CN102367474A - ALDH1A3 gene-related glioblastoma multiforme prognosis kit - Google Patents

Abstract

The invention relates to an ALDH1A3 gene-related glioblastoma multiforme prognosis kit, which comprises a kit of DNA extracted from a tumor tissue sample of a patient with primary glioblastoma multiforme, an operation specification of an optional extracted DNA kit, a kit for analyzing the methylation level of the gene ALDH1A3 in the extracted DNA and an operation specification of an optional kit for the methylation level of the gene ALDH1A3. The invention also provides a molecular marker the ALDH1A3 gene for the glioblastoma multiforme prognosis.

Description

Glioblastoma multiforme existence prognosis test kit with the ALDH1A3 gene-correlation

Technical field

The present invention relates to the medical diagnosis on disease field, particularly relate to and be used for glioblastoma multiforme existence prognosis test kit.

Background technology

Glioblastoma multiforme (glioblastoma multiforme GBM) is the most common primary brain tumors of being grown up, its existence prognosis extreme difference, and patient's median survival interval is about 12-14 month.Although operation, radiation and chemotherapy technology are constantly progressive, this index in the past in 20 years never be improved significantly [1,2].The research group of Esteller [3] has reported that at first the mgmt gene hyper-methylation is to the relation of alkylating agent treatment susceptibility and existence prognosis in the glioblastoma.The mgmt gene methylation state is first molecular marked compound that can be used for assessing the glioblastoma clinical prognosis, and mgmt gene is reticent, and (temozolomide, TMZ) curative effect is relevant with TM.If there is the mgmt gene hyper-methylation in GBM patient, accept alkylating agent TMZ chemotherapy after 2 years survival rates will significantly improve to 46% [4] from 26%.This landmark discovery is the most important progress that glioblastoma treatment field is obtained over nearly 35 years, makes people to the treatment theory of GBM huge transformation take place.

In tumour origin and progression, much more very epigenetics takes place in the dna encoding district of cancer related gene sees the expression silencing [5,6] that especially methylates and cause cancer suppressor gene unusually.In tumour cell cycle, invasion and attack, apoptosis or DNA repair process, the CpG island that is positioned at gene promoter area often methylates unusually and gene transcription reticent relevant [5,6].At present have been found that in GBM dna methylation can cause the expression silencing of several genes, the important cells function that relates to comprise the cell cycle (p16INK4a, p15INK4b), DNA repairs and genomic integrity (MGMT, MLH1), tumor suppression (RB1; VHL, EMP3, RASSF1A; BLU) and tumor invasion and apoptosis (DAPK, TIMP3, CDH1; PCDH-gamma-A11, TMS1/ASC, CASP8) or the like [7-13].

(aldehyde dehydrog enase 1 ALDH1) is one of acetaldehyde dehydrogenase family member to acetaldehyde dehydrogenase 1, and ALDH1A3 is the hypotype of ALDH1.ALDH1 is that oxidation of acetaldehyde is the cytosol enzyme of acetate in the catalysis cell, participates in the detoxification of liver, also is normal stem cell and tumor stem cell (tumorstem cell, TSC) the essential material of growth and differentiation.Research at present shows; ALDH1 can be used as one of the universal marker of TSC such as mammary cancer, SCCHN, lung cancer, colorectal cancer and glioblastoma multiforme [14-18]; It is one of focus of present CSC affinity tag research field, particularly as the affinity tag of breast carcinoma stem cell.In the mammary cancer clinical study, the high expression level of ALDH1 and the generation of mammary cancer, development, transfer and prognosis are closely related.Ginestier etc. confirm that first ALDH1 is a kind of novel breast cancer tumour stem cell markers, and are the independent hazard factors of patient with breast cancer's prognosis, and expressing with tumor grade, oestrogenic hormon or progestogen has certain dependency.Morimoto etc. [19] carry out retrospective study through immunohistochemistry technique to 203 routine patient with breast cancers; Find that also ALDH1 is the characteristic phenotype of mammary cancer aggressive biological behaviour; And have dependency between oestrogenic hormon, progestogen, HER2 and the Ki67, but the analysis of Cox proportional hazard model shows that ALDH1 is not an independent prognostic factor.

The present invention this report ALDH1A3 as in glioblastoma multiforme as the evaluation of new diagnosis and prognosis biomarker and potential therapeutical agent target, its application in the glioblastoma multiforme generation also is provided.

Summary of the invention

Therefore, the present invention relates to the discovery of the methylate expression level relevant with glioblastoma multiforme, and the discovery of the target of exploit person glioblastoma multiforme prognosis.

A kind of glioblastoma multiforme existence prognosis test kit that is used for is provided in an embodiment of the invention, and it is characterized in that said reagent comprises: the test kit of DNA and the test kit working instructions of randomly said extraction DNA are extracted in (1) from primary glioblastoma multiforme patient's neoplasmic tissue sample; (2) test kit of the methylation level of the Gene A LDH1A3 among the said extraction of the analysis DNA and the randomly working instructions of the test kit of the methylation level of said Gene A LDH1A3.

In yet another embodiment of the present invention, a kind of glioblastoma multiforme existence prognosis test kit that is used for is provided, wherein said test kit comprises the test kit that detects CpG site methylation level in the ALDH1A3 gene further.

In yet another embodiment of the present invention, the test kit that detects CpG site methylation level in the ALDH1A3 gene comprises at least one the test kit of methylation level that detects among ALDH1A3 gene CpG site cg19224278, cg23191950 or the cg26509022.Also more in embodiment, the test kit that detects CpG site methylation level in the ALDH1A3 gene comprises and detects among ALDH1A3 gene CpG site cg19224278, cg23191950 or the cg26509022 at least two methylation level.

In further embodiment of the present invention, the test kit of analyzing the methylation level of the Gene A LDH1A3 among the said extraction DNA is the DNA chip chip agent box that methylates.In further embodiment of the present invention, the test kit of CpG site methylation level is the tetra-sodium sequencing kit that methylates in the said detection ALDH1A3 gene.

In embodiment further of the present invention, detect the ALDH1A3 gene test and use PCR primer and sequencing primer to be: GGGTTTTGGGATGGAAG; Vitamin H-ACRTACCCTACTCTTAAATCCAA; And AGGGTTTAGGGGAGAT.

Description of drawings

Fig. 1 is the figure of the distribution situation of the full genomic methylation degree of primary GBM, and Figure 1A is a routine sample of randomly drawing; Figure 1B is the MV of all samples.

Fig. 2 is the figure of the characteristic dna methylation spectrum of the different existence of primary GBM prognosis grouping.

Fig. 3 is the cluster analysis figure of primary GBM according to the most significant 10 genes of methylation level difference.

Fig. 4 is that ALDH1A3 methylates and the survive figure of relation of prognosis of primary GBM patient, and Fig. 4 A is a dna methylation tetra-sodium sequencing result; Fig. 4 B is the rate analytic curve for survival.

Fig. 5 is that MGMT methylates and the survive figure of relation of prognosis of primary GBM patient, and Fig. 5 A is a dna methylation tetra-sodium sequencing result; Fig. 5 B is the rate analytic curve for survival.

Embodiment

Below in conjunction with embodiment the present invention is done further detailed description.

1. patient's data and sample are originated

Amount to 100 routine primary glioblastoma multiformes and go into this research of group, all from glioma therapeutic community of Beijing Tiantan Hospital.All patients undergo surgery year December in January, 2006 to 2008 excision, radiotherapy and alkylating agent chemotherapy, Pathological diagnoses are primary glioblastoma multiforme (according to WHO central nerve neuroma criteria for classification in 2007).

The male sex's 56 examples among all patients, women's 44 examples; Age 17-70 year, average (43.8 ± 14.5) year.According to patient total lifetime (OS), select to organize long lifetime 13 examples (OS＞18 month) at random and organize 20 examples (OS＜9 month) short lifetime and accomplish the complete genome DNA chip that methylates; All the other 67 routine patients are as the individual authentication group.Normal cerebral tissue's contrast sample 3 examples are taken from the Tiantan Hospital's cerebral trauma same period, brain cortex fistulization case.This research is through approval of the Medical Ethics council of hospital and signature patient Informed Consent Form.

2. sample extracting genome DNA

2.1 key instrument

2.2 main agents

Reagent name	PIN	Manufacturer
			QIAamp?DNA?Mini?Ki	#51304	Qiagen
RNase?At	#6053	Merck
			Absolute ethyl alcohol		Beijing chemical reagents corporation

2.3 experimental procedure

Experiment flow is following:

(1) all tumor specimens are inserted the liquid nitrogen freezing preservation immediately after operation, carry out sample frozen section and conventional H E dyeing simultaneously to judge the proportion of tumour cell, and the sample of selecting tumour cell to account for more than 80% carries out next step extraction genomic dna.

(2) tissue grinds: use the mortar of 180 ℃ of pyroprocessing 6h, add the liquid nitrogen precooling, cut-off footpath 0.5cm size tumor tissues is ground to Powdered rapidly, need be interrupted the adding liquid nitrogen for preventing that tissue from melting when grinding.

(3) get 25mg ground organize powder, add 180ul Buffer ATL, add the 20ul Proteinase K again, vibration mixing, 56 ℃ of incubation 3h.

(4) add 4ul RNase A (100mg/ml), incubated at room 2min behind the vibration mixing; Add 200ul Buffer AL, 70 ℃ of incubation 10min behind the vibration mixing; Add the 200ul absolute ethyl alcohol, mixing fully vibrates.

(5) all mixtures are added QIAamp Mini Filter column, the centrifugal 1min of 8000rmp; Add 500ul Buffer AW1, the centrifugal 1min of 8000rmp; Add 500ul Buffer AW2, the centrifugal 3min of 14000rmp; Change collection tube, add 200ul Buffer AE eluted dna, incubated at room 1min, the centrifugal 1min of 8000rmp.

(6) it is quantitative that application NanoDrop ND-1000 uv-spectrophotometric appearance carries out DNA, and detect the DNA integrity with 1% agarose gel electrophoresis, then total DNA sample is stored in-80 ℃.

3.Illumina dna methylation chip

3.1 key instrument

3.2 main agents

3.3 experimental procedure

(1) sulphite is modified the stage:

Concrete experiment flow is referring to Zymo EZ DNA Methylation Kit specification sheets.

1. get dna sample 500ng, add 5ul M-Dilution Buffer, the water of handling with DEPC is made into 50 μ l systems, and fully mixing is hatched 15min for 37 ℃ in the PCR appearance.

2. add 100ul CT Conversion Reagent, fully mixing; In the PCR appearance, carry out following circulation: 95 ℃ of 30sec+50 ℃ of 1h, repeat 16 circulations, finally remain on 4 ℃.

3. assemble filter post and collection tube that test kit provides, add 400ul M-Binding Buffer, add again and hatch the dna sample that finishes, abundant mixing, the centrifugal 30sec of 10000g abandons filtrating.

4. filter and add 100ul M-Wash Buffer, the centrifugal 30sec of 10000g in the post; Add 200ul M-Desolphonation Buffer, incubated at room 15-20min, the centrifugal 30sec of 10000g; Add 200ul M-Wash Buffer, the centrifugal 30sec of 10000g repeats once.

5. change collection tube, add 10ul M-Elution Buffer, the centrifugal 30sec eluted dna of 10000g.

(2) the DNA cloning stage (preparation MSA2 plate):

1. in the MSA2 plate, add 20ul MA1, add the dna sample after 4ul sulphite is modified again, add 4ul 0.1N NaOH, add 1600rpm vortex concussion 1min after the lid may enclose, the centrifugal 1min of 280g, room temperature is hatched 10min for 22 ℃.

2. in MSA2 plate sample, add 68ul MA2, add 75ul MSM, add the mixing at least 10 times of turning upside down after the lid may enclose, the centrifugal 1min of 280g.

3. the MSA2 plate is put into Illumina chip hybridization stove, hatch 22h for 37 ℃.

(3) from the disconnected DNA stage:

1. the MSA2 plate is taken out the centrifugal 1min of 50g from Illumina chip hybridization stove.

2. in MSA2 plate sample, add 50ul FMS, add 1600rpm vortex concussion 1min after the lid may enclose, the centrifugal 1min of 50g under the room temperature.

3. the MSA2 plate is put into the heating appearance, hatch 1h for 37 ℃.

(4) the deposition MSA2 stage:

1. in MSA2 plate sample, add 100ul PM1, add 1600rpm vortex concussion after the lid may enclose

2. the MSA2 plate is put into the heating appearance, hatch 5min for 37 ℃, the centrifugal 1min of 50g under the room temperature.

3. in MSA2 plate sample, add 300ul 100%2-propanol, add the mixing at least 10 times of turning upside down after the lid may enclose, hatch 30min in 4 ℃ of refrigerators.

4. MSA2 plate centrifugal 20min of 3000g under 4 ℃ of environment is placed upside down on filter paper after removing capping, lets suspension liquid flow out gently, and pats 1min gently wall built-up liquid is all flowed out.

5. the MSA2 plate is placed upside down on the top of the shelf 22 ℃ of room temperatures, dry 1h.

(5) suspend again the MSA2 stage:

1. in MSA2 plate sample, add 42ul RA1, the aluminium foil heat-sealing.

2. the MSA2 plate is put Illumina chip hybridization stove, hatch 1h for 48 ℃.

3. the MSA2 plate is taken out 1800rpm vortex concussion 1min, the centrifugal 1min of 280g from hybrid heater.

(6) the chip hybridization stage:

1. prepare Illumina chip hybridization box, add 200ul PB2 in the groove, it is for use to add lid may enclose.

2. the MSA2 plate is put into the heating appearance, hatch 20min for 95 ℃ and make the sample sex change, the centrifugal 1min of 280g.

3. on the chip appearance: in the MSA2 plate sampling this 12ul put dna methylation chip corresponding position, will go up excellent chip and put into the chip hybridization box, in the hybrid heater 48 ℃ hybridization 22h, shaking speed is 5.

(7) the chip wash-out stage:

1. get a glass elution ware and add 200ml WB1, get another glass elution ware and add 200ml PB1.

2. from hybrid heater, take out chip, throw off surperficial pad pasting; Put into WB1 wash-out ware, slowly soft wash-out 1min up and down; Put into PB11 wash-out ware again, slowly soft wash-out 1min up and down.

3. in BeadChip Alignment Fixture, add 150ml PB1, and in BeadChip Alignment Fixture, assemble Flow-Through Chamber.

4. the chip anchor is installed in BeadChip Alignment Fixture; Slowly put into chip on the anchor; Chip surface covers transparent pad, and Alignment Bar is installed again, and the flat that cleans up is placed on the chip; The corresponding chip of opening part coding region, and with the fixing Flow-Through Chamber of metal clip.

5. take off Flow-Through Chamber, with the two ends up and down that scissors is cut transparent pad, stage reagent can pass through smoothly so that next step dyes.

(8) the chip dyeing stage:

1. prepare Chamber Rack and Water Circulator temperature control equipment.

2. when Chamber Rack temperature reaches 44 ℃, Flow-Through Chamber is positioned in the Chamber Rack support.

3. add following reagent in the liquid bath successively to adding of Flow-Through Chamber: 150ul RA1, hatch 30sec, repeat 5 times; 450ul XC1 is hatched 10min; 450ulXC2 is hatched 10min; 200ul TEM is hatched 15min; 450ul 95% methane amide/1mMEDTA is hatched 1min, repeats 1 time; Continue to hatch 5min.

4. according to the temperature of the mark temperature of LTM adjustment Chamber Rack, if the unmarked temperature of LTM then adjust to 37 ℃; Add 450ul XC3, hatch 1min, repeat 1 time.

5. when Chamber Rack temperature reaches 37 ℃, add following reagent: 250ulLTM successively, hatch 10min; 450ul XC3 is hatched lmin, repeats 1 time, waits for 5min; 250ul ATM is hatched l0min; 450ul XC3 is hatched 1min, repeats 1 time, waits for 5min; 250ul LTM is hatched 10min; 450ul XC3 is hatched 1min, repeats 1 time, waits for 5min; 250ul ATM is hatched 10min; 450ul XC3 is hatched 1min, repeats 1 time, waits for 5min; 250ul LTM is hatched 10min; 450ul XC3 is hatched 1min, repeats 1 time, waits for 5min; Continue to hatch 5min.

6. take off Flow-Through Chambers immediately, take and take out chip apart, put into PB1 wash-out ware, slowly soft wash-out up and down 10 times soaks 5min; Put into XC4 wash-out ware again, slowly soft wash-out up and down 10 times soaks 5min.

7. at last in vacuum drier with the dry 50-55min of chip, pressure is 67kPa.

(9) chip scanning and data analysis:

Human Methylation27BeadChip can detect 27578 CpG sites simultaneously by means of Illumina Infinium technology platform, and CpG information has wherein contained＞14000 genes of in ncbi database, describing note.Utilize Illumina BeadArray Reader to carry out chip scanning, all scan images carry out data analysis with BeadStudio software.

The tetra-sodium order-checking 4.DNA methylate

4.1. key instrument

4.2 main agents

Reagent name	PIN	Manufacturer
			EpiTect?Bisulfite?Kit	#59104	Qiagen
PyroMark?Gold?Q96?Reagent	#972804	Qiagen
			Binding?Buffer	#979006	Qiagen
Denaturation?Solution	#979007	Qiagen
			Washing?Buffer?10×	#979008	Qiagen
Annealing?Buffer	#979009	Qiagen
			Taq?DNA?polymerase(Hot?Start)	DR007	TakaRa
dNTP	D4030	TakaRa
			streptavidin?sepharose?HP	#17-5113-01	GE?Healthcare

4.3 experimental procedure

(1) bisulf iotate-treated:

Adopt Qiagen EpiTect Bisulfite Kit to carry out bisulf iotate-treated, the initial amount of dna sample is 1 μ g, and concrete experiment flow is referring to EpiTect Bisulfite Kit Handbook specification sheets.

(2) design of primers:

Each gene test sees the following form with PCR primer and sequencing primer (being labeled as S).

(3) PCR reaction system: the PCR reaction system that each gene pairs is answered sees the following form.

PCR system (40ul)	MGMT	ALDHlA3
				10 * PCR damping fluid	4ul	4ul
?dNTP(2.5mM)	3.2ul	3.2ul
			Primers F (10uM)	0.5ul	0.8ul
Primer R (10uM)	0.5ul	0.8ul
			?Takara?hotstart?Taq	0.5ul(2.5U)	0.5ul(2.5U)
?H 2O	28.7ul	28.7ul
			The bisulf iotate-treated dna sample	2ul	2ul

(4) the tetra-sodium order-checking detects (Pyrosequencing):

1. before use, guarantee that all reagent all reach room temperature condition.

2. in PSQ 96 orifice plates, add the Annealing Buffer that 40 μ l contain 0.5 μ M sequencing primer in advance.

3. use Vertex mixing Sepharose Beads (streptavidin sepharose HP), the Sepharose Beads total amount (every sample 3 μ l) that needs use is transferred in the Eppendorf pipe; In Sepharose Beads, add Binding Buffer, make average each sample that the volume of 40 μ l arranged approximately, the mixture mixing.

4. above mixture is added in the PCR product (40 μ l reaction volume) every sample 40 μ l; With PCR product mixing 5min at normal temperatures, make Beads combine with vitamin H.

5. in Vacuum Prep Workstation, add 180ml high purity water, 70% alcohol, Washing Buffer and Denaturation Buffer in four sample panel successively; Open the pump of Vacuum Prep Workstation, Vacuum Prep Tool is cleaned 30sec in high purity water, then Vacuum Prep Tool is moved on in the PCR plate, grasp Sepharose Beads.

6. pick up the PCR plate, whether most of Beads has been attracted on the Vacuum Prep Tool in inspection; Vacuum Prep Tool is put into 70% ethanol 5sec, move on to 5sec among the Denatureation Buffer then, move on to again among the Washing Buffer and clean 5-10sec; Turn off pump.

7. Vacuum Prep Tool is put into the plate that contains sequencing primer, shake, discharge Sepharose Beads; Use high purity water to clean Vacuum Prep Tool.

PSQ 96 orifice plates that 8. will be placed with sample are heated to 80 ℃ of 2min, and cool to room temperature can carry out the tetra-sodium sequencing reaction again.

5. statistics and bioinformatic analysis

All dna methylation chip utilization Illumina BeadArray Reader scan, and all scan images carry out data analysis with Illumina BeadStudio software platform.The fluorescent signal value of each data point representative that methylates comes from (the M that methylates; Methylated) and non-methylating (U, allelotrope unmethylated) is after going background and standardization; Calculate two allelic fluorescent signal ratio: β=[Max (M according to following formula; 0)]/[Max (U, 0)+Max (M, 0)+100].The detection by quantitative value of the specific CpG of β value or MI value (methylation index) representative site methylation level, span is between 0 (not methylating fully) and 1.0 (exhaustive methylations).Bioinformatic analysis adopts R lingware (http://www.r-project.org).At first the MI value of CpG site disappearance being mended is 0, after the CpG site that filters MI value disappearance >=18 examples, adds up to 495 CpG site MI value disappearance >=18 examples among the 33 routine GBM; Research length is organized the difference of patient's dna methylation level lifetime; The limma software package carries out experience Bayes moderated t check in the employing R language; And, be judged to be difference with p＜0.05 and have statistical significance with Benjamini and Hochberg method correction p value.Statistical analysis mainly adopts SPSS13.0for Windows software.Respectively organize survival time of patients (total lifetime OS with get nowhere PFS lifetime) difference through Kaplan-Meier survival Analysis check, utilize single argument and multivariate COX regression analysis evaluation mark and/or clinical data the predictive value that methylates patient's prognosis lifetime.All methods of inspection are two-way test, are judged to be difference with p＜0.05 and have statistical significance.

6. result

(1) the full genomic methylation chip technology of the distribution situation utilization of the full genomic methylation degree of GBM; This research detects the methylation level (MI value) in 14456 genes, 27578 CpG sites in the 33 routine primary GBM samples altogether; Organize patient's 20 examples (OS＜9 month) comprising organizing patient's 13 examples (OS＞18 month) long lifetime lifetime with lacking, and select 3 routine normal cerebral tissues as contrast.All patients are operation for the first time, do not accept any treatment before the art, and postoperative all adopts the standard care scheme, i.e. 4-6 course of treatment of synchronous chemicotherapy of TMZ and TMZ NACT.We randomly draw the MI value in a sample and whole all CpG sites of sample analysis from the GBM sample, the distribution situation of the full genomic methylation degree of research GBM.As shown in Figure 1, for CpG site in 20006 CpG islands, CpG site MI value＞0.7 of about 74% site MI value＜0.3,16%; For CpG site outside 7574 CpG islands, site MI value＞0.7 of about 69% site MI value＜0.3,20%.The The above results prompting, the hypomethylation state that is in of most genes among the primary GBM only has very little a part of gene the hyper-methylation state to occur; CpG site in the CpG island is in non-methylation state usually, and the incidence that methylates in the CpG site outside the CpG island is higher than the CpG site in the CpG island.Thereby further confirm that primary GBM is the same with other many tumours, have the characteristic of genome hypomethylation and regionality (CpG island) hyper-methylation widely.

(2) this research of dna methylation spectrum of the different existence of GBM prognosis grouping is contrast with 3 routine normal cerebral tissues, has compared the different complete genome DNAs of surviving prognosis grouping of the primary GBM spectrum that methylates.33 all routine MethodsThe cases enrolled are through strict screening criteria, and all patients are operation for the first time, do not accept any treatment before the art, and postoperative all adopts the standard care scheme.Be divided into two groups total lifetime according to the patient: long lifetime group (long-term survival, LTS) 20 examples (OS＞18 month) and short lifetime of group (short-term survival, STS) 13 examples (OS＜9 month).Through the full genomic methylation level that relatively STS organizes and LTS organizes, we find that altogether the methylation level in 1421 CpG sites has significant difference (p＜0.05), and wherein the difference CpG site of p＜0.01 has 217, lays respectively at 210 corresponding genes.We utilize methylation differential gene (p＜0.01) that all cases are carried out cluster analysis then, and the result shows two inferior groups that can well primary GBM be divided into different existence prognosis, represent the different character property spectrum that methylates respectively, and are as shown in Figure 2.

We further select the most significant 10 the CpG sites of methylation level difference between STS group and the LTS group; The gene at place is respectively IL11, RRAD, MS4A6A, SNAPC2, ALDH1A3, ADCY1, MMS19L, NDUFB8, POMC, THSD4; Carry out cluster analysis according to these 10 genes then; The result shows and can the inferior group of difference existence prognosis of primary GBM be made a distinction basically, and is as shown in Figure 3.

(3) methylate lifetime of mark ALDH1A3 and MGMT between the predictive value primary GBM different prognosis grouping gene of methylation level significant difference have 210 (p＜0.01); We carry out functional annotation to all differences gene; And screening key gene wherein is as methylating mark; Utilize dna methylation tetra-sodium sequence measurement then, further check its of predictive value in individual authentication group 67 routine primary GBM patients lifetime.For the ALDH1A3 gene; Be one of the most significant 10 genes of difference between the different prognosis grouping; This gene is at 3 CpG site (cg19224278; There is significant difference in cg23191950 on methylation level cg26509022), the methylation level of LTS group ALDH1A3 gene significantly improves than the STS group.The individual authentication result shows, the methylation state of ALDH1A3 promoter region and the patient prognosis significant correlation of surviving, and as shown in Figure 4, total lifetime of the group that methylates, more non-methylating organized significant prolongation (p=0.028).

In addition, for mgmt gene, the chip that methylates can detect the CpG site of 26 correspondences simultaneously, but the methylation level there was no significant difference of this gene in the different prognosis grouping.Individual authentication is the result also show, in our this group case, the methylation state of MGMT promoter region and the patient prognosis of surviving does not have obvious dependency, and is as shown in Figure 5.

4 mark ALDH1A3 and MGMT the multiple regression analysis that methylate for the existence prognosis

In individual authentication group 67 routine primary GBM patients, we utilize the evaluation of COX multiple regression analysis to methylate KPS scoring before mark ALDH1A3 and MGMT and clinical data age, sex, extent of surgical resection, TMZ chemotherapy, the art to the survive predictive value of prognosis of patient.KPS marked and patient's total lifetime of significant correlation before single argument COX regression analysis showed ALDH1A3 methylation state, extent of surgical resection, TMZ chemotherapy and art; Multivariate COX regression analysis has further confirmed to methylate, and (HR 1.014 for mark ALDH1A3; 95%CI 0.965-1.065; P=0.002) be very important predictor lifetime of primary GBM, and its predictive value is independent of other correlative factors (table 1) such as the preceding KPS scoring of extent of surgical resection, TMZ chemotherapy and art.

The table 1 individual authentication group 67 routine primary GBM patient clinical datas and the COX multiple regression analysis of mark that methylate about total lifetime

These should be understood that specific method, scheme and material that the present invention of disclosure is not limited only to describe, because all can change.Will also be understood that terminology used here only is in order to describe the purpose of specific embodiment scheme, rather than be intended to limit scope of the present invention, scope of the present invention only is subject to appended claim.

Those skilled in the art also will recognize, perhaps can confirm to use to be no more than normal experiment, many Equivalents of described in this article concrete embodiment of the present invention.These Equivalents are intended to be included in the appended claim.

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Glioblastoma multiforme existence prognosis test kit .txt with the ALDH1A3 gene-correlation

Sequence table

< 110>Jiang Tao

< 120>with the glioblastoma multiforme of ALDH1A3 gene-correlation existence prognosis test kit

<130>2011

<140>201110244593.2

<141>2011-08-25

<160>6

<170>PatentIn?version?3.3

<210>1

<211>20

<212>DNA

< 213>artificial sequence

<400>1

gtttyggata?tgttgggata 20

<210>2

<211>20

<212>DNA

< 213>artificial sequence

<400>2

acccaaacac?tcaccaaatc 20

<210>3

<211>17

<212>DNA

< 213>artificial sequence

<400>3

ggatatgttg?ggatagt 17

<210>4

<211>17

<212>DNA

< 213>artificial sequence

<400>4

gggttttggg?atggaag 17

<210>5

<211>24

<212>DNA

< 213>artificial sequence

<400>5

acrtacccta?ctcttaaatc?caac 24

<210>6

<211>16

<212>DNA

< 213>artificial sequence

<400>6

agggtttagg?ggagat 16

Claims (8)

1. one kind is used for glioblastoma multiforme existence prognosis test kit, it is characterized in that said reagent comprises: the test kit of DNA and the test kit working instructions of randomly said extraction DNA are extracted in (1) from primary glioblastoma multiforme patient's neoplasmic tissue sample; (2) test kit of the methylation level of the Gene A LDH1A3 among the said extraction of the analysis DNA and the randomly working instructions of the test kit of the methylation level of said Gene A LDH1A3.

2. test kit according to claim 1, wherein said test kit also comprise the test kit that is positioned at the CpG island methylation level of gene promoter area in the detection ALDH1A3 gene.

3. test kit according to claim 2, wherein said test kit comprise at least one the test kit of methylation level that detects among ALDH1A3 gene CpG site cg19224278, cg23191950 or the cg26509022.

4. test kit according to claim 2, wherein said test kit comprise and detect among ALDH1A3 gene CpG site cg19224278, cg23191950 or the cg26509022 at least two methylation level.

5. test kit according to claim 1, the test kit of wherein analyzing the methylation level of the Gene A LDH1A3 among the said extraction DNA are the DNA chip chip agent boxes that methylates.

6. test kit according to claim 2, the test kit of CpG site methylation level is the tetra-sodium sequencing kit that methylates in the wherein said detection ALDH1A3 gene.

7. test kit according to claim 6 detects the ALDH1A3 gene test and uses PCR primer and sequencing primer to be: GGGTTTTGGGATGGAAG; Vitamin H-ACRTACCCTACTCTTAAATCCAA; And AGGGTTTAGGGGAGAT.

8. molecular marker that is used for glioblastoma multiforme existence prognosis, it is the ALDH1A3 gene.

Images