CN115627294A - CTDNA expression-based multidimensional liver cancer accurate molecular typing risk assessment method - Google Patents

CTDNA expression-based multidimensional liver cancer accurate molecular typing risk assessment method Download PDF

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CN115627294A
CN115627294A CN202211334077.3A CN202211334077A CN115627294A CN 115627294 A CN115627294 A CN 115627294A CN 202211334077 A CN202211334077 A CN 202211334077A CN 115627294 A CN115627294 A CN 115627294A
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王思振
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Wuxi Fanshengzi Biotechnology Co ltd
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Abstract

The invention discloses a CTDNA expression-based multidimensional liver cancer accurate molecular typing risk assessment method, which comprises the following steps: s1, collecting a liver cancer precision molecule sample of a subject; s2, preparing specific primers and probes for detecting or measuring the methylation state or level of the SEPT9 gene in the DNA of a test sample, and determining the expression molecule typing and collecting rate of gene data in the accurate molecular sample of the liver cancer; s3, separating mass-to-charge ratio (mass-to-charge ratio, m/z) according to time sequence or spatial position, and giving molecular weight, molecular formula and structural information of the compound; and S4, determining the risk of multi-dimensional accurate molecular typing and/or survival of the liver cancer of the subject according to the molecular weight, molecular formula and structural information of the gene data in the sample, realizing comprehensive and accurate analysis, realizing absolutely quantitative protein expression, finding the most relevant indexes with the liver cancer, reducing the initial RNA (ribonucleic acid) demand, simplifying operation, shortening period and simultaneously carrying out molecular typing detection.

Description

CTDNA expression-based multidimensional liver cancer accurate molecular typing risk assessment method
Technical Field
The invention belongs to the technical field of early screening/early intervention detection based on liver cancer, and particularly relates to a multidimensional liver cancer accurate molecular typing risk assessment method based on CTDNA expression.
Background
The examination means of early liver cancer is usually B-ultrasonic examination, including black-and-white ultrasound and color ultrasound examination, while color ultrasound is valuable for the examination of early liver cancer, and can find the size, position, blood supply condition of liver tumor, correlation with peripheral organs, local invasion and other conditions. Of course, if the tumor is small, such as 1cm or less, the B-mode ultrasonography may not be able to clearly show the tumor, and the nature, size, number and relation of the liver space occupying lesion to the surrounding tissues and organs can be more clearly confirmed by the nuclear magnetic resonance examination, including the nuclear magnetic resonance enhancement, such as the pleochromatic imaging, and the like, and the enhanced CT examination.
In addition, tumor markers including AFP (alpha fetoprotein) and the like can be closely monitored, so that the early discovery of the liver cancer by the B-mode ultrasonography can play a role in primary screening, and the later diagnosis can be further clarified by CT and nuclear magnetic resonance. For patients with a history of hepatitis B, the replication of hepatitis B DNA must be monitored, active antiviral therapy is given, regular physical examination is carried out, and early liver cancer is detected.
In the existing liver cancer molecular typing, the existing liver cancer molecular typing is based on protein expression, such as an immunohistochemistry method, most of the existing liver cancer molecular typing is artificial, the subjectivity of the existing liver cancer molecular typing easily influences an interpretation result in the interpretation process, in addition, the protein expression cannot be absolutely quantified, in the novel liver cancer risk assessment, the existing liver cancer recurrence risk is calculated based on an RT-PCR method, and the problems of large initial RNA (ribonucleic acid) required amount, complex operation, long time period and incapability of simultaneously carrying out molecular typing detection exist in the calculation process.
Disclosure of Invention
The invention aims to solve the defects in the prior art, and provides a CTDNA expression-based multidimensional liver cancer accurate molecular typing risk assessment method.
In order to achieve the purpose, the invention provides the following technical scheme:
a CTDNA expression-based multidimensional liver cancer accurate molecular typing risk assessment method comprises the following steps:
s1, collecting a liver cancer accurate molecule sample of a subject;
s2, preparing specific primers and probes for detecting or measuring the methylation state or level of SEPT9 genes in the DNA of a tested sample, and determining the expression molecule typing set rate of gene data in the liver cancer accurate molecule sample;
s3, converting vaporized molecules of the sample to be tested into charged ions in a high-vacuum ion source, ionizing, leading out and focusing the charged ions, then enabling the charged ions to enter a mass analyzer, separating mass-to-charge ratios (mass-to-charge ratios, m/z) according to time sequence or spatial positions under the action of a magnetic field or an electric field, and finally detecting the charged ions by an ion detector to give molecular weight, molecular formula and structural information of the compound;
and S4, determining the risk of the multidimensional liver cancer accurate molecular typing and/or survival of the subject according to the molecular weight, molecular formula and structural information of the gene data in the sample.
Wherein, the specific primer and probe for detecting the methylation state of the SEPT9 gene are selected from any one of the following three specific primer and probe combinations: specific primers SAE IN NO. 1-2 and probe SAQ IN NO. 3, specific primers SEM ID NO. 4-5 and probe SEM ID NO. 6, specific primers SEQ ID NO. 7-8 and probe SEQ ID NO. 9.
The gene based on DNA expression is set in polymorphic sites, wherein the polymorphic sites are N +1 sites selected through gene sites related to genetic diseases, and the sites A, the sites B and the sites N +1 are expressed in a table; the genotype is M +1 subtypes appearing in the population at the loci, and subtype A, subtype B and subtype M +1 are replaced in the table; the reference frequency is based on the proportion of each subtype in Chinese population; risk is the relative risk for the polygenic disease according to different genotypes.
The method comprises the following steps of carrying out quantitative analysis on the expression quantity of the multidimensional liver cancer precise molecular gene expressed based on CT DNA, and carrying out molecular typing on a sample through a set threshold, wherein the specific typing method comprises the following steps:
(1) carrying out quantitative analysis on the expression quantity of the liver cancer accurate molecular gene by adopting VRPSolver software;
(2) performing typing model training on the expression quantities of Alexa Fluor dye, FITC, texas Red, cy3 and Cy5 genes of a multi-dimensional liver cancer accurate molecule sample with a clinically definite typing result by adopting a machine learning method, and performing subtype prediction of Next Generation Sequencing (NGS) on the sample based on a model;
(3) further performing secondary classification on patients predicted to be ER + and HER2+ positive; among them, patients with ER + positivity are further classified into luminal A and luminal B, and patients with HER2+ positivity are further classified into HER2 and luminal B-HER 2.
Wherein, the gene data diagnostic substance in the sample can be protein microarray, ELISA diagnostic kit, immunohistochemistry (IHC) kit, next generation sequencing kit, real-time fluorescence quantitative PCR kit, gene chip or combination thereof;
the gene data diagnostic substance in the sample is a second-generation sequencing kit or a real-time fluorescent quantitative PCR kit based on target sequencing;
the gene data diagnostic in the sample is a second-generation sequencing kit based on targeting RNA-seq, which comprises one or more of a total RNA extraction reagent, a reverse transcription reagent and a second-generation sequencing reagent, and a primer with a nucleotide sequence shown in a table 4;
the second generation sequencing reagent is a reagent which can be used for constructing a library Illumina of the target RNA-seq.
Wherein uncalibrated risk values are calculated and identified with a GT, the GT identifying risk values calculated as follows:
GT =0if RSU < 0; RS =20 × (RSU-6.7) if 0 is not less than RSU not more than 100; RS =100if RSU is more than 100, and GT = GT multiplied by 100/max (GT) is ensured to control the GT value between 1 and 99; and taking the maximum GT value of the sample with the measuring age being more than 5 years as a cut-off value according to data acquisition, wherein the sample with the measuring age being less than the cut-off value is a low-risk group.
Wherein, the total number of 21-31 points in the gene data detection result chart of the liver cancer accurate molecule sample is 21-31, each point represents the detection result of one sample of a tested person, wherein, GTH and GTL are risk genotypes, and the damage of organism free radicals is added to gene molecules when the gene molecules carry the risk genotypes of molecular typing.
The risk assessment method further comprises a multidimensional liver cancer accurate molecule typing risk assessment system, the multidimensional liver cancer accurate molecule typing risk assessment system comprises a diagnosis module, the diagnosis module judges by using the molecular weight, molecular formula and structural information of gene data in the sample, and the system further comprises a data input module, a data preprocessing module, a model training module and a model testing module.
Wherein, the kit also comprises the following components: a plasma free DNA extraction reagent and a plasma free DNA methylation conversion reagent, wherein the plasma free DNA methylation conversion reagent is bisulfite, and the tested sample DNA is complete genome, cell-free DNA or circulating tumor DNA.
The invention has the technical effects and advantages that:
the invention provides a CTDNA expression-based multidimensional liver cancer molecular typing risk assessment method, which comprises the steps of collecting a liver cancer molecular sample of a subject, obtaining related index factors, measuring the expression molecular typing collection rate of gene data in the liver cancer molecular sample by using specific primers and probes, separating mass-to-charge ratio (mass-to-charge ratio, m/z) according to time sequence or spatial position, finally detecting by using an ion detector, giving out molecular weight, molecular formula and structural information of a compound, realizing comprehensive and accurate analysis, finding the most relevant indexes to liver cancer, determining the risk of multidimensional liver cancer molecular typing and/or survival of the subject, realizing absolutely quantitative protein expression, finding the most relevant indexes to liver cancer, reducing the initial RNA (ribonucleic acid) requirement, simplifying operation, shortening period and simultaneously carrying out molecular typing detection.
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FIG. 1 is a flow chart of the CT DNA expression-based multi-dimensional liver cancer precise molecular typing risk assessment method.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings of the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
In the description of the present invention, it is to be understood that the terms indicating an orientation or positional relationship are based on the orientation or positional relationship shown in the drawings only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus, should not be construed as limiting the present invention.
In the present invention, unless otherwise explicitly stated or limited, the terms "mounted," "connected," "fixed," and the like are to be construed broadly, e.g., as being permanently connected, detachably connected, or integral; can be mechanically or electrically connected; they may be directly connected or indirectly connected through intervening media, or may be connected through the use of two elements or the interaction of two elements. The specific meanings of the above terms in the present invention can be understood by those of ordinary skill in the art according to the specific conditions of the specification.
The invention provides a CTDNA expression-based multidimensional liver cancer accurate molecular typing risk assessment method as shown in figure 1, which comprises the following steps:
s1, collecting a liver cancer accurate molecule sample of a subject;
s2, preparing specific primers and probes for detecting or measuring the methylation state or level of SEPT9 genes in the DNA of a tested sample, and determining the expression molecule typing set rate of gene data in the liver cancer accurate molecule sample;
s3, converting vaporized molecules of the sample to be tested into charged ions in a high-vacuum ion source, ionizing, leading out and focusing the charged ions, then enabling the charged ions to enter a mass analyzer, separating mass-to-charge ratios (mass-to-charge ratios, m/z) according to time sequence or spatial positions under the action of a magnetic field or an electric field, and finally detecting the charged ions by an ion detector to give molecular weight, molecular formula and structural information of the compound;
and S4, determining the risk of the multidimensional liver cancer accurate molecular typing and/or survival of the subject according to the molecular weight, molecular formula and structural information of the gene data in the sample.
Specifically, the specific primers and probes for detecting the methylation state of the SEPT9 gene are selected from any one of the following three specific primer and probe combinations: specific primers SAE IN NO 1-2 and probe SAQ IN NO 3, specific primers SEM ID NO 4-5 and probe SEM ID NO 6, specific primers SEQ ID NO 7-8 and probe SEQ ID NO 9.
Specifically, the gene based on DNA expression is set in polymorphic sites, wherein the polymorphic sites are N +1 sites selected from gene sites related to genetic diseases, and are represented by site A, site B and site N +1 in a table; the genotype is M +1 subtypes appearing in the population at the loci, and subtype A, subtype B and subtype M +1 are replaced in the table; the reference frequency is based on the proportion of each subtype in Chinese population; risk is the relative risk for the polygenic disease according to different genotypes.
Specifically, the quantitative analysis is carried out on the expression quantity of the multidimensional liver cancer precise molecular gene expressed based on the CT DNA, and the molecular typing is carried out on the sample through a set threshold value, wherein the specific typing method comprises the following steps:
(1) quantitative analysis is carried out on the expression quantity of the liver cancer precision molecular gene by adopting VRPSolver software;
(2) performing typing model training on the expression quantities of Alexa Fluor dye, FITC, texas Red, cy3 and Cy5 genes of a multi-dimensional liver cancer accurate molecule sample with a clinically definite typing result by adopting a machine learning method, and performing subtype prediction of Next Generation Sequencing (NGS) on the sample based on a model;
(3) and further performing secondary classification on the patients predicted to be ER + and HER2+ positive; patients with ER + positivity can be further classified into luminal A and luminal B, and patients with HER2+ positivity can be further classified into HER2 and luminal B-HER 2.
Specifically, the gene data diagnostic substance in the sample can be protein microarray, ELISA diagnostic kit, immunohistochemistry (IHC) kit, next generation sequencing kit, real-time fluorescence quantitative PCR kit, gene chip or combination thereof;
the gene data diagnostic substance in the sample is a second-generation sequencing kit based on target sequencing or a real-time fluorescent quantitative PCR kit;
the gene data diagnostic in the sample is a second-generation sequencing kit based on targeting RNA-seq, which comprises one or more of a total RNA extraction reagent, a reverse transcription reagent and a second-generation sequencing reagent, and a primer with a nucleotide sequence shown in a table 4;
the second generation sequencing reagent is a reagent that can be customized for constructing a library Illumina of the targeted RNA-seq.
Specifically, calculating an uncalibrated risk value, identifying with a GT, the GT identifying the risk value is calculated as follows:
GT =0if RSU < 0; RS =20 × (RSU-6.7) if 0 is not less than RSU not more than 100; RS =100if RSU > 100, ensuring GT value to be controlled between 1-99 according to the formula GT = GT × 100/max (GT); and taking the maximum GT value of the sample with the measuring age being more than 5 years as a cut-off value according to data acquisition, wherein the sample with the measuring age being less than the cut-off value is a low-risk group.
Specifically, in a gene data detection result chart of the liver cancer accurate molecule sample, 21-31 points are provided in total, each point represents a detection result of a tested person sample, wherein GTH and GTL are risk genotypes, and organism free radical damage is aggravated on gene molecules when the risk genotypes of molecular typing are carried.
Specifically, the risk assessment method further comprises a multidimensional liver cancer accurate molecule typing risk assessment system, the multidimensional liver cancer accurate molecule typing risk assessment system comprises a diagnosis module, the diagnosis module judges by using the molecular weight, molecular formula and structural information of gene data in the sample, and the system further comprises a data input module, a data preprocessing module, a model training module and a model testing module.
Specifically, the kit also comprises the following components: a plasma free DNA extraction reagent and a plasma free DNA methylation conversion reagent, wherein the plasma free DNA methylation conversion reagent is bisulfite, and the test sample DNA is complete genome, cell-free DNA or circulating tumor DNA.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered as the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.
It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

Claims (9)

1. A CTDNA expression-based multidimensional liver cancer accurate molecular typing risk assessment method comprises the following steps, and is characterized in that:
s1, collecting a liver cancer precision molecule sample of a subject;
s2, preparing specific primers and probes for detecting or measuring the methylation state or level of the SEPT9 gene in the DNA of a test sample, and determining the expression molecule typing and collecting rate of gene data in the accurate molecular sample of the liver cancer;
s3, converting vaporized molecules of the sample to be tested into charged ions in a high-vacuum ion source, ionizing, leading out and focusing the charged ions, then enabling the charged ions to enter a mass analyzer, separating mass-to-charge ratios (mass-to-charge ratios, m/z) according to time sequence or spatial positions under the action of a magnetic field or an electric field, and finally detecting the charged ions by an ion detector to give molecular weight, molecular formula and structural information of the compound;
and S4, determining the risk of the multidimensional liver cancer accurate molecular typing and/or survival of the subject according to the molecular weight, molecular formula and structural information of the gene data in the sample.
2. The CTDNA expression-based multidimensional liver cancer molecular typing risk assessment method according to claim 1, characterized in that: the specific primers and probes for detecting the methylation state of the SEPT9 gene are selected from any one of the following three specific primer and probe combinations: specific primers SAE IN NO 1-2 and probe SAQ IN NO 3, specific primers SEM ID NO 4-5 and probe SEM ID NO 6, specific primers SEQ ID NO 7-8 and probe SEQ ID NO 9.
3. The CTDNA expression-based multidimensional liver cancer molecular typing risk assessment method according to claim 1, characterized in that: the gene based on DNA expression is set in polymorphic sites, wherein the polymorphic sites are N +1 sites selected through gene sites related to genetic diseases, and are represented by site A, site B and site N +1 in a table; the genotype is M +1 subtypes appearing in the population at the loci, and subtype A, subtype B and subtype M +1 are replaced in the table; the reference frequency is based on the respective proportion of the subtypes in Chinese population; risk is the relative risk for the polygenic disease according to different genotypes.
4. The CTDNA expression-based multidimensional liver cancer molecular typing risk assessment method according to claim 1, characterized in that: quantitative analysis is carried out on the expression quantity of the multidimensional liver cancer precise molecular gene based on CT DNA expression, and molecular typing is carried out on a sample through a set threshold, wherein the specific typing method comprises the following steps:
(1) quantitative analysis is carried out on the expression quantity of the liver cancer precision molecular gene by adopting VRPSolver software;
(2) performing typing model training on the expression quantities of Alexa Fluor dye, FITC, texas Red, cy3 and Cy5 genes of a multi-dimensional liver cancer accurate molecule sample with a clinically definite typing result by adopting a machine learning method, and performing subtype prediction of Next Generation Sequencing (NGS) on the sample based on a model;
(3) and further performing secondary classification on the patients predicted to be ER + and HER2+ positive; among them, patients with ER + positivity are further classified into luminal A and luminal B, and patients with HER2+ positivity are further classified into HER2 and luminal B-HER 2.
5. The CTDNA expression-based multidimensional liver cancer precision molecular typing risk assessment method according to claim 1, which is characterized in that: the gene data diagnostic in the sample can be protein microarray, ELISA diagnostic kit, immunohistochemistry (IHC) kit, next generation sequencing kit, real-time fluorescence quantitative PCR kit, gene chip or combination thereof;
the gene data diagnostic substance in the sample is a second-generation sequencing kit based on target sequencing or a real-time fluorescent quantitative PCR kit;
the gene data diagnostic in the sample is a second-generation sequencing kit based on targeting RNA-seq, which comprises one or more of a total RNA extraction reagent, a reverse transcription reagent and a second-generation sequencing reagent, and a primer with a nucleotide sequence shown in a table 4;
the second generation sequencing reagent is a reagent that can be customized for constructing a library Illumina of the targeted RNA-seq.
6. The CTDNA expression-based multidimensional liver cancer precision molecular typing risk assessment method according to claim 1, which is characterized in that: calculating an uncalibrated risk value, identified by a GT, that identifies the risk value as calculated by:
GT =0if RSU < 0; RS =20 × (RSU-6.7) if 0 is not less than RSU not more than 100; RS =100if RSU > 100, ensuring GT value to be controlled between 1-99 according to the formula GT = GT × 100/max (GT); and taking the maximum GT value of the sample with the measuring age being more than 5 years as a cut-off value according to data acquisition, wherein the sample with the measuring age being less than the cut-off value is a low-risk group.
7. The CTDNA expression-based multidimensional liver cancer molecular typing risk assessment method according to claim 1, characterized in that: in the gene data detection result chart of the liver cancer accurate molecule sample, 21-31 points are provided, each point represents the detection result of a tested person sample, wherein GTH and GTL are risk genotypes, and the gene molecules are subjected to the free radical damage when carrying the risk genotypes of molecular typing.
8. The CTDNA expression-based multidimensional liver cancer precision molecular typing risk assessment method according to claim 1, which is characterized in that: the risk assessment method further comprises a multidimensional liver cancer accurate molecule typing risk assessment system, the multidimensional liver cancer accurate molecule typing risk assessment system comprises a diagnosis module, the diagnosis module judges by using molecular weight, molecular formula and structural information of gene data in the sample, and the system further comprises a data input module, a data preprocessing module, a model training module and a model testing module.
9. The CTDNA expression-based multidimensional liver cancer molecular typing risk assessment method according to claim 1, characterized in that: the kit also comprises the following components: a plasma free DNA extraction reagent and a plasma free DNA methylation conversion reagent, wherein the plasma free DNA methylation conversion reagent is bisulfite, and the tested sample DNA is complete genome, cell-free DNA or circulating tumor DNA.
CN202211334077.3A 2022-10-28 2022-10-28 CTDNA expression-based multidimensional liver cancer accurate molecular typing risk assessment method Pending CN115627294A (en)

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* Cited by examiner, † Cited by third party
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TWI839307B (en) * 2023-05-06 2024-04-11 華聯生物科技股份有限公司 Methods of estimating disease progression and prognosis after treatment in liver cancer patients with a computer

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