WO2020026776A1 - がんの診断のためのプローブ/プライマーライブラリー - Google Patents
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Definitions
- the present invention relates to a method for diagnosing cancer. More specifically, the present invention relates to a probe and primer for detecting a mutation in the TP53 gene, which is useful for early diagnosis of cancer (especially esophageal cancer, stomach cancer, colorectal cancer) recurrence.
- Non-Patent Document 1 includes pre- and post-operative plasma, primary tumor, and peripheral blood mononuclear cells (PBMC) from 44 individuals who have a tumor in the colon and have undergone curative resection. DNA is extracted from the samples, and we are investigating tumor-specific mutations, tumor mutation spectra, and changes in the allele frequency of mutations in ctDNA after curative resection of tumors.
- PBMC peripheral blood mononuclear cells
- Patent Documents 1 and 2 disclose, in a method for monitoring tumor mass, measuring the copy number of a DNA fragment of a gene having a mutation in a blood sample of a patient, and using the copy number as an index of the tumor mass in the patient. Suggest to do.
- Patent Document 3 proposes a method combining the PNA-Clamping method and direct sequencing as a method for detecting a K-ras gene mutation. According to this method, blood is used as a test material in place of cancer tissue, and a patient is examined. Can be evaluated for the therapeutic sensitivity of molecular targeted drugs to the K-ras gene.
- Patent Document 4 discloses a method for non-invasively diagnosing ovarian clear cell adenocarcinoma, in which amplification or deletion in several chromosomal DNA regions is associated with onset, malignancy, prognosis, etc. of ovarian clear cell adenocarcinoma It is proposed to detect these amplifications or deletions based on the finding that they have chromosomal DNA, but describes that chromosomal DNA obtained from patients may be extracted from blood.
- Non-Patent Document 2 discloses that circulating DNA derived from tumor cells contained in peripheral blood at the start of treatment in a patient having high-grade serous ovarian cancer (high-grade serum ovarian carcinoma: HGSOC). : CtDNA) correlated with tumor volume, and the TP53 mutant allele fraction (TP53MAF) after one cycle of chemotherapy was less than 60% lower than before treatment.
- CtDNA TP53 mutant allele fraction
- Non-Patent Document 1 digital PCR (dPCR) is used for detection of ctDNA.
- the dPCR detects a specific sequence with high sensitivity (0.01% -1%) by using a probe complementary to a specific sequence (usually one base) in a DNA fragment amplified by a primer.
- Highly sensitive detection of specific sequences has attracted attention in the medical field, especially in cancer diagnosis, but it is almost the only one that can respond in terms of sensitivity required for diagnosis, cost, test time, and the need for repeated testing.
- Technology is dPCR.
- the specific sequence of interest can be determined from the preceding sequence analysis, public database, literature, etc.
- first design primers and probes It needs to be synthesized.
- specific sequences vary depending on individuals and disease states, and therefore, the types thereof are enormous, but their frequency is low in a population, and the targets to which the synthesized probes can be used are limited.
- commercially available dPCR probes are designed to jump over consecutive nucleotide sequences, that is, are designed intermittently, and have little versatility other than those targeting some high-frequency specific sequences. Therefore, even if a unique mutation derived from a tumor cell is specified in a target patient, existing dPCR probes cannot be used, and it is difficult to make a quick diagnosis.
- the dPCR probe is comprehensively prepared so as to cope with many mutations, and it is more preferable that the dPCR probe be prepared non-intermittently so as to cope with all kinds of mutations.
- the present inventors have focused on mutations in the TP53 gene that are confirmed in about 50% of human cancers. Mutations in the TP53 gene have been identified particularly frequently in lung cancer, stomach cancer, colorectal cancer, breast cancer, and the like, but according to studies by the present inventors, in Japanese esophageal cancer, stomach cancer, and colorectal cancer, 87% are known to have mutations.
- the present inventors actually designed and synthesized 29 sets of dPCR probes and primer sets targeting the TP53 gene for detecting individual case-specific mutations.
- several sets were selected from such a library of probe / primer sets and used for monitoring recurrence of cancer patients. As a result, they found that recurrence could be diagnosed earlier than before by ctDNA analysis, and completed the present invention.
- a library comprising a plurality of probes and / or primers or primer pairs for detecting a cancer-related gene mutation in a region encoding a DNA binding domain of a TP53 gene
- the plurality of probes and / or primers or primer pairs are c.524G> A, c.743G> A, c.818G> A, c.817C> T, c.742C> T, c.844C> T, c.
- the library according to 2 comprising a plurality of probes and / or primers or primer pairs for detecting 578A> C, c.632C> T, c.712T> C, and c.746G> A.
- the library according to 1 or 2 for predicting recurrence or prognosis in a subject after cancer treatment.
- [7] The library according to any one of 1 to 6, wherein the probe and / or the primer or the primer pair is for digital PCR.
- [8] The library according to any one of [1] to [7], wherein the probe and / or the primer or the primer pair comprises an oligonucleotide consisting of any one of SEQ ID NOS: 1 to 116, or a labeled product thereof. .
- a method for analyzing a cancer-related gene mutation in a region encoding the DNA binding domain of the TP53 gene in a subject comprising the following steps (1) to (3): (1) A library comprising a plurality of probes and / or primers or primer pairs, wherein the plurality of probes and / or primers or primer pairs are c.524G> A, c.743G> A, c.818G > A, detect c.817C> T, c.742C> T, c.844C> T, c.637C> T, c.733G> A, c.747G> T, and c.659A> G Preparing a library, including: (2) selecting a probe and / or primer or primer pair for detecting one mutation from the prepared library; (3) The mutation in the nucleic acid obtained from the subject is analyzed by digital PCR using the selected probe and / or primer or primer pair.
- cancer is esophageal cancer, gastric cancer, or colon cancer.
- One mutation selected is a mutation identified in the primary tumor of the subject before treatment, and is included in circulating tumor DNA (ctDNA) derived from tumor cells obtained from the blood of the subject after treatment. 11. The method according to 9 or 10, wherein the mutation is analyzed.
- the method for producing a library comprising the following steps: Providing a probe and / or primer or primer pair for detecting a cancer-associated mutation in the DNA binding domain of the TP53 gene; A step of preparing a probe and / or a primer or a primer pair for detecting a cancer-related mutation in the DNA binding domain of the TP53 gene, which is different from the mutation.
- a set of any one of the following primer pair and probe pair of (1) to (9) for use in digital PCR (1) SEQ ID NO: 1, a primer pair consisting of each sequence of 2, and A probe pair which is an oligonucleotide consisting of the sequences of (2) SEQ ID NO: 5, a primer pair consisting of each sequence of 6, and A probe pair which is an oligonucleotide consisting of the sequences of (3) SEQ ID NO: 9, a primer pair consisting of each sequence of 10, and A probe pair which is an oligonucleotide consisting of the sequences of (4) SEQ ID NO: 13, a primer pair consisting of each of the sequences of 14, and A probe pair which is an oligonucleotide consisting of the sequences of (5) SEQ ID NO: 17, a primer pair consisting of each of the sequences of 18, and A probe pair which is an oligonucleotide consisting of the sequences of (6) SEQ ID NO: 21, a primer pair consisting of each sequence of 22,
- a set of probes and primers composed of any one of the following oligonucleotides or a label thereof for use in digital PCR: c. an oligonucleotide consisting of each of SEQ ID NOs: 1 to 4 for detecting the mutation of 844, c. an oligonucleotide having a sequence of each of SEQ ID NOS: 5 to 8 for detecting the mutation of 637, c. an oligonucleotide consisting of each sequence of SEQ ID NOs: 9 to 12 for detecting the mutation of 659, c. an oligonucleotide consisting of each sequence of SEQ ID NOs: 13 to 16 for detecting the mutation of 527, c.
- an oligonucleotide having a sequence of each of SEQ ID NOS: 17 to 20 for detecting a mutation of 725 c. an oligonucleotide having a sequence of each of SEQ ID NOS: 21 to 24 for detecting a mutation of 614, c. an oligonucleotide consisting of each sequence of SEQ ID NOs: 25 to 28 for detecting the mutation of 499, c. an oligonucleotide consisting of each sequence of SEQ ID NOs: 29 to 32 for detecting a mutation in 298, and c. An oligonucleotide consisting of each sequence of SEQ ID NOs: 33 to 36 for detecting the mutation of 809.
- the present invention also provides the following.
- a library comprising a plurality of probes and / or primers or primer pairs for detecting cancer-related mutations in the DNA binding domain of the TP53 gene.
- Mutations related to cancer are c.298, c.329, c.380, c.399_400 insert, c.475, c.499, c.524, c.527, c.536, exon5 splicesite_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
- the plurality of probes and / or primers or primer pairs are c.524, c.527, c.536, c.578, c.614, c.637, c.659, c.725, c.730. 3.
- the library according to 1 or 2 including a library for detecting a mutation at any position selected from the group consisting of: c.743, c.743, and c.818.
- the library according to 1 or 2 for predicting recurrence or prognosis in a subject after cancer treatment.
- ctDNA circulating tumor DNA
- the probe and / or the primer or the primer pair is a polynucleotide consisting of any one of SEQ ID NOs: 1 to 116, or a labeled product thereof. .
- a method of assisting in predicting recurrence or prognosis in a subject after cancer treatment comprising the following steps: Preparing at least one probe and / or primer or primer pair from the library according to any one of 1 to 6; Using the prepared probe and / or primer or primer pair, the mutation in the nucleic acid obtained from the subject is analyzed by digital PCR. [10] The method according to 9, wherein the cancer is esophageal cancer, gastric cancer, or colon cancer. [11] A probe and / or primer or primer pair for detecting a mutation identified in the primary focus of the subject before treatment is prepared, and circulating tumor (circulating tumor) derived from tumor cells obtained from the blood of the subject after treatment. DNA: ctDNA) to analyze the mutation.
- ⁇ About 90% of gastrointestinal cancer patients can be diagnosed by using the library of the present invention.
- the present invention enables early detection of recurrence after treatment.
- TP53 Location of mutated amino acids of TP53 in 66 cases of esophageal, gastric, or colorectal cancer. Mutation locations often overlap. In such a case, analysis of a plurality of cases is possible with one designed probe / primer set. Location of mutations in the TP53 gene in 66 cases of esophageal, gastric, or colon cancer. Exon II 4-9 of the TP53 gene are underlined. In the examples, the positions of mutations to be detected in which probes and primers for detection were synthesized were marked with *. Operation confirmation results for 29 sets of synthesized primers / probes. DPCR was performed on the primary (or plasma) DNA. In all cases, mutant allele (blue) and wild type allele (red) were detected.
- the present invention provides a library for detecting a mutation associated with cancer.
- the library refers to an aggregate of a plurality of oligonucleotides for detecting a mutation at a plurality of positions. Plurality means at least two or more.
- the library is preferably an aggregate capable of detecting 5 or more mutations, more preferably an aggregate capable of detecting 10 or more mutations, and an aggregate capable of detecting 20 or more mutations. Is more preferable. From the viewpoint that mutations in the portion encoding the DNA binding domain (195 amino acids in length) of the TP53 gene can be detected non-intermittently, the library is preferably capable of detecting all 585 mutations. Libraries are equipped with multiple oligonucleotides to detect multiple mutations, but may be used in a variety of ways.
- the library may be used to analyze mutations at a plurality of locations in one subject.
- the library may be used for analyzing a mutation of interest (one site) for a plurality of subjects, or for analyzing a mutation at a plurality of sites for each of a plurality of subjects.
- a mutation refers to a mutation of a base (sometimes expressed as a nucleotide) unless otherwise specified.
- Oligonucleotides contained in the library include a mutation-containing portion as a primer or a primer pair, a probe for mutation detection, and the like.
- the library may be composed of only an oligonucleotide that can function as a primer, may be composed of only an oligonucleotide that can function as a probe, or may include both.
- a portion of the nucleic acid that may contain the desired mutation is usually amplified by primers using the polymerase chain reaction (PCR), and a probe is used to determine whether the amplification product contains the desired mutation.
- the library preferably contains a set of probes and primer pairs for the target mutation.
- the amplification product refers to DNA amplified by PCR, and is sometimes referred to as amplicon.
- the probe contained in the library may be for detecting a sequence without a desired mutation (wild type) or for detecting a sequence after the desired mutation (mutant). You may.
- the library may have both probes.
- the primers contained in the library are for amplifying a portion containing the position of the target mutation in the nucleic acid in the sample, and are usually a forward primer corresponding to the plus strand and a reverse primer corresponding to the minus strand. Are preferably included in a library.
- a cancer-related mutation refers to a mutation that is frequently observed in DNA from a tumor site of a cancer patient in a population.
- the high frequency of mutation refers to the case where the mutation is observed in 5% or more, preferably 10% or more, more preferably 30% or more of cancer patients.
- the cancer-related mutation is preferably a mutation on the TP53 gene, more preferably a mutation in a portion of the TP53 gene encoding a DNA binding domain.
- the p53 tumor suppressor protein (a 393 amino acid nuclear protein) encoded by the TP53 gene is an important transcriptional regulator that responds to various cellular stresses, such as DNA damage, ultraviolet irradiation, and hypoxia.
- p53 regulates vital cellular processes such as DNA repair, cell cycle progression, angiogenesis and apoptosis, and its activation initiates various molecules and downstream pathways in affected cells obtain. These p53-dependent pathways suppress the growth of damaged cells through cell cycle arrest or apoptosis. Loss or inhibition of p53 function and activity is a contributing factor in many cases of cancer.
- the library is preferably found to be found at a high frequency in our studies in the TP53 gene, c.298, c.329, c.380, c.399_400 insert, c.475, c.499, c.524, c.527, c.536, exon5 @ splicesite_3, c.574, c.578 (including the case of p.His193Arg and the case of 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.
- a plurality of probes and / or primers or primer pairs for detecting any mutation selected from the group consisting of: It should be noted that any one is distinguished from any one, and the number is also arbitrary. The number is not limited to one and may be plural.
- the library is more preferably at least c.524, c.527, c.536, c.578, c.614, c.637, c.659, c.725, c.730, c.743, and c. a plurality of probes and / or primers for detecting a mutation at any position selected from the group consisting of 818, and more particularly for detecting any of the above-described specific mutations at these positions Alternatively, it includes a primer pair.
- the library further preferably comprises a set of probe and primer pairs for detecting all of the mutations at the 11 positions described above, and more particularly for detecting all of the specific mutations at these positions. Including.
- P53 is divided into several domains according to function, but the mutation is most frequently found in the DNA binding domain, accounting for approximately 86% of all mutations. According to the study by the present inventors, 39 mutations (98%) were present in the DNA binding domain among the 40 mutations identified. On the other hand, the DNA binding domain of p53 consists of 196 amino acids, and mutations have been reported for all of them. Therefore, if there is a library of oligonucleotides to cover possible mutations in the DNA binding domain, that is, to detect possible mutations in the DNA binding domain non-intermittently, it is effective for a considerable number of cancer patients It is believed that a diagnosis can be made.
- one particularly preferred library is one that can non-intermittently detect possible mutations in the DNA binding domain.
- Such libraries are directed to coding region mutations for 195 amino acids. Assuming that each set contains a primer pair, a probe for wild type, and a probe for mutant, the library will contain 2,340 theoretical detection target bases. By synthesizing a primer-probe group for these, a library capable of effective diagnosis for nearly 90% of cancer patients having a TP53 mutation can be obtained.
- one particularly preferred library is one that is capable of non-intermittently detecting possible mutations in the DNA binding domain of p53. Specifically, it is a set of 1755 primers / probes capable of detecting 2,340 bases to be theoretically detected.
- Another example is about 800 primer and probe sets for about 800 mutations reported in digestive tract cancers.
- the present inventors when all cancers are targeted (https://cancer.sanger.ac.uk/cosmic), out of 1284 types of TP53 point mutations (a total of 25,376 mutations) In the top 10 reported mutations, 30% of the TP53 mutations (the same applies hereafter), 20 in 40%, 40 in 50%, 80 in 60%, and 232 in 80%.
- the present inventors have synthesized four types of the top ten types and six types of the top twenty types, and have confirmed their usefulness. The ranking of the number of reports compiled by the present inventors is shown below.
- the frequency of appearance (report) differs greatly depending on the mutation.
- One of the particularly preferred libraries is, among the following, a plurality of high-ranking ones, for example, two or more, preferably four or more, more preferably six or more, still more preferably ten or more, even more preferably 20 or more. It is designed to detect more than one kind of mutation.
- the order of mutations targeted by the set of primers and probes used in the Examples section of this specification is as follows.
- chr17: 7579389 c.298C> T (p.Gln100Ter), position 113; chr17: 7579358 c.329G> T (p.Arg110Leu), position 87; chr17: 7578550 c.380C> T (p.Ser127Phe), position 53; chr17: 7578526 c.399_400 insert CAAGATG (p.Phe134fs), not applicable; chr17: 7578455 c.475G> C (p.Ala159Pro), position 104; chr17: 7578431 c.499C> T (p.Gln167Ter), position 105; chr17: 7578406 c.524C> T (p.Arg175His), 1st place; chr17: 7578403 c.527G> A (p.Cys176Tyr), position 34; chr17: 7578394 c.
- “high” means, for example, within 10th (1 to 10th), within 40th (1 to 40th), or within 232th (1 to 232).
- a library capable of detecting at least 30% of TP53 mutations can be designed.
- a library capable of detecting at least 50% of TP53 mutations can be designed.
- a library capable of detecting at least 80% of TP53 mutations can be designed.
- One specific example of a library for detecting a mutation with a high reported number order includes any one of the following three sets, preferably any two sets, and more preferably all three sets: c. an oligonucleotide consisting of any of SEQ ID NOs: 1 to 4 for detecting the mutation of 844, c. an oligonucleotide consisting of any of SEQ ID NOs: 5 to 8 for detecting the mutation of 637, and c. An oligonucleotide consisting of any of SEQ ID NOs: 9 to 12 for detecting the mutation of 659. If the library contains all the sets for detecting mutations whose appearance frequency is within the 10th position represented by these three sets, at least 30% of TP53 mutations can be expected to be detected.
- the library includes, in addition to the above three sets, any one of the following three sets, preferably any two sets, and more preferably all three sets: c. an oligonucleotide consisting of any of SEQ ID NOs: 13 to 16 for detecting the mutation of 527, c. an oligonucleotide consisting of any of SEQ ID NOs: 17 to 20 for detecting the mutation of 725, c. An oligonucleotide consisting of any of SEQ ID NOs: 21 to 24 for detecting the mutation of 614. If the library includes all the sets for detecting mutations whose appearance frequency is within the 40th position represented by these 6 sets, at least 50% of TP53 mutations can be expected to be detected.
- the library includes, in addition to the above six sets, any one of the following three sets, preferably any two sets, and more preferably all three sets: c. an oligonucleotide consisting of any of SEQ ID NOs: 25 to 28 for detecting a mutation in 499, c. an oligonucleotide consisting of any of SEQ ID NOs: 29 to 32 for detecting a mutation in 298, and c. An oligonucleotide consisting of any of SEQ ID NOs: 33 to 36 for detecting the mutation of 809. If the library includes all of the sets for detecting mutations whose appearance frequency is within position 232 represented by these 9 sets, at least 80% of TP53 mutations can be expected to be detected.
- each of the above nine sets is itself useful as a set of probes and primers for detecting a certain mutation.
- Oligonucleotides (probes, primers) having specific sequences included in the above nine sets are useful as such.
- the order of the occurrence frequency of the mutation to be detected is within 10, 10, or 232.
- Any oligonucleotide for detecting mutations with an appearance frequency within the 10th position is useful for detecting at least 30% of TP53 mutations.
- Any of the oligonucleotides for detecting mutations within the 40th rank of occurrence frequency are useful for detecting at least 50% of TP53 mutations.
- Any of the oligonucleotides for detecting mutations in the order of frequency 232 or less are useful for detecting at least 80% of TP53 mutations.
- Each nucleotide in the sequence may be a modified nucleotide. Examples of the modified nucleotide will be described later.
- Probes and / or primers or primer pairs contained in the library can be designed based on sequence information obtained from a primary tumor sample of a cancer patient.
- Patients to be tested for sensitive tumor markers such as ctDNA described below are often in an advanced stage, and the initial treatment is surgery. Therefore, it is relatively easy to obtain sequence information using a solid material obtained by surgery.
- the probes and / or primers or primer pairs contained in the library may be variously designed to allow analysis of mutations in nucleic acids in samples taken from the subject, but may be free in the subject's blood. Preferably, it is designed to be able to analyze the ctDNA present. If ctDNA in blood can be analyzed, tumor-specific mutant DNA will be tracked systemically. In addition, after treatment, it is easy to perform analysis for a long period of time with little burden on the patient, and it is expected that the increase in “intratumoral mass” after treatment can be quickly detected and a rapid transition to secondary treatment can be expected. Note that the analysis includes detecting, quantifying, and the like.
- the subject includes a cancer patient, a person suspected of having cancer, and the like.
- the ratio of the probe and / or the primer or the primer pair contained in the library is low in the presence of a large excess of wild type (sometimes referred to as a non-mutated sequence or a reference sequence). Is designed so that it can be used in a method that can analyze the mutant (sometimes referred to as a mutant sequence).
- the blood concentration of ctDNA (the ratio of the mutated sequence to the non-mutated sequence; sometimes referred to as the allele frequency) is often 1% or less, and it is desirable to be able to perform highly sensitive analysis.
- An example of such a sensitive analytical method is dPCR.
- DPCR (Vogelstein and Kinzler, Proc. Natl. Acad. USA, 96, 9236-9241, 1999) is an extension of the conventional PCR method, and can directly quantify the amount of a target nucleic acid.
- dPCR is suitable for use in combination with enrichment procedures to detect trace mutations.
- the starting material in each PCR reaction is either wild type or mutant. Enrichment is performed from one molecule, with thousands of parallel enrichment reactions occurring simultaneously. After performing multiple PCR reactions from the same starting material, the mutant molecule is isolated and detected. After PCR amplification, the absolute amount of nucleic acid can be calculated by counting the number of chambers containing the final PCR product.
- Various dPCR-based systems that can be used in the present invention are commercially available. The basic methodology of dPCR is described, for example, in Sykes et al., Biotechniques 13 (3): 444-449, 1992.
- the probe and / or the primer or the primer pair be designed to amplify a relatively short nucleic acid fragment as a target sequence and detect a target mutation.
- the size of the amplification product in normal PCR is 100 to 150 bp, and there may be a problem that sensitivity and accuracy are reduced. In order to improve the analytical performance, it is considered important to keep the size of the amplification product as short as possible (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.
- the oligonucleotides contained in the library contain modified nucleotides (sometimes referred to as modified bases).
- Modified nucleotides include diamino-purine analogs (eg, 2'-O-methyl-2,6-diaminopurine), uracil, peptide nucleic acid analogs, biotin-modified analogs, fluorophore-modified analogs, inosine, 7-deazaguanine , 2'-deoxy-2'-fluoro- ⁇ -D-arabino nucleic acid (2'F-ANA) nucleotides, locked nucleic acids (LNAs), ENAs: including 2'-O, 4'-C-ethylene cross-linked nucleic acids . These modifications can increase the difference in Tm between matched or mismatched bases, allowing for highly accurate analysis.
- Locked nucleic acid refers to a class of conformationally restricted nucleotide analogs (e.g., WO 99/14226, Koshkin, AA, et al., Tetrahedron (1998), 54: 3607-3630, and Obika, S. et al., Tetrahedron Lett. (1998), 39: 5401-5404).
- Introducing a locked nucleic acid into an oligonucleotide improves the affinity of the complementary sequence and increases the melting temperature by several steps (Braasch, DA and DR Corey, Chem. Biol. (2001), 8: 1-7) .
- modified nucleotides are 2-amino-dA (2aA) and 5-Methyl-dC (5mC).
- Oligonucleotides included in the library can also include mismatched or unmatched bases.
- One of skill in the nucleic acid arts will be aware of numerous variables such as, for example, oligonucleotide length, oligonucleotide base composition and sequence, ionic strength, and mismatched base content, to determine duplex stability. Can be determined. The stability of a nucleic acid duplex is determined by the melting temperature (sometimes called the denaturation temperature). It can be represented by Tm.
- Probes in the library may be modified near the ends to facilitate analysis.
- a fluorescent substance can be used for the modification.
- the fluorescent substance include 6FAM (sometimes simply referred to as FAM), HEX, Tide Fluor TM 1, ATTO 390, ATTO 425, LC (registered trademark) 480 Cyan500, ATTO 465, ATTO 488, and Tide Fluor TM.
- Probes in the library may also be modified near the end with a quencher (sometimes called a quencher).
- a quencher sometimes called a quencher.
- examples of the quencher Dabcyl, BHQ-0 (registered trademark), BHQ-1 (TM), BHQ-2 (TM), BHQ-3 (TM), TQ TM 1, TQ TM 2, TQ TM 3, TQ TM 4, TQ TM 5, TQ TM 6, TQ TM 7, TAMRA, ATTO540Q, ATTO 575Q, ATTO 580Q, ATTO 612Q, BBQ-650 ( registered trademark).
- D dPCR using the above-described primers and probes can be performed on a scale similar to that of the conventional dPCR method.
- the amounts of nucleic acids, primers and probes in the sample to be used can be appropriately set by those skilled in the art. For example, per dPCR chip, 0.5 to 50 ng of nucleic acids, 0.02 to 2.0 ⁇ M of primers, 0.01 to 2.0 ⁇ M of probes are used. It can be used in the range of 1.0 ⁇ M.
- the composition of the system may include the amounts of nucleic acids, primers and probes, as well as the four dNTPs, buffers, salts, detergents, and enzymes that catalyze the chain extension reaction, as will be apparent to those skilled in the art. it can.
- the library of the invention can be used in a method to assist in predicting relapse or prognosis in a subject after cancer treatment.
- the assisting method refers to a method performed by a person other than a doctor, for example, a laboratory technician, a nurse, a public health nurse, the subject, or the like.
- a method includes preparing at least one probe and / or primer or primer pair from a library of the present invention prepared in advance, and using the prepared probe and / or primer or primer pair. And analyzing the mutation in the nucleic acid obtained from the subject.
- the libraries that can be used in such a method all the descriptions for the libraries already mentioned herein apply.
- the method provided by the present invention is considered to be effective against any cancer, including gastrointestinal cancer such as esophageal cancer, stomach cancer, or colon cancer.
- the method of the present invention can be performed as follows: From the library of the present invention prepared in advance, a set considered to be appropriate (for example, a set of primers and probes for analyzing case-specific mutations detected by mutation analysis of the primary tumor) is obtained and necessary. According to the above, after confirming the operation of the subject using the primary focus DNA, the proportion of mutant DNA in the sample appropriately collected from the subject is monitored.
- the sample is preferably plasma, and the mutation can be analyzed as ctDNA.
- ctDNA may not be detected before treatment, after surgery, or in a follow-up period. From a subject having a certain amount of tumor cells, for example, a subject of Stage II, it can be confirmed that the ctDNA detected before the treatment decreases to 0% after the operation.
- recurrence can be predicted by analyzing the presence or amount of ctDNA. According to the method of the present invention, a decrease in ctDNA associated with tumor reduction due to chemotherapy or radiation therapy can be confirmed for some subjects. According to the method of the present invention, recurrence may be diagnosed earlier as compared with conventional CT diagnosis.
- ctDNA is also excellent in that it is believed to track tumor-specific mutant DNA systemically. In addition, this makes it easy to analyze for a long period of time with little burden on the patient after the treatment, and it is expected that the increase in the “in-vivo tumor volume” after the treatment can be quickly detected and the treatment can be promptly shifted to the second treatment.
- the library of the present invention eliminates the need to design and synthesize primers and probes for each subject when detecting ctDNA.
- the versatile primers and probes, or the library of the present invention containing primers and probes designed to detect mutations non-intermittently early recurrence after cancer treatment can be diagnosed .
- individualized follow-up observation can be easily performed in cancer patients.
- the library of the present invention can also be used in a method for early detection and diagnosis of cancer.
- DNA was extracted from pre-treatment esophageal cancer primary tissue, and gene mutation screening was performed using peripheral blood mononuclear cell DNA as a normal control.
- the analysis was carried out according to the protocol of QuantStudio 3D Digital PCR System (Thermo Fisher Scientific), adjusting the PCR reaction solution to a final concentration of primer 900 nM and probe 250 nM per chip, and using 1 to 20 ng of DNA.
- FIG. 4 shows a flowchart of the ctDNA monitoring experiment.
- a probe that performs fluorescent labeling of wild-type DNA and mutant DNA separately wild-type: HEX, mutant: FAM
- wild-type: HEX, mutant: FAM wild-type DNA
- FAM mutant-type DNA
- ctDNA was not detected before treatment, after surgery, or during the follow-up period.
- ctDNA detected before treatment decreased to 0% after surgery, and ctDNA was not detected during follow-up including postoperative adjuvant chemotherapy. These cases have not recurred to date.
- Stage III cases ctDNA was reduced due to tumor shrinkage due to chemotherapy and radiation therapy, but ctDNA increased with re-growth and died shortly after follow-up was completed (Fig. 5).
- MAF mutant allele frequency
- CF Cisplatin / 5-FU
- DCF Docetaxel / Cisplatin / 5-FU
- CRT chemoradiotherapy
- PTX paclitaxel
- ctDNA Since ctDNA has been elevated since Day 438, ctDNA analysis may be able to diagnose recurrence earlier than CT diagnosis. Complete response (complete response) was obtained by radiochemotherapy for recurrent lesions, but ctDNA was also negatively converted to 0% (Fig. 6). MAF, mutant allele frequency; CF, Cisplatin / 5-FU; CRT, chemoradiotherapy
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Abstract
Description
[1] TP53遺伝子のDNA結合ドメインをコードする領域における、がんに関連する遺伝子変異を検出するための、複数のプローブおよび/またはプライマーもしくはプライマー対で構成されるライブラリーであって、
複数のプローブおよび/またはプライマーもしくはプライマー対が、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、およびc.659A>Gを検出するためのものを含む、ライブラリー。
[2] さらに、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、c.701A>G、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、およびc.641A>Gを検出するための複数のプローブおよび/またはプライマーもしくはプライマー対を含む、1に記載のライブラリー。
[3] さらに、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.503A>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.313G>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.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、およびc.746G>Aを検出するための複数のプローブおよび/またはプライマーもしくはプライマー対を含む、2に記載のライブラリー。
[4] 複数のプローブにより、TP53遺伝子のDNA結合ドメインにおける585箇所のすべての変異を検出できるものである、1~3のいずれか1項に記載のライブラリー。
[5] がんの治療後の被験者における再発または予後の予測のための、1または2に記載のライブラリー。
[6] 腫瘍細胞由来の循環DNA(circulating tumor DNA:ctDNA)を検出するための、1~5のいずれか1項に記載のライブラリー。
[7] プローブおよび/またはプライマーもしくはプライマー対が、デジタルPCR用である、1~6のいずれか1項に記載のライブラリー。
[8] プローブおよび/またはプライマーもしくはプライマー対が、配列番号:1~116のいずれか1つの配列からなるオリゴヌクレオチド、またはその標識物を含む、1~7のいずれか1項に記載のライブラリー。
[9] 以下の(1)~(3)の工程を含む、被験者におけるTP53遺伝子のDNA結合ドメインをコードする領域における、がんに関連する遺伝子変異を分析する方法:
(1)複数のプローブおよび/またはプライマーもしくはプライマー対で構成されるライブラリーであって、複数のプローブおよび/またはプライマーもしくはプライマー対が、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、およびc.659A>Gの変異を検出するためのものを含む、ライブラリーを準備する工程、
(2)準備したライブラリーから、一の変異を検出するためのプローブおよび/またはプライマーもしくはプライマー対を選択する工程;
(3)選択したプローブおよび/またはプライマーもしくはプライマー対を用いて、被験者から得た核酸中の変異をデジタルPCRで分析する。
[10] がんが、食道がん、胃がん、または大腸がんである、9に記載の方法。
[11] 選択される一の変異が、治療前の被験者の原発巣で同定された変異であり、治療後の被験者の血液から得た腫瘍細胞由来の循環DNA(circulating tumor DNA:ctDNA)中の当該変異を分析する、9または10に記載の方法。
[12] 下記の工程を含む、1~8のいずれか1項に記載のライブラリーの製造方法:
TP53遺伝子のDNA結合ドメインにおけるがんに関連する一の変異を検出するための、プローブおよび/またはプライマーもしくはプライマー対を準備する工程;
前記変異とは異なる、TP53遺伝子のDNA結合ドメインにおけるがんに関連する変異を検出するための、プローブおよび/またはプライマーもしくはプライマー対を準備する工程。
[13] デジタルPCRに用いるための、下記の(1)~(9)のいずれか1組のプライマー対およびプローブ対のセット:
(1)配列番号:1、2の各々の配列からなるプライマー対、および
(2)配列番号:5、6の各々の配列からなるプライマー対、および
(3)配列番号:9、10の各々の配列からなるプライマー対、および
(4)配列番号:13、14の各々の配列からなるプライマー対、および
(5)配列番号:17、18の各々の配列からなるプライマー対、および
(6)配列番号:21、22の各々の配列からなるプライマー対、および
(7)配列番号:25、26の各々の配列からなるプライマー対、および
(8)配列番号:29、30の各々の配列からなるプライマー対、および
(9)配列番号:33、34の各々の配列からなるプライマー対、および
[14] デジタルPCRに用いるための、下記のいずれか1組のオリゴヌクレオチド、またはその標識物からなる、プローブおよびプライマーのセット:
c.844の変異を検出するための配列番号:1~4の各々の配列からなるオリゴヌクレオチド、
c.637の変異を検出するための配列番号:5~8の各々の配列からなるオリゴヌクレオチド、
c.659の変異を検出するための配列番号:9~12の各々の配列からなるオリゴヌクレオチド、
c.527の変異を検出するための配列番号:13~16の各々の配列からなるオリゴヌクレオチド、
c.725の変異を検出するための配列番号:17~20の各々の配列からなるオリゴヌクレオチド、
c.614の変異を検出するための配列番号:21~24の各々の配列からなるオリゴヌクレオチド、
c.499の変異を検出するための配列番号:25~28の各々の配列からなるオリゴヌクレオチド、
c.298の変異を検出するための配列番号:29~32の各々の配列からなるオリゴヌクレオチド、および
c.809の変異を検出するための配列番号:33~36の各々の配列からなるオリゴヌクレオチド。
本発明はまた、以下を提供する。
[1] TP53遺伝子のDNA結合ドメインにおける、がんに関連する変異を検出するための、複数のプローブおよび/またはプライマーもしくはプライマー対で構成されるライブラリー。
[2] がんに関連する変異が、c.298 、c.329、c.380、c.399_400 insert、c.475、c.499、c.524、c.527、c.536、exon5 splicesite_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、およびc.993からなる群から選択されるいずれかの位置の変異である、1に記載のライブラリー。
[3] 複数のプローブおよび/またはプライマーもしくはプライマー対が、c.524、c.527、 c.536、 c.578、 c.614、 c.637、 c.659、 c.725、 c.730、 c.743、 およびc.818からなる群より選択されるいずれかの位置の変異を検出するためのものを含む、1または2に記載のライブラリー。
[4] 複数のプローブにより、非間歇的に変異を検出できるものである、1~3のいずれか1項に記載のライブラリー。
[5] がんの治療後の被験者における再発または予後の予測のための、1または2に記載のライブラリー。
[6] 腫瘍細胞由来の循環DNA(circulating tumor DNA:ctDNA)を検出するための、1~5のいずれか1項に記載のライブラリー。
[7] プローブおよび/またはプライマーもしくはプライマー対が、デジタルPCR用である、1~6のいずれか1項に記載のライブラリー。
[8] プローブおよび/またはプライマーもしくはプライマー対が、配列番号:1~116のいずれか1つの配列からなるポリヌクレオチド、またはその標識物である、1~7のいずれか1項に記載のライブラリー。
[9] 以下の工程を含む、がんの治療後の被験者における再発または予後の予測を補助する方法 :
1~6のいずれか1項に記載のライブラリーから、少なくとも一のプローブおよび/またはプライマーもしくはプライマー対を準備し、
準備したプローブおよび/またはプライマーもしくはプライマー対を用いて、被験者から得た核酸中の変異をデジタルPCRで分析 する。
[10] がんが、食道がん、胃がん、または大腸がんである、9に記載の方法。
[11] 治療前の被験者の原発巣で同定された変異を検出するためのプローブおよび/またはプライマーもしくはプライマー対を準備し、治療後の被験者の血液から得た腫瘍細胞由来の循環DNA(circulating tumor DNA:ctDNA)中の当該変異を分析する、
9または10に記載の方法。
[13] 配列番号:1~36のいずれか1つの配列からなるオリゴヌクレオチド、またはその標識物。
[14] 下記のいずれか1組のオリゴヌクレオチド、またはその標識物からなる、プローブおよびプライマーのセット:
c.844の変異を検出するための配列番号:1~4のいずれかの配列からなるオリゴヌクレオチド、
c.637の変異を検出するための配列番号:5~8のいずれかの配列からなるオリゴヌクレオチド、
c.659の変異を検出するための配列番号:9~12のいずれかの配列からなるオリゴヌクレオチド、
c.527の変異を検出するための配列番号:13~16のいずれかの配列からなるオリゴヌクレオチド、
c.725の変異を検出するための配列番号:17~20のいずれかの配列からなるオリゴヌクレオチド、
c.614の変異を検出するための配列番号:21~24のいずれかの配列からなるオリゴヌクレオチド、
c.499の変異を検出するための配列番号:25~28のいずれかの配列からなるオリゴヌクレオチド、
c.298の変異を検出するための配列番号:29~32のいずれかの配列からなるオリゴヌクレオチド、および
c.809の変異を検出するための配列番号:33~36のいずれかの配列からなるオリゴヌクレオチド。
ライブラリーは、複数箇所の変異を検出するための、複数のオリゴヌクレオチドの集合体をいう。複数とは、少なくとも2以上をいう。ライブラリーは、5箇所以上の変異を検出可能な集合体であることが好ましく、10箇所以上の変異を検出可能な集合体であることがより好ましく、20箇所以上の変異を検出可能な集合体であることがさらに好ましい。TP53遺伝子のDNA結合ドメイン(195アミノ酸長)をコードする部分における変異を非間歇的に検出できるとの観点からは、ライブラリーは585箇所のすべての変異を検出できるものであることが好ましい。ライブラリーは複数箇所の変異を検出するために複数のオリゴヌクレオチドを備えるが、用い方は様々であり得る。一人の被験者について目的の変異(1箇所)を分析するために用いられる場合、例えばある患者について、治療前原発巣で検出された変異が、治療経過中に血液中に検出されるか否かを判断するために用いられる場合がある。またライブラリーは、一人の被験者について複数個所の変異を分析するために用いられる場合がある。ライブラリーはさらに、複数の被験者について目的の変異(1箇所)を分析するために用いられる場合、および複数の被験者それぞれについて複数個所の変異を分析するために用いられる場合等がある。なお本発明の説明において変異というときは、特に記載した場合を除き、塩基の(ヌクレオチドの、と表現されることもある。)変異を指す。
chr17:7579358 c.329G>T (p.Arg110Leu)、87位;
chr17:7578550 c.380C>T (p.Ser127Phe)、53位;
chr17:7578526 c.399_400 insert CAAGATG(p.Phe134fs) 、該当なし;
chr17:7578455 c.475G>C (p.Ala159Pro)、104位;
chr17:7578431 c.499C>T (p.Gln167Ter)、105位;
chr17:7578406 c.524C>T (p.Arg175His)、1位;
chr17:7578403 c.527G>A (p.Cys176Tyr)、34位;
chr17:7578394 c.536A>G (p.His179Arg)、13位;
chr17:7578370 exon5 splicesite_3 C>T、該当なし;
chr17:7578275 c.574G>A (p.Gln192Ter)、24位;
chr17:7578271 c.578A>G (p.His193Arg)、20位;
chr17:7578271 c.578A>T (p.His193Leu)、62位;
chr17:7578263 c.586G>A (p. Arg196Ter)、11位;
chr17:7578235 c.614A>G (p.Tyr205Cys)、39位;
chr17:7578212 c.637C>T (p.Arg213Ter)、7位;
chr17:7578190 c.659A>G (p.Tyr220Cys)、10位;
chr17:7577574 c.707A>G (p.Tyr236Cys)、46位;
chr17:7577556 c.725G>T (p.Cys242Phe)、37位;
chr17:7577551 c.730G>A (p.Gly244Ser)、83位;
chr17:7577545 c.736A>T (p.Met246Leu)、455位;
chr17:757753 c.743C>T (p.Arg248Gln)、2位;
chr17:7577517 c.764A>G (p.Ile255Thr)、278位;
chr17:7577500 c.780_780delC (p.Ser260fs)、該当なし;
chr17:7577129 c.809T>G (p.Phe270Cys)、209位;
chr17:7577120 c.818G>A (p.Arg273His)、3位;
chr17:7577099 c.839G>C (p.Arg280Thr)、45位
chr17:7577094 c.844G>A (p.Arg282Trp)、6位;および
chr17:7576853 c.993C>T (p.Gln331Ter)、該当なし(DNA結合ドメイン外)。
c.844の変異を検出するための配列番号:1~4のいずれかの配列からなるオリゴヌクレオチド、
c.637の変異を検出するための配列番号:5~8のいずれかの配列からなるオリゴヌクレオチド、および
c.659の変異を検出するための配列番号:9~12のいずれかの配列からなるオリゴヌクレオチド。これら3セットに代表される出現頻度が10位以内の変異の検出のためのセットをすべて含むライブラリーであれば、少なくとも30%のTP53変異の検出が期待できる。
c.527の変異を検出するための配列番号:13~16のいずれかの配列からなるオリゴヌクレオチド、
c.725の変異を検出するための配列番号:17~20のいずれかの配列からなるオリゴヌクレオチド、
c.614の変異を検出するための配列番号:21~24のいずれかの配列からなるオリゴヌクレオチド。これら6セットに代表される出現頻度が40位以内の変異の検出のためのセットをすべて含むライブラリーであれば、少なくとも50%のTP53変異の検出が期待できる。
c.499の変異を検出するための配列番号:25~28のいずれかの配列からなるオリゴヌクレオチド、
c.298の変異を検出するための配列番号:29~32のいずれかの配列からなるオリゴヌクレオチド、および
c.809の変異を検出するための配列番号:33~36のいずれかの配列からなるオリゴヌクレオチド。これら9セットに代表される出現頻度が232位以内の変異の検出のためのセットをすべて含むライブラリーであれば、少なくとも80%のTP53変異の検出が期待できる。
本発明のライブラリーは、がんの治療後の被験者における再発または予後の予測を補助する方法において用いうる。補助する方法は、医師以外の者、例えば臨床検査技師、看護師、保健師、被験者本人等によって行われる方法を指す。このような方法は、具体的には、予め準備されている本発明のライブラリーから、少なくとも一のプローブおよび/またはプライマーもしくはプライマー対を準備し、準備したプローブおよび/またはプライマーもしくはプライマー対を用いて、被験者から得た核酸中の変異を分析する工程を含む。このような方法に用いることのできるライブラリーについては、本明細書で既に述べたライブラリーについての説明がすべてあてはまる。
予め準備されている本発明のライブラリー中から、適切だと考えられるセット(例えば原発巣の変異解析で検出された症例特異的変異を分析するためのプライマーとプローブのセット)を得て、必要に応じ、被験者の原発巣DNAを用いた動作を確認した後、被験者から適宜採取したサンプル中の変異DNAの割合 mutant allele frequency (%)をモニタリングする。サンプルは、好ましくは血漿であり、変異はctDNAとして分析されうる。
Hypercool Primer&Probeで合成し、動作を確認したプライマーとプローブのセットのうち、代表的なものを下表に示した。合成は、株式会社日本遺伝子研究所(仙台市宮城野区中野一丁目5番地の2)に委託した。なお表に記載した塩基配列中、囲んだ箇所はTm上昇塩基挿入部位(修飾ヌクレオチド部位)であり、*は変異箇所を示す。
個々の症例特異的変異に対しデザイン・合成した29組のprimer/probeについて原発巣(あるいは十分量の血漿)DNAを用い、QuantStudio 3D Digital PCR System (Thermo Fisher Scientific社)にて動作確認を行った。いずれも変異アリル(青)、wild typeアリル(赤)が分離されている(図3)。
治療前食道がん原発巣組織よりDNAを抽出し、末梢血単核球DNAを正常コントロールとし遺伝子変異スクリーニングを施行した。検出された遺伝子変異につきwild type allele(正常アリル)とmutant allele(変異アリル)を別々に標識するdigital PCR probeと変異部位を含む領域を増幅するprimerをデザイン、合成した(日本遺伝子研究所、HypercoolTMテクノロジーを用いた)。
腫瘍細胞量の少ないStage I症例では、治療前、手術後、follow-up期間でctDNAは検出されなかった。Stage II症例では、治療前に検出されたctDNAが術後に0%へと低下し、術後補助化学療法を含めたfollow-up中にctDNAは検出されていない。これらの症例は現在まで再発は見られていない。Stage III症例では化学療法、放射線療法による腫瘍縮小に伴いctDNAの低下が見られたが、再増大とともにctDNAは上昇し、follow-up終了後短期間で死亡した(図5)。MAF, mutant allele frequency; CF, Cisplatin/5-FU; DCF, Docetaxel/Cisplatin/5-FU; CRT, chemoradiotherapy; PTX, paclitaxel
治療前原発巣の変異解析ではTP53変異p.Tyr220Cys(c.659 A>G)が検出された。同変異に対するprimer/probeを合成し、治療経過中に採取した血漿サンプルでctDNAの検出を行った。術前化学療法 (CF: cisplatin/5-FU)によりctDNA量 (MAF)の低下が見られた。術後1年半後 (Day 615)に縦隔リンパ節の再発が確認されたが、CT検査では6か月前 (Day 436)ではリンパ節は検出されず、3か月前(Day 527)でも3mmのリンパ節を認めるのみで再発診断には至らなかった。ctDNAはDay 438より上昇が見られており、ctDNA解析はCT診断に比べ再発を早期に診断できる可能性がある。再発病変に対し放射線化学療法を施行しComplete Response(完全奏効)が得られているが、ctDNAも0%と陰転化している(図6)。MAF, mutant allele frequency; CF, Cisplatin/5-FU; CRT, chemoradiotherapy
SEQ ID NO.:37 TP53のDNA結合ドメインの配列
Claims (14)
- TP53遺伝子のDNA結合ドメインをコードする領域における、がんに関連する遺伝子変異を検出するための、複数のプローブおよび/またはプライマーもしくはプライマー対で構成されるライブラリーであって、
複数のプローブおよび/またはプライマーもしくはプライマー対が、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、およびc.659A>Gを検出するためのものを含む、ライブラリー。 - さらに、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、c.701A>G、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、およびc.641A>Gを検出するための複数のプローブおよび/またはプライマーもしくはプライマー対を含む、請求項1に記載のライブラリー。
- さらに、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.503A>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.313G>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.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、およびc.746G>Aを検出するための複数のプローブおよび/またはプライマーもしくはプライマー対を含む、請求項2に記載のライブラリー。
- 複数のプローブにより、TP53遺伝子のDNA結合ドメインにおける585箇所のすべての変異を検出できるものである、請求項1~3のいずれか1項に記載のライブラリー。
- がんの治療後の被験者における再発または予後の予測のための、請求項1または2に記載のライブラリー。
- 腫瘍細胞由来の循環DNA(circulating tumor DNA:ctDNA)を検出するための、請求項1~5のいずれか1項に記載のライブラリー。
- プローブおよび/またはプライマーもしくはプライマー対が、デジタルPCR用である、請求項1~6のいずれか1項に記載のライブラリー。
- プローブおよび/またはプライマーもしくはプライマー対が、配列番号:1~116のいずれか1つの配列からなるオリゴヌクレオチド、またはその標識物を含む、請求項1~7のいずれか1項に記載のライブラリー。
- 以下の(1)~(3)の工程を含む、被験者におけるTP53遺伝子のDNA結合ドメインをコードする領域における、がんに関連する遺伝子変異を分析する方法:
(1)複数のプローブおよび/またはプライマーもしくはプライマー対で構成されるライブラリーであって、複数のプローブおよび/またはプライマーもしくはプライマー対が、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、およびc.659A>Gの変異を検出するためのものを含む、ライブラリーを準備する工程、
(2)準備したライブラリーから、一の変異を検出するためのプローブおよび/またはプライマーもしくはプライマー対を選択する工程;
(3)選択したプローブおよび/またはプライマーもしくはプライマー対を用いて、被験者から得た核酸中の変異をデジタルPCRで分析する。 - がんが、食道がん、胃がん、または大腸がんである、請求項9に記載の方法。
- 選択される一の変異が、治療前の被験者の原発巣で同定された変異であり、治療後の被験者の血液から得た腫瘍細胞由来の循環DNA(circulating tumor DNA:ctDNA)中の当該変異を分析する、請求項9または10に記載の方法。
- 下記の工程を含む、請求項1~8のいずれか1項に記載のライブラリーの製造方法:
TP53遺伝子のDNA結合ドメインにおけるがんに関連する一の変異を検出するための、プローブおよび/またはプライマーもしくはプライマー対を準備する工程;
前記変異とは異なる、TP53遺伝子のDNA結合ドメインにおけるがんに関連する変異を検出するための、プローブおよび/またはプライマーもしくはプライマー対を準備する工程。 - デジタルPCRに用いるための、下記の(1)~(9)のいずれか1組のプライマー対およびプローブ対のセット:
(1)配列番号:1、2の各々の配列からなるプライマー対、および
(2)配列番号:5、6の各々の配列からなるプライマー対、および
(3)配列番号:9、10の各々の配列からなるプライマー対、および
(4)配列番号:13、14の各々の配列からなるプライマー対、および
(5)配列番号:17、18の各々の配列からなるプライマー対、および
(6)配列番号:21、22の各々の配列からなるプライマー対、および
(7)配列番号:25、26の各々の配列からなるプライマー対、および
(8)配列番号:29、30の各々の配列からなるプライマー対、および
(9)配列番号:33、34の各々の配列からなるプライマー対、および
- デジタルPCRに用いるための、下記のいずれか1組のオリゴヌクレオチド、またはその標識物からなる、プローブおよびプライマーのセット:
c.844の変異を検出するための配列番号:1~4の各々の配列からなるオリゴヌクレオチド、
c.637の変異を検出するための配列番号:5~8の各々の配列からなるオリゴヌクレオチド、
c.659の変異を検出するための配列番号:9~12の各々の配列からなるオリゴヌクレオチド、
c.527の変異を検出するための配列番号:13~16の各々の配列からなるオリゴヌクレオチド、
c.725の変異を検出するための配列番号:17~20の各々の配列からなるオリゴヌクレオチド、
c.614の変異を検出するための配列番号:21~24の各々の配列からなるオリゴヌクレオチド、
c.499の変異を検出するための配列番号:25~28の各々の配列からなるオリゴヌクレオチド、
c.298の変異を検出するための配列番号:29~32の各々の配列からなるオリゴヌクレオチド、および
c.809の変異を検出するための配列番号:33~36の各々の配列からなるオリゴヌクレオチド。
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SG11202100889XA (en) | 2021-03-30 |
AU2019315114A1 (en) | 2021-02-25 |
US20220195527A1 (en) | 2022-06-23 |
JP2020019738A (ja) | 2020-02-06 |
KR20210038607A (ko) | 2021-04-07 |
CA3108248A1 (en) | 2020-02-06 |
JP6544783B1 (ja) | 2019-07-17 |
CN112639134A (zh) | 2021-04-09 |
EP3831962A4 (en) | 2022-04-20 |
EP3831962A1 (en) | 2021-06-09 |
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