Serum surfactant protein D as a predictive biomarker for the efficacy of pirfenidone in patients with idiopathic pulmonary fibrosis: a post-hoc analysis of the phase 3 trial in Japan

Our phase 3 trial of pirfenidone was a prospective, double-blind, randomized, placebo-controlled trial conducted at 73 centers to determine the efficacy and safety of pirfenidone over 52 weeks, prior to the approval for the treatment of IPF in Japan in 2008 [29]. The efficacy and safety results were reviewed by the independent Data and Safety Monitoring Board (DSMB). Patient eligibility, demographic and clinical characteristics, as well as methods, have been described previously [29]. The diagnosis of IPF was in accordance with the American Thoracic Society (ATS)/European Respiratory Society (ERS) consensus statement in 2000 and the “Clinical diagnostic criteria for idiopathic interstitial pneumonia (IIP)” (4th edition) in Japan [30, 31]. Eligible patients were allocated into the following three groups: high dose (1800 mg/day), low dose (1200 mg/day), and placebo, at a ratio of 2:1:2, respectively. In this post-hoc analysis, the high and low dose groups were analyzed together as the pirfenidone group, for simplification.

Peripheral blood samples were collected at weeks 0, 2, 4, 8, 12, 16, 28, 40 and 52. Serum SP-D, SP-A and KL-6 were measured by ELISA using commercial assay kits (SP-D ELISA, Yamasa, Tokyo, Japan; SP-A Test Kokusai-F, Sysmex, Kobe, Japan; ED046, Eizai, Tokyo, Japan, respectively).

The primary endpoint of the original study was the change in vital capacity (VC) at week 52 from baseline. In this post-hoc analysis, the associations between clinical parameters at baseline and disease progression at week 52 were examined in each treatment group. These parameters included patient characteristics, physiological measurements and serum biomarker concentrations. Disease progression was defined by a ≥ 10% relative decline in VC from baseline and/or death. When the VC data could not be obtained because of worsening of respiratory symptoms, including acute exacerbation, the case was also classified as disease progression.

In these analyses, all patients were dichotomized by the median concentration of each biomarker at baseline to the high and low biomarker subgroups. The difference of changes in VC between the pirfenidone and placebo groups at each time point from baseline was investigated by the high and low biomarker subgroups. In addition, we examined the association between biomarker concentrations at baseline and disease progression at week 52, along with progression-free survival (PFS) time, defined as the time from baseline to disease progression.

The effect of pirfenidone on changes in biomarker concentrations at each time point from baseline was assessed using a serial longitudinal measurement of the biomarkers. Serial changes were expressed as relative changes from baseline. In addition, in order to determine whether early changes in biomarker concentrations could predict outcomes, the association of changes in biomarker concentrations at week 16 with disease progression at week 52 and PFS time was investigated. In these analyses, all patients were dichotomized by changes in biomarker concentrations at week 16 to the increase and stable (equal and decrease) biomarker subgroups.

We performed logistic regression analysis to explore the association between clinical parameters at baseline and disease progression at week 52. In this analysis, all parameters were analyzed as continuous variables, except for gender, smoking history (nominal variable), and dyspnea scale (ordinal variable). Factors with a p-value of < 0.10 in the univariate analysis were used as candidates of prognostic factors in the multivariate analysis. We used Fisher’s exact test for the comparisons of the proportion of patients with disease progression at week 52 between the pirfenidone and placebo groups and between the increase and stable biomarker subgroups. In these analyses, patients with worsening respiratory status in which pulmonary function testing could not be conducted and/or who died were classified as disease progression, and other missing data of VC at week 52 were input by the last observation carried forward (LOCF). A mixed model for repeated measurements (MMRM) was applied to calculate the adjusted mean of changes in VC at each time point from baseline. The MMRM consisted of treatment group, time point, interaction between them, and baseline VC as explanatory variables. We plotted survival curves using the Kaplan–Meier method, and the comparison of PFS time between groups was performed using the log-rank test. Summary statistics were calculated for the relative change in biomarker concentrations at each time point, and the comparison between the pirfenidone and placebo groups was performed by Wilcoxon rank-sum test. p values < 0.05 were considered an indication of statistical significance. Because this study was an exploratory post-hoc analysis of a clinical trial, we did not adjust the multiplicity of statistical tests. All statistical analyses were performed with SAS software version 9.2 or higher (SAS Institute Inc., Cary, NC, USA).

The full analysis set of 267 patients were examined in this post-hoc analysis of the phase 3 trial of pirfenidone. The flow diagram was reported previously [29]. Baseline characteristics and clinical data are shown in Table 1, and the number of patients with and without disease progression at week 52 is shown in Additional file 1: Table S1. The result of logistic regression analysis to extract factors that predict disease progression at week 52 is shown in Table 2. In the univariate analysis of the pirfenidone group, body mass index (BMI), smoking history, alveolar–arterial oxygen difference (AaDO₂), %VC and SP-D were extracted with p < 0.10. From the multivariate analysis, BMI, %VC and SP-D were indicated as prognostic factors. In the univariate analysis of the placebo group, BMI, lowest peripheral capillary oxygen saturation (SpO₂) and %VC were extracted with p < 0.10. From the multivariate analysis, BMI and %VC were indicated as prognostic factors.

The distributions of serum concentrations of each biomarker at baseline are shown in Fig. 1. Median concentrations for all patients were as follows: SP-D 202.0 ng/mL (interquartile range [IQR], 138.5–287.0), SP-A 83.0 ng/mL (IQR, 60.9–112.0), and KL-6 1100 U/mL (IQR, 785–1635). There were no numerical differences in these biomarker concentrations between the pirfenidone and placebo groups.

In the original trial, pirfenidone significantly reduced the decline in VC at week 52 and prolonged the PFS time, compared with placebo [29].

The transition of VC over 52 weeks in the pirfenidone and placebo groups by the high and low biomarker subgroups is shown in Fig. 2. Pirfenidone treatment reduced the decline in VC compared with placebo in the low SP-D subgroup; the statistically significant difference was observed from as early as week 16 and continued until week 52, while the high SP-D subgroup showed little difference from placebo. Similar to SP-D, a statistically significant difference was observed between the pirfenidone and placebo groups in the low, but not high, SP-A subgroup from week 16 to 52. In contrast, the high and low KL-6 subgroups did not show any difference.

Table 3 shows the relationship between biomarker concentrations at baseline and disease progression at week 52. Pirfenidone treatment tended to associate with decreased risk of disease progression, compared to placebo in the low SP-D subgroup (p = 0.0949). Consistent results were also obtained using a marginal decline in VC, defined by a ≥ 5% relative decline, in an attempt to achieve more sensitive detection of disease progression (Additional file 1: Table S2) [32].

Figure 3 shows Kaplan–Meier PFS plots created by dichotomizing the patients according to the median concentration of each biomarker at baseline. In the low SP-D subgroup and the low KL-6 subgroup, pirfenidone prolonged PFS.

Figure 4 shows the transition of serum biomarker levels in the pirfenidone and placebo groups. The SP-D level was statistically significantly decreased in the pirfenidone group from week 8 to 52, although the level changed little over time in the placebo group. The SP-A level did not change much over time, and the KL-6 level was numerically elevated in both groups. The levels of SP-A and KL-6 in the pirfenidone group were not significantly different from those in the placebo group.

Conversely, changes in these three biomarker concentrations at week 16 were not associated with the risk of disease progression at week 52 in both the pirfenidone and placebo groups (Additional file 1: Table S3). Similarly, no statistical significance was observed for the association of changes in biomarker concentrations at week 16 with PFS time in both the pirfenidone and placebo groups, except KL-6 in the placebo group (Additional file 2: Fig. S1).