Probe / primer library for cancer diagnosis

Abstract

The objective is to provide a universal method for rapidly detecting cancer-related mutations using dPCR probes. A library is provided for detecting cancer-related mutations in the DNA-binding domain of the TP53 gene, comprising multiple probes and / or primers or primer pairs. This invention enables early diagnosis of post-treatment recurrence of gastrointestinal tumors. Furthermore, it allows for personalized post-treatment follow-up for patients with gastrointestinal tumors.

Description

Technical Field

[0001] This invention relates to methods for diagnosing cancer. More specifically, this invention relates to probes and primers useful for the early diagnosis of cancer recurrence (particularly esophageal cancer, gastric cancer, and colorectal cancer), for detecting mutations in the TP53 gene. Background Technology

[0002] Research has been conducted on the use of circulating tumor DNA (ctDNA) derived from tumor cells in peripheral blood for cancer diagnosis. For example, Non-Patent Literature 1 extracted DNA from pre- and post-operative samples, including plasma, primary tumor cells, and peripheral blood mononuclear cells (PBMCs), from 44 individuals with tumors in the large intestine who underwent radical resection. The study investigated tumor-specific mutations, the mutation spectrum of the tumor, and changes in the allele frequencies of mutations in ctDNA after radical tumor resection. Furthermore, Patent Literature 1 and 2 propose methods for monitoring tumor quantity by measuring the copy number of DNA fragments containing mutated genes in a patient's blood sample, using this copy number as an indicator of the patient's tumor quantity. Additionally, Patent Literature 3 proposes a method combining PNA-Clamping and direct sequencing for detecting K-ras gene mutations, and describes how this method allows the use of blood as a substitute for cancer tissue in the testing material, enabling the evaluation of the therapeutic sensitivity of patients to molecularly targeted drugs targeting the K-ras gene. Furthermore, in Patent Document 4, as a non-invasive method for diagnosing ovarian clear cell adenocarcinoma, a scheme is proposed to detect these amplifications or deletions based on the insight that amplifications or deletions in certain chromosomal DNA regions are related to the incidence, malignancy, and prognosis of ovarian clear cell adenocarcinoma. It is also stated that chromosomal DNA taken from the patient can be extracted from the blood.

[0003] On the other hand, the TP53 gene is known to be one of the tumor suppressor genes. Research is underway to use mutations in the TP53 gene as biomarkers for cancer diagnosis / treatment. For example, Non-Patent Literature 2 reports that in patients with high-grade serious ovarian carcinoma (HGSOC), the amount of circulating tumor DNA (ctDNA) derived from tumor cells in peripheral blood at the start of treatment was correlated with tumor volume. Furthermore, a reduction of less than 60% in the TP53 mutant allele fraction (TP53MAF) after one cycle of chemotherapy was associated with a shortened progression-free survival. These results suggest the potential of ctDNA as an early molecular response marker for HGSOC.

[0004] In Non-Patent Literature 1, digital PCR (dPCR) is used for the detection of ctDNA. dPCR uses probes complementary to a specific sequence (typically one base) in a DNA fragment amplified using primers to detect the specific sequence with high sensitivity (0.01%-1%). The high sensitivity of specific sequence detection is noteworthy in the medical field, particularly in cancer diagnosis, and dPCR is the only technology capable of addressing the sensitivity, cost, testing time, and need for repeated testing required for diagnosis.

[0005] Existing technical documents

[0006] Patent documents

[0007] Patent Document 1: Japanese Patent Publication No. 2011-529691

[0008] Patent Document 2: Japanese Patent Application Publication No. 2016-104010

[0009] Patent Document 3: Japanese Patent Application Publication No. 2012-135290

[0010] Patent Document 4: Japanese Patent Application Publication No. 2016-198027

[0011] Non-patent literature

[0012] Non-patent literature 1: Sato et al, PLoS One, 2016: PMID: 26727500

[0013] Non-patent literature 2: Parkinson et al., PLoS Med 13(12), 2016: e1002198 Summary of the Invention

[0014] The problem the invention aims to solve

[0015] Previously, when detecting ctDNA, target-specific sequences could be determined based on existing sequence analysis, public databases, and literature. However, for high-sensitivity detection using dPCR, primers and probes must first be designed and synthesized. Typically, because specific sequences vary depending on the individual and the disease, their diversity is vast, while their frequency in the general population is low, limiting the scope of applications for which synthesized probes can be used. In fact, commercially available dPCR probes are designed to be sporadic and discontinuous relative to continuous base sequences, lacking universality except for a subset targeting high-frequency specific sequences. Therefore, even if tumor cell-specific mutations are identified in the target patient, existing dPCR probes cannot be used, hindering rapid diagnosis.

[0016] Solution for solving the problem

[0017] Therefore, in order to cope with more mutations, it is desirable to prepare dPCR probes comprehensively, and in order to cope with all mutations, it is even more desirable to prepare dPCR probes continuously.

[0018] On the other hand, the inventors focused on mutations in the TP53 gene, which have been identified in approximately 50% of human cancers. Mutations in the TP53 gene have been identified with a particularly high frequency in lung cancer, gastric cancer, colorectal cancer, breast cancer, etc. According to the inventors' research, mutations are found in 87% of esophageal cancer, gastric cancer, and colorectal cancer in Japanese people.

[0019] Furthermore, the inventors designed and synthesized 29 sets of probes and primers targeting the TP53 gene for dPCR to detect specific mutations in each case. Several sets were selected from a library of such probe / primer sets for recurrence monitoring in cancer patients. The results showed that recurrence could be diagnosed at a earlier stage than before through ctDNA analysis, thus completing this invention.

[0020] The present invention provides the following solution.

[0021] [1] A library for detecting cancer-associated gene mutations in a region encoding the DNA-binding domain of the TP53 gene, comprising multiple probes and / or primers or primer pairs.

[0022] Multiple probes and / or primers or primer pairs include probes and / or primers or primer pairs for detecting c.524G>A, c.743G>A, c.818G>A, c.817C>T, c.742C>T, c.844C>T, c.637C>T, c.733G>A, c.747G>T, and c.659A>G.

[0023] [2] The library according to 1 further includes methods for detecting c.586C>T, c.469G>T, c.536A>G, ​​c.488A>G, c.527G>T, c.818G>T, c.853G>A, c.734G>A, c.722C>T, c.578A>G, c.535C>T, c.856G>A, c.584T>C, c.574C>T, and c.701A. Multiple probes and / or primers or primer pairs of c.614G>A, c.711G>A, c.713G>A, c.743G>T, c.473G>A, c.646G>A, c.832C>T, c.422G>A, c.527G>A, c.455C>T, c.473G>T, c.725G>T, c.833C>T, c.614A>G, and c.641A>G.

[0024] [3]The library according to 2 further comprises those for detecting c.734G>T, c.451C>T, c.797G>A, c.839G>A, c.839G>C, c.707A>G, c.733G>T, c.517G>T, c.404G>A, c.581T>G, c.796G>A, c.517G>A, c.380C>T, c.395A>G, c.824G>A, c.404G>T, c.730G>T, c.577C>T, c.638G>T, c.749C>T, c.772G>A, c.578A>T, c.824G>T, c.736A>G, c.797G>T, c.476C>T, c.725G>A, c.461G>T, c.481G>A, c.731G>A, c.638G>A, c.713G>T, c.715A>G, c.406C>T, c.493C>T, c.536A>T, c.811G>A, c.437G>A, c.438G>A, c.592G>T, c.430C>T, c.711G>T, c.730G>A, c.746G>T, c.610G>T, c.722C>G, c.329G>T, c.745A>T, c.814G>T, c.841G>C, c.396G>T, c.836G>A, c.838A>G, c.799C>T, c.830G>T, c.583A>T, c.832C>A, c.844C>G, c.452C>A, c.548C>G, c.569C>T, c.833C>G, c.396G>C, c.475G>C, c.499C>T, c.427G>A, c.644G>T, c.775G>T, c.700T>C, c.716A>G, c.745A>G, c.841G>A, c.298C>T, c.310C>T, c.820G>T, c.763A>T, c.821T>C, c.464C>A, c.467G>C, c.542G>A, c.580C>T, c.746G>C, c.818G>C, c.845G>A, c.772G>T, c.405C>G, c.541C>T, c.832C>G, c.856G>T, c.329G>C, c.413C>T, c.514G>T, c.584T>A, c.511G>T, c.811G>T, c.375G>T, c.523C>G, c.747G>C, c.394A>G, c.487T>A, c.800G>C, c.853G>T, c.738G>A, c.742C>G, c.785G>T, c.859G>T, c.375G>A, c.454C>T, c.487T>C, c.524G>T, c.725G>C, c.794T>C, c.839G>T, c.848G>C, c.388C>G, c.528C>G, c.535C>A, c.596G>T, c.643A>G , c.722C>A, c.796G>T, c.374C>T, c.377A>G, c.517G>C, c.523C>T, c.530C>T, c.817C>A, c.434T>A, c.463A>C, c.503 A>G, c.535C>G, c.658T>A, c.700T>A, c.743G>C, c.843C>G, c.380C>A, c.400T>C, c.412G>C, c.421T>C, c.472C>T, c. 473G>C, c.587G>C, c.706T>A, c.526T>A, c.526T>C, c.537T>A, c.542G>C, c.659A>C, c.731G>T, c.733G>C, c.843C>A , c.845G>C, c.857A>G, c.661G>T, c.434T>C, c.451C>G, c.490A>G, c.613T>G, c.718A>G, c.808T>C, c.809T>C, c.313 G>T, c.388C>T, c.559G>A, c.623A>T, c.645T>G, c.658T>C, c.796G>C, c.809T>G, c.823T>C, c.840A>T, c.841G>T, c. Multiple probes and / or primers or primer pairs of c.847C>T, c.854A>T, c.328C>T, c.423C>G, c.451C>A, c.472C>G, c.518T>C, c.596G>A, c.755T>C, c.830G>A, c.833C>A, c.842A>G, c.497C>G, c.395A>T, c.470T>G, c.530C>G, c.578A>C, c.632C>T, c.712T>C, and c.746G>A.

[0025] [4] The library according to any one of 1 to 3 is capable of detecting all 585 mutations in the DNA binding domain of the TP53 gene by multiple probes.

[0026] [5] The library described in 1 or 2 is used to predict recurrence or prognosis in subjects after cancer treatment.

[0027] [6] The library according to any one of 1 to 5 is used to detect circulating tumor DNA (ctDNA) derived from tumor cells.

[0028] [7] The library according to any one of 1 to 6, wherein the probe and / or primer or primer pair is used for digital PCR.

[0029] [8] The library according to any one of 1 to 7, wherein the probe and / or primer or primer pair comprises an oligonucleotide or a marker thereof consisting of any one of the sequences in sequence numbers 1 to 116.

[0030] [9] A method for analyzing cancer-related gene mutations in the region encoding the DNA-binding domain of the TP53 gene in a subject, the method comprising the following steps (1) to (3):

[0031] (1) The process of preparing a library, wherein the library consists of multiple probes and / or primers or primer pairs, the multiple probes and / or primers or primer pairs including probes and / or primers or primer pairs for detecting mutations of c.524G>A, c.743G>A, c.818G>A, c.817C>T, c.742C>T, c.844C>T, c.637C>T, c.733G>A, c.747G>T and c.659A>G;

[0032] (2) The process of selecting probes and / or primers or primer pairs from the prepared library to detect a mutation;

[0033] (3) Using the selected probes and / or primers or primer pairs, analyze the mutations in the nucleic acids obtained from the subjects using digital PCR.

[0034]

[10] According to the method described in 9, the cancer is esophageal cancer, gastric cancer or colorectal cancer.

[0035]

[11] The method according to 9 or 10, wherein the selected mutation is a mutation identified in the primary lesion of the subject before treatment, and the mutation is analyzed in circulating tumor DNA (ctDNA) derived from tumor cells obtained from the blood of the subject after treatment.

[0036]

[12] A method for manufacturing a library according to any one of 1 to 8, comprising the following steps:

[0037] The process of preparing probes and / or primers or primer pairs for detecting a cancer-related mutation in the DNA-binding domain of the TP53 gene.

[0038] The process of preparing probes and / or primers or primer pairs for detecting cancer-related mutations in the DNA-binding domain of the TP53 gene that are different from the mutations described herein.

[0039]

[13] A kit of primer pairs and probe pairs from any of the following groups (1) to (9) for digital PCR:

[0040] (1) Primer pairs composed of the sequences of sequence numbers 1 and 2, and primers composed of...

[0041]

[0042] Each oligonucleotide or probe pair consisting of its own sequence;

[0043] (2) Primer pairs composed of the sequences of sequence numbers 5 and 6, and primers composed of...

[0044]

[0045] Each oligonucleotide or probe pair consisting of its own sequence;

[0046] (3) Primer pairs composed of the sequences of sequence numbers 9 and 10, and primers composed of...

[0047]

[0048] Each oligonucleotide or probe pair consisting of its own sequence;

[0049] (4) Primer pairs composed of the sequences of sequence numbers 13 and 14, and primers composed of...

[0050]

[0051] Each oligonucleotide or probe pair consisting of its own sequence;

[0052] (5) Primer pairs composed of the sequences of sequence numbers 17 and 18, and primers composed of...

[0053]

[0054] Each oligonucleotide or probe pair consisting of its own sequence;

[0055] (6) Primer pairs composed of the sequences of sequence numbers 21 and 22, and primers composed of...

[0056]

[0057] Each oligonucleotide or probe pair consisting of its own sequence;

[0058] (7) Primer pairs composed of the sequences of sequence numbers 25 and 26, and primers composed of...

[0059]

[0060] Each oligonucleotide or probe pair consisting of its own sequence;

[0061] (8) Primer pairs composed of the sequences of sequence numbers 29 and 30, and primers composed of...

[0062]

[0063] Each oligonucleotide or probe pair consisting of its own sequence or a marker thereof; and

[0064] (9) Primer pairs composed of the sequences of sequence numbers 33 and 34, and primers composed of...

[0065]

[0066] Each oligonucleotide or probe pair consisting of its own sequence or a label thereof (where the part within the rectangle is the modified nucleotide, and * indicates the nucleotide corresponding to the mutation position.)

[0067]

[14] A kit of probes and primers for digital PCR consisting of oligonucleotides or their markers from any of the following groups:

[0068] The oligonucleotides used to detect mutations in c.844 consist of sequences from sequence numbers 1 to 4.

[0069] The oligonucleotides used to detect mutations in c.637 consist of sequences from sequence numbers 5 to 8.

[0070] The oligonucleotides used to detect mutations in c.659 consist of sequences from sequence numbers 9 to 12.

[0071] The oligonucleotides used to detect mutations in c.527 consist of sequences from sequence numbers 13 to 16.

[0072] The oligonucleotides used to detect mutations in c.725 consist of sequences from sequence numbers 17 to 20.

[0073] The oligonucleotides used to detect mutations in c.614 consist of sequences from sequence numbers 21 to 24.

[0074] The oligonucleotides used to detect mutations in c.499 consist of sequences from sequence numbers 25 to 28.

[0075] The oligonucleotides used to detect mutations in c.298 consist of sequences from sequence numbers 29 to 32, and

[0076] The oligonucleotides used to detect mutations in c.809 consist of sequences from sequence numbers 33 to 36.

[0077] The present invention also provides the following solutions.

[0078] [1] A library for detecting cancer-associated mutations in the DNA-binding domain of the TP53 gene, comprising multiple probes and / or primers or primer pairs.

[0079] [2] According to the library described in 1, the cancer-related mutations are mutations selected from any position in the group consisting of c.298, c.329, c.380, c.399_400 insertion, c.475, c.499, c.524, c.527, c.536, exon 5 splice site_3, c.574, c.578, c.586, c.614, c.637, c.659, c.707, c.725, c.730, c.736, c.743, c.764, c.780, c.809, c.818, c.839, c.844 and c.993.

[0080] [3] The library according to 1 or 2, wherein the plurality of probes and / or primers or primer pairs comprise probes and / or primers or primer pairs for detecting mutations at any position selected from the group consisting of c.524, c.527, c.536, c.578, c.614, c.637, c.659, c.725, c.730, c.743 and c.818.

[0081] [4] The library according to any one of 1 to 3 is capable of detecting mutations uninterruptedly by multiple probes.

[0082] [5] The library described in 1 or 2 is used to predict recurrence or prognosis in subjects after cancer treatment.

[0083] [6] The library according to any one of 1 to 5 is used to detect circulating tumor DNA (ctDNA) derived from tumor cells.

[0084] [7] The library according to any one of 1 to 6, wherein the probe and / or primer or primer pair is used for digital PCR.

[0085] [8] The library according to any one of 1 to 7, wherein the probe and / or primer or primer pair is a polynucleotide or a marker thereof consisting of any one of the sequences in sequence numbers 1 to 116.

[0086] [9] A method for assisting in predicting recurrence or prognosis in subjects after cancer treatment, comprising the following steps:

[0087] Prepare at least one probe and / or primer or primer pair from the library described in any one of 1 to 6.

[0088] Using prepared probes and / or primers or primer pairs, mutations in nucleic acids obtained from the subject were analyzed by digital PCR.

[0089]

[10] According to the method described in 9, the cancer is esophageal cancer, gastric cancer or colorectal cancer.

[0090]

[11] The method according to 9 or 10, wherein probes and / or primers or primer pairs are prepared for detecting mutations identified in the primary lesion of the subject before treatment, and the mutations are analyzed in circulating tumor DNA (ctDNA) derived from tumor cells obtained from the blood of the subject after treatment.

[0091]

[13] An oligonucleotide or a marker thereof, which consists of any one of the sequences in sequence numbers 1 to 36.

[0092]

[14] A probe and primer kit comprising oligonucleotides or their markers from any of the following groups:

[0093] The oligonucleotides used to detect mutations in c.844 consist of any sequence from sequence numbers 1 to 4.

[0094] The oligonucleotides used to detect mutations in c.637 consist of any sequence from sequence numbers 5 to 8.

[0095] The oligonucleotides used to detect mutations in c.659 consist of any sequence from sequence numbers 9 to 12.

[0096] The oligonucleotides used to detect mutations in c.527 consist of any sequence from sequence numbers 13 to 16.

[0097] The oligonucleotides used to detect mutations in c.725 consist of any sequence from sequence numbers 17 to 20.

[0098] The oligonucleotides used to detect mutations in c.614 consist of any sequence from sequence numbers 21 to 24.

[0099] The oligonucleotides used to detect mutations in c.499 consist of any sequence from sequence numbers 25 to 28.

[0100] The oligonucleotides used to detect mutations in c.298 consist of any sequence from sequence numbers 29 to 32, and

[0101] The oligonucleotides used to detect mutations in c.809 consist of any sequence from sequence numbers 33 to 36.

[0102] The effects of the invention

[0103] By utilizing the library of this invention, it is possible to diagnose approximately 90% of patients with gastrointestinal tumors.

[0104] According to the present invention, early detection of recurrence after treatment can be achieved.

[0105] According to the present invention, early detection of gastrointestinal tumors can be expected. Attached Figure Description

[0106] Figure 1 This involves identifying the mutated amino acid position of TP53 in 66 cases of esophageal, gastric, or colorectal cancer. The mutation positions are often repeated. In such cases, a single probe / primer kit can be designed to analyze multiple cases.

[0107] Figure 2 These are the locations of TP53 gene mutations in 66 cases of esophageal, gastric, or colorectal cancer. Exons 4–9 of the TP53 gene are underlined. Additionally, * marks the mutation locations for which probes and primers were synthesized in the examples for detection.

[0108] Figure 3 This is the working confirmation result of the 29 synthesized primer / probe sets. dPCR was performed on the DNA from the primary lesion (or plasma). Mutant alleles (blue) and wild-type alleles (red) were detected in all samples.

[0109] Figure 4 This is a flowchart of the ctDNA monitoring experiment. For the disease mutations detected through mutation analysis of the primary lesion, probes targeting wild-type DNA and mutant DNA were prepared respectively. After confirming the use of primary lesion DNA, plasma was collected from the patient during treatment, and the proportion of mutant DNA, i.e., the mutant allele frequency (%), was measured. Compared with the mutant DNA in the plasma before treatment (1), a reduction in mutant DNA (blue) was observed after treatment (2)(3).

[0110] Figure 5 This involves monitoring TP53 mutant DNA in the plasma of patients with stage I to IV esophageal cancer. MAF stands for Mutant Allele Frequency; CF stands for Cisplatin / 5-FU; DCF stands for Docetaxel / Cisplatin / 5-FU; CRT stands for Chemoradiotherapy; and PTX stands for Paclitaxel.

[0111] Figure 6 This involves ctDNA monitoring in recurrent cases of stage IIA esophageal cancer. MAF stands for mutational allele frequency; CF for cisplatin / 5-FU; and CRT for chemoradiotherapy. Detailed Implementation

[0112] This invention provides a library for detecting cancer-related mutations.

[0113] [library]

[0114] A library is a collection of multiple oligonucleotides used to detect multiple mutations. "Multiple" means at least two or more. Preferably, the library is a collection capable of detecting five or more mutations, more preferably a collection capable of detecting ten or more mutations, and even more preferably a collection capable of detecting twenty or more mutations. From the viewpoint of being able to continuously detect mutations in the portion of the coding DNA-binding domain (195 amino acids in length) of the TP53 gene, the library is preferably capable of detecting all 585 mutations. The library contains multiple oligonucleotides for detecting multiple mutations, but the method of use can be varied. When used to analyze a target mutation (one mutation) in a single subject, for example, it may be used to determine whether a mutation detected in the primary lesion before treatment is detected in the blood during treatment for a particular patient. Furthermore, there are cases where the library is used to analyze multiple mutations in a single subject. Moreover, there are cases where the library is used to analyze a target mutation (one mutation) in multiple subjects, and cases where the library is used to analyze multiple mutations separately in multiple subjects, etc. It should be noted that when mutation is mentioned in the description of this invention, unless otherwise specified, it refers to mutation of bases (sometimes described as mutation of nucleotides).

[0115] The oligonucleotides contained in a library include primers or primer pairs for amplifying portions containing mutations, and probes for detecting mutations. Libraries may consist only of oligonucleotides that function as primers, oligonucleotides that function only as probes, or both. Mutation detection is typically performed using polymerase chain reaction (PCR) in nucleic acids. Primers that may contain the target mutation are amplified using primer pairs, and probes are used to detect the presence of the target mutation in the amplification products. Therefore, libraries preferably contain a set of probes and primer pairs targeting the target mutation. It should be noted that the amplification product refers to the DNA amplified by PCR, sometimes also called an amplicon.

[0116] The probes contained in the library can be either probes for detecting sequences without the target mutation (wild-type) or probes for detecting sequences with the target mutation (mutant type). The library can also contain both types of probes.

[0117] The primers contained in the library are used to amplify the portion of the nucleic acid in the sample, including the location of the target mutation. They are usually preferably contained in the library in the form of primer pairs, with a forward primer corresponding to the positive strand and a reverse primer corresponding to the negative strand.

[0118] The primers and probes included in the library are used to detect cancer-related mutations. Cancer-related mutations are defined as mutations observed at a high frequency in a population of DNA originating from tumor sites in cancer patients. A high frequency mutation means that the mutation can be observed in 5% or more, preferably 10% or more, and more preferably 30% or more of cancer patients.

[0119] Cancer-related mutations are preferably mutations on the TP53 gene, and more preferably mutations on the portion of the TP53 gene that encodes the DNA-binding domain.

[0120] The p53 tumor suppressor protein (a 393-amino acid nucleoprotein) encoded by the TP53 gene is a crucial transcriptional regulator in response to various cellular stresses, such as DNA damage, ultraviolet radiation, and hypoxia. p53 controls vital cellular processes essential for life, including DNA repair, cell cycle progression, angiogenesis, and apoptosis. Its activation can initiate various molecular and downstream pathways in affected cells. These p53-dependent pathways inhibit the proliferation of damaged cells through cell cycle arrest or apoptosis. In numerous cancer cases, the loss or inhibition of p53 function and activity is a contributing factor.

[0121] The library preferably contains a plurality of probes and / or primers or primer pairs, wherein the plurality of probes and / or primers or primer pairs are used to detect, as confirmed by the researchers, the TP53 gene, insertions selected from c.298, c.329, c.380, c.399_400, c.475, c.499, c.524, c.527, c.536, exon 5 splice site_3, c.574, etc. Mutations at any position in the group consisting of c.578 (including the cases of p.His193Arg and p.His193Leu), c.586, c.614, c.637, c.659, c.707, c.725, c.730, c.736, c.743, c.764, c.780, c.809, c.818, c.839, c.844, and c.993. More specifically, the library contains multiple probes and / or primers or primer pairs used to detect mutations in the TP53 gene that have been observed at high frequency according to the work of the authors, namely, selected c.298C>T, c.329G>T, c.380C>T, c.399_400 inserted CAAGATG, c.475G>C, c.499C>T, c.524G>A, c.527G>A, c.536A>G, ​​exon 5 splice site_3, c. Any mutation in the group consisting of c.574G>A, c.578A>G, c.578A>T, c.586C>T, c.614A>G, c.637C>T, c.659A>G, c.707A>G, c.725G>T, c.730G>A, c.736A>T, c.743G>A, c.764T>C, c.780_780delC, c.809T>G, c.818G>A, c.839G>C, c.844C>T, and c.993C>T. It should be noted that "any case" is different from "any one case"; the number of mutations is arbitrary, not limited to one, and can be multiple.

[0122] The library more preferably includes a plurality of probes and / or primers or primer pairs for detecting mutations at any position selected from at least the group consisting of c.524, c.527, c.536, c.578, c.614, c.637, c.659, c.725, c.730, c.743, and c.818. More specifically, the plurality of probes and / or primers or primer pairs are used to detect any of the aforementioned specific mutations at these positions.

[0123] The library is further preferably composed of a set of probes and primer pairs, which are used to detect all of the above 11 mutations. More specifically, the set of probes and primer pairs is used to detect all of the above specific mutations at these positions.

[0124] p53 can be divided into several domains based on function, but the DNA-binding domain has been found to have the highest mutation frequency, accounting for approximately 86% of all mutations. According to our research, of the 40 identified mutations, 39 (98%) were found in the DNA-binding domain. Furthermore, the p53 DNA-binding domain consists of 196 amino acids, and mutations have been reported in all of them. Therefore, it is reasonable to assume that a library of oligonucleotides covering possible mutations in the DNA-binding domain—that is, a library for the uninterrupted detection of such mutations—could be used for the effective diagnosis of a large number of cancer patients.

[0125] Therefore, one particularly preferred type of library is one capable of uninterrupted detection of possible mutations in the DNA-binding domain. Such a library targets mutations in the coding region of 195 amino acids. If each kit contains primer pairs, probes for the wild type, and probes for the mutant type, the library contains 2340 theoretically detectable bases. If primer / probe sets targeting these bases are synthesized, it can become a library capable of effectively diagnosing nearly 90% of cancer patients with TP53 mutations. Therefore, one particularly preferred type of library is one capable of uninterrupted detection of possible mutations in the p53 DNA-binding domain. Specifically, it is a kit of 1755 primers / probes capable of detecting 2340 theoretically detectable bases. Another example is a kit of approximately 800 primers / probes targeting approximately 800 mutations reported in digestive system cancers. Furthermore, according to the research of the present inventors, considering all cancers (https: / / cancer.sanger.ac.uk / cosmic), among 1284 point mutations of TP53 (out of a total of 25376 mutations), 30% of the top 10 reported mutations correspond to TP53 mutations (hereinafter the same), 40% correspond to the top 20, 50% correspond to the top 40, 60% correspond to the top 80, and 80% correspond to the top 232. Currently, the present inventors have synthesized 4 of the top 10 and 6 of the top 20 mutations, and their usefulness has been confirmed. The following shows the ranking of reported cases compiled by the present inventors.

[0126] [Table 1]

[0127]

[0128]

[0129] Thus, the frequency of occurrence (reporting) varies considerably depending on the mutation. One particularly preferred library is designed to detect multiple, for example, two or more, preferably four or more, more preferably six or more, further preferably ten or more, and even more preferably twenty or more of the top-ranked mutations. The ranking of the target mutations in the primer and probe sets used in the embodiments of this specification is as follows.

[0130] chr17: 7579389c.298C>T(p.Gln100Ter), 113th bit;

[0131] chr17: 7579358c.329G>T(p.Arg110Leu), 87th position;

[0132] chr17: 7578550c.380C>T(p.Ser127Phe), 53rd position;

[0133] chr17:7578526c.399_400 Insert CAAGATG (p.Phe134fs), not applicable;

[0134] chr17: 7578455c.475G>C (p.Ala159Pro), 104th bit;

[0135] chr17: 7578431c.499C>T(p.Gln167Ter), 105th position;

[0136] chr17: 7578406c.524C>T(p.Arg175His), 1st position;

[0137] chr17: 7578403c.527G>A(p.Cys176Tyr), 34th position;

[0138] chr17: 7578394c.536A>G(p.His179Arg), 13th position;

[0139] chr17: 7578370 exon 5 splice site_3C>T, not applicable;

[0140] chr17: 7578275c.574G>A(p.Gln192Ter), 24th position;

[0141] chr17: 7578271c.578A>G(p.His193Arg), 20th position;

[0142] chr17: 7578271c.578A>T(p.His193Leu), 62nd position;

[0143] chr17: 7578263c.586G>A(p.Arg196Ter), 11th position;

[0144] chr17: 7578235c.614A>G(p.Tyr205Cys), 39th position;

[0145] chr17: 7578212c.637C>T(p.Arg213Ter), 7th position;

[0146] chr17: 7578190c.659A>G(p.Tyr220Cys), 10th position;

[0147] chr17: 7577574c.707A>G(p.Tyr236Cys), 46th position;

[0148] chr17: 7577556c.725G>T(p.Cys242Phe), 37th position;

[0149] chr17: 7577551c.730G>A(p.Gly244Ser), 83rd position;

[0150] chr17: 7577545c.736A>T(p.Met246Leu), 455th bit;

[0151] chr17: 757753c.743C>T(p.Arg248Gln), 2nd position;

[0152] chr17: 7577517c.764A>G(p.Ile255Thr), bit 278;

[0153] chr17: 7577500c.780_780delC(p.Ser260fs), Not applicable;

[0154] chr17: 7577129c.809T>G(p.Phe270Cys), 209th position;

[0155] chr17: 7577120c.818G>A(p.Arg273His), 3rd position;

[0156] chr17: 7577099c.839G>C(p.Arg280Thr), 45th bit

[0157] chr17: 7577094c.844G>A(p.Arg282Trp), 6th position; and

[0158] chr17: 7576853c.993C>T (p.Gln331Ter), Not applicable (outside the DNA binding domain).

[0159] Regarding the ranking of frequency of occurrence, higher rankings refer to, for example, the top 10 (ranked 1-10), the top 40 (ranked 1-40), or the top 232 (ranked 1-232). If mutations ranking within the top 10 of frequency are selected as targets, a library capable of detecting at least 30% of TP53 mutations can be designed. Furthermore, if mutations ranking above 40 of frequency are selected as targets, a library capable of detecting at least 50% of TP53 mutations can be designed. And if mutations ranking above 232 of frequency are selected as targets, a library capable of detecting at least 80% of TP53 mutations can be designed.

[0160] A specific example of a library used to detect mutations with the highest number of reported mutations includes any one of the following three sets, preferably any two sets, and more preferably all three sets:

[0161] The oligonucleotides used to detect mutations in c.844 consist of any sequence from sequence numbers 1 to 4.

[0162] The oligonucleotides used to detect mutations in c.637 consist of any sequence from sequence numbers 5 to 8, and

[0163] The oligonucleotides used to detect c.659 mutations consist of any sequence from sequence numbers 9 to 12. If the library contains all the sets represented by these three sets for detecting mutations occurring within 10 positions, at least 30% of TP53 mutations can be expected to be detected.

[0164] In addition to the three sets mentioned above, the library also includes any one of the following three sets, any two sets (preferably), and all three sets (preferably):

[0165] Oligonucleotides consisting of any sequences from sequence numbers 13 to 16 are used to detect the c.527 mutation; oligonucleotides consisting of any sequences from sequence numbers 17 to 20 are used to detect the c.725 mutation.

[0166] The oligonucleotides used to detect c.614 mutations consist of any sequence from sequence numbers 21 to 24. If the library contains all the sets represented by these 6 sets for detecting mutations occurring within 40 positions, at least 50% of TP53 mutations can be expected to be detected.

[0167] In addition to the 6 sets mentioned above, the library also includes any 1 set from the following 3 sets, any 2 sets preferred, and all 3 sets preferred:

[0168] The oligonucleotides used to detect mutations in c.499 consist of any sequence from sequence numbers 25 to 28.

[0169] The oligonucleotides used to detect mutations in c.298 consist of any sequence from sequence numbers 29 to 32, and

[0170] The oligonucleotides used to detect c.809 mutations consist of any sequence from sequence numbers 33 to 36. If the library contains all the sets represented by these 9 sets for detecting mutations occurring at a frequency of up to position 232, then at least 80% of TP53 mutations can be expected to be detected.

[0171] Each of the nine sets described above is useful in itself as a probe and primer set for detecting a specific mutation. Additionally, the oligonucleotides (probes, primers) composed of specific sequences included in the nine sets are useful in themselves. The mutations to be detected by the oligonucleotides are ranked within the top 10, 40, or 232 positions. Oligonucleotides used to detect mutations with a frequency within the top 10 positions are useful for detecting at least 30% of TP53 mutations. Oligonucleotides used to detect mutations ranked within the top 40 positions are useful for detecting at least 50% of TP53 mutations. Oligonucleotides used to detect mutations ranked within the top 232 positions are useful for detecting at least 80% of TP53 mutations.

[0172] It should be noted that each nucleotide in the sequence can be a modified nucleotide. Examples of modified nucleotides will be given later.

[0173] The specific location and content of the mutation, as well as the nucleotide sequences of the probe and primers, are also shown in the tables in the examples of this specification. Figure 2 And in the sequence list.

[0174] The probes and / or primers or primer pairs contained in the library can be designed based on sequence information obtained from primary tumor samples from cancer patients. Most patients who become test subjects for sensitive tumor markers such as ctDNA (described later) are in advanced stages, with surgery as their initial treatment. Therefore, obtaining sequence information using solid materials obtained during surgery is relatively easy.

[0175] Various designs are possible for the probes and / or primers or primer pairs contained in the library, enabling the analysis of mutations in nucleic acids in samples collected from the subject. Ideally, the design should also allow for the analysis of cell-free ctDNA (ctDNA) present in the subject's blood. This is because analyzing ctDNA in the blood allows for the systematic tracking of tumor-specific mutated DNA. Furthermore, it is less burdensome for patients after treatment and facilitates long-term analysis, allowing for early detection of post-treatment increases in "tumor load" and rapid initiation of secondary treatment. It should be noted that the analysis includes detection and quantification. Additionally, subjects include cancer patients and suspected cancer patients.

[0176] From the perspective of analyzing the ctDNA of a subject, the probes and / or primers or primer pairs contained in the library can be designed to be used in methods that can analyze a low proportion of mutant (sometimes called mutant sequences) in the presence of a large excess of wild-type (also called non-mutated sequences or reference sequences). This is because, in cancer diagnosis, the concentration of ctDNA in the blood (the ratio of mutant sequences to non-mutated sequences, sometimes called allele frequency) is mostly below 1%, and high-sensitivity analysis is desired. An example of such a high-sensitivity analytical method is dPCR.

[0177] dPCR (Vogelstein and Kinzler, Proc. Natl. Acad. USA, 96, 9236-9241, 1999) is an extension of existing PCR methods that can directly quantify the amount of target nucleic acids. dPCR is suitable for use in conjunction with a concentration step to detect minute amounts of mutations. In dPCR, because the DNA sample is diluted to a single molecule, the starting material in each PCR reaction can be either wild-type or mutant. Concentration can be performed from a single molecule, and thousands of parallel concentration reactions can be performed simultaneously. After multiple PCR reactions from the same starting material, mutant molecules can be isolated and detected. After PCR amplification, the chambers containing the final PCR product are counted, thereby allowing the calculation of the absolute amount of nucleic acid. Various dPCR-based systems that can be used in this invention are commercially available. The basic methodology of dPCR is described, for example, in Sykes et al., Biotechniques 13(3): 444-449, 1992.

[0178] From the perspective of analyzing ctDNA, probes and / or primers or primer pairs are preferably designed to detect target mutations by amplifying short nucleic acid fragments as target sequences. Typical PCR amplification products are 100–150 bp in size, sometimes leading to reduced sensitivity and accuracy. It is generally considered important to minimize the size of amplification products to improve analytical performance (Antonov J, et al. Lab Invest. 2005 Aug; 85(8): 1040-50, Kong H, et al. Sci Rep. 2014 Nov 28; 4: 7246, Florent Mouliere et al. PLOS ONE September 2011 Volume 6 Issue 9e23418). However, methods have been developed, for example, to analyze amplification products based on shorter amplification products, such as those less than 70 bp, by including modified nucleotides in the oligonucleotides of primers and probes. Primers and probes used in this method are known to be Hypercool Primer & Probe. TM (Japan Gene Research Institute Co., Ltd., 2, 1-5 Nakano, Miyagino-ku, Sendai City) has disclosed a design method for it (http: / / ngrl.co.jp / wp / wp-content / uploads / 2015 / 01 / eb3607d6c1eb4174f10bb3bde67844fe.pdf). Furthermore, regarding the case of detecting a low proportion of mutant sequences in the presence of a large excess of non-mutant sequences, for example, Japanese Patent Publication No. 2010-535031 can be referred to. This technique is based on a method of treating the reaction mixture at a critical denaturation temperature or a temperature below the melting temperature Tm of the non-mutant sequence.

[0179] In a preferred embodiment, the oligonucleotides contained in the library include modified nucleotides (sometimes also referred to as modified bases). Modified nucleotides include: diaminopurine analogs (e.g., 2'-O-methyl-2,6-diaminopurine), uracil, peptide nucleic acid analogs, biotin-modified analogs, fluorophore-modified analogs, inosine, 7-denitroguanine, 2'-deoxy-2'-fluoro-β-D-arabinose (2'F-ANA) nucleotides, locked nucleic acids (LNAs), ENAs: 2'-O,4'-C-ethylene-bridged nucleic acids, etc. These modifications can increase the difference in Tm between matched or mismatched bases, enabling high-precision analysis.

[0180] Locked nucleic acids are a class of nucleotide analogs with restricted stereostructure (see, for example, WO99 / 14226, Koshkin, AA, et al., Tetrahedron (1998), 54: 3607-3630, and Obika, S. et al., Tetrahedron Lett. (1998), 39: 5401-5404). Introducing locked nucleic acids into oligonucleotides increases the affinity for complementary sequences, resulting in a stepwise increase in melting temperature (Braasch, DA and DR Corey, Chem. Biol. (2001), 8: 1-7).

[0181] Particularly preferred examples of modified nucleotides are 2-amino-dA (2aA) and 5-methyl-dC (5mC).

[0182] The oligonucleotides contained in the library may also contain mismatched or unmatched bases. Those skilled in the art of nucleic acid technology can determine double-strand stability by considering multiple variables such as oligonucleotide length, base composition and sequence, ionic strength, and the percentage of mismatched bases. The stability of nucleic acid double strands is expressed by the melting temperature (sometimes called denaturation temperature) and Tm.

[0183] To facilitate analysis, the ends of probes in the library can be modified. Modification can be achieved using fluorescent substances. Examples of fluorescent substances include: 6FAM (sometimes abbreviated as FAM), HEX, and Tide Fluor. TM 1. ATTO 390, ATTO 425, LC (Registered Trademark) 480, Cyan 500, ATTO 465, ATTO 488, Tide Fluor TM 2. ATTO 495, ATTO 514, ATTO 520, TET, JOE, CAL Fluor 540, Yakima Yellow, ATTO 532, ATTO Rho6G, LC (registered trademark) Yellow 555, CAL Fluor 560, ATTO 542, Quasar (registered trademark) 570, Cy3, ATTO 550, TAMRA, Tide Fluor TM 3. ATTO 565, ATTO Rho3B, ROX, ATTO Rho11, ATTO Rho12, Cy3.5, ATTOThio12, ATTO Rho101, LC (registered trademark) Red610, CAL Fluor 610, Tide Fluor TM4. ATTO 590, TexasRed (SR101), ATTO 594, ATTO Rho13, ATTO 610, LC (registered trademark) Red670, ATTO 620, LC (registered trademark) Red640, ATTO Rho14, ATTO 633, ATTO 647, ATTO 647N, Quasar (registered trademark) 670, Tide Fluor TM 5. Cy5, ATTO 655, ATTO Oxa12, ATTO 665, Tide Fluor TM 6. Cy5.5, ATTO 680, LC (registered trademark) Red705, Quasar (registered trademark) 705, ATTO 700, ATTO 725, ATTO 740, Tide Fluor TM 7. Tide Fluor TM 8. By using different fluorescent substances in the probes for wild-type and the probes for mutants, it is possible to analyze both simultaneously.

[0184] The probes in the library can also be modified near their tips with matting agents (sometimes called quenchers). Examples of matting agents include: Dabcyl, BHQ-0 (registered trademark), BHQ-1 (registered trademark), BHQ-2 (registered trademark), BHQ-3 (registered trademark), and TQ. TM 1. TQ TM 2. TQ TM 3. TQ TM 4. TQ TM 5. TQ TM 6. TQ TM 7. TAMRA, ATTO540Q, ATTO575Q, ATTO 580Q, ATTO 612Q, BBQ-650 (registered trademarks).

[0185] dPCR using the primers and probes described above can be performed on the same scale as existing dPCR methods. Those skilled in the art can appropriately set the amounts of nucleic acid, primers, and probes in the sample used; for example, the amounts of nucleic acid (0.5–50 ng), primers (0.02–2.0 μM), and probes (0.01–1.0 μM) per dPCR chip can be used. In addition to the amounts of nucleic acid, primers, and probes, the system composition, as will be apparent to those skilled in the art, may include four dNTPs, a buffer, a salt, a surfactant, and an enzyme catalyzing the chain elongation reaction.

[0186] [Using the document library]

[0187] The library of this invention can be used in methods to assist in predicting recurrence or prognosis in subjects after cancer treatment. "Assisting in..." refers to methods performed by persons other than physicians, such as clinical testing technicians, nurses, public health nurses, or the subject themselves. Specifically, this method includes the following steps: preparing at least one probe and / or primer or primer pair from a pre-prepared library of this invention; and using the prepared probe and / or primer or primer pair to analyze mutations in nucleic acids obtained from the subject. For libraries capable of being used in this method, the description of the library already set forth in this specification applies.

[0188] The method provided by this invention can be considered effective against all cancers, including digestive tract tumors such as esophageal cancer, gastric cancer, or colorectal cancer.

[0189] In a preferred embodiment, the method of the present invention can be implemented as follows:

[0190] Suitable kits (e.g., kits of primers and probes for analyzing case-specific mutations detected in mutation analysis of primary lesions) are obtained from a pre-prepared library of the present invention. After confirming the use of primary lesion DNA from the subject as needed, the proportion of mutated DNA, i.e., the frequency (%) of mutated alleles, in appropriately collected samples from the subject is monitored. The sample is preferably plasma, and mutations can be analyzed using ctDNA.

[0191] In cases involving subjects with low tumor cell counts, such as stage I patients, ctDNA may sometimes be undetectable before treatment, after surgery, or during follow-up. In subjects with a certain level of tumor cell count, such as stage II patients, it can be confirmed that ctDNA detected before treatment decreases to 0% post-surgery. Furthermore, by analyzing the presence or amount of ctDNA during follow-up, including post-operative adjuvant chemotherapy, recurrence can be predicted. Using the method of this invention, based on the individual patient, a decrease in ctDNA accompanying tumor shrinkage caused by chemotherapy or radiation therapy can be confirmed. Compared to existing CT diagnostic methods, the method of this invention offers the potential for earlier diagnosis of recurrence.

[0192] For ctDNA analysis, it is also excellent in systematically tracking the inherent mutated DNA of tumors. In addition, it reduces the burden on patients after treatment and facilitates long-term analysis, allowing for early detection of post-treatment increases in "tumor mass" and rapid initiation of secondary treatment.

[0193] With the library of this invention, it is not necessary to design and synthesize primers and probes for each subject when detecting ctDNA. By using the library of this invention, which contains highly universal primers and probes, or primers and probes designed to continuously detect mutations, early diagnosis of cancer recurrence after treatment is possible. In addition, personalized post-treatment follow-up can be easily implemented for cancer patients.

[0194] The library of this invention can also be used in methods for the early detection and diagnosis of cancer.

[0195] The following examples are provided to illustrate the invention in further detail, but the invention is not limited to any of the examples.

[0196] Example

[0197] [The creation of the paperback]

[0198] The following shows representative examples of primer and probe kits synthesized and confirmed using Hypercool Primer & Probe. Synthesis was commissioned to Nippon Genome Research Institute Co., Ltd. (2, 1-5 Nakano, Miyagino-ku, Sendai City). It should be noted that in the base sequences listed in the table, the portions within the rectangular boxes represent Tm-rising base insertion sites (modified nucleotide sites), and * indicates mutation sites.

[0199] [Table 2-1]

[0200] The occurrence rates of TP53 primer / probe sequences in 1284 point mutations of TP53 (a total of 25376 mutations) across all cancers are represented by the proportions of the first 10, 40, and 232 primer / probe positions relative to the theoretically detectable TR52 mutations.

[0201] The first 10 primers / probes contained in the sample can detect 30% of TP53 mutations.

[0202]

[0203] [Table 2-2]

[0204] The first 40 positions contain primers / probes (capable of detecting 50% of TP53 mutations).

[0205]

[0206] [Table 2-3]

[0207] The primers / probes contained in the first 232 positions (can detect 80% of TP53 mutations)

[0208]

[0209] [Confirmation of primer / probe functionality]

[0210] Twenty-nine primer / probe sets designed / synthesized for each case-specific mutation were validated using DNA from the primary lesion (or sufficient plasma) via the QuantStudio 3D Digital PCR System (Thermo Fisher Scientific). Mutant alleles (blue) and wild-type alleles (red) were isolated in all cases. Figure 3 ).

[0211] [Monitoring of circulating tumor DNA using a case-specific TP53 mutation probe]

[0212] DNA was extracted from the primary esophageal cancer lesion tissue before treatment, and peripheral blood mononuclear cell DNA was used as a normal control for gene mutation screening. For the detected gene mutations, digital PCR probes that labeled wild-type alleles (normal alleles) and mutant alleles (mutant alleles) and primers that amplified regions containing mutation sites were designed and synthesized (using the Japan Gene Research Institute and Hypercool™ technology).

[0213] Blood samples were collected from patients before treatment, after chemotherapy, surgery, radiation therapy, and during post-treatment follow-up. Plasma was separated from each sample by centrifugation, and cell-free DNA was extracted from the plasma. Plasma DNA extraction was performed using the QIAamp (registered trademark) Circulating Nucleic Acid Kit (QIAGEN). The cell-free DNA in plasma is primarily wild-type DNA derived from normal cells; mutant DNA derived from cancer cells is typically less than 1%, and is often undetectable. Therefore, DNA from the primary lesion was used to confirm the functionality of synthesized primers / probes in digital PCR. After confirming the separation and measurement of wild-type and mutant alleles, analysis was performed based on the plasma samples. Analysis was performed according to the QuantStudio 3D Digital PCR System (Thermo Fisher Scientific) protocol, adjusting the PCR reaction solution to a final concentration of 900 nM primers and 250 nM probe per chip, using 1–20 ng of DNA.

[0214] The flowchart of the ctDNA monitoring experiment is shown below. Figure 4For case-specific mutations detected through mutation analysis of the primary lesion, probes for wild-type DNA and mutant DNA, respectively fluorescently labeled (wild-type: HEX, mutant: FAM), were prepared. After validation using the primary lesion DNA, the proportion of mutant DNA in plasma samples, i.e., the mutant allele frequency (%), was collected and measured during treatment. Example: Compared with the mutant DNA in plasma before treatment (1), a reduction in mutant DNA (blue) was observed in post-treatment (2) and (3).

[0215] [Monitoring of TP53 mutant DNA in plasma of stage I–IV esophageal cancer cases]

[0216] In stage I cases with low tumor cell counts, ctDNA was undetectable before treatment, after surgery, and during follow-up. In stage II cases, ctDNA detected before treatment decreased to 0% after surgery, and was undetectable during follow-up, including after adjuvant chemotherapy. No recurrence has been observed in these cases to date. In stage III cases, a decrease in ctDNA was observed associated with tumor shrinkage induced by chemotherapy and radiation therapy; however, ctDNA levels rose again as the tumor grew larger, and death occurred shortly after the end of follow-up. Figure 5 MAF, mutation allele frequency; CF, cisplatin / 5-FU; DCF, docetaxel / cisplatin / 5-FU; CRT, chemotherapy and radiotherapy; PTX, paclitaxel.

[0217] [ctDNA monitoring for stage IIA esophageal cancer]

[0218] In the mutation analysis of the primary lesion prior to treatment, the TP53 mutation p.Tyr220Cys (c.659A>G) was detected. Primers / probes for the same mutation were synthesized and ctDNA was detected in plasma samples collected during treatment. A decrease in ctDNA levels (MAF) was observed after preoperative chemotherapy (CF: cisplatin / 5-FU). Mediastinal lymph node recurrence was observed 1.5 years postoperatively (day 615), but no lymph nodes were detected on CT scans 6 months prior (day 436), and only a 3 mm lymph node was found 3 months prior (day 527), not meeting the diagnostic criteria for recurrence. An increase in ctDNA was observed after day 438, suggesting that ctDNA analysis may be able to diagnose recurrence earlier than CT diagnosis. Complete remission (full response) was achieved with radiochemotherapy for the recurrent lesion, and ctDNA also turned negative, reaching 0% ( Figure 6 MAF, mutation allele frequency; CF, cisplatin / 5-FU; CRT, chemotherapy-radiotherapy.

[0219] Sequence List Readable Text

[0220] SEQ ID NO.: Sequences of primers or probes for dPCR, 1–36

[0221] The sequence of the DNA binding domain of SEQ ID NO.: 37TP53 sequence list <110> School legal person Iwate Medical University (Iwate Medical University) <120> Probe / primer library for cancer diagnosis <130> 191551K <150> JP 2018-143912 <151> 2018-07-31 <160> 37 <170> PatentIn version 3.5 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 1 gggacggaac agctttgag 19 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 2 cccctttctt gcggagattc 20 <210> 3 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Wild-type probe <400> 3 cctctgtgcg ccggtct 17 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> mutant probes <400> 4 tcctctgtgc gccagtct 18 <210> 5 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 5 tgtggagtat ttggatgaca ga 22 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 6 agttgcaaac cagacctca 19 <210> 7 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Wild-type probe <400> 7 accaccacac tatgtcgaaa ag 22 <210> 8 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> mutant probes <400> 8 accaccacac tatgtcaaaa ag 22 <210> 9 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 9 tgtggagtat ttggatgaca ga 22 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 10 agttgcaaac cagacctcag 20 <210> 11 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Wild-type probe <400> 11 tggtgcccta tgagccg 17 <210> 12 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> mutant probes <400> 12 tggtgccctg tgagcc 16 <210> 13 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 13 gtcacagcac atgacgga 18 <210> 14 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 14 caccatcgct atctgagca 19 <210> 15 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Wild-type probe <400> 15 ttgtgaggcg ctgcccc 17 <210> 16 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> mutant probes <400> 16 ttgtgaggcg ctaccccc 18 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 17 gttggctctg actgtaccac 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 18 ccagtgtgat gatggtgagg 20 <210> 19 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Wild-type probe <400> 19 tgtaacagtt cctgcatggg c 21 <210> 20 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> mutant probes <400> 20 tgtaacagtt ccttcatggg c 21 <210> twenty one <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> twenty one agtggaagga aatttgcgtg 20 <210> twenty two <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> twenty two accaccacac tatgtcgaaa ag 22 <210> twenty three <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> Wild-type probe <400> twenty three tggagtattt ggatgacaga aaca 24 <210> twenty four <211> twenty three <212> DNA <213> Artificial Sequence <220> <223> mutant probes <400> twenty four tggagtgttt ggatgacaga aac 23 <210> 25 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 25 ccatggccat ctacaagca 19 <210> 26 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 26 ctcaccatcg ctatctgagc a 21 <210> 27 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Wild-type probe <400> 27 cctccgtcat gtgctgtga 19 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mutant probes <400> 28 acctccgtca tgtgctatga 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 29 gcccctgtca tcttctgtcc 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 30 ccagaatgca agaagcccag 20 <210> 31 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Wild-type probe <400> 31 ccctggtagg ttttctggga ag 22 <210> 32 <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> mutant probes <400> 32 tgccctggta ggttttctag gaag 24 <210> 33 <211> twenty three <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 33 tcctgagtag tggtaatcta ctg 23 <210> 34 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 34 cccaggacag gcacaaac 18 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Wild-type probe <400> 35 acctcaaagc tgttccgtcc 20 <210> 36 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> mutant probes <400> 36 cctcacagct gttccgtcc 19 <210> 37 <211> 2940 <212> DNA <213> Homo sapiens <400> 37 gggctgggga cctggagggc tggggggctg gggggctgag gacctggtcc tctgactgct 60 cttttcaccc atctacagtc ccccttgccg tcccaagcaa tggatgattt gatgctgtcc 120 ccggacgata ttgaacaatg gttcactgaa gacccaggtc cagatgaagc tcccagaatg 180 ccagaggctg ctccccccgt ggcccctgca ccagcagctc ctacaccggc ggcccctgca 240 ccagccccct cctggcccct gtcatcttct gtcccttccc agaaaaccta ccagggcagc 300 tacggtttcc gtctgggctt cttgcattct gggacagcca agtctgtgac ttgcacggtc 360 agttgccctg aggggctggc ttccatgaga cttcaatgcc tggccgtatc cccctgcatt 420 tcttttgttt ggaactttgg gattcctctt caccctttgg cttcctgtca gtgttttttt 480 atagtttacc cacttaatgt gtgatctctg actcctgtcc caaagttgaa tattcccccc 540 ttgaatttgg gcttttatcc atcccatcac accctcagca tctctcctgg ggatgcagaa 600 cttttctttt tcttcatcca cgtgtattcc ttggcttttg aaaataagct cctgaccagg 660 cttggtggct cacacctgca atcccagcac tctcaaagag gccaaggcag gcagatcacc 720 tgagcccagg agttcaagac cagcctgggt aacatgatga aacctcgtct ctacaaaaaa atacaaaaaa ttagccaggc atggtggtgc acacctatag tcccagccac ttaggaggct 840 gaggtggga gatcacttga ggccaggaga tggaggctgc agtgagctgt gatcacacca ctgtgctcca gcctgagtga cagagcaaga ccctatctca aaaaaaaaa aaaaaaaaga aagctcctga ggtgtagcg ccaactctct ctagctcgct agtgggttgc aggaggtgct tacgcatgtt tgtttctttg ctgccgtctt ccagttgctt tatctgttca cttgtgccct 1080 gactttcaac tctgtctcct tcctcttcct acagtactcc cctgccctca acaagatgtt ttgccaactg gccaagacct gccctgtgca gctgtgggtt gattccacac ccccgcccgg cacccgcgtc cgcgccatgg ccatctacaa gcagtcacag cacatgacgg aggttgtgag gcgctgcccc caccatgagc gctgctcaga tagcgatggt gagcagctgg ggctggagag acgacagggc tggttgccca gggtccccag gcctctgatt cctcactgat tgctcttagg 1380 tctggcccct cctcagcatc ttatccgagt ggaggaat ttgcgtgtgg agtatttgga tgacagaaac acttttcgac atagtgtggt ggtgccctat gagccgcctg aggtctggtt 1500 tgcaactggg gtctctggga ggaggggtta agggtggttg tcagtggccc tccaggtgag 1560 cagtagggg gctttctcct gctgcttatt tgacctccct ataaccccat gagatgtgca 1620 aagtaaatgg gtttaactat tgcacagttg aaaaaactga agcttacaga ggctaagggc 1680 ctcccctgct tggctgggcg cagtggctca tgcctgtaat cccagcactt tgggaggcca 1740 aggcaggcgg atcacgaggt tgggagatcg agaccatcct ggctaacggt gaaaccccgt 1800 ctctactgaa aaatacaaaa aaaatagc cgggcgtggt gctgggcacc tgtagtccca 1860 gctactcggg aggctgagga aggagaatgg cgtgaacctg ggcggtggag cttgcagtga 1920 gctgagatca cgccactgca ctccagcctg ggcgacagag cgagattcca tctcaaaaaa 1980 aaaaaaaaaa ggcctcccct gcttgccaca ggtctcccca aggcgcactg gcctcatctt 2040 gggcctgtgt tatctcctag gttggctctg actgtaccac catccactac aactacatgt 2100 gtaacagttc ctgcatgggc ggcatgaacc ggaggcccat cctcaccatc atcacactgg 2160 aagactccag gtcaggagcc acttgccacc ctgcacactg gcctgctgtg ccccagcctc 2220 tgcttgcctc tgacccctgg gcccacctct taccgatttc ttccatacta ctacccatcc 2280 acctctcatc acatccccgg cggggaatct ccttactgct cccactcagt tttcttttct 2340 ctggctttgg gacctcttaa cctgtggctt ctcctccacc tacctggagc tggagcttag 2400 gctccagaaa ggacaagggt ggttgggagt agatggagcc tggtttttta aatgggacag 2460 gtaggacctg atttccttac tgcctcttgc ttctcttttc ctatcctgag tagtggtaat 2520 ctactgggac ggaacagctt tgaggtgcgt gtttgtgcct gtcctgggag agaccggcgc 2580 acagaggaag agaatctccg caagaaaggg gagcctcacc acgagctgcc cccagggagc 2640 actaagcgag gtaagcaagc aggacaagaa gcggtggagg agaccaaggg tgcagttatg 2700 cctcagattc acttttatca cctttccttg cctctttcct agcactgccc aacaacacca 2760 gctcctctcc ccagccaaag aagaaaccac tggatggaga atatttcacc cttcaggtac 2820 taagtcttgg gacctcttat caagtggaaa gtttccagtc taacactcaa aatgccgttt 2880 tcttcttgac tgttttacct gcaattgggg catttgccat cagggggcag tgatgcctca 2940

Claims

1. The use of a library in the preparation of reagents for detecting cancer-associated gene mutations in a region encoding the DNA-binding domain of the TP53 gene, said library comprising multiple probes and / or multiple primer pairs, Multiple probes and / or multiple primer pairs include probes and / or primer pairs for detecting c.524G>A, c.743G>A, c.818G>A, c.817C>T, c.742C>T, c.844C>T, c.637C>T, c.733G>A, c.747G>T, and c.659A>G. The library is used to prepare reagents for predicting recurrence or prognosis in subjects after cancer treatment. The cancer is esophageal cancer.

2. According to claim 1, the library further comprises a collection for detecting c.586C>T, c.469G>T, c.536A>G, ​​c.488A>G, c.527G>T, c.818G>T, c.853G>A, c.734G>A, c.722C>T, c.578A>G, c.535C>T, c.856G>A, c.584T>C, c.574C>T, and c.7 Multiple probes and / or multiple primer pairs of c.814G>A, c.711G>A, c.713G>A, c.743G>T, c.473G>A, c.646G>A, c.832C>T, c.422G>A, c.527G>A, c.455C>T, c.473G>T, c.725G>T, c.833C>T, c.614A>G, and c.641A>G.

3. According to claim 2, the library further comprises a collection for detecting c.734G>T, c.451C>T, c.797G>A, c.839G>A, c.839G>C, c.707A>G, c.733G>T, c.517G>T, c.404G>A, c.581T>G, c.796G>A, c.517G>A, c.380C>T, c.395A>G, c.824G>A, c.404G>T, c.730G>T, c.577C>T, c.638G>T, c.749C>T, c.772G>A, c.578A>T, c.824G>T, c.736A>G, ​​and c.797G>T. , c.476C>T, c.725G>A, c.461G>T, c.481G>A, c.731G>A, c.638G>A, c.713G> T, c.715A>G, c.406C>T, c.493C>T, c.536A>T, c.811G>A, c.437G>A, c.438G> A, c.592G>T, c.430C>T, c.711G>T, c.730G>A, c.746G>T, c.610G>T, c.722C >G, c.329G>T, c.745A>T, c.814G>T, c.841G>C, c.396G>T, c.836G>A, c.838A >G, c.799C>T, c.830G>T, c.583A>T, c.832C>A, c.844C>G, c.452C>A, c.548 C>G, c.569C>T, c.833C>G, c.396G>C, c.475G>C, c.499C>T, c.427G>A, c.644 G>T, c.775G>T, c.700T>C, c.716A>G, ​​c.745A>G, c.841G>A, c.298C>T, c.31 0C>T, c.820G>T, c.763A>T, c.821T>C, c.464C>A, c.467G>C, c.542G>A, c.58 0C>T, c.746G>C, c.818G>C, c.845G>A, c.772G>T, c.405C>G, c.541C>T, c.8 32C>G, c.856G>T, c.329G>C, c.413C>T, c.514G>T, c.584T>A, c.511G>T, c.8 11G>T, c.375G>T, c.523C>G, c.747G>C, c.394A>G, c.487T>A, c.800G>C, c. 853G>T, c.738G>A, c.742C>G, c.785G>T, c.859G>T, c.375G>A, c.454C>T, c.487T>C, c.524G>T, c.725G>C, c.794T>C, c.839G>T, c.848G>C, c.388C>G, c.528C>G, c.535C>A, c.596G>T, c.643A>G , c.722C>A, c.796G>T, c.374C>T, c.377A>G, c.517G>C, c.523C>T, c.530C>T, c.817C>A, c.434T>A, c.463A>C, c.503 A>G, c.535C>G, c.658T>A, c.700T>A, c.743G>C, c.843C>G, c.380C>A, c.400T>C, c.412G>C, c.421T>C, c.472C>T, c. 473G>C, c.587G>C, c.706T>A, c.526T>A, c.526T>C, c.537T>A, c.542G>C, c.659A>C, c.731G>T, c.733G>C, c.843C>A , c.845G>C, c.857A>G, c.661G>T, c.434T>C, c.451C>G, c.490A>G, c.613T>G, c.718A>G, c.808T>C, c.809T>C, c.313 G>T, c.388C>T, c.559G>A, c.623A>T, c.645T>G, c.658T>C, c.796G>C, c.809T>G, c.823T>C, c.840A>T, c.841G>T, c. Multiple probes and / or multiple primer pairs of c.847C>T, c.854A>T, c.328C>T, c.423C>G, c.451C>A, c.472C>G, c.518T>C, c.596G>A, c.755T>C, c.830G>A, c.833C>A, c.842A>G, c.497C>G, c.395A>T, c.470T>G, c.530C>G, c.578A>C, c.632C>T, c.712T>C, and c.746G>A.

4. According to claim 1, the library is used to prepare reagents for detecting circulating tumor DNA (ctDNA) derived from tumor cells.

5. The use according to any one of claims 1 to 4, wherein, Probes and / or primer pairs are used for digital PCR.

6. The use according to any one of claims 1 to 4, wherein, The probe and / or primer pair contains an oligonucleotide or a marker thereof consisting of any one of the sequences in sequence numbers 1 to 36.

7. Use of a library in the preparation of a reagent for analyzing cancer-related gene mutations in the region encoding the DNA-binding domain of the TP53 gene in a subject by means of the following steps (1) to (3): (1) The process of preparing a library, wherein the library consists of multiple probes and / or multiple primer pairs, the multiple probes and / or multiple primer pairs including probes and / or primer pairs for detecting mutations of c.524G>A, c.743G>A, c.818G>A, c.817C>T, c.742C>T, c.844C>T, c.637C>T, c.733G>A, c.747G>T and c.659A>G; (2) The process of selecting probes and / or primer pairs from the prepared library to detect a mutation; (3) Using the selected probes and / or primer pairs, analyze the mutations in the nucleic acids obtained from the subjects using digital PCR; The library is used to prepare reagents for predicting recurrence or prognosis in subjects after cancer treatment. The cancer is esophageal cancer.

8. The use according to claim 7, wherein, One of the selected mutations was identified in the primary lesion of the subject before treatment, and the library was used to prepare reagents for analyzing the mutation in circulating tumor DNA (ctDNA) derived from tumor cells obtained from the blood of the subject after treatment.

9. The use according to any one of claims 1 to 4 and 7 to 8, wherein the library comprises a set of probes and primers consisting of oligonucleotides or markers thereof from any group 1 to 4: The oligonucleotides used to detect mutations in c.844 consist of sequences from sequence numbers 1 to 4. The oligonucleotides used to detect mutations in c.637 consist of sequences from sequence numbers 5 to 8. The oligonucleotides used to detect mutations in c.659 consist of sequences from sequence numbers 9 to 12. The oligonucleotides used to detect mutations in c.527 consist of sequences from sequence numbers 13 to 16. The oligonucleotides used to detect mutations in c.725 consist of sequences from sequence numbers 17 to 20. The oligonucleotides used to detect mutations in c.614 consist of sequences from sequence numbers 21 to 24. The oligonucleotides used to detect mutations in c.499 consist of sequences from sequence numbers 25 to 28. The oligonucleotides used to detect mutations in c.298 consist of sequences from sequence numbers 29 to 32, and The oligonucleotides used to detect mutations in c.809 consist of sequences from sequence numbers 33 to 36.

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