TW201802247A - Method of detecting a risk of cancer - Google Patents

Abstract

Method of detecting a risk of cancer, which comprises: measuring the expression level of the cancer markers in a blood sample from a subject and a no cancer control group; calculating the basic expression level of each cancer marker in the no cancer control group; calculating the magnification of expression of each cancer marker by dividing the expression level of each cancer marker in the subject and individual in the no cancer control group by the basic expression level of each cancer marker; making the increased and decreased magnification have the same weight by using the natural logarithm; and calculating cancer risk index by summing the natural logarithm of magnification. The cancer risk index is used to evaluate a risk of cancer in a subject and the prognosis of subject's response to a cancer therapy.

Description

Methods to detect the risk of cancer

The invention relates to a method for detecting the risk of cancer; in particular, it relates to a method for detecting the risk of cancer by using 18 cancer gene markers.

In recent years, molecular medical technology has gradually developed cancer genetic markers for the diagnosis and prediction of cancer risk in blood samples. These markers can be used for early detection to improve patient survival, including before symptoms are clinically detectable and undergo treatment. Time and when recovering. Most of these technologies select cancer-related genomes and observe the differences between cancer patients and the general population. Usually, Pearson's correlation coefficient is used to establish the probability conversion mechanism or judgment mechanism, and then the subject's gene performance Introduce the aforementioned probability conversion mechanism or judgment mechanism for inspection.

However, the above method only uses the Pearson correlation coefficient, which is a linear correlation coefficient and is used to reflect the statistics of the degree of linear correlation between two variables. This method has the following disadvantages: (1) The method needs to consider whether the gene expression patterns are similar, and it is easy to misjudge that the gene expressions are in the normal fluctuation range but the gene expression patterns are similar, without considering the gene expression amount. Whether the height is reasonable; (2) the method ignores the differences among individuals in order to consider the distribution of the single gene in the population, and (3) the single gene has insufficient efficacy.

Therefore, the current cancer genetic markers used for the diagnosis and prediction of cancer risk in blood samples lack a method that uses the gene expression of a population as a base value to reduce individual effects, and weights genes.

In view of this, the present invention provides a method for detecting the risk of cancer, the steps comprising: (a) obtaining a specimen sample of a body fluid containing nucleic acid from a subject; (b) measuring a MRNA expression of a cancer gene marker set consisting of beta-globin (HBB), hemoglobin A1 (HBA1), and suppressor of cytokine signalling 3 , SOCS3), adrenomedullin (ADM), secretory leukocyte protease inhibitor-1 (SLP1), cluster of differentiation 68 (CD68), S100 calcium-binding protein P ( S100 calcium binding protein P (S100P), DNA damage inducible transcript 3 (DDIT3), cathepsin Z (CTSZ), Bcl-2-associated protein x, Bax), amyloid precursor protein (APP), transketolase (TKT), G protein-coupled estrogen receptor (GPER), heme-binding protein And heme-binding protein-related proteins (haptoglobin and haptoglobin-related (HPR | HP), FK506 Binding Protein 3 (FKBP3), Acid phosphatase 5 (ACP5), lysosomal protein transmembrane 4 alpha (LAPTM4A), and chemokine receptor (CXCR4); (c) calculation The subject's cancer risk index, the formula of which is sum [LN (individual gene expression / basic expression of individual genes)], includes: (i) dividing the expression of an individual gene by the expression of the basic gene of another gene to Calculate the performance magnification of each gene; (ii) take the natural logarithm of the magnification so that the ascent or declining multiple has the same weight; (iii) the person with the increased gene expression confirms that there is a risk of cancer, and the person with the decreased gene expression does not Take into account; and (iv) the weight of the individual whose genetic performance increases is the subject's cancer risk index; (d) the basic expression of the individual gene is a nucleic acid-containing body fluid taken from a non-cancer control group The geometric mean of the expression levels of the corresponding cancer genes in the specimen of the subject; and (e) calculating the cancer risk index of the non-cancer control group in step (c); wherein the cancer risk index based on the subject is lower than the non-cancer control index; The median cancer risk index of the control group represents that the subject has a low risk of developing cancer; based on the subject's cancer risk index is higher than the 99% confidence interval of the cancer risk index of the non-cancer control group (CI) means that the subject has a high risk of developing cancer.

The invention further provides a method for assessing a patient's risk of recurrence of cancer treatment. The steps include: (a) obtaining a specimen of a body fluid containing nucleic acid from a subject; (b) Measure the mRNA expression of a cancer gene marker set consisting of HBB, HBA1, SOCS3, ADM, SLP1, CD68, S100P, DDIT4, CTSZ, BAX, APP, TKT, GPER, HPR | HP , FKBP3, ACP5, LAPTM4A, and CXCR4; (c) Calculate the subject's cancer risk index, the formula is sum [LN (individual gene expression / individual gene expression)), including: (i) Divide the expression of individual genes by the basic expression of another gene to calculate the performance ratio of each gene; (ii) then take the natural logarithm of the ratio so that the ascending or descending multiple has the same weight; (iii) the gene expression The riser confirms that there is a risk of cancer, and the decline in gene expression is not considered; and (iv) the total weight of the individual's belonging is the cancer risk index of the subject; (d) the basic expression of the individual gene is taken as A geometric mean of the expression levels of corresponding cancer genes from a sample of a nucleic acid-containing body fluid in a non-cancer control group; and (e) calculating the cancer risk index of the non-cancer control group in step (c); Patient's cancer risk index is below The median cancer risk index of the non-cancer control group represents that the patient has a low risk of cancer recurrence; based on the patient's cancer risk index is higher than the 99% confidence interval (CI) of the cancer risk index of the non-cancer control group This means that the patient has a high risk of cancer recurrence; and the number of the non-cancer control group needs to be more than 20 people.

In one embodiment of the present invention, step (b) is measured by a microarray wafer, a real-time PCR, a northern blot method, or an in situ hybridization measurement.

In one embodiment of the present invention, the cancer is breast cancer, gastric cancer, bladder cancer or liver cancer.

In one embodiment of the present invention, the specimen is a peripheral blood mononuclear cell (PBMC) sample.

Therefore, the present invention provides a method for detecting the risk of cancer. The method includes providing a blood sample of a subject and a non-cancer control group, and measuring the expression of 18 cancer gene markers, wherein the 18 cancer gene markers include HBB, HBA1, SOCS3, ADM, SLP1, CD68, S100P, DDIT4, CTSZ, BAX, APP, TKT, GPER, HPR | HP, FKBP3, ACP5, LAPTM4A, and CXCR4, first use the non-cancer control group The basic expression of an individual gene, and then divide the individual gene expression of the individual and the non-cancer control group by the basic expression of the individual gene to calculate the performance ratio of each gene; take the natural logarithm to increase the multiple or The descending multiples have the same weight; However, the multiple of the logarithm is the cancer risk index, which is used to assess the risk of a cancer and the response and prognosis of a patient after receiving cancer treatment.

The embodiments of the present invention will be further described below. The examples listed below are intended to clarify the present invention and are not intended to limit the scope of the present invention. Any person skilled in the art will not depart from the spirit and scope of the present invention. Some changes and retouching can be done, so the scope of protection of the present invention shall be determined by the scope of the attached patent application.

The first figure is a cancer risk index data chart of a single test performed by a single individual in the method for detecting the risk of cancer of the present invention.

The second figure is a cancer risk index curve continuously detected by gastric cancer patients in the method for detecting the risk of cancer according to the present invention.

The third figure is a diagram of cancer risk index data for verifying liver cancer using the method for detecting the risk of cancer of the present invention using other wafers.

The invention provides a method for detecting the risk of cancer. The method includes providing a blood sample of a single body and a non-cancer control group, and measuring the performance of 18 cancer gene markers, and using the gene expression of the population as a base value to reduce Individual effects and gene weighted methods are used to calculate the cancer sub-index, which is used to assess the risk of a cancer and the response and prognosis of a patient after receiving cancer treatment.

definition

The term "genetic marker" as used herein refers to a change in the expression level of mRNA that can indicate the increased risk of a specific disease or abnormality.

As used herein, the term "gene expression level" refers to a protein or functional RNA product produced by a gene.

The term "non-cancer control group" as used herein refers to an individual who has been confirmed not to have cancer.

The term "microarray chip" as used herein refers to a microarray chip that can detect gene expression, which can be AFFYMETRIX, ILLUMINA, AGILENT, and Hualiansheng Micro-Array Wafers from Biotech Co., Ltd., but is not limited to this.

Example 1 Method for detecting the risk of cancer of the present invention

In the present invention, weighting is achieved by different expression amounts of different genes in peripheral blood mononuclear cells (PBMC) to achieve the purpose of non-cancer control group and cancer component group.

1.1 Collecting the genome

First, the present invention studies genes with special expression (overexpression or underexpression) in peripheral blood mononuclear cells when cancer occurs; and observes these genes in detection tools (mRNA microarray wafers or real-time polymerase chain reaction (real -time PCR) system or other mRNA analysis methods), if the gene expression falls within the non-linear detection range, decrease the weight (30%, 40%, 50%, 60%, 70% or other can get better The proportion of analysis results) is preferably reduced by 50%.

The eighteen gene markers most relevant to cancer of the present invention include beta-globulin (HBB), hemoglobin A1 (HBA1), and suppressor of cytokine-3 signalling 3 (SOCS3), adrenomedullin (ADM), secretory leukocyte protease inhibitor-1 (SLP1), cluster of differentiation 68 (CD68), S100 calcium binding protein P (S100 calcium binding protein P, S100P), DNA damage inducible transcript 3 (DDIT3), cathepsin Z (CTSZ), Bcl-2 associated X protein (Bcl-2-associated protein x, Bax), amyloid precursor protein (APP), transketolase (TKT), G protein-coupled estrogen receptor (GPER), heme Binding protein and heme-binding protein-related protein (haptoglobin and haptoglobin-related (HPR | HP), FK506 Binding Protein 3 (FKBP3) ), Acid phosphatase 5, ACP5, lysosomal protein transmembrane 4 alpha (LAPTM4A), and chemokine receptor (CXCR4) .

1.2 Calculate the basic expression of individual genes

In the present invention, the geometric mean (or weighted average of the performance distribution) of a single gene in different individuals in the non-cancer control group is used as the geometric mean of the genes.

1.3 Calculating Individual Cancer Risk Index

In the present invention, all genes of a single individual in all samples are divided by the basic gene expression of individual genes to obtain the magnification ratio of the gene. The magnification is taken as the natural logarithm so that the ascending magnification or the descending magnification has the same weight; inspect the genes of the ascending magnification and finally add The weight of a gene whose genetic performance is increased in the total individual is the individual's cancer risk index.

1.4 Grouping

The present invention calculates the cancer risk index distribution of the non-cancer control group and the cancer risk index distribution of the cancer group separately.

Single test for a single individual: The individual's cancer risk index is below the median (or average) of the normal distribution area of the non-cancer control group, which is a low-risk cancer, and the 99% confidence interval (CI) in the normal distribution area of the non-cancer control group. The above are at high risk for cancer, and the two are at intermediate risk for cancer.

Continuous testing of a single individual: The individual's cancer risk index appears continuously (2, 3, 4 or more times) in the normal distribution area of the non-cancer control group or the distribution area of cancer patients, which is the corresponding performance.

Example 2 Verification of the method for detecting the risk of cancer of the present invention

In order to confirm that the 18 gene markers can be accurately used for detecting, diagnosing cancer, and evaluating the effect of cancer treatment and the prediction of prognosis, the blood samples of the control group of cancer patients and non-cancer patients are used to measure the performance of the 18 gene markers. Calculate the cancer risk index for individual samples.

2.1 Research Sample

Control group samples: Peripheral blood mononuclear cell (PBMC) samples from 26 normal individuals diagnosed without cancer were collected by the present invention, and all of them obtained the consent of the donated samples. Each sample was tested at least twice, and the present invention performed a total of 55 test results.

Cancer samples: The present invention collects peripheral blood mononuclear cell (PBMC) samples from one patient with gastric cancer, two patients with bladder cancer, and one patient with breast cancer. These patients are respectively diagnosed before cancer recurrence, cancer recurrence diagnosis, treatment period or treatment. After the test, they all obtained the consent of the donor sample.

2.2 RNA extraction

The present invention uses a TRlzol® reagent (INVITROGEN, USA) to extract RNA according to a commercial operation manual. After the extraction, the concentration and purity of RNA are measured by a Nano Drop spectrophotometer (THERMO FISHER SCIENTIFIC, USA). The ratio of / OD280 and OD260 / OD230 estimates RNA purity, where the ratio of OD260 / OD280 is between 1.8 and 2.0; the ratio of OD260 / OD230 is not less than 2.0, which indicates that the purity of RNA is good; and analysis by microfluidic electrophoresis Instrument (model 2100, AGILENT, USA) evaluates RNA fragment size, qualitative and quantitative analysis.

2.3 Human Oligonucleotide DNA microarray (HOA) analysis

The present invention uses a human expression profile chip v6 (Hualian Biotechnology Co., Ltd., Taiwan) containing 32,679 probes, and each probe is designed as a human expression profile gene probe with a long-chain 60-base sense-strand Needles; 32,741 probes refer to the annotated genes of the internationally recognized RefSeq and Ensembl sequence databases, and also contain 938 control group probes.

2.4 Chip analysis

In the present invention, fluorescently labeled anti-strand RNA (aRNA) is prepared by using OneArray® amino-labeled aRNA amplification reagent (Hualian Biotechnology Co., Ltd., Taiwan) and Cy5 stain (Qiyi Medical Equipment Corporation, USA) via 1 μg of RNA. Subject matter. The hybridization system (Hualian Biotechnology Co., Ltd., Taiwan) was used to hybridize the fluorescently labeled anti-strand RNA (aRNA) target to the human expression profile chip in a hybridization buffer (Hualian Biotechnology Co., Ltd., Taiwan). on. After 16 hours of hybridization, the non-specifically bound target was washed, and then a DNA wafer scanner (model G2565CA, AGILENT, USA) was used for wafer scanning, and GenePix 4.1 software analysis (Molecular Device) was used to analyze Cy5 fluorescence signals. strength.

Each single sample is tested at least twice for reproducibility exceeding 0.975, and the resulting signal strength is input to the Rosetta Parser System (Rosetta Biosoft, Inc., USA) for data processing and standardization of the data for median The 75th percentile of the number. Error-weighted approaches were used to evaluate the errors of the samples at the same time. The comparison of the change in the expression level and the p value of the paired samples is calculated by evaluating the differentially expressed gene analysis. The criteria for determining the differential expression of the gene are the change in the expression level ≧ 2 or ≦ -2 and the p value <0.05. Further analysis.

2.5 Cancer Risk Index Analysis

The invention extracts the expression values of 18 genes in the non-cancer control group, and takes the geometric mean of the individual genes in the experimental population as the basic expression; as shown in Tables 1 and 2, all samples (including the non-cancer control group) And cancer group) All genes of a single individual are divided by the basic gene expression of each individual, and the magnification of the gene can be obtained. Take the natural logarithm of the magnification so that the ascending or descending magnification has the same weight. Finally, add the weight of the individual to the individual. Cancer Risk Index.

Those in the non-cancer control group's normal distribution area with a confidence interval of more than 99% (that is, the cancer risk index of 6.25) are high-risk cancer patients or cancer patients; those with a cancer risk index of 3 (median) or lower are low-risk cancer patients; Between the two (3 and 6.25) are moderate risk. Cancer risk index in cancer tissues: a. The cancer risk index before diagnosis of a gastric cancer patient was 8.52, 6.08, 8.75, 18.09, and 10.58. The cancer risk index before chemotherapy was 12.77, and the cancer risk index during chemotherapy was 7.90, 6.48, 6.31. , 5.27 and 8.45; b. The cancer risk index of two bladder cancer patients was 11.63 and 13.31, 10.24, and 12.30, respectively; c. The cancer risk index of a breast cancer patient during treatment was 11.88, and the cancer risk index one month after treatment was 2.40. The inspection interval of the above samples is 2 to 8 weeks.

The method for detecting the risk of cancer of the present invention As shown in the first figure, breast cancer, gastric cancer, and bladder cancer patients use the 18 gene markers of the present invention to obtain the best detection performance. The effective indicators are: 88.2% sensitivity and 91% specificity ( specificity), 90.4% accuracy.

The method for detecting the risk of cancer according to the present invention The cancer risk index results of gastric cancer patients are continuously detected. As shown in the second figure, the cancer risk index of gastric cancer patients before recurrence is diagnosed and before chemotherapy gradually increases with time, from chemotherapy to computer tomography. The scan showed that the cancer risk index of stable cancer status gradually decreased over time, confirming that cancer patients using the 18 gene markers of the present invention can be used to detect the presence of cancer and assess the patient's prognostic response to cancer treatment.

Example 3 Validation of the method for detecting the risk of cancer of the present invention using other wafers

The method of the present invention can be applied to the measurement of the mRNA expression of the 18 cancer gene marker sets in order to verify different experimental methods.

First, the present invention is via Shi M, Chen MS, Sekar K, Tan CK et al. A blood-based three-gene signature for the non-invasive detection of early human hepatocellular carcinoma. Eur J Cancer 2014 Mar; 50 (5): All gene expressions of the Affymetrix Human Genome U133 Plus 2.0 Array gene chip disclosed in the 928-36 paper, to obtain the mRNA expressions of the 18 cancer genes described in the present invention, and use the method described in Example 2 to carry out the cancer of the present invention. Risk index analysis.

The cancer risk index distribution is shown in the third figure, which contains 10 control group samples (as shown in Table 3) and 10 liver cancer samples (as shown in Table 4). The confidence interval is 99% in the normal distribution area of the non-cancer control group. (I.e., cancer risk index 2.9) Those above are high-risk cancer patients or cancer patients; those with a cancer risk index below 1.0 (median) are low-risk cancer patients; those between (1 and 2.9) are medium People at risk. Its effective indicators are: 100% sensitivity, 60% specificity, 80% accuracy. It was confirmed that the method of the present invention can be used to detect the risk of various cancers by using the mRNA expression obtained by different experimental methods.

Based on the above, the method for detecting the risk of cancer of the present invention uses the measurement of the expression of 18 cancer gene markers in blood samples of one individual and a non-cancer control group, and then uses the gene expression of the population as a base value The cancer risk index is calculated by reducing individual effects and weighting genes. The cancer risk index is used to assess the risk of a cancer and the response and prognosis of a patient after receiving cancer treatment. Therefore, the method of the present invention can be applied to preventive healthcare medicine to evaluate the potential risk of cancer; the evaluation of the effectiveness of cancer treatment to assist in assessing whether the current treatment is effective; and to be used to track the prognosis of cancer to assess the risk of cancer recurrence.

Claims (8)

A method for detecting the risk of cancer, comprising the steps of: (a) obtaining a specimen of a body fluid containing nucleic acid from a subject; (b) measuring the expression level of mRNA of a cancer gene marker set, the cancer gene marker The group consists of β-globin (HBB), hemoglobin A1 (HBA1), suppressor of cytokine signalling 3 (SOCS3), adrenomedullin (ADM), Secretory leukocyte protease inhibitor-1 (SLP1), surface antigen differentiation cluster 68 (CD68), S100 calcium binding protein P (S100P), DNA damage-induced metastasis Factor 3 (DNA damage inducible transcript 3, DDIT3), cathepsin Z (CTSZ), Bcl-2-associated protein x (Bax), amyloid precursor protein (amyloid precursor protein, APP), transketolase (TKT), G protein-coupled estrogen receptor (GPER), heme-binding protein And heme-binding protein-related proteins (haptoglobin and haptoglobin-related (HPR | HP), FK506 Binding Protein 3 (FKBP3), acid phosphatase 5, ACP5), lysosomal-associated protein across Membrane 4 alpha (lysosomal protein transmembrane 4 alpha, LAPTM4A) and chemokine receptor (CXCR4); (c) calculate the subject's cancer risk index, the formula is sum [LN (individual gene Expression amount / basic expression amount of individual genes)], including: (i) dividing the expression amount of another gene by the expression amount of another gene to calculate the expression ratio of each gene; (ii) taking the ratio as natural Logarithmic weights have the same weighting ascending or descending multiples; (iii) those with increased gene expression confirm that there is a risk of cancer, those with reduced gene expression are not considered; and (iv) the weights of individuals with increased gene expression are Is the subject's cancer risk index; (d) the basic expression of the individual gene is the geometric mean of the expression of the corresponding cancer gene from a sample of nucleic acid-containing body fluid taken from a non-cancer control group; and (e) step (c) Calculate the cancer risk index of the non-cancer control group; where the cancer risk index based on the subject is lower than the median cancer risk index of the non-cancer control group, it means that the subject has a low risk of developing cancer; based on A subject whose cancer risk index is higher than the 99% confidence interval (CI) of the cancer risk index of the non-cancer control group indicates that the subject has a high risk of developing cancer. The method according to item 1 of the patent application scope, wherein step (b) is measured by a microarray chip, a real-time PCR, a northern dot method, or an in situ hybridization measurement. The method of claim 1, wherein the cancer is breast cancer, gastric cancer, bladder cancer or liver cancer. The method according to item 1 of the scope of patent application, wherein the specimen is a peripheral blood mononuclear cell (PBMC) sample. A method for assessing a patient's risk of recurrence of cancer treatment. The steps include: (a) obtaining a specimen of a body fluid containing nucleic acid from a patient; and (b) measuring the mRNA expression of a cancer gene marker set. Cancer genetic markers are composed of HBB, HBA1, SOCS3, ADM, SLP1, CD68, S100P, DDIT4, CTSZ, BAX, APP, TKT, GPER, HPR | HP, FKBP3, ACP5, LAPTM4A, and CXCR4; (c) calculations The patient's cancer risk index, the formula is sum [LN (individual gene expression / basic expression of individual genes)], including: (i) taking the expression of another gene divided by the expression of the basic gene of another gene to calculate Show the magnification of each gene; (ii) Take the natural logarithm of the magnification so that the ascending or descending multiple has the same weight; (iii) those with increased gene expression confirm that there is a risk of cancer, and those with reduced gene expression are not allowed Consider; and (iv) the weight of the individual whose genetic performance is increased is the cancer risk index of the patient; (d) the basic expression of the individual gene is a specimen containing nucleic acid-containing body fluid taken from a non-cancer control group Sample phase Cancer gene should show the geometric mean amount; (e) to step (c) calculating cancer risk index of the non-cancer control group; The cancer risk index based on the patient is lower than the median cancer risk index of the non-cancer control group, which means that the patient has a low risk of cancer recurrence; the cancer risk index based on the patient is higher than the cancer of the non-cancer control group. A risk index with a 99% confidence interval (CI) indicates that the patient has a high risk of cancer recurrence. The method according to item 5 of the scope of patent application, wherein step (b) is measured by a microarray chip, real-time PCR, northern dot method or in situ hybridization. The method according to item 5 of the application, wherein the cancer is breast cancer, gastric cancer, bladder cancer or liver cancer. The method according to item 5 of the scope of patent application, wherein the specimen is a peripheral blood mononuclear cell (PBMC) sample.