Method for assistance in predicting risk of side effects in chemotherapy for pancreatic cancer

Abstract

To provide simple and efficient means for predicting the risk of side effects in chemotherapy for pancreatic cancer.SOLUTION: The single nucleotide polymorphism of any of (a) to (c): (a) a single nucleotide polymorphism identified by rs1980576 in APCDD1 L gene or a single nucleotide polymorphism in linkage disequilibrium or genetic linkage with the single nucleotide polymorphism; (b) a single nucleotide polymorphism identified by rs2272761 in R3HCC1 gene or a single nucleotide polymorphism in linkage disequilibrium or genetic linkage with the single nucleotide polymorphism; and (c) a single nucleotide polymorphism identified by rs9425343 in EDEM3 gene or a single nucleotide polymorphism in linkage disequilibrium or genetic linkage with the single nucleotide polymorphism, which are present on the genomic DNA in a biological sample taken from a subject, is analyzed, the genotype for the single nucleotide polymorphism is determined, and based on the determined genotype, the prediction of the risk of side effects when chemotherapy for pancreatic cancer is performed is assisted.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for assisting in predicting the risk of side effects when chemotherapy is administered to pancreatic cancer. [Background technology]

[0002] Pancreatic cancer has an extremely poor prognosis. Current standard chemotherapy regimens for pancreatic cancer are mFOLFIRINOX (5-fluorouracil + leucovorin + irinotecan + oxaliplatin) or GEM / nab-PTX (gemcitabine + nab-paclitaxel). While FOLFIRINOX is considered to have a superior response rate, the frequency of side effects is such that even modified FOLFIRINOX (mFOLFIRINOX), a regimen with modified dosage, is only used in approximately 10% of cases in Japan. Predicting the side effects of mFOLFIRINOX therapy before treatment could potentially improve pancreatic cancer prognosis by providing appropriate anticancer drug treatment tailored to each individual patient, known as personalized medicine.

[0003] Some forms of personalized medicine have already been put into practical use. For example, when administering a specific anticancer drug to treat gastrointestinal cancers, genetic testing can predict the side effects and effectiveness of the drug, helping to determine treatment strategies. A correlation has been found between irinotecan's side effects and polymorphisms in the UGT1A1 gene. The drug's package insert also notes that 4 / 5 (80%) patients with UGT1A1 polymorphisms (*6 homozygote, *28 homozygote, and compound heterozygote) experienced neutropenia (G3 or higher), while the odds ratio for other UGT1A1 polymorphisms (10 / 50 (20%)) was 15. Therefore, caution is advised when using this drug. However, side effects from irinotecan are still observed even in patients who are not considered high-risk based on UGT1A1 polymorphisms (*28, *6). The inventors have addressed this issue and disclosed a method for analyzing specific single nucleotide polymorphisms in the regions encoding the APCDD1L gene, R3HCC1 gene, MKKS gene, EDEM3 gene, or ACOX1 gene present on genomic DNA, determining whether the variant type is homozygous, the reference type is homozygous, or the reference type is heterozygous, thereby assisting in predicting the risk of side effects caused by irinotecan (see Patent Document 1).

[0004] Since mFOLFIRINOX therapy for pancreatic cancer contains the aforementioned irinotecan, it is recommended that the dosage be determined according to UGT1A1 gene polymorphisms. However, the frequency of side effects remains high, and in contrast to its active use in Europe and the United States, the use of mFOLFIRINOX therapy is currently limited in Japan. Furthermore, to date, the only method in practical use to predict side effects in mFOLFIRINOX therapy for pancreatic cancer is the measurement of UGT1A1 gene polymorphisms, but side effects are currently observed even in patients who are not considered high-risk.

[0005] Furthermore, since there are racial differences in the frequency of genetic polymorphisms, such as UGT1A1*28 being common in Europe and the United States but low in Japan, and conversely, *6 being common in Europe and the United States but high in Japan, analysis using cases in Japan is necessary in addition to reports from Europe and the United States. In fact, it is known that the genetic polymorphism in R3HCC1, which is the subject of the present invention, is biased toward Asians.

[0006] In this context, there is a need to improve the safety of mFOLFIRINOX therapy for pancreatic cancer, which is said to have a high response rate, by making it possible to predict the side effects of this therapy before treatment, thereby improving the prognosis of pancreatic cancer. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2016 / 132736 Brochure Summary of the Invention [Problem to be solved by the invention]

[0008] In chemotherapy for pancreatic cancer, there is a need for personalized medicine, which enables prediction of the risk of side effects in individual patients and provides treatment with anticancer drugs appropriate for each cancer patient. Therefore, an object of the present invention is to provide a simple and efficient means for predicting the risk of side effects in chemotherapy for pancreatic cancer by analyzing single nucleotide polymorphisms in a predetermined region of a base sequence encoding a specific gene. [Means for solving the problem]

[0009] As a result of intensive research to solve the above-mentioned problems, the inventors have found that single nucleotide polymorphisms in the regions encoding the APCDD1L gene, the R3HCC1 gene, or the EDEM3 gene are factors that assist in predicting the risk of side effects from mFOLFIRINOX therapy, and have completed the present invention.

[0010] That is, the present invention is as follows. [1] Present on genomic DNA in a biological sample collected from a subject, (a) a single nucleotide polymorphism identified by rs1980576 in the APCDD1L gene or a single nucleotide polymorphism in linkage disequilibrium or genetic linkage with the single nucleotide polymorphism; (b) a single nucleotide polymorphism identified by rs2272761 in the R3HCC1 gene or a single nucleotide polymorphism in linkage disequilibrium or genetic linkage with the single nucleotide polymorphism; (c) a single nucleotide polymorphism identified by rs9425343 in the EDEM3 gene or a single nucleotide polymorphism in linkage disequilibrium or genetic linkage with the single nucleotide polymorphism; A method for assisting in predicting the risk of side effects when mFOLFIRINOX therapy, FOLFIRINOX therapy, or modified regimens thereof are administered to pancreatic cancer, by analyzing any one of the single nucleotide polymorphisms (a) to (c) above, determining the genotype at the single nucleotide polymorphism, and based on the determined genotype. [2] The single nucleotide polymorphism identified by rs1980576 in the APCDD1L gene is (a') a single nucleotide polymorphism in which the reference type is adenine and the variant type is guanine at base 186 of the base sequence encoding the APCDD1L gene set forth in SEQ ID NO: 1; The single nucleotide polymorphism identified by rs2272761 in the R3HCC1 gene is (b') a single nucleotide polymorphism in which the reference type is guanine and the variant type is adenine at base 919 of the base sequence encoding the R3HCC1 gene set forth in SEQ ID NO: 2; The single nucleotide polymorphism identified by rs9425343 in the EDEM3 gene is (c') a single nucleotide polymorphism in which the reference type is thymine and the variant type is guanine at base 2459 of the base sequence encoding the EDEM3 gene set forth in SEQ ID NO: 3; The method according to [1] above, characterized in that [3] The method according to [1] or [2] above, wherein the method assists in predicting a high risk of side effects when mFOLFIRINOX therapy, FOLFIRINOX therapy, or a modified regimen thereof is administered to pancreatic cancer when the patient has a heterozygous or variant form in the single nucleotide polymorphism of (a) or (a'), a homozygous variant form in the single nucleotide polymorphism of (b) or (b'), or a homozygous variant form in the single nucleotide polymorphism of (c) or (c'), and assists in predicting a low risk of side effects when mFOLFIRINOX therapy, FOLFIRINOX therapy, or a modified regimen thereof is administered to pancreatic cancer when the patient has a homozygous reference form in the single nucleotide polymorphism of (a) or (a'), a homozygous reference form in the single nucleotide polymorphism of (b) or (b'), or a homozygous reference form in the single nucleotide polymorphism of (c) or (c'). [4] The method according to any one of [1] to [3] above, wherein the side effect is neutropenia. [5] A probe set for use in assisting in predicting the risk of side effects when mFOLFIRINOX therapy, FOLFIRINOX therapy, or modified regimens thereof are administered to pancreatic cancer, comprising an oligonucleotide that hybridizes under stringent conditions to a contiguous region of 5 to 50 bases containing any of the single nucleotide polymorphisms (a) to (c): (a) a single nucleotide polymorphism identified by rs1980576 in the APCDD1L gene or a single nucleotide polymorphism in linkage disequilibrium or genetic linkage with the single nucleotide polymorphism; (b) a single nucleotide polymorphism identified by rs2272761 in the R3HCC1 gene or a single nucleotide polymorphism in linkage disequilibrium or genetic linkage with the single nucleotide polymorphism; or (c) a single nucleotide polymorphism identified by rs9425343 in the EDEM3 gene or a single nucleotide polymorphism in linkage disequilibrium or genetic linkage with the single nucleotide polymorphism. [6] The single nucleotide polymorphism identified by rs1980576 in the APCDD1L gene is (a') a single nucleotide polymorphism in which the reference type is adenine and the variant type is guanine at base 186 of the base sequence encoding the APCDD1L gene set forth in SEQ ID NO: 1; The single nucleotide polymorphism identified by rs2272761 in the R3HCC1 gene is (b') a single nucleotide polymorphism in which the reference type is guanine and the variant type is adenine at base 919 of the base sequence encoding the R3HCC1 gene set forth in SEQ ID NO: 2; The single nucleotide polymorphism identified by rs9425343 in the EDEM3 gene is (c') a single nucleotide polymorphism in which the reference type is thymine and the variant type is guanine at base 2459 of the base sequence encoding the EDEM3 gene set forth in SEQ ID NO: 3; The probe set according to [5] above, [7] A reference probe corresponding to the reference type in (a) or (a') and a variant probe corresponding to a variant type at the single nucleotide polymorphism; a reference probe corresponding to the reference type in (b) or (b') and a variant probe corresponding to a variant type at the single nucleotide polymorphism; or a reference probe corresponding to the reference type in (c) or (c') and a variant probe corresponding to a variant type at the single nucleotide polymorphism; The probe set according to [5] or [6] above, [Effects of the Invention]

[0011] According to the present invention, it is possible to assist in predicting the risk of side effects in mFOLFIRINOX therapy, FOLFIRINOX therapy, or modified regimens thereof for pancreatic cancer. By predicting side effects in individual patients using such a method, it is possible to provide pancreatic cancer patients with the opportunity to select a treatment with a higher efficacy rate and to perform treatment with an anticancer drug appropriate for each pancreatic cancer patient, i.e., so-called personalized medicine. DETAILED DESCRIPTION OF THE INVENTION

[0012] (Method for assisting in predicting the risk of side effects) The method for assisting in prediction of the risk of side effects when mFOLFIRINOX therapy, FOLFIRINOX therapy, or modified regimens thereof are administered to pancreatic cancer herein includes: present on genomic DNA in a biological sample collected from a subject, (a) a single nucleotide polymorphism identified by rs1980576 in the APCDD1L gene or a single nucleotide polymorphism in linkage disequilibrium or genetic linkage with the single nucleotide polymorphism; (b) a single nucleotide polymorphism identified by rs2272761 in the R3HCC1 gene or a single nucleotide polymorphism in linkage disequilibrium or genetic linkage with the single nucleotide polymorphism; (c) a single nucleotide polymorphism identified by rs9425343 in the EDEM3 gene or a single nucleotide polymorphism in linkage disequilibrium or genetic linkage with the single nucleotide polymorphism; The present invention is a method for assisting in the prediction of the risk of side effects when mFOLFIRINOX therapy, FOLFIRINOX therapy, or modified regimens thereof are administered to pancreatic cancer, by analyzing any one of the single nucleotide polymorphisms (a) to (c) in the above, determining the genotype at the single nucleotide polymorphism, and, based on the determined genotype, is also referred to as the "method for assisting in the prediction of the risk of side effects."

[0013] The FOLFIRINOX therapy is a chemotherapy in which three types of anticancer drugs, 5-fluorouracil (5-FU), irinotecan hydrochloride hydrate (CPT-11), and oxaliplatin (L-OHP), are administered in combination with l-leucovorin (levofolinate calcium: l-LV), a 5-FU enhancer, and is one of the standard treatments for pancreatic cancer. One embodiment of the FOLFIRINOX therapy is as follows: (1) 85 mg / m 2 (body surface area) of oxaliplatin administered intravenously for 2 hours (2) Then 200 mg / m 2(body surface area) of leucovorin administered intravenously for 2 hours and 30 minutes after the leucovorin infusion at 180 mg / m 2 (body surface area) of irinotecan administered intravenously for 90 minutes (3) Next, 400 mg / m 2 (body surface area) of 5-FU intravenously, followed by 2400 mg / m 2 (body surface area) of 5-FU administered intravenously for 46 hours One example of such a treatment is a cycle of (1) to (3) above, which is repeated every two weeks.

[0014] In addition, as an embodiment of the mFOLFIRINOX therapy, the dose of irinotecan in (2) above is 150 mg / m 2 (body surface area) and 400 mg / m 2 An example of such a treatment is one in which the following (1) to (3') are considered as one cycle, excluding intravenous injection of 5-FU (body surface area), and this cycle is repeated every two weeks. (1) 85 mg / m 2 (body surface area) of oxaliplatin administered intravenously for 2 hours (2') Then 200 mg / m 2 (body surface area) of leucovorin administered intravenously for 2 hours and 30 minutes after the leucovorin infusion at 150 mg / m 2 (body surface area) of irinotecan administered intravenously for 90 minutes (3') Then 2400 mg / m 2 (body surface area) of 5-FU administered intravenously for 46 hours

[0015] As a modified regimen of the above-mentioned FOLFIRINOX therapy or mFOLFIRINOX therapy, the dose of oxaliplatin is set to 50 to 85, 65, or 50 mg / m 2 (body surface area), and the dose of irinotecan may be reduced to 90-150, 90, or 120 mg / m 2 The dose of 5-FU may be reduced to 1200-2400, 1200, or 1800 mg / m (body surface area). 2 (body surface area) may be reduced.

[0016] The dosage of each drug in the above FOLFIRINOX therapy, mFOLFIRINOX therapy, and their modified regimens can be adjusted by referring to "Points to Note When Using Combination Chemotherapy (FOLFIRINOX) for Unresectable Pancreatic Cancer," Notification No. 1220-7 from the Evaluation and Licensing Division, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare, dated December 20, 2013: URL www.mhlw.go.jp / web / t_doc?dataId=00tb9751&dataType=1&pageNo=1, and the paper by Ozaki et al. (Cancer Chemotherapy and Pharmacology, volume 81, pages 1017-1023 (2018)).

[0017] The biological sample collected from the subject is not particularly limited as long as it contains the subject's genomic DNA, and examples thereof include blood collected from the subject and blood-related samples derived therefrom (blood, serum, plasma, etc.), body fluids such as lymph, sweat, tears, saliva, urine, feces, ascites, and cerebrospinal fluid, as well as crushed and extracted cells, tissues, or organs, with blood-related samples being preferred.

[0018] The extraction means for extracting genomic DNA from a biological sample collected from a subject is not particularly limited, and is preferably a means that can directly separate, purify, and recover DNA components from the biological sample.

[0019] The single nucleotide polymorphism identified by rs1980576 in the (a) APCDD1L gene is a genetic polymorphism at the genomic position indicated by Chr.20:58470611 on Build GRCh38, and is a single nucleotide polymorphism at the 417th base of NCBI accession number NM_153360.2 (updated May 4, 2019). Specifically, (a') a single nucleotide polymorphism at the 186th base of the base sequence encoding the APCDD1L gene set forth in SEQ ID NO: 1, in which the reference type is adenine and the variant type is guanine, is preferred. Note that the "rs" in rs1980576 above is the reference number in the NCBI SNP database (www.ncbi.nlm.nih.gov / SNP / ). Hereinafter, in the present invention, specific single nucleotide polymorphisms may be referred to by the rs number in the NCBI SNP database.

[0020] The single nucleotide polymorphism specified by rs2272761 in the (b) R3HCC1 gene is a genetic polymorphism at the genomic position indicated as Chr.8:23291427 on Build GRCh38, and is preferably a single nucleotide polymorphism at the 1003rd base of NCBI accession number NM_001136108.2 (updated October 21, 2018), specifically, (b') a single nucleotide polymorphism in which the reference type is guanine and the variant type is adenine at the 919th base of the base sequence encoding the R3HCC1 gene set forth in SEQ ID NO: 2.

[0021] The above (C) single nucleotide polymorphism identified by rs9425343 in the EDEM3 gene is preferably a genetic polymorphism at a genomic position indicated as Chr.1:184694403 on Build GRCh38, which is a single nucleotide polymorphism at the 2720th base of NCBI accession number NM_025191.3 (updated June 23, 2018), or (c') a single nucleotide polymorphism at the 2459th base of the base sequence encoding the EDEM3 gene set forth in SEQ ID NO: 3, in which the reference type is thymine and the variant type is guanine.

[0022] As used herein, "linkage disequilibrium" refers to a population genetic phenomenon in which a non-random correlation is observed between alleles or genetic markers (polymorphisms) at multiple loci in a population of organisms, i.e., the frequency of a specific combination (haplotype) of these alleles is significantly higher, and "genetic linkage" refers to a genetic phenomenon in which a specific combination of alleles is inherited from parent to child without following Mendel's law of assortment.

[0023] In the present specification, side effects are not particularly limited, but include neutropenia, leukopenia, diarrhea, vomiting, general fatigue, loss of appetite, hair loss, etc., with neutropenia being preferred.

[0024] As used herein, methods for analyzing single nucleotide polymorphisms can be any known method for analyzing single nucleotide polymorphisms, including real-time PCR, direct sequencing, TaqMan® PCR, Invader®, Luminex®, quenching primer / probe (QP), MALDI-TOF, and molecular beacon methods. Specifically, examples include a method in which a nucleic acid fragment containing a single nucleotide polymorphism site to be measured is amplified by PCR using primers via an amplification reaction using genomic DNA from a biological sample collected from a subject (usually a human subject) as a template, and hybridization between the resulting nucleic acid fragment and a pair of probes corresponding to a reference type and a variant type is detected; and a method in which a probe or primer specific to the single nucleotide polymorphism site is used in the PCR amplification process to detect a reference type and a variant type.

[0025] (probe set) As used herein, the probe set used to assist in predicting the risk of side effects when mFOLFIRINOX therapy, FOLFIRINOX therapy, or modified regimens thereof are administered to pancreatic cancer includes: (a) a single nucleotide polymorphism identified by rs1980576 in the APCDD1L gene or a single nucleotide polymorphism in linkage disequilibrium or genetic linkage with the single nucleotide polymorphism; (b) a single nucleotide polymorphism identified by rs2272761 in the R3HCC1 gene or a single nucleotide polymorphism in linkage disequilibrium or genetic linkage with the single nucleotide polymorphism; or (c) a single nucleotide polymorphism identified by rs2272761 in the E The probe set may be used to assist in predicting the risk of side effects when mFOLFIRINOX therapy, FOLFIRINOX therapy, or modified regimens thereof are administered to pancreatic cancer, and may include an oligonucleotide that hybridizes under stringent conditions to a region of 5 to 50 consecutive bases that includes a single nucleotide polymorphism identified by rs9425343 in the DEM3 gene or any one of single nucleotide polymorphisms (a) to (c) that is in linkage disequilibrium or genetic linkage with the single nucleotide polymorphism, and is hereinafter also referred to as "the probe set in question."

[0026] The probe used in the present probe set can be a probe consisting of an oligonucleotide that hybridizes under stringent conditions with a sequence of 5 to 50 consecutive bases, preferably 10 to 40 bases, and more preferably 15 to 30 bases, including the single nucleotide polymorphism site to be analyzed. Furthermore, by using an oligoprobe synthesized using an artificial nucleic acid such as locked nucleic acid (LNA) as the probe, it is possible to obtain a probe that specifically hybridizes even with a short base.

[0027] Stringent conditions refer to conditions under which so-called specific hybrids are formed but nonspecific hybrids are not formed. Specific examples include conditions under which hybrids are formed at 45°C in a solution containing 6×SSC (10×SSC is a solution containing 1.5 M NaCl and 0.15 M trisodium citrate) and 50% formamide, followed by washing with 2×SSC at 50°C (Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6), and conditions under which hybrids are formed at 54°C in a solution containing 3×SSC / 0.3×SDS, followed by sequential washing with wash solution A (10×SSC / 1% SDS solution), wash solution B (20×SSC), and wash solution C (5×SSC) (see JP 2011-250726 A).

[0028] The probe may be immobilized on a carrier, such as a planar substrate or a bead-like spherical carrier, specifically the carrier described in Japanese Patent Application Laid-Open No. 2011-250726. The probe for detecting the reference type and the probe for detecting the variant type may be immobilized on the same carrier or on different carriers.

[0029] The primer used when analyzing a single nucleotide polymorphism using the above-mentioned probe may be a primer consisting of an oligonucleotide that can amplify at least 5 consecutive bases including the single nucleotide polymorphism site to be analyzed as a nucleic acid fragment using genomic DNA as a template, and examples of such primers include a primer consisting of an oligonucleotide that can amplify at least 5 consecutive bases, preferably 10 to 500 bases, more preferably 20 to 200 bases, and even more preferably 50 to 100 bases, including the single nucleotide polymorphism site specified by rs1980576 in the APCDD1L gene, the single nucleotide polymorphism site specified by rs2272761 in the R3HCC1 gene, or the single nucleotide polymorphism site specified by rs9425343 in the EDEM3 gene.

[0030] Furthermore, when amplifying at least five consecutive bases including the single nucleotide polymorphism site to be analyzed, the amplified sequence can be identified by using pre-labeled primers or by using labeled nucleotides as substrates in the amplification reaction. The labeling substance is not particularly limited, but examples thereof include radioisotopes, fluorescent dyes, and organic compounds such as digoxigenin (DIG) and biotin.

[0031] The probe or primer can be obtained by chemical synthesis using a nucleic acid synthesizer, such as a DNA synthesizer or a fully automated nucleic acid synthesizer.

[0032] When the amplified nucleic acid fragments have a label, the nucleic acid fragments hybridized to each probe can be measured by detecting the label. For example, when a fluorescent dye is used as a label, the nucleic acid fragments hybridized to the probe can be measured by measuring the fluorescence intensity derived from the fluorescent dye. Specifically, the ratio of the nucleic acid fragments hybridized to the probe detecting the reference type to the nucleic acid fragments hybridized to the probe detecting the variant type can be calculated from the output value when the label in the probe detecting the reference type is detected and the output value when the label in the probe detecting the variant type is detected.

[0033] More specifically, the determination value can be calculated by dividing the intensity value derived from the nucleic acid fragment hybridized to the probe corresponding to the variant type by the average of the intensity value derived from the nucleic acid fragment hybridized to the probe corresponding to the variant type and the intensity value derived from the nucleic acid fragment hybridized to the probe corresponding to the reference type. This determination value approximates the normalized value of the abundance of the variant type contained in the nucleic acid fragment. Therefore, based on the magnitude of this determination value, it is possible to analyze single nucleotide polymorphisms in a subject and determine whether the subject has a homozygous variant type, a homozygous reference type, or a heterozygous reference type.

[0034] When using this judgment value, it is preferable to set two thresholds (threshold A and threshold B) in advance to analyze a single nucleotide polymorphism in a subject and determine whether the subject has a variant type as a homozygote, a reference type as a homozygote, or a heterozygote. Here, threshold A and threshold B have a relationship of (threshold A>threshold B). That is, if the judgment value calculated as described above exceeds threshold A, it is determined that the subject has a variant type as a homozygote; if the judgment value is equal to or less than threshold A and exceeds threshold B, it is determined that the subject has a heterozygote; and if the judgment value is equal to or less than threshold B, it is determined that the subject has a reference type as a homozygote.

[0035] These thresholds A and B are set for the single nucleotide polymorphisms described above. The method for setting thresholds A and B is not particularly limited, but includes calculating the determination value as described above using a sample whose genotype has been determined in advance, and calculating the probability density as a normal distribution for each of the cases where the variant type is homozygous, where the reference type is homozygous, and where the variant type is heterozygous. At this time, the intersection point where the probability densities overlap (the position where the probability densities change between their respective maximum values) is determined, and the average value is calculated for each of the cases where the variant type is homozygous, where the reference type is homozygous, and where the variant type is heterozygous. The threshold for cases where the variant type is homozygous and where the variant type is heterozygous can be calculated as the average value of (the average value when the variant type is homozygous and the average value when the variant type is heterozygous) and the average value of the intersection point. Similarly, the threshold for cases where the variant type is heterozygous and where the reference type is homozygous can be calculated as the average value of (the average value when the variant type is heterozygous and the average value when the reference type is homozygous) and the average value of the intersection point.

[0036] By using the above method to analyze a single nucleotide polymorphism specified by rs1980576 in the APCDD1L gene, a single nucleotide polymorphism specified by rs2272761 in the R3HCC1 gene, or a single nucleotide polymorphism specified by rs9425343 in the EDEM3 gene, or a single nucleotide polymorphism that is in linkage disequilibrium or genetic linkage with the single nucleotide polymorphism, and determining whether the variant type is homozygous, or the reference type is homozygous or heterozygous, it is possible to assist in predicting the risk of side effects when mFOLFIRINOX therapy, FOLFIRINOX therapy, or modified regimens thereof are administered to pancreatic cancer. For example, a single nucleotide polymorphism identified by rs1980576 in the APCDD1L gene, a single nucleotide polymorphism identified by rs2272761 in the R3HCC1 gene, or a single nucleotide polymorphism identified by rs9425343 in the EDEM3 gene, or the relationship between the single nucleotide polymorphism and a single nucleotide polymorphism in linkage disequilibrium or genetic linkage, is examined in patients who experienced and did not experience side effects when mFOLFIRINOX therapy, FOLFIRINOX therapy, or modified regimens thereof were previously administered to pancreatic cancer. Next, by examining the single nucleotide polymorphisms of the target patient and comparing them with the data of the patient previously examined, it is possible to assist in predicting the risk of side effects when the target patient is administered mFOLFIRINOX therapy, FOLFIRINOX therapy, or modified regimens thereof to treat pancreatic cancer.

[0037] Specifically, when the single nucleotide polymorphism of (a) or (a') has a heterozygous or variant type as a homozygous form, when the single nucleotide polymorphism of (b) or (b') has a variant type as a homozygous form, or when the single nucleotide polymorphism of (c) or (c') has a variant type as a homozygous form, it assists in predicting that there is a high risk of side effects when mFOLFIRINOX therapy, FOLFIRINOX therapy, or a modified regimen thereof is performed for pancreatic cancer; and when the single nucleotide polymorphism of (a) or (a') has a homozygous reference type, when the single nucleotide polymorphism of (b) or (b') has a heterozygous or reference type as a homozygous form, or when the single nucleotide polymorphism of (c) or (c') has a homozygous reference type, it can assist in predicting that there is a low risk of side effects when mFOLFIRINOX therapy, FOLFIRINOX therapy, or a modified regimen thereof is performed for pancreatic cancer.

[0038] Furthermore, a combination of two or more, or three or more of the above single nucleotide polymorphisms (a) to (c), or a combination of two or more, or three or more of the above single nucleotide polymorphisms (a') to (c'), may assist in predicting a low risk of side effects when mFOLFIRINOX therapy, FOLFIRINOX therapy, or a modified regimen thereof is administered to pancreatic cancer.

[0039] The probe set of the present invention is not particularly limited as long as it contains the above-mentioned probe, but may also contain the above-mentioned primer, a buffer solution for analyzing single nucleotide polymorphisms, reagents such as enzymes, and instructions for assisting in predicting that there is a low risk of side effects when mFOLFIRINOX therapy, FOLFIRINOX therapy, or modified regimens thereof are performed for pancreatic cancer.

[0040] Other embodiments of the present invention include the following. 1. An application of a reagent for analyzing the genotype of a single nucleotide polymorphism present on genomic DNA in a biological sample collected from a subject in the manufacture of a probe set for use in assisting in predicting the risk of side effects when mFOLFIRINOX therapy, FOLFIRINOX therapy, or a modified regimen thereof is administered to pancreatic cancer, wherein the single nucleotide polymorphism is any of the single nucleotide polymorphism of (a) or (a'); the single nucleotide polymorphism of (b) or (b'); or the single nucleotide polymorphism of (c) or (c'); and wherein the single nucleotide polymorphism of (a) or (a') has a heterozygous or variant form as a homozygous form, the single nucleotide polymorphism of (b) or (b') has a variant form as a homozygous form, or the single nucleotide polymorphism of (c) or (c') and assists in predicting that the risk of side effects will be low when mFOLFIRINOX therapy, FOLFIRINOX therapy, or a modified regimen thereof is administered to pancreatic cancer when the patient has a homozygous reference type in the single nucleotide polymorphism of (a) or (a'), a heterozygous or homozygous reference type in the single nucleotide polymorphism of (b) or (b'), or a homozygous reference type in the single nucleotide polymorphism of (c) or (c'). [Example]

[0041] (Analysis target) Genomic DNA was prepared from peripheral blood samples from 30 patients with pancreatic cancer who underwent mFOLFIRINOX therapy (excluding patients at high risk of irinotecan side effects due to UGT1A1 gene polymorphisms, i.e., *6 homozygotes, *28 homozygotes, and compound heterozygotes). All patients were Japanese. (Preparation of genomic DNA) Genomic DNA was prepared from peripheral blood collected from subjects in EDTA-containing tubes using the sodium iodide method (Wang et al., Nucleic Acids Res 34:195-201(2014)). The prepared DNA was dissolved in 10 mM Tris-HCl buffer (pH 8.0) containing 1 mM EDTA·2Na and stored at 4°C or -20°C until use.

[0042] The single nucleotide polymorphism rs1980576 in the region encoding the APCDD1L gene, the single nucleotide polymorphism rs2272761 in the region encoding the R3HCC1 gene, and the single nucleotide polymorphism rs9425343 in the region encoding the EDEM3 gene were verified using the TaqMan® probe method in the clinical samples analyzed above. Genotyping was performed on 10 ng of genomic DNA using TaqMan SNP Assays_Human (Applied Biosystems), LightCycler® 480 Probe Master (Roche Diagnostics), Universal Probe Library (Roche Diagnostics), and LightCycler480 System II (Roche Diagnostics). After incubation at 95°C for 10 minutes, 55 cycles of PCR were performed (92°C for 15 seconds and 60°C for 60 seconds per cycle), and the fluorescence of the PCR product was measured.

[0043] (result) The results of a statistical analysis of the frequency of each gene polymorphism and the frequency of side effects (neutropenia of grade 3 or higher) in patients receiving mFOLFIRINOX therapy for pancreatic cancer are shown in Table 1. In Table 1, the CA trend test is the result of the Cochran-Armitage trend test, and the Fisher's exact test is the result of Fisher's exact test. In Table 1, the odds ratio is calculated using Fisher's exact test.

[0044] [Table 1]

[0045] For the single nucleotide polymorphism rs1980576 in the APCDD1L gene coding region, the CA trend test yielded a P value of 0.035, and the Fisher's exact test yielded an odds ratio (A / A vs. A / G, G / G) of 6.52 with a P value of 0.046. Therefore, a significant linear trend (correlation) and frequency difference were observed between the single nucleotide polymorphism rs1980576 and the incidence of side effects associated with mFOLFIRINOX therapy for pancreatic cancer. Furthermore, it was found that the risk of side effects in pancreatic cancer mFOLFIRINOX cases was predicted to be low in cases of reference homozygotes (A / A), and high in cases of heterozygotes (A / G) or variant homozygotes.

[0046] For the single nucleotide polymorphism rs2272761 in the R3HCC1 gene coding region, the CA trend test yielded a P value of 0.013, and the Fisher's exact test yielded an odds ratio (G / G vs G / A, A / A) of 9.81 with a P value of 0.009. Therefore, a significant linear trend and difference in frequency was observed between the single nucleotide polymorphism rs2272761 and the incidence of side effects associated with mFOLFIRINOX therapy for pancreatic cancer. Furthermore, it was found that the risk of side effects in pancreatic cancer mFOLFIRINOX patients was predicted to be low in cases with a reference homozygous (G / G) or heterozygous (A / G), and high in cases with a variant homozygous.

[0047] In the FOLFIRI therapy described in Patent Document 1, the cutoff for the single nucleotide polymorphism rs2272761 in the region encoding the R3HCC1 gene was a reference homozygous (G / G) cutoff, indicating a low risk of side effects, i.e., it could be used for exclusion diagnosis. Furthermore, the frequency of side effects was not high enough to indicate a high risk of side effects in the heterozygous (G / A) and variant homozygous (A / A) cutoffs. However, when assisting in the prediction of the risk of side effects in pancreatic cancer mFOLFIRINOX cases, it was revealed that the cutoff for the variant homozygous (A / A) cutoff indicates a high risk of side effects, i.e., it could be used for definitive diagnosis.

[0048] For the single nucleotide polymorphism rs9425343 in the EDEM3 gene coding region, the CA trend test showed a P value of 0.001, and the Fisher's exact test showed an inf. odds ratio (T / T vs. T / G, G / G) of 0.005. Therefore, a highly significant linear trend and frequency difference was observed between the single nucleotide polymorphism rs9425343 and the incidence of side effects associated with mFOLFIRINOX therapy for pancreatic cancer. In other words, it was found that the risk of side effects in pancreatic cancer mFOLFIRINOX cases can be predicted by predicting that the reference homozygous variant (T / T) has a low risk of side effects, while the variant homozygous variant (G / G) has a high risk of side effects.

[0049] In the FOLFIRI therapy described in Patent Document 1, the risk of side effects was high in the case of reference homozygotes (T / T) and low in the case of variant homozygotes (G / G). However, in the case of the mFOLFIRINOX therapy, the opposite result was surprisingly obtained, that the risk of side effects was low in the case of reference homozygotes (T / T).

[0050] [Reference example] In the above Examples, three types of single nucleotide polymorphisms and the prediction of the risk of side effects in mFOLFIRINOX therapy were investigated. The same three types of single nucleotide polymorphisms and the prediction of the risk of side effects in Gem / nab-PTX therapy for pancreatic cancer treatment were also investigated.

[0051] The relationship between three types of single nucleotide polymorphisms (rs1980576, rs2272761, rs9425343) and side effects (neutropenia) was investigated in the same manner as in the above examples, except that the analysis subjects were 41 cases that underwent Gem / nab-PTX therapy. The results are shown in Table 2.

[0052] [Table 2]

[0053] As is clear from Table 2, in Gem / nab-PTX therapy, no significant correlation was observed between any of the genetic polymorphisms and the occurrence of side effects. [Industrial Applicability]

[0054] INDUSTRIAL APPLICABILITY The present invention can be used in the medical field because it can assist in predicting the risk of side effects when mFOLFIRINOX therapy, FOLFIRINOX therapy, or modified regimens thereof are administered to pancreatic cancer.

Claims

1. present on genomic DNA in a biological sample collected from a subject, (b) A method for analyzing a single nucleotide polymorphism identified by rs2272761 in the R3HCC1 gene, determining the genotype at the single nucleotide polymorphism, and, based on the determined genotype, assisting in predicting the risk of side effects when mFOLFIRINOX therapy, FOLFIRINOX therapy, or a modified regimen thereof is administered to pancreatic cancer.

2. The single nucleotide polymorphism identified by rs2272761 in the R3HCC1 gene is (b') The method according to claim 1, characterized in that the reference type is guanine and the variant type is adenine at the 919th base of the base sequence encoding the R3HCC1 gene set forth in SEQ ID NO:

2.

3. The method according to claim 1 or 2, characterized in that when a variant type in the single nucleotide polymorphism of (b) or (b') is homozygous, it assists in predicting that there is a high risk of side effects when mFOLFIRINOX therapy, FOLFIRINOX therapy, or a modified regimen thereof is performed for pancreatic cancer, and when a heterozygous or reference type in the single nucleotide polymorphism of (b) or (b') is homozygous, it assists in predicting that there is a low risk of side effects when mFOLFIRINOX therapy, FOLFIRINOX therapy, or a modified regimen thereof is performed for pancreatic cancer.

4. The method according to any one of claims 1 to 3, wherein the side effect is neutropenia.

5. (b) a probe set for use in the method according to any one of claims 1 to 4, comprising an oligonucleotide that hybridizes under stringent conditions to a region of 10 to 50 consecutive bases containing a single nucleotide polymorphism identified by rs2272761 in the R3HCC1 gene.

6. The single nucleotide polymorphism identified by rs2272761 in the R3HCC1 gene is (b') The probe set according to claim 5, characterized in that the 919th base of the base sequence encoding the R3HCC1 gene described in SEQ ID NO: 2 is a single nucleotide polymorphism in which the reference type is guanine and the variant type is adenine.

7. a reference probe corresponding to the reference type in (b) or (b') and a variant probe corresponding to the variant type in the single nucleotide polymorphism; The probe set according to claim 5 or 6.

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