CN102199664B - Test kit for esophageal cancer treatment selection and/or prognostic evaluation - Google Patents
Test kit for esophageal cancer treatment selection and/or prognostic evaluation Download PDFInfo
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Abstract
The invention discloses a test kit for esophageal cancer treatment selection and/or prognostic evaluation. The test kit is used for testing one or more reagents represented by the following genetic group; and the genetic group is composed of two or more genes from the following genes: BMP-8, CD133, CD29, Car1, CL5, Endomus2, Nectin3, Nectin1, Kai1, PPARGCA, WAVE-1, WAVE-3, BMP9, CMG1, 1L22R and VEGF-D. By detecting and testing the expressions of a plurality of genes, the effectiveness of clinical treatment is evaluated, so as to determine who has poor prognosis and who is not suitable for using the regularly treatment. The test kit has the advantages of simple test method, good accuracy, low cost and easy to be accepted by patients.
Description
Technical field
The present invention relates to a kind of detection kit, specifically a kind of detection kit for esophageal carcinoma therapy Scheme Choice and/or prognosis evaluation.
Background technology
The esophageal carcinoma is to occur in one of the highest tumour of upper gi tract grade malignancy.Its epithelium that comes from oesophagus is broadly divided into gland cancer and squamous cell carcinoma.Think at present the generation of the esophageal carcinoma may be with smoking, be addicted to drink, Gastroesophageal reflux disease, infection, food habits (maror of eating as usual), chewing tobacco, age and other X factor be relevant.
Although the esophageal carcinoma in the world overall incidence is lower, be a kind of malignant tumour occurred frequently in China, Iceland, Ireland, Japan, Korea S and Britain.Esophageal carcinoma incidence is lower in the U.S., and only there were new cases more than 15,000 in the U.S. in 2007, and in Britain nearly 8,000 new cases was arranged, and this is so that the esophageal carcinoma becomes the 9th malignant tumour occurred frequently of Britain.And there are 500,000 esophageal carcinoma new cases in the whole world every year approximately, especially at nearest 20 years, the esophageal carcinoma present lasting ascendant trend.The esophageal carcinoma incidence of China occupies and beats the world, and secondly is some African country and Japan.
The esophageal carcinoma is the very poor malignant tumour of a kind of prognosis, and often transfer has appearred in tumour when patient's first visit, and the result causes the curative effect of patient with esophageal carcinoma very poor, and five year survival rate is less than 5%.And in Britain, annual the depositing of patient with esophageal carcinoma is about 30%, and five year survival rate is 8%.Another reason that causes esophageal carcinoma poor prognosis is to have serious medical care problem so that the patient can not undergo surgery or assisting therapy.Therefore cause Britain in 2007 that nearly 7600 patient with esophageal carcinoma death are arranged, and there is the death above 400,000 in the whole world.
Do not have at present a kind of effective means that the prognosis of patient with esophageal carcinoma is detected, and can assist the doctor to select reasonably targetedly treatment plan.
Summary of the invention
The object of the invention is to propose a kind of detection kit that can be used for accurately and rapidly esophageal carcinoma therapy Scheme Choice and/or prognosis evaluation.
Another object of the present invention is to propose a kind of gene chip for esophageal carcinoma therapy Scheme Choice and/or prognosis evaluation.
The 3rd purpose of the present invention is to propose the new purposes in preparation esophageal carcinoma therapy Scheme Choice and/or prognosis evaluation detection kit of a gene group.
Invention thinking of the present invention is: the contriver identifies one group of gene, and the variation of its expression level has significant clinical meaning.Finally identify 16 genes (comprising two house-keeping genes) and prepare detection kit through a large amount of statistical analysis of later stage again, the expression level of these genes is carried out can assessing patient's prognosis well after the analysis-by-synthesis.Use this test kit can adversary's postoperative or the biopsy patient carry out fast clinical detection, thereby help the clinicist that patient's prognosis is well assessed.In addition, which patient is this detected result can also identify and be not suitable for conventional treatment plan, thereby the suggestion patient selects more suitable individualized treatment scheme.At last, these molecular marked compounds may be as the treatment of potential drug target for tumour in the future.
In order to realize the foregoing invention purpose, concrete technical scheme of the present invention is:
A kind of detection kit for esophageal carcinoma therapy Scheme Choice and/or prognosis evaluation, described test kit comprises the reagent that detects following genetic expression;
Described gene comprises:
BMP-8, CD133, CD29, Car1, CL5, Endomus2, Nectin3, Nectin1, Kai1, PPARGCA, WAVE-1, WAVE-3, BMP9, CMG1, IL22R and VEGF-D.
Described gene also comprises house-keeping gene: GAPDH and CK19.
Described reagent detects agents useful for same for the described gene of claim 1 being carried out immunohistochemical methods.
Described test kit specifically consists of:
（1）Hot-start Q-master mix (Abgene)；
(2) for detection of the primer of the described gene of claim 1;
(3) general probe of FAM mark.
Described primer sequence is shown in SEQ ID No.1 to 36.
Primer for detection of above-mentioned each genetic expression is characterized in that, described primer sequence is shown in SEQ ID No.1 to 36.
A kind of gene chip for esophageal carcinoma therapy Scheme Choice and/or prognosis evaluation, described gene chip comprises solid phase carrier and probe, described probe is hybridized with following gene and/or its complementary sequence; Described gene comprises:
BMP-8, CD133, CD29, Car1, CL5, Endomus2, Nectin3, Nectin1, Kai1, PPARGCA, WAVE-1, WAVE-3, BMP9, CMG1, IL22R and VEGF-D.
One gene group is for the preparation of the purposes of esophageal carcinoma therapy Scheme Choice and/or prognosis evaluation detection kit, and described gene group comprises following gene:
BMP-8, CD133, CD29, Car1, CL5, Endomus2, Nectin3, Nectin1, Kai1, PPARGCA, WAVE-1, WAVE-3, BMP9, CMG1, IL22R and VEGF-D.
One gene group is for the preparation of the purposes of esophageal carcinoma therapy Scheme Choice and/or prognosis evaluation gene chip, and described gene group comprises following gene:
BMP-8, CD133, CD29, Car1, CL5, Endomus2, Nectin3, Nectin1, Kai1, PPARGCA, WAVE-1, WAVE-3, BMP9, CMG1, IL22R and VEGF-D.
The name of above-mentioned each gene all comes from confessed name in www.NCBI.LM.NIH.gov database or the document, is well known to a person skilled in the art.
Beneficial effect of the present invention:
Test kit of the present invention is by detection and evaluation to a plurality of gene expression doses, assess the curative effect of clinical treatment, thereby the prognosis of judging which patient is relatively poor, and which patient is not suitable for is used conventional treatment plan, provides auxiliary reference for the doctor selects treatment plan.Kit test method of the present invention is easy, accuracy is strong, and cost is lower, is easy to be accepted by extensive patients.
Description of drawings
Fig. 1 is that the molecular marked compound of CTF8 pattern is divided into two groups with patient with esophageal carcinoma.0 is " good prognosis " group; 1 is " poor prognosis " group, has significant significant difference (p＜0.00001) between two groups.
Fig. 2 is that the molecular marked compound of CTF8.12 pattern is divided into three groups with patient with esophageal carcinoma.1 is " good prognosis " group; 2 is " in the prognosis " group; 3 is " poor prognosis " group, has significant significant difference (p＜0.00001) between three groups.
Fig. 3 is the relation of lymph node status and esophageal carcinoma prognosis.0 is node-negative metastasis; 1 is the nodus lymphoideus transferring rate positive.
Fig. 4 be TNM by stages with the relation of esophageal carcinoma prognosis.2 is by stages StageII of TNM; 3 is by stages StageIII of TNM; 4 is by stages StageIV of TNM.
Fig. 5 is the relation of tumour cell differentiation state and esophageal carcinoma prognosis.1 is differentiated; 2 are senior middle school's differentiation; 3 is middle differentiation; 4 are differentiation in low; 5 are low differentiation.
Fig. 6 is the relation of tumor embolism and esophageal carcinoma prognosis.0 for tumor embolism occurring; 1 is without tumor embolism.
Fig. 7 is the relation of histological type and esophageal carcinoma prognosis.1 is squamous cell carcinoma; 2 is gland cancer.
Fig. 8 is that the molecular marked compound of CTF8 pattern is divided into two groups with the patient with esophageal carcinoma of node-negative metastasis.0 is " good prognosis " group; 1 is " poor prognosis " group.
Fig. 9 is that the molecular marked compound of CTF8 pattern is divided into two groups with the patient with esophageal carcinoma of the nodus lymphoideus transferring rate positive.0 is " good prognosis " group; 1 is " poor prognosis " group.
Embodiment
The acquisition of embodiment 1 goal gene
1, collection, storage and the record of tissue
The test organization sample is collected by the contriver, the discarded tissue samples (comprising healthy tissues, cancer beside organism and tumor tissues) after the Operation of Esophageal Carcinoma excision and to be stored in-80 ℃ of refrigerators for subsequent use.
2, the structure in the sample process of human esophageal carcinoma and cDNA storehouse
Human esophageal carcinoma carries out frozen tissue section with the permanent frozen sheet cutter of low temperature (Leica), preserve partially sliced in order to histologic analysis, it is as follows that the tissue of about 30 parts of sections is used for extracting the RNA(method), after RNA being carried out quantitatively, with equivalent RNA construction cDNA storehouse.
3, organize the extraction of RNA and synthesizing of cDNA
Frozen tissue is cut into 5-10 μ m thickness, and wherein a part is used for immunohistochemical analysis (Jiang et al 2003a).After the RNA that is organized in the ice precooling that gets 20-30 part section extracts in the reagent (RNA isolation reagent) and to carry out homogenate with homogenizer.The concentration of RNA detects (Jiang et al 2003a) with ultraviolet spectrophotometer.Take the total RNA of 3 μ g as template, Oligo-dT is primer, and (Promega) carries out reverse transcription with the RT test kit, and with-actin primer do confidential reference items detect synthesized the quality of cDNA.The PCR primer designs with Beacon Designer (California, USA), and is synthetic at Invitrogen Ltd and Sigma company.Test the agarose of used molecular biology grade and DNA Ladder all available from Invitrogen, conventional PCR reagent and quantitative PCR reagent are respectively available from AbGene and Biorad.
4, the quantitative analysis of molecular marked compound
The detection of testing gene expression level among the above-mentioned synthetic cDNA is adopted based on the Real-time quantitative PCR (Nazarenko et al 1997 and Jiang et al 2003a and 2003b) after the improvement of Amplifuor principle.(version 2 at first to adopt Beacon Designer software, Biosoft, Palo Alto, California, USA) design PCR primer, wherein a primer (normally antisense primer) end adds a Z sequence (5 ' actgaacctgaccgtaca ' 3, sequence and general Z probe sequence are complementary).Be used for-Taqman test kit that actin detects is available from Perkin-Elmer.
Reaction system is as follows: Hot-start Q-master mix, special upstream primer 10pmol, contain the downstream primer 1pmol of Z sequence, the probe of FAM mark (Intergen Inc.) 10pmol and about 50ngRNA.Reaction is at IcyclerIQ (Bio-Rad, Hemel Hamstead, England, UK) carry out on, concrete reaction conditions is as follows: 94 ℃ of sex change 12 min at first, then carry out 50 thermal cyclings, comprise 94 ℃ of sex change 15s, 55 ℃ of renaturation 40s, 72 ℃ are extended 20s (Jiang et al 2003b, 2003c).Mark increases simultaneously with sample to be tested in adopting in the reaction, thereby determines the transcriptional level of goal gene in the sample.The result represents in two ways: a kind of is the transcriptional level of equivalent RNA, and another kind is the relative ratio of goal gene and GAPDH gene or CK19 gene expression dose.
5, carrying out molecular marked compound according to the phraseology of goal gene selects
At first use Minitab software (Minitab Inc., State College, PA16801, USA) that the goal gene transcriptional level of sample is analyzed.
Determine final selected gene: according to the expression level of alternative gene and whether long-term ill group can be come with other group differentiation and select at last, screening conditions: the clinical datas such as clinical stages, histological type, therapeutic modality and survival time of collecting the patient, if the expression level by alternative gene can be distinguished these clinical datas of patient well, especially can distinguish the different survival patient, then be defined as final selected gene.By Excel(Microsoft Office 2007 version) software divides into groups and simple statistical analysis, uses SPSS(SPSS Inc., Chicago, Illinois, US again) software is to making further statistical analysis patient's lifetime.
The preparation of test kit: after determining final selected gene marker, concrete gene marker sees Table 1, according to these marker preparation detection kit.At first prepare all for detection of the reagent of genetic expression, then with its automatic point sample in 96 orifice plates for detection of clinical and cell sample.Test kit after the laboratory prepares, be stored in-20 ℃ for subsequent use.
6, the evaluation of molecular marked compound
After experimental analysis, determine that finally 16 genes as the molecular marked compound of judging patient with esophageal carcinoma prognosis and curative effect evaluation, see Table 1.BMP-8, CD133, CD29, Car1, CL5, Endomus2, Nectin3, Nectin1, Kai1, PPARGCA, WAVE-1, WAVE-3, BMP9, CMG1, IL22R and VEGF-D, the contrast of GAPDH house-keeping gene and the contrast of CK19 house-keeping gene.
Table 1 is as 16 gene markers of esophageal cancer genetic marker
7, the checking of the relation of esophageal carcinoma molecular marked compound and prognosis
(1) according to two fold classification method and the result verification of 16 molecular marked compound assess patient prognosis:
No matter selected marker gene is up-regulated or down-regulated expression, all is considered as unconventionality expression, divides " poor prognosis " group into if having more than 8 abnormal gene expressions then with the patient, organizes if be less than or equal 8 abnormal gene expressions then divide " good prognosis " into.
As shown in Figure 1, the patient can be divided into " good prognosis group (representing with 0 among the figure) " and " poor prognosis group (representing with 1 among the figure) " according to these molecular marked compound expression levels.The marker pattern called after CTF8 of good prognosis group, namely detected result is the CTF8 pattern, empirical tests patient is 39.3 months mean survival time (MST), in contrast to this, and " poor prognosis " marker, empirical tests patient only is 15.3 months (p＜0.00001) mean survival time (MST).In following up a case by regular visits to, 78.3% " good prognosis " group can be survived to following up a case by regular visits to end, and " poor prognosis " group only has 12.5%.The expression level input SPSS software of patient's survival time, survival condition and marker is carried out survival analysis just can divide into groups can draw mean survival time (MST) simultaneously.
Patient's clinical data is comprised the appearance of tumor embolism, the nodus lymphoideus transferring rate situation, neoplasm staging, TNM by stages, differentiation state, making the COX multiplicity behind histological type and the CTF8 input SPSS can draw, CTF8 is an independently Prognostic Factors (p＜0.001), the appearance of same tumor embolism also can be used as an independently Prognostic Factors (p=0.013), and other factors such as nodus lymphoideus transferring rate situation (p=0.227), neoplasm staging (p=0.255), TNM is (p=0.313) by stages, differentiation state (p=0.162) and histological type (p=0.78) then can not be as Prognostic Factors independently.
(2) according to three group categories method and result verification of 16 molecular marked compound assess patient prognosis:
In order to distinguish in further detail patient's prognosis, we adopt the CTF8.12 pattern, and the patient is divided into three groups, according to being less than 8 abnormal gene expressions, 8～12 abnormal gene expressions divide the patient into " good prognosis group, " organizing in the prognosis " and " poor prognosis group more than 12 abnormal gene expressions.
As shown in Figure 2, by detecting 16 molecular marked compounds the patient is divided into " good prognosis group (representing with 1 among the figure) ", " group in the prognosis (representing with 2 among the figure) " and " poor prognosis group (representing with 3 among the figure) ".Show according to the Kaplan-Meier statistical analysis, have significant significant difference (p＜0.00001) these three groups of patients' lifetime.Wherein 87% " good prognosis group " patient can be survived to following up a case by regular visits to end, its median survival interval is 44.6 months, 35% " organizing in the prognosis " patient is survived to following up a case by regular visits to end, median survival interval is 20.1 months, finish all without surviving to following up a case by regular visits to and own " poor prognosis group ", and median survival interval only is 6.5 months.
Show according to multiplicity equally, CTF8.12 is an independently Prognostic Factors (p＜0.00001), the appearance, nodus lymphoideus transferring rate situation, neoplasm staging, TNM that patient's clinical data is comprised tumor embolism by stages, make the COX multiplicity behind differentiation state, histological type and the CTF8 input SPSS and can draw.And other all clinical and pathological factors all can not as Prognostic Factors independently (lymph node status p=0.314, tumor embolism p=0.074, neoplasm staging p=0.076, TNM is p=0.231 by stages, differentiation state p=0.403, the p=0.891 of types of organization).
8, clinical and pathological factor and prognostic value thereof
According to statistical analysis, three group categories method CTF8.12 patterns of prognosis are independently Prognostic Factors (p＜0.00001), and the clinical and pathological factor such as the lymph node status of prior art (Fig. 3), neoplasm staging (Fig. 4), tumour cell differentiation state (Fig. 5), tumor embolism (Fig. 6) and histological type (Fig. 7) all can not come as Prognostic Factors independently the prognosis of assess patient.
9, the result of test kit of the present invention
We are with patient's lymph node status and 16 molecular marked compound joint assessment patients prognosis, as shown in Figure 8, among the node-negative metastasis patient, " good prognosis " group patient of 85.6% is survived to following up a case by regular visits to end, median survival interval is 43.3 months, and " poor prognosis " of all node-negative metastasis organizes the patient all without survival, and median survival interval only is 4 months." good prognosis " group patient of the 70% nodus lymphoideus transferring rate positive is survived to following up a case by regular visits to end, and median survival interval is 28.5 months." poor prognosis " group patient of the 16.7% nodus lymphoideus transferring rate positive is survived to following up a case by regular visits to end, and median survival interval is 18.4 months, as shown in Figure 9.
10, conclusion
The invention provides a kind of new detection kit of assessing the patient with esophageal carcinoma prognosis, simultaneously the present invention can also assess the possibility of patient with esophageal carcinoma postoperative chemotherapy failure.
It is a difficult problem for the prognosis evaluation of patient with esophageal carcinoma always.At present mainly adopt more postoperative clinical and pathology methods to come evaluate its prognosis, wait such as lymph node status, neoplasm staging, TNM by stages.This research finds that TNM has certain values aspect the evaluate its prognosis with the appearance of tumor embolism by stages really, and tumour cell differentiation state, lymph node status, histological type etc. also have certain values equally aspect evaluate its prognosis, but most of all not statistically significants of these factors or only have very little statistical significance.And 16 molecular marked compounds that we find are compared with these factors and are had obvious advantage (shown in Fig. 4-8).Statistical analysis shows, two groups and three group categories methods all have obvious statistical significance aspect prognosis evaluation, and 16 molecular marked compounds can be used as independently Prognostic Factors.
The using method of embodiment 2 test kits
1, reaction system: reaction is carried out in 96 low-profile PCR plate, need to do three parallel reactors for each marker gene, also need detect in addition two house-keeping genes as confidential reference items.The reaction system of each reaction (every hole) is as follows: Hot-start Q-master mix (8 μ l), upstream primer (10pmol/ μ l, 1 μ l), downstream primer (1pmol/ μ l, 1 μ l), general Z probe (the 10pmol/ μ l of FAM mark, 1 μ l), PCR level H
2O 1 μ l, sample to be tested cDNA or house-keeping gene 4 μ l.
Wherein sample to be tested cDNA be extracted as art technology people unit known technology, the cDNA after the extraction is with PCR level H
2Getting 4 μ l after the O dissolving joins in the reaction system.
2, reaction conditions: reaction is carried out in Bio-Rad Q-PCR thermo cycler, and concrete reaction conditions is as follows: 94 ℃ 12 minutes, then carry out 50 circulations, in each circulation 94 ℃ 15 seconds, 55 ℃ of 40 seconds 72 ℃ of seconds.In the time of the sample amplification, according to confidential reference items transcript is carried out quantitatively.The result represents in two ways: a kind of is the transcript degree that measures out according to average total RNA, and another kind is the relative ratio of goal gene and GAPDH gene or CK19 gene dosage.
Sequence table
＜110〉tension force is built
Jiang Wenguo
＜120〉be used for the detection kit of esophageal carcinoma therapy Scheme Choice and/or prognosis evaluation
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Claims (2)
1. detection kit that is used for esophageal carcinoma therapy Scheme Choice and/or prognosis evaluation is characterized in that described test kit consists of:
（1）Hot-start Q-master mix；
(2) primer of the following gene of detection:
BMP-8, CD133, CD29, Car1, CL5, Endomus2, Nectin3, Nectin1, Kai1, PPARGCA, WAVE-1, WAVE-3, BMP9, CMG1, IL22R and VEGF-D, and house-keeping gene: GAPDH and C K19;
Described primer sequence is shown in SEQ ID No.1 to 36;
(3) general probe of FAM mark.
2. for detection of the primer of each genetic expression described in the claim 1, it is characterized in that described primer sequence is shown in SEQ ID No.1 to 36.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 201110072082 CN102199664B (en) | 2011-03-24 | 2011-03-24 | Test kit for esophageal cancer treatment selection and/or prognostic evaluation |
| PCT/CN2011/002037 WO2012126162A1 (en) | 2011-03-24 | 2011-12-06 | Test kit for selection of esophageal cancer treatment protocols and/or prognostic evaluation |
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| CN 201110072082 CN102199664B (en) | 2011-03-24 | 2011-03-24 | Test kit for esophageal cancer treatment selection and/or prognostic evaluation |
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| CN102199664B (en) * | 2011-03-24 | 2013-03-20 | 张力建 | Test kit for esophageal cancer treatment selection and/or prognostic evaluation |
| CN104034902B (en) * | 2014-05-09 | 2015-11-25 | 赤峰学院 | Utilize the kit of 4 albumen associated prediction esophageal cancer patients prognosis |
| CN109182479A (en) * | 2018-07-27 | 2019-01-11 | 广州医科大学附属第医院 | A kind of real-time fluorescence quantitative PCR Universal fluorescence probe and its application |
| CN116904590A (en) * | 2023-02-21 | 2023-10-20 | 芜湖胤星医学检验实验室有限公司 | Esophageal cancer gene detection primer, probe and kit based on digital PCR platform |
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| CN101608218A (en) * | 2008-06-20 | 2009-12-23 | 上海主健生物工程有限公司 | Esophageal cancer genetic test kit |
| CN101671729B (en) * | 2009-09-07 | 2012-09-05 | 张力建 | Gene group for selection and/or prognostic evaluation of lung cancer treatment scheme, gene chip and detection kit thereof |
| CN102199664B (en) * | 2011-03-24 | 2013-03-20 | 张力建 | Test kit for esophageal cancer treatment selection and/or prognostic evaluation |
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