Antinuclear antibodies seroconversion and endoscopic response to infliximab in biologic-naive Crohn’s disease: a Chinese single-center, retrospective study
Article information
Abstract
Background/Aims
The development of antinuclear antibodies (ANAs) during infliximab treatment has been observed in previous clinical trials. To evaluate the clinical significance of ANA seroconversion and its potential association with the formation of antibodies against infliximab.
Methods
This retrospective study included 130 Crohn’s disease patients undergoing infliximab therapy. ANA titers were measured at baseline and after 6 months of treatment. Inverse probability of treatment weighting was applied to control for confounding variables.
Results
Among the 111 patients with negative baseline ANA, 36 (32.4%) developed ANA positivity after 6 months of infliximab treatment. After adjustment with inverse probability of treatment weighting, a significantly higher proportion of patients in the ANA non-seroconversion group achieved endoscopic remission at 6 months compared to those with ANA seroconversion (64.5% vs. 35.5%: adjusted odds ratio [aOR], 3.61; 95% confidence interval [CI], 1.30–10.02; P= 0.014). At 18 months, patients in the non-seroconversion group also exhibited higher rates of both endoscopic response (57.5% vs. 42.3%: aOR, 3.38; 95% CI, 1.15–9.96; P= 0.027) and endoscopic remission (60.0% vs. 40.1%: aOR, 2.91; 95% CI, 1.06–8.01; P= 0.039) compared to the seroconversion group. Additionally, patients who developed ANA seroconversion had a higher rate of detectable antibodies against infliximab at both 6 and 18 months. A multivariable analysis identified female sex, older age at diagnosis, and lower serum albumin levels as independent predictors of ANA seroconversion at 6 months.
Conclusions
ANA non-seroconversion at 6 months was associated with higher rates of endoscopic remission and a lower likelihood of developing antibodies against infliximab.
INTRODUCTION
Crohn’s disease (CD) is a chronic, progressive condition with diverse clinical manifestations. Traditionally, its treatment involved corticosteroids, 5-aminosalicylates, and non-targeted immunosuppressive agents, such as thiopurines or methotrexate, primarily aimed at alleviating disease-related symptoms. However, over the past two decades, novel therapies with distinct mechanisms of action have been approved for the treatment of moderate-to-severe CD. The first biologic agents developed, tumor necrosis factor-alpha (TNF-α) inhibitors, proved effective in treating CD, ushering in a new era of management [1]. The subsequent introduction of anti-α4β7 integrin inhibitors, anti-interleukin 12/interleukin 23p40 antibodies, and Janus kinase inhibitors has further expanded available therapeutic options [2].
Anti-TNF-α inhibitors remain a cornerstone of current treatment regimens. A recent network meta-analysis of biologic-naive patients demonstrated that infliximab, with or without azathioprine, was associated with a higher likelihood of achieving clinical remission in moderate-to-severe CD compared to other treatment options [3]. However, it is estimated that 10% to 30% of patients will not respond to initial therapy (primary non-response), and 23% to 46% will experience a loss of response over time (secondary loss of response) [4].
It is well established that anti-TNF-α therapy can induce the development of antinuclear antibodies (ANA) and anti-double-stranded DNA (anti-dsDNA) antibodies in chronic inflammatory conditions, including rheumatoid arthritis, spondyloarthropathy, and CD [5,6]. Although an association between ANA seroconversion and lower infliximab trough levels has been suggested [7], and anti-dsDNA antibody levels correlate with clinical symptoms of a lupus-like syndrome (LLS) [8], the formation of ANAs and their potential clinical significance in CD remain unclear.
This study aims to assess the impact of ANA seroconversion, induced by infliximab, on secondary loss of response and the formation of antibodies to infliximab (ATI) during therapy in CD patients.
METHODS
1. Study Design and Patients
This retrospective study was conducted at a tertiary referral center specializing in inflammatory bowel disease (IBD), and ethical approval was obtained from the Ethics Committee of Renji Hospital (IBD KY2021-168-B). The requirement for informed consent was waived by the Ethics Committee. The study included patients diagnosed with CD in accordance with international guidelines, who received infliximab therapy for moderate-to-severe CD between January 2018 and December 2020.
The inclusion criteria were as follows: (1) adult patients (≥18 years) with active CD who initiated infliximab as their first biologic therapy; (2) all patients received an induction regimen of at least 5 mg/kg infliximab at weeks 0, 2, and 6, followed by maintenance therapy every 8 weeks; (3) a minimum follow-up period of 6 months, with endoscopic examinations conducted at baseline and at 6 months; (4) serum ANA and infliximab antibody levels were assessed at baseline and at 6 months; (5) active CD was defined as a Simple Endoscopic Score for Crohn’s Disease (SES-CD) of ≥ 3. Exclusion criteria included: (1) patients in endoscopic remission at baseline; (2) those with a history of malignancy, Sjogren syndrome, systemic lupus erythematosus, recent surgical interventions, pregnant women, and cases with insufficient documentation; (3) patients who had received treatment with any other biologics at baseline.
2. Determinations of Autoantibodies, ATI and Infliximab Concentration
ANA were detected using indirect immunofluorescence, with a cutoff value of 1:80 for ANA positivity, as recommended by the manufacturer and consistent with thresholds reported in the literature. Serum ANA titers were measured at baseline and again at 6 months. ANA seroconversion was defined as the development of autoantibodies in patients who were initially ANA-negative during follow-up. Patients with an ANA titer of 1:80 were classified as low ANA titers, while those with titers greater than 1:80 were categorized as high ANA titers. Infliximab serum concentrations were quantified using enzyme-linked immunosorbent assay (ELISA), and ATI were also measured by ELISA (Herui Biotechnology Co., Ltd, Suzhou, China). Infliximab trough levels and ATI were measured at 6 months and 18 months, respectively. Immunogenicity was defined as an anti-drug antibody titer of ≥ 10 AU/mL.
3. Variables Measures and Outcomes
The collected data included patient demographics (age, sex), date of diagnosis, phenotype based on the Montreal classification, concurrent use of immunomodulator, and medical and surgical treatment history. Biomarker results, such as C-reactive protein (CRP), dsDNA, and albumin, were recorded when available. Endoscopic response was defined as a ≥ 50% reduction in the total SES-CD score from baseline, with a score greater than 2. Endoscopic remission was defined as a total SES-CD score of ≤ 2 points [9,10].
The primary endpoint was the association between ANA seroconversion and endoscopic response and remission to infliximab therapy at 6 months. The secondary endpoint included the association between ANA seroconversion and endoscopic response and remission at 18 months, as well as the relationship between ANA, infliximab trough levels, and ATI.
4. Statistical Analysis
Continuous variables were reported as medians with interquartile ranges (IQRs), while categorical variables were expressed as frequencies and percentages. The chi-square test or Fisher exact test was used for comparisons of categorical variables, and the Mann-Whitney U test was employed for comparing continuous variables.
Inverse probability of treatment weighting (IPTW) was used to estimate the probability of individuals with ANA seroconversion at 6 months after received infliximab treatment, using propensity scores derived from a multivariable logistic regression model. The propensity-score model incorporated baseline characteristics of the participants, considering both clinical relevance and subject characteristics. These variables included sex, age at diagnosis, disease duration, disease behavior, location according to the Montreal classification, perianal fistula, CRP (mg/L), serum albumin (g/L), anti-dsDNA (IU/mL), concurrent use of immunomodulator, prior use of immunomodulatory therapies, and SES-CD score.
To identify independent predictors of ANA seroconversion, univariate logistic regression was first performed on unweighted data. Variables with a P-value less than 0.05 were subsequently included in a multivariable model to assess factors independently associated with ANA seroconversion. All statistical tests were two-sided, and a P-value of less than 0.05 was considered statistically significant. Statistical analyses were conducted using Stata software (version 17.0, StataCorp LP, College Station, TX, USA).
RESULTS
1. Study Population
The initial study population comprised 130 patients with CD. The median age was 25 years (IQR, 20–32 years), with 35 patients (26.9%) being female. The median disease duration was 3 months (IQR, 1–17 months). A total of 30 patients (23.1%) had previously received immunomodulators, and 8 patients (7.2%) had concurrent use of immunomodulator. A subgroup of 118 patients received infliximab maintenance therapy and were evaluated for long-term therapeutic outcomes at 18 months. Prior to infliximab treatment, 19 of the 130 patients (14.6%) had positive ANA, while 111 patients tested negative for ANA.
Among the 111 ANA-negative patients, 36 developed ANA positivity after 6 months of infliximab treatment, yielding a seroconversion rate of 32.4%. The distribution of ANA positivity was as follows: 20 patients (55.6%) at a 1:80 dilution, 11 (30.6%) at 1:160, 4 (11.1%) at 1:320, and 1 (2.8%) at 1:640. Among 75 patients who were ANA-negative at 6 months, we found that at 18 months, 47 patients remained negative, 12 patients were not tested, and 16 developed ANA positivity. The distribution of ANA positivity was as follows: 6 patients at a 1:80 dilution, 9 at 1:160, and 1 at 1:1,280. Among 36 patients who were ANA positive at 6 months, 25 patients remained positive at 18 months, while 6 patients were not tested. Notably, 5 cases with a dilution of 1:80 experienced a shift to negative status at 18 months among the 30 cases of ANA-positive patients at 6 months.
Baseline characteristics of the 111 ANA-negative patients are presented in Table 1. Before adjusting for propensity scores, no significant differences were observed between the seroconverted and non-seroconverted groups in terms of disease duration, disease behavior, perianal fistula, CRP, anti-dsDNA, concurrent use of immunomodulator, prior immunomodulator use, and SES-CD score. However, significant differences were observed for sex (seroconverted group 15 [41.7%] vs. non-seroconverted group 13 [17.3%]; P=0.006), median age (seroconverted group 27.5 years [IQR, 23.0–36.0 years] vs. non-seroconverted group 23.0 years [IQR, 18.0–30.0 years]; P=0.005), disease location according to the Montreal classification (P=0.049), and serum albumin (seroconverted group 38.0 g/L [IQR, 32.5–40.4 g/L] vs. non-seroconverted group 40.3 g/L [IQR, 36.8–44.7 g/L]; P=0.002). After IPTW adjustment, no statistically significant differences between the 2 groups were observed.
Characteristics of the Patients before and after IPTW
2. Association between ANA Seroconversion and Endoscopic Outcomes to Infliximab Therapy
After adjusting for propensity scores using IPTW, no significant difference was observed in endoscopic response at 6 months between the ANA non-seroconversion and seroconversion groups (52.1% vs. 47.9%: adjusted odds ratio [aOR], 1.19; 95% confidence interval [CI], 0.32–4.44; P=0.799). However, a higher proportion of patients in the ANA non-seroconversion group achieved endoscopic remission at 6 months compared to the seroconversion group (64.5% vs. 35.5%: aOR, 3.61; 95% CI, 1.30–10.02; P=0.014).
At 18 months, the rate of endoscopic response was significantly higher in the ANA non-seroconversion group (57.5%) than in the seroconversion group (42.3%) (aOR, 3.38; 95% CI, 1.15–9.96; P=0.027). Similarly, a greater proportion of patients in the ANA non-seroconversion group achieved endoscopic remission at 18 months compared to those in the seroconversion group (60.0% vs. 40.1%: aOR, 2.91; 95% CI, 1.06–8.01; P=0.039) (Fig. 1).
Association between antinuclear antibodies (ANA) seroconversion and endoscopic outcome using inverse probability of treatment weighting analysis in patients with Crohn’s disease.
Furthermore, patients were divided into 2 subgroups: a high-titer ANA group (n=20) and a low-titer ANA group (n=16). At 6 months, patients with high-titer ANA were more likely to demonstrate complete endoscopic response compared to those with low-titer ANA (97.6% vs. 64.2%: aOR, 0.04; 95% CI, 0.01–0.50; P=0.012). Likewise, patients in the high-titer ANA group were more likely to achieve complete endoscopic remission at 6 months than those in the low-titer ANA group (72.6% vs. 14.9%: aOR, 0.07; 95% CI, 0.01–0.41; P=0.004). However, no significant differences were observed in endoscopic response at 12 months between the 2 groups (56.9% in the high-titer vs. 62.4% in the low-titer ANA groups: aOR, 1.25; 95% CI, 0.22–7.12; P=0.798). Similarly, there was no significant difference in the rate of endoscopic remission at 12 months (52.1% in the high-titer vs. 42.9% in the low-titer ANA groups: aOR, 0.69; 95% CI, 0.12–3.93; P=0.677).
3. Relationship of ANA with ATI
After adjusting for IPTW, among ANA-negative patients at baseline, those who experienced ANA seroconversion had a similar rate of detectable infliximab antibodies at 6 months compared to those without seroconversion (23.8% vs. 19.5%: aOR, 1.29; 95% CI, 0.38–4.31; P=0.683). However, at 18 months, patients with ANA seroconversion exhibited a significantly higher rate of detectable infliximab antibodies compared to those without seroconversion (41.4% vs. 8.5%: aOR, 7.59; 95% CI, 2.13–27.07; P=0.002) (Fig. 2).
Association between antinuclear antibodies (ANA) seroconversion and antibodies against infliximab using inverse probability of treatment weighting analysis in patients with Crohn’s disease.
4. Correlation of Infliximab trough Levels and ANA
Among ANA-negative patients at baseline, those who experienced ANA seroconversion had significantly lower median infliximab trough levels at 6 months compared to those without seroconversion (0.7 μg/mL [IQR, 0.4–1.9 μg/mL] vs. 2.6 μg/mL [IQR, 0.8–5.1 μg/mL]; P <0.001). Similarly, at 18 months, patients with ANA seroconversion continued to exhibit significantly lower median infliximab trough levels compared to those without seroconversion (1.4 μg/mL [IQR, 0.4–2.9 μg/mL] vs. 3.1 μg/mL [IQR: 1.2–6.3 μg/mL]; P=0.014).
5. Factors-Associated with ANA Seroconversion at 6 Months
The univariate analysis revealed that female sex, higher age at diagnosis, lower albumin levels, and lower infliximab trough levels at 6 months (P<0.05). In a multivariable model, female sex (OR, 3.93; 95% CI, 1.38–11.22; P=0.033), older age at diagnosis (OR, 1.08; 95% CI, 1.03–1.14; P=0.009), and lower baseline albumin levels (OR, 0.91; 95% CI, 0.84–0.99; P=0.028) were identified as independent predictors of ANA seroconversion at 6 months (Table 2).
Univariable and Multivariable Factors-Associated with ANA Seroconversion at 6 Months
DISCUSSION
The utilization of TNF-α antagonists has significantly improved the therapeutic landscape for CD; however, a noteworthy loss of response is encountered during maintenance therapy. This phenomenon can be attributed to various mechanisms, including patient-dependent drug pharmacokinetics (such as low albumin or high body weight) and the emergence of anti-drug antibodies due to immunogenicity [11]. While therapeutic drug monitoring has been proposed as a strategy to manage this loss of response, its practicality, efficacy, and cost-effectiveness are not universally available across medical centers. In conjunction with immunogenicity, another pertinent issue in this context is the induction of ANA and dsDNA autoantibodies during infliximab treatment.
In our CD cohort, only 14.6% of patients exhibited ANA at baseline, a prevalence comparable to that in healthy control populations [12]. Vermeire et al. [6] described a cumulative ANA incidence at 24 months in 71 of 125 (56.8%). Most patients develop autoantibodies soon after the first infusion. With a follow-up of 12 months, the majority of patients remain ANA positive, with only a minority becoming seronegative. Our data revealed a seroconversion rate of 32.4% at 6 months, consistent with prior studies where ANA was detected in 20% to 45% of IBD patients receiving anti-TNF-α therapy.
In the field of rheumatology, it has been documented that patients undergoing infliximab therapy for rheumatoid arthritis, who develop ANA, exhibit elevated Disease Activity Scores (DAS 28) at 52 weeks compared to those who do not develop ANAs [13]. Similarly, the transition to antinuclear antibody negativity has been associated with a lower disease flare in individuals with systemic lupus erythematosus [14]. Our study is a first large-scale clinical investigation in CD patients to examine the correlations between autoantibodies and the clinical efficacy of infliximab based endoscopic evaluation, trough serum infliximab levels, and anti- infliximab antibodies. In contrast to previous studies where the time interval between the first and last ANA tests varied among patients, we specifically investigated the impact of ANA conversion on the efficacy of infliximab in patients with CD at both 6 and 18 months. Importantly, our findings indicated that the baseline ANA status did not influence the endoscopic response of infliximab treatment in CD patients at 6 months (52.1% in the non-seroconversion groups vs. 47.9% in the seroconversion groups: aOR, 1.19; 95% CI, 0.32-4.44; P=0.799). However, ANA seroconversion at 6 months emerged as a predictor of secondary loss of endoscopic response at 18 months, marked by a lower incidence of endoscopic response (57.5% in the non-seroconversion groups vs. 42.3% in the seroconversion groups: aOR, 3.38; 95% CI, 1.15–9.96; P=0.027).
The recommended approach for initiating infliximab therapy typically involves a combination regimen with azathioprine for a duration of 6 to 12 months, as this strategy has demonstrated enhanced pharmacokinetic benefits [15]. However, due to intolerance to azathioprine, a substantial number of Asian patients receive monotherapy with infliximab [16,17]. Considering the balance between risks and benefits, an alternative strategy could involve the delayed addition of thiopurine before the occurrence of loss of response to infliximab in the treatment of CD.
For patients experiencing ANA conversion during infliximab monotherapy, optimizing the combination of azathioprine in advance, could be more beneficial than waiting until the point where infliximab concentration becomes unmeasurable. Conversely, for CD patients using infliximab who do not undergo ANA seroconversion, it might be predicted that they can continue infliximab treatment for a prolonged period and have a lower likelihood of experiencing secondary loss of endoscopic response. Therefore, ANA non-seroconversion could serve as a predictor of sustained and durable response to infliximab.
Therapeutic drug monitoring is widely employed for monitoring and guiding therapy in patients with IBD. Wilson et al. [18] found that HLADQA1*05 is independently associated with a high risk of infliximab antibody formation, as well as infliximab loss of response and treatment discontinuation. Vermeire et al. [6] demonstrated that ANA-positive patients are more prone to developing significant ATI titers compared to patients who remain ANA-negative with a borderline significant relationship. Theodoraki et al. [7] revealed that in IBD patients undergoing maintenance therapy with anti-TNF-α treatment, ANA positivity is associated with lower infliximab trough levels. Notably, there were variations in the time intervals between the first and last ANA tests among patients in our study. Our data suggests that infliximab trough concentration in patients experiencing ANA seroconversion is lower than in those without ANA seroconversion both at 6 months and at 18 months. In terms of antibody generation, patients with ANA seroconversion showed a heightened likelihood of developing antibodies compared to those without ANA seroconversion, whether at 6 or 18 months.
Our multivariate analysis revealed that female sex, older age at diagnosis, and lower baseline albumin levels were independently associated with ANA seroconversion. The sex disparity may be attributed to estrogen-mediated immune modulation, as studies suggest estrogens enhance B-cell activation and autoantibody production through CD40 ligand overexpression [19]. The precise mechanisms underlying the generation of autoantibodies during anti-TNF-α therapy have not been clearly defined. Some authors suggest that the emergence of autoantibody seroconversion could result from the effect of anti-TNF-α blockade. This blockade might impede the induction of cytotoxic T lymphocyte responses that traditionally suppress autoreactive B-cells, potentially fostering humoral autoimmunity [20]. Conversely, an alternative perspective posits that the seroconversion of autoantibodies is closely tied to an increase in apoptosis of lymphocytes and monocytes. This process triggers the release of nuclear autoantigens, possibly driven by the presence of anti-TNF-α agents [21-23].
This study also has some limitations that should be acknowledged. Firstly, ANA encompass a diverse range of autoantibodies that bind to various antigens, such as Sm, Ro, La, RNP, and dsDNA [24]. However, we did not explore the relationship between specific ANA subtypes and infliximab efficacy in detail. Secondly, there have been reports of an incidence of 5% of induced LLS in such patients [5], and levels of dsDNA antibodies are known to be associated with clinical symptoms of LLS [25]. In our study, however, we did not present data regarding the occurrence of LLS. Secondly, due to the retrospective nature of the study, it was not possible to accurately obtain information on coexisting autoimmune diseases clinical response (Crohn’s Disease Activity Index, Harvey-Bradshaw Index) and biochemical response (fecal calprotectin). Lastly, the follow-up period was relatively short (18 months), highlighting the need for a more extended long-term follow-up.
In conclusion, ANA seroconversion at 6 months may indicate potential loss of endoscopic response to anti-TNF-α therapy at 18 months, decreased infliximab concentrations and an increased risk of anti-drug antibody formation. serial ANA testing at both baseline and 6 months provides valuable insights. In contrast, ANA non-seroconversion seems to predict a sustained response to infliximab. Moreover, the ANA assay is a routine, easily accessible laboratory test that can be conveniently performed in everyday clinical practice. Monitoring ANA titers has proven useful as a biomarker for treatment optimization and predicting loss of response during anti-TNF-α therapy.
Notes
Funding Source
This study was sponsored by Key Support Project of Shenkang Three-Year Action Plan for Promoting Clinical Skills and Clinical Innovation in Municipal Hospitals (SHDC2024CRI013).
Conflict of Interest
No potential conflict of interest relevant to this article was reported.
Data Availability Statement
All data generated or analyzed during this study are included in this published article.
Author Contributions
Conceptualization: Tong J, Jun S, Cao Z. Data curation: Tong J, Xu X, Lu J, Qiao Y. Investigation: Tong J, Cao Y, Xu X, Zhu M, Dai Z. Formal analysis: Tong J, Cao Y, Zhu M, Wang T, Sun Y. Writing - original draft: Tong J. Writing - review and editing: all authors. Funding acquisition: Tong J. Approval of final manuscript: all authors.