Method for determining risk of adverse event

Abstract

Provided is a method for determining the risk of an adverse event during multidisciplinary therapy. This method for determining the risk of an adverse event during multidisciplinary therapy includes measuring Anaerostipes hadrus in a specimen collected from a subject.

Description

How to determine the risk of adverse events

[0001] The present invention relates to a method for determining the risk of adverse events during multidisciplinary treatment.

[0002] Neoadjuvant chemotherapy combined with surgery is a promising treatment strategy for advanced esophageal cancer. However, neoadjuvant chemotherapy can cause serious adverse events, such as diarrhea and febrile neutropenia. These adverse events can lead to the discontinuation or reduction of anticancer drugs, making it difficult to achieve sufficient antitumor effects. They can also cause a decline in immune function and nutritional status, which can lead to a decrease in patients' quality of life (QOL). Therefore, reducing the adverse events associated with chemotherapy is an important issue.

[0003] It has been reported that the onset of febrile neutropenia and severe diarrhea are significantly reduced by having patients take synbiotics during DCF therapy (a triple-drug combination therapy of docetaxel, cisplatin, and 5-fluorouracil), a neoadjuvant chemotherapy for esophageal cancer (Non-Patent Document 1). On the other hand, if the occurrence of adverse events can be predicted before treatment, preventive measures such as considering the amount of anticancer drugs to be used and the concomitant use of synbiotics can be considered in advance.

[0004] Anaerostipes hadrus is a dominant bacterium in the intestine and is known to produce butyrate by utilizing lactic acid and acetic acid in the intestine. It has also been reported that in healthy individuals, deficiency of the inositol catabolism and butyrate biosynthesis pathway in A. hadrus is associated with increased body weight and metabolic disease risk in the host (Non-Patent Document 2). However, the relationship between A. hadrus and adverse events during treatment, such as chemotherapy, is unknown.

[0005] Motoori M, et al. Clin Nutr, 2017, 36: 93-99Zeevi D, et al. Nature, 2019, 568: 43-48

[0006] For effective treatment of a disease, it is desirable to start treatment after assessing the risk of onset or aggravation of adverse events during treatment. Therefore, an objective of the present invention is to provide a method for assessing the risk of adverse events during multidisciplinary treatment.

[0007] As a result of extensive research focusing on intestinal bacteria, the present inventors have found that there is a correlation between Anaerostipes hadrus, a type of intestinal bacteria, and the occurrence or severity of adverse events during treatment, and that Anaerostipes hadrus can be used as an indicator to determine the risk of adverse events during multidisciplinary treatment.

[0008] That is, the present invention provides the following [1] to

[12] . [1] A method for determining the risk of an adverse event during multidisciplinary treatment, comprising measuring Anaerostipes hadrus in a sample collected from a subject. [2] The method according to [1], wherein the risk of an adverse event is the risk of onset or aggravation of the adverse event. [3] The method according to [1] or [2], wherein the multidisciplinary treatment is a treatment selected from drug therapy, surgical therapy, radiation therapy, and combinations thereof. [4] The method according to any of [1] to [3], wherein the multidisciplinary treatment is drug therapy. [5] The method according to any of [1] to [4], wherein the adverse event is at least one selected from the group consisting of febrile neutropenia and diarrhea. [6] The method according to any of [1] to [5], wherein the sample is a stool sample from a subject. [7] The method according to any one of [1] to [6], further comprising: comparing the number of Anaerostipes hadrus bacteria in the sample with a standard value; and determining that the risk of an adverse event during multidisciplinary treatment is high if the number of bacteria is equal to or less than the standard value. [8] The method according to any one of [1] to [6], further comprising: comparing the occupancy rate of Anaerostipes hadrus in the total bacteria in the sample with a standard value; and determining that the risk of an adverse event during multidisciplinary treatment is high if the occupancy rate is equal to or less than the standard value. [9] The method according to any one of [1] to [6], further comprising: comparing the occupancy rate of Anaerostipes hadrus in the total bacteria in the sample with a standard value; and determining that the risk of an adverse event during multidisciplinary treatment is high if the occupancy rate is equal to or less than the standard value. 7.7

[10] The method according to any one of [1] to [6], further comprising determining that the risk of febrile neutropenia during multidisciplinary therapy is high when the number of Anaerostipes hadrus bacteria in the sample is 10 or less per 1 g of the sample, or when the occupancy rate of Anaerostipes hadrus in the total bacteria in the sample is 0.25% or less. 7.6

[10] The method of any one of [1] to [6], further comprising determining that the risk of diarrhea during multidisciplinary treatment is high when the number of Anaerostipes hadrus bacteria is 0.084% or less, or when the occupancy rate of Anaerostipes hadrus in the total bacteria in the sample is 0.084% or less.

[11] A kit for carrying out the method of any one of [1] to

[10] , comprising a reagent and protocol for measuring Anaerostipes hadrus in a sample.

[12] A method for screening for an agent for reducing the risk of adverse events during multidisciplinary treatment, using the number or occupancy rate of Anaerostipes hadrus bacteria as an index.

[0009] According to the present invention, the risk of developing or aggravating adverse events during multidisciplinary treatment can be easily determined, which allows for the selection of treatment to be performed, adjustment of drug dosage, preventive measures against adverse events, and other measures to deal with adverse events depending on the level of risk, thereby enabling the selection of an appropriate treatment regimen tailored to each individual patient.

[0010] Figure 1 shows a comparison of A. hadrus bacterial counts based on the presence or absence of febrile neutropenia. Gr. 0 (Grade 0) indicates patients who did not develop febrile neutropenia, and Gr. 3 (Grade 3) indicates patients who did. Baseline indicates before DCF therapy, and Day 8 indicates day 8 of the first cycle of DCF therapy. *p<0.05, **p<0.01. Figure 1 shows a comparison of A. hadrus bacterial counts based on the presence or absence of febrile neutropenia in the synbiotic-administered group. Gr. 0 (Grade 0) indicates patients who did not develop febrile neutropenia, and Gr. 3 (Grade 3) indicates patients who did develop febrile neutropenia. Baseline indicates before DCF therapy, and Day 8 indicates day 8 of the first cycle of DCF therapy. A. hadrus bacterial counts based on the severity of diarrhea Figure 1 shows a comparison of A. hadrus bacterial counts by severity of diarrhea in the synbiotic-administered groups. Gr. 0-2 (grades 0-2) represent patients with relatively mild diarrhea, while Gr. 3-4 (grades 3-4) represent patients with extremely severe diarrhea. Baseline represents the data before DCF therapy, and Day 8 represents the eighth day of one cycle of DCF therapy. **p<0.01, ***p<0.001. Figure 1 shows a comparison of A. hadrus bacterial counts by severity of diarrhea in the synbiotic-administered groups. Gr. 0-2 (grades 0-2) represent patients with relatively mild diarrhea, while Gr. 3-4 (grades 3-4) represent patients with extremely severe diarrhea. Baseline represents the data before DCF therapy, and Day 8 represents the eighth day of one cycle of DCF therapy. *p<0.05.

[0011] "Anaerostipes hadrus (Eubacterium hadrum)" is a gram-positive bacterium and a type of human intestinal bacterium. In this specification, "Anaerostipes hadrus" refers to bacteria belonging to Anaerostipes hadrus.

[0012] As used herein, "multidisciplinary therapy" includes drug therapy using drugs such as anticancer drugs, surgical therapy such as surgery, radiation therapy, and combinations thereof. As used herein, "during treatment" includes during and after treatment.

[0013] As used herein, the term "adverse event" refers to an undesirable or unintended sign, symptom, or illness that occurs in a patient during or after treatment, regardless of whether or not it is causally related to the treatment. Examples of adverse events and their severity include adverse events and their grades included in the Common Terminology Criteria for Adverse Events (CTCAE) v4.0 published by the National Cancer Institute (NCI).

[0014] According to CTCAE v4.0, the severity of adverse events is classified into the following grades 1 to 5 or some of them. Grade 1: Mild; no symptoms or mild symptoms; clinical or laboratory findings only; no treatment required. Grade 2: Moderate; minimal / local / non-invasive treatment required; limitation in activities of daily living other than self-care appropriate for age. Grade 3: Severe or medically significant, but not immediately life-threatening; requiring hospitalization or prolonged hospitalization; disabling / incapacitating; limitation in activities of daily living other than self-care. Grade 4: Life-threatening; requiring emergency treatment. Grade 5: Death due to adverse event. In this specification, no event is defined as Grade 0.

[0015] As used herein, "risk of an adverse event" refers to the risk of developing an adverse event or the risk of an adverse event becoming severe. "Risk of developing an adverse event" refers to the possibility of developing an adverse event. That is, when an individual belongs to a high-risk group, it means that the individual is predicted to have a high probability of developing an adverse event, and when an individual belongs to a low-risk group, it means that the individual is predicted to have a low probability of developing an adverse event. Furthermore, "risk of an adverse event becoming severe" refers to the possibility of an adverse event becoming severe. That is, when an individual belongs to a high-risk group, it means that the individual is predicted to have a high probability of developing an adverse event and that the adverse event will become severe, and when an individual belongs to a low-risk group, it means that the individual is predicted to have a low probability of developing an adverse event, or that even if the individual develops an adverse event, the adverse event will be unlikely to become severe.

[0016] In this specification, "determining" includes the concepts of "detecting," "examining," "measuring," "predicting," and "diagnosis," but does not include medical procedures such as diagnosis by a doctor.

[0017] In the method of the present invention for assessing the risk of adverse events during multidisciplinary treatment, Anaerostipes hadrus in a sample is used as an indicator. The method includes measuring A. hadrus in the sample. Specifically, the method includes measuring the number of A. hadrus bacteria in a sample collected from a subject. Alternatively, the method includes measuring the occupancy rate of A. hadrus in the total bacteria in the sample collected from the subject. Here, the occupancy rate of A. hadrus in the total bacteria in the sample means the ratio of the number of A. hadrus bacteria to the total number of bacteria in the sample, and is a value equivalent to the occupancy rate of A. hadrus in the intestinal bacterial flora of the subject from whom the sample was derived.

[0018] The subject is not particularly limited, but includes those who require assessment of the risk of adverse events during multidisciplinary treatment, such as patients who are scheduled to receive multidisciplinary treatment, patients who are currently receiving multidisciplinary treatment, etc. Examples of specimens include biological samples derived from subjects, such as gastrointestinal contents such as intestinal fluid and feces, with feces being preferred because it places less strain on the subject.

[0019] The means for measuring the number of A. hadrus bacteria in a sample is not particularly limited, but preferred methods include, for example, RT-PCR and sequencing based on the base sequence of the 16S rRNA gene of A. hadrus, with RT-PCR being more preferred.

[0020] Here, the RT-PCR method will be described. An analytical method using the RT-PCR method can be carried out, for example, by (1) extracting RNA of enterobacteria in a sample, (2) synthesizing cDNA from the extracted RNA by reverse transcription and performing PCR using a nucleic acid fragment (primer) that hybridizes to the A. hadrus -derived cDNA, and (3) detecting the DNA fragment amplified in step (2). By combining the nucleic acid fragment with a template cDNA derived from the sample and performing an amplification reaction, a DNA fragment (PCR product) specific to A. hadrus can be obtained. By observing the PCR product over time and determining the number of PCR cycles at which a certain amount of DNA is reached, it is possible to quantify the number of A. hadrus bacteria in the sample.

[0021] The amplified PCR product can be monitored over time by labeling the PCR product with an intercalating fluorescent dye such as SYBR® Green I and measuring the fluorescence intensity at each PCR stage. Intercalating dyes have the property of increasing their fluorescence intensity upon intercalation into double-stranded nucleic acids, allowing accurate measurement of the PCR product generated by PCR from A. hadrus cDNA, and SYBR Green I is particularly suitable.

[0022] By determining the PCR cycle number (hereinafter referred to as Cq value) at which a certain arbitrarily set fluorescence intensity (DNA amount) is reached, it is possible to quantify A. hadrus in the sample. Alternatively, fluorescent dye-labeled TaqMan (registered trademark) probes, Molecular Beacons, etc. can also be used. TaqMan probes and Molecular Beacons are probes in which a fluorescent dye and a quencher are bound to an oligonucleotide having homology to the internal sequence of the region amplified by PCR, and are used in combination with the PCR reaction. The interaction between the fluorescent dye bound to the probe and the quencher emits fluorescence in response to the PCR amplification reaction, so the amplified PCR product can be observed over time by measuring the fluorescence intensity at each PCR stage.

[0023] The number of A. hadrus bacteria in a sample can be determined from a calibration curve of the logarithm of the number of bacteria measured by the DAPI counting method, culture method, etc. and the Cq value. That is, a calibration curve is prepared in advance, in which the logarithm of the number of A. hadrus bacteria is plotted on the horizontal axis and the Cq value is plotted on the vertical axis, and the Cq value obtained as a result of the PCR reaction is applied to the calibration curve to measure the number of A. hadrus bacteria in the sample.

[0024] When measuring the number of A. hadrus bacteria in a sample, primers that can specifically hybridize to and amplify a region of the 16S rRNA gene of A. hadrus that is conserved within the species A. hadrus and is not conserved in species other than A. hadrus may be used in the PCR reaction. Primers for measuring the number of A. hadrus bacteria include, but are not limited to, the primers of SEQ ID NOs: 3 and 4.

[0025] The means for measuring the occupancy rate of A. hadrus in the total bacteria in a sample is not particularly limited, but preferred methods include measurement based on the base sequence of the 16S rRNA gene of enterobacteria, such as RT-PCR and sequencing, and of these, measurement by sequencing (e.g., 16S rRNA gene amplicon analysis) is more preferred.

[0026] Here, 16S rRNA gene amplicon analysis will be described. 16S rRNA gene amplicon analysis can be performed, for example, by (1) extracting genomic DNA from enterobacteria in a sample, (2) using the extracted genomic DNA as a template to perform PCR using nucleic acid fragments (primers) that hybridize to the 16S rRNA gene of the enterobacteria, (3) determining the base sequence of the DNA fragments amplified in step (2), and (4) analyzing the sequence data obtained in step (3). The nucleic acid fragments can be combined with the template genomic DNA derived from the sample and subjected to an amplification reaction to obtain DNA fragments (PCR products) derived from the 16S rRNA gene of the enterobacteria. Furthermore, after sequencing the PCR products and removing error sequences, the sequence data is compiled as amplicon sequence variants (ASVs), and a known database is referenced to assign phylogenetic information to each ASV, allowing the type and abundance of enterobacteria contained in the sample to be analyzed. Based on the information obtained, it is possible to calculate the occupancy rate of A. hadrus relative to the total intestinal bacteria.

[0027] The region of the 16S rRNA gene of enterobacteria amplified by the PCR is preferably a region amplified using primers that hybridize to a conserved region universally conserved among bacterial species, and that contains a variable region that is not conserved among bacterial species. The variable region may include at least one of the V1 to V9 regions of the 16s rRNA gene, preferably a region including V1 and V2 or a region including V3 and V4. The primers may optionally include an adapter sequence for sequencing and / or an index sequence for sample identification. Examples of such primers include universal primers commonly used in the field for amplifying bacterial 16S rRNA genes, such as the primers set forth in SEQ ID NOs: 1 and 2.

[0028] The base sequence of the amplified PCR product can be determined by known methods, but can also be rapidly determined using a next-generation sequencer such as the MiSeq platform (Illumina). Analysis of sequence data can be performed using analysis software such as QIIME2 (Quantitative Insights Into Microbial Ecology 2), and sequence errors can be removed using the DADA2 (Divisive Amplicon Denoising Algorithm 2) plug-in of QIIME2. The acquired sequence data can be classified into ASVs based on sequence identity, and the species of each ASV can be determined by referring to known databases such as SILVA and Greengenes. From the abundance ratio of each ASV species determined in this way and the corresponding sequence read number, the proportion of A. The occupancy rate of hadrus can be calculated.

[0029] As shown in the Examples below, a significant negative correlation was observed between the prevalence of A. hadrus in the intestinal flora of esophageal cancer patients after triple therapy (DCF therapy) of docetaxel, cisplatin, and 5-fluorouracil and the severity of febrile neutropenia or diarrhea (Table 2). Furthermore, patients who did not develop febrile neutropenia during DCF therapy had significantly higher A. hadrus bacterial counts both before and after treatment compared with patients who developed febrile neutropenia (Figure 1). Even in an analysis of only the group receiving synbiotic therapy in addition to DCF therapy, patients who did not develop febrile neutropenia during the therapy tended to have higher A. hadrus bacterial counts both before and after treatment compared with patients who developed febrile neutropenia (Figure 2). Here, synbiotic therapy has been reported to significantly alleviate febrile neutropenia and severe diarrhea caused by DCF therapy (Non-Patent Document 1), and uses Lacticaseibacillus paracasei strain Shirota (LcS) and Bifidobacterium breve strain Yakult (BbrY) (Bifidobacterium breve YIT 12272 (FERM BP-11320)) as probiotics and galactooligosaccharides as prebiotics. LcS is a strain known as Lactobacillus casei YIT9029 (FERM BP-1366) prior to the reclassification of Lactobacillus bacteria in 2020 (Zheng J et al. Int J Syst Evol Microbiol. 2020 Apr; 70(4): 2782-2858), and was deposited at the National Institute of Advanced Industrial Science and Technology (AIST) Patent Organism Depositary Center (currently the National Institute of Technology and Evaluation) Patent Microorganism Depositary Center) on January 12, 1981. The results of the logistic analysis suggested that low A. hadrus bacterial counts and occupancy rates before DCF therapy are risk factors for the development of febrile neutropenia.Furthermore, patients who did not develop diarrhea or whose diarrhea was mild (≤6 bowel movements per day compared to baseline) during DCF therapy had significantly higher A. hadrus bacterial counts both before and after treatment compared with patients who developed severe diarrhea (≥7 bowel movements per day compared to baseline, fecal incontinence, and hospitalization or emergency treatment required) (Figure 3). Even in the analysis of only the group receiving synbiotic therapy in addition to DCF therapy, patients who did not develop diarrhea or whose diarrhea was mild tended to have higher A. hadrus bacterial counts both before and after treatment compared with patients who developed severe diarrhea (Figure 4). Logistic regression analysis suggested that low A. hadrus bacterial counts and occupancy before DCF therapy are risk factors for the development of severe diarrhea.

[0030] On the other hand, multidisciplinary treatments, including not only drug therapy such as DCF therapy but also surgery, radiation therapy, and combinations of these, generally damage the gastrointestinal mucosa, especially the intestinal mucosa. Furthermore, damage to the host's immune function can also occur, leading to bacterial translocation, in which intestinal bacteria migrate into the body. This is known to cause adverse events and infectious complications.

[0031] Therefore, A. hadrus in a sample can be used as an indicator for determining the risk of adverse events not only during drug therapy such as DCF therapy but also during multidisciplinary therapy.

[0032] Multidisciplinary treatments include drug therapy, surgical therapy, radiation therapy, and combinations thereof, preferably drug therapy, more preferably drug therapy using an anticancer drug, and even more preferably drug therapy using docetaxel, cisplatin, and 5-fluorouracil (DCF therapy). DCF therapy is known as a treatment for esophageal cancer. Adverse events for which risk assessment is required include, but are not limited to, blood and lymphatic system disorders, cardiac disorders, ear and labyrinth disorders, endocrine disorders, eye disorders, gastrointestinal disorders, general / systemic disorders and administration site disorders, hepatobiliary system disorders, immune system disorders, metabolic and nutritional disorders, musculoskeletal system and connective tissue disorders, nervous system disorders, psychiatric disorders, kidney and urinary tract disorders, reproductive system and breast disorders, respiratory, thoracic and mediastinal disorders, skin and subcutaneous tissue disorders, and vascular disorders. The method of the present invention is preferably used for risk assessment of at least one adverse event selected from the group consisting of blood and lymphatic system disorders and gastrointestinal disorders, and more specifically, is preferably used for risk assessment of at least one adverse event selected from the group consisting of febrile neutropenia and diarrhea, i.e., for risk assessment of at least one adverse event selected from the group consisting of febrile neutropenia risk and diarrhea risk.

[0033] The severity of febrile neutropenia is classified into the following grades according to CTCAE v4.0: Grade 1: - (undefined) Grade 2: - (undefined) Grade 3: absolute neutrophil count <1,000 / mm 3and a fever of 38.3°C or higher even once, or a fever of 38.0°C or higher that lasts for more than one hour. Grade 4: Life-threatening; emergency treatment required. Grade 5: Death. According to CTCAE v4.0, the severity of diarrhea is classified into the following grades. Note that baseline refers to bowel habits (stool frequency) in daily life. Grade 1: An increase in bowel movements of <4 times / day compared to baseline; a mild increase in colostomy output compared to baseline. Grade 2: An increase in bowel movements of 4-6 times / day compared to baseline; a moderate increase in colostomy output compared to baseline. Grade 3: An increase in bowel movements of 7 or more times / day compared to baseline; fecal incontinence; hospitalization required; a severe increase in colostomy output compared to baseline; limitations in self-care activities of daily living. Grade 4: Life-threatening; emergency treatment required. Grade 5: Death.

[0034] In the method of the present invention, the risk of adverse events during multidisciplinary treatment can be determined using the number of A. hadrus bacteria in a sample as an index. Specifically, it can be determined that the lower the number of A. hadrus bacteria in a sample, the higher the risk of adverse events during multidisciplinary treatment, and the higher the number of A. hadrus bacteria in a sample, the lower the risk of adverse events during multidisciplinary treatment. Such risk determination is preferably performed by comparing the number of A. hadrus bacteria in a sample with a reference value (cutoff value) previously set according to the level of risk. The reference value can be set, for example, by analyzing the results of a follow-up survey of multiple subjects whose A. hadrus bacteria counts have been confirmed in advance to determine the occurrence and / or severity of adverse events during multidisciplinary treatment using a statistical analysis method. Examples of statistical analysis methods include receiver operating characteristic (ROC) analysis, and for example, a value that can distinguish between groups at high risk of adverse events can be used as the reference value. When the number of A. hadrus bacteria is equal to or less than the reference value, the risk of adverse events during multidisciplinary treatment can be determined to be high. On the other hand, when the number of A. hadrus bacteria in the sample is greater than the reference value, the risk of adverse events during multidisciplinary treatment can be determined to be low.

[0035] In the method of the present invention, the risk of developing an adverse event during multidisciplinary treatment can be determined using the number of A. hadrus bacteria in a sample as an index. Specifically, it can be determined that the lower the number of A. hadrus bacteria in a sample, the higher the risk of developing an adverse event during multidisciplinary treatment, and the higher the number of A. hadrus bacteria in a sample, the lower the risk of developing an adverse event during multidisciplinary treatment. Such risk determination is preferably performed by comparing the number of A. hadrus bacteria in a sample with a reference value set in advance according to the level of risk. The reference value can be appropriately set by those skilled in the art, as described above. When the number of A. hadrus bacteria in a sample is equal to or less than the reference value, it can be determined that the risk of developing an adverse event during multidisciplinary treatment is high. On the other hand, when the number of A. hadrus bacteria in a sample is higher than the reference value, it can be determined that the risk of developing an adverse event during multidisciplinary treatment is low. In one example, if the adverse event is febrile neutropenia, the number of A. hadrus bacteria in the sample can be determined. The number of bacteria of Hadrus per 1g of sample was 10 7.7 When the number of A. hadrus bacteria in the sample is less than 10 per 1 g of the sample, the risk of developing febrile neutropenia during multidisciplinary treatment is determined to be high. 7.7 In another example, if the adverse event is diarrhea, the risk of developing febrile neutropenia during multidisciplinary treatment can be determined to be low when the number of A. hadrus bacteria in the sample is 10 or more per 1 g of the sample. 7.6 When the number of A. hadrus bacteria in the sample is less than 10 per 1 g of sample, the risk of diarrhea during multidisciplinary treatment is judged to be high. 7.6 When the number of cases is greater than or equal to 1, it can be determined that the risk of developing diarrhea during multidisciplinary treatment is low. Determining the risk of developing an adverse event during multidisciplinary treatment is particularly preferably applied when the adverse event is febrile neutropenia.

[0036] Furthermore, in the method of the present invention, the risk of aggravation of adverse events during multidisciplinary treatment can be determined using the number of A. hadrus bacteria in the sample as an index. Specifically, it can be determined that the lower the number of A. hadrus bacteria in the sample, the higher the risk of aggravation of adverse events during multidisciplinary treatment, and the higher the number of A. hadrus bacteria in the sample, the lower the risk of aggravation of adverse events during multidisciplinary treatment. Such risk determination is preferably performed by comparing the number of A. hadrus bacteria in the sample with a reference value set in advance according to the level of risk. The reference value can be appropriately set by those skilled in the art, as described above. When the number of A. hadrus bacteria in the sample is equal to or less than the reference value, it can be determined that the risk of aggravation of adverse events during multidisciplinary treatment is high. On the other hand, when the number of A. hadrus bacteria in the sample is higher than the reference value, it can be determined that the risk of aggravation of adverse events during multidisciplinary treatment is low. In one example, if the adverse event is febrile neutropenia, the number of A. hadrus bacteria in the sample can be determined. The number of bacteria of Hadrus per 1g of sample was 10 7.7 When the number of A. hadrus bacteria in the sample is less than 10 per 1 g of the sample, the risk of febrile neutropenia becoming severe during multidisciplinary treatment can be determined to be high. 7.7 When the number of A. hadrus bacteria in a sample is more than 10 per 1 g of the sample, it can be determined that the risk of febrile neutropenia becoming severe during multidisciplinary treatment is low. 7.7 When the number of A. hadrus bacteria in the sample is less than 10 per 1 g of the sample, it can be determined that there is a high possibility that febrile neutropenia during multidisciplinary treatment will be equivalent to the above-mentioned Grade 3. 7.7 In another example, if the adverse event is diarrhea, it can be determined that the number of A. hadrus bacteria in the sample is 10 or more per 1 g of the sample. 7.6 When the number of A. hadrus bacteria in the sample is less than 10 per 1 g of sample, the risk of diarrhea becoming severe during multidisciplinary treatment is judged to be high. 7.6 When the number of A. hadrus bacteria in a sample is more than 10 per 1 g of the sample, it can be determined that the risk of diarrhea becoming severe during multidisciplinary treatment is low. 7.6When the number of A. hadrus bacteria in the sample is less than 10 per 1 g of the sample, it can be determined that the diarrhea during multidisciplinary treatment is likely to be equivalent to grade 3 or higher, for example, grade 3 to 4. 7.6 When the number of adverse events is more than 1, it can be determined that there is a high possibility that diarrhea during multidisciplinary treatment will correspond to the above-mentioned grades 0 to 2. The determination of the risk of aggravation of an adverse event is particularly suitably applied when the adverse event is diarrhea.

[0037] Alternatively, in the method of the present invention, the risk of adverse events during multidisciplinary treatment may be assessed using the occupancy rate of A. hadrus in the total bacteria in the sample as an index. Specifically, the lower the occupancy rate of A. hadrus in the total bacteria in the sample, the higher the risk of adverse events during multidisciplinary treatment, and the higher the occupancy rate of A. hadrus in the total bacteria in the sample, the lower the risk of adverse events during multidisciplinary treatment. Such risk assessment is preferably performed by comparing the occupancy rate of A. hadrus in the total bacteria in the sample with a reference value previously set according to the level of risk. The reference value can be set, for example, by analyzing the results of a follow-up survey of the occurrence and / or severity of adverse events during multidisciplinary treatment for multiple subjects whose occupancy rate of A. hadrus in the total bacteria has been confirmed in advance using a statistical analysis method. Examples of statistical analysis methods include receiver operating characteristic (ROC) analysis, and a value that can distinguish between high-risk groups for adverse events can be used as the reference value. When the occupancy rate of A. hadrus in the total bacteria in the sample is equal to or lower than the reference value, the risk of adverse events during multidisciplinary treatment can be determined to be high. On the other hand, when the occupancy rate of A. hadrus in the total bacteria in the sample is higher than the reference value, the risk of adverse events during multidisciplinary treatment can be determined to be low.

[0038] In the method of the present invention, the risk of developing an adverse event during multidisciplinary treatment can be determined using the occupancy rate of A. hadrus in the total bacteria in the sample as an index. Specifically, the lower the occupancy rate of A. hadrus in the total bacteria in the sample, the higher the risk of developing an adverse event during multidisciplinary treatment, and the higher the occupancy rate of A. hadrus in the total bacteria in the sample, the lower the risk of developing an adverse event during multidisciplinary treatment. Such risk determination is preferably performed by comparing the occupancy rate of A. hadrus in the total bacteria in the sample with a predetermined reference value set according to the level of risk. The reference value can be appropriately set by those skilled in the art, as described above. If the occupancy rate of A. hadrus in the total bacteria in the sample is equal to or less than the reference value, the risk of developing an adverse event during multidisciplinary treatment can be determined to be high. On the other hand, if the occupancy rate of A. hadrus in the total bacteria in the sample is higher than the reference value, the risk of developing an adverse event during multidisciplinary treatment can be determined to be low. In one example, if the adverse event is febrile neutropenia, the risk of developing febrile neutropenia during multidisciplinary treatment can be determined to be high if the occupancy rate of A. hadrus among the total bacteria in the sample is 0.25% or less, and the risk of developing febrile neutropenia during multidisciplinary treatment can be determined to be low if the occupancy rate of A. hadrus among the total bacteria in the sample is higher than 0.25%. In another example, if the adverse event is diarrhea, the risk of developing diarrhea during multidisciplinary treatment can be determined to be high if the occupancy rate of A. hadrus among the total bacteria in the sample is 0.084% or less, and the risk of developing diarrhea during multidisciplinary treatment can be determined to be low if the occupancy rate of A. hadrus among the total bacteria in the sample is higher than 0.084%. Determining the risk of developing an adverse event during multidisciplinary treatment is particularly suitable when the adverse event is febrile neutropenia.

[0039] Furthermore, in the method of the present invention, the risk of aggravation of adverse events during multidisciplinary treatment can be determined using the occupancy rate of A. hadrus in the total bacteria in the sample as an index. Specifically, the lower the occupancy rate of A. hadrus in the total bacteria in the sample, the higher the risk of aggravation of adverse events during multidisciplinary treatment, and the higher the occupancy rate of A. hadrus in the total bacteria in the sample, the lower the risk of aggravation of adverse events during multidisciplinary treatment. Such risk determination is preferably performed by comparing the occupancy rate of A. hadrus in the total bacteria in the sample with a predetermined reference value set according to the level of risk. The reference value can be appropriately set by those skilled in the art, as described above. If the occupancy rate of A. hadrus in the total bacteria in the sample is equal to or less than the reference value, the risk of aggravation of adverse events during multidisciplinary treatment can be determined to be high. On the other hand, if the occupancy rate of A. hadrus in the total bacteria in the sample is higher than the reference value, the risk of aggravation of adverse events during multidisciplinary treatment can be determined to be low. In one example, if the adverse event is febrile neutropenia, when the occupancy rate of A. hadrus in the total bacteria in the sample is 0.25% or less, it can be determined that the risk of febrile neutropenia becoming severe during multidisciplinary treatment is high, and when the occupancy rate of A. hadrus in the total bacteria in the sample is higher than 0.25%, it can be determined that the risk of febrile neutropenia becoming severe during multidisciplinary treatment is low. More specifically, when the occupancy rate of A. hadrus in the total bacteria in the sample is 0.25% or less, it can be determined that the febrile neutropenia during multidisciplinary treatment is likely to be equivalent to Grade 3, and when the occupancy rate of A. hadrus in the total bacteria in the sample is higher than 0.25%, it can be determined that the febrile neutropenia during multidisciplinary treatment is likely to be equivalent to Grade 0. In another example, if the adverse event is diarrhea, when the occupancy rate of A. hadrus in the total bacteria in the sample is higher than 0.25%, it can be determined that the febrile neutropenia during multidisciplinary treatment is likely to be equivalent to Grade 0. When the occupancy rate of A. hadrus in the total bacteria in the sample is higher than 0.084%, the risk of diarrhea becoming severe during multidisciplinary treatment can be determined to be low.More specifically, when the occupancy rate of A. hadrus in the total bacteria in the sample is 0.084% or less, it can be determined that there is a high possibility that diarrhea during multidisciplinary treatment will correspond to the above-mentioned grade 3 or higher, for example, grade 3 to 4, and when the occupancy rate of A. hadrus in the total bacteria in the sample is higher than 0.084%, it can be determined that there is a high possibility that diarrhea during multidisciplinary treatment will correspond to the above-mentioned grade 0 to 2. The determination of the risk of aggravation of an adverse event is particularly suitably applied when the adverse event is diarrhea.

[0040] The method of the present invention is preferably carried out before multimodal therapy or at an early stage of multimodal therapy (for example, during or after one course of multimodal therapy including multiple courses), and more preferably before multimodal therapy.

[0041] According to the method of the present invention, the risk of adverse events during multidisciplinary treatment can be determined early, particularly before treatment, making it possible to formulate a more appropriate treatment plan for each patient, which in turn leads to an improvement in the patient's quality of life and therapeutic efficacy. For patients determined to have a low risk of adverse events during multidisciplinary treatment, the planned multidisciplinary treatment can be implemented, and for patients determined to have a high risk of adverse events during multidisciplinary treatment, measures to prevent or alleviate adverse events, such as reducing the drug dose or using synbiotics in combination, can be implemented.

[0042] To carry out the method of the present invention, it is preferable to use a kit containing a protocol for measuring A. hadrus in a sample. The kit includes an A. hadrus measurement reagent and a protocol (which describes the A. hadrus measurement method, criteria for determining the risk of adverse events during multidisciplinary treatment, factors that affect the measurement results and the degree of their influence, etc.). The criteria can be used to make a determination as in the above-mentioned method. Here, examples of the A. hadrus measurement reagent include the aforementioned reagents for measuring the number of enterobacteria, reagents for RNA detection, reagents for DNA detection, primers that can specifically detect A. hadrus, and primers that can detect enterobacteria.

[0043] Furthermore, from the above, it is believed that agents that reduce the risk of adverse events during multidisciplinary treatment can be screened by using fluctuations in the number or occupancy rate of A. hadrus bacteria, preferably fluctuations in the number of bacteria, as an index. Here, the concept of "fluctuations in the number of bacteria" used as an index includes cases where the number of A. hadrus bacteria increases after administration of the test substance, where the increase in the number of A. hadrus bacteria is promoted by comparing before and after administration, and where the decrease in the number of A. hadrus bacteria is suppressed by comparing before and after administration. In other words, a test substance that increases the number of A. hadrus bacteria in vitro or in vivo, promotes the increase in the number of bacteria, or suppresses the decrease in the number of bacteria is determined to have an effect of reducing the risk of adverse events during multidisciplinary treatment. Furthermore, the concept of "fluctuations in the occupancy rate" used as an index includes cases where the occupancy rate of A. hadrus increases after administration of the test substance, and cases where the increase in the occupancy rate of A. hadrus is suppressed by comparing before and after administration. This concept includes cases where an increase in the A. hadrus occupancy rate is promoted, or a decrease in the A. hadrus occupancy rate is inhibited by comparing before and after administration. That is, a test substance that increases the A. hadrus occupancy rate in vitro or in vivo, promotes the increase in the occupancy rate, or inhibits the decrease in the occupancy rate is determined to have an effect of reducing the risk of adverse events during multidisciplinary treatment. For example, a test substance is administered to humans or experimental animals such as mice, rats, or rabbits, and compared with unadministered humans or experimental animals to determine whether the test substance changes the number or occupancy rate of A. hadrus bacteria in the sample. When the test substance is determined to have increased the number of A. hadrus bacteria, promoted the increase in the number of bacteria, or inhibited the decrease in the number of bacteria, the test substance can be used as an agent for reducing the risk of adverse events during multidisciplinary treatment. Alternatively, the test substance can be used as an agent for reducing the risk of adverse events during multidisciplinary treatment. When a test substance is determined to have increased the occupancy rate of hadrus, promoted the increase in occupancy rate, or inhibited the decrease in occupancy rate, the test substance can be used as an agent for reducing the risk of adverse events during multidisciplinary treatment.

[0044] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0045] Example 1 1. Methods (1) Analysis Subjects Eighty-one esophageal cancer patients aged 20 to 80 years who were scheduled to undergo neoadjuvant chemotherapy were enrolled in the study. Of these, 73 patients (38 in the prophylactic antibiotic group and 35 in the synbiotic group) from whom feces were collected at the time of enrollment and on the 8th day after the start of DCF therapy were analyzed. All patients who participated in this study provided informed consent prior to participation.

[0046] (2) Treatment DCF therapy is docetaxel 70 mg / m 2 and cisplatin 70 mg / m 2 On Day 1, 5-fluorouracil 700 mg / m 2 Patients in the prophylactic antibiotic group received levofloxacin 500 mg orally once daily from Day 5 to Day 15 of each chemotherapy course. Patients in the synbiotic group received 600 mL of Racol NF enteral liquid (Otsuka Pharmaceutical Factory, Inc.) daily from 3 days before the start of each chemotherapy course until Day 12, and probiotics including Lacticaseibacillus paracasei strain Shirota (Lacticaseibacillus paracasei YIT 9029: LcS) (Lactobacillus casei YIT 9029 (FERM BP-1366) before the reclassification of Lactobacillus in 2020) and Bifidobacterium breve strain from 3 days before the start of each chemotherapy course until the end of chemotherapy. 3 × 10 live cells of Yakult (BbrY) (Bifidobacterium breve YIT 12272 (FERM BP-11320)) 8 They took 3 g of Yakult BL Intestinal Regulator (Yakult Honsha Co., Ltd.) containing 10 or more cells per day, and 15 mL of Oligomate S-HP (Yakult Pharmaceutical Co., Ltd.) (containing 5 g of galactooligosaccharides) per day as a prebiotic.

[0047] (3) Evaluation of chemotherapy-induced toxicity The toxicity of febrile neutropenia and diarrhea, which are adverse events of chemotherapy, was evaluated using the criteria of the Common Terminology Criteria for Adverse Events v4.0 (CTCAE v4.0) published by the National Cancer Institute (NCI).

[0048] (4) Collection of Fecal Samples At the time of study enrollment and on Day 8 of the first chemotherapy course, approximately 1.0 g of fresh stool was collected with a collection spoon into a pre-weighed tube containing 2 mL of RNALater (Ambion) and φ5 mm zirconia beads. After shaking and suspending, the sample was stored at 4 °C until further processing. The primary processing of fecal samples used for nucleic acid extraction was performed as follows. After measuring the weight of the fecal sample, RNALater was added to make a 10-fold volume. This was shaken at 1,048 rpm for 10 minutes using a ShakeMaster Auto (Biomedical science). For RNA extraction, 40 μL of the fecal suspension was transferred to a new 2 mL tube containing 1 mL of PBS, centrifuged (4 °C, 13,000 × g, 5 minutes), and the supernatant was removed by decantation. This pellet was stored at -80 °C until RNA extraction. For DNA extraction, 200 μL of the suspension was transferred to a new 2 mL tube containing 1 mL of PBS and vortexed. This was centrifuged (4°C, 13,000 × g, 5 minutes), and 1 mL of the supernatant was removed. Another 1 mL of PBS was added, the mixture was suspended, and centrifuged (4°C, 13,000 × g, 5 minutes), and 1 mL of the supernatant was removed. The resulting 200 μL suspension was stored at -30°C until DNA extraction.

[0049] (5) 16S rRNA gene amplicon analysis using a next-generation sequencer. DNA extraction from fecal samples was performed using the QIAamp DNA Stool Mini Kit (QIAGEN GmbH) according to the manual. 16S rRNA gene amplicon analysis was performed as follows: The V1-V2 region of the 16S rRNA gene of each sample was amplified using the 27Fmod2 forward primer and the 338R reverse primer (SEQ ID NOs: 1 and 2) on an ABI 7500 Real-Time PCR System (Thermo Fisher Scientific). The PCR reaction solution (50 μL) was a mixture of 2x SYBR Premix Ex Taq II (50 μL, Takara Bio), Nuclease-Free Water (22 μL), each primer (100 nM, 1 μL), and template DNA (10 ng / mL, 1 μL). The PCR reaction was performed by heating at 95°C for 30 seconds for initial denaturation, followed by 25 cycles of 95°C for 5 seconds, 55°C for 30 seconds, and 72°C for 40 seconds. The amplified product was purified using the AMPure XP Kit (Beckman Coulter Genomics) and quantified using the Quant-iT PicoGreen dsDNA Kit (Invitrogen). Equal amounts of the amplified products from each sample were mixed to prepare libraries, which were then sequenced on a MiSeq platform (Illumina) using MiSeq Reagent Kits v2 (Illumina). As a result, 2,621,476 amplicon sequence reads were obtained (9,158-36,003 reads per sample).

[0050] (6) Processing of 16S rRNA gene sequence data QIIME2 (ver. 2019.10, https: / / qiime2.org / ) was used to process the sequence data. After performing quality control of the sequence data using the DADA2 plugin, the sequence data was compiled into ASVs (amplicon sequence variants), and the occupancy rate of each ASV for each subject was calculated. The SILVA 138 database (https: / / www.arb-silva.de / ) was used as the database for assigning lineage information.

[0051] (7) Design and validation of Anaerostipes hadrus species-specific primers. A multiple alignment of A. hadrus and related species was constructed using ClustalX using 16S rRNA sequences obtained from the DDBJ / GenBank / EMBL database. Based on this alignment, a primer set (SEQ ID NOs: 3 and 4) consisting of the AH7F forward primer and the AH1R reverse primer specific to the 16S rRNA gene of A. hadrus was designed using Primer3plus software (http: / / www.bioinformatics.nl / cgi-bin / primer3plus / primer3plus.cgi). Using this primer set, quantitative RT-PCR was performed using RNA extracted from cultured A. hadrus as a template on a QuantStudio 12K Flex Real-Time PCR System (Thermo Fisher). Quantitative RT-PCR was performed using a Qiagen OneStep RT-PCR kit (Qiagen). The PCR reaction solution (10 μL) was prepared by mixing 1× Qiagen OneStep RT-PCR Buffer (2 μL), 0.5× Q-solution (2 μL), dNTP Mixture (400 μM each), 100,000-fold diluted SYBR Green I (Molecular Probes), Qiagen OneStep RT-PCR enzyme mix (0.4 μL), AH7F forward primer and AH1R reverse primer (0.6 μM), and template RNA (5 μL). The RT-PCR reaction was performed under the following conditions: reverse transcription reaction at 50°C for 30 minutes, initial denaturation at 95°C for 15 minutes, and 40 cycles of 94°C for 5 seconds, 55°C for 30 seconds, and 72°C for 50 seconds. As a result, the A. hadrus type strain YIT10092 T (DSM3319 TIt was confirmed that an amplification product was obtained when RNA extracted from the designed primers was used. In addition, using the Primer-Blast program (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ), it was confirmed that no nonspecific amplification products due to the designed primers were present in existing databases. Furthermore, the specificity of the designed primers was confirmed by comparing RNA (10 5 As a result of carrying out quantitative RT-PCR using the 100-kDa IgG-1000 (equivalent to 1000 kDa cells) under the above conditions, it was confirmed that the 100-kDa IgG-1000 (equivalent to 1000 kDa cells) did not cross-react with any of the bacterial species.

[0052] (8) Quantification of A. hadrus bacterial counts by RT-qPCR. Total RNA was extracted from fecal samples using a modified AGPC (acid guanidinium thiocyanate-phenol-chloroform extraction) method. Thawed samples were resuspended in a mixture of 346.5 μL of RLT Buffer (Qiagen), 3.5 μL of β-mercaptoethanol (Sigma-Aldrich), and 100 μL of Tris-EDTA buffer. Glass beads (0.1 mm, 300 mg, BioSpec Products) were added, and the mixture was shaken at 1,048 rpm for 5 minutes using a ShakeMaster Auto (Biomedical science). Water-saturated phenol (500 μL) was added, and the mixture was incubated at 60°C for 10 minutes. Then, chloroform-isoamyl alcohol (24:1, 100 μL) was added, and the mixture was stirred and centrifuged (4°C, 14,000 × g, 5 minutes). A 470 μL aliquot of the supernatant was taken, and the same amount of chloroform-isoamyl alcohol (24:1) was added and stirred. A 400 μL aliquot of the supernatant was collected, and RNA was precipitated by isopropanol precipitation. The RNA was then recovered and dissolved in 200 μL of nuclease-free water (Ambion). A. hadrus in the sample was quantified using YIF-SCAN (registered trademark), which is based on the principle of quantitative RT-PCR (RT-qPCR). For RT-qPCR, the above-mentioned species-specific primers (SEQ ID NOS: 3 and 4) targeting 16S rRNA for A. hadrus and the reaction conditions were used. The threshold cycle (Cq) values ​​in the linear range of the assay were substituted into the standard curve to determine the corresponding bacterial count in each sample. From this, the bacterial count in each sample was determined. The standard curve was created using Cq values ​​and the standard strain A. hadrus YIT10092, whose bacterial count had been determined by DAPI staining. T (DSM3319 T The limit of detection (LOD) was determined as the smallest number of bacteria in the standard curve (10 -2 cells / reaction).

[0053] The sequences of the primers used in (5), (7) and (8) above are shown in Table 1 below.

[0054]

[0055] (9) Sequencing of RT-qPCR Amplified Products To confirm whether the amplified products were derived from the target microorganism, the bacterial species amplified by RT-qPCR was estimated by sequence analysis. RT-qPCR products (10 μL) were purified using a MultiScreen® Filter Plate (Merck Millipore). Cycle sequencing reactions were performed using the BigDye® Terminator version 3.1 Cycle Sequencing kit (Applied Biosystems) according to the manufacturer's instructions. The amplified products were purified by ethanol precipitation, dissolved in formamide (1 μL), denatured, and sequenced using an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems). Comparison of the resulting rRNA sequences for assignment to specific species was performed using the BLAST program from NCBI (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0056] (10) Statistical Analysis Statistical analysis was performed using R (ver. 3.6.0) (https: / / www.r-project.org / ). Continuous variables were expressed as median (interquartile range). The non-parametric Mann-Whitney U test was used to test for differences between two groups. Differences in the ASV composition of the intestinal microbiota between groups were evaluated using ANOVA-Like Differential Expression Analysis (ALDEx) analysis with centered log-ratio transformed data. The aldex function (ALDEx2 package) was used for ALDEx analysis. For RT-qPCR analysis results, samples below the lower limit of quantification were substituted with half the lower limit of quantification and used for analysis. In both analyses, p<0.05 was considered significant.

[0057] 2. Results (1) Comparison of intestinal flora after DCF therapy based on the onset and severity of adverse events The composition of ASV (amplicon sequence variant) detected 8 days after the start of DCF therapy in the prophylactic antibiotic group (antibiotics group) and the synbiotics group (synbiotics group) was compared based on the severity of febrile neutropenia, an adverse event of DCF therapy. The occupancy rate of ASV15 was significantly higher in patients who did not develop febrile neutropenia during one course of DCF therapy (grade 0) compared with patients who developed febrile neutropenia (grade 3). As a result of assignment using SILVA as the reference database, ASV15 was estimated to be Anaerostipes hadrus. The correlation between the occupancy rate of ASV15, which was suggested to be associated with adverse events, and the severity of febrile neutropenia and diarrhea was analyzed, and the Spearman correlation coefficient was calculated. As a result, a significant negative correlation was found between ASV15 and the severity of febrile neutropenia and diarrhea (Table 2).

[0058]

[0059] (2) Changes in A. hadrus bacterial counts due to DCF therapy No significant difference was observed between the antibiotics group and the synbiotics group in the fecal A. hadrus bacterial counts before DCF therapy. Eight days after the start of DCF therapy, the antibiotics group showed a tendency for A. hadrus bacterial counts to decrease compared to before DCF therapy (p = 0.11), but the synbiotics group showed no decrease in A. hadrus bacterial counts after DCF therapy. Although DCF therapy and administration of prophylactic antibiotics reduced A. hadrus bacterial counts, oral administration of synbiotics inhibited this decrease in bacterial counts.

[0060] (3) Comparison of A. hadrus bacterial counts depending on the presence or absence of febrile neutropenia In the antibiotics and synbiotics groups, patients who did not develop febrile neutropenia during one course of DCF therapy (grade 0) had significantly higher A. hadrus bacterial counts both before and after DCF therapy compared with patients who developed febrile neutropenia (grade 3) (Figure 1). Even in the analysis of the synbiotics group alone, patients who did not develop febrile neutropenia (grade 0) tended to have higher A. hadrus bacterial counts both before and after DCF therapy compared with patients who developed febrile neutropenia (grade 3) (Figure 2). Therefore, it was suggested that febrile neutropenia was alleviated in patients with high intestinal A. hadrus bacterial counts before treatment.

[0061] (4) Comparison of A. hadrus bacterial counts by severity of diarrhea In the antibiotics and synbiotics groups, patients who developed extremely severe diarrhea during one course of DCF therapy (grade 3-4: increased bowel movements 7 or more times per day compared to baseline; fecal incontinence present; hospitalization or emergency treatment required) were compared with patients with less severe diarrhea (grade 0-2: no diarrhea or increased bowel movements <6 times per day compared to baseline). The A. hadrus bacterial counts were significantly higher in patients with less severe diarrhea both before and after DCF therapy (Figure 3). Even in the analysis of only the synbiotics group, patients with less severe diarrhea (grade 0-2) tended to have higher A. hadrus bacterial counts both before and after DCF therapy compared with patients with extremely severe diarrhea (grade 3-4) (Figure 4). Therefore, even before treatment, intestinal A. It was suggested that diarrhea was reduced in patients with high numbers of S. hadrus bacteria.

[0062] (5) The Importance of Intestinal A. hadrus Bacterial Counts Before DCF Therapy in the Suppression of Fever Neutropenia After DCF Therapy with Synbiotics When comparing baseline characteristics based on the presence or absence of febrile neutropenia in the synbiotics group, slight differences in patient gender were observed, but no significant differences were observed in patient age, BMI, dysphagia score, tumor location, stage, white blood cell count, neutrophil count, total lymphocyte count, or serum albumin. Therefore, to examine risk factors for adverse events in DCF therapy under synbiotic therapy, logistic regression was performed using patient gender and A. hadrus bacterial counts. The results are shown in Table 3. In Table 3, CI represents the confidence interval, OR represents the odds ratio, and the cutoff value for A. hadrus bacterial counts was calculated using receiver operating characteristic (ROC) analysis. In univariate analysis, the A. hadrus bacterial count before DCF therapy was significantly associated with a reduced risk of developing febrile neutropenia (OR, 0.13; 95% CI, 0.02-0.66, p=0.023). Furthermore, in multivariate analysis including patient gender, the A. hadrus bacterial count before DCF therapy was significantly associated with a reduced risk of developing febrile neutropenia (OR, 0.11; 95% CI, 0.01-0.60, p=0.019). The cutoff value was 10 per gram of specimen. 7.7 There were 100 pieces.

[0063]

[0064] (6) Importance of intestinal A. hadrus occupancy rate before DCF therapy in the suppression of febrile neutropenia after DCF therapy with synbiotics. To examine risk factors for adverse events in DCF therapy under synbiotic therapy, logistic regression was performed using patient gender and A. hadrus occupancy rate. The results are shown in Table 4. In Table 4, CI indicates confidence interval, OR indicates odds ratio, and the cutoff value for A. hadrus occupancy rate was calculated by receiver operating characteristic (ROC) analysis. In univariate analysis, A. hadrus occupancy rate before DCF therapy tended to be associated with a reduced risk of developing febrile neutropenia (OR, 0.24; 95% CI, 0.05-1.02, p = 0.060). Furthermore, in multivariate analysis including patient gender, A. hadrus occupancy rate before DCF therapy was also significantly associated with a reduced risk of developing febrile neutropenia. Hadrus occupancy tended to be associated with a decreased risk of developing febrile neutropenia (OR, 0.20; 95% CI, 0.03-0.95, p=0.051), with a cutoff value of 0.25%.

[0065]

[0066] (7) Importance of intestinal A. hadrus bacterial count before DCF therapy in the suppression of diarrhea after DCF therapy with synbiotics. When baseline characteristics were compared between the synbiotics group and the synbiotics group based on the severity of diarrhea, no significant differences were observed in patient age, sex, BMI, dysphagia score, tumor location, stage, white blood cell count, neutrophil count, total lymphocyte count, and serum albumin. Therefore, to examine risk factors for adverse events in DCF therapy under synbiotic therapy, logistic regression was performed using the A. hadrus bacterial count. The results are shown in Table 5. In Table 5, CI represents the confidence interval, OR represents the odds ratio, and the cutoff value for A. hadrus bacterial count was calculated by receiver operating characteristic (ROC) analysis. In univariate analysis, the A. hadrus bacterial count before DCF therapy was significantly associated with the risk of adverse events. The number of S. hadrus bacteria tended to be associated with a decreased risk of diarrheal severity (OR, 0.14; 95% CI, 0.01-1.13, p=0.11). 7.6 There were 100 pieces.

[0067]

[0068] (8) Importance of the intestinal A. hadrus occupancy rate before DCF therapy in the suppression of diarrhea after DCF therapy with synbiotics. To examine risk factors for adverse events in DCF therapy under synbiotic therapy, logistic regression was performed using the A. hadrus occupancy rate. The results are shown in Table 6. In Table 6, CI indicates confidence interval, OR indicates odds ratio, and the cutoff value for A. hadrus occupancy rate was calculated by receiver operating characteristic (ROC) analysis. In univariate analysis, the A. hadrus occupancy rate before DCF therapy tended to be associated with a reduced risk of diarrhea severity (OR, 0.05; 95% CI, 0.00-0.42, p = 0.014). The cutoff value was 0.084%.

[0069]

[0070] From the above, A. hadrus can be used to determine the risk of adverse events during multidisciplinary treatment, particularly during drug therapy.

Claims

1. A method for determining the risk of adverse events during multidisciplinary treatment, comprising measuring Anaerostipes hadrus in a sample collected from a subject.

2. The method according to claim 1, wherein the risk of an adverse event is the risk of onset or aggravation of the adverse event.

3. The method according to claim 1, wherein the multimodal therapy is a therapy selected from drug therapy, surgical therapy, radiation therapy, and combinations thereof.

4. The method of claim 1, wherein the multimodal therapy is drug therapy.

5. The method of claim 1, wherein the adverse event is at least one selected from the group consisting of febrile neutropenia and diarrhea.

6. The method described in claim 1, wherein the adverse event is febrile neutropenia.

7. 10. The method of claim 1, wherein the sample is a fecal sample from the subject.

8. The method according to any one of claims 1 to 7, further comprising comparing the number of Anaerostipes hadrus bacteria in the sample with a standard value, and determining that the risk of adverse events during multidisciplinary treatment is high when the number of bacteria is equal to or less than the standard value.

9. The method according to any one of claims 1 to 7, further comprising comparing the occupancy rate of Anaerostipes hadrus in the total bacteria in the sample with a reference value, and determining that the risk of adverse events during multidisciplinary treatment is high when the occupancy rate is equal to or lower than the reference value.

10. The number of Anaerostipes hadrus bacteria in the sample is 10 per 1 g of sample. ７．７ The method according to any one of claims 1 to 7, further comprising determining that the risk of febrile neutropenia during multidisciplinary therapy is high when the number of Anaerostipes hadrus bacteria is 0.25% or less, or when the occupancy rate of Anaerostipes hadrus in the total bacteria in the sample is 0.25% or less.

11. The number of Anaerostipes hadrus bacteria in the sample is 10 per 1 g of sample. ７．６ The method according to any one of claims 1 to 5 and 7, further comprising determining that the risk of diarrhea during multidisciplinary treatment is high when the number of Anaerostipes hadrus bacteria in the sample is 0.084% or less, or when the occupancy rate of Anaerostipes hadrus in the total bacteria in the sample is 0.084% or less.

12. A kit for carrying out the method according to any one of claims 1 to 7, comprising a reagent and a protocol for measuring Anaerostipes hadrus in a sample.

13. A screening method for an agent for reducing the risk of adverse events during multidisciplinary treatment, using the number or occupancy rate of Anaerostipes hadrus as an index.