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Effect of pre-existing anti-PEG antibodies on PEG-ASNase
bound, were used as the antigen (RAPP Polymere, Tuebingen, Germany). Mean fluorescent intensities (MFI) of duplicate deter- minations were calculated for anti-PEG IgG and IgM levels. Based on the MFI, determined in the reference cohort, cut-points of 8 (anti-PEG IgG) and 2 (anti-PEG IgM) were defined to classify sam- ples as positive or negative. A detailed description of the method for the determination of anti-PEG antibodies and its performance characteristics is included in the Online Supplementary Data along with a description of the measurement of PEG-ASNase activity and total IgG and IgM.
Statistics
Statistical analyses were performed using SAS® Version 9.4 (SAS Institute Inc., Cary, NC, USA) and RStudio Version 1.2.5033 (RStudio: Integrated Development for R. RStudio, Inc., Boston, MA, USA. URL: http://www.rstudio.com/). Kruskal-Wallis one way analysis of variance on ranks, all pairwise multiple comparison procedures (Dunn method, Holm-Sidak method), the Mann- Whitney rank sum test, Wilcoxon signed-rank test, χ2 test, McNemar χ2 test with continuity correction, Fisher exact test, Pearson correlation and logistic regression were used as indicated.
Results
Anti-PEG IgG and anti-PEG IgM antibody levels
The MFI for anti-PEG IgG was between 0.65 and 67.4, whereas that for anti-PEG IgM was between 0.13 and 30.8. Overall, anti-PEG IgG levels correlated with anti-PEG IgM levels (r=0.68, P<0.005, Pearson correlation). However, high anti-PEG IgG levels did not necessarily coincide with
high anti-PEG IgM levels and vice versa (Online Supplementary Tables S3 and S4). On average the lowest levels of anti-PEG IgG and IgM were determined in the ref- erence cohort and the highest levels in children with pri- mary ALL prior to their first dose of PEG-ASNase (ALL- cohort 1) (Figure 1).
In ALL-cohort 1 anti-PEG IgG and IgM levels were sig- nificantly lower after the administration of PEG-ASNase. This difference was statistically significant in an unpaired analysis, when all samples available after the first adminis- tration were included (n=1,183, median days after admin- istration: 13; range: 1-15; P<0.001, Mann-Whitney rank sum test) and in a paired analysis, when only the first sam- ple taken after administration was chosen for the pairwise comparison (n=646, median days after administration: 7; range: 1-15; P<0.001, Wilcoxon signed-rank test). In addi- tion, anti-PEG IgG and IgM levels were also significantly lower in ALL-cohorts 2 and 3, which were analyzed after PEG-ASNase administration (P<0.001, Kruskal-Wallis one- way analysis of variance on ranks, all pairwise multiple comparison procedures [Dunn method]) (Figure 1). The prevalence of anti-PEG antibodies was correspondingly lower in samples/patients analyzed after administration of PEG-ASNase (Figure 2C-E). In ALL-cohort 1 13.9% of sam- ples were positive for anti-PEG IgG and 29.1% positive for anti-PEG IgM prior to the administration of PEG-ASNase. After administration of PEG-ASNase the prevalence dropped to 4.2% for anti-PEG IgG and 4.5% for anti-PEG IgM. This represented a significant reduction in prevalence by PEG-ASNase administration (P<0.0001, McNemar χ2 test with continuity correction) (Figure 2B, C). Among
AB
Figure 1. Box plots of anti-PEG antibodies in the reference and acute lymphocytic leukemia cohorts. (A, B) Box plots of anti-PEG IgG (A) and anti-PEG IgM (B) mean fluorescent intensities (MFI) determined in the reference and the acute lymphocytic leukemia (ALL) cohorts. The boxes represent the first and third quartiles, the lines in the box the represent the medians, the whiskers the first quartile – (1.5 x the interquartile range between the first and third quartiles [IQR]) and the third quartile + (1.5 x IQR), and the dots the outliers. The dashed reference lines represent the cut-points for anti-PEG IgG (MFI = 8) (A) and anti-PEG IgM (MFI = 2) (B).
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