CN108893533B - Kit for predicting or assisting in predicting risk of radiation pneumonitis after lung radiation - Google Patents
Kit for predicting or assisting in predicting risk of radiation pneumonitis after lung radiation Download PDFInfo
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Abstract
The invention relates to the field of lung cancer treatment, and particularly provides a kit for predicting or assisting in predicting the risk of radiation pneumonitis after lung radiation, wherein the kit comprises a system for detecting genotypes of genetic biomarkers, which can evaluate whether a breast tumor of a patient can cause radiation pneumonitis of grade 2 or above after being subjected to radiation treatment, and 31 genetic biomarkers can be used as detection and evaluation targets. The kit can accurately and effectively pre-judge whether the breast tumor of the patient can cause radiation pneumonia of grade 2 or above grade 2 after receiving radiation treatment, is simple and convenient to use, easy to operate and reliable in result, and has very important guiding significance for monitoring and treating the patient after radiation treatment.
Description
Technical Field
The invention relates to the field of lung cancer treatment, in particular to a kit for predicting or assisting in predicting the risk of radiation pneumonitis after lung radiation.
Background
Lung cancer is the first and second highest tumors in males and females in China, and is the most lethal tumor in China. Radiotherapy is an important technical means for tumor treatment, and with the progress of technology, the treatment effect of radiotherapy for lung cancer can be similar to that of surgical treatment, and meanwhile, the radiotherapy has small damage to the body of a patient, the life quality of the treated patient is good, and the average treatment cost is lower than that of surgical treatment. With the development of national economy, it is expected that more medical institutions will introduce advanced radiotherapy equipment, and meanwhile, the demand of people for life quality is improved, so that radiotherapy will gradually play an increasingly important role in the field of tumor treatment.
In the field of accurate lung cancer treatment, technological progress, such as advanced therapeutic equipment and advanced image delineation technology, endows a radiation oncologist with personalized treatment and accurate radiotherapy capabilities. These precision treatments are dose-based on clinical parameters and anatomical information. One of the biggest challenges in radiotherapy is how to deal with the radiation resistance of tumor hypoxic cells, i.e. the sensitivity of the tumor itself to radiation. The accurate information related to radiotherapy, such as whether a patient has complications (e.g. lung and kidney injury caused by radiotherapy) after a certain dose of radiotherapy, cannot be obtained from clinical parameters and anatomical information. Therefore, reliable radiation dose biomarkers are currently lacking. Currently, for radiation therapy of lung cancer, the greatest limiting factor is radiation lung injury. The injury is caused by radiation damage to surrounding normal tissues, including acute radiation pneumonitis and chronic radiation pulmonary fibrosis. Therefore, the problems to be solved by radiotherapy of lung cancer in clinic at present are: how to predict whether the patient will have high risk of 2-grade or above 2-grade radiation pneumonitis in advance.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a kit for predicting or assisting in predicting the risk of radiation pneumonitis after lung radiation, so as to alleviate the defect in the prior art that a product capable of accurately and effectively predicting whether a breast tumor of a patient can cause radiation pneumonitis of grade 2 or above grade 2 after being subjected to radiation treatment is lacked.
The second purpose of the invention is to provide the application of the system for detecting the genotype of the genetic biomarker in the preparation of a kit, and the third purpose of the invention is to provide the application of the system for detecting the genotype of the genetic biomarker and a vector which is recorded with the following weight coefficient and an evaluation method in the preparation of a kit for diagnosing or assisting in diagnosing the radiation pneumonitis, so as to relieve the technical problems of the prior art that the biomarker for predicting the occurrence of the radiation pneumonitis and the evaluation method are lacked.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
the invention provides a kit for predicting or assisting in predicting the risk of radiation pneumonitis after lung radiation, comprising a system for detecting the genotype of genetic biomarkers as follows:
the physical coordinates are referenced to human reference genome version 37.
Further, the kit further comprises a carrier which is recorded with the following weight coefficients and evaluation methods:
detecting the genotype of the genetic biomarker locus of the patient, comparing the genotype with the human reference genome version 37, and when the genotype is wild homozygous, the original assignment is 0; when the hybrid is a wild hybrid type or a variant hybrid type, the original value is 1; when the mutation is homozygotic, the original assignment is 2, the original assignment of each site is multiplied by the weight coefficient of the site to serve as a final assignment, and the final assignments of all the sites are summed to serve as a calculation score;
if the calculated score is greater than 0.5, the patient is predicted to develop grade 2 or greater radiation pneumonitis.
Further, the system for detecting the genotype of the genetic biomarker is a reagent and/or an instrument required for detecting the genotype of the genetic biomarker by a gene sequencing method;
the reagents consist of primers or probes that detect the genotype of the genetic biomarker and other reagents required for gene sequencing.
Further, the system for detecting the genotype of the genetic biomarker is a reagent and/or an instrument required for detecting the genotype of the genetic biomarker by a gene chip technology;
the reagent consists of a probe for detecting the genotype of the genetic biomarker and other reagents required for carrying out the gene chip detection.
Further, the system for detecting the genotype of the genetic biomarker is a reagent and/or an instrument required for detecting the genotype of the genetic biomarker by a fluorescence PCR method;
the reagent consists of a probe for detecting the genotype of the genetic biomarker and other reagents required for carrying out fluorescence PCR detection;
preferably, the system for detecting the genotype of the genetic biomarker is a reagent and/or an instrument required for detecting the genotype of the genetic biomarker by a bead chip technology;
the reagent consists of a probe for detecting the genotype of the genetic biomarker and other reagents required for carrying out the detection of the bead chip technology;
preferably, the system for detecting the genotype of a genetic biomarker is a reagent and/or an instrument required for detecting the genotype of the genetic biomarker by a competitive allele-specific PCR technique;
the reagent consists of a primer for detecting the genotype of the genetic biomarker and other reagents required for detection by a competitive allele-specific PCR technology.
The invention provides the application of a system for detecting the genotype of a genetic biomarker in the preparation of a kit, wherein the kit has the function of predicting or assisting in predicting the risk of the radiation pneumonitis after lung radiation; the genetic biomarkers are as follows:
| site of the body | Chromosome number | Physical coordinates | Genotype(s) |
| RP2-01 | 15 | 96898166 | [A/G] |
| RP2-02 | 2 | 213937020 | [T/G] |
| RP2-03 | 20 | 3009669 | [T/C] |
| RP2-04 | 6 | 83598005 | [T/C] |
| RP2-05 | 16 | 11347858 | [A/G] |
| RP2-06 | 2 | 76871844 | [A/G] |
| RP2-07 | 13 | 101723882 | [T/C] |
| RP2-08 | 8 | 115464957 | [A/G] |
| RP2-09 | 7 | 97234588 | [T/G] |
| RP2-10 | 6 | 32684257 | [A/G] |
| RP2-11 | 12 | 46122002 | [A/G] |
| RP2-12 | 22 | 21410886 | [T/C] |
| RP2-13 | 3 | 172587874 | [A/G] |
| RP2-14 | 1 | 171386073 | [A/G] |
| RP2-15 | 7 | 84365672 | [T/C] |
| RP2-16 | 8 | 27197773 | [A/G] |
| RP2-17 | 1 | 150958836 | [T/C] |
| RP2-18 | 1 | 16452998 | [A/G] |
| RP2-19 | 2 | 213936394 | [A/C] |
| RP2-20 | 15 | 36005740 | [T/C] |
| RP2-21 | 9 | 89594294 | [A/G] |
| RP2-22 | 6 | 161557940 | [A/G] |
| RP2-23 | 15 | 81870629 | [A/G] |
| RP2-24 | 19 | 13155755 | [A/G] |
| RP2-25 | 8 | 139327776 | [T/C] |
| RP2-26 | 13 | 110833090 | [A/G] |
| RP2-27 | 10 | 133960262 | [A/G] |
| RP2-28 | 2 | 167120956 | [A/G] |
| RP2-29 | 8 | 47196063 | [A/G] |
| RP2-30 | 7 | 44224098 | [A/G] |
| RP2-31 | 14 | 97134921 | [A/G] |
；
The physical coordinates are referenced to human reference genome version 37.
Further, the system for detecting the genotype of a genetic biomarker is a reagent and/or an apparatus required for detecting the genotype of the genetic biomarker by a gene sequencing method,
the reagent consists of a primer or a probe for detecting the genotype of the genetic biomarker and other reagents required for gene sequencing;
or, the system for detecting the genotype of the genetic biomarker is a reagent and/or an apparatus required for detecting the genotype of the genetic biomarker by a gene chip technology,
the reagent consists of a probe for detecting the genotype of the genetic biomarker and other reagents required for carrying out gene chip detection;
or, the system for detecting the genotype of the genetic biomarker is a reagent and/or an instrument required for detecting the genotype of the genetic biomarker by a fluorescence PCR method,
the reagent consists of a probe for detecting the genotype of the genetic biomarker and other reagents required for carrying out fluorescence PCR detection;
or, the system for detecting the genotype of the genetic biomarker is a reagent and/or an apparatus required for detecting the genotype of the genetic biomarker by a bead chip technology,
the reagent consists of a probe for detecting the genotype of the genetic biomarker and other reagents required for carrying out the detection of the bead chip technology;
or, the system for detecting the genotype of a genetic biomarker is a reagent and/or an apparatus required for detecting the genotype of the genetic biomarker by a competitive allele-specific PCR method,
the reagent consists of a primer for detecting the genotype of the genetic biomarker and other reagents required for detection by a competitive allele-specific PCR method.
The invention provides a system for detecting the genotype of a genetic biomarker and application of a vector in preparing a kit for diagnosing or assisting in diagnosing radiation pneumonitis, wherein the vector is recorded with the following weight coefficients and evaluation methods:
detecting the genotype of the above locus of the patient, comparing with the version 37 of the human reference genome, and if the genotype is wild homozygous, the original assignment is 0; when the hybrid is a wild hybrid type or a variant hybrid type, the original value is 1; when the mutation is homozygotic, the original assignment is 2, the original assignment of each site is multiplied by the weight coefficient of the site to serve as a final assignment, and the final assignments of all the sites are summed to serve as a calculation score;
if the calculated score is greater than 0.5, the patient is predicted to develop grade 2 or greater radiation pneumonitis.
Further, the system for detecting the genotype of a genetic biomarker is a reagent and/or an apparatus required for detecting the genotype of the genetic biomarker by a gene sequencing method,
the reagent consists of a primer or a probe for detecting the genotype of the genetic biomarker and other reagents required for gene sequencing;
or, the system for detecting the genotype of the genetic biomarker is a reagent and/or an apparatus required for detecting the genotype of the genetic biomarker by a gene chip technology,
the reagent consists of a probe for detecting the genotype of the genetic biomarker and other reagents required for carrying out gene chip detection;
or, the system for detecting the genotype of the genetic biomarker is a reagent and/or an instrument required for detecting the genotype of the genetic biomarker by a fluorescence PCR method,
the reagent consists of a probe for detecting the genotype of the genetic biomarker and other reagents required for carrying out fluorescence PCR detection;
or, the system for detecting the genotype of the genetic biomarker is a reagent and/or an apparatus required for detecting the genotype of the genetic biomarker by a bead chip technology,
the reagent consists of a probe for detecting the genotype of the genetic biomarker and other reagents required for carrying out the detection of the bead chip technology;
or, the system for detecting the genotype of a genetic biomarker is a reagent and/or an apparatus required for detecting the genotype of the genetic biomarker by a competitive allele-specific PCR method,
the reagent consists of a primer for detecting the genotype of the genetic biomarker and other reagents required for detection by a competitive allele-specific PCR method.
Further, the genetic biomarkers are as follows:
compared with the prior art, the invention has the beneficial effects that:
the invention provides a kit for predicting or assisting in predicting the risk of radiation pneumonitis after lung radiation, which comprises a system for detecting the genotype of a genetic biomarker, wherein the system can evaluate whether a breast tumor of a patient can cause radiation pneumonitis of grade 2 or more than grade 2 after being treated by radiation, and 31 genetic biomarkers can be used as detection and evaluation targets in total. The kit can accurately and effectively pre-judge whether the breast tumor of the patient can cause radiation pneumonia of grade 2 or above grade 2 after receiving radiation treatment, is simple and convenient to use, easy to operate and reliable in result, and has very important guiding significance for monitoring and treating the patient after radiation treatment.
Drawings
FIG. 1 is a statistical chart of the risk score of radiation pneumonitis predicted by using the genetic biomarkers of the present invention before radiotherapy for 72 lung cancer patients in example 4 of the present invention.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.
The invention provides a kit for predicting or assisting in predicting the risk of radiation pneumonitis after lung radiation, which comprises a system for detecting the genotype of a genetic biomarker, wherein the genetic biomarker is as follows:
the physical coordinates are referenced to human reference genome version 37.
The kit for predicting or assisting in predicting the risk of the pneumonia after lung radiation comprises a system for detecting the genotype of genetic biomarkers, wherein the genotype of the genetic biomarkers can be evaluated whether a breast tumor of a patient can cause the pneumonia with grade 2 or above grade 2 after being treated by radioactive radiation, and 31 genetic biomarkers can be used as detection and evaluation targets. The kit can accurately and effectively pre-judge whether the breast tumor of the patient can cause radiation pneumonia of grade 2 or above grade 2 after receiving radiation treatment, is simple and convenient to use, easy to operate and reliable in result, and has very important guiding significance for monitoring and treating the patient after radiation treatment.
The lung radiation refers to that all or part of the lung is irradiated in the radiation field in the radiotherapy adopted in the breast tumor therapy such as lung cancer, breast cancer, esophageal cancer, mediastinal tumor, thymus tumor and the like.
Note that the genetic biomarkers are marked as 31 single nucleotide polymorphism sites in total from RP2-01 to RP2-31 in the above table.
The meaning of each row in the table is specifically: taking the line of the RP2-01 locus as an example, the chromosome number represents the specific position of the human chromosome number of the RP2-01 locus, namely the RP2-01 locus is located on the human chromosome 11; the physical coordinates are the specific position of the RP2-01 locus on chromosome 11, namely sr94950910, which is obtained by taking the human reference genome version 37 (GRCh37) as a reference; the genotype represents the type of base that may appear at the RP2-01 site, and A/G indicates that the type of base that may appear at the site is A or G. The genotypes likely to occur at this site are AA, AG and GG.
In one embodiment of the present invention, the kit further comprises a carrier in which the following weight coefficients and evaluation methods are described:
detecting the genotype of the patient at the genetic biomarker locus, and comparing the genotype with the genotype of the human reference genome version 37 (GRCh37), wherein the original assignment is 0 when the genotype is wild homozygous; when the hybrid is a wild hybrid type or a variant hybrid type, the original value is 1; when the mutation is homozygotic, the original assignment is 2, the original assignment of each site is multiplied by the weight coefficient of the site to serve as a final assignment, and the final assignments of all the sites are summed to serve as a calculation score;
if the calculated score is greater than 0.5, the patient is predicted to develop grade 2 or greater radiation pneumonitis. Multiplying the calculated score by 100% provides the probability that the patient will develop grade 2 or greater radiation pneumonitis.
It should be noted that the original assignment of the three genotypes of the wild homozygous type, the wild heterozygous type or the variant heterozygous type refers to an initial value before evaluation, the original assignment, i.e., the initial value, is a custom value, the initial value is multiplied by a corresponding weight coefficient, and the obtained score is used as a final assignment. Mathematically, to show how important a number of numbers have in the total, different scaling factors are given, respectively, which is a weighting. The weighted assignment coefficient is the weight, also called weight and weight. The weight coefficient is used for representing the importance degree of a certain index item in the index item system, and representing the influence of the change of the index item on the result under the condition that other index items are not changed.
Taking an RP2-01 locus as an example, detecting the genotype of the locus, and comparing the genotype with a human reference genome version 37, wherein a) the locus is wild homozygous, the original assignment of the RP2-01 locus is 0, and the original assignment is multiplied by a weight coefficient-0.039615128 of the locus to obtain a final assignment of 0; b) the hybrid is a wild hybrid or a variant hybrid, the original assignment of the RP2-01 site is 1, and the original assignment is multiplied by the weight coefficient-0.039615128 of the site to obtain the final assignment of-0.039615128; c) for the homozygous variant, the original assignment at the RP2-01 locus was 2, which was multiplied by the-0.039615128 weight coefficient for that locus to give the final assignment of-0.079230256.
Taking an RP2-02 locus as an example, detecting the genotype of the locus, and comparing the genotype with a human reference genome version 37, wherein a) the locus is wild homozygous, the original assignment of the RP2-02 locus is 0, and the original assignment is multiplied by a weight coefficient 0.000122236 of the locus to obtain a final assignment of 0; b) the hybrid is a wild hybrid or a variant hybrid, the original assignment of the RP2-02 site is 1, and the original assignment is multiplied by the weight coefficient 0.000122236 of the site to obtain the final assignment 0.000122236; c) for the variant homozygous type, the original assignment of the RP2-02 locus is 2, and the original assignment is multiplied by the weight coefficient 0.000122236 of the locus to obtain the final assignment 0.000244472.
The other 29 sites also obtain respective final assignments according to the methods of the RP2-01 site and the RP2-02 site, and the final assignments of the RP2-01 to the RP2-31 are added to obtain a calculated score.
In addition, the wild homozygous means that the genotypes of the two copies of the locus are identical to the human reference genome version 37 and are homozygous; wild heterozygote and variant heterozygote mean that one of the two copies of the genotype at the site is identical to the genotype of human reference genome version 37, and the other copy is not; variation homozygous means that the genotypes of the two copies of the locus are identical, but both are not identical to the genotype of human reference genome version 37, and are variation homozygous. When the genotype of the site does not conform to the wild homozygote, the wild heterozygote, the variant heterozygote and the variant homozygote as defined above, the original value of the site is 0, i.e., the site participates in the evaluation as the wild homozygote. In rare cases, it is possible that the variant base at the site in the sample does not match the common variant base type, in which case the original assignment for the site is 0. For example, RP2-01, where the possible base at the site is A or G, and the possible genotypes are AA, AG and GG, and if a particular case occurs, where a mutation occurs at the site, the resulting base is T or C, i.e., any base other than A/G, the assignment at the site is 0, and the assignment at the site is 0 regardless of which of TA, TG, TT, CA, CG and CC the genotype the mutation generates.
A regression containing two or more independent variables is commonly referred to as a multiple linear regression, and is expressed as: y ═ β 0+ β 1X1+ … + β pxp + e, or expressed in matrix form as y ═ X β + e.
In the above formula, y is a dependent variable, β is a regression coefficient, and X is an independent variable. The algorithm is to solve the regression coefficient, so that: (y-X. beta.)2And minimum.
Least squares regression has little bias if there is a relatively significant linear relationship between the response variable and the predictor variable, and particularly has a small variance if the number of observations, n, is much greater than the predictor variable, p. But if n and p are relatively close, then an overfitting is likely to occur; if n < p, least squares regression yields no meaningful results. In addition, many variables in the multiple linear regression model may be independent of response variables; multiple collinearity phenomena may also occur: i.e., significant correlation between multiple predictor variables. These conditions increase the complexity of the model and impair the interpretability of the model. At this time, variable selection (feature selection) is required.
In the given data, the total number of independent variables is p ≈ 700000 and the number of dependent variables n <100, obviously n < < p.
Aiming at the problems, the invention applies Rob Tibshirani and the like to propose a contraction method (shrinkage method) to establish a multiple linear regression model. The shrinkage method is also known as regularization. Mainly ridge regression and lasso regression. The estimation of some coefficients is made 0 by adding a penalty constraint to the least squares estimation. The invention applies an elastic net (elastic net) to deal with the problems, and the method integrates two regularization methods of ridge regression and lasso regression, and is represented as follows:
elastic net has a significant effect on cases where p is much larger than n, or severe multicollinearity. For elastic net, when alpha is close to 1, elastic net behaves very close to lasso, but degenerations or strange manifestations caused by extreme correlations are removed. In general, elastic net is a good compromise between ridge regression and lasso, and when alpha varies from 0 to 1, the sparseness of the objective function (in the case of coefficients of 0) also increases monotonically from 0 to the sparseness of lasso.
The coordinate descent method is a fast calculation method for lasso (the fastest calculation method for lasso at present), and has the basic key points that: each parameter is optimized while keeping the other parameters fixed, looping until the coefficients stabilize. This calculation is performed on the lattice point values of lambda.
In one embodiment of the present invention, the weight coefficient and the evaluation method are obtained by the following algorithm:
firstly, performing original assignment on all polymorphic sites related to a single sample, wherein the assignment algorithm is as follows: comparing the genotype of the locus with the human reference genome version 37 (GRCh37), and when the locus is wild homozygous, originally assigning a value of 0; when the site is a wild heterozygous or a variant heterozygous, the original value is 1; when the site is homozygous for the variation, the original assignment is 2.
The original assignment is arbitrary and does not represent any biological significance, but the overall principle of assignment is that the original assignment can distinguish between wild homozygous, wild heterozygous or variant heterozygous and variant homozygous. And then, performing the above operations on all samples to finally obtain a matrix of P × N orders, wherein P is the total number of the polymorphic sites counted by each sample, and N is the total sample number. The matrix will be used as an input file. The grade of the radiation pneumonitis for all samples is used to create a second matrix file, which is 1 × N, where N is the total number of samples. And taking the two matrix files as independent variables and dependent variables respectively, and introducing the independent variables and the dependent variables into a GLMNET R program package for operation, wherein the final result of the operation is a regression coefficient table, namely the weight coefficient of each site.
The algorithm can establish a multiple linear regression mathematical model for predicting the radioactive damage of the lung based on the information of the single nucleotide polymorphism sites.
In conjunction with the above algorithm, it should be understood that the above is provided only one of a plurality of weight coefficient tables and evaluation methods, and those skilled in the art can obtain a plurality of weight coefficient tables and corresponding evaluation methods according to the screening method of the algorithm.
For example, the present invention provides an evaluation method:
detecting the genotype of the patient's genetic biomarker locus, and in comparison to human reference genome version 37 (GRCh37), the original assignment is 0 when it is wild homozygous; when the hybrid is a wild hybrid type or a variant hybrid type, the original value is 1; when the mutation is homozygotic, the original assignment is 2, the original assignment of each site is multiplied by the weight coefficient of the site to serve as a final assignment, and the final assignments of all the sites are summed to serve as a calculation score; if the calculated score is greater than 0.5, the patient is predicted to develop grade 2 or greater radiation pneumonitis.
According to the algorithm, the original assignments of the wild type homozygote type, the wild heterozygote type or the variant heterozygote type and the variant homozygote type can be changed, as long as the three original assignments are different from each other, the numerical value does not represent any biological significance, and the overall principle of the original assignments is that the wild type homozygote type, the wild heterozygote type or the variant heterozygote type and the variant homozygote type can be distinguished, so that the purpose of further calculating the weight coefficient is achieved. It is not limited to the wild homozygous original assignment provided by the present invention being 0; the original assignment of the wild heterozygous or the variant heterozygous is 1; the original assignment of homozygous variant is 2, a calculation. And substituting the three different original assignments into a corresponding weight coefficient table to obtain a corresponding evaluation method.
In one embodiment of the present invention, the system for detecting the genotype of a genetic biomarker is a reagent and/or an apparatus required for detecting the genotype of a genetic biomarker by a gene sequencing method;
the reagents consist of primers or probes that detect the genotype of the genetic biomarker and other reagents required for performing gene sequencing. The kit can realize the sample treatment before the genotype detection of 31 single nucleotide polymorphism sites in a blood sample of a patient, directly apply the treated sample to a gene sequencing instrument to obtain genotype data, and directly obtain the risk value of the patient for generating 2-grade or more than 2-grade radiation pneumonia after the patient receives radiation treatment by utilizing a carrier containing a weight coefficient and an evaluation method.
The gene sequencing can be any one of the 1 generation, 2 generation or 3 generation sequencing methods, such as single base extension sequencing, as long as the sequencing range covers the 31 single nucleotide polymorphism sites provided by the invention. The substance for detecting the genotype of the genetic biomarker comprises other reagents and/or detection instruments such as all specific amplification primers, specific probes and the like involved in detecting the genotype of 31 single nucleotide polymorphism sites by gene sequencing.
In one embodiment of the present invention, the system for detecting the genotype of a genetic biomarker is a reagent and/or an instrument required for detecting the genotype of the genetic biomarker by a gene chip technology;
the reagent consists of a probe for detecting the genotype of the genetic biomarker and other reagents required for carrying out the gene chip detection. The kit can realize the sample treatment before the genotype detection of 31 single nucleotide polymorphism sites in a blood sample of a patient, directly apply the treated sample to a gene chip detection instrument to obtain genotype data, and directly obtain the risk value of the patient for generating 2-grade or more than 2-grade radiation pneumonia after the patient receives radiation treatment by utilizing a carrier containing a weight coefficient and an evaluation method.
The range of the sites detected by the gene chip covers the 31 single nucleotide polymorphism sites provided by the invention. The substance for detecting the genotype of the genetic biomarker comprises all specific probes, other reagents and/or detection instruments related to the detection of the genotype of 31 single nucleotide polymorphism sites by using a gene chip.
In one embodiment of the present invention, the system for detecting the genotype of a genetic biomarker is a reagent and/or an instrument required for detecting the genotype of the genetic biomarker by a fluorescent PCR method;
the reagents consist of probes for detecting the genotype of genetic biomarkers and other reagents required for fluorescent PCR detection. The kit can realize the sample treatment before the genotype detection of 31 single nucleotide polymorphism sites in a blood sample of a patient, directly apply the treated sample to a fluorescence PCR instrument to obtain genotype data, and directly obtain the risk value of the patient for generating 2-grade or more than 2-grade radiation pneumonia after receiving radiation treatment by utilizing a carrier containing a weight coefficient and an evaluation method.
The fluorescent PCR method can be, for example, but not limited to, TaqMan probe method and high resolution melting curve analysis method. The range of sites detected by fluorescence PCR covers 31 single nucleotide polymorphism sites provided by the invention. The substance for detecting the genotype of the genetic biomarker comprises all specific probes, other reagents and/or detection instruments involved in the detection of the genotype of 31 single nucleotide polymorphism sites by using fluorescent PCR.
In one embodiment of the present invention, the system for detecting the genotype of a genetic biomarker is a reagent and/or an instrument required for detecting the genotype of the genetic biomarker by a bead chip technology;
the reagent consists of a probe for detecting the genotype of the genetic biomarker and other reagents required for carrying out the detection of the bead chip technology. The kit can realize the sample treatment before the genotype detection of 31 single nucleotide polymorphism sites in a blood sample of a patient, directly apply the treated sample to a microbead chip instrument to obtain genotype data, and directly obtain the risk value of the patient for generating 2-grade or more than 2-grade radiation pneumonia after the patient receives radiation treatment by utilizing a carrier containing a weight coefficient and an evaluation method.
The bead chip technology may be, for example, but not limited to, Illumina bead xpress. The range of the sites detected by the bead chip technology covers the 31 single nucleotide polymorphism sites provided by the invention. The substance for detecting the genotype of the genetic biomarker comprises all specific probes, other reagents and/or detection instruments involved in the detection of the genotype of 31 single nucleotide polymorphism sites by using fluorescent PCR.
In one embodiment of the present invention, the system for detecting the genotype of a genetic biomarker is a reagent and/or an instrument required for detecting the genotype of the genetic biomarker by a competitive allele-specific PCR method;
the reagents consist of probes to detect the genotype of genetic biomarkers and other reagents required to perform competitive allele-specific PCR detection. The kit can realize the sample treatment before the genotype detection of 31 single nucleotide polymorphism sites in a blood sample of a patient, directly apply the treated sample to a fluorescence PCR instrument to obtain genotype data, and directly obtain the risk value of the patient for generating 2-grade or more than 2-grade radiation pneumonia after receiving radiation treatment by utilizing a carrier containing a weight coefficient and an evaluation method.
The range of sites detected by competitive allele-specific PCR covers the 31 single nucleotide polymorphic sites provided by the present invention. The substance for detecting the genotype of the genetic biomarker comprises all specific probes, other reagents and/or detection instruments involved in the detection of the genotype of 31 single nucleotide polymorphism sites by using fluorescent PCR.
The invention also provides the application of the system for detecting the genotype of the genetic biomarker in the preparation of the kit, and the function of the kit is to predict or assist in predicting the risk of the radiation pneumonitis after lung radiation; the genetic biomarkers were as follows:
the physical coordinates are referenced to human reference genome version 37.
In one embodiment of the present invention, the system for detecting the genotype of a genetic biomarker is a reagent and/or an apparatus required for detecting the genotype of the genetic biomarker by a gene sequencing method,
the reagent consists of a primer or a probe for detecting the genotype of the genetic biomarker and other reagents required for gene sequencing;
or, the system for detecting the genotype of the genetic biomarker is a reagent and/or an apparatus required for detecting the genotype of the genetic biomarker by a gene chip technique,
the reagent consists of a probe for detecting the genotype of the genetic biomarker and other reagents required for carrying out gene chip detection;
or, the system for detecting the genotype of a genetic biomarker is a reagent and/or an instrument required for detecting the genotype of the genetic biomarker by a fluorescent PCR method,
the reagent consists of a probe for detecting the genotype of the genetic biomarker and other reagents required for carrying out fluorescence PCR detection;
alternatively, the system for detecting the genotype of the genetic biomarker is a reagent and/or an instrument required for detecting the genotype of the genetic biomarker by a bead chip technology. The reagent consists of a probe for detecting the genotype of the genetic biomarker and other reagents required for carrying out the detection of the bead chip technology;
alternatively, the system for detecting the genotype of a genetic biomarker is a reagent and/or an instrument required for detecting the genotype of the genetic biomarker by a competitive allele-specific PCR method. The reagents consist of probes for detecting the genotype of genetic biomarkers and other reagents required for detection by competitive allele-specific PCR.
The invention finally provides a system for detecting the genotype of the genetic biomarker and application of a vector which is recorded with the following weight coefficient and evaluation method in preparing a kit for diagnosing or assisting in diagnosing the radiation pneumonitis:
detecting the genotype of the above locus of the patient, comparing with the version 37 of the human reference genome, and if the genotype is wild homozygous, the original assignment is 0; when the hybrid is a wild hybrid type or a variant hybrid type, the original value is 1; when the mutation is homozygotic, the original assignment is 2, the original assignment of each site is multiplied by the weight coefficient of the site to serve as a final assignment, and the final assignments of all the sites are summed to serve as a calculation score;
if the calculated score is greater than 0.5, the patient is predicted to develop grade 2 or greater radiation pneumonitis.
In one embodiment of the present invention, the system for detecting the genotype of a genetic biomarker is a reagent and/or an apparatus required for detecting the genotype of a genetic biomarker by a gene sequencing method,
the reagent consists of a primer or a probe for detecting the genotype of the genetic biomarker and other reagents required for gene sequencing;
or, the system for detecting the genotype of the genetic biomarker is a reagent and/or an apparatus required for detecting the genotype of the genetic biomarker by a gene chip technology,
the reagent consists of a probe for detecting the genotype of the genetic biomarker and other reagents required for carrying out gene chip detection;
or, the system for detecting the genotype of a genetic biomarker is a reagent and/or an instrument required for detecting the genotype of the genetic biomarker by a fluorescent PCR method,
the reagents consist of probes for detecting the genotype of genetic biomarkers and other reagents required for fluorescent PCR detection.
Alternatively, the system for detecting the genotype of the genetic biomarker is a reagent and/or an instrument required for detecting the genotype of the genetic biomarker by a bead chip technology. The reagent consists of a probe for detecting the genotype of the genetic biomarker and other reagents required for carrying out the detection of the bead chip technology.
Alternatively, the system for detecting the genotype of a genetic biomarker is a reagent and/or an instrument required for detecting the genotype of the genetic biomarker by a competitive allele-specific PCR method. The reagents consist of probes for detecting the genotype of genetic biomarkers and other reagents required for detection by competitive allele-specific PCR.
In one embodiment of the invention, the genetic biomarkers are as follows:
in order to facilitate understanding of the technical solutions provided by the present invention, the technical solutions provided by the present invention are further described below with reference to the embodiments. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents and apparatus used are those which are not specified by the manufacturer and are conventional products commercially available.
EXAMPLE 1 clinical sample Collection
The clinical blood samples come from a certain Beijing hospital, the samples all obtain the informed consent of the inventor, and 72 samples are obtained from lung cancer patients before radiotherapy. Blood collection was performed according to the clinical routine, and 1ml of venous blood was collected and stored in a conventional EDTA anticoagulant tube for less than one week.
Example 2 identification of genotype of samples
And extracting the genome DNA of the collected blood sample by using a DNA extraction kit.
72 parts of genomic DNA extracted from 72 samples in example 1 were sequenced by a gene sequencing method to obtain the genotypes of the 31 SNP sites of the present invention.
Example 3 calculation of Risk score
Comparing the genotypes of the 31 sites detected in the sample in example 2 with the human reference genome version 37 (GRCh37), and when the two copies of the site are identical, the original assignment of the site is 0; when the result of the site comparison is that one copy is consistent, the original assignment of the site is 1; when the alignment of this site results in both copies being mutated, the original assignment of this site is 2. The original assignment for each locus is then multiplied by the locus's weight factor (see table below) and the resulting value is the calculated score for that locus. And adding the calculated scores of the 31 sites of one sample, wherein the sum is the corresponding risk value of the sample. When the sum is greater than 0.5, the patient is judged to have grade 2 or above grade 2 radiation pneumonitis. Multiplying the risk value by 100% is the probability of the patient being at risk for developing grade 2 or above grade 2 radiation pneumonitis.
Example 4 statistics of results
The risk scores of the 72 samples with radiation pneumonitis in example 3 were counted, and the results are shown in fig. 1, and 17 lung cancer patients with radiation pneumonitis of grade 2 or more after surgery are predicted to be present.
The 72 patients were followed up with actual postoperative follow-up and investigated for the presence and grade of radiation pneumonitis, with the results shown in the following table:
from the above table, 17 of 72 patients obtained radiation pneumonitis of grade 2 or above, and the sample number of the patient is consistent with the result of prediction of the present invention, which indicates that 31 SNP sites provided by the present invention can be used as genetic biomarkers for predicting or assisting in predicting the risk of radiation pneumonitis after lung radiation, and the kit provided by the present invention can effectively predict the risk of radiation pneumonitis.
While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
Claims (1)
1. The system for detecting the genotype of the genetic biomarker and a carrier which is recorded with the following weight coefficient and evaluation method are applied to the preparation of the kit for diagnosing the radiation pneumonitis together:
detecting the genotype of the above locus of the patient, comparing with the version 37 of the human reference genome, and if the genotype is wild homozygous, the original assignment is 0; when the hybrid is a wild hybrid type or a variant hybrid type, the original value is 1; when the mutation is homozygotic, the original assignment is 2, the original assignment of each site is multiplied by the weight coefficient of the site to serve as a final assignment, and the final assignments of all the sites are summed to serve as a calculation score;
if the calculated score is greater than 0.5, the patient is predicted to develop grade 2 or greater radiation pneumonitis;
the system for detecting the genotype of the genetic biomarker is a reagent and an instrument required for detecting the genotype of the genetic biomarker by a gene sequencing method,
the reagent consists of a primer or a probe for detecting the genotype of the genetic biomarker and other reagents required for gene sequencing;
or, the system for detecting the genotype of the genetic biomarker is a reagent and an apparatus required for detecting the genotype of the genetic biomarker by a gene chip technology,
the reagent consists of a probe for detecting the genotype of the genetic biomarker and other reagents required for carrying out gene chip detection;
or, the system for detecting the genotype of the genetic biomarker is a reagent and an apparatus required for detecting the genotype of the genetic biomarker by a fluorescence PCR method,
the reagent consists of a probe for detecting the genotype of the genetic biomarker and other reagents required for carrying out fluorescence PCR detection;
or, the system for detecting the genotype of the genetic biomarker is a reagent and an instrument required for detecting the genotype of the genetic biomarker by a bead chip technology,
the reagent consists of a probe for detecting the genotype of the genetic biomarker and other reagents required for carrying out the detection of the bead chip technology;
or, the system for detecting the genotype of a genetic biomarker is a reagent and an apparatus required for detecting the genotype of the genetic biomarker by a competitive allele-specific PCR method,
the reagent consists of a primer for detecting the genotype of the genetic biomarker and other reagents required for detecting by a competitive allele-specific PCR method;
the genetic biomarkers are as follows:
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