CN114107498B - Colorectal cancer blood detection marker and application thereof - Google Patents
Colorectal cancer blood detection marker and application thereof Download PDFInfo
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Abstract
The invention discloses a colorectal cancer blood detection marker and application thereof, belonging to the field of tumor detection. The colorectal cancer blood detection marker is a combination of BEND5 and TFPI2 genes, and the marker or a methylation detection reagent thereof can be used for preparing colorectal cancer or precancerous lesion detection kits. The marker has high sensitivity and specificity in colorectal cancer detection, the sensitivity of the combination detection of BEND5 and TFPI2 methylation in a plasma sample is more than 71% on colorectal cancer in stage I/II, the sensitivity of the combination detection of BEND5 and TFPI2 methylation in colorectal cancer in stage III/IV is more than 89%, and meanwhile, false positives generated by other digestive tract cancers such as gastric cancer, esophageal cancer and the like can be effectively avoided by the combination. The invention provides a new idea for noninvasive diagnosis of colorectal cancer.
Description
Technical Field
The invention relates to the field of tumor detection, in particular to a colorectal cancer blood detection marker and application thereof.
Background
Abnormal DNA methylation may occur at very early stages of colorectal cancer occurrence, and to date, several colorectal cancer methylation biomarkers have been identified, including SDC2, NDRG4, BMP3, VIM, SFRP2, septin 9, and the like.
Although many markers were found, there was little real commercialization, the first fecal-based colorectal cancer detection product, "cologard", whose detection targets were hemoglobin, KRAS mutations, and two methylation genes (NDRG 4 and BMP 3), was 92% sensitive to colorectal cancer, with a specificity of 87%. Studies have shown that the current commercial blood test markers for colorectal cancer have a higher acceptance than colorectal cancer fecal testing, only septin 9, a Meta assay (Diagnostic accuracy of methylated SEPT for blood-based colorectal cancer detection: a systematic review and Meta-analysis, clin trans-gastro-entol 2017;8 (1): e216) result in a septin 9 overall sensitivity of the colorectal cancer test of 72%, a specificity of 92%, a sensitivity increase of 94% when tested in combination with FIT, but a specificity decrease of 68%, and septin 9 sensitivity of 45% for stage I colorectal cancer, a polyp sensitivity of 15%, making it less suitable for use in screening tests for colorectal cancer, thus requiring further searching for methylation markers suitable for blood and combinations thereof.
Disclosure of Invention
The invention aims to solve the problem that the existing colorectal cancer blood detection effect is not ideal, and provides a colorectal cancer blood detection marker and application thereof.
The aim of the invention is achieved by the following technical scheme:
a colorectal cancer blood detection marker that is a combination of BEND5 and TFPI2 genes.
A reagent for detecting methylation level of the above marker is a reagent for detecting methylation level of BEND5 and TFPI2 genes. The methylation level detection method comprises at least one of the following steps: methylation-specific PCR, bisulfite sequencing, methylation-specific microarray, whole genome methylation sequencing, pyrosequencing, methylation-specific high performance liquid chromatography, digital PCR, methylation-specific high resolution dissolution profile, methylation-sensitive restriction endonuclease, and fluorescent quantitative PCR.
In some embodiments, the reagent for detecting methylation levels of the above-described markers comprises primers and/or probes for detecting methylation levels of the BEND5 and TFPI2 genes. Further, the reagent further comprises a primer and/or a probe for detecting the methylation level of a reference gene, preferably an ACTB gene.
In some embodiments, the sequences of the primers, probes to detect the methylation level of the BEND5, TFPI2 or ACTB genes are as follows:
band 5 upstream primer: GTAGCGCGTAGTAGACGTTGTT the number of the individual pieces of the plastic,
band 5 downstream primer: CCCAACACCGTAACGAAACT the number of the individual pieces of the plastic,
BEND5 probe: CGGGTTTCGGTCGGGTAGTTTAG;
TFPI2 upstream primer: TTAGGTTTCGTTTCGGCGG the number of the individual pieces of the plastic,
TFPI2 downstream primer: ACAACCCCAAAAAACGAACGAAATC the number of the individual pieces of the plastic,
TFPI2 probe: TCTACTCCAAACGACCCGAATACC;
ACTB upstream primer: AAGGTGGTTGGGTGGTTGTTTTG the number of the individual pieces of the plastic,
ACTB downstream primer: AATAACACCCCCACCCTGC the number of the individual pieces of the plastic,
ACTB probe: GGAGTGGTTTTTGGGTTTG.
Further, the probe is a Taqman probe, wherein the 5 'end of the probe is provided with a reporter group, and the 3' end of the probe is provided with a quenching group.
The application of the marker or the reagent in preparing a colorectal cancer or precancerous lesion detection kit. Preferably, the precancerous lesion is a colorectal adenoma, such as tubular adenoma, villous adenoma, and villous tubular adenoma. In other embodiments, the precancerous lesions also include adenomatosis, inflammatory bowel disease-related dysplasia, traditional serrated adenomas, and broad-base serrated adenomas/polyps, and the like. Precancerous lesions include, but are not limited to, other intestinal diseases.
A colorectal cancer or precancerous lesion detection kit comprising the above-described reagent.
The detection sample of the reagent, the application or the kit is a blood sample, including whole blood, serum, plasma and blood cells.
The invention has the advantages and beneficial effects that: the inventor finds that the methylation level of three genes of ITGA4, ZNF829 and BEND5 in colorectal cancer tissue samples is obviously higher than that of normal tissue samples in early-stage researches, and further finds that ITGA4 and BEND5 have high sensitivity and specificity to colorectal cancer/precancerous lesions in plasma samples, and the sensitivity of single gene methylation detection to adenoma in the progressive stage is more than 50%, the sensitivity to colorectal cancer is more than 70% and the specificity is more than 95%. Furthermore, the invention discovers that the combination detection of the BEND5 gene and the TFPI2 gene can effectively improve the sensitivity, and simultaneously keep high specificity, the sensitivity of the combination detection of the BEND5 and the TFPI2 methylation in a plasma sample is more than 71 percent on colorectal cancer of stage I/II, the sensitivity of the combination detection of the BEND5 and the TFPI2 methylation on colorectal cancer of stage III/IV is more than 89 percent, and meanwhile, the combination can effectively avoid false positives generated by other digestive tract cancer types such as gastric cancer, esophageal cancer and the like, and has high specificity. The invention provides a new idea for noninvasive diagnosis of colorectal cancer.
Detailed Description
The following examples are provided to further illustrate the present invention and should not be construed as limiting the invention, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principles of the invention are intended to be equivalent substitutes.
The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
Example 1
1. Extraction of DNA template:
when the sample used was a whole blood sample, the whole blood sample volume was 1 ml using a blood/cell/tissue genomic DNA extraction kit (catalog number: DP 304) from Tiangen Biochemical technology (Beijing) limited, and the extraction procedure was as described in the kit specification.
When the sample used was a plasma sample, cfDNA extraction was performed using a nucleic acid extraction reagent from marchand Ai Misen life technologies, ltd (jaw arm 20210740), the volume of plasma used was 600 μl, for specific operation see kit instructions.
2. Conversion of sulfite
The extracted whole blood genome DNA or plasma cfDNA is subjected to bisulphite conversion, and the nucleic acid conversion kit is a nucleic acid purification reagent (Ehan mechanical preparation 20500843) of Wohan Ai Misen life technology Co., ltd, and specific experimental operation is described in the specification of the kit.
3. Methylation-specific PCR reactions
And (3) carrying out methylation-specific PCR reaction on the DNA subjected to bisulfite conversion to detect the methylation state of a single target gene or a combination of genes in a sample, wherein ACTB is used as an internal reference gene, a reporter group at the 5 'end of a Taqman probe of the target gene is FAM, a quencher group at the 3' end of the Taqman probe of the target gene is MGB, a reporter group at the 5 'end of the ACTB is VIC, and a quencher group at the 3' end of the Taqman probe of the target gene is BHQ1. Primer probe sequences of the respective target genes and the reference genes are shown in Table 1.
TABLE 1
In a 50. Mu.L PCR reaction system, the names and the amounts of the reagents added are as follows: buffer (5×), 5 μl; dNTPs (dATP, dTTP, dCTP and dGTP each 2.5 mM), 10-15mM; upstream and downstream primers of the target or internal reference, each added at 6-12. Mu.M; taqman probes for the target or reference, each added at 6-12. Mu.M; hotspot DNA polymerase, 1.5-5U; DNA template, 5 [ mu ] L; the portion of insufficient volume is made up with purified water. Three wells were made for each sample.
And (3) quality control: the negative control and the positive control are synchronously detected during each detection, the negative control template is purified water, the positive control template is artificial plasmid containing target amplification region, and the concentration is 10 3 Copy/microliter.
The PCR reaction conditions are shown in Table 2 below.
TABLE 2
4. Data analysis
Ct value reading: after the PCR is completed, the baseline is adjusted, the fluorescence value before the minimum Ct value of the sample in one PCR is advanced by 1-2 cycles is set as the baseline value, and the threshold value is set at the inflection point of the S-shaped amplification curve.
Under the conditions that the negative control is not amplified, the positive control has obvious index increase period, and the Ct value of the reference gene in the sample to be detected is less than or equal to 35, the test is effective, the Ct value of the target gene of the sample can be read, otherwise, the test is ineffective, the detection is required to be carried out again, and the Ct value (Ct value of the target gene/gene combination) of the amplification curve corresponding to each sample FAM signal is read under the condition that the test is effective.
Result analysis and interpretation method: when detecting a single target gene, if Ct value of the target gene in at least 2 holes in 3 multiple holes of a sample is less than or equal to 40, the target gene is considered to be methylation positive in the sample, otherwise, the target gene is considered to be methylation negative in the sample. When detecting a combination of target genes, when all target genes are methylation negative, then the combination of target genes is considered methylation negative in the sample, otherwise the target genes are considered methylation positive in the sample. Comparing the methylation detection result of the sample with the pathological result, and calculating the sensitivity and specificity of the methylation detection. Sensitivity is the proportion of methylation positives in samples with positive pathological results, and specificity is the proportion of methylation negatives in samples with negative pathological results.
Experimental example 1
30 cases of colorectal cancer patients and 50 cases of colorectal cancer patients were collected from a south-middle-arm hospital at the university of Wuhan, 30 cases of healthy human plasma samples from blood donation sources were subjected to anonymization treatment for all patients. The sensitivity of the three genes in adenoma and cancer samples and the specificity in healthy human samples were calculated by extracting DNA from plasma samples, performing bisulfite conversion on the extracted DNA, performing methylation-specific PCR reaction using the converted DNA as a template, and examining the methylation levels of the ITGA4 gene, the ZNF829 gene and the BEND5 gene in various plasma samples, respectively, as shown in Table 3.
TABLE 3 sensitivity and specificity data for three genes in adenoma, cancer, healthy plasma
As can be seen from Table 3, the methylation of three genes, ITGA4, ZNF829 and BEND5, has the sensitivity of over 50% for 30 colorectal blood plasma samples, the sensitivity of not less than 70% for 50 colorectal blood plasma samples, the specificity of the two genes, ITGA4 and BEND5, in 30 healthy human blood plasma samples is better and is more than 95%, the specificity of the ZNF829 gene in 30 healthy human blood plasma samples is lower and is 83.33%, and a larger sample size verification is required for the specificity of the three genes.
Experimental example 2
Samples of healthy human whole blood from blood donation were collected from a southern hospital at the university of martial arts (60 cases, different from healthy persons in experimental example 1), and anonymized treatment was performed on all patients. Whole blood sample DNA extraction was performed as described in example 1, bisulfite conversion was performed on the extracted DNA, methylation-specific PCR reaction was performed using the converted DNA as a template, and methylation levels of the ITGA4 gene, the ZNF829 gene and the BEND5 gene in 60 samples were examined, and the methylation positive rates of these three genes in healthy human samples were counted, and the specificity was calculated, and the results were shown in Table 4.
Table 4 specificity of three genes in 60 healthy human Whole blood samples
As shown in Table 4, the methylation of three genes of ITGA4, ZNF829 and BEND5 was slightly reduced in 60 healthy human whole blood samples compared with the data in experimental example 1, but remained consistent overall. The specificity of ITGA4 and BEND5 is still not less than 90% and the specificity of ZNF829 is only 80%, indicating that ZNF829 gene may be interfered by some substances in blood, resulting in false positive in healthy human samples.
Based on the above results, two genes, ITGA4 and BEND5, were selected for further validation.
Experimental example 3
85 cases of colorectal cancer patient plasma samples were collected from the south-middle-arm university Hospital, 21 cases of colorectal cancer patient plasma samples at stage I/II and 64 cases of colorectal cancer plasma samples at stage III/IV, 54 cases of healthy human plasma samples (the cancer sample and the healthy human sample in Experimental example 3 are different from Experimental example 1), and anonymization treatment was performed on all patients. Plasma sample DNA extraction was performed as described in example 1, bisulfite conversion was performed on the extracted DNA, methylation-specific PCR reaction was performed using the converted DNA as a template, and the joint detection of ZNF829 and BEND5 genes, and the methylation levels of these two genes in each type of plasma sample when each of these genes was jointly detected with one of BMP3, NDRG4, SDC2, TFPI2, and Septin 9 genes, were examined, and the sensitivity in cancer samples at the time of joint detection, and the specificity in healthy human samples were calculated, respectively, and the results are shown in table 5.
TABLE 5 sensitivity and specificity data for the combined detection of two genes in cancer, healthy plasma
As can be seen from Table 5, the joint methylation detection of the two genes ITGA4 and BEND5, and one of the two genes, has a large difference in sensitivity and specificity when combined with one of the five genes NDRG4, BMP3, SDC2, TFPI2 and Septin 9, in total: the detection sensitivity of the three combinations of ITGA4+ TFPI2, BEND5+ TFPI2 and BEND5+ Septin 9 in 21 cases of colorectal cancer in stage I/II is highest and is above 70%; the four combinations of ITGA4+TFPI2, ITGA4+Septin 9, BEND5+TFPI2 and BEND5+Septin 9 have the highest detection sensitivity in 64 colorectal cancer samples in stage III/IV, which is above 85 percent; however, the combination of ITGA4+Septin 9 and BEND5+Septin 9 had lower specificity in 54 healthy human plasma, which was less than 90%. Taken together, the sensitivity and specificity of the two combinations itga4+tfpi2 and bend5+tfpi2 are both superior.
Experimental example 4
10 cases of plasma from gastric cancer patients and 10 cases of plasma from esophageal cancer patients were collected in the south-middle-arm university Hospital and all patients were anonymized. Plasma sample DNA extraction, bisulfite conversion of the extracted DNA, methylation-specific PCR reaction using the converted DNA as a template were performed as described in example 1, and methylation positive rates of two gene combinations, itga4+tfpi2 and bend5+tfpi2, in gastric cancer patients and esophageal cancer patients were examined, as shown in table 6.
Table 6 methylation positive Rate of Gene combinations in gastric and esophageal cancer samples
As shown in table 6, the combination itga4+tfpi2 was detected as methylation positive in 4 out of 10 gastric cancer samples, and was detected as methylation positive in 2 out of 10 esophageal cancer samples; while the combination of bend5+tfpi2 was methylation-positive in only 1 out of 10 gastric cancer samples and was methylation-positive in none of the esophageal cancer samples. The results show that the combination of ITGA4 and TFPI2 is likely to be easily interfered by other digestive tract cancers when colorectal cancer is detected, and BEND5 and TFPI2 is less affected, and can still keep higher specificity under the interference of other digestive tract samples.
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Claims (9)
1. A colorectal cancer blood detection marker, characterized by: the marker is a combination of BEND5 and TFPI2 genes;
the sequences of the primers and probes for detecting the methylation level of the marker are as follows:
band 5 upstream primer: GTAGCGCGTAGTAGACGTTGTT the number of the individual pieces of the plastic,
band 5 downstream primer: CCCAACACCGTAACGAAACT the number of the individual pieces of the plastic,
BEND5 probe: CGGGTTTCGGTCGGGTAGTTTAG;
TFPI2 upstream primer: TTAGGTTTCGTTTCGGCGG the number of the individual pieces of the plastic,
TFPI2 downstream primer: ACAACCCCAAAAAACGAACGAAATC the number of the individual pieces of the plastic,
TFPI2 probe: TCTACTCCAAACGACCCGAATACC.
2. A reagent for detecting the methylation level of the marker of claim 1, characterized in that: comprising primers and/or probes for detecting methylation levels of the BEND5 and TFPI2 genes; the sequences of the primers and probes for detecting the methylation levels of the BEND5 and TFPI2 genes are as follows:
band 5 upstream primer: GTAGCGCGTAGTAGACGTTGTT the number of the individual pieces of the plastic,
band 5 downstream primer: CCCAACACCGTAACGAAACT the number of the individual pieces of the plastic,
BEND5 probe: CGGGTTTCGGTCGGGTAGTTTAG;
TFPI2 upstream primer: TTAGGTTTCGTTTCGGCGG the number of the individual pieces of the plastic,
TFPI2 downstream primer: ACAACCCCAAAAAACGAACGAAATC the number of the individual pieces of the plastic,
TFPI2 probe: TCTACTCCAAACGACCCGAATACC.
3. A reagent according to claim 2, wherein: the methylation level detection method comprises at least one of the following steps: methylation-specific PCR, bisulfite sequencing, methylation-specific microarray, whole genome methylation sequencing, pyrosequencing, methylation-specific high performance liquid chromatography, digital PCR, methylation-specific high resolution dissolution profile, methylation-sensitive restriction endonuclease, and fluorescent quantitative PCR.
4. A reagent according to claim 2, wherein: primers and/or probes for detecting the methylation level of a reference gene are also included.
5. The reagent according to claim 4, wherein: the reference gene is ACTB gene.
6. The reagent according to claim 5, wherein: the sequences of the primers and probes for detecting the methylation level of the ACTB gene are as follows:
ACTB upstream primer: AAGGTGGTTGGGTGGTTGTTTTG the number of the individual pieces of the plastic,
ACTB downstream primer: AATAACACCCCCACCCTGC the number of the individual pieces of the plastic,
ACTB probe: GGAGTGGTTTTTGGGTTTG.
7. Use of a marker as claimed in claim 1 or a reagent as claimed in any one of claims 2 to 6 in the manufacture of a kit for colorectal cancer or precancerous lesions detection.
8. A colorectal cancer or precancerous lesion detection kit, characterized in that: comprising the agent of any one of claims 2-6.
9. The reagent according to any one of claims 2 to 6 or the kit according to claim 8, wherein: the detection sample is a blood sample, including whole blood, serum, plasma and blood cells.
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