JP2022187310A - Method for assistance in predicting risk of side effects in chemotherapy for pancreatic cancer - Google Patents

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 The present invention relates to a method for assisting prediction of the risk of developing side effects in the case of chemotherapy for pancreatic cancer.

Pancreatic cancer is a cancer type with an extremely poor prognosis. Currently, mFOLFIRINOX (5-fluorouracil + leucovorin + irinotecan + oxaliplatin) therapy or GEM/nab-PTX (gemcitabine + nab-paclitaxel) therapy is standard for pancreatic cancer chemotherapy. FOLFIRINOX therapy is said to have a better response rate, but in Japan, even modified FOLFIRINOX (hereinafter also referred to as “mFOLFIRINOX”) therapy, which is a regimen with modified doses, etc., is selected in about 10% in terms of the frequency of side effects. Stay. By making it possible to predict the side effects of mFOLFIRINOX therapy before treatment, it is desired to provide appropriate anticancer drug treatment for individual cancer patients, so-called personalized medicine, and improve the prognosis of pancreatic cancer. ing.

Part of personalized medicine has already been put into practical use. For example, in the administration of a predetermined anticancer drug for gastrointestinal cancer or the like, the presence or absence of side effects and effects are predicted by genetic testing, and are used to determine treatment strategies. Regarding the side effects of irinotecan, a correlation with polymorphisms in the UGT1A1 gene has been found, and the package insert of the drug also states that UGT1A1 gene polymorphisms (*6 homozygous, *28 homozygous, compound heterozygous) cases are 4/5 ( 80%) showed neutropenia (G3 or higher), and the odds ratio for other UGT1A1 gene polymorphism cases (10/50 (20%) of the above side effects) was 15. It is stated to However, side effects due to irinotecan are still observed even in a group of patients considered to be non-high-risk based on UGT1A1 gene polymorphisms (*28, *6). The present inventors have addressed this problem by analyzing a predetermined single nucleotide polymorphism in the region encoding the APCDD1L gene, R3HCC1 gene, MKKS gene, EDEM3 gene, or ACOX1 gene present on genomic DNA, Disclosed is a method for assisting prediction of the risk of side effects caused by irinotecan by determining whether the variant type is homozygous, the reference type is homozygous, or the heterozygous type is present (Patent Document 1) reference).

By the way, since mFOLFIRINOX therapy for pancreatic cancer contains the above-mentioned irinotecan, it is recommended to determine the dosage according to the UGT1A1 gene polymorphism, but the frequency of side effects is still high. In contrast, the selection of mFOLFIRINOX therapy in Japan is currently limited. In addition, until now, only the measurement of the UGT1A1 gene polymorphism has been put into practical use as a method of predicting in advance the side effects of mFOLFIRINOX therapy for pancreatic cancer, but even in a non-high-risk patient group, Currently, side effects are observed.

In addition, UGT1A1*28 has a high frequency in Europe and the United States, but a low frequency in Japan. Conversely, *6 has a low frequency in Europe and the United States, but a high frequency in Japan. Therefore, it is necessary to analyze not only reports from Europe and the United States but also cases from Japan. In fact, it is known that genetic polymorphisms in R3HCC1 targeted by the present invention are biased toward Asians.

Under these circumstances, there is a need to improve the safety of mFOLFIRINOX therapy and improve the prognosis of pancreatic cancer by making it possible to predict the side effects of mFOLFIRINOX therapy for pancreatic cancer, which is said to have an excellent response rate, before treatment. ing.

International Publication No. 2016/132736 pamphlet

In chemotherapy for pancreatic cancer, it is possible to predict the risk of occurrence of side effects in individual patients, and treatment with appropriate anticancer drugs for individual cancer patients, so-called personalized medicine, is required. Accordingly, an object of the present invention is to provide a simple and efficient method for predicting the risk of side effects in chemotherapy for pancreatic cancer by analyzing a single nucleotide polymorphism in a predetermined region of a nucleotide sequence encoding a specific gene. to provide a means of

As a result of intensive studies to solve the above problems, the present inventors have found that a single nucleotide polymorphism in the region encoding the APCDD1L gene, R3HCC1 gene, or EDEM3 gene is a factor that assists in predicting the risk of side effects caused by mFOLFIRINOX therapy. I found a certain thing and completed the present invention.

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;
Analyzing any single nucleotide polymorphism of (a) to (c), determining the genotype in the single nucleotide polymorphism, and based on the determined genotype, mFOLFIRINOX therapy for pancreatic cancer, FOLFIRINOX A method to help predict the risk of developing side effects when a therapy or modified regimen thereof is administered.
[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 the 186th base of the nucleotide sequence encoding the APCDD1L gene set forth in SEQ ID NO: 1;
The single nucleotide polymorphism identified at 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 the 919th base of the base sequence encoding the R3HCC1 gene set forth in SEQ ID NO: 2;
The single nucleotide polymorphism identified at 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 the 2459th base of the nucleotide sequence encoding the EDEM3 gene set forth in SEQ ID NO: 3.
The method according to [1] above, characterized in that:
[3] When the single nucleotide polymorphism of (a) or (a′) has a heterozygous or variant type as homozygous, the single nucleotide polymorphism of (b) or (b′) has a homozygous variant type As, or when the variant type is homozygous in the single nucleotide polymorphism of (c) or (c'), mFOLFIRINOX therapy for pancreatic cancer, FOLFIRINOX therapy, or those Aids prediction that the risk of occurrence of side effects is high when the modified regimen is performed, and if the single nucleotide polymorphism of (a) or (a') has the reference type as homozygous, the (b ) or (b') when the single nucleotide polymorphism has a heterozygous or reference type as homozygous, or the single nucleotide polymorphism of (c) or (c') has a homozygous reference type In some cases, the above [1] or [2], which is characterized by assisting prediction that the risk of side effects is low when mFOLFIRINOX therapy, FOLFIRINOX therapy, or a modified regimen thereof is performed for pancreatic cancer ] method described.
[4] The method according to any one of [1] to [3] above, wherein the side effect is neutropenia.
[5] (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) identified by rs2272761 in the R3HCC1 gene A single nucleotide polymorphism 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 linkage dissociation with the single nucleotide polymorphism An oligonucleotide that hybridizes under stringent conditions with a continuous region of 5 to 50 bases containing any of the single nucleotide polymorphisms (a) to (c) of the single nucleotide polymorphisms in equilibrium or in genetic linkage A probe set for use in aiding prediction of the risk of developing side effects when mFOLFIRINOX therapy, FOLFIRINOX therapy, or a modified regimen thereof is performed for pancreatic cancer, comprising:
[6] the single nucleotide polymorphism identified at 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 the 186th base of the nucleotide sequence encoding the APCDD1L gene set forth in SEQ ID NO: 1;
The single nucleotide polymorphism identified at 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 the 919th base of the base sequence encoding the R3HCC1 gene set forth in SEQ ID NO: 2;
The single nucleotide polymorphism identified at 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 the 2459th base of the nucleotide sequence encoding the EDEM3 gene set forth in SEQ ID NO: 3.
The probe set according to [5] above, characterized in that:
[7] A reference type probe corresponding to the reference type in (a) or (a′) above, a variant probe corresponding to the variant type in the single nucleotide polymorphism, the reference type in (b) or (b′) above A reference type probe corresponding to a variant type probe corresponding to a variant type in the single nucleotide polymorphism, or a reference type probe corresponding to the reference type in (c) or (c') above, and a reference type probe corresponding to the single nucleotide polymorphism a variant-type probe corresponding to the variant-type;
The probe set according to [5] or [6] above, characterized in that:

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to assist prediction of the risk of side effects in mFOLFIRINOX therapy, FOLFIRINOX therapy, or their modified regimens for pancreatic cancer. By predicting side effects in individual patients using such a method, it is possible to provide an opportunity for selecting a treatment with a higher response rate by pancreatic cancer patients, and to treat individual pancreatic cancer patients with appropriate anticancer drugs. Treatment, so-called personalized medicine, can be performed.

(Method for Assisting Prediction of Occurrence Risk of Side Effects)
The method for assisting prediction of the risk of side effects when mFOLFIRINOX therapy, FOLFIRINOX therapy, or a modified regimen thereof is performed for pancreatic cancer herein,
present on genomic DNA in a biological sample taken 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;
Analyzing any single nucleotide polymorphism of (a) to (c), determining the genotype in the single nucleotide polymorphism, and based on the determined genotype, mFOLFIRINOX therapy for pancreatic cancer, FOLFIRINOX It is a method for assisting prediction of the risk of occurrence of side effects when a therapy or a modified regimen thereof is performed, and is hereinafter also referred to as "method for assisting prediction of the risk of occurrence of side effects."

The above FOLFIRINOX therapy consists of three types of anticancer agents, 5-fluorouracil (5-FU), irinotecan hydrochloride hydrate (CPT-11), and oxaliplatin (L-OHP), and a 5-FU enhancer. This chemotherapy is administered in combination with a certain l-leucovorin (levofolinate calcium: l-LV), and is one of the standard treatments for pancreatic cancer. In one embodiment of such FOLFIRINOX therapy,
(1) 85 mg/m ² (body surface area) oxaliplatin infusion for 2 hours; (2) 200 mg/m ² (body surface area) leucovorin infusion for 2 hours; 180 mg/m ² (body surface area) irinotecan infusion 90 minutes (3) followed by 400 mg/m ² (body surface area) 5-FU intravenous injection followed by 2400 mg/m ² (body surface area) 5-FU A 46-hour cycle of FU continuous intravenous infusion (1) to (3) can be repeated every two weeks.

In addition, as an embodiment of mFOLFIRINOX therapy, the dose of irinotecan is reduced to 150 mg/m ² (body surface area) in (2) above, and 400 mg/m ² (body surface area) of 5-FU is administered in (3). The following treatments (1) to (3') excluding intravenous injection are treated as one cycle, and this cycle is repeated every two weeks.
(1) an infusion of 85 mg/m ² (body surface area) of oxaliplatin for 2 hours (2′) followed by an infusion of 200 mg/m ² (body surface area) of leucovorin for 2 hours, and an intravenous infusion of said leucovorin; 30 minutes later 150 mg/m ² (body surface area) irinotecan infusion for 90 minutes (3′) followed by 2400 mg/m ² (body surface area) 5-FU continuous intravenous infusion for 46 hours

As modified regimens of the FOLFIRINOX therapy or mFOLFIRNOX therapy, the dose of oxaliplatin may be reduced to 50-85, 65, or 50 mg/m ² body surface area, and the dose of irinotecan may be reduced to 90-150, The dose of 5-FU may be reduced to 90, or 120 mg/m ² (body surface area) and may be reduced to 1200-2400, 1200, 1800 mg/m ² (body surface area).

The dosage of each drug in the above FOLFIRINOX therapy, mFOLFIRINOX therapy and their modified regimens is described in "Considerations for the use of combination chemotherapy (FOLFIRINOX method) indicated for unresectable pancreatic cancer" December 2013 20, PFSB/ELD Notification No. 1220-7, Notification by Director of Evaluation Division, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labor and Welfare: URL www.mhlw.go.jp/web/t_doc?dataId=00tb9751&dataType=1&pageNo=1” and Ozaki et al.’s paper (Cancer Chemotherapy and Pharmacology volume 81, pages 1017-1023 (2018)).

The biological sample collected from the subject is not particularly limited as long as it contains the subject's genomic DNA, and blood collected from the subject and blood-related samples derived therefrom (blood, serum and blood plasma, etc.), body fluids such as lymph, sweat, tears, saliva, urine, feces, ascites and cerebrospinal fluid, as well as cell, tissue or organ fragmentation and extracts, and blood-related samples are preferred. be able to.

The extraction means for extracting genomic DNA from a biological sample collected from a subject is not particularly limited, and a means capable of directly separating, purifying and recovering DNA components from the biological sample is preferred.

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

The single nucleotide polymorphism identified by rs2272761 in the above (b) R3HCC1 gene is a genetic polymorphism at the position on the genome indicated as Chr.8:23291427 on Build GRCh38, NCBI accession number NM_001136108.2 (update Date October 21, 2018) single nucleotide polymorphism at the 1003rd base, specifically, (b') reference type at the 919th base of the base sequence encoding the R3HCC1 gene described in SEQ ID NO: 2 is guanine and the variant type is adenine.

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

As used herein, "linkage disequilibrium" means that in a population of organisms, non-random correlations are found between alleles or genetic markers (polymorphisms) at multiple loci, i.e., specific combinations thereof ( "Genetic linkage" refers to the population genetic phenomenon in which the frequency of a haplotype) is significantly elevated, and "genetic linkage" refers to the phenomenon in which certain allele combinations are passed together from parent to child without following Mendel's law of independence. It means an inherited genetic phenomenon.

In the present specification, side effects are not particularly limited, but include neutropenia, leukopenia, diarrhea, vomiting, general malaise, anorexia, hair loss, etc. Preferred examples include neutropenia. can.

As used herein, as a method for analyzing single nucleotide polymorphisms, known methods for analyzing single nucleotide polymorphisms can be used, such as real-time PCR, direct sequencing, TaqMan (registered trademark) PCR, Invader (registered trademark) method, Luminex (registered trademark) method, quenching primer/probe (QP) method, MALDI-TOF method, molecular beacon method, and the like. Specifically, a nucleic acid fragment containing a single nucleotide polymorphism site to be measured is amplified using primers by an amplification reaction using the genomic DNA of a biological sample collected from a subject (usually a human subject) as a template. A method of detecting hybridization between a nucleic acid fragment obtained by amplifying by PCR and a pair of probes corresponding to a reference type and a variant type, or in the above PCR amplification process, a single nucleotide polymorphism site-specific Examples include methods for detecting reference types and variant types using probes or primers.

(probe set)
In the present specification, as a probe set for use in the prediction of the risk of side effects when mFOLFIRINOX therapy, FOLFIRINOX therapy, or a modified regimen thereof is performed for pancreatic cancer, (a) in the APCDD1L gene A single nucleotide polymorphism identified by rs1980576 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 the single nucleotide polymorphism A single nucleotide polymorphism in linkage disequilibrium or genetic linkage with the type, or (c) a single nucleotide polymorphism identified by rs9425343 in the EDEM3 gene or a single nucleotide in linkage disequilibrium or genetic linkage with the single nucleotide polymorphism mFOLFIRINOX for pancreatic cancer, comprising an oligonucleotide that hybridizes under stringent conditions with a continuous 5-50 base region containing any of polymorphisms (a) to (c) Any probe set may be used as long as it is used to assist in predicting the risk of developing side effects when a therapy, FOLFIRINOX therapy, or a modified regimen thereof is performed, and is hereinafter also referred to as "probe set."

The probes used in the present probe set hybridize under stringent conditions with a sequence of 5 to 50 consecutive bases, preferably 10 to 40 bases, more preferably 15 to 30 bases containing the single nucleotide polymorphism site to be analyzed. Mention may be made of probes consisting of dyeing oligonucleotides. Moreover, by using an oligo probe synthesized using an artificial nucleic acid such as Locked Nucleic Acid (LNA) as a probe, it is possible to obtain a probe that specifically hybridizes even with a short base.

Stringent conditions refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. The solution containing 50% formamide is referred to as 10×SSC), and after hybridization is formed in a solution containing 50% formamide at 45° C., the conditions are such that the solution is washed with 2×SSC at 50° C. (Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6) or in a solution containing 3×SSC/0.3×SDS at 54° C., followed by washing solution A (10×SSC/ 1% SDS solution), washing solution B (20×SSC) and washing solution C (5×SSC) (see JP-A-2011-250726).

In addition, the probe may be used by immobilizing it on a carrier, and examples of the carrier include a planar substrate and a bead-like spherical carrier. Mention may be made of the carriers mentioned. Further, the probes for detecting the reference type and the probes for detecting the variant type may be immobilized on the same carrier or may be immobilized on different carriers.

As a primer used when analyzing a single nucleotide polymorphism using the above probe, an oligo capable of amplifying at least 5 consecutive bases containing a single nucleotide polymorphism site to be analyzed as a nucleic acid fragment using genomic DNA as a template. A single nucleotide polymorphism site identified by rs1980576 in the APCDD1L gene, a single nucleotide polymorphism site identified by rs2272761 in the R3HCC1 gene, or a single nucleotide polymorphism site identified by rs9425343 in the EDEM3 gene. A primer consisting of an oligonucleotide capable of amplifying at least 5 consecutive bases containing the site, preferably 10 to 500 bases, more preferably 20 to 200 bases, and still more preferably 50 to 100 bases can be used.

In addition, when amplifying at least 5 consecutive bases containing 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. becomes possible. Although the labeling substance is not particularly limited, examples thereof include radioactive isotopes, fluorescent dyes, and organic compounds such as digoxigenin (DIG) and biotin.

The above probes or primers can be obtained, for example, by chemical synthesis using a nucleic acid synthesizer. As a nucleic acid synthesizer, a DNA synthesizer, a fully automatic nucleic acid synthesizer, or the like can be used.

When the amplified nucleic acid fragment has a label, the nucleic acid fragment hybridized to each probe can be measured by detecting the label. For example, when a fluorescent dye is used as the label, the nucleic acid fragment hybridized to the probe can be measured by measuring the fluorescence intensity derived from the fluorescent dye. Specifically, in order to calculate the ratio of the nucleic acid fragment hybridized to the probe for detecting the reference type and the nucleic acid fragment hybridized to the probe for detecting the variant type, the label on the probe for detecting the reference type is detected. It can be calculated from the output value when the probe for detecting the variant type is detected and the output value when the label is detected in the probe for detecting the variant type.

More specifically, the intensity value derived from the nucleic acid fragment hybridized to the probe corresponding to the variant type is combined with 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. A determination value can be calculated by dividing by the average value of intensity values derived from soybean nucleic acid fragments. This determination value approximates the value obtained by normalizing the amount of variants contained in the nucleic acid fragment. Therefore, depending on the height of the judgment value, the single nucleotide polymorphism in the subject is analyzed and it is determined whether the variant type is homozygous, the reference type is homozygous, or heterozygous. can do.

When using the determination value, analyze the single nucleotide polymorphism in the subject to determine whether the variant type is homozygous, the reference type is homozygous, or heterozygous Therefore, it is preferable to set two levels of thresholds (threshold A and threshold B) in advance. Here, threshold A and threshold B are assumed to have a relationship of (threshold A>threshold B). That is, when the judgment value calculated as described above exceeds the threshold A, it is judged that the variant type is homozygous. If the determination value is equal to or less than the threshold value B, it can be determined that the reference type is homozygous.

These threshold A and threshold B are set for the single nucleotide polymorphism described above. The method for setting the threshold A and the threshold B is not particularly limited. A method of calculating the probability density as a normal distribution can be used for each of cases in which the reference type is homozygous or heterozygous. At this time, the intersection point where the probability densities overlap each other (the position where the magnitude of the probability density is switched, between the respective maximum values) is obtained, and if the variant type is homozygous, the reference type is homozygous. Find the average value for each of the cases where the Then, as a threshold value for cases where the variant type is homozygous and heterozygous, (average value when the variant type is homozygous and heterozygous ) and the average value of the intersection points. Similarly, the thresholds for heterozygote and homozygous reference type are (average value for heterozygote and average value for heterozygote ) and the average value of the intersection points.

The single nucleotide polymorphism identified by rs1980576 in the APCDD1L gene by the above method, the single nucleotide polymorphism identified by rs2272761 in the R3HCC1 gene, or the single nucleotide polymorphism identified by rs9425343 in the EDEM3 gene, or the single nucleotide polymorphism By analyzing single nucleotide polymorphisms in linkage disequilibrium or genetic linkage and determining whether the variant type is homozygous, the reference type is homozygous, or heterozygous, It can assist in predicting the risk of side effects when mFOLFIRINOX therapy, FOLFIRINOX therapy, or their modified regimens are administered to pancreatic cancer. For example, the single nucleotide polymorphism identified by rs1980576 in the APCDD1L gene in patients who did not experience side effects when mFOLFIRINOX therapy, FOLFIRINOX therapy, or a modified regimen thereof was performed in advance for pancreatic cancer , the single nucleotide polymorphism identified by rs2272761 in the R3HCC1 gene, or the single nucleotide polymorphism identified by rs9425343 in the EDEM3 gene, or the single nucleotide polymorphism and the single nucleotide polymorphism in linkage disequilibrium or genetic linkage Examine relationships. Next, by examining the single nucleotide polymorphism of the target patient and comparing it with the data of the patient examined in advance, mFOLFIRINOX therapy, FOLFIRINOX therapy, or their modified regimen for pancreatic cancer in the target patient It can assist in predicting the risk of occurrence of side effects in the event that it is performed.

Specifically, when the single nucleotide polymorphism of (a) or (a′) has a hetero or variant type as homozygous, the single nucleotide polymorphism of (b) or (b′) has a variant type is homozygous, or if the single nucleotide polymorphism of (c) or (c') has a variant type as homozygous, mFOLFIRINOX therapy for pancreatic cancer, FOLFIRINOX therapy, Or if the single nucleotide polymorphism of (a) or (a') has a homozygous reference type, the If the single nucleotide polymorphism of (b) or (b') has a homozygous hetero or reference type, or if the single nucleotide polymorphism of (c) or (c') has a homozygous reference type can assist in predicting that the risk of developing side effects is low when mFOLFIRINOX therapy, FOLFIRINOX therapy, or their modified regimens are performed for pancreatic cancer.

Furthermore, in a single nucleotide polymorphism combining two or more of (a) to (c), or three or more, or two or more of (a') to (c'), or three or more of (a') to (c'), pancreatic It may also help predict a low risk of side effects when mFOLFIRINOX therapy, FOLFIRINOX therapy, or modified regimens thereof are administered to cancer.

The probe set is not particularly limited as long as it contains the above probes, but the above primers, buffer solutions for analyzing single nucleotide polymorphisms, reagents such as enzymes, mFOLFIRINOX therapy for pancreatic cancer, FOLFIRINOX Instructions may be included to assist in predicting a low risk of side effects from the therapy, or a modified regimen thereof.

Further, other embodiments of the present invention include the following.
A reagent for analyzing the genotype of single nucleotide polymorphisms present on genomic DNA in a biological sample collected from a subject was subjected to mFOLFIRINOX therapy, FOLFIRINOX therapy, or a modified regimen thereof for pancreatic cancer. Application in the manufacture of a probe set for use in predicting the risk of side effects in cases where the single nucleotide polymorphism is the single nucleotide polymorphism of (a) or (a′); or (b') the single nucleotide polymorphism; the single nucleotide polymorphism of (c) or (c'); When the variant type is homozygous, when the single nucleotide polymorphism of (b) or (b') has the variant type as homozygous, or the single nucleotide of (c) or (c') If the variant type is homozygous in the polymorphism, mFOLFIRINOX therapy for pancreatic cancer, FOLFIRINOX therapy, or assists in predicting that the risk of side effects is high when performing a modified regimen thereof. , When the single nucleotide polymorphism of (a) or (a′) has a homozygous reference type, the heterozygous or reference type is homozygous in the single nucleotide polymorphism of (b) or (b′) If you have, or if you have the reference type as homozygous in the single nucleotide polymorphism of (c) or (c'), mFOLFIRINOX therapy for pancreatic cancer, FOLFIRINOX therapy, or modifications thereof Applications that assist in predicting a low risk of side effects when following a regimen.

(Analysis object)
Genomic DNA was prepared from the peripheral blood of 30 pancreatic cancer mFOLFIRINOX therapy cases (high-risk cases of irinotecan side effects due to UGT1A1 gene polymorphism, ie *6 homozygous, *28 homozygous, compound heterozygous cases excluded). All cases are Japanese.
(Preparation of genomic DNA)
Genomic DNA was prepared based on the sodium iodide method (Wang et al., Nucleic Acids Res 34:195-201 (2014)) using the subject's peripheral blood collected in EDTA-containing tubes. 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.

For 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, the TaqMan (registered trademark) probe method described above Verification was performed with clinical samples to be analyzed. TaqMan SNP Assays_Human (manufactured by Applied Biosystems) and LightCycler (registered trademark) 480 Probe Master (manufactured by Roche Diagnostics), Universal Probe Library (manufactured by Roche Diagnostics), and LightCycler 480 System II (manufactured by Roche Diagnostics) were applied to 10 ng of genomic DNA. performed genotyping. After incubation at 95°C for 10 minutes, PCR was performed for 55 cycles (15 seconds at 92°C and 60 seconds at 60°C per cycle), and the fluorescence of the PCR products was measured.

(result)
Table 1 shows the results of statistical analysis of the frequency of each gene polymorphism and the frequency of side effects (grade 3 or higher neutropenia) in pancreatic cancer mFOLFIRINOX therapy cases. CA trend test in Table 1 is the result of Cochrane-Armitage trend test, and Fisher's exact test is the result of Fisher's exact test. In Table 1, Odd ratio is the odds ratio calculated by Fisher's exact test.

In the single nucleotide polymorphism rs1980576 in the region encoding the APCDD1L gene, the P value in the CA trend test is 0.035, and the odd ratio (A/A vs. A/G, G/G) in the Fisher's exact test is 6.0. 52, P Value was 0.046. Therefore, there was a significant linear trend (correlation) and frequency difference between the single nucleotide polymorphism rs1980576 and the frequency of side effects of mFOLFIRINOX therapy for pancreatic cancer. In addition, regarding the risk of side effects in mFOLFIRINOX cases of pancreatic cancer, the risk of side effects is low in the case of reference homozygous (A/A), and the risk of side effects is low in heterozygous (A/G) or variant homozygous. It turned out that it can assist the prediction that it is high.

In the single nucleotide polymorphism rs2272761 in the region encoding the R3HCC1 gene, the P value in the CA trend test is 0.013, and the odd ratio (G/G vs G/A, A/A) in the Fisher's exact test is 9.0. 81, P Value was 0.009. Therefore, there was a significant linear trend and frequency difference between the single nucleotide polymorphism rs2272761 and the side effect frequency of mFOLFIRINOX therapy for pancreatic cancer. In addition, regarding the risk of side effects in mFOLFIRINOX cases of pancreatic cancer, the risk of side effects is low in the case of reference homozygous (G/G) or heterozygous (A/G), and the risk of side effects is high in the case of variant homozygous. It was found that it can assist the prediction of

In the FOLFIRI therapy described in Patent Document 1, the risk of occurrence of side effects is low when the cutoff for the single nucleotide polymorphism rs2272761 in the region encoding the R3HCC1 gene is homozygous (G/G), i.e. exclusion It was possible to use it for diagnosis. Furthermore, in the heterozygous (G/A) and variant homozygous (A/A) cases, the frequency of side effects was not observed to the extent that the risk of occurrence of side effects was high. However, when assisting the prediction of the risk of side effects in mFOLFIRINOX cases of pancreatic cancer, the risk of side effects is high in case of variant homozygous (A/A) as a cutoff, that is, it can be used for definitive diagnosis. One thing became clear.

In the single nucleotide polymorphism rs9425343 in the region encoding the EDEM3 gene, the P Value in the CA trend test is 0.001, and the odd ratio (T/T vs T/G, G/G) in the Fisher's exact test is Inf. (infinite), with a P Value of 0.005. Therefore, there was a highly significant linear trend and frequency difference between the single nucleotide polymorphism rs9425343 and the side effect frequency with mFOLFIRINOX therapy for pancreatic cancer. In other words, with regard to the risk of side effects in mFOLFIRINOX cases of pancreatic cancer, the risk of side effects is low in the case of homozygous reference type (T/T) and high in the case of homozygous variant (G/G). It was found that it can assist the prediction of

In the FOLFIRI therapy of Patent Document 1, the risk of side effects is high in the case of reference type homozygous (T / T), and the risk of side effects is low in the case of variant homozygous (G / G). In the case of the mFOLFIRINOX therapy, it was surprising to find the opposite result, with a lower risk of side effects occurring in reference type homozygous (T/T).

[Reference example]
In the above example, three types of single nucleotide polymorphisms and the prediction of the risk of side effects in mFOLFIRINOX therapy were investigated. Risk prediction was also examined.

The relationship between the three types of single nucleotide polymorphisms (rs1980576, rs2272761, rs9425343) and side effects (neutropenia) was analyzed in the same manner as in the above example, except that 41 cases in which Gem/nab-PTX therapy was performed were analyzed. Examined. Table 2 shows the results.

As is clear from Table 2, in the Gem/nab-PTX therapy, no significant correlation was observed between any gene polymorphism and occurrence of side effects.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to assist in predicting the risk of side effects when mFOLFIRINOX therapy, FOLFIRINOX therapy, or a modified regimen thereof is performed for pancreatic cancer, and thus can be used in the medical field. be.

Claims (7)

present on genomic DNA in a biological sample taken 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;
Analyzing any single nucleotide polymorphism of (a) to (c), determining the genotype in the single nucleotide polymorphism, and based on the determined genotype, mFOLFIRINOX therapy for pancreatic cancer, FOLFIRINOX A method to help predict the risk of developing side effects when a therapy or modified regimen thereof is administered. The single nucleotide polymorphism identified at 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 the 186th base of the nucleotide sequence encoding the APCDD1L gene set forth in SEQ ID NO: 1;
The single nucleotide polymorphism identified at 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 the 919th base of the base sequence encoding the R3HCC1 gene set forth in SEQ ID NO: 2;
The single nucleotide polymorphism identified at 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 the 2459th base of the nucleotide sequence encoding the EDEM3 gene set forth in SEQ ID NO: 3.
2. The method of claim 1, wherein: When the single nucleotide polymorphism of (a) or (a′) has a homozygous hetero or variant type, the single nucleotide polymorphism of (b) or (b′) has a homozygous variant type. or if the variant type is homozygous in the single nucleotide polymorphism of (c) or (c'), mFOLFIRINOX therapy for pancreatic cancer, FOLFIRINOX therapy, or a modified regimen thereof If the single nucleotide polymorphism of (a) or (a') has a homozygous reference type, the above (b) or ( If the single nucleotide polymorphism in b′) has a heterozygous or reference type as homozygous, or if the single nucleotide polymorphism in (c) or (c′) above has a homozygous reference type 3. The method according to claim 1 or 2, which aids prediction of a low risk of side effects when mFOLFIRINOX therapy, FOLFIRINOX therapy, or a modified regimen thereof is administered to pancreatic cancer. The method according to any one of claims 1 to 3, wherein the side effect is neutropenia. (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 type or linkage disequilibrium with the single nucleotide polymorphism or single nucleotide polymorphism in genetic linkage, or (c) single nucleotide polymorphism identified by rs9425343 in the EDEM3 gene or linkage disequilibrium with the single nucleotide polymorphism or genetic Pancreas comprising an oligonucleotide that hybridizes under stringent conditions with a continuous 5-50 base region containing any of the single nucleotide polymorphisms (a) to (c) of the linked single nucleotide polymorphisms A probe set for use in assisting prediction of the risk of side effects in mFOLFIRINOX therapy, FOLFIRINOX therapy, or a modified regimen thereof for cancer. The single nucleotide polymorphism identified at 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 the 186th base of the nucleotide sequence encoding the APCDD1L gene set forth in SEQ ID NO: 1;
The single nucleotide polymorphism identified at 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 the 919th base of the base sequence encoding the R3HCC1 gene set forth in SEQ ID NO: 2;
The single nucleotide polymorphism identified at 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 the 2459th base of the nucleotide sequence encoding the EDEM3 gene set forth in SEQ ID NO: 3.
6. The probe set according to claim 5, characterized by: A reference type probe corresponding to the reference type in (a) or (a'), a variant type probe corresponding to the variant type in the single nucleotide polymorphism, and a reference type in (b) or (b') above A reference type probe, a variant type probe corresponding to the variant type in the single nucleotide polymorphism, or a reference type probe corresponding to the reference type in (c) or (c′) above, and a variant type in the single nucleotide polymorphism corresponding variant probes;
7. The probe set according to claim 5 or 6, characterized by: