Introduction

A proliferation-inducing ligand (APRIL), also known as TNFSF13, is a member of the tumor necrosis factor family that is known to bind to two receptors, B-cell maturation antigen (BCMA) and transmembrane activator and CAML interactor.Patient cohort and serum collection

The records of 432 patients diagnosed with colorectal cancer who underwent primary resection between 1990 and 2009 were reviewed. Sampling of biomaterials was approved by the local ethics committee (AZ 99/110). Serum samples were collected at the time of the operation, but before any invasive manipulation. The observation time in this collected cohort was the interval between diagnosis and last contact (death or last follow-up). The survival time interval was calculated between operation and last contact.

Overview ELISA

To test whether the newly produced antibody hAPRIL.133 recognizes APRIL in the presence of the serum, we generated two different standard curves. We used APRIL, which was produced by transfection of a plasmid construct leading to expression of wt APRIL in HEK293T cells. This APRIL was diluted in either PBS+fetal calf serum 10%+human serum 20% (Sigma, cat# H4522), or in PBS+BSA 1%. These two standard curves were tested using several commercially available antibodies or ELISA kits and the newly designed hAPRIL.133-based ELISA. The sensitivity of the hAPRIL.133 ELISA was calculated as the analyte concentration resulting in an absorbance significantly higher than that of dilution media (PBS–fetal calf serum–human seum). hAPRIL.133-based ELISA. The anti-APRIL hAPRIL.133 was used for the detection of endogenous levels of APRIL in the serum of our patient cohort. ELISA plates were coated with 100 μl of 0.5 μg/ml BCMA-Fc (EBC0512081; R&D, Abingdon, UK), in coating buffer (0.2 m sodium phosphate (11.8 g Na2PO4, 16.1 g NaH2PO4, H2O ad 1 l, pH=6.5) and incubated overnight at 4 °C. Later, plates were blocked to prevent unspecific binding using 150 μl PBS–BSA 1% for 1 h at 37 °C. Next, 100 μl of samples and standard curves concentrations were incubated for 2 h at room temperature. After the incubation time, 100 μl of the anti-APRIL hAPRIL.133 were added to the plates at a concentration of 1 μg/ml diluted in PBS–BSA 1%, this was done for 1 h at 37 °C. The following step included the addition of a 100 μl of goat anti-mouse IgG (H&L; Southern Biotech, cat# 1031-05, Uithoorn, The Netherlands) diluted 1:1000 in PBS–BSA 1% for 1 h at 37 °C. In between every step, plates were washed three times with PBS–Tween 0.2% (80 g NaCl, 11.6 g Na2PO4, 2 g KH2PO4, 2 g KCl H2O ad 1 l, pH=6.5+0.2% Tween). The enzymatic development reaction was done using TMB substrate (3,3′,5,5-tetramethylbenzidine). The reaction was stopped by adding an equal amount of 1 m hydrochloric acid to the reaction volume. This yielded a yellow color that was measured in an ELISA reader at 450 nm. The two different APRIL standard curves were tested in the following ELISA procedures. Biolegend (Uithoorn, The Netherlands) MAX ELISA Kit with PRE-coated Plates (cat# 439307). We followed the protocol provided by the manufacturer. ELISA performed by coating with BCMA-Fc and detection with APRILY-5 bio. ELISA plates were coated with 100 μl of 0.5 μg/ml BCMA-Fc (EBC0512081; R&D), in coating buffer (previously described) and incubated overnight at 4 °C. Plates were blocked using 150 μl PBS–BSA 1% for 1 h at 37 °C. Hundred microliter of samples or APRIL standard curves were incubated for 2 h at room temperature. Next, the commercially available APRILY-5 biotinylated (ALX-804-801-C100, Enzo Life Sciences BVBA, Antwerpen, Belgium) was added to the wells (100 μl of 1 μg/ml diluted in PBS–BSA 1%) and incubated for 1 h at 37 °C. Subsequently, 100 μl of Streptavidin-horseradish peroxidase (cat# 890803, R&D) diluted 1:1000 in PBS–BSA 1% was added for 1 h at 37 °C. In between every step, the plates were washed the times with PBS–Tween 0.2%. The enzymatic substrate reaction was carried out using TMB. ELISA using coated Sascha-2 anti-APRIL Ab and detection with biotinylated APRILY-5. ELISA plates were coated with 100 μl of anti-APRIL antibody Sascha-2 (804-804-C100, Enzo Life Sciences BVBA, Antwerpen, Belgium) in coating buffer (previously described) and incubated overnight at 4 °C. Later, plates were blocked using 150 μl PBS–BSA 1% for 1 h at 37 °C. Hundred microliter of samples or APRIL standard curves were incubated for 2 h at room temperature. Next, the commercially available biotinylated APRILY-5 was added to the plates (100 μl of 1 μg/ml diluted in PBS–BSA 1%) and incubated 1 h at 37 °C. After, 100 μl of Streptavidin-horseradish peroxidase diluted 1:1000 in PBS–BSA 1% was added for 1 h at 37 °C. The enzymatic substrate reaction was carried out using TMB.

Statistics

As a cutoff for high versus low APRIL serum levels we used the mean for colorectal and colon cancer patients (8.24 ng/ml and 8.89 ng/ml, respectively), whereas for rectal cancer patients we used the median expression level (7.51 ng/ml). We used a χ2 test to evaluate the frequencies of APRIL high and low serum level patients in the different subgroups for clinicopathologic parameters. Kaplan–Meier curves were used to assess the impact on overall survival rate. The follow-up times in the total cohort are minimum 4 days, median 1257 days and maximum of 6278 days. The significance of APRIL expression in the survival analysis was assessed by the log-rank test. We used a Cox proportional hazards model to test the individual and simultaneous influence on cumulative survival. All covariates found to be significant in the univariate analysis were tested in a multivariate model. The analysis of Wilcoxon–Mann–Whitney was the nonparametric tests used to analyze whether the distribution of APRIL expression differs in patient populations. All tests were two sided. A P-value of <0.05 was considered to indicate statistical significance marked at times as (**); a P-value of <0.01 was marked as (***). All analyses were performed with the use IBM SPSS 20 (Amsterdam, The Netherlands).