Serum Concentrations of Infliximab and IL-6 for Predicting One-Year Discontinuation of Infliximab Treatment Owing to Secondary Non-response in Patients with Rheumatoid Arthritis

Abstract

Secondary non-response to infliximab (IFX) occurs in some patients with rheumatoid arthritis (RA). Although therapeutic drug monitoring (TDM) is a useful tool to optimize IFX therapy, it is unclear whether it can help to identify the risk of secondary non-response. This study aimed to explore the utility of serum levels of IFX or other biomarkers to predict IFX discontinuation owing to secondary non-response. A single-center, retrospective study was conducted using the Kyoto University Rheumatoid Arthritis Management Alliance cohort database between 2011 and 2020. Serum IFX levels were measured using liquid chromatography-tandem mass spectrometry. An electrochemiluminescence assay was used to quantify serum levels of tumor necrosis factor-α and interleukin-6 and detect anti-drug antibodies. Eighty-four out of 310 patients were eligible for this study. The cutoff levels of biomarkers were determined by receiver operating characteristic analysis. IFX persistence was similar between groups stratified using IFX levels, tumor necrosis factor-α levels, interleukin-6 levels, and anti-drug antibodies positivity. The group with lower IFX and higher interleukin-6 levels had the worst therapy persistence (p = 0.017) and the most frequent disease worsening (90.0%, p < 0.001). Evaluating both interleukin-6 and IFX levels, not just IFX alone, enabled us to identify patients at risk of discontinuing IFX treatment. These findings support the utility of measuring IFX and interleukin-6 levels for successful maintenance therapy for RA.

INTRODUCTION

Infliximab (IFX), an anti-tumor necrosis factor-α (anti-TNF-α) monoclonal antibody, is highly effective in controlling disease activity in rheumatoid arthritis (RA).^1,2) However, some patients fail to maintain an initial response, also termed as secondary non-response.³⁾ A previous study analyzing data from two Canadian registries reported that the rates of secondary non-response were 68.3 and 55.7% among all anti-TNF-α non-responders within each follow-up period (2632 and 1114 person-years, respectively).⁴⁾ Among the patients with secondary non-response, 87.0 and 60.9%, respectively, discontinued the treatment after 12 months of anti-TNF-α initiation.⁴⁾ In another cohort study, during the first year, 55% of the primary responders ceased IFX therapy predominantly owing to secondary non-response.⁵⁾ As secondary non-response can lead to subsequent discontinuation of treatment, the current challenge is to predict and avoid such discontinuation.

To date, several studies have reported a correlation between the trough levels of IFX and clinical response. In randomized controlled trials (RCTs) such as the ATTRACT and RISING studies, greater improvement in the responses according to the criteria of the American College of Rheumatology (ACR) or European Alliance of Associations for Rheumatology (EULAR) were found in patients with higher trough serum concentrations of IFX at week 54.^6,7) Observational studies have also reported a correlation between an increase in serum IFX concentration and clinical improvement.^8,9) Several factors can affect the pharmacokinetics of IFX. The formation of anti-drug antibodies (ADAs) reduces serum drug levels.^10,11) In a previous study, 43% of RA patients treated with IFX developed ADAs by one year of follow-up after therapy initiation.¹²⁾ Additionally, baseline TNF-α levels might affect serum IFX levels.¹³⁾ Interindividual variation in IFX levels can affect clinical responses.

Monitoring serum IFX concentrations has been useful for therapy optimization in clinical practice.¹⁴⁾ In our previous study, we demonstrated the potential application of therapeutic drug monitoring (TDM) of IFX to facilitate the identification of secondary non-responders at the sampling point.¹⁵⁾ Recently, the NOR-DRUM study assessed the effectiveness of IFX TDM for achieving remission in patients starting treatment (part A) and ensuring disease control in patients on maintenance treatment (part B).¹⁶⁾ The results showed that treatment with TDM was a more effective strategy than treatment without TDM in sustaining disease control without disease worsening.¹⁷⁾ Thus, TDM might contribute to adjusting the treatment with IFX. However, no data exist on the use of TDM to identify patients at risk of secondary non-response to IFX.

In the present study, using real-world cohort data, we aimed to explore the utility of serum IFX levels and other serological factors to predict the need for IFX discontinuation owing to secondary non-response.

PATIENTS AND METHODS

Patients

A single-center, retrospective study was conducted using the Kyoto University Rheumatoid Arthritis Management Alliance (KURAMA) cohort database. The database was established in 2011 by the Center for Rheumatic Diseases at Kyoto University Hospital, as described previously.¹⁸⁾ For the present study, we analyzed cohort data from the KURAMA database, collected between January 1, 2011, and December 31, 2020. All patients fulfilled the revised 1987 ACR or 2010 ACR/EULAR classification criteria for RA.^19,20) Written informed consent to participate in the study was obtained from all patients. All data were de-identified and analyzed anonymously. The present study adhered to the principles of the Declaration of Helsinki and was approved by the Medical Ethics Committee of Kyoto University Graduate School and Faculty of Medicine (Approval Number: R0357).

Data Collection and Evaluation of Disease Activity

The patient data at the sampling point (baseline) were obtained including age, body weight, sex, RA disease duration, IFX treatment duration, IFX dose, weekly methotrexate dose, oral glucocorticoid use, other conventional synthetic disease-modifying antirheumatic drug (csDMARD) use, levels of C-reactive protein (CRP), erythrocyte sedimentation rate at one hour (ESR_1h), anti-cyclic citrullinated peptide antibody (ACPA), and rheumatoid factor (RF). Actarit, aurothiomalate, auranofin, bucillamine, iguratimod, leflunomide, mizoribine, salazosulfapyridine, cyclosporine, and tacrolimus were considered other csDMARDs. As disease activities of RA, we evaluated tender joint count (TJC), swollen joint count (SJC), clinical disease activity index (CDAI), simplified disease activity index (SDAI), and disease activity score-28 for rheumatoid arthritis with erythrocyte sedimentation rate (DAS28-ESR). The health assessment questionnaire-disability index (HAQ-DI) was also used as a measure of functionality. Levels of RF and ACPA higher than 15 and 4.5 U/mL, respectively, were defined as positive results.

Clinical Events

In the KURAMA cohort, the reasons for drug discontinuation were classified into four major categories: 1) lack of effectiveness, 2) disease remission, 3) toxic adverse events, and 4) non-toxic reasons. There is a tendency to identify primary responses within the first 6 months of treatment.³⁾ In the present study, the primary outcome was time to discontinuation of IFX owing to secondary non-response, which was defined as the loss of response after 6 months (24 weeks) of treatment. Disease worsening and intensification of IFX treatment were assessed as secondary outcomes. Disease worsening was defined as an increase in DAS28-ESR of 1.2 or more from the minimum scores in individuals, resulting in moderate to severe disease activity (DAS28-ESR >3.2) by EULAR criteria. Intensification of IFX treatment was defined as a dose escalation (of at least 100 mg) and/or a shorter interval between infusions (of at least 14 d at least twice consecutively) compared with the dose and the interval of time between administrations employed at the time of the individual minimum DAS28-ESR scores.

Measurement of Serum IFX, TNF-α, and Interleukin (IL)-6 Levels

Trough serum IFX levels were measured using a liquid chromatography-electrospray ionization-triple quadrupole mass spectrometer (Nexera × 2 and LCMS-8060, Shimadzu, Kyoto, Japan), as previously reported.²¹⁾ To obtain the signature peptides from the fragment antigen-binding (Fab) region of IFX, serum samples were pretreated using the nSMOL™ Antibody BA Kit (Shimadzu) according to the provided protocol.

Serum TNF-α and IL-6 levels were analyzed using commercially available V-PLEX Human kits (Meso Scale Discovery (MSD), Rockville, MD, U.S.A.) according to the manufacturer's instructions. This assay detected only free TNF-α; TNF-α complexed with IFX was not detected (data not shown). The electrochemiluminescence (ECL) signal from the solution was measured using a MESO QuickPlex SQ120 plate reader (Meso Scale Discovery). MSD Discovery Workbench software (Meso Scale Discovery) was used for analysis. Only the first sample collected during the study period was analyzed for each patient.

Detection of ADAs in Serum

For the detection of serum antibodies against IFX, we developed an ECL assay on the Meso Scale Discovery^® platform, as previously reported.¹⁵⁾ We employed a multi-tiered approach, which included a screening assay for the identification of ADA-positive samples without acidification (drug-sensitive) and a confirmatory assay for determining ADA specificity. The ECL signal from the solution was measured using MESO QuickPlex SQ120 (Meso Scale Discovery).

Statistical Analysis

Statistical analysis was performed using GraphPad Prism v7.0 (GraphPad Software Inc., La Jolla, CA, U.S.A.) and JMP^® Pro15 (SAS Institute, Inc., Cary, NC, U.S.A.). Continuous data are expressed as mean ± standard deviation (S.D.) or median (min to max) and analyzed using nonparametric tests (Wilcoxon rank-sum test). Categorical data expressed as percentages were analyzed using the chi-squared test or Fisher's exact test. A p-value ≤ 0.05 was considered statistically significant. The Kaplan–Meier method was performed to evaluate the time to IFX discontinuation owing to ineffectiveness. Patients who received IFX consistently for at least one year from baseline, or had less than one year of observation from baseline without discontinuation, were censored. Those patients who discontinued IFX therapy for reasons other than insufficient efficacy were also censored. Receiver operating characteristic (ROC) analysis was conducted to find the optimum TNF-α and IL-6 cutoff points, indicating the need for discontinuation of IFX owing to lack of effectiveness within one year.

RESULTS

Patients Enrolled in the Study

We assessed the eligibility of 310 patients with RA who received intravenous administration of IFX during the study period and were registered in the KURAMA cohort database (Fig. 1). First, 219 patients were excluded from this analysis because their serum samples were not available (serum samples were not obtained during the study period or immediately after administration). All patients were required to have completed induction therapy (defined as 16 weeks after the initiation of IFX). Primary non-responders who discontinued IFX therapy within 24 weeks after the initiation of therapy were ineligible for our analysis. Patients who participated in a clinical trial during the study period were also excluded (n = 3). In the end, 84 patients were enrolled in the study.

Fig. 1. Flowchart of Inclusion/Exclusion of Patients in the Study

IFX: infliximab.

Trough Levels of IFX and Other Biomarkers

In the 84 enrolled patients, the median trough serum level of IFX was 1.62 µg/mL (interquartile range: 0.31–5.23). A trough level of IFX ≥0.32 µg/mL was set as the cutoff value, based on the previous report.¹⁵⁾ Based on the cutoff value, the patients were divided into two groups, and their characteristics are summarized in Table 1. At the sampling point, patients in the lower-IFX group (IFX levels <0.32 µg/mL) had a significantly worse disease status as indicated by IL-6, TJC, SJC, CRP, ESR_1h, RF positivity and level, CDAI, SDAI, and DAS28-ESR values.

Table 1. Baseline Demographics and Clinical Characteristics of Patients Enrolled in This Study

Characteristics at the sampling point (baseline)	IFX ≥ 0.32 µg/mL (N = 63)	IFX < 0.32 µg/mL (N = 21)	p-Value
Age, mean (S.D.), (years)	60.3 (14.0)	56.2 (15.7)	0.23
Body weight, mean (S.D.), (kg)	55.5 (9.4)	57.6 (10.2)	0.44
Women, n/N (%)	50/63 (79.4)	16/21 (76.2)	0.76
Disease duration, mean (S.D.), (years)	12.0 (11.5)	8.5 (10.5)	0.11
The time from IFX initiation until blood sampling, median (min–max), (d)	791 (154–5073)	462 (185–3913)	0.64
IFX dose, mean (S.D.), (mg)	265.4 (100.2)	255.7 (109.3)	0.48
Weekly methotrexate dose, mean (S.D.), (mg/week)	7.5 (3.1)	8.6 (3.5)	0.10
Oral glucocorticoid use, n/N (%)	14/63 (22.2)	2/21 (9.5)	0.17
csDMARDs use, n/N (%)	10/63 (15.9)	4/21 (19.1)	0.74
Trough IFX level, mean (S.D.), (µg/mL)	5.21 (9.74)	0.01 (0.07)	< 0.001
TNF-α level, mean (S.D.), (pg/mL)	4.0 (1.4)	16.9 (40.4)	0.12
IL-6 level, mean (S.D.), (pg/mL)	2.3 (4.0)	9.1 (18.4)	0.011
ADA-positive, n/N (%)	0/62 (0.0)	9/21 (42.9)	< 0.001
RF-positive, n/N (%)	32/56 (57.1)	16/18 (88.9)	0.009
RF, median (min–max), (IU/mL)	16.5 (8.0–387.9)	33.3 (8.0–563.9)	0.002
ACPA-positive, n/N (%)	29/42 (69.1)	12/13 (92.3)	0.066
ACPA, median (min–max), (U/mL)	28.8 (0.5–902)	36.5 (0.6–500)	0.23
CRP level, mean (S.D.), (mg/dL)	0.25 (0.71)	1.06 (1.07)	< 0.001
ESR_1h, mean (S.D.), (mg/dL)	18.2 (12.0)	30.4 (18.7)	0.008
TJC, mean (S.D.)	0.68 (1.53)	1.74 (2.10)	0.004
SJC, mean (S.D.)	0.58 (1.38)	1.11 (1.45)	0.009
CDAI, mean (S.D.)	4.01 (5.29)	7.27 (6.10)	0.014
SDAI, mean (S.D.)	4.17 (5.61)	8.31 (6.50)	0.003
DAS28-ESR, mean (S.D.)	2.45 (0.99)	3.41 (1.23)	0.003
HAQ-DI, mean (S.D.)	0.55 (0.73)	0.39 (0.46)	0.74

The patients were divided into two groups: low-IFX (<0.32 µg/mL) and high-IFX (≥0.32 µg/mL). Demographics and clinical characteristics at the sampling point (baseline) are represented as mean ± standard deviation (S.D.) for continuous data and numbers (percentages) for categorical data. To compare patient characteristics between the groups, the Wilcoxon rank-sum test and Fisher’s exact test were used for continuous and categorical variables, respectively. csDMARDs include actarit, aurothiomalate, auranofin, bucillamine, iguratimod, leflunomide, mizoribine, salazosulfapyridine, cyclosporine, and tacrolimus. ACPA: anti-cyclic citrullinated peptide antibody, ADA: anti-drug antibody, CDAI: clinical disease activity index, csDMARDs: conventional synthetic disease-modifying antirheumatic drugs, CRP: C-reactive protein, DAS28-ESR: 28 joint disease activity score incorporating erythrocyte sedimentation rate, ESR_1h: erythrocyte sedimentation rate at one hour, HAQ-DI: physical disability by health assessment questionnaire-disability index, IFX: infliximab, IL-6: interleukin-6, RF: rheumatoid factor, SDAI: simplified disease activity index, SJC: swollen joint count, TJC: tender joint count, TNF-α: tumor necrosis factor-alpha.

Associations between IFX levels and ADA positivity, TNF-α levels, and IL-6 levels are shown in Supplementary Fig. 2. Due to the limited amount of blood samples, ADA analysis was conducted for 83 patients, and TNF-α and IL-6 were measured for 82 patients. Using ROC analysis, we established levels of 5.82 pg/mL for TNF-α and 1.69 pg/mL for IL-6 as optimized cutoff values indicating the need for discontinuation of therapy with IFX owing to lack of effectiveness (Supplementary Fig. 1). The distribution of patients according to IFX, TNF-α and IL-6 levels, and ADA positivity are also shown in Supplementary Fig. 2. All the ADA-positive patients had IFX levels lower than the corresponding cutoff value. Of the enrolled patients, 8.5% had higher levels of both IFX and TNF-α, and 18.3% had higher levels of both IFX and IL-6, than their corresponding cutoff values. Compared to the high IFX/low TNF-α group, the high IFX/high TNF-α group received similar doses of IFX (261.9 (99.5) vs. 292.9 (109.7) mg on average (S.D.), respectively; p = 0.36) while serum drug levels tended to be lower (5.6 (10.3) vs. 2.5 (2.4) µg/mL on average (S.D.), respectively; p = 0.17).

Prediction of Discontinuation of IFX Therapy

The patients were divided into two groups based on the cutoff values of IFX, TNF-α and IL-6 levels, and ADA positivity. Time to discontinuation of IFX treatment during one year after sampling was compared between the two groups using Kaplan–Meier survival analysis with a log-rank test (Fig. 2). No significant differences were observed between groups for any factors.

Fig. 2. One-Year IFX Persistence Rates in Patients with RA by (a) IFX Levels, (b) ADA Positivity, (c) TNF-α Levels, and (d) IL-6 Levels

The x-axis represents months after blood sampling. The y-axis represents the rate of IFX persistence. Cutoff values for classification were determined by ROC analyses for TNF-α and IL-6 levels. Patients who had over one-year IFX persistence and less than one year of observation without discontinuation or who discontinued IFX therapy for reasons other than insufficient efficacy were censored. ADA: anti-drug antibody, IFX: infliximab, IL-6: interleukin-6, ROC: receiver operating characteristic, TNF-α: tumor necrosis factor-alpha.

Next, the patients were divided into four groups using combinations of IFX levels and ADA positivity, TNF-α levels, or IL-6 levels (Fig. 3). There were no significant differences in time to IFX discontinuation between the groups obtained by combining IFX levels and ADA positivity or TNF-α levels. When patients were divided according to their serum concentrations of both IFX and IL-6 levels, the patients with lower IFX and higher IL-6 levels had the worst persistence (log-rank p-value = 0.017, 68.2% at 12 months). The patient demographics in each of the four groups, categorized based on IFX and IL-6 levels, are shown in Supplementary Table 1.

Fig. 3. Persistence of IFX Therapy in the Four Groups Classified by Serum IFX Levels in Combination with (a) ADA Positivity, (b) TNF-α Levels, or (c) IL-6 Levels

The x-axis represents months after blood sampling. The y-axis represents the rate of IFX persistence. Cutoff values for classification were determined by ROC analyses for TNF-α and IL-6 levels. Patients who had over one-year IFX persistence and less than one year of observation without discontinuation or who discontinued IFX therapy for reasons other than insufficient efficacy were censored. ADA: anti-drug antibody, IFX: infliximab, IL-6: interleukin-6, ROC: receiver operating characteristic, TNF-α: tumor necrosis factor-alpha.

Other Clinical Events

In the four groups classified based on IFX and IL-6 levels, disease worsening or intensification of IFX treatment during one year after blood sampling was assessed (Table 2). In the low IFX/high IL-6 group, disease worsening was significantly more frequent (90.0%) when compared to that in the other three groups (chi-square p-value <0.001). Regarding the proportion of patients who required IFX intensification, there were no significant differences between the four groups (chi-square p-value = 0.54).

Table 2. Comparison of the Proportion of Patients with Disease Worsening or Treatment Intensification

	Occurrence of disease worsening n/N (%)	Intensification of IFX treatment n/N (%)
High IFX/Low IL-6	10/39 (25.6)	13/44 (30.0)
Low IFX/Low IL-6	1/9 (11.1)	1/10 (10.0)
High IFX/High IL-6	4/13 (30.8)	3/15 (20.0)
Low IFX/High IL-6	9/10 (90.0)	3/11 (27.3)
Total	24/71 (33.8)	20/80 (25.0)
Chi-square p-value	< 0.001	0.54

The patients were classified into four groups based on serum IFX and IL-6 concentrations. The number of patients (%) with worsening of DAS28-ESR or intensification of IFX therapy during one year from serum sampling is shown. Disease worsening was defined as an increase in the individual DAS28-ESR score of 1.2 or more resulting in moderate to severe disease activity (DAS28-ESR >3.2) by EULAR criteria. Intensification of IFX treatment was defined as a dose escalation (at least 100 mg) and/or a shorter interval between infusions (at least 14 d at least twice consecutively) from those at the time of the individual minimum DAS28-ESR scores. Patients with missing data on DAS28-ESR and IFX dosage were excluded from the analysis. The chi-squared test was used to investigate statistical significance. DAS28-ESR: 28 joint disease activity score incorporating erythrocyte sedimentation rate, EULAR: European Alliance of Associations for Rheumatology, IFX: infliximab, IL-6: interleukin-6.

DISCUSSION

TDM of IFX is being embraced in clinical practice as an objective tool to assess therapeutic efficiency in the management of patients with RA. The efficacy of IFX therapy is correlated with its trough levels.^6–9,15) Studies demonstrating this association used standard tools such as DAS28-ESR,²²⁾ to measure disease activity and evaluate RA severity. In clinical practice, the length of time that patients remain on anti-rheumatic therapy is another important measure of effectiveness.²³⁾ Our previous claims database study indicated that the clinical responses reported from RCTs might not always reflect the persistence rates in real-world settings.²⁴⁾ Efficacy assessment based on disease activities might not fully correlate with the likelihood of treatment persistence. In fact, the present study found no significant differences for the one-year persistence between groups divided based on baseline serum IFX concentrations, whereas serum IFX levels were associated with DAS28-ESR at the given point. IFX levels alone could not distinguish patients who subsequently discontinued IFX therapy owing to secondary non-response.

Assessing serum IL-6 levels has been suggested as a tool for measuring disease activity. The lowest disease activity was observed in the group with higher IFX and lower IL-6 serum levels than the corresponding cutoff values at week 54.²⁵⁾ Furthermore, patients with low baseline serum IL-6 levels showed a good response to IFX dose escalation, resulting in increased serum IFX levels and reduced disease activity.²⁶⁾ However, only IL-6 level could not predict the continuation of treatment in the present study, but may rather reflect effectiveness at blood sampling. As a novel finding in this study, patients with lower IFX and higher IL-6 serum levels than the corresponding cutoff values during treatment, showed a relatively lower continuation rate for one year, while the patients in other groups hardly discontinued IFX therapy owing to loss of response. Monitoring both IFX and IL-6 levels can be useful to identify patients prone to maintain therapeutic response, thereby avoiding therapy discontinuation owing to loss of response.

Among other biomarkers that can affect the response to IFX therapy, TNF-α plays a dominant role in RA pathobiology.^27,28) In patients with RA, serum levels of TNF-α at baseline affect the serum levels of IFX.¹³⁾ The presence of ADA can also reduce IFX's half-life via formation of immune complexes.^10,11) However, in this study, serum levels of TNF-α or ADA positivity in combination with IFX serum levels were not associated with differences in drug persistence. Our assay detected only free TNF-α, and TNF-α complexed with IFX was not detected. A possible explanation for this observation is that free TNF-α levels in serum and ADA positivity could be surrogates for serum IFX levels. Because TNF-α is one of the factors that induce IL-6 release, IFX can downregulate IL-6 production by neutralizing TNF-α.^29,30) Although the TNF-IL-6 axis plays a major role in RA, the TNF-independent IL-6 axis may also contribute to disease progression in a subset of patients with RA.³¹⁾ These data suggest that measuring serum levels of IFX and IL-6 may prove more beneficial in therapeutic management of RA than measuring TNF-α or ADA.

Several limitations of the present study should be noted. First, the sample size was relatively small (n = 84). Since this was a retrospective observational study, many patients whose serum samples were not stored had to be excluded. Second, the event rates were low because we selected a difficult endpoint: time to discontinuation owing to secondary non-response. The overall rate of IFX discontinuation at one year after sampling was 8.4%. Even in the group of patients with low IFX and high IL-6 serum levels, the discontinuation rate at one year was 31.8%. Third, the possibility of immortal time bias should be considered. As this was a retrospective study, serum samples were collected at random time points during the course of maintenance therapy. The duration of IFX treatment at blood sampling varied among patients, and this might have affected the likelihood to discontinue IFX. However, there were no significant differences in the duration of IFX treatment among the four groups based on IFX and IL-6 levels (Supplementary Table 1). Thus, we postulate that the risk of immortal time bias is low. Finally, participants were limited to our single center, and the generalizability of the findings to other populations is not clear.

In conclusion, the serum levels of IFX, TNF-α, IL-6, and ADAs were not independent predictive factors for IFX discontinuation in patients with RA. However, in these patients, the combination of serum concentrations of IFX and IL-6 can predict one-year IFX persistence after blood sampling. The present findings support the clinical utility of measuring both IFX and IL-6 levels for assessing the effectiveness of long-term maintenance therapy for RA.

Acknowledgments

The authors are grateful to Dr. Akiko Ishii-Watabe, Dr. Hiroko Shibata, and Ms. Kazuko Nishimura, National Institute of Health Sciences, for advice on ADA analysis, and thank all the medical staff of the Rheumatic Disease Center, Kyoto University Hospital.

Conflict of Interest

The Department of Advanced Medicine for Rheumatic Diseases is supported by two local governments (Nagahama City, Shiga and Toyooka City, Hyogo, Japan) and five pharmaceutical companies (Mitsubishi Tanabe Pharma Co., Chugai Pharmaceutical Co., Ltd., UCB Japan Co., Ltd., Asahi Kasei Pharma Corp., and AYUMI Pharmaceutical Co.). The KURAMA cohort study is supported by a Grant from Daiichi Sankyo Co., Ltd. A.Y. received a research Grant from Shimadzu Corporation and AYUMI Pharmaceutical Co. A.O. reports Grants from Pfizer Inc., Bristol-Myers Squibb., and Advantest and personal fees from Asahi Kasei Pharma Corp., Chugai Pharmaceutical Co., Ltd., Eli Lilly Japan K.K., Ono Pharmaceutical Co., Mitsubishi Tanabe Pharma Co., Eisai Co., Ltd., Abbvie Inc., Takeda Pharmaceutical Co., Ltd., and Daiichi Sankyo Co., Ltd. M. Hashimoto received research Grants and speaker fees from Abbvie, Asahi Kasei, Astellas, Ayumi, Bristol Meyers, Chugai, EA Pharma, Eisai, Daiichi Sankyo, Eli Lilly, Nihon Shinyaku, Novartis Pharma, and Tanabe Mitsubishi. T.F. received speaking fees from Eisai Co., Ltd., Asahi Kasei Pharma Corp., Abbvie Inc., and Janssen Pharmaceutical K.K. K. Murata received a speaking fee and/or consulting fees from Eisai Co., Ltd., Chugai Pharmaceutical Co., Ltd., Asahi Kasei Pharma Corp., Bristol-Myers Squibb, Mitsubishi Tanabe Pharma Co., Janssen Pharmaceutical K.K. and Daiichi Sankyo Co., Ltd. M.T. received research Grants and/or speaker fees from Abbvie Inc., Asahi Kasei Pharma Corp., Astellas Pharma Inc., Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Eisai Co., Ltd., Eli Lilly Japan K.K., Janssen Pharmaceutical K.K., Kyowa Kirin Co., Ltd., Pfizer Inc., Taisho Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Co., Teijin Pharma, Ltd., and UCB Japan Co., Ltd. S. Matsuda received speaker fees from Pfizer Inc., Chugai Pharmaceutical Co., Ltd., Asahi Kasei Pharma Corp., Eisai Co., Ltd., Mitsubishi Tanabe Pharma Corp., and Teijin Pharma, Ltd. A.M. received honoraria from AbbVie G.K., Chugai Pharmaceutical Co., Ltd., Eli Lilly Japan K.K., Eisai Co., Ltd., Pfizer Inc., Bristol-Myers Squibb., Mitsubishi Tanabe Pharma Co., Astellas Pharma Inc., and Gilead Sciences Japan, and received research Grants from AbbVie G.K., Asahi Kasei Pharma Corp., Chugai Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Co., and Eisai Co., Ltd.

Supplementary Materials

This article contains supplementary materials.

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