Prognostic value of plasminogen activator inhibitor‐1 in biomarker exploration using multiplex immunoassay in patients with metastatic renal cell carcinoma treated with axitinib

Abstract Background and Aims Vascular endothelial growth factor‐directed therapies play a significant role in patients with metastatic renal cell carcinoma (mRCC). Biomarkers for predicting treatment efficacy and resistance are required to develop personalized medicine. We evaluated multiple serum cytokine levels in patients with mRCC treated with axitinib to explore predictive biomarkers. Methods From September 2012 to October 2015, serum samples were collected from 44 patients with mRCC before treatment and 4 weeks after axitinib initiation. Bio‐Plex Pro Human Cancer Biomarker Panels 1 and 2 were used to measure levels of 34 serum biomarkers related to angiogenesis and cell proliferation. Results Patients with partial response or stable disease had significantly decreased serum plasminogen activator inhibitor‐1 (PAI‐1) level from pre‐treatment to 4 weeks after axitinib initiation compared with those with progressive disease (P = .022). The median progression‐free survival (PFS) and median overall survival (OS) in patients with increased serum PAI‐1 level from pre‐treatment to 4 weeks after axitinib initiation were significantly shorter than those with decreased serum PAI‐1 level (P = .027 and P = .026, respectively). Increased serum PAI‐1 level from pre‐treatment to 4 weeks after axitinib initiation was an independent prognostic marker for shorter PFS and OS in multivariate analyses (P = .015 and P = .032, respectively). The immunohistochemical staining intensity of PAI‐1 in tumor specimens was significantly associated with Fuhrman grade and presence of distant metastasis (P = .026 and P = .010, respectively). Conclusions The initial change in serum PAI‐1 level in the early stage of axitinib treatment could be a useful prognostic biomarker in patients with mRCC.

Conclusions: The initial change in serum PAI-1 level in the early stage of axitinib treatment could be a useful prognostic biomarker in patients with mRCC.

K E Y W O R D S
metastatic renal cell carcinoma, molecular-targeted therapy, plasminogen activator inhibitor-1, serum biomarker

| INTRODUCTION
In 2017, the age-adjusted incidence and mortality rates of renal cell carcinoma (RCC) in Japanese men were 11.5 and 2.8 per 100 000 personyears, respectively. 1

Distant metastasis is observed in approximately 20%
to 30% of patients with RCC at the time of initial diagnosis. 2 Although current first-line treatment for patients with metastatic RCC (mRCC) is either an immune-checkpoint inhibitor (ICI) or vascular endothelial growth factor (VEGF)-directed multitargeted tyrosine kinase inhibitors (TKIs), 3 TKIs improved overall survival (OS) in patients with mRCC with a median value of 8.5 to 14.4 months from 2002 to 2008. 4 Although the treatment paradigm for mRCC is currently shifting from TKIs to ICIs with or without concurrent use of TKIs, personalized biomarker-guided sequential or combination therapies for predicting the efficacy and adverse effects of TKIs are still strongly required for patients with mRCC. 3 For appropriate use of TKIs in individual patients, useful biomarkers which can be measured during treatment to predict treatment effect, resistance, and prognosis are strongly required. As strategies to predict the treatment effect and prognosis during treatment, serum TKI level can be measured. 5 Pre-treatment evaluation of genetic polymorphisms of drug-metabolizing enzymes and transporters can predict the serum TKI level. 5 In addition, serum VEGF-C, sVEGFR-2, and sVEGFR-3 levels, [6][7][8] and the number of endothelial cells in circulating blood 9 have been reported to be biomarkers that correlate with treatment effect and prognosis. However, other potential biomarkers relevant to personalized therapy including TKIs and immunotherapies have not been investigated.
Axitinib is a TKI selective for VEGFR-1, -2, and -3. Patients with mRCC treated with axitinib as second-line therapy had a significantly longer progression-free survival (PFS) than those treated with sorafenib in a randomized, multicenter phase III trial. 10 In this study, we aimed to analyze various potentially prognostic serum cytokines involved in cancer angiogenesis and cell proliferation using the multiplex immunoassay method before treatment and 4 weeks after axitinib initiation in patients with mRCC. We comprehensively explored biomarkers which can predict the clinical effect and prognosis in patients with mRCC treated with axitinib.

| Quantitative analysis of serum biomarkers
Serum samples were centrifuged at 3000 revolutions per min for 10 minutes, and stored at −80 C prior to analysis. Beads array analysis using the Bio-Plex Pro Cancer Biomarker assay kit1 and kit2 (Bio-Rad, Hercules, California) was performed to measure 34 cytokines and tumor growth factors.
Briefly, the capture antibody-coupled beads were first incubated with antigen standards, quality control samples, and serum samples in 96-well plates, followed by incubation with biotinylated detection antibodies. Samples were diluted 1:4 using sample diluent. After washing the unbound biotinylated detection antibodies, the beads were incubated with a reporter streptavidinphycoerythrin (SA-PE) conjugate. Following the removal of excess SA-PE, the beads were passed through the 2-laser flow cytometer Bio-Plex array reader (Bio-Plex 200 system, Bio-Rad), which measures the fluorescence of the bead and the bound SA-PE. Details of the procedure have been described previously. 11 Assay incubations were performed at room temperature. All washes were performed using the Bio-Plex Pro wash station. Data acquisition was performed using Bio-Plex manager TM 6.0. Using the automatic calibration curve optimization function, the recovery rate was regressed to be in the range of approximately 70% to 130%. All samples were assayed in duplicate.

| Immunohistochemistry staining
Tumor specimens obtained by radical nephrectomy or biopsy were fixed in 20% formalin, embedded in paraffin, and evaluated for expression of PAI-1. Specimens were sliced into 3 μm sections and immunohistochemically analyzed using anti-PAI-1 antibody (#66705, Abcam, Cambridge, UK). Peroxidase and 3,3-diaminobenzidine (DAB) were used as labeling enzyme and chromogenic substrate, respectively. Immunohistochemistry (IHC) staining was assessed using an automated quantitative pathology imaging system workstation (Mantra, PerkinElmer, Waltham, Massachusetts). DAB-positive cells were detected, and the staining intensity was scored using inForm ver. 2.3 software (PerkinElmer). Five representative areas were photographed with a 400-fold field of view, and nuclei were automatically recognized. Staining intensity was measured radially from the nucleus, and DAB staining was recognized around the cell membrane ( Figure S1). The positive threshold for staining intensity per cell was defined as ≥25% of the maximum staining intensity. The percentage of cells exceeding the threshold was counted, and the average value of the five visualized areas was scored as the final IHC staining intensity.

| Statistical analysis
The Kolmogorov-Smirnov test was used for nonparametric analysis of the serum biomarkers because of their nonnormal distribution. The relationships between serum biomarker level, treatment response, IHC staining intensity, and pathological parameters were evaluated using the Mann-Whitney U test. Bonferroni's correction was applied in the multiple comparison. Fisher's exact test was used to examine the proportion of patients between groups. The Kaplan-Meier method was used to plot time-to-event curves, and statistical significance was estimated using the log-rank test. The Cox proportional hazard model was used to determine independent prognostic factors of PFS and OS. P < .05 was considered as statistically significant. All statistical analyses were performed using SPSS statistics version 23 (IBM, New York).
In contrast, the median serum level of sEGFR and PRL significantly increased from pre-treatment to 4 weeks after axitinib initiation (P = .032 and P = .010, respectively; Table 2). Using Bonferroni's correction, only sTIE2, sVEGFR-2, and PRL were significantly decreased or increased. The number of patients for each serum biomarker who exhibited a decrease or increase in level is shown in Table 2.

| Relationship between serum biomarker levels and treatment response
The treatment responses of 42 patients treated with axitinib were partial remission (PR) in 16 (38.1%) patients, stable disease (SD) in 20 (47.6%), and progressive disease (PD) in 6 (14.3%). Two patients were excluded because of unknown response. The median serum PDGF-AB/BB and sVEGFR-2 levels at baseline were significantly higher in the six patients with PD than in the 36 patients with PR or SD (P = .040 and P = .003, respectively); however, the baseline median serum PAI-1 level was significantly lower in the patients with PD than those with PR or SD (P = .048) (Table S1). Using Bonferroni's correction, there was no significant relationship.
The proportion of patients with decreased serum level of PAI-1 and IL-18 from pre-treatment to 4 weeks after axitinib initiation was significantly higher in patients with PR or SD compared to those with PD (P = .022 and P = .022, respectively; Table S2). The proportion of patients with decreased serum levels of endoglin, IL-6, and VEGF-A from pre-treatment to 4 weeks after axitinib initiation was significantly higher in patients with PR than those with SD or PD (P = .011, F I G U R E 1 Kaplan-Meier curves comparing, A, progression-free survival, and B, overall survival in patients with decreased or increased serum plasminogen activator inhibitor-1 (PAI-1) level from pre-treatment to 4 weeks after axitinib initiation T A B L E 3 Cox proportional hazard model to predict the shorter progression-free survival using baseline clinical parameter and change in the serum biomarker level from pre-treatment to 4 weeks after initiation of axitinib (Continues) P = .025, and P = .029, respectively; Table S2). Using Bonferroni's correction, there was no significant relationship.

| Relationship between serum biomarker levels and PFS and OS
The presence of lymph node swelling on initial imaging studies (cN1) and baseline serum leptin level lower than the median were independent factors related to worse PFS in multivariate analysis (P < .001 and P = .026; Table S3). No independent factor related to OS was found using baseline serum biomarker level (Table S4).
Patients with increased serum PAI-1 level from pre-treatment to 4 weeks after axitinib initiation had significantly shorter PFS and OS than those with decreased serum PAI-1 (15.0 months vs 5.1 months, P = .027 and 34.9 months vs 14.2 months, P = .026, respectively; Figure 1A,B).
The presence of lymph node swelling on initial imaging studies (cN1) and increased serum PAI-1 level from pre-treatment to 4 weeks after axitinib initiation were independent prognostic factors for shorter PFS (P < .001 and P = .015, respectively; Table 3). Increased serum PAI-1 level from pre-treatment to 4 weeks after axitinib initiation was also an independent prognostic marker for shorter OS (P = .032; Table 4).

| Relationship between IHC staining intensity and clinical parameters
Of the 44 patients enrolled in this study, 41 (93.2%) underwent radical nephrectomy and 3 (6.8%) underwent tumor biopsy. IHC analysis using PAI-1 antibody was available in 39 specimens from 36 nephrectomies and 3 biopsies. The median IHC staining intensity of PAI-1 was significantly higher in patients with metastatic disease at the time of diagnosis than those with nonmetastatic disease (P = .010; Table 5), as well as in patients with Fuhrman grade ≥ 3 tumors than in those with grade ≤ 2 (P = .026; Table 5). There was no significant relation-

| CONCLUSIONS
The initial changes in serum PAI-1 level at the early stage of axitinib treatment could be a useful prognostic biomarker in patients with mRCC.

ACKNOWLEDGMENT
The authors thank Yoko Mitobe, Yukiko Sugiyama, and Yuka Izumida for their contribution of clinical sample collections and preparations.

CONFLICT OF INTEREST
The authors declare no conflicts of interest.

TRANSPARENCY STATEMENT
The corresponding author, Takamitsu Inoue, affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.