:
(1) detect
rICTORthe genotypic Auele Specific Primer pair of rs6878291 pleomorphism site on gene;
(2) polymerase chain reaction and sequencing reaction assembly, comprise: 1U Taq enzyme 100 μ l and 10 × Taq damping fluid 1mL, 2mM dNTP mixed solution 1ml, 1U shrimp alkaline phosphotase 100 μ l, 1U exonuclease 100 μ l, BigDye v3.1 reagent and supporting damping fluid 1ml thereof, 120mM ethylenediamine tetraacetic acid (EDTA) 1ml, 95% methane amide 1ml, dehydrated alcohol and ultrapure water.
3. test kit according to claim 2, is characterized in that described Auele Specific Primer is to being: forward primer is shown in SEQ ID NO.1, and reverse primer is shown in SEQ ID NO.2.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2016106643A1 (en) * | 2014-12-31 | 2016-07-07 | 深圳华大基因股份有限公司 | Primers for detecting related gene mutations of non-small-cell lung cancer medications and detection method |
| CN112121170A (en) * | 2020-09-16 | 2020-12-25 | 合肥金域医学检验实验室有限公司 | Lung cancer targeted drug and chemotherapy drug genome and application thereof in clinical drug treatment of lung cancer |
Citations (1)
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| CN102286615A (en) * | 2011-07-06 | 2011-12-21 | 复旦大学 | Molecular marker for predicting curative effect of platinum chemo-treatment on later non-small cell lung cancer and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286615A (en) * | 2011-07-06 | 2011-12-21 | 复旦大学 | Molecular marker for predicting curative effect of platinum chemo-treatment on later non-small cell lung cancer and application thereof |
Non-Patent Citations (2)
| Title |
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| 王诗铭等: "mTOR通路关键基因rictor基因多态性与晚期非小细胞肺癌含铂化疗临床效果的关联分析", 《中国遗传学会第九次全国会员代表大会暨学术研讨会论文摘要汇编（2009-2013）》 * |
| 王诗铭等: "mTOR通路关键基因rictor基因多态性与晚期非小细胞肺癌含铂化疗临床效果的关联分析", 《中国遗传学会第九次全国会员代表大会暨学术研讨会论文摘要汇编（2009-2013）》, 18 September 2013 (2013-09-18), pages 169 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016106643A1 (en) * | 2014-12-31 | 2016-07-07 | 深圳华大基因股份有限公司 | Primers for detecting related gene mutations of non-small-cell lung cancer medications and detection method |
| CN112121170A (en) * | 2020-09-16 | 2020-12-25 | 合肥金域医学检验实验室有限公司 | Lung cancer targeted drug and chemotherapy drug genome and application thereof in clinical drug treatment of lung cancer |
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