CN114107498A - Colorectal cancer blood detection marker and application thereof - Google Patents
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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 a colorectal cancer or precancerous lesion detection kit. The marker has high sensitivity and specificity for colorectal cancer detection, and in a plasma sample, BEND5 and TFPI2 methylation combination detects that the sensitivity of the combined detection on colorectal cancer at the stage I/II is more than 71 percent, and the sensitivity of the combined detection on colorectal cancer at the stage III/IV is more than 89 percent, and meanwhile, the combination can effectively avoid false positive generated by other digestive tract cancer species such as gastric cancer, esophageal cancer and the like. 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
Colorectal cancer is one of the most common malignant tumors in China and even all over the world, and mainly comprises two main types of colon cancer and rectal cancer. The national cancer statistical data published by the national cancer center in 2019 show that 37.63 new cases of colorectal cancer are found in 2015 years in China, and 19.10 new cases of colorectal cancer-dead patients are located at the third position and the fifth position of the morbidity of malignant tumors. The prognosis of colorectal cancer is related to the severity of disease diagnosis, and the 5-year survival rate of colorectal cancer patients in the United states is 67%, while the current survival rate in China is only 31%. Between 2004 and 2013, the incidence of colorectal cancer in the united states has decreased by 3% per year, the overall mortality has decreased by an average of 2.7% per year, and the contribution of early screening therein has exceeded 50%.
Sporadic colorectal cancer progresses mainly in the order and pattern of normal-adenoma-carcinoma, early findings can significantly reduce mortality. Colonoscopy is the gold standard for colorectal cancer diagnosis, but its compliance in the average risk population is low due to the invasiveness and complexity of the bowel preparation process. Fecal Occult Blood Test (FOBT) and Fecal Immunochemical Test (FIT) are non-invasive, but they are not sufficiently sensitive, especially for stage I colorectal cancer and advanced adenoma. Due to the lack of symptoms of colorectal cancer at an early stage, the weak consciousness of cancer screening of the masses and the rejection of the existing detection technology, the early diagnosis rate of colorectal cancer in China is only about 10%, and more than 80% of patients reach the middle and late stages when the diagnosis is confirmed.
Aberrant DNA methylation may occur at a very early stage in colorectal cancer development and to date several colorectal cancer methylation biomarkers have been identified including SDC2, NDRG4, BMP3, VIM, SFRP2, Septine9 and the like.
Although many markers were found, there are very few, truly commercial products, the first fecal-based colorectal cancer test product, "Cologuard", which targets hemoglobin, KRAS mutations and two methylation genes (NDRG4 and BMP3), has a sensitivity of 92% for colorectal cancer and a specificity of 87%. Research shows that the acceptance of the general public for colorectal cancer blood detection is higher than that of colorectal cancer feces detection, and only Septine9 is a blood detection marker which is commercialized at present, and a Meta analysis (Diagnostic acquisition of methylated SEPT9 for blood-based color cancer detection: a systematic review and Meta-analysis. clin Transl Gastranterol.2017; 8(1): e216.) shows that the overall sensitivity of Septine9 for colorectal cancer detection is 72% and the specificity is 92%, when combined with FIT detection, the sensitivity is improved to 94% but the specificity is reduced to 68%, and in addition, the sensitivity of Septine9 for colorectal cancer at stage I is 45% and the sensitivity of Septine9 for polyp at stage I is 15%, so that the Septine9 is not suitable for colorectal cancer detection, and therefore, a methylation marker which is further suitable for blood and a combination thereof need to be searched.
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 purpose of the invention is realized by the following technical scheme:
a colorectal cancer blood detection marker which is a combination of BEND5 and TFPI2 genes.
One reagent for detecting the methylation level of the marker is a reagent for detecting the methylation level of BEND5 and TFPI2 genes. The methylation level detection method comprises at least one of the following methods: 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 melting curve, methylation-sensitive restriction endonuclease, and fluorescent quantitative PCR.
In some embodiments, the reagent for detecting the methylation level of the marker comprises primers and/or probes for detecting the methylation levels of BEND5 and TFPI2 genes. Furthermore, the reagent also comprises a primer and/or a probe for detecting the methylation level of an internal reference gene, and the internal reference gene is preferably ACTB gene.
In some embodiments, the sequences of primers, probes to detect methylation levels of the BEND5, TFPI2, or ACTB genes are as follows:
BEND5 upstream primer: GTAGCGCGTAGTAGACGTTGTT the flow of the air in the air conditioner,
BEND5 downstream primer: CCCAACACCGTAACGAAACT the flow of the air in the air conditioner,
BEND5 Probe: CGGGTTTCGGTCGGGTAGTTTAG, respectively;
TFPI2 upstream primer: TTAGGTTTCGTTTCGGCGG the flow of the air in the air conditioner,
TFPI2 downstream primer: ACAACCCCAAAAAACGAACGAAATC the flow of the air in the air conditioner,
TFPI2 probe: TCTACTCCAAACGACCCGAATACC, respectively;
ACTB upstream primer: AAGGTGGTTGGGTGGTTGTTTTG the flow of the air in the air conditioner,
ACTB downstream primer: AATAACACCCCCACCCTGC the flow of the air in the air conditioner,
ACTB probe: GGAGTGGTTTTTGGGTTTG are provided.
Furthermore, the probe is a Taqman probe, and the 5 'end of the Taqman probe is provided with a reporter group and the 3' end of the Taqman probe is provided with a quenching group.
The marker or the reagent is applied to the preparation of a colorectal cancer or precancerous lesion detection kit. Preferably, the precancerous condition is colorectal adenoma, such as tubular adenoma, villous adenoma, and villous tubular adenoma. In other embodiments, the precancerous condition further includes adenomatosis, inflammatory bowel disease-associated dysplasia, traditional serrated adenomas, and broad-base serrated adenomas/polyps, among others. Precancerous lesions include, but are not limited to, other intestinal diseases.
A kit for detecting colorectal cancer or precancerous lesions comprises the reagent.
The detection sample of the reagent, the application or the kit is a blood sample, and comprises 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 is obviously higher than that of a normal tissue sample in a colorectal cancer tissue sample in a previous study, and further finds that ITGA4 and BEND5 have high sensitivity and specificity on colorectal cancer/precancerous lesion in a plasma sample, and the single-gene methylation detection has sensitivity of more than 50% on advanced adenoma, sensitivity of more than 70% on colorectal cancer and specificity of more than 95%. Furthermore, the invention finds that the combination detection of BEND5 gene and TFPI2 gene can effectively improve sensitivity and maintain high specificity, in a plasma sample, the combination detection of BEND5 and TFPI2 methylation has the sensitivity of more than 71% to colorectal cancer at stage I/II and more than 89% to colorectal cancer at stage III/IV, and meanwhile, the combination can effectively avoid false positive generated by other digestive tract cancer species 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 intended to further illustrate the present invention and should not be construed as limiting the present invention, and any other changes, modifications, substitutions, combinations and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and shall be included within the scope of the present invention.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1
1. Extraction of DNA template:
when the sample used was a whole blood sample, a blood/cell/tissue genomic DNA extraction kit (catalog No. DP304) from Tiangen Biochemical technology (Beijing) Ltd was used, and the volume of the whole blood sample used was 1 ml, and the extraction procedure was as described in the kit instructions.
When the sample is a plasma sample, the plasma cfDNA is extracted by using nucleic acid extraction reagent (heihan instruments 20210740) of the armysson life science ltd, the volume of the used plasma is 600 μ L, and the specific operation is described in the kit specification.
2. Conversion of sulphites
And (3) carrying out bisulfite conversion on the extracted whole blood genome DNA or plasma cfDNA, wherein the nucleic acid conversion kit is a nucleic acid purification reagent (20500843, Ehan instruments) of Wuhan Amison Life technologies, Ltd.
3. Methylation specific PCR reactions
And carrying out methylation specific PCR reaction on the DNA converted by the bisulfite to detect the methylation state of a single target gene or a gene combination in a sample, wherein ACTB is used as an internal reference gene, a reporter group at the 5 'end of a target gene Taqman probe is FAM, a quenching group at the 3' end is MGB, a reporter group at the 5 'end of the ACTB Taqman probe is VIC, and a quenching group at the 3' end is BHQ 1. The primer probe sequences of each target gene and the reference gene are shown in Table 1.
TABLE 1
In a 50. mu.L PCR reaction system, the names and the amounts of the added reagents are as follows: buffer (5 ×), 5 μ L; dNTPs (2.5 mM each of dATP, dTTP, dCTP and dGTP), 10-15 mM; adding 6-12 mu M of each of the upstream and downstream primers of the target or the internal reference; taqman probes for a target or an internal reference, each of which is added with 6-12 mu M; hotspot DNA polymerase, 1.5-5U; DNA template, 5. mu.L; the volume-deficient fraction was made up with purified water. Three replicates were made for each sample.
Quality control: the negative control and the positive control are synchronously detected in each detection, the negative control template is purified water, the positive control template is artificially synthesized plasmid containing a target amplification area, and the concentration is 103Copy/microliter.
The PCR reaction conditions are shown in Table 2 below.
TABLE 2
4. Data analysis
Ct value reading: after the PCR is finished, adjusting a base line, setting a fluorescence value of the sample in the primary PCR before the minimum Ct value is advanced by 1-2 cycles as a base line value, and setting a threshold value at the inflection point of an S-shaped amplification curve.
And under the conditions that the negative control is not amplified, the positive control has obvious index increment and the Ct value of the reference gene in the sample to be detected is less than or equal to 35, the experiment is effective, the Ct value of the target gene of the sample can be read, otherwise, the experiment is invalid, the detection needs to be carried out again, and the Ct value (the 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 experiment is effective.
Results analysis and interpretation methods: when a single target gene is detected, if the Ct value of the target gene in at least 2 of 3 multiple wells of the 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, the combination of target genes is considered methylation negative in the sample when all target genes are methylation negative, otherwise the target genes are considered methylation positive in the sample. And comparing the methylation detection result of the sample with the pathological result, and calculating the sensitivity and specificity of the methylation detection. The sensitivity is the proportion of methylation positivity in the sample with positive pathological result, and the specificity is the proportion of methylation negativity in the sample with negative pathological result.
Experimental example 1
Plasma samples of colorectal advanced adenoma patients, 30 plasma samples of colorectal cancer patients and 30 plasma samples of healthy persons who donated blood sources were collected from southern hospital, Wuhan university, and anonymized for all patients. DNA extraction from plasma samples, bisulfite conversion of the extracted DNA, methylation-specific PCR reaction using the converted DNA as a template were carried out according to the method described in example 1, and the methylation levels of ITGA4 gene, ZNF829 gene and BEND5 gene in each type of plasma samples were examined, and the sensitivity of these three genes in adenoma and cancer samples and the specificity in healthy human samples were calculated, respectively, and the results are shown in Table 3.
TABLE 3 sensitivity and specificity data of three genes in adenoma, cancer, healthy plasma
As can be seen from Table 3, the methylation of the three genes ITGA4, ZNF829 and BEND5 has a sensitivity of more than 50% for 30 colorectal plasma samples and no less than 70% for 50 colorectal plasma samples, the specificity of the two genes ITGA4 and BEND5 is better and more than 95% for 30 healthy human plasma samples, and the specificity of the ZNF829 gene is lower and 83.33% for 30 healthy human plasma samples, so that a larger amount of sample verification needs to be performed on the specificity of the three genes.
Experimental example 2
60 whole blood samples of healthy persons from donated blood sources (different from the healthy persons in experimental example 1) were collected from the southern hospital in Wuhan university, and anonymization was performed on all patients. DNA extraction from whole blood samples, bisulfite conversion of the extracted DNA, methylation-specific PCR reaction using the converted DNA as a template were carried out as described in example 1, and the methylation levels of ITGA4 gene, ZNF829 gene and BEND5 gene in 60 samples were examined, and the methylation positive rates of these three genes in healthy human samples were counted, respectively, to calculate the specificity, and the results are shown in Table 4.
TABLE 4 specificity of three genes in 60 healthy human whole blood samples
| Name of gene | Number of positive | Specificity of |
| ITGA4 | 5 | 91.67％ |
| ZNF829 | 12 | 80.00％ |
| BEND5 | 6 | 90.00％ |
As shown in Table 4, the methylation of the three genes ITGA4, ZNF829 and BEND5 showed slightly lower specificity in the whole blood samples of 60 healthy persons compared to the data in Experimental example 1, but remained generally the same. The specificity of ITGA4 and BEND5 is still not lower than 90%, and the specificity of ZNF829 is only 80%, indicating that ZNF829 gene may be interfered by some substances in blood, thereby causing false positive in healthy human samples.
Based on the above results, two genes, i.e., ITGA4 and BEND5, were selected for further verification.
Experimental example 3
85 plasma samples of colorectal cancer patients were collected from southern hospital, Wuhan university, 21 plasma samples of stage I/II colorectal cancer patients, 64 plasma samples of stage III/IV colorectal cancer patients, and 54 plasma samples of healthy persons (the cancer sample and the healthy person sample in Experimental example 3 are different from those in Experimental example 1), and anonymization treatment was performed on all patients. DNA extraction of plasma samples, bisulfite conversion of the extracted DNA, methylation-specific PCR reaction using the converted DNA as a template, examination of the methylation levels in various types of plasma samples in the combined assay of ZNF829 and BEND5 genes and the combined assay of these two genes with one of the BMP3, NDRG4, SDC2, TFPI2 and Septin 9 genes, and calculation of the sensitivity in cancer samples and the specificity in healthy human samples in the combined assay, respectively, were carried out as described in example 1, and the results are shown in Table 5.
TABLE 5 Combined detection of sensitivity and specificity data in cancer, healthy plasma
As can be seen from table 5, the joint methylation detection of the two genes ITGA4 and BEND5, and the joint detection of one of the two genes and one of the five genes NDRG4, BMP3, SDC2, TFPI2 and Septin 9 have great differences in sensitivity and specificity, and in total: the detection sensitivity of three combinations of ITGA4+ TFPI2, BEND5+ TFPI2 and BEND5+ Septin 9 in 21 cases of colorectal cancer at stage I/II is the highest, and all the detection sensitivities are more than 70%; the detection sensitivity of the four combinations of ITGA4+ TFPI2, ITGA4+ Septin 9, BEND5+ TFPI2 and BEND5+ Septin 9 in 64 colorectal cancer samples at stage III/IV is the highest, and all the detection sensitivities are more than 85%; however, the specificity of the combination of ITGA4+ Septin 9 and BEND5+ Septin 9 in the plasma of 54 healthy people is lower, and both are lower than 90%. Taken together, the sensitivity and specificity of both the combination ITGA4+ TFPI2 and BEND5+ TFPI2 were superior.
Experimental example 4
10 cases of plasma of gastric cancer patients and 10 cases of plasma of esophageal cancer patients were collected at the southwestern hospital of Wuhan university, and anonymization treatment was performed on all patients. The methylation positive rates of the combinations of the two genes ITGA4+ TFPI2 and BEND5+ TFPI2 in gastric and esophageal cancer patients were examined by performing DNA extraction of plasma samples, bisulfite conversion of the extracted DNA, and methylation-specific PCR using the converted DNA as a template according to the method described in example 1, as shown in Table 6.
TABLE 6 methylation Positive rates of Gene combinations in gastric and esophageal cancer samples
As shown in table 6, the combination of ITGA4+ TFPI2 was detected as methylation positive in 4 out of 10 gastric cancer samples and in 2 out of 10 esophageal cancer samples; the combination BEND5+ TFPI2 showed only 1 of 10 samples of gastric cancer, but no methylation in esophageal cancer samples. The above results indicate that the combination of ITGA4+ TFPI2 may be susceptible to interference from other digestive tract cancers when detecting colorectal cancer, while BEND5+ TFPI2 is less affected and still maintains high specificity under interference from other digestive tract samples.
Sequence listing
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Claims (10)
1. A marker for blood detection of colorectal cancer, characterized in that: the marker is the combination of BEND5 and TFPI2 genes.
2. A reagent for detecting the methylation level of the marker of claim 1.
3. The reagent according to claim 2, characterized in that: the methylation level detection method comprises at least one of the following methods: 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 melting curve, methylation-sensitive restriction endonuclease, and fluorescent quantitative PCR.
4. The reagent according to claim 2, characterized in that: comprises primers and/or probes for detecting methylation levels of BEND5 and TFPI2 genes.
5. The reagent according to claim 4, characterized in that: also comprises a primer and/or a probe for detecting the methylation level of the internal reference gene.
6. The reagent according to claim 5, characterized in that: the reference gene is ACTB gene.
7. The reagent according to claim 4 or 6, characterized in that: the sequences of primers and probes for detecting the methylation level of BEND5, TFPI2 or ACTB genes are as follows:
BEND5 upstream primer: GTAGCGCGTAGTAGACGTTGTT the flow of the air in the air conditioner,
BEND5 downstream primer: CCCAACACCGTAACGAAACT the flow of the air in the air conditioner,
BEND5 Probe: CGGGTTTCGGTCGGGTAGTTTAG, respectively;
TFPI2 upstream primer: TTAGGTTTCGTTTCGGCGG the flow of the air in the air conditioner,
TFPI2 downstream primer: ACAACCCCAAAAAACGAACGAAATC the flow of the air in the air conditioner,
TFPI2 probe: TCTACTCCAAACGACCCGAATACC, respectively;
ACTB upstream primer: AAGGTGGTTGGGTGGTTGTTTTG the flow of the air in the air conditioner,
ACTB downstream primer: AATAACACCCCCACCCTGC the flow of the air in the air conditioner,
ACTB probe: GGAGTGGTTTTTGGGTTTG are provided.
8. Use of a marker according to claim 1 or a reagent according to any one of claims 2 to 7 in the manufacture of a kit for the detection of colorectal cancer or pre-cancerous lesions.
9. A colorectal cancer or precancerous lesion detection kit, characterized in that: comprising an agent according to any one of claims 2 to 7.
10. The reagent according to any one of claims 2 to 7, the use according to claim 8 or the kit according to claim 9, characterized in that: the detection sample is a blood sample and comprises whole blood, serum, plasma and blood cells.
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