WO2017110764A1 - Test method for predicting effectiveness and safety of multi-kinase inhibitor, test kit, and biomarker - Google Patents

Abstract

Provided are a test method for predicting the effect of administering Regorafenib, and a test kit. Measurements are taken of the CCL-5 concentration and the VEGF-A concentration in a specimen collected from a patient, whereby the effectiveness of treatment on the patient and the risk of adverse events are estimated.

Description

Test method, test kit, and biomarker for predicting efficacy and safety of multikinase inhibitor

The present invention relates to a method for predicting the effectiveness and safety of a multikinase inhibitor targeting tumor cells, and a test kit. Among multikinase inhibitors, the present invention particularly relates to the effectiveness of regorafenib, a method for predicting the risk of side effects, biomarkers used therefor, and kits.

Chemotherapy using an anticancer drug is a treatment method that is widely used together with surgery and radiation therapy as a tumor treatment method. Chemotherapy using anticancer drugs involves treating cancer cells that may spread throughout the body or cancer cells that have the potential to metastasize by administering anticancer drugs orally or by injection. It is aimed.

Anticancer drugs include platinum agents, alkylating agents, plant alkaloids that act on mitotic cells, and specific properties of cancer cells at the molecular level. There are various types such as targeted drugs. However, side effects often occur because any anticancer agent acts on normal cells. Symptoms that often appear as side effects include decreased white blood cells and platelets, vomiting / nausea, hair loss, general malaise, and stomatitis. Since side effects occur with most anticancer agents, anticancer agents with reduced side effects and pharmaceuticals that reduce side effects have been developed. As a result, side effects have been reduced compared to before, but the side effects caused by the administration of anticancer agents are a major problem in chemotherapy.

Also, it is known that the types and severity of side effects are not only different depending on the anticancer agent used, but also vary greatly from individual to individual. Furthermore, it is known that not only side effects but also adverse events whose causal relationship is not clear. Furthermore, because there are individual differences in the effects of anticancer agents, even for the same type of tumor, some patients may not be very effective and may have strong side effects. Therefore, if the anticancer drug administration can be expected to have a significant effect and whether serious side effects or adverse events occur can be determined before administering the anticancer drug, Anticancer drugs with few side effects and adverse events can be selected. If the effectiveness and safety of drug administration can be predicted, it will be a great advantage for patients.

In recent years, analysis has been conducted on the relationship between markers in blood that change due to anticancer drug treatment and the effectiveness of treatment (Patent Documents 1 and 2, Non-Patent Documents 1 and 2). Patent Document 1 discloses a method for predicting the risk of side effects of docetaxel used for treatment of refractory breast cancer, non-small cell lung cancer, ovarian cancer, etc. by detecting single nucleotide polymorphisms present on SLCO1B3 gene and ABCC2 gene. Is described. Patent Document 2 describes a method for predicting side effects by analyzing the correlation between side effects caused by fluorouracil and cisplatin used in a wide variety of treatments and cytokine gene polymorphisms.

Non-patent document 1 describes that the therapeutic effect of epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) administration on non-small cell lung cancer patients is correlated with serum HGF concentration. It is disclosed. Non-Patent Document 2, FOLFIRI therapy for patients with colorectal cancer (Fol nic acid, F luorouracil, Iri notecan) performs a therapy plus bevacizumab, analyzes the correlation between efficacy and biomarker for therapeutic It is described. In particular, it has been disclosed that changes in serum VEGF-A concentration correlate with therapeutic efficacy.

However, the analysis by genetic polymorphism disclosed in Patent Documents 1 and 2 requires that DNA is extracted from serum and PCR is performed, so that protein in serum is directly measured by immunoassay represented by ELISA. Compared to the method, there are many processes and complicated.

Also, as disclosed in the above prior art documents, the risk of side effects caused by administration of anticancer agents has been analyzed for specific anticancer agents and chemotherapy regimens. However, since many anticancer agents do not have markers for examining the efficacy and side effects before treatment, it is the actual situation that the efficacy of treatment and the strength of side effects are being examined while performing chemotherapy.

Regorafenib is a multikinase inhibitor that inhibits multiple protein kinase activities. Legorafenib is an angiogenesis-related kinase (VEGFR1-3, TIE2), a tumor microenvironment-related stromal kinase (PDGFRβ, FGFR), a tumorigenesis-related kinase (KIT, RET, RAF-1, BRAF) Have been shown to inhibit multiple protein kinase activities.

In Japan, it was approved on March 25, 2013 for the efficacy and effects of unresectable advanced / recurrent colorectal cancer. In clinical practice, it is common to use single-agent therapy as the last line when progression of relapsed colorectal cancer that cannot be curatively resected has progressed against existing standard chemotherapy. On August 20, 2013, it was also approved for gastrointestinal stromal tumor (GIST) that worsened after chemotherapy.

Regorafenib is a side effect (including abnormal laboratory values) in 465 cases (93.0%) of 500 cases (including 65 Japanese) in a global phase III clinical trial for patients with colorectal cancer Was recognized. The number of cases of incidence of major side effects (incidence rate) was 225 cases of hand-foot syndrome (45.0%), 169 cases of diarrhea (33.8%), 152 cases of decreased appetite (30.4%), and 145 cases of fatigue (29 0.0%), dysphonia 142 cases (28.4%), hypertension 139 cases (27.8%), and rash 113 cases (22.6%).

In an international joint phase III clinical trial for patients with gastrointestinal stromal tumors, 130 (98.5%) of 132 cases (including 12 Japanese) had side effects (including abnormal laboratory values). Was recognized. The number of cases of incidence of major side effects (incidence rate) was 87 cases (65.9%) for hand-foot syndrome, 64 cases (48.5%) for hypertension, 53 cases (40.2%) for diarrhea, 44 cases for dysphonia (33) 3%), 39 cases (29.5%) of fatigue, 38 cases (28.8%) of rash, and 28 cases (21.2%) of decreased appetite.

In addition, as serious side effects, limb syndrome, toxic epidermal necrolysis (TEN), mucocutaneous eye syndrome (Stevens-Johnson syndrome), erythema multiforme, fulminant hepatitis, liver failure, liver dysfunction, Jaundice, bleeding (gastrointestinal bleeding, hemoptysis, pulmonary bleeding, intraperitoneal bleeding, vaginal bleeding, cerebral bleeding, nasal bleeding, hematuria, etc.), interstitial lung disease, thromboembolism (myocardial ischemia, myocardial infarction, etc.), hypertension, hypertension Crisis, post-plastic leukoencephalopathy, gastrointestinal perforation, gastrointestinal fistula, thrombocytopenia may occur.

Furthermore, when these side effects were compared between Japanese and others, it was clear that the incidence of Japanese was high. Liver dysfunction, which is one of the serious side effects, has been reported in Japanese deaths, and strict follow-up during treatment is recommended in the appropriate use guide. Although research on predictive markers for the efficacy and side effects of regorafenib was conducted in an international phase III clinical trial, no useful results were obtained in actual clinical practice (Non-patent Document 3).

JP 2009-240232 A JP 2007-006744 A

The object of the present invention is to extract a marker for predicting the effect of regorafenib and a marker relating to the onset of an adverse event, and to use it for actual clinical treatment. It is another object of the present invention to provide an inspection method and an inspection kit using the marker.

The present invention relates to the following test method, test kit, and biomarker related to the effect prediction of regorafenib.

(1) A test method for predicting the effect of administration of Regorafenib, wherein the test method comprises measuring CCL-5 concentration in a sample collected from a patient.
(2) The test method according to (1), wherein the sample is blood, plasma, serum, urine, ascites, or pleural effusion.
(3) The inspection method according to (1) or (2), wherein the sample is one before the start of treatment.
(4) The test method according to (3), wherein when the CCL-5 concentration is lower than a predetermined value, it is determined that improvement in progression-free survival (PFS) and overall survival (OS) can be expected Method.
(5) The test method according to any one of (1), (2), or (4), wherein a VEGF-A concentration in a patient sample is measured before the start of treatment and early after the start of the first treatment. A test method for determining that an improvement in PFS and OS can be expected when the VEGF-A concentration after the start of treatment tends to be lower than that before the start of treatment.
(6) The test method according to (5), wherein the VEGF-A concentration in the patient sample at the time of the exacerbation of the primary disease is measured and increased compared to the VEGF-A concentration early after the start of the first treatment Is an inspection method for determining that improvement of PFS and OS can be expected.
(7) The test method according to any one of (1) to (6), wherein at least one of Ang-2, bFGF, and CCL-2 in a patient sample before the start of treatment is measured, A test method to determine the risk of adverse events.
(8) A test kit for predicting the effect of regorafenib administration, comprising a reagent for measuring CCL-5 concentration in a patient sample.
(9) The test kit according to (8), further comprising a reagent for measuring a VEGF-A concentration.
(10) The test kit according to (8) or (9), comprising a reagent for measuring at least one concentration of Ang-2, bFGF, and CCL-2.
(11) The kit according to any one of (8) to (10), wherein the reagent is an immunoassay reagent.
(12) A biomarker for predicting the effect of regorafenib administration, which is CCL-5 and / or VEGF-A.
(13) A biomarker for predicting an adverse event caused by administration of regorafenib, wherein the biomarker comprises at least one of CCL-5, VEGF-A, Ang-2, bFGF, and CCL-2.

The present invention provides a marker for predicting the effects of regorafenib and a marker for the onset of adverse events, which can be used for actual clinical treatment. Specifically, by measuring serum CCL-5 (Chemokine (CC motif) ligand-5) in advance, it is highly responsive to treatment and has a low risk of serious adverse events and side effects. A patient can be selected and treated. Furthermore, by comparing the concentrations of VEGF-A (vascular endothelial growth factor-A, vascular endothelial growth factor-A) in the serum on the 21st day before and after treatment, the effect can be predicted more accurately at an early stage of treatment. be able to.

The figure which shows the regorafenib sensitivity in a cultured cell strain. The figure which shows the change of VEGF-A secretion amount by the regorafenib in a cultured cell strain. The figure which shows the result of CCL-5 density | concentration in a serum before a treatment start, and a survival analysis. FIG. 3 (A) shows the ROC curve of tumor shrinkage and CCL-5 value. FIG. 3 (B) shows CCL-5 ≦ 59.96 (ng / mL), CCL-5> 59.96 (ng / mL). The progression-free survival period is shown in FIG. 3 (C). The figure which analyzed the change of the VEGF-A value before a treatment start, and after a start, and progression-free survival. FIG. 4 (A) shows the relationship between the decrease on day 21 after the start of VEGF-A treatment and progression-free survival, and FIG. 4 (B) shows the decrease on day 21 after the start of treatment with VEGF-A and the primary disease. The relationship between the increase in exacerbation and progression-free survival is shown. The figure which analyzed progression-free survival time combining CCL-5 density | concentration before treatment start and VEGF-A density | concentration after treatment start. FIG. 5 (A) shows that the CCL-5 concentration before the start of treatment is less than or equal to the cut-off value, the VEGF-A concentration after the start of treatment decreases, and the CCL-5 concentration before the start of treatment is higher than the cut-off value. The comparison with the group in which the VEGF-A density | concentration after a treatment start increases is shown. FIG. 5B further shows a group not classified into both. The figure which analyzed the total survival time combining the CCL-5 density | concentration before a treatment start, and the VEGF-A density | concentration after a treatment start. FIG. 6A shows that the CCL-5 concentration before the start of treatment is less than or equal to the cut-off value, the VEGF-A concentration after the start of treatment decreases, and the CCL-5 concentration before the start of treatment is higher than the cut-off value. The comparison with the group in which the VEGF-A density | concentration after a treatment start increases is shown. FIG. 6B further shows a group that is not classified into both. The figure which analyzed the progression-free survival time combining CCL-5 density | concentration before a treatment start, and the VEGF-A density | concentration after a treatment start and a disease-exacerbation stage. FIG. 7A shows a group in which the CCL-5 concentration before the start of treatment is below the cut-off value, the VEGF-A concentration after the start of treatment decreases, and the VEGF-A concentration in the exacerbation stage increases, and the CCL before the start of treatment. The comparison is made with the group in which the -5 concentration is higher than the cut-off value, the VEGF-A concentration is increased after the start of the treatment, and the VEGF-A concentration is decreased in the exacerbation stage. FIG. 7B further shows a group that is not classified into both. The figure which analyzed the whole survival time combining the CCL-5 density | concentration before a treatment start, and the VEGF-A density | concentration after the start of treatment, and a disease-exacerbation stage. FIG. 8A shows a group in which the CCL-5 concentration before the start of treatment is below the cut-off value, the VEGF-A concentration after the start of treatment decreases, the VEGF-A concentration in the exacerbation stage increases, and the CCL before the start of treatment. The comparison is made with the group in which the -5 concentration is higher than the cut-off value, the VEGF-A concentration is increased after the start of the treatment, and the VEGF-A concentration is decreased in the exacerbation stage. FIG. 8B further shows a group not classified into both.

In the present invention, an adverse event refers to any undesirable or unintended sign, symptom, or illness that has occurred in a patient to whom a drug has been administered, including those in which the causal relationship with the drug is not clear. It is not limited to side effects with known causal relationships. Further, progression-free survival (Progression Free; Survival, PFS) and overall survival (Overall Survival; OS) are defined as follows. The progression-free survival period is from the date of start of administration to the date on which the exacerbation (PD) is determined or the death date due to any cause (regardless of cause of death), whichever comes first. Surviving cases that are not judged to be exacerbated are treated as censored on the last day when it is confirmed that there is no exacerbation. In other words, if an exacerbation is not confirmed when the event is canceled due to an adverse event, it is treated as being terminated.

“Overall survival period (OS)” means the total survival period from the start date of administration to the date of death due to any cause. In surviving cases, the date of confirmation of final survival is censored, and in the case of inability to follow up, the censored date is determined to be the last date of confirmation of survival before becoming untraceable.

In the present invention, the sample refers to blood, plasma, serum, urine, ascites, pleural effusion. This is because it is already known that the marker of the present invention is contained in these samples (Non-Patent Documents 4 to 8). In particular, blood, plasma, and serum are preferable as samples because the biomarker of the present invention can be measured with high sensitivity. The sample before starting treatment refers to a sample before starting treatment with regorafenib for patients who have become refractory or intolerant to standard chemotherapy. Moreover, although the sample of the 21st day is used for the sample at the early stage after the start of the first treatment in the Examples, the first cycle is one cycle (one cycle is provided with a one week rest period after oral administration for 3 weeks every day). Any sample may be used as long as it is a sample. Considering the effect of drug administration, the samples on the 14th to 28th days are preferable.

In addition, although the patient sample was examined here by ELISA, any measurement method may be used as long as the protein amount of the biomarker in the sample can be measured. Measurement is preferably performed by immunoassay because measurement sensitivity is high and testing can be performed relatively easily. In addition to ELISA, for example, radioimmunoassay (RIA), fluorescence immunoassay (FIA), fluorescence polarization immunoassay (FPIA), chemiluminescence immunoassay (CLIA), and the like can be used.

The “cut-off value” is a value that can satisfy both high diagnostic sensitivity (adverse event rate) and high diagnostic specificity when a disease is determined based on the value. For example, a value showing a high positive rate in an individual who has developed an adverse event and a high negative rate in an individual who has not developed an adverse event can be set as the cutoff value. Since the specific cut-off value varies depending on the sample and assay system used, it can be appropriately determined according to each assay system. The cut-off value calculation method may be determined by a method known in this field (for example, see Non-Patent Documents 9 to 11).

The kit of the present invention can contain an antibody for detecting CCL-5 and VEGF-A, which are biomarkers found in the present invention, a reagent for detecting this, and the like. The detection reagent includes a secondary antibody, a substrate agent, and a labeling substance (for example, fluorescent dye, enzyme). Moreover, these elements can also be mixed beforehand as needed. Further, it may include a solid phase such as a microtiter plate, a reaction container, a buffer for diluting a washing solution or an antibody, a positive control, a negative control, an instruction describing a protocol, and the like.

Hereinafter, the present invention will be specifically described.
(1) Analysis using cultured cells First, candidate factors as biomarkers were narrowed down by preclinical experiments and biological information analysis. Regorafenib is a multikinase inhibitor, as described above, and has been suggested to act on cancer cell environmental signals related to signal transduction and angiogenesis. However, it was not clear which of these factors was directly affected by regorafenib treatment. Therefore, the present inventors analyzed changes in factors secreted from cancer cells detected by microarray gene expression analysis or ELISA method, in addition to candidates selected from factors involved in angiogenesis and inflammatory cytokines. .

First, it was analyzed whether the cultured cell lines have different sensitivities to regorafenib. HCT-15, HCT-116 and HT-29 available from ATCC were used as human colon cancer cell lines.

The above colon cancer cell lines were seeded at a cell density of 9000 cells / mL using 10% FBS-RPMI medium, 24 hours later, regorafenib was added to a concentration of 0 μM, 1 μM, and 3 μM and cultured for 72 hours. Cell proliferation was analyzed by MTS assay (Promega). The results are shown in FIG.

As shown in FIG. 1, cell growth is suppressed in a concentration-dependent manner by adding regorafenib. In particular, the HT-29 and HCT-116 cell lines are extremely strong and suppress cell growth and are highly sensitive to regorafenib. On the other hand, even when regorafenib is added at 3 μM, the HCT-15 cell line is a cell line that is less resistant to cell growth than other cell lines and has resistance to regorafenib. It became clear.

Using these three types of colorectal cancer cell lines having different sensitivities to regorafenib, changes in factors secreted from cancer cells were analyzed by adding regorafenib by ELISA. As a result, VEGF-A was found as a factor having a significant change in the secretion amount in correlation with the regorafenib sensitivity of the cells (FIG. 2).

Regorafenib was added to colorectal cancer cell lines HCT-15, HCT-116, and HT-29, and VEGF-A concentration secreted into the culture medium was measured 48 hours later by ELISA (Quantikine ELISA kit from R & D Systems) ). The VEGF-A concentration is shown with the VEGF-A concentration in the culture medium being 100% when cultured without adding regorafenib in each cultured cell line.

In regorafenib-resistant colorectal cancer cells (HCT-15), VEGF-A secretion increases after regorafenib treatment. On the other hand, it was clarified that the amount of secretion in legolafenib-sensitive colon cancer cells (HT-29, HCT-116) was strongly reduced after treatment with regorafenib.

Information on the expression of VEGF-A in colon cancer cells can be obtained from Oncomine database (https://www.oncomine.org/resource/login.html). According to this, expression of VEGF-A is increased in colon cancer cells. In addition, according to Kumar et al. (Non-patent Document 12), it is shown that the concentration of VEGF-A is significantly higher in the serum of colorectal cancer patients than in the healthy human serum, and the VEGF-A level is higher in advanced cancer. . These facts strongly suggest that serum VEGF-A in patients with advanced colorectal cancer to whom regorafenib is administered depends on colorectal cancer.

A prospective study using actual patient sera, predicting that changes in serum levels of cytokines, especially VEGF-A, could be a good marker for regorafenib sensitivity, combining these findings with the data obtained Advanced. In addition to VEGF-A and the like, changes in other angiogenesis cytokines such as IL-8 and humoral factors also involved in inflammation related to cancer malignancy were also examined. In addition to VEGF-A, CCL-2, CCL-5, IL-8 (interleukin-8), PlGF (Placent Growth Factor), Ang-1 (angiopoietin-1), Ang-2 (angiopoietin-2), bFGF ( Analysis was performed using patient serum using basic fibroblast growth factor), SDF-1 (stromal cell-derived factor 1), and PDGFβ (platelet-derived growth factor beta) as candidate factors.

(2) Analysis using patient serum [subject]
In the present invention, patients with advanced recurrent colorectal cancer who were refractory or intolerant to standard chemotherapy were subjected to analysis by administering regorafenib according to the following selection criteria.

Selection criteria A case histologically diagnosed as colorectal cancer.
2. Cases in which refractory or intolerance has been confirmed by standard chemotherapy using fluorouracil (5-FU), oxaliplatin, irinotecan, and molecular targeted drugs (bevacizumab, cetuximab, panitumumab).
3. Cases with measurable lesions (according to New Guideline for Evaluation of Treatment Effect for Solid Cancer, New Response Evaluation Criteria in Solid Tumors; RECIST ver.1.1).
4). Cases where PS (Performance Status; ECOG (Eastern Cooperative Oncology Group) standard), which is an index of systemic symptoms, is 0 to 1.
5). Patients who are expected to survive for 12 weeks or more from the start date of administration.
6). A case where written consent was obtained from the patient himself / herself for participation in the study.

Exclusion criteria Cases with active double cancer (simultaneous double cancer or metachronous double cancer with a disease-free period of 5 years or less. However, it is equivalent to Carcinoma in situ (localized cancer) judged to be cured by local treatment or intramucosal cancer. Lesions are not included in active double cancer).
2. Complications that may cause serious problems in the study (interstitial pneumonia, pulmonary fibrosis, severe emphysema, intestinal paralysis, bowel obstruction, uncontrolled diabetes, liver cirrhosis, uncontrolled hypertension, myocardium within 3 months Patients with a history of infarction, heart disease, poorly controlled angina or arrhythmia).
3. Cases where participation in the study is considered difficult due to clinically problematic psychiatric and neurological symptoms.
4). Cases judged by doctors to be inappropriate for registration.

Specifically, 54 registered patients were analyzed from May 2013 to December 2014. The anti-tumor effect was 51.9% for patients with confirmed disease control (Disease control, DC) and 35.5% for patients with confirmed tumor reduction (Tumor shrinkage, TS). Median progression-free survival was 97 days and overall survival was median 264 days.

[Treatment]
Continue to use regorafenib until exacerbation of the disease and onset of adverse events that are difficult to control. In general, for adults, 160 mg of regorafenib is orally administered once daily for 3 weeks after meals, and then withdrawn for 1 week. This is repeated as one cycle. The dose may be reduced according to the patient's condition.

[Inspection method]
1. Serological measurement items and measurement method Before starting the first treatment of regorafenib, on the 21st day after the start of the first treatment, about 20 mL of blood is collected at the time of treatment discontinuation, and the serum is separated and stored at −80 ° C. Candidate cytokines were measured by ELISA.

The amount of cytokines such as CCL-5 and VEGF-A in the serum was determined according to the attached protocol by the sandwich ELISA method using Quantikine ELISA kit of R & D Systems.

First, the buffer attached to the kit was added to a 96-well plate coated with an antibody against each cytokine, and then a serum sample and a cytokine preparation for preparing a calibration curve (dilution series) were added and reacted at room temperature for 2 hours. . Thereafter, after washing three times with a washing buffer, an HRP-labeled antibody against each cytokine was added, and further reacted at room temperature for 1-2 hours. Thereafter, after washing three times with a washing buffer, a coloring solution was added, and after reacting at room temperature for 20-25 minutes, a reaction stop solution was added, and OD450 and OD570 (background) were measured using a plate reader. . Each sample was tested in triplicate, and the cytokine concentration was calculated based on the calibration curve for the value obtained by subtracting the background.

2. Anti-tumor evaluation, survival period RECIST ver. Evaluate with 1.1. Survival time was calculated using the Kaplan-Meier method.

3. Assessment of adverse events CTCAE ver. The severity was classified according to 4.0 (Common Terminology Criteria for Adverse Events v4.0; Adverse Event Common Terminology Criteria v4.0, Japanese translation JCOG version).

[analysis method]
Analyzes were conducted in two groups: disease control (DC) and non-morbidity control (Non-DC) or tumor reduction (TS) and non-tumor reduction (Non-TS), and predictors of antitumor effects were selected. It was. Regarding adverse events, predictors that correlate with adverse events were selected based on grades according to CTCAE 4.0. We also analyzed the correlation between the occurrence of adverse events and therapeutic effects.

Regarding the above-mentioned candidate factors, ELISA was conducted at 3 points before the start of the first treatment of regorafenib, 21 days after the start of the first treatment, and at the time of the decision to stop the treatment, and whether each candidate factor was correlated with the occurrence of an adverse event was analyzed. .

[result]
We analyzed 54 patients who received regorafenib monotherapy for metastatic colorectal cancer. The subject patient background is shown in Table 1. The targeted 54 patients have been treated at the Cancer Institute Ariake Hospital since 2013 and obtained informed consent.

Among the analyzed cytokines, it was CCL-5 and VEGF-A that were correlated with efficacy and adverse events. CCL-5 and VEGF-A levels in the sample are related to efficacy and incidence of adverse events that are particularly common in Japanese, ie hand-foot syndrome, hypertension, liver dysfunction, hyperbilirubinemia, thrombocytopenia It became clear that it was an effective predictive marker. The results will be described in detail below.

[Anti-tumor effect and cytokine analysis]
(1) Correlation between serum CCL-5 concentration before start of treatment and tumor shrinkage, survival time By ROC (receiver operating characteristic curve) analysis on serum CCL-5 value and tumor shrinkage (TS) before start of treatment, CCL- Significant tumor reduction was obtained when 5 ≦ 59.96 (ng / mL, cut-off value). FIG. 3 (A) shows an ROC curve created for tumor shrinkage and serum CCL-5 concentration. From this result, if the CCL-5 concentration in the serum before the start of treatment is CCL-5 ≦ 59.96 (ng / mL), a tumor reducing effect can be expected by administration of regorafenib. Since the cut-off value is calculated using a standard curve, it is unlikely that the cut-off value will deviate significantly from this value, but it may vary depending on the measurement method and the patient's disease state grade. In that case, it is preferable to recalculate the cutoff value as appropriate. According to Student's T-test, CCL-5 low (mean value 50.5 ± 23.8 ng / mL) was compared to CCL-5 high (mean value 65.8 ± 23.2 ng / mL) Reduction can be expected (p = 0.030). In the survival analysis, CCL-5 ≦ 59.96 (ng / mL, cut-off value), progression-free survival (PFS, FIG. 3 (B)), and overall survival (OS, FIG. 3 (C)). A statistically significant improvement trend was observed. Therefore, it is possible to predict tumor shrinkage, progression-free survival, and overall survival by measuring CCL-5 before the start of treatment.

(2) Correlation between changes in VEGF-A concentration and survival time Analysis of changes in cytokine levels before and after treatment reveals that the change in VEGF-A concentration before and after treatment correlates with survival time It became. If the VEGF-A concentration decreases from 21 days after the start of treatment than before treatment, the progression-free survival averages 146 days, whereas if increased, the progression-free survival averages 62 days (FIG. 4A).

In addition, the VEGF-A concentration decreased on the 21st day, and the pattern of increase during exacerbation of the original disease improved the progression-free survival period, and the average value of the progression-free survival period was 146 days. On the other hand, the VEGF-A concentration increased on the 21st day, and the pattern of decrease during the exacerbation of the primary disease had a progression-free survival period of 55 days (FIG. 4 (B)). There was a statistically significant correlation between the VEGF-A concentration on day 21 after the start of treatment and the concentration at the time of exacerbation of the original disease. Moreover, although it was not statistically significant, it was recognized that the same tendency was shown also in the whole survival period. That is, it became clear that the change in serum VEGF-A level after the start of treatment is a factor predicting the therapeutic effect.

(3) Correlation between changes in CCL-5 concentration and VEGF-A and survival time From the results of (1) and (2) above, CCL-5 values in serum before treatment start and VEGF-A after treatment start As a result of combining the changes in concentration, further improvement in progression-free survival and overall survival was clarified.

(3-1) Combination analysis of CCL-5 concentration before treatment start and VEGF-A concentration on the 21st day after the start of treatment The CCL-5 concentration before the start of the treatment is below the cut-off value, and the 21st day after the start of the treatment The group of patients with reduced VEGF-A concentration (Good group) and its opposite CCL-5 concentration before the start of treatment was higher than the cutoff value, and the VEGF-A concentration on the 21st day after the start of treatment increased. The patient group (Poor group) was compared. As a result, it was revealed that the progression-free survival period was significantly improved in the Good group (FIG. 5A).

Moreover, the patient group which is not classified into the above was analyzed as a Medium group. From the Kaplan-Meier curve, the progression-free survival period of the Medium group was a result intermediate between the Good group and the Poor group (FIG. 5B). Mean values of progression-free survival in the Good group, Medium group, and Poor group were 188 days, 69 days, and 70 days, respectively.

Furthermore, an analysis was performed on the overall survival time. As described above, the CCL-5 concentration before the start of treatment was below the cut-off value, and the VEGF-A concentration decreased on the 21st day after the start of treatment (Good group), and the opposite electrode before the start of treatment A comparison was made with a patient group (Poor group) in which the CVEGF-5 concentration was higher than the cut-off value and the VEGF-A concentration increased on the 21st day after the start of treatment (FIG. 6 (A)). Moreover, the patient group which does not belong to either was made into the Medium group, and the whole survival time was compared (FIG.6 (B)). The overall survival was similar to progression-free survival. The mean overall survival was 414 days in the Good group, compared to 187 days in the Medium group and 239 days in the Poor group.

(3-2) Combined analysis of CCL-5 concentration before the start of treatment and VEGF-A concentration on the 21st day after the start of treatment and VEGF-A concentration in the stage of exacerbation of the disease CCL-5 concentration before the start of treatment is below the cut-off value A group of patients (Good group) in which the VEGF-A concentration on the 21st day after the start of treatment is decreased and the VEGF-A concentration in the stage of exacerbation of disease is increased, and the CCL before the start of the treatment, which is the opposite electrode Compared with the patient group (Poor group) in which the VEGF-A concentration was higher than the cutoff value, the VEGF-A concentration increased on the 21st day after the start of treatment, and the VEGF-A concentration in the exacerbation period was decreased (FIG. 7 (A)). As a result, it was clear that the progression-free survival period was significantly improved in the Good group. Other than these were analyzed as a Medium group. As a result, from the Kaplan-Meier curve, the progression-free survival period of the Medium group was an intermediate result (FIG. 7B). Mean values of progression-free survival in the Good group, Medium group, and Poor group were 232 days, 70 days, and 53 days, respectively.

Furthermore, an analysis was performed on the overall survival time. Similar to the above, the CCL-5 concentration before the start of treatment is below the cut-off value, the VEGF-A concentration on the 21st day after the start of treatment decreased, and the VEGF-A concentration during the exacerbation stage increased The patient group (Good group) and its opposite CCL-5 concentration before the start of treatment were higher than the cut-off value, the VEGF-A concentration increased on the 21st day after the start of the treatment, and VEGF- A comparison was made with a patient group (Poor group) in which the A concentration decreased (FIG. 8 (A)). Moreover, the group which does not belong to neither was made into the Medium group, and the whole survival time was compared (FIG.8 (B)). The overall survival was similar to progression-free survival. The mean overall survival was 414 days in the Good group, compared to 239 days in the Medium group and 192 days in the Poor group.

From the above results, serum CCL-5 and VEGF-A can be used alone or in combination as a marker for predicting the effect of regorafenib monotherapy before treatment or at the beginning of treatment.

(4) Analysis of adverse events and cytokines An analysis was conducted to determine whether there was a correlation between the onset of adverse events and serum levels of cytokines before the start of treatment. The endpoints for adverse events were regorafenib, the most common hand-foot syndrome, hypertension, liver dysfunction, hyperbilirubinemia, and thrombocytopenia. Table 2 shows cytokine concentrations that are highly correlated with adverse events.

In the table, AE indicates an adverse event (Adverse Event), and T-Bil indicates the total amount of bilirubin. AST is an abbreviation for aspartate aminotransferase and ALT is an abbreviation for alanine aminotransferase, both of which are test values serving as indicators of liver function. HT represents hypertension, and HFS represents hand-foot syndrome. Ang-2, bFGF, and CCL-2 are as described above.

It was the values of Ang-2, bFGF, and CCL-2 that showed statistically significant differences between cytokine serum levels before the start of treatment and adverse events. When Ang-2 was high, hyperbilirubinemia (grade 3 ≦) was high. Moreover, AST (grade3 ≦) and ALT (grade3 ≦) were high at high bFGF, and a correlation between bFGF and liver function was observed. In addition, bFGF was low and hypertension (grade 3 ≦) was high, and CCL-2 was low and limb syndrome (grade 2 ≦) was high. The occurrence of these adverse events is largely related to withdrawal and weight loss during treatment. By measuring the concentrations of these cytokines before the start of treatment, one can predict the onset of a specific adverse event, so that regorafenib can be dosed while monitoring the patient's condition more carefully. That is, Ang-2, bFGF, and CCL-2 concentrations before the start of treatment can be predictive markers for the onset of adverse events involved in treatment performance.

Further, CCL-5 ≦ 59.96 (ng / mL, cut-off value) before starting treatment and adverse events, which were useful in determining the antitumor effect of regorafenib, were analyzed by chi-square test. The results are shown in Table 3. In the table, PLT represents thrombocytopenia, OR represents an odds ratio, and other abbreviations are the same as those in Table 2.

When the CCL-5 concentration was below the cut-off value (CO), the onset of hand-foot syndrome (grade1 ≦) and thrombocytopenia (grade1 ≦) were significantly higher. Hand-foot syndrome (HFS) grade 1 ≦ is 9.1 times more likely to develop when CCL-5 is below the cut-off value, and thrombocytopenia (PLT) grade 1 ≦ is about 4.2 times more likely to occur. It was. However, it did not correlate with the onset of serious cases. In addition, hyperbilirubinemia (grade 2 ≦) was slightly more frequent.

Further, change in CCL-5 concentration before and after treatment, combination of CCL-5 concentration before treatment start (CO value or less) and change in VEGF-A concentration after treatment start, or VEGF concentration on the 21st day after treatment start The correlation between changes in AEs and adverse events was analyzed by chi-square test. The results are shown in Table 4.

Regarding the correlation between changes in CCL-5 concentration before and after treatment and adverse events, AST values of grade 2 ≦ were particularly correlated when CCL-5 concentration decreased after treatment, and liver function abnormalities were frequently developed. Combination of CCL-5 concentration (below CO value) before treatment start and change in VEGF-A concentration after treatment start is associated with the development of hyperbilirubinemia (grade2 ≦) and thrombocytopenia (grade1 ≦, grade2 ≦) It became clear that Similar results were obtained with VEGF alone. That is, when the VEGF-A concentration on the 21st day after the start of treatment decreased, hyperbilirubinemia (grade1 ≦), hyperbilirubinemia (grade2 ≦), and thrombocytopenia (grade2 ≦) were significantly more frequent. It was.

From the above results, CCL-5 and VEGF-A can be used as predictive markers for the effect of pretreatment or early treatment onset of adverse events with high incidence in Japanese in regorafenib monotherapy in salvage therapy.

Also, the correlation between these adverse events and CCL-5 and VEGF-A is not inconsistent with the correlation between progression-free survival and overall survival. Using the CCL-5 concentration before treatment and the VEGF-A concentration at the early stage of treatment as an indicator of the effectiveness of regorafenib, and carefully observing the adverse events correlated with the CCL-5 and VEGF-A concentrations, Treatment can be performed. Moreover, an adverse event can be predicted by measuring the Ang-2, bFGF, and CCL-2 concentrations before starting treatment.

In the present invention, regorafenib was analyzed in detail, but application to other multikinase inhibitors such as sorafenib and sunitinib which are multikinase inhibitors having similar structures and activities of the compounds can also be expected.

As shown in the present invention, anticancer drug treatment is performed only on patients who are effective in treatment by selecting patients who respond well to anticancer drug administration and chemotherapy. The risk of serious side effects can be avoided.

Claims (13)

1. A test method for predicting the effect of administration of regorafenib,
   A test method comprising measuring CCL-5 concentration in a sample collected from a patient.
2. The inspection method according to claim 1,
   The test method, wherein the sample is blood, plasma, serum, urine, ascites, or pleural effusion.
3. The inspection method according to claim 1 or 2,
   An inspection method, wherein the sample is one before the start of treatment.
4. The inspection method according to claim 3,
   If the CCL-5 concentration is lower than the specified value,
   A test method for determining that improvement in progression-free survival (PFS) and overall survival (OS) can be expected.
5. The inspection method according to any one of claims 1, 2, or 4,
   Measure the VEGF-A concentration in the patient sample before the start of treatment and early after the start of the first treatment,
   When the VEGF-A concentration after the start of treatment is decreasing compared to before the start of treatment,
   Inspection method for determining that improvement of PFS and OS can be expected.
6. The inspection method according to claim 5,
   Measure VEGF-A concentration in patient sample at the time of exacerbation
   If there is an increase compared to the early VEGF-A concentration after initial treatment,
   Inspection method for determining that improvement of PFS and OS can be expected.
7. The inspection method according to any one of claims 1 to 6,
   Measuring at least one of Ang-2, bFGF, CCL-2 in a patient sample prior to treatment;
   A test method to determine the risk of adverse events.
8. A test kit for predicting the effects of regorafenib administration,
   A test kit comprising a reagent for measuring CCL-5 concentration in a patient sample.
9. The test kit according to claim 8, wherein
   Furthermore, a test kit comprising a reagent for measuring a VEGF-A concentration.
10. The test kit according to claim 8 or 9, wherein
    A kit comprising a reagent for measuring the concentration of at least one of Ang-2, bFGF and CCL-2.
11. A kit according to any one of claims 8 to 10,
    A kit, wherein the reagent is an immunoassay reagent.
12. A biomarker for predicting the effects of regorafenib administration,
    A biomarker characterized by being CCL-5 and / or VEGF-A.
13. A biomarker for predicting adverse events due to regorafenib administration,
    A biomarker comprising at least one of CCL-5, VEGF-A, Ang-2, bFGF, and CCL-2.

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