CN102367474B - 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 kit with ALDH1A3 gene-correlation

Technical field

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

Background technology

Glioblastoma multiforme (glioblastoma multiforme, GBM) is the most common primary brain tumors of being grown up, its poor prognosis of surviving, 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 first reported the relation of mgmt gene hyper-methylation to alkylating agent treatment susceptibility and existence prognosis in glioblastoma.Mgmt gene methylation state is first molecular marked compound that can be used for assessing glioblastoma clinical prognosis, and mgmt gene is reticent relevant with Temozolomide (temozolomide, TMZ) curative effect.If there is 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 occur.

In tumour origin and progression, it is extremely very common to there is epigenetics in the DNA encoding district of cancer related gene, especially methylates and causes the expression silencing [5,6] of cancer suppressor gene.In tumour cell cycle, invasion and attack, apoptosis or DNA repair process, be positioned at CpG island, gene promoter area aberrant methylation often relevant to the Transcriptional Silencing of gene [5,6].At present in GBM, have been found that DNA methylation can cause the expression silencing of several genes, the important cells function relating to comprises that cell cycle (p16INK4a, p15INK4b), DNA repair 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) etc. [7-13].

(aldehyde dehydrog enase 1, ALDH1) is one of acetaldehyde dehydrogenase family member to acetaldehyde dehydrogenase 1, and ALDH1A3 is a hypotype of ALDH1.ALDH1 is the cytosol enzyme that in catalysis cell, oxidation of acetaldehyde is acetic acid, participates in the detoxification of liver, is also the essential material of normal stem cell and tumor stem cell (tumorstem cell, TSC) growth and differentiation.Research at present shows, ALDH1 can be used as one of the universal marker of the TSC such as mammary cancer, squamous cell carcinoma of the head and neck, lung cancer, colorectal cancer and glioblastoma multiforme [14-18], it is one of focus of current CSC marker research field, particularly as the marker of breast carcinoma stem cell.In 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 ALDH1 is a kind of novel breast cancer tumour stem cell markers first, and are the independent hazard factors of patient with breast cancer's prognosis, express and have certain dependency with tumor grade, oestrogenic hormon or progestogen.Morimoto etc. [19] carry out retrospective study by immunohistochemistry technique to 203 routine patient with breast cancers, also find that ALDH1 is the feature phenotype of mammary cancer aggressive biological behaviour, and between oestrogenic hormon, progestogen, HER2 and Ki67, having dependency, is not prognostic factor independently but the analysis of Cox proportional hazard model shows ALDH1.

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 glioblastoma multiforme occurs is also provided.

Summary of the invention

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

Provide in an embodiment of the invention a kind of for glioblastoma multiforme existence prognosis kit, it is characterized in that described reagent comprises: (1) extracts the test kit of DNA and the test kit working instructions of the described DNA of extraction optionally from primary glioblastoma multiforme patient's neoplasmic tissue sample; (2) analyze test kit and the working instructions of the test kit of the methylation level of described Gene A LDH1A3 optionally of the methylation level of the Gene A LDH1A3 in described extraction DNA.

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

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

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

In further embodiment of the present invention, detection ALDH1A3 gene test with PCR primer and sequencing primer is: GGGTTTTGGGATGGAAG; Vitamin H-ACRTACCCTACTCTTAAATCCAA; And AGGGTTTAGGGGAGAT.

Accompanying drawing explanation

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 mean value 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 that primary GBM is according to the cluster analysis figure of the most significant 10 genes of methylation level difference.

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

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

Embodiment

Below in conjunction with embodiment, the present invention is described in further detail.

1. patient's data and Specimen origin

Amount to 100 routine primary glioblastoma multiformes and enter this research of group, all from Beijing Tiantan Hospital's Treatment for Glioma center.All patients year December in January, 2006 to 2008, undergo surgery excision, radiotherapy and alkylating agent chemotherapy, Pathological diagnoses is primary glioblastoma multiforme (according to WHO central nerve neuroma criteria for classification in 2007).

The male sex's 56 examples in all patients, women's 44 examples; Age 17-70 year, average (43.8 ± 14.5) year.According to patient's Overall survival (OS), random select to organize 13 examples (OS > 18 months) long lifetime and organize 20 examples (OS < 9 months) short lifetime to complete the complete genome DNA chip that methylates; All the other 67 routine patients are as 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.Patient's Informed Consent Form is ratified and signed to this research by the Medical Ethics council of hospital.

2. sample extracting genome DNA

2.1 key instrument

2.2 main agents

Reagent name	Production code member	Manufacturer
			QIAamp DNA Mini Ki	#51304	Qiagen
RNase At	#6053	Merck
			Dehydrated alcohol		Beijing chemical reagents corporation

2.3 experimental procedure

Experiment flow is as follows:

(1) all tumor specimens are inserted immediately liquid nitrogen freezing preservation after operation, carry out the proportion that sample frozen section and conventional H E dye to judge tumour cell simultaneously, 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 Liquid nitrogen precooler, cut-off footpath 0.5cm size tumor tissues is ground to rapidly Powdered, for preventing that tissue need be interrupted and add liquid nitrogen while melting grinding.

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

(4) add 4ul RNase A (100mg/ml), vibration mixes rear incubated at room 2min; Add 200ul Buffer AL, vibration mixes rear 70 ℃ of incubation 10min; Add 200ul dehydrated alcohol, fully vibration mixes.

(5) all mixtures are added to 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 instrument carries out DNA, and detect DNA integrity with 1% agarose gel electrophoresis, then total DNA sample is stored in to-80 ℃.

3.Illumina DNA methylation chip

3.1 key instrument

3.2 main agents

3.3 experimental procedure

(1) sulphite is modified the stage:

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

1. get DNA sample 500ng, add 5ul M-Dilution Buffer, with the water that DEPC processes, be made into 50 μ l systems, fully mix, in PCR instrument, hatch 15min for 37 ℃.

2. add 100ul CT Conversion Reagent, fully mix; In PCR instrument, 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, then add and hatch complete DNA sample, fully mix, the centrifugal 30sec of 10000g, abandons filtrate.

4. filter and in post, add 100ul M-Wash Buffer, the centrifugal 30sec of 10000g; 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 MSA2 plate, add 20ul MA1, then the DNA sample after adding 4ul sulphite to modify, add 4ul 0.1N NaOH, add a cover 1600rpm vortex concussion 1min after sealing, the centrifugal 1min of 280g, 22 ℃ of room temperatures are hatched 10min.

2. in MSA2 plate sample, add 68ul MA2, add 75ul MSM, add a cover after sealing and turn upside down and mix at least 10 times, the centrifugal 1min of 280g.

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

(3) from the disconnected DNA stage:

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

2. in MSA2 plate sample, add 50ul FMS, add a cover the rear 1600rpm vortex concussion of sealing 1min, the centrifugal 1min of 50g under room temperature.

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

(4) the precipitation MSA2 stage:

1. in MSA2 plate sample, add 100ul PM1, add a cover the rear 1600rpm vortex concussion of sealing

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

3. in MSA2 plate sample, add 300ul 100%2-propanol, add a cover after sealing and turn upside down and mix at least 10 times, in 4 ℃ of refrigerators, hatch 30min.

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

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

(5) the Eddy diffusion MSA2 stage:

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

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

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

(6) the chip hybridization stage:

1. prepare Illumina chip hybridization box, in groove, add 200ul PB2, add a cover sealing stand-by.

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

3. chip loading: in MSA2 plate, sample this 12ul and put DNA methylation chip corresponding position, upper excellent chip is put into chip hybridization box, 48 ℃ of hybridization 22h in hybrid heater, 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 film on surface; Put into WB1 wash-out ware, slowly soft upper and lower wash-out 1min; Put into again PB11 wash-out ware, slowly soft upper and lower wash-out 1min.

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

4. in BeadChip Alignment Fixture, chip fixing bracket is installed, chip is slowly put on anchor, chip surface covers transparent pad, Alignment Bar is installed again, the flat cleaning up is placed on chip, the corresponding chip of opening part coding region, and with the fixing Flow-Through Chamber of metal clip.

5. next step dyeing stage reagent takes off Flow-Through Chamber, with scissors, cuts the two ends up and down of transparent pad, so that can pass through smoothly.

(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 Chamber Rack support.

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

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

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

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

7. finally in vacuum drier, chip is dried to 50-55min, pressure is 67kPa.

(9) chip scanning and data analysis:

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

Methylate tetra-sodium order-checking of 4.DNA

4.1. key instrument

4.2 main agents

Reagent name	Production code member	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 specific experiment flow process 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
			Bisulf iotate-treated DNA sample	2ul	2ul

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

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

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

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

4. above mixture is added in PCR product (40 μ l reaction volume) to every sample 40 μ l; PCR product is mixed to 5min at normal temperatures, Beads is combined with vitamin H.

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

6. pick up PCR plate, check whether most of Beads has been attracted on Vacuum Prep Tool; Vacuum Prep Tool is put into 70% ethanol 5sec, then move on to 5sec in Denatureation Buffer, then move on in 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.

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

5. statistics and bioinformatic analysis

All DNA methylation chips use Illumina BeadArray Reader to 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 the non-(U that methylates, unmethylated) allelotrope, after going background and standardization, according to following formula, calculate two allelic fluorescent signal ratio: β=[Max (M, 0)]/[Max (U, 0)+Max (M, 0)+100].β value or MI value (methylation index) represent the detection by quantitative value of specific CpG site methylation level, and span is between 0 (not methylating completely) and 1.0 (exhaustive methylations).Bioinformatic analysis adopts R lingware (http://www.r-project.org).First the MI value complement of JiangCpG site disappearance is 0, after the CpG site that filters MI value disappearance >=18 examples, adds up to 495 GeCpG site MI value disappearance >=18 examples in 33 routine GBM; Research length is organized the difference of patient's DNA methylation level lifetime, adopt limma software package in R language to carry out Empirical Bayes moderated t check, and proofread and correct p value by Benjamini and Hochberg method, with p < 0.05, be judged to be difference and there is statistical significance.Statistical analysis mainly adopts SPSS13.0for Windows software.By Kaplan-Meier survival Analysis check, respectively organize patient's (Overall survival OS and Progression free survival phase PFS) difference lifetime, utilize single argument and multivariate COX regression analysis evaluation methylate mark and/or the predictive value of clinical data to the prognosis of survival of patients phase.All methods of inspection are two-way test, be judged to be difference have statistical significance with p < 0.05.

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 in 33 routine primary GBM samples the methylation level (MI value) in 14456 genes, 27578 GeCpG sites altogether, comprising organizing patient's 13 examples (OS > 18 months) and organize patient's 20 examples (OS < 9 months) short lifetime long lifetime, and select 3 routine normal cerebral tissues in contrast.All patients are for the first time operation, preoperatively do not accept any treatment, the postoperative standard care scheme that all adopts, i.e. 4-6 the course for the treatment of of TMZ Synchronous Radio chemotherapy and TMZ adjuvant chemotherapy.We randomly draw the MI value in a sample and whole all CpG of sample analysis site from GBM sample, the distribution situation of the full genomic methylation degree of research GBM.As shown in Figure 1, for the CpG site MI value > 0.7 of 20006 ，Yue74% site, NeiCpG site, GeCpG island MI value < 0.3,16%; For 7574 MI value < 0.3，20% site, ，Yue69% site, WaiCpG site, GeCpG island MI value > 0.7.The above results prompting, in primary GBM most genes in hypomethylation state, only have very little a part of gene to occur hyper-methylation state; NeiCpG site, CpG island conventionally the incidence that methylates in WaiCpG site, non-methylation state ，Er CpG island higher than NeiCpG site, CpG island.Thereby further confirm, primary GBM is the same with other many tumours, there is the feature of genome hypomethylation and regionality (CpG island) hyper-methylation widely.

(2) DNA methylations of the different existence of GBM prognosis grouping are composed this research to take 3 routine normal cerebral tissues are contrast, have compared the complete genome DNA methylome of the different prognosis grouping of surviving of primary GBM.33 all routine MethodsThe cases enrolled are through strict screening criteria, and all patients are operation for the first time, preoperative any treatment, the postoperative standard care scheme that all adopts do not accepted.According to patient's Overall survival, be divided into two groups: length group (long-term survival lifetime, LTS) 20 examples (OS > 18 months) and short group lifetime (short-term survival, STS) 13 examples (OS < 9 months).By the full genomic methylation level that relatively STS group and LTS organize, we find that the methylation level in 1421 GeCpG sites has significant difference (p < 0.05) altogether, wherein the difference CpG site of p < 0.01 has 217, lays respectively at 210 corresponding genes.Then we utilize methylation differential gene (p < 0.01) to carry out cluster analysis to all cases, result shows two subgroups that can well primary GBM be divided into different existence prognosis, represent respectively different characteristic methylomes, as shown in Figure 2.

We further select the most significant 10 GeCpG sites of methylation level difference between STS group and LTS group, the gene at place is respectively IL11, RRAD, MS4A6A, SNAPC2, ALDH1A3, ADCY1, MMS19L, NDUFB8, POMC, THSD4, then according to these 10 genes, carry out cluster analysis, result shows that the prognosis subgroup base region of the difference of primary GBM can being survived separate, as shown in Figure 3.

(3) methylate lifetime of mark ALDH1A3 and MGMT between predictive value primary GBM different prognosis grouping the gene of methylation level significant difference have 210 (p < 0.01), we carry out functional annotation to all differences gene, and screening key gene is wherein as the mark that methylates, then utilize DNA methylation tetra-sodium sequence measurement, further in the routine primary GBM patient of individual authentication group 67, check its of predictive value lifetime.For ALDH1A3 gene, one of the most significant 10 genes of difference between different prognosis grouping, this gene is at 3 GeCpG site (cg19224278, cg23191950, cg26509022) in methylation level, all have significant difference, the methylation level of LTS group ALDH1A3 gene significantly improves compared with STS group.Individual authentication result shows, the methylation state of ALDH1A3 promoter region and survival of patients prognosis significant correlation, as shown in Figure 4, the more non-group significant prolongation (p=0.028) that methylates of the Overall survival of the group that methylates.

In addition, for mgmt gene, the chip that methylates can detect 26 corresponding CpG sites simultaneously, but the methylation level there was no significant difference of this gene in different prognosis grouping.Individual authentication result also shows, in our this group case, the methylation state of MGMT promoter region and survival of patients prognosis are without obvious dependency, as shown in Figure 5.

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

In the routine primary GBM patient of individual authentication group 67, we utilize the evaluation of COX multiple regression analysis methylate mark ALDH1A3 and MGMT and clinical data age, sex, extent of surgical resection, TMZ chemotherapy, the predictive value of preoperative KPS scoring to survival of patients prognosis.Single argument COX regression analysis shows ALDH1A3 methylation state, extent of surgical resection, TMZ chemotherapy and preoperative KPS scoring and patient's Overall survival significant correlation, 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 extent of surgical resection, TMZ chemotherapy and preoperative KPS scoring.

The routine primary GBM patient clinical data of table 1 individual authentication group 67 and the COX multiple regression analysis of mark about Overall survival that methylate

The present invention who should be understood that disclosure is not limited only to specific method, scheme and the material described, because these all can change.Will also be understood that terminology used here is only used to describe the object of specific embodiment scheme, rather than be intended to limit the scope of the invention, scope of the present invention is only limited to appended claim.

Those skilled in the art also will recognize, or can confirm to use and be no more than normal experiment, in this article many Equivalents of described specific embodiment of the present invention.These Equivalents are intended to comprise in the appended claims.

Reference:

[1]Stupp R，Dietrich PY，Ostermann Kraljevic S，Pica A，Maillard I，Maeder P，et al.Promising survival for patients with newly diagnosed glioblastoma multiforme treated with concomitant radiation plus temozolomide followed by adjuvant temozolomide.J Clin Oncol 2002；20：1375-82.

[2]Walker MD，Green SB，Byar DP，Alexander E Jr，Batzdorf U，Brooks WH，et al.Randomized comparisons of radiotherapy and nitrosoureas for the treatment of malignant glioma after surgery.N Engl J Med 1980；303：1323-9.

[3]Esteller M，Garcia-Foncillas J，Andion E，Goodman SN，Hidalgo OF，Vanaclocha V，et al.Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents.N Engl J Med 2000；343：1350-4.

[4]Stupp R，Mason WP，van den Bent MJ，Weller M，Fisher B，Taphoorn MJ，et al.Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma.N Engl J Med 2005；352：987-96.

[5]Esteller M.CpG island hypermethylation and tumor suppressor genes：a booming present，a brighter future.Oncogene 2002；21：5427-40.

[6]Jones PA，Baylin SB.The fundamental role of epigenetic events in cancer.Nat Rev Genet 2002；3：415-28.

[7]Baylin SB，Herman JG，Graff JR，Vertino PM，Issa JP.Alterations in DNA methylation：a fundamental aspect ofneoplasia.Adv Cancer Res 1998；72：141-96.

[8]Esteller M，Corn PG，Baylin SB，Herman JG.A gene hypermethylation profile of human cancer.Cancer Res 2001；61：3225-9.

[9]Horiguchi K，Tomizawa Y，Tosaka M，Ishiuchi S，Kurihara H，Mori M，Saito N.Epigenetic inactivation of RASSF1A candidate tumor suppressor gene at 3p21.3 in brain tumors.Oncogene 2003；22：7862-5.

[10]Nakamura M，Watanabe T，Klangby U，Asker C，Wiman K，Yonekawa Y，et al.p14ARF deletion and methylation in genetic pathways to glioblastomas.Brain Pathol2001；11：159-68.

[11]Stone AR，Bobo W，Brat DJ，Devi NS，Van Meir EG，Vertino PM.Aberrant methylation and downregulation of TMS1/ASC in human glioblastoma.Am J Pathol2004；165：1151-61.

[12]Waha A，Guntner S，Huang TH，Yan PS，Arslan B，Pietsch T，Wiestler OD.Epigenetic silencing of the protocadherin family member PCDH-gamma-A11 in astrocytomas.Neoplasia 2005；7：193-9.

[13]Martinez R，Setien F，Voelter C，Casado S，Quesada MP，Schackert G，Esteller M.CpG island promoter hypermethylation of the pro-apoptotic gene caspase-8 is a common hallmark of relapsed glioblastoma multiforme.Carcinogenesis 2007；28：1264-8.

[14]Ginestier，Hur MH，Charafe-Jauffret E，et al.ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome.Cell Stem Cell 2007；1：555-67.

[15]Chen YC，Chen YW，Hsu HS，et al.Aldehyde dehydrogenase 1 is a putative marker for cancer stem cells in head and neck squamous cancer.Biochem Biophys Res Commun 2009；385：307-13.

[16]Jiang F，Qiu Q，Khanna A，et al.Aldehyde dehydrogenase 1 is a tumor stem cell-associated marker in lung cancer.Mol Cancer Res 2009；7：330-8.

[17]Huang EH，Hynes MJ，Zhang T，et al.Aldehyde dehydrogenase 1 is a marker for normal and malignant human colonic stem cells(SC)and tracks SC overpopulation during colon tumorigenesis.Cancer Res 2009；69：3382-9.

[18]Rasper M，

A，Piontek G，Teufel J，Brockhoff G，Ringel F，Heindl S，Zimmer C，Schlegel J.Aldehyde dehydrogenase 1 positive glioblastoma cells show brain tumor stem cell capacity.Neuro Oncol.2010；12：1024-33.

[19]Morimoto K，Kim SJ，Tanei T，et al.Stem cell marker aldehyde dehydrogenase 1positive breast cancers are characterized by negative estrogen receptor，positive human epidermal growth factor receptor type 2，and high Ki67 expression.Cancer Sci 2009；100：1062-8

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

Sequence table

<110>Jiang Tao

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

<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 (7)

1. test kit, for the preparation of the application in the medicine of glioblastoma multiforme existence prognosis, is characterized in that described test kit comprises: (1) extracts the test kit of DNA and the test kit working instructions of the described DNA of extraction optionally from primary glioblastoma multiforme patient's neoplasmic tissue sample; (2) analyze test kit and the working instructions of the test kit of the methylation level of described Gene A LDH1A3 optionally of the methylation level of the Gene A LDH1A3 in described extraction DNA.

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

3. application according to claim 2, wherein said test kit comprises at least one the test kit of methylation level detecting in ALDH1A3 gene CpG site cg19224278, cg23191950 or cg26509022.

4. application according to claim 2, wherein said test kit comprises the methylation level that detects in ALDH1A3 gene CpG site cg19224278, cg23191950 or cg26509022 at least two.

5. application according to claim 1, the test kit of wherein analyzing the methylation level of the Gene A LDH1A3 in described extraction DNA is the DNA chip chip agent box that methylates.

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

7. application according to claim 6, detection ALDH1A3 gene test with PCR primer and sequencing primer is: GGGTTTTGGGATGGAAG; Vitamin H-ACRTACCCTACTCTTAAATCCAA; And AGGGTTTAGGGGAGAT.

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