Patients Methods and study design
We performed a retrospective analysis of routine requests for TDM analysis of infliximab and adalimumab (also including samples sent by other hospitals) received as a diagnostic service by the Unit of Clinical Pharmacology at the Luigi Sacco University Hospital (Milan, Italy), between July 2020 and July 2021. Patients aged 6–18 years previously diagnosed with CD or UC according to the European Society Pediatric Gastroenterology Hepatology and Nutrition (ESPGHAN) revised Porto criteria (29), and treated with infliximab or adalimumab were considered in this retrospective analysis. In these patients an anti-TNFα therapy has been started according ESPGHAN guidelines (30, 31). Demographic and clinical data, including age at diagnosis, localization and behavior of disease according to the Paris classification (32), and any previous therapies, were collected. For each patient, clinical activity was assessed, using the Pediatric Crohn’s Disease Activity Index (PCDAI) and Pediatric Ulcerative Colitis Activity Index (PUCAI) for patients with CD and UC respectively, at the start of anti-TNFα therapy and the end of follow up. Disease was considered in clinical remission if indexes were less than or equal to 10. PCDAI > 40 and PUCAI > 65 at diagnosis were used to define severe disease. The responsiveness was defined as: 1- sustained clinical remission (PCDAI ≤ 10 points) without steroids over all visits starting from week 14 for infliximab and week 8 for adalimumab to the last follow up (up to 5 years maximum) for both; 2- sustained biologic remission (defined by CRP ≤ 0.5 mg/dL and fecal calprotectin ≤ 150ug/g) from weeks 14 (for infliximab) and from weeks 8 (for adalimumab) to the last follow up (up to 5 years maximum).
We retrieved also data on serum hemoglobin, C reactive protein (CRP), erythrocyte sedimentation rate (ESR), albumin and fecal calprotectin carried out in all patients. Information on the clinical management of the patients (i.e., treatment intensification, agents like steroids, non-steroid anti-inflammatory drugs, i.e. mesalazine, azathioprine) were also collected.
Infliximab was given intravenously in standard dosing (5 mg/kg) weight-based during the induction (infusion at weeks 0, 2 and 6). Subsequently the dosing regimen were decided by the clinician on target trough concentrations or clinical status (during maintenance phase, every 4/8 weeks) (33). Adalimumab was administered via subcutaneous injection on day 1 at 160 and 80 mg, for body weight ≥ 40 or ≤ 40 kg, respectively (induction phase); followed by every other week at 40 or 20 mg for body weight ≥ 40 or ≤ 40 kg, respectively (maintenance phase). Adalimumab treatment may be escalated when there is clinical indication of an inadequate response by reducing the time between injections to once a week.
For the analysis on efficacy clinical outcomes, patients were stratified into two groups according to their treatment: group 1 treated with infliximab (IFX) and group 2 with adalimumab (ADM). The two groups were analyzed together (IFX + ADM) to assess the association of HLA-DQA1 variants (HLA-DQA1*05 allele and SNP rs2097432) with immunogenicity risk in IBD cohort.
Assessment of plasma drug concentrations
Serum samples for anti-TNFα measurements, infliximab and adalimumab, was performed by a lateral flow immunochromatographic assay with the Ridaquick Monitoring assay (R-Biopharm AG, Darmstadt, Germany). The concentration of the two anti-TNFα was measured at trough, before the next scheduled administration. A target therapeutic trough concentration window of 3–7 mg/L is recommended for infliximab (33), and 5–10 mg/L for adalimumab in IBD (11). ADAs were measured when anti-TNFα plasma levels were less than or equal to 1.0 mg/L with an enzyme-linked immunosorbent assays (ELISA) based method with Ridascreen kit (R-Biopharm AG, Darmstadt, Germany). The assay was performed according to the manufacturer’s guidelines. Samples were measured in a 1/25 dilution (LLOQ 2.5 ng/mL; HLOQ 125 ng/mL) or a 1/200 dilution (LLOQ 20 ng/mL; HLOQ 1000 ng/mL). Only for infliximab, a cutoff of 119 ng/mL was determined to discriminate between high and low ADA concentrations, a parameter that may help clinicians to make treatment decisions (34).
Genotyping
Genomic DNA was isolated from peripheral blood cells using an automated DNA extraction system (EZ1 Advanced XL, Qiagen, Hilden, Germany) according to the manufacturer’s instructions. DNA concentration and purity were evaluated by absorbance methodology using a NanoDrop 1000 Spectrophotometer V3.7 (Thermo Fisher Scientific, Waltham, Massachusetts). All subjects were genotyped for three TNFα gene polymorphisms (-238G > A, -308G > A, and − 857C > T) and one SNP (rs2097432) in the HLA-DQA1 region. All genotypes were determined by Real-Time PCR using the LightSNiP (TIB-MolBiol, Berlin, Germany) on a LightCycler 480 (Roche, Basel, Switzerland), according to the manufacturer’s instructions. Data analysis was performed as described in the LightCycler 480 Instrument operator’s manual. Genotyping performance was estimated through use, in each analysis, of known-genotype internal quality controls.
The HLA-DQA1*05 allele was identified using a TaqMan allelic discrimination assay with a Genvinset® HLA Celiac Plus diagnostic kit (provided by Nuclear Laser Medicine srl, Milan, Italy), according to the manufacturer’s instructions.
Ethics Statement
Data used for this study were collected for clinical purposes and were previously anonymized, according to the requirements set by the Italian Data Protection Code (leg. Decree 196/2003) and by the general authorizations issued by the Data Protection Authority. All patients were registered in the IBD Registry of the Italian Society of Pediatric Gastroenterology, Hepatology and Nutrition (SIGENP). The methodology of the registry has been previously described in detail (35). The study was conducted in accordance with the Declaration of Helsinki and all patients and parents and/or legal guardian of subjects who are under 18 provided written informed consent.
Statistical methods
Quantitative values were given as mean ± standard deviation or as the median and interquartile range (IQR) based on the distribution of the data verified through the Kolmogorov Smirnov test, whereas qualitative variables were described as counts and relative frequency (%) of each category. Comparisons between groups were made with a chi-square test for qualitative variables and with a t-test or Mann–Whitney test for quantitative data. Significance was set at p < 0.05. Associations between quantitative variables were assessed with the Pearson correlation coefficient (Pearson’s r). All genotypes were tested for Hardy–Weinberg equilibrium using χ2 test. Statistical comparisons for genotype frequencies between the two groups were tested by χ2 test. Statistical significance was defined at two-sided p values < 0.05. We used the odds ratio (OR) with 95% confidence intervals (95% CI) as a measure of the effect of risk genotype on each endpoint. Associations between clinical response and demographic and clinic-pathological characteristics, chemotherapy regimens and genotypes were assessed using the χ2 test, Fisher’s exact test or Wilcoxon signed rank test, as appropriate.