CN102251030A - Kit for predetermining treatment effect of iressa on patient with non-small-cell lung cancer by using FAS-1377G/A - Google Patents

Abstract

The invention discloses a kit for predetermining treatment effect of iressa on patient with non-small-cell lung cancer by using FAS-1377G/A. The invention provides special primers which form a primer pair used for amplifying a segment containing FAS-1377G/A, in particular a primer pair formed by a DNA fragment represented by a sequence 1 and a DNA segment represented by a sequence 2. The use of the special primers to help to predict the treatment effect of iressa on non-small-cell lung cancer in the patient has the following advantages: 1, a detection sample may from nucleated cells of any normal or tumor tissue, so almost all patient can obtain a detection sample; 2, FAS-1377G/A belongs to hereditary variation, and the detection result is free from any influence of the sampling time and place; 3, the detection process flow is relative simple and convenient, the equipment requirement is low and the cost is low; and 4, the sensibility is much higher than that of genetic mutation, the detection result is more beneficial to the patient receiving the prediction, and thus, the kit can better meet current sanitary and economic requirements of China.

Description

Use the test kit of FAS-1377G/A prediction nonsmall-cell lung cancer patient Gefitinib curative effect

Technical field

The present invention relates to a kind of test kit of the FAS-1377G/A of application prediction nonsmall-cell lung cancer patient Gefitinib curative effect.

Background technology

Lung cancer is one of the most common, that mortality ratio is the highest malignant tumour, and its incidence and mortality is also increasing year by year.Wherein nonsmall-cell lung cancer (NSCLC) accounts for more than 80% of whole lung cancer, and most of patient has been late period when diagnosis.Chemotherapy once was the topmost treatment means of nonsmall-cell lung cancer in late period, even if but best chemotherapy regimen, at present also 50%, 1 year survival rate of less than only about 40% of the total effective rate of treatment.

The appearance of target therapeutic agent has changed the treatment present situation of nonsmall-cell lung cancer, has improved survival time of patients significantly.Gefitinib (Gefitinib wherein, ZD1839) be go on the market the earliest, the nonsmall-cell lung cancer target therapeutic agent the most widely of clinical application at present, belong to a kind of selectivity, reversibility EGFR (EGF-R ELISA) tyrosine kinase inhibitor, go on the market in the many countries that comprise China at present, commodity are called Iressa (Iressa).

Gefitinib is treated the objective efficient widely different of nonsmall-cell lung cancer, and bibliographical information does not wait from 8.9-69%.Wherein in the IDEAL1 clinical study, Japanese efficient be 27%, the American then only is 12%.And in EAP research, Chinesely efficiently be about 30%.Below all point out the clinical efficacy of Gefitinib treatment nonsmall-cell lung cancer to exist significant individual difference.Because patients with advanced NSCLC is very limited lifetime, therefore select the effective pharmacological agent of most probable particularly important as early as possible to prolonging patient's existence.Gefitinib is that the target therapeutic agent of representative is relatively expensive in addition, and price is tens of times of common chemotherapy, also requires the patient who selects most probable to benefit to accept targeted therapy from the angle of health economics.Therefore, seeking Gefitinib outcome prediction index, instruct the individuation targeted therapy, is the key clinical problem that needs to be resolved hurrily at present.

Be most commonly used at present predict that the biomarker of Gefitinib curative effect is the sudden change of EGFR gene, be mainly the base deletion (G2235-A2249 disappearance) of exons 19 and the point mutation (L858R) of exon 21.Biomarker retrospective analysis at the international multicenter III of ISEL phase clinical study is found, existing 16 routine NSCLC patients of EGFR transgenation to accept efficient after the Gefitinib treatment is 37.5%, and the efficient of 116 routine patient's Gefitinib of EGFR gene wild-type only is 2.6%.But the EGFR transgenation is as the biomarker of Gefitinib outcome prediction, have certain deficiency: 1. Tu Bian detection needs tumor tissues, and most of lung cancer has been late period when initial diagnosis, lose the chance that operation obtains tumor specimen, even if small part patient might biopsy obtain sample, also can limit because of recall rate finite sum biopsy specimen amount, the detection that is difficult to suddenly change can be satisfied sudden change at present clinically and detecting the patient's ratio that requires and be less than for 20% (being about 17% in the ISEL research); 2. transgenation is subjected to the influence of tumour heterogeneity, and same patient is at the tumor specimen of different sites or the acquisition of disease different steps, and its sudden change detected result may there are differences, and therefore detected the possibility of result can not be represented whole body and omnidistance tumour characteristics; 3. the flow process complexity that detects of sudden change, the requirement height of plant and instrument, common laboratory is difficult to carry out, and it is also high to detect cost simultaneously, has influenced its clinical application widely; 4. suddenly change as the outcome prediction index, its susceptibility not high (in the ISEL research is 68.8%), and at the expensive like this medicine of Gefitinib, the clinical guidance meaning that susceptibility is high is bigger.

Based on the weak point of above EGFR transgenation, the clinical demand of developing better outcome prediction biomarker and detection method thereof seems more urgent.

Summary of the invention

The test kit that the purpose of this invention is to provide a kind of FAS-1377G/A of application prediction nonsmall-cell lung cancer patient Gefitinib curative effect.

The invention provides the primer special of auxiliary prediction nonsmall-cell lung cancer patient Gefitinib curative effect, is that pcr amplification comprises that the sequence 3 of sequence table is right from the used primer of the dna fragmentation of 5 ' terminal the 501st deoxyribonucleotide (FAS-1377G/A).It is right that described primer special can be the used primer of the dna fragmentation of Nucleotide shown in the sequence 3 that pcr amplification comprises sequence table.Described primer special specifically can be right for the primer of being made up of dna fragmentation shown in the sequence 2 of dna fragmentation shown in the sequence 1 of sequence table and sequence table.

The present invention also protects the application of described primer special in the test kit of the auxiliary prediction of preparation nonsmall-cell lung cancer patient Gefitinib curative effect.

The present invention also protects described primer special and the application of restriction enzyme BstU I in the test kit of the auxiliary prediction of preparation nonsmall-cell lung cancer patient Gefitinib curative effect.

The present invention also protects a kind of test kit of auxiliary prediction nonsmall-cell lung cancer patient Gefitinib curative effect, comprises described primer special.Described test kit also can comprise restriction enzyme BstU I.

Genomic dna with nonsmall-cell lung cancer patient to be measured is a template, carries out pcr amplification with described primer special, cuts the product of described pcr amplification with restriction enzyme BstU I enzyme, obtains enzyme and cuts product; If enzyme is cut the fragment that product is 104bp and 18bp, described patient's genotype is GG; If enzyme is cut the fragment that product is 122bp, described patient's genotype is AA; If enzyme is cut the fragment that product is 122bp, 104bp and 18bp, described patient's genotype is GA.If patient to be measured is non-AA genotype (GG genotype or a GA genotype), patient to be measured treats effective patient for candidate's application Gefitinib; If patient to be measured is the AA genotype, the patient that patient to be measured fails to respond to any medical treatment for candidate's application Gefitinib.

The contriver discovers that FAS gene mononucleotide polymorphic (FAS-1377G/A) is a kind of Biological indicators of measurable Gefitinib treatment nonsmall-cell lung cancer curative effect.FAS gene (TNF receptor superfamily, member 6) belongs to the TNF receptor family, is called APT1, CD95, FAS1, APO-1, FASTM, ALPS1A, TNFRSF6 sometimes again.The sequence number of FAS gene in NCBI GENBANK is NG_009089.2 (range:5001..30255).Mononucleotide polymorphic FAS-1377G/A is positioned at FAS upstream region of gene (promoter region), sees the sequence 3 of sequence table.The rs of FAS-1377G/A number is rs2234767.

Detect as Gefitinib outcome prediction index with the FAS-1377G/A gene polymorphic, can overcome the part shortcoming of existing prediction index (EGFR transgenation) to a certain extent: 1. the sample that detects of FAS-1377G/A can be derived from the karyocyte of any normal or tumor tissues, the easiest method is that the 2ml peripheral blood gets final product, and therefore nearly all patient all can obtain to detect sample; 2. FAS-1377G/A belongs to heritable variation, and detected result is not subjected to the influence of tumour heterogeneity, whenever obtains sample in life the patient, perhaps takes from any position sample, and its detected result should be consistent; 3. the FAS-1377G/A testing process is easy relatively, equipment requirements is low, with low cost, help clinical expansion; 4. FAS-1377G/A is as the outcome prediction index, and its susceptibility is significantly higher than transgenation, and detected result is higher to the certainty that the prediction patient benefits, and therefore is more suitable for the current health economics requirement of China.

The contrast of EGFR transgenation prediction curative effect and FAS-1377G/A prediction curative effect sees Table 1.

The contrast of table 1EGFR transgenation prediction curative effect and FAS-1377G/A prediction curative effect

	The EGFR transgenation	FAS-1377G/A
			Detect sample	Tumor tissues	Hemocyte, healthy tissues, tumor tissues etc. all can
Can obtain to detect patient's ratio of sample	?＜20％	100％
			The consistence that the different sites sample detects	May be inconsistent	Consistent
The consistence that the various disease stage is detected	May be inconsistent	Consistent
			Detection needs the time	3-5 days	3-5 days
Testing cost	About 2000 yuan/example	About 400 yuan/example
			Detection method	Dna sequencing or ARMS method	Restriction fragment length polymorphic (PCR-RFLP)
The susceptibility of prediction curative effect	?66.7％	90.7％

Description of drawings

Fig. 1 is the sample restriction enzyme digestion and electrophoresis figure of part.

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is to buy from routine biochemistry reagent shop and obtains.

Embodiment, according to the auxiliary prediction of FAS-1377G/A Gefitinib curative effect

One, the preparation special primer is right

Upstream primer: 5 '-TGTGTGCACAAGGCTGGC GC-3 ' (sequence 1 of sequence table is introduced a base mismatch A → G, as follows line);

Downstream primer: 5 '-TGCATCTGTCACTGCACTTACCACCA-3 ' (sequence 2 of sequence table).

Special primer is to being made up of the downstream primer shown in the sequence 2 of upstream primer shown in the sequence 1 of sequence table and sequence table.

Two, the special primer of applying step one is to detecting patient's FAS-1377G/A

87 patients (the NSCLC patient of informed consent) carry out following detection respectively:

1, gathers patient's anti-freezing peripheric venous blood 2ml, extract genomic dna.

2, the genomic dna that extracts with step 1 is a template, to carrying out pcr amplification, obtains pcr amplification product (122bp) with the special primer of step 1 preparation.

PCR reaction system (25 μ l): contain 0.1 μ g genomic dna, 0.2mmol/L dNTP, 1.5mmol/L MgCl ₂, 0.5mol/L upstream primer, 0.5mol/L downstream primer, 1U Taq polysaccharase and 1 * reaction buffer.

PCR reaction conditions: 95 ℃ of pre-sex change 2min; 94 ℃ of sex change 30sec, 61 ℃ of renaturation 30sec, 72 ℃ are extended 30sec, carry out 35 circulations; 72 ℃ are extended 7min.

3, pcr amplification product and the restriction enzyme BstU I with step 2 is incubated overnight for 60 ℃ jointly, obtains enzyme and cuts product.

4, the enzyme of step 3 is cut product and carry out 3% agarose gel electrophoresis, with the systematic observation of GDS-8000 gel imaging analysis and write down the result.

87 patients' pcr amplification product shows three kinds of banding patterns, and partial results is seen Fig. 1.Swimming lane 1 to 3 among Fig. 1 has shown three kinds of banding patterns respectively.

5, each pcr amplification product with step 2 checks order, and with the electrophorogram result of step 4 contrast.

The result shows: enzyme is cut the banding pattern (104bp+18bp that product shows swimming lane 3; Do not have in the 18bp electrophorogram to show) pcr amplification product sequencing result unanimity, pcr amplification product has only a kind of, all shown in the sequence 3 of sequence table (is G from the 151st Nucleotide of 5 ' end) is with this patient's genotype called after GG; Enzyme is cut the pcr amplification product sequencing result unanimity that product shows the banding pattern (122bp) of swimming lane 2, and pcr amplification product has only a kind of, and all shown in the sequence 3 of sequence table (is A from 5 ' terminal the 151st Nucleotide) is with this patient's genotype called after AA; Enzyme is cut the banding pattern (122bp+104bp+18bp that product shows swimming lane 1; Do not have in the 18bp electrophorogram to show) pcr amplification product sequencing result unanimity, pcr amplification product has two kinds, all shown in the sequence 3 of sequence table, a kind of pcr amplification product is G from 5 ' terminal the 151st Nucleotide, another kind of pcr amplification product is A from 5 ' terminal the 151st Nucleotide, with this patient's genotype called after GA.

Based on FAS-1377G/A, 16 patients are the AA genotype, and 71 patients are non-AA genotype (being GG genotype or GA genotype).

Three, the patient uses the curative effect of Gefitinib

87 routine patients are carried out with Gefitinib treatment, and therapeutic modality is: 250mg, and oral, once a day, until progression of disease or the toxicity that can not tolerate occurs.

Adopt RECIST standard (Therasse P, Arbuck SG, Eisenhauer EA, et al.New guidelines to evaluate the response to treatment in solid tumors.J Natl Cancer Inst 2000; 92:205-16.), the curative effect of periodic evaluation Gefitinib: the tumour completely dissolve is for alleviating (CR) fully, the major diameter sum of tumour is dwindled and is surpassed 30% and be defined as part and alleviate (PR), the major diameter sum of tumour is dwindled less than 30% or is increased less than 20% and is defined as stable disease (SD), and the major diameter sum of tumour increases that to surpass 20% be progression of disease (PD); The patient who CR, PR, SD was surpassed 24 weeks is defined as treatment effectively, and the patient of 24 weeks of SD less than or PD is defined as and fails to respond to any medical treatment.54 routine Gefitinib treatment effectively (62.1%) among the 87 routine patients, 33 examples fail to respond to any medical treatment (37.9%).

Four, FAS-1377G/A and patient use the association analysis of the curative effect of Gefitinib

Step 2 is carried out association analysis based on the somatotype result of FAS-1377G/A and the therapeutic evaluation result of step 3, see Table 2.

Table 2 association analysis result

P value=0.005.

Based on the result of association analysis, can draw the result of following auxiliary prediction curative effect: if patient to be measured is non-AA genotype, patient to be measured treats effective patient for candidate's application Gefitinib; If patient to be measured is the AA genotype, the patient that patient to be measured fails to respond to any medical treatment for candidate's application Gefitinib.

The effective patient's number of the effective non-AA genotype patient number ÷ actual therapeutic of the susceptibility=treatment * 100%=49 ÷ 54 * 100%=907% of prediction curative effect.

Claims (7)

1. the primer special of auxiliary prediction nonsmall-cell lung cancer patient Gefitinib curative effect is that pcr amplification comprises that the sequence 3 of sequence table is right from the used primer of the dna fragmentation of 5 ' terminal the 501st deoxyribonucleotide.

2. the primer shown in claim 1 is right, it is characterized in that: described primer special is that pcr amplification comprises that the used primer of the dna fragmentation of Nucleotide shown in the sequence 3 of sequence table is right.

3. primer special as claimed in claim 2 is characterized in that: the primer that described primer special is made up of dna fragmentation shown in the sequence 2 of dna fragmentation shown in the sequence 1 of sequence table and sequence table is right.

4. the application of arbitrary described primer special in the test kit of the auxiliary prediction of preparation nonsmall-cell lung cancer patient Gefitinib curative effect in the claim 1 to 3.

5. arbitrary described primer special and the restriction enzyme BstU I application in the test kit of the auxiliary prediction of preparation nonsmall-cell lung cancer patient Gefitinib curative effect in the claim 1 to 3.

6. the test kit of auxiliary prediction nonsmall-cell lung cancer patient Gefitinib curative effect comprises arbitrary described primer special in the claim 1 to 3.

7. test kit as claimed in claim 6 is characterized in that: described test kit also comprises restriction enzyme BstU I.

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