TWI682175B - Method of gastric cancer diagnosis - Google Patents

Abstract

The present invention provides a method for diagnosing gastric cancer using biomarkers, which includes the steps of (1) obtaining a blood sample of a body; (2) detecting the concentration of sex hormone-binding globulin (SHBG) in the sample; And (3) judging the content of SHBG in the specimen, where the concentration of SHBG in the specimen is higher than the concentration of SHBG in a control, then the individual has gastric cancer.

Description

A method for diagnosing gastric cancer

The present invention relates to the field of a method for diagnosing gastric cancer, and particularly to the field of a method for diagnosing gastric cancer using biomarkers in blood.

In 2012, gastric cancer was one of the top five cancers in the world. Men are twice as likely to get stomach cancer as women. GC is also the third leading cause of cancer deaths, causing about 800,000 deaths in 2012. Current treatment strategies for gastric cancer have a five-year survival rate of approximately 30% in the first to third stages of gastric cancer, and even less than 5% in the fourth stage.

In order to reduce the social burden caused by gastric cancer, in Asian countries with a high incidence of gastric cancer, such as Japan and South Korea, gastric cancer will be screened in advance. In the 1960s, Japan had used barium radiography and endoscopy on a large scale to perform gastric cancer screening nationwide, and claimed to reduce the mortality rate of gastric cancer by 50%. In South Korea, people over the age of 40 have also been established to conduct biennial gastric cancer screening.

However, barium contrast or endoscopy is an invasive detection method, which is not only expensive, but also requires experienced radiologists and endoscopy physicians. Therefore, these methods are not suitable for countries with limited resources.

Furthermore, using these methods for large-scale screening of gastric cancer is not cost-effective, especially in areas where the incidence of gastric cancer is not high. Therefore, it is important to develop cheap, simple, low-invasive, sensitive, and specialized screening tools. The "blood biomarker" has the above advantages.

However, the number of clinically available blood biomarkers for gastric cancer is very limited. There is currently no recommended blood biomarker for the diagnosis of early gastric cancer. Although there are some indexes, such as serum pepsinogen, serum carcinoembryonic antigen, CA125 index, etc., can be used for the management of gastric cancer, but the sensitivity and specificity of these biomarkers are highly variable, which often affects judgment.

At present, more and more studies use proteomics to develop suitable blood biomarkers, because proteomics can show the dynamic relationship between the genome and the environment, and it is directly related to the pathogenesis of the disease. Therefore, the present invention utilizes proteomic technology to develop novel blood gastric cancer biomarkers.

The purpose of the present invention is to develop a suitable blood gastric cancer biomarker by using the technology of protein body, and the biomarker is verified as a valid and sensitive blood biomarker for early diagnosis of gastric cancer through verification and confirmation steps.

In order to achieve the above object, the present invention is achieved by the following techniques. The invention provides a gastric cancer detection kit, comprising: a reagent for detecting the concentration of sex hormone-binding globulin (SHBG) and a control substance, wherein the control substance is derived from a non-gastric cancer sample.

The reagent for detecting the concentration of SHBG is an ELISA detection reagent or an immuno-ink dot detection reagent.

The present invention also provides a method for diagnosing gastric cancer, which includes the following steps: (1) obtaining a blood sample of a subject; (2) detecting the expression level of SHBG protein in the sample; and (3) comparing the SHBG in the sample The amount of protein expression is the same as the amount of SHBG protein expression of a reference substance, where the amount of SHBG protein expression of the sample is higher than the amount of SHBG protein expression of the reference substance, then the individual has gastric cancer.

The SHBG contains a SEQ ID NO: 1 amino acid sequence IALGGLLFPASNLR.

The blood sample includes plasma, serum, whole blood, or a combination thereof; preferably, the blood sample is plasma.

The expression level of SHBG protein in the sample is at least 1.5 times that of the control group. Preferably, the expression level of SHBG protein in the sample is twice or more than that of the control group.

The method provided by the present invention can be used in the detection kit provided by the present invention. The method includes the steps of: (1) obtaining a blood sample of a body; (2) detecting the SHBG protein expression level in the sample using the reagent for detecting the SHBG performance level; and (3) comparing the sample with the control substance In the SHBG performance level in the sample, where the SHBG expression level of the sample is higher than the SHBG performance level of the control, the individual has gastric cancer.

The blood sample includes plasma, serum, whole blood, or a combination thereof; preferably, the blood sample is plasma.

The SHBG contains a SEQ ID NO: 1 amino acid sequence IALGGLLFPASNLR.

The invention uses proteomics to verify and confirm the performance of SHBG by ELISA to verify SHBG as a blood biomarker for early diagnosis of gastric cancer.

The invention can also use stomach tissue as a test specimen for diagnosing gastric cancer.

experimental design

The invention takes blood samples from 76 gastric cancer patients and 86 healthy people. The age of gastric cancer patients is 40-90; the type of gastric cancer is adenocarcinomatous type. All samples are divided into three independent groups: Discovery, verification, and validation. Screening group (cohort): select 24 patients according to the patient's age, course, and gender, and divide into four groups of different periods (group), each group has 6 groups, and the ratio of men to women in each group 2:1. The control group consisted of six men and three women. Verification group: There are 24 patients and nine control subjects. The western blot method was used to verify the candidate blood biomarkers. Confirmation group: contains 50 patients (22 of which are younger than 60 years old from the screening group and verification group) and 68 control subjects, using ELISA to test the blood in the verification group Confirm the biomarker. The experiment outline design is shown in Figure 1.

Protein concentration determination

In this example, the Pierce BCA test was used to quantify protein concentration. Each sample and BSA were repeated in triplicate. The samples and BSA and 100 μ L were mixed reaction reagent, and then incubated at 37 ° C for 30 minutes environment. The ELISA reader reads samples at a wavelength of 562 nm. A linear standard curve was prepared using BSA, and the sample OD value was substituted into the standard curve formula to calculate the protein concentration of the sample to be measured.

Immune depletion of albumin ( Immunodepletion of albumin )

In this example, HSA CaptureSelect ^™ Proteomics Depletion Product was used to deplete albumin. Healthy subjects and patients take 400 μ g plasma protein samples were resuspended in 50 μ L PBS, then mixed with 100 μ L HSA antibody, and then incubated in rotary shaking 4 ° C for one hour. The incubated mixture was transferred to a Multi-spin separation column kit and centrifuged at 13,500 g at 4°C for one minute, and then an albumin-depleted plasma sample was collected. In order to confirm the efficiency of albumin consumption, the original sample and the sample after albumin consumption were run side by side at the same time on SDS-PAGE. The result is shown in Figure 2. It can be seen that the albumin has been completely depleted.

Protein digestion

In this example, the In-Solution Tryptic Digestion and Guanidination Kit was used to digest the albumin-depleted sample into peptides. After the reduction step, mixing 15 μ L of digestion buffer, 1.5 μ L of reducing buffer and 3 μ L of albumin depleted sample. Ultrapure water mixture was adjusted to 27 μ L was then incubated at 95 ° C 5 min. Take 3 μ L of buffer containing alkyl iodide acetyl amine (alkylation buffer) was added to the mixture, protected from light and incubated at room temperature for 20 minutes. In the digestion step, the trypsin activation 1 μ L were added to the mixture, and then incubated at 37 ° C 3 hours. Subsequently plus 1 μ L trypsin activated and incubated at 30 ° C overnight. The final mixture was 32 μL .

Afterwards, a ZipTip C18 micro column (ZTC18S960, Merck Millipore, California) was used for desalting treatment to avoid excess salt in the sample after protein digestion and digestion to interfere with the subsequent mass spectrometry analysis.

LC-MS/MS

Peptide sample was injected into the capture column and then separated in a chromatography column (25 cm x 75 μm id BEH130 C18); mobile phase gradient: mobile phase increased from 0% to 85% in 120 minutes (buffer A: 0.1 % Formic acid in water; buffer B: 0.1% formic acid in acetonitrile); flow rate: 300nL/min.

Then use the Orbitrap mass spectrometer to capture the top ten strong peptide ions of the signal. Full scan positive ion mode; scanning range: m/z 350-1600; resolution: 240,000. All Orbitrap measurements are performed in the context of lock mass to obtain higher accuracy.

Protein identification

Use PEAKS Studio to analyze Orbitrap raw data. In the protein database Uniprot-Human search and identify the protein of the mass spectrometry analysis result. Label-free quantification (LFQ) was used to quantify protein intensity.

Protein separation

SDS-PAGE was used to separate the proteins in the original plasma.

Immunoblot

In the verification cohort1, the performance of four proteins in plasma were individually measured by immunoblotting: Apolipoprotein C1 (APOC1), gelsolin (GSN), SHBG, and complement component C4-A (C4A). Finally, the performance of the four proteins in each sample was measured.

ELISA

Use the Human SHBG Quantikine ELISA Kit to confirm SHBG.

data analysis

Mann–Whitney U- test was used to analyze the relative abundance/intensity of the protein in the control group and the gastric cancer patient group from LFQ and immunoblot. Kruskal–Wallis was used to analyze the dot signals in four different patient groups and three different age groups. Calculate the average standard deviation of plasma SHBG performance measured by ELISA.

result

All the operation objects are divided into three independent groups for searching biomarkers. Table 1 is the characteristic data of the subjects in each experimental group. Table 1 Characteristic data of subjects in an experimental group

Screening of candidate biomarkers ( Discovery )

The invention detects biomarkers in the blood of 24 gastric cancer patients and 9 healthy subjects in the Discovery group, especially the biomarkers in plasma. Each plasma sample was trypsinized after albumin consumption, followed by desalting, and then run LS-MS/MS. The LS-MS/MS raw data of 24 gastric cancer patients and 9 healthy subjects were imported into PEAKS 7 software and analyzed by LFQ. The results showed that the four proteins (C4A, GSN, SHBG, and APOC1) with higher MS signals in the patient group also had relatively denser abundances in their hot zone maps, as shown in Figure 3.

verification( Verification )

Quantify APOC1, GSN, SHBG, and C4A in the blood samples of the verification group (Verification cohort) by Western blot method to confirm the MS analysis results. In this example, the sample with the lowest concentration of SHBG in the control group was selected as the comparison standard. Among the four proteins, only SHBG protein consistently showed high performance in the patient group of the validation group (as shown in Figure 4). From Figure 4A to Figure 4D, it can be seen that only SHBG protein has a significant difference in performance between the control group and the patient group, and the SHBG performance in the patient group is at least 1.5 times that of the control group, so SHBG was selected for further confirmation . The patients were divided into the first stage to the fourth stage according to the number of gastric cancer stages. As shown in the results of FIG. 4E, the performance of SHBG in the four stages was higher than that of the control group. Therefore, it was initially confirmed that SHBG can be used as a biomarker for gastric cancer detection.

ELISA Confirm in the blood SHBG Performance( Validation )

In the Discovery group and the verification group of gastric cancer patients, the expression level of SHBG protein in plasma has a consistent high expression level. Therefore, in this example, in the validation cohort (control group: 68; patient group: 50), the absolute quantitative measurement of plasma SHBG concentration by ELISA was used. Recently, it has been found that the SHBG performance of women has a gradual decline before the age of 60, and then steadily increases at the age of 60, while the SHBG performance of men will increase with age. In this confirmation example, the results of ELISA are stratified according to gender and age, and only subjects younger than 60 years of age will be counted, which is to reduce the cause of change. Perform the A Priori test according to the data of the verification group (mean and standard deviation). Setting of prior test conditions: calculated Effect size d = 1.1316042; α err prob = 0.05; Power (1-β err prob) = 0.95. These values ensure that at least 22 samples from each group are included in the analysis and become an effective analysis.

The blood SHBG concentration of gastric cancer patients is about twice as high as that of the control group (as shown in Figure 5A). After age stratification (20-40 years old, and 41-60 years old), the SHBG concentration remained significantly higher in the two age groups of patients (as shown in Figure 5B). When grouped by gender, the SHBG concentration also has a significantly high performance in the male and female patient groups (as shown in Figure 5C). Finally, it was confirmed that compared with the control group, the SHBG concentration also had a significantly high expression level in each gastric cancer stage, but the SHBG concentration did not increase with the increase of gastric cancer stages (as shown in Figure 5D).

After the above detailed description, it has been fully shown that the present invention has progress in implementation, and is a new invention that has not been seen before, which fully meets the requirements of the invention patent, and the application is submitted according to law. However, the above is only the preferred embodiment of the present invention, and it should not be used to limit the scope of the implementation of the creation, that is, the equal changes and modifications made according to the patent scope of the invention, should all fall within the scope of the invention patent .

Figure 1 shows a summary of the experimental design of the present invention.

Figure 2 shows a comparison chart of albumin consumption before and after consumption. N11d, N6Fd, P2d, and P42d: samples after albumin consumption; N11, N6F, P2, and P42: original samples.

Figure 3 shows the hot zone diagram of C4A, GSN, SHBG, and APOC1 compared to the reference.

Figure 4 shows the Western blot method results of APOC1, GSN, SHBG, and C4A in the patient group and control group; A: APOC1 signal strength of the blot in the patient group and control group; B: GSN in the patient Ink dot signal intensity of group and control group; C: Ink dot signal intensity of SHBG in patient group and control group; D: Ink dot signal intensity of C4A in patient group and control group; E: SHBG in four gastric cancer stages Western blots of numbers and control groups.

Figure 5 shows the results of ELISA analysis of SHBG performance in the cohort; A: Comparison of SHBG performance between gastric cancer patients and control group; B: Comparison of SHBG performance between age-stratified gastric cancer patients and control group; C : Comparison of SHBG performance of gastric cancer patients with sex group and control group; D: Comparison of SHBG performance of four gastric cancer stages with control group.

<110> Taipei Medical University <120> A method for diagnosing gastric cancer <130> 1071007 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 14 <212> PRT <213> Homo sapiens <400> 1 Ile Ala Leu Gly Gly Leu Leu Phe Pro Ala Ser Asn Leu Arg 1 10 5 5 5 5 10 10

Claims (9)

A gastric cancer detection kit includes: a reagent for detecting the expression level of sex hormone-binding globulin (SHBG) protein and a control product, wherein the control product is from a non-gastric cancer sample. A method for diagnosing gastric cancer, comprising the following steps: (1) detecting the expression level of sex hormone-binding globulin (SHBG) in a blood sample; and (2) comparing the performance of SHBG in the blood sample The amount is the same as the SHBG expression of a reference substance, where the SHBG expression of the blood sample is higher than the SHBG expression of the reference substance, the individual has gastric cancer, and the control substance is from a sample of a non-gastric cancer individual. The method as described in item 2 of the patent application scope, wherein the SHBG comprises a SEQ ID NO: 1 amino acid sequence IALGGLLFPASNLR. The method according to item 2 of the patent application scope, wherein the blood sample comprises plasma, serum, whole blood, or a combination thereof. The method as described in item 2 of the patent application scope, wherein the detection method in step (2) includes immuno-ink dot method and/or ELISA. The method as described in item 2 of the patent application scope, wherein the SHBG expression amount in the blood sample is at least 1.5 times the SHBG expression amount of the control substance. The method as described in item 2 of the patent application scope, wherein the individuals in steps (1) and (3) are human beings. The method as described in item 7 of the patent application scope, wherein the human age is not greater than 60 years old. The method as described in item 7 of the patent application scope, wherein the gender of the human being is the same gender.

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