JP7462035B2 - Probe composition for detecting 11 types of cancer - Google Patents

Description

This application relates to a composition for detecting cancer gene methylation, in particular a probe composition that specifically recognizes DNA sequences after bisulfite treatment, and its application to detecting changes in free DNA methylation levels in 11 types of tumors, including esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, and endometrial cancer, based on a high-throughput sequencing (NGS) method.

High-throughput sequencing (NGS) technology is a revolutionary innovation in the field of modern genomics research, which can perform sequence analysis on tens to millions of DNA molecules simultaneously, marking the arrival of the post-genomic era. By controlling the depth of sequencing, different goals such as de novo sequencing and resequencing can be achieved, and genome, transcriptome and methylome sequences can be analyzed through different initial processes.

At present, the technologies of clinical gene detection are mainly polymerase chain reaction (PCR), fluorescence in situ hybridization (FISH) and gene chip technology. PCR equipment is low in price, highly sensitive, simple and fast to operate, and has a high clinical popularity, but due to technical limitations, it can only detect a small number of genes at the same time. FISH has high sensitivity, but is difficult to operate. The throughput of gene chips is higher than both of the above, and can detect a large number of genes at the same time. However, the drawback is that it can only detect known genes or mutations, has low accuracy, and has high false positives. NGS technology has the advantages of high throughput (detecting a large number of known and unknown genes and mutations at the same time), accurate results (higher probability than gene chips), fast detection speed, low detection cost per gene, etc., and is currently gradually applied to fields such as clinical disease detection and monitoring. With the further reduction of sequencing costs in the future, NGS will inevitably gradually replace other high-throughput technologies such as gene chips.

At present, the total price of general whole genome sequencing is high, and if the sequencing depth is increased to detect rare mutations, the final price is difficult for consumers to bear. Therefore, capture sequencing of target sequences has become mainstream, and the technology designs capture probes for genomic regions of interest according to the demand for detection, enriches the target fragment DNA through the principle of complementary hybridization, and then performs NGS detection. Such a policy can be flexibly formulated according to the purpose of research or detection, and can select only a small amount of gene regions, increase the sequencing depth, and effectively discover the mutation status of the target region, with very high sensitivity and accuracy.

In the process of cancer development and progression, genetic information undergoes a series of changes, including DNA mutations, insertions/deletions, chromosome structure mutations, copy number variations, and changes in epigenetic information. In the process of cancer progression, DNA sequence mutations occur randomly, and only when mutations occur in important growth control genes will they lead to the development of malignant tumors. Many gene expression abnormalities are derived from epigenetic changes, generally from changes in DNA methylation levels. Research has shown that changes in gene methylation levels occur earlier than gene mutations, and by tracking and detecting changes in gene methylation, the development of cancer can be predicted early. In recent years, with the development of genomics, the epigenomes of more than 30 types of cancer have been studied. The results show that although DNA methylation is not dominant in any of various cancers, changes in gene methylation modification modes can of course change the development trends of cells and tumor phenotypes, which have an important impact on the development and progression of many cancers.

At the beginning of 2018, the Cancer Genomic Profile (TCGA) published 27 comprehensive analysis articles, and conducted the most comprehensive pan-cancer genome analysis to date on 30 types of cancer data over more than 10 years. After collating and analyzing various data such as chromosomal mutations, DNA methylation, RNA and protein, it was found that 33 anatomical types of cancer can be divided into 28 subtypes based on molecular characteristics. A molecular subtype includes 25 types of cancer in the traditional sense. This result indicates that cancers originating from different organs have common molecular characteristics. At the same time, cancers originating from the same organ may have different genomic profiles. As can be seen, in the near future, the development of cancer screening and diagnostic markers will inevitably introduce more pan-cancer concepts, and we should not only study anatomical cancers, but also develop pan-cancer markers that can study cancer at the molecular level and cover a certain molecular typing.

Liquid biopsy is a method of in vitro diagnosis, which adopts non-invasive blood testing to monitor circulating tumor cells (CTCs) or circulating tumor DNA (ctDNA) released into the blood by tumors or metastatic lesions. This technology can effectively reduce the harm caused by invasion, and realize sampling of each part of the tumor and all metastatic lesions, overcome the heterogeneity of the tumor (however, the standard tissue biopsy currently used only reflects the characteristics of a certain part of the tumor), and realize real-time monitoring, has higher sensitivity, and can predict the site where the lesion occurs based on genomic information, which may effectively extend the survival time of patients. Based on these advantages, liquid biopsy can be used in the aspects of early diagnosis of tumors, auxiliary determination of disease stage, prognosis and recurrence monitoring, drug guidance, etc. Currently, the most commonly used liquid biopsy is free DNA.

Free DNA (cfDNA) is endogenous DNA that exists in the circulating blood and is released outside the cells and partially degraded. Research has shown that during the development and development of tumor tissue, the tumor cells release DNA into the plasma after apoptosis, and after decomposition, form free tumor DNA (ctDNA). The molecular genetic characteristics of ctDNA (e.g., gene mutation, microsatellite instability, and tumor suppressor gene promoter methylation, etc.) are consistent with tumor tissue DNA. In the early screening and detection of multiple cancers, peripheral blood collection is more convenient than other clinical detection means, is easily distributed in core hospitals, and is easily accepted by asymptomatic people due to its non-invasive characteristics. Therefore, detecting changes in the ctDNA methylation level in plasma is one of the important means for early screening and diagnosis of multiple cancers.

Target sequence capture technology can be combined with NGS to monitor cfDNA mutations and changes in methylation levels, enabling applications in various areas such as early tumor screening, susceptibility gene monitoring, companion diagnosis, personalized medicine, and prognosis monitoring. At present, several companies in China and abroad have launched cancer detection panels of different scales and for different application scenarios, and some of the panels have obtained FDA or CFDA approval numbers. For example, FoundationOne CDx launched by Foundation Medicine can cover 324 genes, IMPACT launched by Memorial Sloan Kettering Cancer Center (MSK) can cover 468 types of cancer-related genes, Burning Rock Biotech has launched the "Human EGFR/ALK/BRAF/KRAS Gene Mutation Joint Detection Kit," and Novogene has launched the "Human EGFR, KRAS, BRAF, PIK3CA, ALK, ROS1 Gene Mutation Detection Kit." Singlera Genomics also launched a product called "ColonES" in 2018 to detect methylation levels in colorectal cancer.

Therefore, in response to the current lack of detection products for multiple cancers and technical limitations, the present application provides a probe composition that can be used for early screening of 11 types of cancer, including esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, and endometrial cancer. The probe composition 1) can be used for early screening of asymptomatic people in a non-invasive manner and for prognostic detection of cancer patients, reducing the harm caused by invasive detection; 2) increases the sequencing depth, making the breadth of detected genes superior to conventional technologies and products, and has the advantages of high throughput, fast detection speed, and low detection cost for each gene; 3) can achieve sampling of each part of the tumor and all metastatic lesions, overcoming the heterogeneity of the tumor; 4) has higher sensitivity and accuracy, can realize real-time monitoring, and may even be able to predict the site where a lesion will occur based on genomic information, effectively prolonging the survival time of patients.

Specifically, the present application relates to the following:
1. A probe composition comprising a probe targeting a specific region of any of esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer and endometrial cancer, the cancer-specific region being selected from any of Seq ID No. 1-62.
2. The probe composition according to claim 1, further comprising a probe targeting any of the pan-cancer specific regions, wherein the pan-cancer specific regions are selected from any of Seq ID Nos. 63-64.
3. The probe composition according to claim 1, further comprising a probe targeting any one of tissue-specific regions, the tissue-specific region being selected from any one of Seq ID Nos. 65-117.
4. The probe composition according to item 3, wherein Seq ID Nos. 66-67, 79, 81-82, 97-102, and 116-117 in the tissue-specific region are tissue-specific target regions of the esophagus.
5. The probe composition according to item 3, wherein Seq ID Nos. 81, 82, and 97-102 in the tissue-specific region are tissue-specific target regions of the stomach.
6. The probe composition according to item 3, wherein Seq ID Nos. 83, 87-90, and 110 in the tissue-specific region are tissue-specific target regions of the colorectum.
7. The probe composition according to item 3, wherein Seq ID Nos. 65, 70-72, 76, 91-92, and 103-104 in the tissue-specific region are lung tissue-specific target regions.
8. The probe composition according to item 3, wherein Seq ID Nos. 73-74 and 107 in the tissue-specific region are tissue-specific target regions for the liver.
9. The probe composition according to item 3, wherein Seq ID No. 111-115 in the tissue-specific region is a pancreatic tissue-specific target region.
10. The probe composition according to item 3, wherein Seq ID Nos. 80, 84, 93, and 95 in the tissue-specific region are tissue-specific target regions of the prostate.
11. The probe composition according to item 3, wherein Seq ID Nos. 78 and 84-86 in the tissue-specific region are tissue-specific target regions of the mammary gland.
12. The probe composition according to claim 3, wherein in the tissue-specific region, Seq ID Nos. 77, 96, and 105 are tissue-specific target regions for the ovary.
13. The probe composition according to item 3, wherein Seq ID Nos. 68-69, 75, and 109 in the tissue-specific region are tissue-specific target regions of the uterine cervix.
14. The probe composition according to claim 3, wherein Seq ID Nos. 94, 106, and 108 in the tissue-specific region are tissue-specific target regions of the endometrium.
15. The probe composition according to any one of paragraphs 1 to 14, comprising a low methylation probe hybridizing to the specific region where the bisulfite converted CG is not methylated, and a high methylation probe hybridizing to the specific region where the entire bisulfite converted CG is methylated.
16. The probe composition according to item 15, wherein each probe has a length of 40 to 60 bp.
17. The probe composition according to item 16, wherein the length of each probe is 45-56 bp, preferably 50-56 bp, and more preferably 50 bp.
18. The probe composition according to claim 15, characterized in that the hypomethylated probe comprises any of the probes Seq ID No. 118-214 targeting a cancer-specific region, any of the probes Seq ID No. 215-216 targeting a pan-cancer-specific region, and any of the probes Seq ID No. 217-274 targeting a tissue-specific region.
19. The probe composition according to claim 15, characterized in that the hypermethylated probe comprises any of the probes Seq ID No. 275-371 targeting cancer-specific regions, any of the probes Seq ID No. 372-373 targeting pan-cancer-specific regions, and any of the probes Seq ID No. 374-431 targeting tissue-specific regions.
20. A kit comprising the probe composition according to any one of items 1-19.
21. Use of the probe composition according to any one of items 1 to 19 in the manufacture of a kit for detecting esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer and endometrial cancer.

The present application further provides a method for detecting 11 types of cancer, including esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, and endometrial cancer, by utilizing the probe composition.

The present application also provides a method for simultaneously detecting changes in the methylation levels of the above 11 types of cancer using the kit.

Due to the limitations of conventional cancer detection technologies, there is a need to develop probe compositions that have the following advantages:
1. Liquid biopsy belongs to non-invasive tumor detection and can be applied to asymptomatic people and patient populations where tissue samples cannot be obtained.
2. It can simultaneously detect changes in the methylation levels of 11 common types of cancer in China, covering more than 80% of the cancer-affected population.
3. To increase the accuracy of detection, the average sequencing depth is more than 5000X for each cancer.
4. Subjects can complete screening for all high-incidence cancers in one go, improving the detection efficiency, and the average price of each marker is lower than that of traditional single-marker detection on the market.
5. For companies, it allows them to complete screening of major cancers with only one penalty, saving the cost of probe synthesis, simplifying the experimental flow, and facilitating the operation of experimenters.
6. In principle, it may be used to monitor the prognosis of cancer patients.

FIG. 1 shows an operational flow of the present invention.

The present application provides a probe composition for cancer gene methylation detection. Based on a high-throughput sequencing (NGS) method, it detects the changes in the methylation levels of free DNA in 11 types of tumors, including esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, and endometrial cancer. It can simultaneously detect the changes in the methylation levels of 11 types of common cancers in a non-invasive manner, with high sensitivity and accuracy, deep sequencing depth, and low cost, and is applicable to asymptomatic people and patient populations who cannot obtain tissue samples, and cancer monitoring of the prognosis of cancer patients.

Definitions Unless specifically limited elsewhere herein, all other technical and scientific terms used herein are commonly understood by one of ordinary skill in the art to which this application belongs.

The probe is a single-stranded or double-stranded DNA with a length of tens to hundreds or even thousands of base pairs, and can hydrogen bond (hybridize) with complementary unlabeled single-stranded DNA or RNA in the sample to be measured to form a double-stranded complex (hybrid) by utilizing the high accuracy of molecular denaturation, annealing, and complementary base pairing. After washing away the unpaired probe, the hybridization reaction result can be detected using a detection system such as autoradiography or an enzyme-linked reaction. In this application, the region that is complementary bound or hybridized to the probe is the specific target region. Multiple probes are combined into a probe composition.

A cancer-specific region means that there is a significant difference in the methylation level of the region in a few cancer types compared to normal control tissue.

A pan-cancer specific region means that there is a significant difference in the methylation level of the region compared to normal control tissue in most cancer types.

A tissue-specific region means that the methylation level of that region in a specific tissue is significantly different compared to other tissues.

DNA methylation refers to the occurrence of the methylation process of the fifth carbon atom on cytosine in CpG dinucleotides, which, as a stable modified state, can be inherited by the action of DNA methyltransferase to the newly born progeny DNA during the DNA replication process, and is an important epigenetic mechanism. During DNA methylation, the methylation of gene promoter regions can cause transcriptional silencing of tumor suppressor genes, and thus is closely related to the occurrence of tumors. Abnormal methylation includes hypermethylation of tumor suppressor genes and DNA repair genes, hypomethylation of repetitive DNA, and loss of imprinting of some genes, which are associated with the occurrence of various tumors.

As used herein, Panel refers to the probe composition used herein.

The technical solutions of the present application are described in detail below.
In one specific embodiment of the present application, the probe composition is a probe targeting any cancer-specific region of esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, and endometrial cancer, the cancer-specific region including a probe selected from any of Seq ID No. 1-62, a probe targeting any pan-cancer specific region selected from any of Seq ID No. 63-64, and a probe targeting any tissue-specific region selected from any of Seq ID No. 65-117. In the tissue-specific region, Seq ID No. 66-67, 79, 81-82, 97-102, 116-117 are tissue-specific target regions of the esophagus, Seq ID No. 81, 82, 97-102 are tissue-specific target regions of the stomach, and Seq ID No. 83, 87-90, 110 are tissue-specific target regions of the colorectum.

In one specific embodiment of the present application, the probe composition is a probe that targets a specific region of any of esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, and endometrial cancer, and the cancer-specific region includes a probe selected from any of Seq ID No. 1-62, a probe that targets any of the pan-cancer specific regions selected from any of Seq ID No. 63-64, and a probe that targets any of the tissue-specific regions selected from any of Seq ID No. 65-117. Seq ID No. 65, 70-72, 76, 91-92, 103-104 in the tissue-specific region are lung tissue-specific target regions.

In one specific embodiment of the present application, the probe composition is a probe that targets a specific region of any of esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, and endometrial cancer, and the cancer-specific region includes a probe selected from any of Seq ID No. 1-62, a probe that targets any of pan-cancer specific regions selected from any of Seq ID No. 63-64, and a probe that targets any of tissue-specific regions selected from any of Seq ID No. 65-117. In the tissue-specific region, Seq ID No. 73-74 and 107 are tissue-specific target regions of the liver, and Seq ID No. 111-115 are tissue-specific target regions of the pancreas.

In one specific embodiment of the present application, the probe composition is a probe that targets a specific region of any of esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, and endometrial cancer, and the cancer-specific region includes a probe selected from any of Seq ID No. 1-62, a probe that targets any of the pan-cancer specific regions selected from any of Seq ID No. 63-64, and a probe that targets any of the tissue-specific regions selected from any of Seq ID No. 65-117. Seq ID No. 80, 84, 93, and 95 in the tissue-specific region are tissue-specific target regions of the prostate.

In one specific embodiment of the present application, the probe composition is a probe targeting a specific region of any of esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, and endometrial cancer, and the cancer-specific region includes a probe selected from any of Seq ID No. 1-62, a probe targeting any of pan-cancer specific regions selected from any of Seq ID No. 63-64, and a probe targeting any of tissue-specific regions selected from any of Seq ID No. 65-117. In the tissue-specific region, Seq ID No. 78, 84-86 are tissue-specific target regions of the mammary gland, Seq ID No. 77, 96, and 105 are tissue-specific target regions of the ovary, and Seq ID No. 68-69, 75, and 109 are tissue-specific target regions of the cervix, and Seq ID No. 94, 106, and 108 are tissue-specific target regions of the endometrium.

In one specific embodiment of the present application, the probe composition includes a low methylation probe hybridized to the specific region where the bisulfite converted CG is not methylated, and a high methylation probe hybridized to the specific region where the entire bisulfite converted CG is methylated. The low methylation probe includes any of the probes Seq ID No. 118-214 targeting the cancer specific region, any of the probes Seq ID No. 215-216 targeting the pan cancer specific region, and any of the probes Seq ID No. 217-274 targeting the tissue specific region. The high methylation probe includes any of the probes Seq ID No. 275-371 targeting the cancer specific region, any of the probes Seq ID No. 372-373 targeting the pan cancer specific region, and any of the probes Seq ID No. 374-431 targeting the tissue specific region.

As shown in Table 1 below, Seq ID No. 118, Seq ID No. 119 and Seq ID No. 120 are all hypomethylated probes targeting the target region indicated by Seq ID No. 1, Seq ID No. 275, Seq ID No. 276 and Seq ID No. 277 are all hypermethylated probes targeting the target region indicated by Seq ID No. 1, Seq ID No. 121 and Seq ID No. 122 are all hypomethylated probes targeting the target region indicated by Seq ID No. 2, Seq ID No. 278 and Seq ID No. 279 are all hypermethylated probes targeting the target region indicated by Seq ID No. 2. Seq ID No. 123 and Seq ID No. 124 are both hypomethylated probes targeting the target region shown in Seq ID No. 3, and Seq ID No. 280 and Seq ID No. 281 are both hypermethylated probes targeting the target region shown in Seq ID No. 3. Seq ID No. 125 is a hypomethylated probe targeting the target region shown in Seq ID No. 4, and Seq ID No. 282 is a hypermethylated probe targeting the target region shown in Seq ID No. 4. Seq ID No. 126 and Seq ID No. 127 are both hypomethylated probes targeting the target region shown in Seq ID No. 3. Seq ID No. 128 is a hypomethylated probe targeted to the target region shown in Seq ID No. 6, Seq ID No. 285 is a hypermethylated probe targeted to the target region shown in Seq ID No. 6, Seq ID No. 129 is a hypomethylated probe targeted to the target region shown in Seq ID No. 7, Seq ID No. 286 is a hypermethylated probe targeted to the target region shown in Seq ID No. 7, Seq ID No. 130 and Seq ID No. Seq ID No. 130 is a low methylation probe targeting the target region shown in Seq ID No. 8, and Seq ID No. 287 and Seq ID No. 288 are high methylation probes targeting the target region shown in Seq ID No. 8. Seq ID No. 132, Seq ID No. 133 and Seq ID No. 134 are low methylation probes targeting the target region shown in Seq ID No. 9, and Seq ID No. 289, Seq ID No. 290 and Seq ID No. 291 are high methylation probes targeting the target region shown in Seq ID No. 9. Seq ID No. 135 is a low methylation probe targeting the target region shown in Seq ID No. 9. Seq ID No. 136 is a hypomethylated probe targeted to the target region designated Seq ID No. 11, Seq ID No. 293 is a hypermethylated probe targeted to the target region designated Seq ID No. 11, Seq ID No. 137 is a hypomethylated probe targeted to the target region designated Seq ID No. 12, Seq ID No. 294 is a hypermethylated probe targeted to the target region designated Seq ID No. 12, Seq ID No. 138 is a hypomethylated probe targeted to the target region designated Seq ID No. 12, Seq ID No. 295 is a hypermethylated probe targeted to the target region designated Seq ID No. 12, Seq ID No. 139 is a hypomethylated probe targeted to the target region designated Seq ID No. 136, Seq ID No. 294 is a hypermethylated probe targeted to the target region designated Seq ID No. 136, Seq ID No. 137 is a hypomethylated probe targeted to the target region designated Seq ID No. 12, Seq ID No. 295 is a hypermethylated probe targeted to the target region designated Seq ID No. 12, Seq ID No. 139 is a hypomethylated probe targeted to the target region designated Seq ID No. 136, Seq ID No. 292 is a hypermethylated probe targeted to the target region designated Seq ID No. 10, Seq ID No. 139 is a hypomethylated probe targeted to the target region designated Seq ID No. 1 Seq ID No. 13 is a hypomethylated probe targeting the target region shown in Seq ID No. 13, and Seq ID No. 295 is a hypermethylated probe targeting the target region shown in Seq ID No. 13. Seq ID No. 139 and Seq ID No. 140 are both hypomethylated probes targeting the target region shown in Seq ID No. 14, and Seq ID No. 296 and Seq ID No. 297 are both hypermethylated probes targeting the target region shown in Seq ID No. 14. Seq ID No. 141 and Seq ID No. 142 are both hypomethylated probes targeting the target region shown in Seq ID No. 15, and Seq ID No. 298 and Seq ID No. Seq ID No. 143 and Seq ID No. 144 are both low methylated probes targeting the target region shown in Seq ID No. 16, Seq ID No. 300 and Seq ID No. 301 are both high methylated probes targeting the target region shown in Seq ID No. 16, Seq ID No. 145 and Seq ID No. 146 are both low methylated probes targeting the target region shown in Seq ID No. 17, Seq ID No. 302 and Seq ID No. 303 are both low methylated probes targeting the target region shown in Seq ID No. 17, Seq ID No. 303 are both high methylated probes targeting the target region shown in Seq ID No. 17, Seq ID No. 304 and Seq ID No. 305 are both low methylated probes targeting the target region shown in Seq ID No. 17, Seq ID No. 306 and Seq ID No. 307 are both low methylated probes targeting the target region shown in Seq ID No. 17, Seq ID No. 308 and Seq ID No. 309 are both low methylated probes targeting the target region shown in Seq ID No. 18, Seq ID No. 309 and Seq ID No. 310 are both low methylated probes targeting the target region shown in Seq ID No. 18, Seq ID No. 301 and Seq ID No. 302 are both low methylated probes targeting the target region shown in Seq ID No. 18, Seq ID No. 303 and Seq ID No. 311 are both low methylated probes targeting the target region shown in Seq ID No. 18, Seq ID No. 304 and Seq ID No. 312 are both low methylated probes targeting the target region shown in Seq ID No. 18, Seq ID No. Seq ID No. 147 is a low methylation probe targeted to the target region shown in Seq ID No. 18, and Seq ID No. 304 is a high methylation probe targeted to the target region shown in Seq ID No. 18. Seq ID No. 148 and Seq ID No. 149 are both low methylation probes targeted to the target region shown in Seq ID No. 19, and Seq ID No. 305 and Seq ID No. 306 are both high methylation probes targeted to the target region shown in Seq ID No. 19. Seq ID No. 150 is a low methylation probe targeted to the target region shown in Seq ID No. 19. Seq ID No. 151 is a hypomethylated probe targeted to the target region represented by Seq ID No. 21, Seq ID No. 308 is a hypermethylated probe targeted to the target region represented by Seq ID No. 21, Seq ID No. 152 is a hypomethylated probe targeted to the target region represented by Seq ID No. 22, Seq ID No. 309 is a hypermethylated probe targeted to the target region represented by Seq ID No. 22. Seq ID No. 153, Seq ID No. 304 is a hypomethylated probe targeted to the target region represented by Seq ID No. 22, Seq ID No. 305 is a hypermethylated probe targeted to the target region represented by Seq ID No. 22. Seq ID No. 154 and Seq ID No. 155 are all hypomethylated probes targeting the target region shown in Seq ID No. 23, and Seq ID No. 310, Seq ID No. 311, and Seq ID No. 312 are all hypermethylated probes targeting the target region shown in Seq ID No. 23. Seq ID No. 156 and Seq ID No. 157 are all hypomethylated probes targeting the target region shown in Seq ID No. 24, and Seq ID No. 313 and Seq ID No. 314 are all hypermethylated probes targeting the target region shown in Seq ID No. 24. Seq ID No. 158 and Seq ID No. 159 are both hypomethylated probes targeting the target region shown in Seq ID No. 25, and Seq ID No. 315 and Seq ID No. 316 are both hypermethylated probes targeting the target region shown in Seq ID No. 25. Seq ID No. 160 and Seq ID No. 161 are both hypomethylated probes targeting the target region shown in Seq ID No. 26, and Seq ID No. 317 and Seq ID No. 318 are both hypermethylated probes targeting the target region shown in Seq ID No. 26. Seq ID No. 162 is both hypomethylated probes targeting the target region shown in Seq ID No. 26. Seq ID No. 163 and Seq ID No. 164 are both hypomethylated probes targeting the target region represented by Seq ID No. 28, Seq ID No. 320 and Seq ID No. 321 are both hypermethylated probes targeting the target region represented by Seq ID No. 28, Seq ID No. 165 and Seq ID No. 166 are both hypomethylated probes targeting the target region represented by Seq ID No. 29, Seq ID No. 322 and Seq ID No. 323 are both hypomethylated probes targeting the target region represented by Seq ID No. 29, Seq ID No. 324 and Seq ID No. 325 are both hypermethylated probes targeting the target region represented by Seq ID No. 29, Seq ID No. 165 and Seq ID No. 166 are both hypomethylated probes targeting the target region represented by Seq ID No. 29, Seq ID No. 326 and Seq ID No. 327 are both hypomethylated probes targeting the target region represented by Seq ID No. 29, Seq ID No. 328 and Seq ID No. 329 are both hypermethylated probes targeting the target region represented by Seq ID No. 29, Seq ID No. 329 and Seq ID No. 330 are both hypomethylated probes targeting the target region represented by Seq ID No. 29, Seq ID No. 331 and Seq ID No. 332 are both hypomethylated probes targeting the target region represented by Seq ID No. 29, Seq ID No. 333 and Seq ID No. 334 are both hypomethylated probes targeting the target region represented by Seq ID No. 29, Seq ID No. 335 and Seq ID No. 336 are both hypomethylated probes targeting the target region represented by Seq ID No. 29, Seq ID No. 3 Seq ID No. 167 is a low methylation probe targeted to the target region shown in Seq ID No. 30, and Seq ID No. 324 is a high methylation probe targeted to the target region shown in Seq ID No. 30. Seq ID No. 168 and Seq ID No. 169 are low methylation probes targeted to the target region shown in Seq ID No. 31, and Seq ID No. 325 and Seq ID No. 326 are high methylation probes targeted to the target region shown in Seq ID No. 31. Seq ID No. 170 is a low methylation probe targeted to the target region shown in Seq ID No. 30. Seq ID No. 327 is a low methylated probe targeting the target region shown in Seq ID No. 32. Seq ID No. 171 and Seq ID No. 172 are both low methylated probes targeting the target region shown in Seq ID No. 33. Seq ID No. 328 and Seq ID No. 329 are both high methylated probes targeting the target region shown in Seq ID No. 33. Seq ID No. 173 is a low methylated probe targeting the target region shown in Seq ID No. 34. Seq ID No. 330 is a high methylated probe targeting the target region shown in Seq ID No. 34. Seq ID No. 174 and Seq ID No. 175 are both hypomethylated probes targeting the target region shown in Seq ID No. 35, and Seq ID No. 331 and Seq ID No. 332 are both hypermethylated probes targeting the target region shown in Seq ID No. 35. Seq ID No. 176 is a hypomethylated probe targeting the target region shown in Seq ID No. 36, and Seq ID No. 333 is a hypermethylated probe targeting the target region shown in Seq ID No. 36. Seq ID No. 177 and Seq ID No. 178 are both hypomethylated probes targeting the target region shown in Seq ID No. 35. Seq ID No. 179 and Seq ID No. 180 are both hypomethylated probes targeted to the target region shown in Seq ID No. 38, and Seq ID No. 336 and Seq ID No. 337 are both hypermethylated probes targeted to the target region shown in Seq ID No. 38. Seq ID No. 181 is a hypomethylated probe targeted to the target region shown in Seq ID No. 39, and Seq ID No. 338 is a hypermethylated probe targeted to the target region shown in Seq ID No. 38. Seq ID No. 182 and Seq ID No. 183 are both low methylated probes targeting the target region represented by Seq ID No. 40, and Seq ID No. 339 and Seq ID No. 340 are both high methylated probes targeting the target region represented by Seq ID No. 40. Seq ID No. 184 is a low methylated probe targeting the target region represented by Seq ID No. 41, and Seq ID No. 341 is a high methylated probe targeting the target region represented by Seq ID No. 41. Seq ID No. 185 is a low methylated probe targeting the target region represented by Seq ID No. 41. Seq ID No. 187 is a hypomethylated probe targeted to the target region represented by Seq ID No. 44 and Seq ID No. 344 is a hypermethylated probe targeted to the target region represented by Seq ID No. 44. Seq ID No. 188 and Seq ID No. 342 are hypomethylated probes targeted to the target region represented by Seq ID No. 42 and hypermethylated probes targeted to the target region represented by Seq ID No. 42. Seq ID No. 186 is a hypomethylated probe targeted to the target region represented by Seq ID No. 43 and Seq ID No. 343 is a hypermethylated probe targeted to the target region represented by Seq ID No. 43. Seq ID No. 187 is a hypomethylated probe targeted to the target region represented by Seq ID No. 44 and Seq ID No. 344 is a hypermethylated probe targeted to the target region represented by Seq ID No. 44. Seq ID No. 189 is a low methylated probe targeting the target region shown in Seq ID No. 45, and Seq ID No. 345 and Seq ID No. 346 are high methylated probes targeting the target region shown in Seq ID No. 45. Seq ID No. 190 is a low methylated probe targeting the target region shown in Seq ID No. 46, and Seq ID No. 347 is a high methylated probe targeting the target region shown in Seq ID No. 46. Seq ID No. 191 is a low methylated probe targeting the target region shown in Seq ID No. 47, and Seq ID No. 348 is a high methylated probe targeting the target region shown in Seq ID No. 47. Seq ID No. 192 is a hypomethylated probe targeted to the target region represented by Seq ID No. 48, and Seq ID No. 349 is a hypermethylated probe targeted to the target region represented by Seq ID No. 48. Seq ID No. 193 is a hypomethylated probe targeted to the target region represented by Seq ID No. 49, and Seq ID No. 350 is a hypermethylated probe targeted to the target region represented by Seq ID No. 49. Seq ID No. 194 is a hypomethylated probe targeted to the target region represented by Seq ID No. 50, and Seq ID No. 351 is a hypermethylated probe targeted to the target region represented by Seq ID No. 50. Seq ID No. 195 and Seq ID No. 196 are both hypomethylated probes targeting the target region shown in Seq ID No. 51, and Seq ID No. 352 and Seq ID No. 353 are both hypermethylated probes targeting the target region shown in Seq ID No. 51. Seq ID No. 197 is a hypomethylated probe targeting the target region shown in Seq ID No. 52, and Seq ID No. 354 is a hypermethylated probe targeting the target region shown in Seq ID No. 52. Seq ID No. 198 and Seq ID No. 199 are both hypomethylated probes targeting the target region shown in Seq ID No. 52. Seq ID No. 200 is a hypomethylated probe targeted to the target region shown in Seq ID No. 54, Seq ID No. 357 is a hypermethylated probe targeted to the target region shown in Seq ID No. 54, Seq ID No. 201 and Seq ID No. 202 are hypomethylated probes targeted to the target region shown in Seq ID No. 55, Seq ID No. 358 and Seq ID No. 359 are hypomethylated probes targeted to the target region shown in Seq ID No. 54, ...0 is a hypomethylated probe targeted to the target region shown in Seq ID No. 54, Seq ID No. 200 is a hypomethylated probe targeted to the target region shown in Seq ID No. 54, Seq ID No. 357 is a hypermethylated probe targeted to the target region shown in Seq ID No. 54, Seq ID No. 200 is a hypomethylated probe targeted to the target region shown in Seq ID No. 54, Seq ID No. 200 is a hypomethylated probe targeted to the target region shown in Seq ID No. 54, Seq ID No. Seq ID No. 203 and Seq ID No. 204 are both low methylated probes targeting the target region shown in Seq ID No. 56, and Seq ID No. 360 and Seq ID No. 361 are both high methylated probes targeting the target region shown in Seq ID No. 56. Seq ID No. 205 and Seq ID No. 206 are both low methylated probes targeting the target region shown in Seq ID No. 57, and Seq ID No. 362 and Seq ID No. 363 are both high methylated probes targeting the target region shown in Seq ID No. 57. Seq ID No. 207 and Seq ID No. 208 are both hypomethylated probes targeting the target region shown in Seq ID No. 58, and Seq ID No. 364 and Seq ID No. 365 are both hypermethylated probes targeting the target region shown in Seq ID No. 58. Seq ID No. 209 and Seq ID No. 210 are both hypomethylated probes targeting the target region shown in Seq ID No. 59, and Seq ID No. 366 and Seq ID No. 367 are both hypermethylated probes targeting the target region shown in Seq ID No. 59. Seq ID No. 211 is both hypomethylated probes targeting the target region shown in Seq ID No. 59. Seq ID No. 368 is a low methylated probe targeting the target region indicated by Seq ID No. 60, and Seq ID No. 369 is a high methylated probe targeting the target region indicated by Seq ID No. 61, Seq ID No. 212 and Seq ID No. 213 are both low methylated probes targeting the target region indicated by Seq ID No. 61, and Seq ID No. 369 and Seq ID No. 370 are both high methylated probes targeting the target region indicated by Seq ID No. 61, Seq ID No. 214 is a low methylated probe targeting the target region indicated by Seq ID No. 62, and Seq ID No. 371 is a high methylated probe targeting the target region indicated by Seq ID No. 62. Seq ID No. 215 is a hypomethylated probe targeted to the target region designated Seq ID No. 63, and Seq ID No. 372 is a hypermethylated probe targeted to the target region designated Seq ID No. 63. Seq ID No. 216 is a hypomethylated probe targeted to the target region designated Seq ID No. 64, and Seq ID No. 373 is a hypermethylated probe targeted to the target region designated Seq ID No. 64. Seq ID No. 217 is a hypomethylated probe targeted to the target region designated Seq ID No. 65, and Seq ID No. 374 is a hypermethylated probe targeted to the target region designated Seq ID No. 65. Seq ID No. 218 is a hypomethylated probe targeted to the target region designated Seq ID No. 66, and Seq ID No. 375 is a hypermethylated probe targeted to the target region designated Seq ID No. 66. Seq ID No. 219 is a hypomethylated probe targeted to the target region designated Seq ID No. 67, and Seq ID No. 376 is a hypermethylated probe targeted to the target region designated Seq ID No. 67. Seq ID No. 220 is a hypomethylated probe targeted to the target region designated Seq ID No. 68, and Seq ID No. 377 is a hypermethylated probe targeted to the target region designated Seq ID No. 68. Seq ID No. 221 is a hypomethylated probe targeted to the target region designated Seq ID No. 69, and Seq ID No. 378 is a hypermethylated probe targeted to the target region designated Seq ID No. 69. Seq ID No. 222 is a hypomethylated probe targeted to the target region designated Seq ID No. 70, and Seq ID No. 379 is a hypermethylated probe targeted to the target region designated Seq ID No. 70. Seq ID No. 223 is a hypomethylated probe targeted to the target region designated Seq ID No. 71, and Seq ID No. 380 is a hypermethylated probe targeted to the target region designated Seq ID No. 71. Seq ID No. 224 is a hypomethylated probe targeted to the target region designated Seq ID No. 72, and Seq ID No. 381 is a hypermethylated probe targeted to the target region designated Seq ID No. 72. Seq ID No. 225 is a hypomethylated probe targeted to the target region designated Seq ID No. 73, and Seq ID No. 382 is a hypermethylated probe targeted to the target region designated Seq ID No. 73. Seq ID No. 226 is a hypomethylated probe targeted to the target region designated Seq ID No. 74, and Seq ID No. 383 is a hypermethylated probe targeted to the target region designated Seq ID No. 74. Seq ID No. 227 is a hypomethylated probe targeted to the target region represented by Seq ID No. 75, and Seq ID No. 384 is a hypermethylated probe targeted to the target region represented by Seq ID No. 75. Seq ID No. 228 and Seq ID No. 229 are both hypomethylated probes targeted to the target region represented by Seq ID No. 76, and Seq ID No. 385 and Seq ID No. 386 are both hypermethylated probes targeted to the target region represented by Seq ID No. 76. Seq ID No. 230 is a hypomethylated probe targeted to the target region represented by Seq ID No. 77, and Seq ID No. 385 is a hypermethylated probe targeted to the target region represented by Seq ID No. 76. Seq ID No. 231 is a hypomethylated probe targeted to the target region represented by Seq ID No. 78, and Seq ID No. 388 is a hypermethylated probe targeted to the target region represented by Seq ID No. 78, Seq ID No. 232 is a hypomethylated probe targeted to the target region represented by Seq ID No. 79, and Seq ID No. 389 is a hypermethylated probe targeted to the target region represented by Seq ID No. 79, Seq ID No. 233 is a hypomethylated probe targeted to the target region represented by Seq ID No. 80, and Seq ID No. 389 is a hypermethylated probe targeted to the target region represented by Seq ID No. 79, Seq ID No. 234 is a hypomethylated probe targeted to the target region represented by Seq ID No. 80, and Seq ID No. 387 is a hypermethylated probe targeted to the target region represented by Seq ID No. 77, Seq ID No. 231 is a hypomethylated probe targeted to the target region represented by Seq ID No. 78, and Seq ID No. 388 is a hypermethylated probe targeted to the target region represented by Seq ID No. 78, Seq ID No. 232 is a hypomethylated probe targeted to the target region represented by Seq ID No. 79, and Seq ID No. 389 is a hypermethylated probe targeted to the target region represented by Seq ID No. 79, Seq ID No. 233 is a hypomethylated probe targeted to the target region represented by Seq ID No. 80, and Seq ID No. Seq ID No. 390 is a hypermethylated probe targeted to the target region represented by Seq ID No. 80. Seq ID No. 234 is a hypomethylated probe targeted to the target region represented by Seq ID No. 81, and Seq ID No. 391 is a hypermethylated probe targeted to the target region represented by Seq ID No. 81. Seq ID No. 235 is a hypomethylated probe targeted to the target region represented by Seq ID No. 82, and Seq ID No. 392 is a hypermethylated probe targeted to the target region represented by Seq ID No. 82. Seq ID No. 236 is a hypomethylated probe targeted to the target region represented by Seq ID No. 83, and Seq ID No. 392 is a hypermethylated probe targeted to the target region represented by Seq ID No. 82. Seq ID No. 237 is a hypomethylated probe targeted to the target region represented by Seq ID No. 84, and Seq ID No. 394 is a hypermethylated probe targeted to the target region represented by Seq ID No. 84, Seq ID No. 238 is a hypomethylated probe targeted to the target region represented by Seq ID No. 85, and Seq ID No. 395 is a hypermethylated probe targeted to the target region represented by Seq ID No. 85, Seq ID No. 239 is a hypomethylated probe targeted to the target region represented by Seq ID No. 86, and Seq ID No. 395 is a hypermethylated probe targeted to the target region represented by Seq ID No. 85, Seq ID No. 239 is a hypomethylated probe targeted to the target region represented by Seq ID No. 86, and Seq ID No. 394 is a hypermethylated probe targeted to the target region represented by Seq ID No. 84, Seq ID No. 239 is a hypomethylated probe targeted to the target region represented by Seq ID No. 86, and Seq ID No. 395 is a hypermethylated probe targeted to the target region represented by Seq ID No. 86, Seq ID No. 240 is a hypomethylated probe targeted to the target region represented by Seq ID No. 87, and Seq ID No. 397 is a hypermethylated probe targeted to the target region represented by Seq ID No. 87. Seq ID No. 241 is a hypomethylated probe targeted to the target region represented by Seq ID No. 88, and Seq ID No. 398 is a hypermethylated probe targeted to the target region represented by Seq ID No. 88. Seq ID No. 242 is a hypomethylated probe targeted to the target region represented by Seq ID No. 89, and Seq ID No. 397 is a hypermethylated probe targeted to the target region represented by Seq ID No. 87. Seq ID No. 399 is a hypermethylated probe targeted to the target region represented by Seq ID No. 89. Seq ID No. 243 is a hypomethylated probe targeted to the target region represented by Seq ID No. 90, and Seq ID No. 400 is a hypermethylated probe targeted to the target region represented by Seq ID No. 90. Seq ID No. 244 is a hypomethylated probe targeted to the target region represented by Seq ID No. 91, and Seq ID No. 401 is a hypermethylated probe targeted to the target region represented by Seq ID No. 91. Seq ID No. 245 is a hypomethylated probe targeted to the target region represented by Seq ID No. 92, and Seq ID No. 402 is a hypermethylated probe targeted to the target region represented by Seq ID No. 92. Seq ID No. 402 is a hypermethylated probe targeted to the target region represented by Seq ID No. 92. Seq ID No. 246 is a hypomethylated probe targeted to the target region represented by Seq ID No. 93, and Seq ID No. 403 is a hypermethylated probe targeted to the target region represented by Seq ID No. 93. Seq ID No. 247 is a hypomethylated probe targeted to the target region represented by Seq ID No. 94, and Seq ID No. 404 is a hypermethylated probe targeted to the target region represented by Seq ID No. 94. Seq ID No. 248 is a hypomethylated probe targeted to the target region represented by Seq ID No. 95, and Seq ID No. 405 is a hypermethylated probe targeted to the target region represented by Seq ID No. 96. Seq ID No. 249 is a hypomethylated probe targeted to the target region represented by Seq ID No. 96, and Seq ID No. 406 is a hypermethylated probe targeted to the target region represented by Seq ID No. 96. Seq ID No. 250 is a hypomethylated probe targeted to the target region represented by Seq ID No. 97, and Seq ID No. 407 is a hypermethylated probe targeted to the target region represented by Seq ID No. 97. Seq ID No. 251 is a hypomethylated probe targeted to the target region represented by Seq ID No. 98, and Seq ID No. 405 is a hypermethylated probe targeted to the target region represented by Seq ID No. 95. Seq ID No. 249 is a hypomethylated probe targeted to the target region represented by Seq ID No. 96, and Seq ID No. 406 is a hypermethylated probe targeted to the target region represented by Seq ID No. 96. Seq ID No. 250 is a hypomethylated probe targeted to the target region represented by Seq ID No. 97, and Seq ID No. 407 is a hypermethylated probe targeted to the target region represented by Seq ID No. 97. Seq ID No. 251 is a hypomethylated probe targeted to the target region represented by Seq ID No. 98, and Seq ID No. Seq ID No. 253 is a hypomethylated probe targeted to the target region represented by Seq ID No. 100 and Seq ID No. 410 is a hypermethylated probe targeted to the target region represented by Seq ID No. 100. Seq ID No. 254 is a hypomethylated probe targeted to the target region represented by Seq ID No. 101 and Seq ID No. 408 is a hypermethylated probe targeted to the target region represented by Seq ID No. 98. Seq ID No. 252 is a hypomethylated probe targeted to the target region represented by Seq ID No. 99 and Seq ID No. 409 is a hypermethylated probe targeted to the target region represented by Seq ID No. 99. Seq ID No. 253 is a hypomethylated probe targeted to the target region represented by Seq ID No. 100 and Seq ID No. 410 is a hypermethylated probe targeted to the target region represented by Seq ID No. 100. Seq ID No. 254 is a hypomethylated probe targeted to the target region represented by Seq ID No. 101 and Seq ID No. 409 is a hypermethylated probe targeted to the target region represented by Seq ID No. 100. Seq ID No. 411 is a hypermethylated probe targeting the target region represented by Seq ID No. 101. Seq ID No. 255 and Seq ID No. 256 are both hypomethylated probes targeting the target region represented by Seq ID No. 102, and Seq ID No. 412 and Seq ID No. 413 are both hypermethylated probes targeting the target region represented by Seq ID No. 102. Seq ID No. 257 is a hypomethylated probe targeting the target region represented by Seq ID No. 103, and Seq ID No. 414 is a hypermethylated probe targeting the target region represented by Seq ID No. 103. Seq ID No. 258 is a hypomethylated probe targeted to the target region designated Seq ID No. 104, and Seq ID No. 415 is a hypermethylated probe targeted to the target region designated Seq ID No. 104. Seq ID No. 259 is a hypomethylated probe targeted to the target region designated Seq ID No. 105, and Seq ID No. 416 is a hypermethylated probe targeted to the target region designated Seq ID No. 105. Seq ID No. 260 is a hypomethylated probe targeted to the target region designated Seq ID No. 106, and Seq ID No. 417 is a hypermethylated probe targeted to the target region designated Seq ID No. 106. Seq ID No. 261 is a hypomethylated probe targeted to the target region designated Seq ID No. 107, and Seq ID No. 418 is a hypermethylated probe targeted to the target region designated Seq ID No. 107. Seq ID No. 262 is a hypomethylated probe targeted to the target region designated Seq ID No. 108, and Seq ID No. 419 is a hypermethylated probe targeted to the target region designated Seq ID No. 108. Seq ID No. 263 is a hypomethylated probe targeted to the target region designated Seq ID No. 109, and Seq ID No. 420 is a hypermethylated probe targeted to the target region designated Seq ID No. 109. Seq ID No. 264, Seq ID No. 265, and Seq ID No. 266 are all hypomethylated probes targeting the target region shown in Seq ID No. 110, and Seq ID No. 421, Seq ID No. 422, and Seq ID No. 423 are all hypermethylated probes targeting the target region shown in Seq ID No. 110. Seq ID No. 267 is a hypomethylated probe targeting the target region shown in Seq ID No. 111, and Seq ID No. 424 is a hypermethylated probe targeting the target region shown in Seq ID No. 111. Seq ID No. 268 is a hypomethylated probe targeting the target region shown in Seq ID No. 111. Seq ID No. 425 is a low methylated probe targeting the target region designated Seq ID No. 112, Seq ID No. 269 is a low methylated probe targeting the target region designated Seq ID No. 113, Seq ID No. 426 is a high methylated probe targeting the target region designated Seq ID No. 113, Seq ID No. 270 and Seq ID No. 271 are both low methylated probes targeting the target region designated Seq ID No. 114, Seq ID No. 427 and Seq ID No. 428 are both low methylated probes targeting the target region designated Seq ID No. 114, Seq ID No. 429 is a high methylated probe targeting the target region designated Seq ID No. 112, Seq ID No. 230 is a low methylated probe targeting the target region designated Seq ID No. 112, Seq ID No. 232 is a high methylated probe targeting the target region designated Seq ID No. 112, Seq ID No. 233 is a low methylated probe targeting the target region designated Seq ID No. 113, Seq ID No. 234 is a high methylated probe targeting the target region designated Seq ID No. 113, Seq ID No. 235 is a low methylated probe targeting the target region designated Seq ID No. 114, Seq ID No. 236 is a high methylated probe targeting the target region designated Seq ID No. 114, Seq ID No. 237 is a high methylated probe targeting the target region designated Seq ID No. 114, Seq ID No. 238 is a high methylated probe targeting the target region designated Seq ID No. 114, Seq ID No. 239 is a low methylated probe targeting the target region designated Seq ID No. 112, Seq ID No. 240 Seq ID No. 272 is a low methylation probe targeted to the target region designated Seq ID No. 115, and Seq ID No. 429 is a high methylation probe targeted to the target region designated Seq ID No. 115, Seq ID No. 273 is a low methylation probe targeted to the target region designated Seq ID No. 116, and Seq ID No. 430 is a high methylation probe targeted to the target region designated Seq ID No. 116, Seq ID No. 274 is a low methylation probe targeted to the target region designated Seq ID No. 117, and Seq ID No. 431 is a high methylation probe targeted to the target region designated Seq ID No. 117, The hypermethylated probe targets the target region indicated by 117. Table 1 shows the target sequences targeted by the probes.

In one specific embodiment of the present application, the length of each probe in the above probe composition is 40 to 60 bp, preferably 45 to 56 bp, preferably 50 to 56 bp, and more preferably 50 bp.

The present application further provides a kit.

In one specific embodiment of the present application, the kit includes a probe composition described in any of the above embodiments.

The present application further provides uses of the probe composition.

In one specific embodiment of the present application, the probe composition is used to manufacture a kit for detecting esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, and endometrial cancer.

The present application further provides a chip.

In one specific embodiment of the present application, the probe composition described in any of the above embodiments is immobilized on the chip.

The present application further provides a method for detecting 11 types of cancer, including esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, and endometrial cancer, by utilizing the probe composition.

In one specific embodiment of the present application, the detection method employs a hybridization capture method to enrich cfDNA, and uses NGS technology to detect methylation sites closely related to cancer. It covers the six malignant tumors with the highest incidence in China (lung cancer, gastric cancer, colorectal cancer, liver cancer, breast cancer, and esophageal cancer), and pancreatic cancer, which has a very high malignancy. It also includes other three types of female and male tumors (cervical cancer, ovarian cancer, endometrial cancer, and prostate cancer). Finally, it provides information for early screening and early diagnosis of multiple cancers based on the change levels of gene methylation in the detected plasma cfDNA.

The present application also provides a method for simultaneously detecting changes in the methylation levels of the above 11 types of cancer using the kit.

Specifically, the present application relates to an in vitro method for detecting eleven types of cancer in a subject, the method including the steps of collecting a subject sample, extracting and purifying DNA in the sample, constructing a DNA library for sequencing from the purified DNA sample, converting the constructed DNA library with bisulfite, amplifying the bisulfite-converted DNA library by pre-PCR, performing hybridization capture on the pre-PCR amplified sample using a probe composition, amplifying the hybridization capture products using PCR, performing high-throughput next-generation sequencing on the hybridization capture products after PCR amplification, analyzing the sequencing data to determine the methylation level of the sample, and interpreting the disease status of the patient based on the methylation level of the sample.

Specifically, the subject is suspected of having cancer. Specifically, the subject sample collected is a plasma sample. Specifically, the conversion uses a bisulfite treatment.

Specifically, the probe composition includes two probes targeting pan-cancer-specific regions, n probes targeting cancer-specific regions, and m probes targeting tissue-specific regions. The probe composition includes a low methylation probe hybridized to the cancer-specific region, pan-cancer-specific region, and tissue-specific region in which the bisulfite-converted CG is not methylated, and a high methylation probe hybridized to the cancer-specific region, pan-cancer-specific region, and tissue-specific region in which the entire bisulfite-converted CG is methylated. The length of each probe in the probe composition is 40 to 60 bp. For example, the length of each probe in the probe composition is 45 to 56 bp, preferably 50 to 56 bp, and more preferably 50 bp.

Specifically, the n probes in the probe composition target a cancer-specific region, n being any integer selected from 1-192, and the cancer-specific region being selected from any of Seq ID Nos. 1-62. The m probes in the probe composition target a tissue-specific region, m being any integer selected from 1-116, and the tissue-specific region being selected from any of Seq ID Nos. 65-117. The low methylation probe includes any of probes Seq ID Nos. 118-214 targeting a cancer-specific region, any of probes Seq ID Nos. 215-216 targeting a pan-cancer-specific region, and any of probes Seq ID Nos. 217-274 targeting a tissue-specific region. The high methylation probe includes any of probes Seq ID Nos. 217-274 targeting a cancer-specific region. These include any of probes Seq ID No. 275-371 targeting pan-cancer specific regions, any of probes Seq ID No. 372-373 targeting tissue specific regions, and any of probes Seq ID No. 374-431 targeting tissue specific regions.

Specifically, the interpretation includes the steps of (1) comparing and interpreting the pan-cancer specific region database to determine whether the subject has cancer, (2) comparing and interpreting the cancer specific region database to determine whether the subject has cancer of one of several suspected cancers, and (3) comparing and interpreting the tissue specific region database to determine the site of the subject's cancer. The step (1) includes determining whether the methylation level of the pan-cancer specific region Seq ID No. 63 is 55% or more and determining whether the methylation level of the pan-cancer specific region Seq ID No. 64 is 60% or more, and interpreting the patient as having cancer if the methylation level of Seq ID No. 63 is 55% or more and the methylation level of Seq ID No. 64 is 60% or more. The step (2) includes determining that the patient is suffering from any of the tissue-specific cancers when the methylation levels of the regions targeted by n1 probes among the n probes targeting the cancer-specific region are equal to or greater than the respective thresholds and n1/n≧20%, preferably n1/n≧30%. The step (3) includes further analyzing the tissues targeted by the m1 probes that are equal to or greater than the respective thresholds when the methylation levels of the regions targeted by m1 probes among the m probes targeting the tissue-specific region are equal to or greater than the respective thresholds, counting the number of probes that are equal to or greater than the threshold for each tissue, and determining that the tissue suffering from the patient's cancer is the tissue with the largest number of probes that have a methylation level equal to or greater than the threshold. The methylation thresholds for each target region are also shown in Table 1 below.

Example 1:
As shown in FIG. 1, the implementation flow of the present invention is specifically as follows.

1.1. Extraction and purification of cfDNA 1.1.1. Preparation of plasma samples:
The blood samples were centrifuged at 2000g at 4°C for 10 min and the plasma was transferred to a new centrifuge tube. The plasma samples were centrifuged at 16000g at 4°C for 10 min and the following steps were performed based on the collection tube type used; the collection tube type used in this experiment is other.

1.1.2. Dissolution and binding 1.1.2.1. Prepare the solution/bead mixture to be bound according to the table below and then mix thoroughly to homogeneity.

An appropriate volume of plasma sample was added.
1.1.2.2. The plasma sample and binding solution/bead mixture were mixed thoroughly and homogeneously.
1.1.2.3. The cfDNA was bound to the magnetic beads after thorough binding in a Rotisserie for 10 min.
1.1.2.4. The binding tube was placed on the magnetic stand for 5 min until the solution became clear and the magnetic beads were completely adsorbed to the magnetic stand.
1.1.2.5. Carefully discard the supernatant with a pipette, keep the tube on the magnetic stand for a few minutes, and remove the remaining supernatant with a pipette.

1.1.3. Washing 1.1.3.1. The beads were resuspended in 1 ml of washing solution.
1.1.3.2. Transfer the resuspension to a new non-adsorbed 1.5 ml centrifuge tube. Leave the combined tube behind.
1.1.3.3. The centrifuge tube containing the bead suspension was placed on the magnetic stand for 20 s.
1.1.3.4. The resulting supernatant was aspirated from the wash bind tube, the remaining washed beads were recollected in the resuspension, and the lysis/bind tube was discarded.
1.1.3.5. Place the tube on the magnetic stand for 2 min until the solution was clear and the beads had clumped to the magnetic stand, then remove the supernatant with a 1 ml pipette.
1.1.3.6. The tube was left on the magnetic stand and as much residual liquid as possible was removed with a 200 μL pipette.
1.1.3.7. The tube was removed from the magnetic stand and 1 ml of Wash Solution was added and vortexed for 30 s.
1.1.3.8. Place on magnetic stand for 2 min until solution becomes clear and beads clump to the magnetic stand, then remove supernatant with 1 ml pipette.
1.1.3.9. The tube was left on the magnetic stand and any remaining liquid was completely removed with a 200 μL pipette.
1.1.3.10. The tube was removed from the magnetic stand and 1 ml of 80% ethanol was added and vortexed for 30 s.
1.1.3.11. Place on magnetic stand for 2 min until solution becomes clear and remove supernatant with 1 ml pipette.
1.1.3.12. The tube was left on the magnetic stand and residual liquid was removed with a 200 μL pipette.
1.1.3.13. The above steps 1.1.3.10. to 1.1.3.12. were repeated once using 80% ethanol, and the supernatant was removed as much as possible.
1.1.3.14. The tube was left on the magnetic stand and the beads were allowed to air dry for 3-5 minutes.

1.1.4. Elution of cfDNA 1.1.4.1. Eluents were added according to the table below.

1.1.4.2. Vortex for 5 min, place on magnetic stand for 2 min until solution becomes clear, and aspirate off cfDNA in the supernatant.
1.1.4.3. Use the purified cfDNA immediately or transfer the supernatant to a new centrifuge tube and store at -20°C.

1.2. gDNA Fragmentation and Purification:
1.2.1. Depending on Qubit concentration, 2 μg gDNA was made up to 125 μl with water and added to a 130 μl shearing tube in a Covaris and the program was set to 50 W, 20%, 200 cycles, 250 s.

1.2.2. After the fragmentation was completed, 1 μl of the sample was used to detect the fragments using Agilent 2100, and after successful fragmentation, the main peak detected in the sample was about 150 bp-200 bp.
The cfDNA samples were subjected to fragment detection on the Agilent 2100 and directly subjected to Qubit to prepare them for subsequent experiments.

1.3. End repair, adding an "A" tail to the 3' end.
1.3.1.20 ng of fragmented gDNA or cfDNA was placed in a PCR tube, and nuclease-free water was added to a volume of 50 μl. The following reagents were then added and vortexed to mix uniformly.

1.3.2. The reaction was run in a PCR machine with the following program: hot lid temperature 85°C.

1.4. Linker Ligation and Purification:
1.4.1. Pre-diluted the linker to the appropriate concentration, referring to the table below:

1.4.2. Prepare the following reagents according to the table below, gently pipette to mix evenly, and briefly centrifuge.

1.4.3. The reaction was run on a PCR machine using the following program: No hot lid.

1.4.4. Experiments were performed with purified magnetic beads according to the following system (Agencourt AMPure XP magnetic beads were brought to room temperature in advance, shaken to mix uniformly, and prepared for use):

1.4.4.1. Gently pipette 6 times to mix evenly.
1.4.4.2. Incubate at room temperature stationary for 5-15 min, then place the PCR tube on a magnetic stand for 3 min to clarify the solution.
1.4.4.3. The supernatant was removed, and the PCR tube was placed on the magnetic stand. 200 μl of 80% ethanol solution was added to the PCR tube, and the tube was allowed to stand for 30 s.
1.4.4.4. Remove the supernatant, then add 200 μl of 80% ethanol solution to the PCR tube, leave it for 30 s, and then completely remove the supernatant (it is recommended to use a 10 μl pipette to remove the ethanol solution remaining at the bottom).
1.4.4.5. Allow to stand at room temperature for 3-5 minutes to completely evaporate the remaining ethanol.
1.4.4.6.22 μl of nuclease-free water was added, the PCR tube was removed from the magnetic stand, and the magnetic beads were resuspended by gently pipetting, avoiding the creation of air bubbles, and allowed to stand at room temperature for 2 min.
1.4.4.7. The PCR tube was placed on a magnetic stand for 2 min to clarify the solution.
1.4.4.8. 20 μl of the supernatant was aspirated with a pipette and transferred to a new PCR tube.

1.5 Bisulfite Treatment and Purification:
1.5.1. Remove and dissolve the necessary reagents in advance. Add each reagent according to the table below.

1.5.2. The liquid turned blue when added to the DNA protection buffer, gently pipetted to mix evenly, then split into two tubes and placed in the PCR machine.
1.5.3 The following program was set and run: Hot lid 105°C.

1.5.4. Centrifuge briefly and combine the identical samples from the two tubes into identical clean 1.5 ml centrifuge tubes.
1.5.5. Add 310 μl of Buffer BL to each sample (if the sample amount is less than 100 ng, add 1 μl of Carrier RNA (1 μg/μl)), vortex to mix evenly, and briefly centrifuge.
1.5.6. 250 μl of absolute ethanol was added to each sample, vortexed for 15 s to mix evenly, briefly centrifuged, and the mixture was loaded onto the corresponding prepared spin column.
1.5.7.1 min was allowed to stand, then centrifuged for 1 min, the liquid in the collection tube was transferred to the spin column again, centrifuged for 1 min, and the liquid in the centrifuge tube was discarded.
1.5.8.Add 500 μl of Buffer BW (be careful not to add absolute ethanol), centrifuge for 1 min, and discard the waste liquid.
1.5.9. Add 500 μl of Buffer BD (be careful to add absolute ethanol), cap the tube and leave at room temperature for 15 min. Centrifuge for 1 min and discard the liquid after centrifugation.
1.5.10. Add 500 μl of buffer BW (be careful whether to add absolute ethanol), centrifuge for 1 min, discard the liquid after centrifugation, and repeat once, for a total of two times.
1.5.11. Add 250 μl of absolute ethanol, centrifuge for 1 min, place the spin column into a new 2 ml collection tube and discard all remaining liquid.
1.5.12. Place the spin column into a clean 1.5 ml centrifuge tube and add 20 μl of nuclease-free water to the center of the spin column membrane, loosely cap the tube, let sit at room temperature for 1 min, and centrifuged for 1 min.
1.5.13. The liquid in the collection tube was transferred back into the spin column, left at room temperature for 1 min, and centrifuged for 1 min.

1.6. Pre-amplification and purification before hybridization:
1.6.1. Prepare the reaction system according to the table below, mix uniformly by pipetting, and briefly centrifuge.

1.6.2. The PCR program was run with the following settings: Hot lid 105°C.

1.6.3. The number of PCR cycles was adjusted according to the amount of DNA input, and the baseline data was shown as follows:

1.6.4. After the reaction was completed, 50 μl of Agencourt AMPure XP magnetic beads were added to the PCR tube and mixed uniformly by pipetting to avoid the formation of air bubbles (Agencourt AMPure XP was mixed uniformly at room temperature in advance and balanced).
1.6.5. Incubate at room temperature for 5-15 min and then place the PCR tube on a magnetic stand for 3 min to clarify the solution.
1.6.6. The supernatant was removed, and the PCR tube was placed on the magnetic stand. 200 μl of 80% ethanol solution was added to the PCR tube and allowed to stand for 30 s.
1.6.7. Remove the supernatant, then add 200 μl of 80% ethanol solution to the PCR tube, leave it for 30 s, and then completely remove the supernatant (it is recommended to use a 10 μl pipette to remove the ethanol solution remaining at the bottom).
1.6.8. The mixture was left to stand at room temperature for 5 minutes to completely evaporate the remaining ethanol.
1.6.9.30 μl of nuclease free water was added, the centrifuge tube was removed from the magnetic stand, and using a pipette, the magnetic beads were resuspended by gently pipetting up and down.
1.6.10. Let stand at room temperature for 2 min, then place the 200 μl PCR tube on a magnetic stand for 2 min to clarify the solution.
1.6.11. Pipette the supernatant into a new 200 μl PCR tube (keep in icebox), mark the reaction tube with the sample number, and prepare it for the next reaction.
1.6.12.1 μl sample was taken to perform library concentration measurement using Qubit and the library concentration was recorded.
1.6.13. Library fragment lengths were measured using an Agilent 2100 on 1 μl samples and library lengths were between approximately 270 bp-320 bp.

1.7. Hybridization of sample with probe:
1.7.1. The sample library and various Hyb Blockers were mixed homogeneously according to the following scheme and designated as B:

1.7.2. The prepared sample and HybBlocker mixture was placed into a vacuum concentration centrifuge, the PCR tube cap was opened, the centrifuge was started, the vacuum pump switch was opened, and concentration was started.
1.7.3. The aspirated and dried sample was dissolved again in about 9 μl of nuclease-free water to a total volume of 10 μl, gently pipetted to mix uniformly, centrifuged briefly, and then placed on ice for use, designated as B.
1.7.4. Thaw the Hyb buffer solution at room temperature. After thawing, precipitates were formed. After mixing uniformly, place in a water bath at 65°C to preheat. After complete dissolution (no precipitate or turbidity), place 20 μl of Hyb buffer solution in a new 200 μl PCR tube, cap the tube, label it as A, and continue to incubate in a water bath at 65°C for use.

1.7.5. The following hypomethylated probes were synthesized by iGeneTech Biotech (Beijing) Co., Ltd.
a) any of probes Seq ID Nos. 118-214 targeting cancer-specific regions, b) any of probes Seq ID Nos. 215-216 targeting pan-cancer-specific regions, and c) any of probes Seq ID Nos. 217-274 targeting tissue-specific regions.
In addition, the following hypermethylated probes were synthesized.
d) any of probes Seq ID Nos. 275-371 that target a cancer-specific region, e) any of probes Seq ID Nos. 372-373 that target a pan-cancer-specific region, and f) any of probes Seq ID Nos. 374-431 that target a tissue-specific region.
In addition, the probe composition was prepared in a ratio of a:b:c:d:e:f=1:1:1:1:1:1:1.

1.7.6.5 μl of RNase blocker and 2 μl of probe composition were placed in a 200 μl PCR tube, gently pipetted to mix evenly, centrifuged briefly, and then placed on ice for preparation for use, designated as C.
1.7.7. PCR instrument parameters were set to hot lid 100°C, 95°C, 5 min, hold at 65°C.
1.7.8. Place PCR tube B in the PCR machine and run the above program.
1.7.9. When the temperature of the PCR instrument was reduced to 65°C, PCR tube A was placed in the PCR instrument and incubated, and the hot lid of the PCR instrument was turned on.
1.7.10. After 5 min, C was placed in the PCR and incubated, then the hot lid of the PCR machine was turned on.
1.7.11. After placing PCR tube C in the PCR machine for 2 min, adjust the pipette to 13 μl, aspirate 13 μl of Hyb buffer from PCR tube A and transfer it to PCR tube C, aspirate all the samples in PCR tube B and transfer them to PCR tube C, gently pipette 10 times to mix thoroughly and evenly, avoiding the generation of a large amount of air bubbles, seal the tube cap, put the hot lid on the PCR machine, and incubate at 65°C overnight (16-24 h).

1.8. Capture of target region DNA library:
1.8.1. Preparation of magnetic beads for capture 1.8.1.1. Magnetic beads (Dynabeads MyOne Streptavidin T1 magnetic beads) were removed at 4° C. and resuspended by vortexing and shaking.
1.8.1.2. 50 μl of magnetic beads were placed into a new PCR tube and placed on the magnetic stand for 1 min to clarify the solution and the supernatant was removed.
1.8.1.3. Remove the PCR tube from the magnetic stand and add 200 μL of binding buffer and mix evenly by gently pipetting multiple times to resuspend the magnetic beads.
1.8.1.4. Place on magnetic stand for 1 min and remove supernatant.
1.8.1.5. Steps 3-4 were repeated twice to wash the magnetic beads a total of three times.
1.8.1.6. Remove the PCR tube from the magnetic stand and add 200 μL of Binding Buffer and gently pipette up and down 6 times to resuspend the magnetic beads and prepare for use.

1.8.2. Capture of target DNA library 1.8.2.1. Place hybridization product PCR tube C in the PCR instrument, add 200 μL of prepared capture magnetic beads to post-hybridization product PCR tube C, pipette 6 times to mix evenly, and place on a rotisserie to bind at room temperature for 30 min (rotation speed is preferably 10 rpm or less).
1.8.2.2. The PCR tube was placed on a magnetic stand for 2 min to clarify the solution and the supernatant was removed.
1.8.2.3. Add 200 μL of Wash Buffer 1 into PCR tube C, gently pipette 6 times to mix evenly, place on a rotisserie to wash for 15 min (rotation speed is preferably 10 rpm or less), then briefly centrifuge, place the PCR tube on a magnetic stand for 2 min to clarify the solution, and remove the supernatant.
1.8.2.4. 200 μl of washing buffer 2 preheated at 65° C. was added, gently pipetted six times to mix evenly, placed on a mixer and incubated at 65° C. for 10 min, and washed at a rotation speed of 800 rpm.
1.8.2.5. Centrifuge briefly, place the PCR tube on a magnetic stand for 2 min, and remove the supernatant. Repeat washing with Wash Buffer 2 twice for a total of 3 times. Finally, remove Wash Buffer 2 completely.
1.8.2.6. The PCR tube was placed on the magnetic stand, 200 μl of 80% ethanol was added to the PCR tube, and the tube was left to stand for 30 s, after which the ethanol solution was completely removed and the tube was dried at room temperature for 2 min.
1.8.2.7. Add 30 μL of nuclease-free water to the PCR tube, remove the PCR tube from the magnetic stand, and gently pipette up and down 6 times to resuspend the magnetic beads and prepare for use.

1.9. Post-capture amplification and purification 1.9.1. Prepare a reaction system according to the table below to concentrate the captured library, mix uniformly by gentle pipetting, and then briefly centrifuge.

1.9.2. Set up the following program, place the samples in the PCR instrument and run the program: Hot lid 105°C.

1.9.3. After PCR was completed, 55 μl of Agencourt AMPure XP magnetic beads were added to the samples and mixed evenly by gently pipetting.
1.9.4. Incubate at room temperature for 5 min and place the PCR tube on a magnetic stand for 3 min to clarify the solution.
1.9.5. Remove the supernatant and, while still placing the PCR tube on the magnetic stand, add 200 μl of 80% absolute ethanol and let stand for 30 s.
1.9.6. The supernatant was removed, and then 200 μl of 80% absolute ethanol was added to the PCR tube. After standing for 30 s, the supernatant was completely removed.
1.9.7. The remaining ethanol was allowed to evaporate completely by leaving it at room temperature for 5 minutes.
1.9.8.25 μl of nuclease-free water was added, the PCR tube was removed from the magnetic stand, gently pipetted to mix evenly to resuspend the magnetic beads, and the tube was left at room temperature for 2 min.
1.9.9. The PCR tube was placed on a magnetic stand for 2 min to clarify the solution.
1.9.10. Pipette 23 μl of supernatant into a 1.5 ml centrifuge tube and label with sample information.
1.9.11.1 μl of library was taken and quantified using Qubit and the library concentration was recorded.
1.9.12. 1 μl of sample was taken and library fragment lengths were measured using Agilent 2100.
1.9.13. Sequencing was performed using the Illumina high-throughput sequencing platform.

1.10. The analytical flow of methylation bioinformation is generally to check the sequencing quality using quality control software such as trimmomatic and remove low-quality reads, then compare the clean data after quality control with the reference genome using comparison software such as Bismarker, and extract the corresponding methylation sites using an R package such as methykit. Finally, the methylation ratio of each target region on the panel was calculated.

Example 2
Taking the pathologically evaluated sample as an example, the Panel detection of the present application is adopted, and peripheral blood is collected by the method of Example 1. A library is constructed and sequenced by the Illumina platform. The sequencing data is processed by the bioinformation analysis flow to obtain the methylation level, and the results are shown in Table 19 below (Table 19 shows the target regions above the methylation threshold that were detected).

Pattern recognition classification identification is performed on the detected samples. First, if the methylation levels of the pan-cancer specific markers TBX15 and CRYGD genes are determined to be 55% and 60% or more, the sample is preliminarily determined to be a sample suffering from cancer. Next, if the methylation levels of the cancer specific markers OTX1, SFRP2, CDO1, TRIM15, ALX4, and CCNA1 are all determined to be equal to or higher than the respective thresholds shown in Table 1 (as shown in Table 19 above), the sample is determined to be a sample suffering from any of the following 11 types of cancer (esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, and endometrial cancer). Finally, the tissue-specific markers are determined to be equal to or higher than the respective thresholds shown in Table 19, and six target regions Seq ID No. 81, Seq ID No. 82, Seq ID No. 83, Seq ID No. 84, Seq ID No. 85, Seq ID No. 86, Seq ID No. 87, Seq ID No. 88, Seq ID No. 89, Seq ID No. 90, Seq ID No. 91, Seq ID No. 92, Seq ID No. 93, Seq ID No. 94, Seq ID No. 95, Seq ID No. 96, Seq ID No. 97, Seq ID No. 98, Seq ID No. 99, Seq ID No. 99, Seq ID No. 91, Seq ID No. 94, Seq ID No. 95, Seq ID No. 96, Seq ID No. 97, Seq ID No. 99, Seq ID No. 99, Seq ID No. 98, Seq ID No. 99, Seq ID No. If it is found that Seq ID No. 82, Seq ID No. 97, Seq ID No. 98, Seq ID No. 99 and Seq ID No. 100 are above their respective methylation thresholds, and the methylation of other tissue-specific markers is not above their respective thresholds, and therefore, among the tissue-specific markers above their respective methylation thresholds, stomach tissue-specific markers are the most abundant, then the sample is finally determined to be a sample affected by gastric cancer.
The patient's blood was collected again 48 hours after the operation, and the Panel detection of the present application was adopted to collect peripheral blood by the method of Example 1. A library was constructed and sequenced by the Illumina platform, and the sequencing data was processed by the above bioinformation analysis flow, and all the sequencing data was analyzed by the method of pattern recognition, and the results showed that the gene methylation levels in the above table were restored to normal levels.

Example 3
Taking colorectal cancer samples as an example, the Panel detection of the present application is adopted, and peripheral blood is collected by the method of Example 1. A library is constructed and sequenced by the Illumina platform. The sequencing data is processed by the above bioinformation analysis flow to obtain the methylation level, and the results are shown in Table 20 below (Table 20 shows the target regions above the methylation threshold that were detected).

The detected samples are subjected to pattern recognition classification identification. First, if the methylation levels of the pan-cancer specific markers TBX15 and CRYGD genes are determined to be 55% and 60% or more, the sample is preliminarily determined to be a sample suffering from cancer. Next, if the methylation levels of the cancer specific markers TRH, CDO1, ELMO1, GFRA1, CCNA1, and SALL1 are all determined to be equal to or higher than the respective thresholds shown in Table 1 (as shown in Table 20 above), the sample is determined to be a sample suffering from any of the following 11 types of cancer (esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, and endometrial cancer). Finally, the tissue-specific markers are determined to be equal to or higher than the respective thresholds shown in Table 20, and the six target regions Seq ID No. 63, Seq ID No. 64, Seq ID No. 65, Seq ID No. 66, Seq ID No. 67, Seq ID No. 68, Seq ID No. 69, Seq ID No. 70, Seq ID No. 71, Seq ID No. 72, Seq ID No. 73, Seq ID No. 74, Seq ID No. 75, Seq ID No. 76, Seq ID No. 77, Seq ID No. 78, Seq ID No. 79, Seq ID No. 80, Seq ID No. 81, Seq ID No. 82, Seq ID No. 83, Seq ID No. 84, Seq ID No. 85, Seq ID No. 86, Seq ID No. 87, Seq ID No. 89, Seq ID No. 89, Seq ID No. 81, Seq ID No. 82, Seq ID No. 8 If it is found that Seq ID No. 64, Seq ID No. 87, Seq ID No. 88, Seq ID No. 89, and Seq ID No. 90 are above their respective methylation thresholds, and the methylation of other tissue-specific markers is not above their respective thresholds, and therefore, among the tissue-specific markers above their respective methylation thresholds, colorectal tissue-specific markers are the most abundant, then the sample is finally determined to be a sample affected by colorectal cancer.
The patient's blood was collected again 48 hours after the operation, and the Panel detection of the present application was adopted to collect peripheral blood by the method of Example 1. A library was constructed and sequenced, and the sequencing data was processed by the above bioinformation analysis flow, and all the sequencing data was analyzed by the method of pattern recognition, and the results showed that the gene methylation levels in the above table were restored to normal levels.

Example 4
Taking lung cancer samples as an example, the Panel detection of the present application is adopted, and peripheral blood is collected by the method of Example 1. A library is constructed and sequenced by the Illumina platform. The sequencing data is processed by the above-mentioned bioinformation analysis flow to obtain the methylation level, and the results are as shown in Table 21 below (Table 21 shows the target regions above the methylation threshold that were detected).

Pattern recognition classification identification is performed on the detected samples. First, if the methylation levels of the pan-cancer specific markers TBX15 and CRYGD genes are determined to be 55% and 60% or more, the sample is preliminarily determined to be a sample suffering from cancer. Next, if the methylation levels of the cancer specific markers RUNX3, APC, HOXA11 AS, PHOX2A, and GALR1 are all determined to be equal to or higher than the respective thresholds shown in Table 1 (as shown in Table 21 above), the sample is determined to be a sample suffering from any of the following 11 types of cancer (esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, and endometrial cancer). Finally, tissue-specific markers are determined, and five lung tissue-specific target regions Seq ID No. 71, Seq ID No. 61, Seq ID No. 62, Seq ID No. 63, Seq ID No. 64, Seq ID No. 65, Seq ID No. 66, Seq ID No. 67, Seq ID No. 69, Seq ID No. 70, Seq ID No. 71, Seq ID No. 72, Seq ID No. 73, Seq ID No. 74, Seq ID No. 75, Seq ID No. 76, Seq ID No. 77, Seq ID No. 78, Seq ID No. 79, Seq ID No. 80, Seq ID No. 81, Seq ID No. 82, Seq ID No. 83, Seq ID No. 84, Seq ID No. 85, Seq ID No. 86, Seq ID No. 87, Seq ID No. 89, Seq ID No. 89, Seq ID No. 89, Seq ID No. 81, Seq ID If it is found that Seq ID No. 72, Seq ID No. 76, Seq ID No. 91 and Seq ID No. 92 are above their respective methylation thresholds, and the methylation of other tissue-specific markers is not above their respective thresholds, and therefore, among the tissue-specific markers above their respective methylation thresholds, the lung tissue-specific markers are the most abundant, then the sample is finally determined to be a sample suffering from lung cancer.
The patient's blood was collected again 48 hours after the operation, and the Panel detection of the present application was adopted to collect peripheral blood by the method of Example 1. A library was constructed and sequenced, and the sequencing data was processed by the above bioinformation analysis flow, and all the sequencing data was analyzed by the method of pattern recognition, and the results showed that the gene methylation levels in the above table were restored to normal levels.

Example 5
Taking liver cancer samples as an example, the Panel detection of the present application is adopted, and peripheral blood is collected by the method of Example 1. A library is constructed and sequenced by the Illumina platform. The sequencing data is processed by the above-mentioned bioinformation analysis flow to obtain the methylation level, and the results are as shown in Table 22 below (Table 22 shows the target regions above the methylation threshold that were detected).

The detected samples were subjected to pattern recognition classification identification. First, the methylation levels of the pan-cancer specific markers TBX15 and CRYGD genes were determined to be 55% and 60% or more, respectively, to preliminarily determine that the sample was affected by cancer. Second, the methylation levels of the cancer specific markers RASSF1, APC, TRIM15, HOXA11 AS, and CCND2 were determined to be equal to or higher than the respective thresholds shown in Table 1 (as shown in Table 22 above), to determine that the sample was affected by any of the following 11 types of cancer (esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, and endometrial cancer). Finally, the tissue-specific markers were determined to be equal to or higher than the respective thresholds shown in Table 22, and two liver tissue-specific target regions Seq ID No. 73 and Seq ID No. 61 were determined to be equal to or higher than the respective thresholds shown in Table 22. If it is found that 74 are above their respective methylation level thresholds and the methylation of other tissue-specific markers is not above their respective thresholds, and therefore liver tissue-specific markers are the most abundant among the tissue-specific markers above their respective methylation thresholds, then the sample is finally determined to be a sample suffering from liver cancer.
The patient's blood was collected again 48 hours after the operation, and the Panel detection of the present application was adopted to collect peripheral blood by the method of Example 1. A library was constructed and sequenced, and the sequencing data was processed by the above bioinformation analysis flow, and all the sequencing data was analyzed by the method of pattern recognition, and the results showed that the gene methylation levels in the above table were restored to normal levels.

Example 6
Taking esophageal cancer samples as an example, the Panel detection of the present application is adopted, and peripheral blood is collected by the method of Example 1. A library is constructed and sequenced by the Illumina platform. The sequencing data is processed by the above-mentioned bioinformation analysis flow to obtain the methylation level, and the results are as shown in Table 23 below (Table 23 shows the target regions above the methylation threshold that were detected).

Pattern recognition classification identification is performed on the detected samples. First, if the methylation levels of the pan-cancer specific markers TBX15 and CRYGD genes are determined to be 55% and 60% or more, the sample is preliminarily determined to be a sample affected by cancer. Next, if the methylation levels of the cancer specific markers CPE, TFAP2E, TRH, C11orf21, and EDNRB are all determined to be equal to or higher than the respective thresholds shown in Table 1 (as shown in Table 23 above), the sample is determined to be a sample affected by any of the following 11 types of cancer (esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, and endometrial cancer). Finally, the tissue-specific markers are determined to be equal to or higher than the respective thresholds shown in Table 23, and the four target regions Seq ID No. 66, Seq ID No. 67, Seq ID No. 69, Seq ID No. 70, Seq ID No. 71, Seq ID No. 72, Seq ID No. 73, Seq ID No. 74, Seq ID No. 75, Seq ID No. 76, Seq ID No. 77, Seq ID No. 78, Seq ID No. 79, Seq ID No. 80, Seq ID No. 81, Seq ID No. 82, Seq ID No. 83, Seq ID No. 84, Seq ID No. 85, Seq ID No. 86, Seq ID No. 87, Seq ID No. 88, Seq ID No. 89, Seq ID No. 90, Seq ID No. 91, Seq ID No. 92, Seq ID No. 93, Seq ID No. 94, Seq ID No. 95, Seq ID No. 96 If it is found that Seq ID No. 79 and Seq ID No. 80 are above their respective methylation level thresholds, and the methylation of other tissue-specific markers is not above their respective thresholds, and therefore, among the tissue-specific markers above their respective methylation thresholds, the esophageal tissue-specific markers are the most abundant, then the sample is finally determined to be a sample affected by esophageal cancer.
The patient's blood was collected again 48 hours after the operation, and the Panel detection of the present application was adopted to collect peripheral blood by the method of Example 1. A library was constructed and sequenced, and the sequencing data was processed by the above bioinformation analysis flow, and all the sequencing data was analyzed by the method of pattern recognition, and the results showed that the gene methylation levels in the above table were restored to normal levels.

Example 7
Taking pancreatic cancer samples as an example, the Panel detection of the present application is adopted, and peripheral blood is collected by the method of Example 1. A library is constructed and sequenced by the Illumina platform. The sequencing data is processed by the above-mentioned bioinformation analysis flow to obtain the methylation level, and the results are as shown in Table 24 below (Table 24 shows the target regions above the methylation threshold that were detected).

Pattern recognition classification identification is performed on the detected samples. First, if the methylation levels of the pan-cancer specific markers TBX15 and CRYGD genes are determined to be 55% and 60% or more, the sample is preliminarily determined to be a sample suffering from cancer. Next, if the methylation levels of the cancer specific markers HOPX, SFRP2, GFRA1, HOXB4, and SALL1 are all determined to be equal to or higher than the respective thresholds shown in Table 1 (as shown in Table 24 above), the sample is determined to be a sample suffering from any of the following 11 types of cancer (esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, and endometrial cancer). Finally, the tissue-specific markers are determined to be equal to or higher than the respective thresholds shown in Table 24, and five target regions Seq ID No. 111, Seq ID No. 112, Seq ID No. 113, Seq ID No. 114, Seq ID No. 115, Seq ID No. 116, Seq ID No. 117, Seq ID No. 118, Seq ID No. 119, Seq ID No. 120, Seq ID No. 121, Seq ID No. 122, Seq ID No. 123, Seq ID No. 124, Seq ID No. 125, Seq ID No. 126, Seq ID No. 127, Seq ID No. 128, Seq ID No. 129, Seq ID No. 130, Seq ID No. 131, Seq ID No. 132, Seq ID No. 133, Seq ID No. 134, Seq ID No. 135, Seq ID No. 136, Seq ID No. 137, S If it is found that Seq ID No. 112, Seq ID No. 113, Seq ID No. 114 and Seq ID No. 115 are equal to or above their respective methylation level thresholds, and the methylation of other tissue-specific markers is not equal to or above their respective thresholds, and therefore pancreatic tissue-specific markers are the most abundant among the tissue-specific markers equal to or above their respective methylation thresholds, then the sample is finally determined to be a sample suffering from pancreatic cancer.
The patient's blood was collected again 48 hours after the operation, and the Panel detection of the present application was adopted to collect peripheral blood by the method of Example 1. A library was constructed and sequenced, and the sequencing data was processed by the above bioinformation analysis flow, and all the sequencing data was analyzed by the method of pattern recognition, and the results showed that the gene methylation levels in the above table were restored to normal levels.

Example 8
Taking breast cancer samples as an example, the Panel detection of the present application is adopted, and peripheral blood is collected by the method of Example 1. A library is constructed and sequenced by the Illumina platform. The sequencing data is processed by the above-mentioned bioinformation analysis flow to obtain the methylation level, and the results are as shown in Table 25 below (Table 25 shows the target regions above the methylation threshold that were detected).

Pattern recognition classification identification is performed on the detected samples. First, if the methylation levels of the pan-cancer specific markers TBX15 and CRYGD genes are determined to be 55% and 60% or more, the sample is preliminarily determined to be a sample suffering from cancer. Next, if the methylation levels of the cancer specific markers TRIM15, RUNX3, TFAP2E, RASSF1, SFRP2, and CCND2 are all determined to be equal to or higher than the respective thresholds shown in Table 1 (as shown in Table 25 above), the sample is determined to be a sample suffering from any of the following 11 types of cancer (esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, and endometrial cancer). Finally, the tissue-specific markers are determined to be equal to or higher than the respective thresholds shown in Table 25, and the three target regions Seq ID No. 78, Seq ID No. 100, Seq ID No. 1100, Seq ID No. 120, Seq ID No. 130, Seq ID No. 140, Seq ID No. 150, Seq ID No. 160, Seq ID No. 170, Seq ID No. 180, Seq ID No. 190, Seq ID No. 200, Seq ID No. 210, Seq ID No. 220, Seq ID No. 230, Seq ID No. 240, Seq ID No. 250, Seq ID No. 260, Seq ID No. 270, Seq ID No. 280, Seq ID No. 290, Seq ID No. 300, Seq ID No. 310, Seq ID No. 320, Seq ID No. 330, Seq ID No. 340, Seq ID If it is found that Seq ID No. 85 and Seq ID No. 86 are above their respective methylation thresholds, and the methylation of other tissue-specific markers is not above their respective thresholds, and therefore, among the tissue-specific markers above their respective methylation thresholds, the breast tissue-specific markers are the most abundant, then the sample is finally determined to be a sample affected by breast cancer.
The patient's blood was collected again 48 hours after the operation, and the Panel detection of the present application was adopted to collect peripheral blood by the method of Example 1. A library was constructed and sequenced, and the sequencing data was processed by the above bioinformation analysis flow, and all the sequencing data was analyzed by the method of pattern recognition, and the results showed that the gene methylation levels in the above table were restored to normal levels.

Those skilled in the art can make various corresponding modifications and variations based on the above technical solutions and concepts, and all these modifications and variations should be included within the scope of protection of the claims of this application.

Claims (10)

probes targeting esophageal, gastric, colorectal, lung, liver, pancreatic, prostate, breast, ovarian, cervical and endometrial cancer specific regions, probes targeting pan-cancer specific regions, and probes targeting tissue specific regions;
The cancer-specific region is Seq ID No. 1-62;
The pan-cancer specific region is Seq ID No. 63-64;
The tissue-specific region is Seq ID No. 65-117;
Probe composition. Seq ID No. 66-67, 79, 81-82, 97-102, 116-117 in the tissue-specific region are tissue-specific target regions of the esophagus, or Seq ID No. 81, 82, 97-102 in the tissue-specific region are tissue-specific target regions of the stomach, or Seq ID No. 83, 87-90, 110 in the tissue-specific region are tissue-specific target regions of the colorectum, or Seq ID No. 65, 70-72, 76, 91-92, 103-104 in the tissue-specific region are tissue-specific target regions of the lung, or Seq ID No. 73-74, 107 in the tissue-specific region are tissue-specific target regions of the liver ...65, 70-72, 76, 91-92, 103-104 in the tissue-specific region are tissue-specific target regions of the lung, or Seq ID No. 73-74, 107 in The probe composition according to claim 1, wherein Seq ID Nos. 111-115 are tissue-specific target regions of the pancreas, or Seq ID Nos. 80, 84, 93, and 95 in the tissue-specific region are tissue-specific target regions of the prostate, or Seq ID Nos. 78, 84-86 in the tissue-specific region are tissue-specific target regions of the mammary gland, or Seq ID Nos. 77, 96, and 105 in the tissue-specific region are tissue-specific target regions of the ovary, or Seq ID Nos. 68-69, 75, and 109 in the tissue-specific region are tissue-specific target regions of the cervix, or Seq ID Nos. 94, 106, and 108 in the tissue-specific region are tissue-specific target regions of the endometrium. The probe composition according to any one of claims 1-2, comprising a low-methylation probe hybridized to the specific region in which the bisulfite-converted CG is not methylated, and a high-methylation probe hybridized to the specific region in which the bisulfite-converted CG is entirely methylated. The probe composition according to claim 3, wherein each probe has a length of 40 to 60 bp. The probe composition according to claim 4, wherein the length of each probe is 45 to 56 bp, preferably 50 to 56 bp, and more preferably 50 bp. The probe composition according to claim 3, characterized in that the hypomethylated probes include probes Seq ID No. 118-214 targeting cancer-specific regions, probes Seq ID No. 215-216 targeting pan-cancer-specific regions, and probes Seq ID No. 217-274 targeting tissue-specific regions. The probe composition according to claim 3, characterized in that the hypermethylated probes include probes Seq ID No. 275-371 targeting cancer-specific regions, probes Seq ID No. 372-373 targeting pan-cancer-specific regions, and probes Seq ID No. 374-431 targeting tissue-specific regions. A kit comprising the probe composition according to any one of claims 1 to 7. Use of the probe composition according to any one of claims 1 to 7 in the manufacture of a kit for detecting esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer and endometrial cancer. Probes targeting specific regions of esophageal, gastric, colorectal, lung, liver, pancreatic, prostate, breast, ovarian, cervical and endometrial cancer; Use of probes targeting pan-cancer specific regions and probes targeting tissue specific regions for detecting esophageal, gastric, colorectal, lung, liver, pancreatic, prostate, breast, ovarian, cervical and endometrial cancer :
wherein the cancer-specific region is Seq ID No. 1-62;
The pan-cancer specific region is Seq ID No. 63-64;
The tissue-specific region is Seq ID No. 65-117 .