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WO2021169874A1 - Composition de sonde pour la détection de tumeurs d'organe à trois lumières - Google Patents

Composition de sonde pour la détection de tumeurs d'organe à trois lumières Download PDF

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Publication number
WO2021169874A1
WO2021169874A1 PCT/CN2021/077062 CN2021077062W WO2021169874A1 WO 2021169874 A1 WO2021169874 A1 WO 2021169874A1 CN 2021077062 W CN2021077062 W CN 2021077062W WO 2021169874 A1 WO2021169874 A1 WO 2021169874A1
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Prior art keywords
seq
probe
cancer
target region
region shown
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Chinese (zh)
Inventor
韩晓亮
李永君
吴宁宁
郭媛媛
王建铭
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Biochain (beijing) Science & Technology Inc
Biochain Beijing Science and Technology Inc
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Biochain (beijing) Science & Technology Inc
Biochain Beijing Science and Technology Inc
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Publication of WO2021169874A1 publication Critical patent/WO2021169874A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer

Definitions

  • This application relates to a cancer gene methylation detection composition, in particular to a probe composition that specifically recognizes the DNA sequence after Bisulfite treatment, and detection based on high-throughput sequencing (NGS) method
  • NGS high-throughput sequencing
  • NGS High-throughput sequencing
  • This technology can simultaneously sequence dozens to millions of DNA molecules, marking the arrival of the post-genome era.
  • different goals such as de novo sequencing and resequencing can be achieved, and the sequence of the genome, transcriptome, and methylome can also be analyzed through different pre-processing.
  • PCR polymerase chain reaction
  • FISH fluorescence in situ hybridization
  • gene chip technology has low price, high sensitivity, simple and fast operation, and high clinical popularity.
  • FISH fluorescence in situ hybridization
  • the gene chip throughput is higher than the former two, and it can detect a large number of genes at the same time.
  • the limitation is that it can only detect known genes or mutations, with low accuracy and high false positives.
  • the NGS technology has the characteristics of high throughput (detecting a large number of known and unknown genes and mutations at the same time), accurate results (higher accuracy than gene chips), fast detection speed, and low cost of detection for each gene. Now Has been gradually applied to clinical disease detection and monitoring and other fields. With the further reduction of sequencing costs in the future, NGS will inevitably gradually replace other high-throughput technologies such as gene chips.
  • target sequence capture and sequencing has become a more mainstream choice.
  • This technology is based on detection requirements, designing capture probes for the genomic region of interest, enriching the target fragment DNA through the principle of hybridization and complementation, and subsequently performing NGS detection.
  • This strategy can be flexibly customized according to the purpose of research or detection, selecting only a small number of gene regions, increasing the depth of sequencing, and effectively discovering the variation of the target region, with high sensitivity and accuracy.
  • Liquid biopsy is a method of in vitro diagnosis. It uses non-invasive blood testing to monitor circulating tumor cells (CTC) or circulating tumor DNA (ctDNA) released into the blood by tumors or metastases. This technology can effectively reduce invasiveness. It can realize the sampling of all parts of the tumor and all metastases, overcome tumor heterogeneity (and the current standard tissue biopsy can only reflect the characteristics of a certain part of the tumor), and realize real-time monitoring with higher sensitivity , It is even possible to predict the location of the lesion based on genomic information, which can effectively prolong the survival time of patients. Based on these advantages, liquid biopsy can be used for early diagnosis of tumors, auxiliary staging, prognosis and recurrence monitoring, medication guidance and other aspects. Currently the most commonly used liquid biopsy of free DNA.
  • CtDNA Cell-free DNA
  • cfDNA is a partially degraded endogenous DNA that is free and extracellular in circulating blood. Studies have shown that in the process of tumor tissue development, after tumor cell apoptosis, DNA will be released into plasma, and after degradation, free tumor DNA (ctDNA) will be formed.
  • the molecular genetic characteristics of CtDNA (such as gene mutation, microsatellite instability, and tumor suppressor gene promoter methylation, etc.) are consistent with tumor tissue DNA.
  • the collection of peripheral blood is simpler than other clinical detection methods, easy to promote to the grassroots, and because of its non-invasive characteristics, it is easier to be accepted by asymptomatic people. Therefore, the detection of changes in the level of ctDNA methylation in plasma can become one of the important methods for early screening and diagnosis of multiple cancers.
  • Using target sequence capture technology combined with NGS to monitor cfDNA variation and methylation level changes can realize early tumor screening, susceptibility gene monitoring, companion diagnosis, personalized medication, prognostic monitoring and other applications.
  • many companies at home and abroad have launched cancer detection panels of different scales for different application scenarios. Some panels have obtained FDA or CFDA approval numbers.
  • FoundationOne CDx launched by Foundation Medicine covers 324 genes
  • IMPACT launched by Memorial Sloan Kettering Cancer Research Center (MSK) covers 468 cancer-related genes
  • Burning Rock Medicine launched "human EGFR/ALK/BRAF/KRAS gene mutations” Joint detection kit”, “Human EGFR, KRAS, BRAF, PIK3CA, ALK, ROS1 gene mutation detection kit” launched by Nuohe Zhiyuan, etc.
  • Kunyuan Gene also launched the product "Chang Lesi” to detect the methylation level of colorectal cancer in 2018.
  • this application provides a probe composition that can be used for early screening of esophageal cancer, gastric cancer, colorectal cancer and other three types of luminal organ tumors.
  • the probe composition can: 1) be used in a non-invasive way for early screening of asymptomatic people and prognostic detection of cancer patients, reducing the harm caused by invasive detection, 2) increasing the depth of sequencing and making The breadth of gene detection is better than existing technologies and products, with high throughput, faster detection speed, and low cost of detection for each gene. 3) It can sample all parts of the tumor and all metastases. Overcome tumor heterogeneity, and 4) have higher sensitivity and accuracy, can realize real-time monitoring, and it is even possible to predict the location of lesions through genomic information to effectively prolong the survival of patients.
  • this application involves the following:
  • a probe composition comprising: a probe targeting a specific region of any cancer among esophageal cancer, gastric cancer, and colorectal cancer, wherein the cancer-specific region is selected from Seq ID No.: 1 Any of -62.
  • the probe composition according to item 1 further comprising: any probe targeting a pan-cancer specific region, the pan-cancer specific region selected from Seq ID No.: 63-64 Arbitrarily.
  • the probe composition according to item 3 characterized in that Seq ID Nos.: 65-69, 75-80, and 82-83 in the tissue-specific region are tissue-specific target regions of the esophagus.
  • the probe composition according to item 3 characterized in that Seq ID Nos.: 68, 69, 75-80 in the tissue-specific region are tissue-specific target regions of the stomach.
  • the probe composition according to item 3 characterized in that Seq ID No.: 70-74 and 81 in the tissue-specific region are tissue-specific target regions of the colorectal.
  • a hypomethylation probe that hybridizes to the specific region that has been converted by bisulfite and does not contain CG methylation
  • hypomethylation probe includes a probe that targets a cancer-specific region.
  • Seq ID No. any of 84-180, targeting Seq ID No. of probes for pan-cancer specific regions: any of 181-182, and Seq ID No. of probes for tissue-specific regions: any of 183-204.
  • a kit comprising the probe composition according to any one of items 1-11.
  • This application also provides a method for detecting three types of luminal organ tumors, including esophageal cancer, gastric cancer and colorectal cancer, by using the probe composition.
  • This application also provides a method for simultaneously detecting changes in the methylation level of the above three cancers by using the kit.
  • Liquid biopsy is a non-invasive tumor detection, which can be applied to asymptomatic and patient groups who cannot obtain tissue samples.
  • the average sequencing depth exceeds 5000X.
  • the screening of all high-incidence cancers can be completed at one time, which improves the detection efficiency.
  • the average price of each marker is lower than the existing single-marker detection in the market.
  • one Panel can complete the screening of major cancers, which saves the cost of probe synthesis, simplifies the experimental process, and facilitates the operation of experimenters.
  • FIG 1 is the implementation process of this application
  • This application provides a probe composition for cancer gene methylation detection.
  • NGS high-throughput sequencing
  • the probe is a single-stranded or double-stranded DNA with a length of tens to hundreds or even thousands of base pairs, which can take advantage of molecular denaturation, renaturation and high accuracy of base complementary pairing, and can be complementary to the sample to be tested
  • the unlabeled single-stranded DNA or RNA is hydrogen-bonded (hybridized) to form a double-stranded complex (hybrid).
  • detection systems such as autoradiography or enzyme-linked reaction can be used to detect the results of the hybridization reaction.
  • the region that complementarily binds or hybridizes with the probe is the specific target region. Multiple probes are combined into a probe composition.
  • a cancer-specific region refers to a significant difference in the methylation level of this region compared with normal control tissues in a small number of cancer types.
  • pan-cancer specific region refers to a significant difference in the methylation level of this region compared with normal control tissues in most cancer types.
  • Tissue-specific region refers to the significant difference in the methylation level of the region in a specific tissue compared with other tissues.
  • DNA methylation refers to the methylation process that occurs at the 5th carbon atom of cytosine in CpG dinucleotides. As a stable modification state, DNA methyltransferase can follow DNA The duplication process of dna is inherited to the new generation DNA, which is an important epigenetic mechanism. When DNA is methylated, the methylation of the gene promoter region can lead to the silence of tumor suppressor gene transcription, so it is related to the occurrence of tumors. close. Abnormal methylation includes hypermethylation of tumor suppressor genes and DNA repair genes, hypomethylation of repetitive sequence DNA, and loss of imprinting of certain genes, which are related to the occurrence of a variety of tumors.
  • Panel refers to the probe composition used in this article.
  • a probe composition includes: a probe targeting a specific region of any cancer among esophageal cancer, gastric cancer, and colorectal cancer, wherein the cancer-specific region Selected from any of Seq ID No.: 1-62; probes that target any of the pan-cancer specific regions, the pan-cancer specific region selected from any of Seq ID No.: 63-64; and Target any probe in a tissue-specific region, the tissue-specific region is selected from any of Seq ID No.: 65-83.
  • Seq ID No.: 65-69, 75-80, 82-83 in the tissue-specific regions are tissue-specific target regions of the esophagus.
  • a probe composition includes: a probe targeting a specific region of any cancer among esophageal cancer, gastric cancer, and colorectal cancer, wherein the cancer-specific region Selected from any of Seq ID No.: 1-62; probes that target any of the pan-cancer specific regions, the pan-cancer specific region selected from any of Seq ID No.: 63-64; and Target any probe in a tissue-specific region, the tissue-specific region is selected from any of Seq ID No.: 65-83.
  • Seq ID No. of the tissue-specific regions: 68, 69, 75-80 are tissue-specific target regions of the stomach.
  • a probe composition includes: a probe targeting a specific region of any cancer among esophageal cancer, gastric cancer, and colorectal cancer, wherein the cancer-specific region Selected from any of Seq ID No.: 1-62; probes that target any of the pan-cancer specific regions, the pan-cancer specific region selected from any of Seq ID No.: 63-64; and Target any probe in a tissue-specific region, the tissue-specific region is selected from any of Seq ID No.: 65-83.
  • Seq ID No. of the tissue-specific regions: 70-74 and 81 are tissue-specific target regions of the colorectal.
  • the above-mentioned probe composition includes a hypomethylated probe which hybridizes to the bisulfite-converted specific region without CG methylation, and high methylation A probe that hybridizes to the specific region where the bisulfite-converted CG is fully methylated.
  • the hypomethylation probes include probes targeting cancer-specific regions Seq ID No.: any of 84-180, and probes targeting pan-cancer specific regions Seq ID No.: 181-182 Any, and Seq ID No. of probes targeting tissue-specific regions: Any of 183-204.
  • the hypermethylation probes include probes targeting cancer-specific regions, Seq ID No.: any of 205-301, and probes targeting pan-cancer specific regions, Seq ID No.: 302-303, Seq ID No.: any of 304-325 probes targeting tissue-specific regions.
  • Seq ID No. 84, Seq ID No. 85 and Seq ID No. 86 are all hypomethylated probes that target the target region shown in Seq ID No. 1
  • Seq ID No. 205 , Seq ID No. 206 and Seq ID No. 207 are all hypermethylated probes that target the target region shown in Seq ID No. 1.
  • Seq ID No. 87 and Seq ID No. 88 are hypomethylated probes that target the target region shown in Seq ID No. 2
  • Seq ID No. 208 and Seq ID No. 209 are both targeted to Seq ID No. .2 Hypermethylated probes in the target region shown.
  • Seq ID No. 90 are hypomethylated probes that target the target region shown in Seq ID No. 3, and Seq ID No. 210 and Seq ID No. 211 are both targeted to Seq ID No. .3 Hypermethylated probes in the target region shown.
  • Seq ID No. 91 is a hypomethylated probe targeting the target region shown in Seq ID No. 4
  • Seq ID No. 212 is a hypermethylated probe targeting the target region shown in Seq ID No. 4.
  • Seq ID No. 92 and Seq ID No. 93 are hypomethylated probes that target the target region shown in Seq ID No. 5, and Seq ID No. 213 and Seq ID No. 214 are both targeted to Seq ID No. .3 Hypermethylated probes in the target region shown.
  • Seq ID No. 94 is a hypomethylated probe that targets the target region shown in Seq ID No. 6, and Seq ID No. 215 is a hypermethylated probe that targets the target region shown in Seq ID No. 6.
  • Seq ID No. 95 is a hypomethylated probe that targets the target region shown in Seq ID No. 7, and Seq ID No. 216 is a hypermethylated probe that targets the target region shown in Seq ID No. 7.
  • Seq ID No. 96 and Seq ID No. 97 are hypomethylated probes that target the target region shown in Seq ID No. 8, and Seq ID No. 217 and Seq ID No. 218 are both targeted to Seq ID No.
  • Seq ID No. 100 is all hypomethylated probes that target the target region shown in Seq ID No. 9, Seq ID No. 219, Seq ID No. 220 and Seq ID No. 221 are all hypermethylated probes that target the target region shown in Seq ID No. 9.
  • Seq ID No. 101 is a hypomethylated probe targeting the target region shown in Seq ID No. 10
  • Seq ID No. 222 is a hypermethylated probe targeting the target region shown in Seq ID No. 10.
  • Seq ID No. 102 is a hypomethylated probe targeting the target region shown in Seq ID No. 11, and Seq ID No. 223 is a hypermethylated probe targeting the target region shown in Seq ID No. 11.
  • Seq ID No. 103 is a hypomethylated probe targeting the target region shown in Seq ID No. 12, and Seq ID No. 224 is a hypermethylated probe targeting the target region shown in Seq ID No. 12.
  • Seq ID No. 104 is a hypomethylated probe targeting the target region shown in Seq ID No. 13, and Seq ID No. 225 is a hypermethylated probe targeting the target region shown in Seq ID No. 13.
  • Seq ID No. 105 and Seq ID No. 106 are both hypomethylated probes that target the target region shown in Seq ID No. 14, and Seq ID No. 226 and Seq ID No. 227 are both targeted to Seq ID No.
  • Seq ID No. 109 and Seq ID No. 110 are both hypomethylated probes that target the target region shown in Seq ID No. 16, and Seq ID No. 230 and Seq ID No. 231 are both targeted to Seq ID No.
  • Seq ID No. 111 and Seq ID No. 112 are hypomethylated probes that target the target region shown in Seq ID No. 17, and Seq ID No. 232 and Seq ID No.
  • Seq ID No. 113 is a hypomethylated probe targeting the target region shown in Seq ID No. 18, and Seq ID No. 234 is a hypermethylated probe targeting the target region shown in Seq ID No. 18.
  • Seq ID No. 114 and Seq ID No. 115 are both hypomethylated probes that target the target region shown in Seq ID No. 19, and Seq ID No. 235 and Seq ID No. 236 are both targeted to Seq ID No.
  • Seq ID No. 116 is a hypomethylated probe targeting the target region shown in Seq ID No. 20, and Seq ID No.
  • Seq ID No. 20 is a hypermethylated probe targeting the target region shown in Seq ID No. 20.
  • Seq ID No. 117 is a hypomethylated probe targeting the target region shown in Seq ID No. 21, and Seq ID No. 238 is a hypermethylated probe targeting the target region shown in Seq ID No. 21.
  • Seq ID No.118 is a hypomethylated probe targeting the target region shown in Seq ID No.22, and Seq ID No.239 is a hypermethylated probe targeting the target region shown in Seq ID No.22.
  • Seq ID No. 119, Seq ID No. 120, and Seq ID No. 121 are all hypomethylated probes that target the target region shown in Seq ID No. 23, Seq ID No. 240, Seq ID No.
  • Seq ID No. 242 are all hypermethylated probes that target the target region shown in Seq ID No. 23.
  • Seq ID No. 122 and Seq ID No. 123 are both hypomethylated probes that target the target region shown in Seq ID No. 24, and Seq ID No. 243 and Seq ID No. 244 are both targeted to Seq ID No.
  • Seq ID No. 124 and Seq ID No. 125 are both hypomethylated probes that target the target region shown in Seq ID No. 25, and Seq ID No. 245 and Seq ID No. 246 are both targeted to Seq ID No.
  • Seq ID No. 126 and Seq ID No. 127 are both hypomethylated probes that target the target region shown in Seq ID No. 26, and Seq ID No. 247 and Seq ID No. 248 are both targeted to Seq ID No.
  • Seq ID No. 128 is a hypomethylated probe targeting the target region shown in Seq ID No. 27, and Seq ID No. 249 is a hypermethylated probe targeting the target region shown in Seq ID No. 27.
  • Seq ID No. 129 and Seq ID No. 130 are hypomethylated probes that target the target region shown in Seq ID No. 28, and Seq ID No. 250 and Seq ID No. 251 are both targeted to Seq ID No.
  • the hypermethylated probe in the target region shown in .28. Seq ID No. 131 and Seq ID No. 132 are hypomethylated probes that target the target region shown in Seq ID No. 29, and Seq ID No. 252 and Seq ID No. 253 are both targeted to Seq ID No.
  • Seq ID No. 133 is a hypomethylated probe that targets the target region shown in Seq ID No. 30, and Seq ID No. 254 is a hypermethylated probe that targets the target region shown in Seq ID No. 30.
  • Seq ID No. 134 and Seq ID No. 135 are hypomethylated probes that target the target region shown in Seq ID No. 31, and Seq ID No.
  • Seq ID No. 136 is a hypomethylated probe targeting the target region shown in Seq ID No. 32
  • Seq ID No. 257 is a hypermethylated probe targeting the target region shown in Seq ID No. 32.
  • Seq ID No. 137 and Seq ID No. 138 are hypomethylated probes that target the target region shown in Seq ID No. 33
  • Seq ID No. 258 and Seq ID No. 259 are both targeted to Seq ID No.
  • Seq ID No. 139 is a hypomethylated probe targeting the target region shown in Seq ID No.
  • Seq ID No. 34 is a hypermethylated probe targeting the target region shown in Seq ID No. 34.
  • Seq ID No. 140 and Seq ID No. 141 are both hypomethylated probes that target the target region shown in Seq ID No. 35, and Seq ID No. 261 and Seq ID No. 262 are both targeted to Seq ID No.
  • Seq ID No. 142 is a hypomethylated probe targeting the target region shown in Seq ID No. 36
  • Seq ID No. 263 is a hypermethylated probe targeting the target region shown in Seq ID No. 36.
  • Seq ID No. 144 are hypomethylated probes that target the target region shown in Seq ID No. 37, and Seq ID No. 264 and Seq ID No. 265 are both targeted to Seq ID No.
  • Seq ID No. 145 and Seq ID No. 146 are hypomethylated probes that target the target region shown in Seq ID No. 38, and Seq ID No. 266 and Seq ID No. 267 are both targeted to Seq ID No.
  • Seq ID No. 147 is a hypomethylated probe targeting the target region shown in Seq ID No. 39
  • Seq ID No. 268 is a hypermethylated probe targeting the target region shown in Seq ID No. 39.
  • Seq ID No. 148 and Seq ID No. 149 are both hypomethylated probes that target the target region shown in Seq ID No. 40, and Seq ID No. 269 and Seq ID No. 270 are both targeted to Seq ID No.
  • Seq ID No. 150 is a hypomethylated probe targeting the target region shown in Seq ID No. 41
  • Seq ID No. 271 is a hypermethylated probe targeting the target region shown in Seq ID No. 41.
  • Seq ID No. 151 is a hypomethylated probe targeting the target region shown in Seq ID No. 42
  • Seq ID No. 272 is a hypermethylated probe targeting the target region shown in Seq ID No. 42.
  • Seq ID No. 152 is a hypomethylated probe targeting the target region shown in Seq ID No. 43
  • Seq ID No. 273 is a hypermethylated probe targeting the target region shown in Seq ID No. 43.
  • Seq ID No. 153 is a hypomethylated probe targeting the target region shown in Seq ID No. 44
  • Seq ID No. 274 is a hypermethylated probe targeting the target region shown in Seq ID No. 44.
  • Seq ID No. 154 and Seq ID No. 155 are hypomethylated probes that target the target region shown in Seq ID No. 45
  • Seq ID No. 275 and Seq ID No. 276 are both targeted to Seq ID No.
  • Seq ID No.156 is a hypomethylated probe targeting the target region shown in Seq ID No.46
  • Seq ID No.277 is a hypermethylated probe targeting the target region shown in Seq ID No.46.
  • Seq ID No. 157 is a hypomethylated probe targeting the target region shown in Seq ID No. 47
  • Seq ID No. 278 is a hypermethylated probe targeting the target region shown in Seq ID No. 47.
  • Seq ID No. 158 is a hypomethylated probe targeting the target region shown in Seq ID No. 48
  • Seq ID No. 279 is a hypermethylated probe targeting the target region shown in Seq ID No. 48.
  • Seq ID No.159 is a hypomethylated probe targeting the target region shown in Seq ID No.49
  • Seq ID No.280 is a hypermethylated probe targeting the target region shown in Seq ID No.49
  • Seq ID No. 160 is a hypomethylated probe targeting the target region shown in Seq ID No. 50
  • Seq ID No. 281 is a hypermethylated probe targeting the target region shown in Seq ID No. 50
  • Seq ID No. 161 and Seq ID No. 162 are hypomethylated probes that target the target region shown in Seq ID No. 51
  • Seq ID No. 282 and Seq ID No. 283 are both targeted to Seq ID No.
  • Seq ID No. 163 is a hypomethylated probe targeting the target region shown in Seq ID No. 52
  • Seq ID No. 284 is a hypermethylated probe targeting the target region shown in Seq ID No. 52.
  • Seq ID No.164 and Seq ID No.165 are hypomethylated probes that target the target region shown in Seq ID No.53, and Seq ID No.285 and Seq ID No.286 are both targeted to Seq ID No.
  • Seq ID No.166 is a hypomethylated probe targeting the target region shown in Seq ID No.54
  • Seq ID No.287 is a hypermethylated probe targeting the target region shown in Seq ID No.54.
  • Seq ID No. 168 are hypomethylated probes that target the target region shown in Seq ID No. 55, and Seq ID No. 288 and Seq ID No. 289 are both targeted to Seq ID No.
  • Seq ID No. 169 and Seq ID No. 170 are both hypomethylated probes that target the target region shown in Seq ID No. 56, and Seq ID No. 290 and Seq ID No. 291 are both targeted to Seq ID No.
  • Seq ID No. 171 and Seq ID No. 172 are hypomethylated probes that target the target region shown in Seq ID No. 57, and Seq ID No. 292 and Seq ID No.
  • Seq ID No. 173 and Seq ID No. 174 are both hypomethylated probes that target the target region shown in Seq ID No. 58, and Seq ID No. 294 and Seq ID No. 295 are both targeted to Seq ID No.
  • Seq ID No. 175 and Seq ID No. 176 are hypomethylated probes that target the target region shown in Seq ID No. 59, and Seq ID No. 296 and Seq ID No. 297 are both targeted to Seq ID No.
  • Seq ID No. 177 is a hypomethylated probe targeting the target region shown in Seq ID No. 60
  • Seq ID No. 298 is a hypermethylated probe targeting the target region shown in Seq ID No. 60
  • Seq ID No. 178 and Seq ID No. 179 are hypomethylated probes that target the target region shown in Seq ID No. 61
  • Seq ID No. 299 and Seq ID No. 300 are both targeted to Seq ID No.
  • Seq ID No. 180 is a hypomethylated probe targeting the target region shown in Seq ID No. 62
  • Seq ID No. 301 is a hypermethylated probe targeting the target region shown in Seq ID No. 62.
  • Seq ID No. 181 is a hypomethylated probe targeting the target region shown in Seq ID No. 63
  • Seq ID No. 302 is a hypermethylated probe targeting the target region shown in Seq ID No. 63
  • Seq ID No. 182 is a hypomethylated probe targeting the target region shown in Seq ID No. 64
  • Seq ID No. 303 is a hypermethylated probe targeting the target region shown in Seq ID No. 64
  • Seq ID No. 183 is a hypomethylated probe targeting the target region shown in Seq ID No. 65
  • Seq ID No. 304 is a hypermethylated probe targeting the target region shown in Seq ID No. 65
  • Seq ID No. 184 is a hypomethylated probe targeting the target region shown in Seq ID No.
  • Seq ID No. 165 is a hypermethylated probe targeting the target region shown in Seq ID No. 66.
  • Seq ID No. 185 is a hypomethylated probe targeting the target region shown in Seq ID No. 67
  • Seq ID No. 306 is a hypermethylated probe targeting the target region shown in Seq ID No. 67.
  • Seq ID No. 186 is a hypomethylated probe targeting the target region shown in Seq ID No. 68
  • Seq ID No. 307 is a hypermethylated probe targeting the target region shown in Seq ID No. 68.
  • Seq ID No. 187 is a hypomethylated probe targeting the target region shown in Seq ID No. 69, and Seq ID No.
  • Seq ID No. 308 is a hypermethylated probe targeting the target region shown in Seq ID No. 69.
  • Seq ID No.188 is a hypomethylated probe targeting the target region shown in Seq ID No.70
  • Seq ID No.309 is a hypermethylated probe targeting the target region shown in Seq ID No.70.
  • Seq ID No. 189 is a hypomethylated probe targeting the target region shown in Seq ID No. 71
  • Seq ID No. 310 is a hypermethylated probe targeting the target region shown in Seq ID No. 71.
  • Seq ID No. 190 is a hypomethylated probe targeting the target region shown in Seq ID No. 72
  • Seq ID No. 311 is a hypermethylated probe targeting the target region shown in Seq ID No. 72.
  • Seq ID No. 191 is a hypomethylated probe targeting the target region shown in Seq ID No. 73
  • Seq ID No. 312 is a hypermethylated probe targeting the target region shown in Seq ID No. 73
  • Seq ID No. 192 is a hypomethylated probe targeting the target region shown in Seq ID No. 74
  • Seq ID No. 313 is a hypermethylated probe targeting the target region shown in Seq ID No. 74
  • Seq ID No. 193 is a hypomethylated probe that targets the target region shown in Seq ID No. 75
  • Seq ID No. 314 is a hypermethylated probe that targets the target region shown in Seq ID No. 75.
  • Seq ID No. 194 is a hypomethylated probe targeting the target region shown in Seq ID No. 76
  • Seq ID No. 315 is a hypermethylated probe targeting the target region shown in Seq ID No. 76.
  • Seq ID No. 195 is a hypomethylated probe targeting the target region shown in Seq ID No. 77
  • Seq ID No. 316 is a hypermethylated probe targeting the target region shown in Seq ID No. 77.
  • Seq ID No. 196 is a hypomethylated probe targeting the target region shown in Seq ID No. 78
  • Seq ID No. 317 is a hypermethylated probe targeting the target region shown in Seq ID No. 78.
  • Seq ID No.197 is a hypomethylated probe targeting the target region shown in Seq ID No.79
  • Seq ID No.318 is a hypermethylated probe targeting the target region shown in Seq ID No.79.
  • Seq ID No. 198 and Seq ID No. 199 are hypomethylated probes that target the target region shown in Seq ID No. 80
  • Seq ID No. 319 and Seq ID No. 320 are both targeted to Seq ID No.
  • Seq ID No. 200, Seq ID No. 201, and Seq ID No. 202 are all hypomethylated probes that target the target region shown in Seq ID No. 81, Seq ID No. 321, Seq ID No.
  • Seq ID No. 323 are all hypermethylated probes that target the target region shown in Seq ID No. 81.
  • Seq ID No. 203 is a hypomethylated probe targeting the target region shown in Seq ID No. 82
  • Seq ID No. 324 is a hypermethylated probe targeting the target region shown in Seq ID No. 82.
  • Seq ID No. 204 is a hypomethylated probe targeting the target region shown in Seq ID No. 83
  • Seq ID No. 325 is a hypermethylated probe targeting the target region shown in Seq ID No. 83.
  • Table 1 shows the target sequence targeted by the probe.
  • the length of each probe in the above-mentioned probe composition is 40-60 bp, preferably 45-56 bp, preferably 50-56 bp, and more preferably 50 bp.
  • the application also provides a kit.
  • the kit includes the probe composition described in any of the above embodiments.
  • the application also provides the use of the probe composition.
  • the above-mentioned probe composition is used to prepare a kit for detecting esophageal cancer, gastric cancer and colorectal cancer.
  • the application also provides a chip.
  • the probe composition described in any of the above embodiments is immobilized on the chip.
  • This application also provides a method for detecting three types of luminal organ tumors, including esophageal cancer, gastric cancer and colorectal cancer, by using the probe composition.
  • the detection method uses hybrid capture to enrich cfDNA, and uses NGS technology to detect methylation sites that are highly related to cancer. It covers the three most common malignant tumors of luminal organs in my country (esophageal cancer, gastric cancer and colorectal cancer). Finally, based on the detection of gene methylation changes in plasma cfDNA, it provides information for early screening and early diagnosis of multiple cancers.
  • This application also provides a method for simultaneously detecting changes in the methylation level of the above-mentioned three types of luminal organ tumors by using the kit.
  • this application relates to three in vitro cancer detection methods for subjects, including the following steps: collecting a sample of the subject; extracting and purifying the DNA in the sample; constructing a DNA library for sequencing based on the purified DNA sample; Bisulfite transforms the constructed DNA library; pre-PCR amplifies the bisulfite-converted DNA library; uses the probe composition to hybridize and capture the sample amplified by pre-PCR; Products captured by hybridization; perform high-throughput next-generation sequencing on the products amplified by PCR and captured by hybridization; analyze the sequencing data to determine the methylation level of the sample; judge based on the methylation level of the sample The condition of the patient.
  • the subject is suspected of having cancer.
  • the sample collected from the subject is a plasma sample.
  • the conversion is treatment with bisulfite.
  • the probe composition includes: 2 probes targeting a pan-cancer specific region, n probes targeting a cancer-specific region, and m probes targeting a tissue-specific region.
  • the probe composition includes: a hypomethylation probe that hybridizes to the cancer-specific region, pan-cancer specific region, and tissue-specific region that are converted by bisulfite and do not contain CG methylation , And a hypermethylation probe that hybridizes to the cancer-specific region, pan-cancer-specific region, and tissue-specific region where the bisulfite-converted CG is fully methylated.
  • the length of each probe in the probe composition is 40-60 bp.
  • the length of each probe in the probe composition is 45-56 bp, preferably 50-56 bp, and more preferably 50 bp.
  • the n probes in the probe composition target a cancer-specific region, where n is any integer selected from 1-192; wherein, the cancer-specific region is selected from Seq ID No.: Any of 1-62.
  • the m probes in the probe composition target the tissue-specific region, where m is an integer selected from 1-116; wherein, the tissue-specific region is selected from Seq ID No. : Any of 65-83.
  • the hypomethylation probes include probes targeting cancer-specific regions Seq ID No.: any of 84-180, and probes targeting pan-cancer specific regions Seq ID No.: 181-182 Any, and Seq ID No. of probes targeting tissue-specific regions: Any of 183-204.
  • the hypermethylation probes include probes targeting cancer-specific regions, Seq ID No.: any of 205-301, and probes targeting pan-cancer specific regions, Seq ID No.: 302-303, Seq ID No.: any of 304-325 probes targeting tissue-specific regions.
  • the interpretation includes the following steps: (1) compare the pan-cancer specific region database and perform interpretation to confirm whether the subject has cancer; (2) compare the cancer specific region database and perform interpretation To confirm that the subject's cancer is one of several suspected cancers; (3) Compare the tissue-specific area database and perform interpretation to confirm the subject's cancer site.
  • the step (1) includes the following interpretation: judging whether the methylation level of the pan-cancer specific region Seq ID No.: 63 is greater than or equal to 55%, and the pan-cancer specific region Seq ID No.: 64 If the methylation level of is greater than or equal to 60%, it is judged that the patient has cancer.
  • the step (2) includes the following interpretation: if among the n probes targeting the cancer-specific region, the methylation level of the region targeted by n1 probes is greater than or equal to the respective threshold, and n1/n ⁇ 20%, preferably n1/n ⁇ 30%, then it is judged that the patient has any one of the tissue-specific cancers.
  • the step (3) includes the following interpretation: among the m probes targeting the tissue-specific region, the methylation level of the region targeted by m1 probes is greater than or equal to the respective threshold, and then further analysis is greater than or equal to the respective threshold.
  • Threshold m1 probes target tissues and count the number of probes in each tissue that are greater than or equal to the threshold, and judge that the tissue in which the patient suffers from cancer is the tissue with the largest number of probes with methylation level greater than or equal to the threshold. Table 1 below also shows the methylation threshold of each target region.
  • Agilent 2100 performs fragment detection, and Qubit is directly used in subsequent experiments.
  • Component volume Connector connection product 110 ⁇ l Agencourt AMPure XP beads 110 ⁇ l
  • DNA protection buffer added to the liquid turns blue. Mix gently by pipetting, and then divide into two tubes on the PCR machine.
  • the number of PCR cycles is adjusted according to the amount of DNA input.
  • the reference data is as follows:
  • the length of the library is about 270bp-320bp.
  • Component volume Pre-amplified product 750ng corresponding volume Hyb human blocker 5 ⁇ l Junction blocker 6 ⁇ l Enhancer 5 ⁇ l
  • Hyb buffer solution at room temperature to melt, there will be precipitation after melting, and place it in a 65°C water bath to preheat it after mixing. After completely dissolved (no precipitation and turbidity), take 20 ⁇ l Hyb buffer solution and place In the new 200 ⁇ l PCR tube, close the tube cap, mark it as A, and continue to incubate it in a 65°C water bath for later use.
  • Methylation bio-information analysis process It is roughly as follows: Use quality control software such as trimmomatic to check the quality of sequencing, remove low-quality reads, and then use comparison software such as Bismarker to compare the clean data after quality control to the reference genome, and use R packages such as methykit to extract the corresponding Methylation site. Finally, calculate the methylation ratio of each target area on the Panel.
  • quality control software such as trimmomatic to check the quality of sequencing, remove low-quality reads
  • comparison software such as Bismarker to compare the clean data after quality control to the reference genome
  • R packages such as methykit
  • pan-cancer specific markers TBX15 and CRYGD genes greater than or equal to 55% and 60%, and then preliminarily determine that the sample is a sample suffering from cancer;
  • the methylation levels of cancer-specific markers OTX1, SFRP2, CDO1, TRIM15, ALX4, and CCNA1 are greater than or equal to the respective thresholds shown in Table 1 (as shown in Table 19 above), and then the sample is further judged as suffering from the following 11 types
  • a sample of any cancer esophageal cancer, stomach cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, and endometrial cancer
  • interpret tissue-specific markers Based on the target regions that are greater than or equal to their respective thresholds in Table 19, it can be seen that there are 6 target regions specific to gastric tissue Seq ID No.
  • Seq ID No. 69, Seq ID No. 75, Seq ID No. 76, Seq ID No. 77 and Seq ID No. 78 are greater than or equal to their respective thresholds of methylation levels, and no other tissue-specific markers have methylation greater than or equal to their respective thresholds, so they are greater than or equal to their respective methylation thresholds Among the tissue-specific markers, gastric tissue-specific markers are the most, and the sample is finally judged to be a sample with gastric cancer.
  • the patient’s blood was drawn again 48 hours after the operation, and the peripheral blood was collected using the Panel test of the application according to the method of Example 1.
  • the database was constructed and sequenced on the Illumina platform; the sequencing data was subjected to the above-mentioned biological information analysis process and passed pattern recognition By analyzing all the sequencing data, the results show that the gene methylation level in the above table has returned to the normal level.
  • Seq ID No. 64, Seq ID No. 71, Seq ID No. 72 , Seq ID No. 73, Seq ID No. 74 are greater than or equal to their respective thresholds of methylation levels, and no other tissue-specific markers have methylation greater than or equal to their respective thresholds, so they are greater than or equal to their respective methylation thresholds.
  • threshold tissue-specific markers colorectal tissue-specific markers are the most, and the sample is finally judged to be a sample with colorectal cancer.
  • a sample of esophageal cancer was detected by the Panel of this application, and peripheral blood was collected according to the method of Example 1; the database was built and sequenced on the Illumina platform; the sequencing data was subjected to the above-mentioned biological information analysis process to obtain the methylation level.
  • the results are as follows 21 (Table 21 shows the detected target regions greater than or equal to the methylation threshold).

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Abstract

La présente divulgation concerne une une composition de sonde. En effectuant une méta-analyse sur des données de méthylation d'une base de données TCGA et d'une base de données GEO, la composition obtient une région de capture de 4,29 Kbp, comprenant jusqu'à 37 gènes de changement de méthylation et des régions d'ADN non codantes hautement liées aux cancers. Le procédé peut mettre en œuvre un test en une seule fois pour un changement de niveau de méthylation de tumeurs d'organe à trois lumières telles que le cancer de l'œsophage, le cancer gastrique et le cancer colorectal, et peut être utilisé pour un dépistage précoce pour des personnes ne portant aucun symptôme et pour des tests de pronostic pour des patients atteints d'un cancer.
PCT/CN2021/077062 2020-02-25 2021-02-20 Composition de sonde pour la détection de tumeurs d'organe à trois lumières Ceased WO2021169874A1 (fr)

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