Low immunoglobulin levels increase the risk of severe hypogammaglobulinemia in granulomatosis with polyangiitis patients receiving rituximab

Background Randomized controlled trials and retrospective studies in ANCA-associated vasculitis (AAV) concurred that rituximab (RTX) is effective to induce and maintain remission. Infections and hypogammaglobulinemia during RTX were usually infrequent and uncomplicated. But in the Tromsø study cohort, 45 % of patients with granulomatosis with polyangiitis (GPA) developed hypogammaglobulinemia during RTX maintenance leading to its discontinuation in 62 %. Methods To explain these differences in outcome when using RTX in AAV to maintain remission, we used statistical structural methods to compare the Tromsø study cohort with other published cohorts. Results GPA patients’ characteristics of the Tromsø study cohort were not so different compared with other cohorts. Rates of hypogammaglobulinemia and discontinuation of RTX seemed closely related to the cut-off used and to the levels of immunoglobulin (Ig) at baseline. Combination of low IgG serum levels at baseline (7.7 g/L) and low cut-off to define hypogammaglobulinemia in the Tromsø study cohort explained the high rate of hypogammaglobulinemia and discontinuation of RTX. Conclusions Patients’ characteristics in the Tromsø study cohort were not skewed, apart from IgG levels. Low IgG level at baseline seemed to contribute the most to hypogammaglobulinemia and its complications.


Background
Granulomatosis with polyangiitis (GPA) is an ANCAassociated vasculitis (AAV) affecting usually the small and medium vessels and is closely related to proteinase3-ANCA (PR3-ANCA). GPA often involves the upper and lower airways tracts and kidneys and was a fatal disease prior to the use of cyclophosphamide (CYC) [1].
Rituximab (RTX) is a chimeric monoclonal antibody against CD20 that depletes B cells [2] and is effective in inducing and maintaining remission in AAV patients in randomized controlled studies [3][4][5]. In two retrospective studies of remission maintenance with RTX, severe infections and hypogammaglobulinemia were frequent adverse events: 26-29 % had severe infections and 41-45 % had hypogammaglobulinemia [6][7][8]. However interpretation of the risk of hypogammaglobulinemia and its practical implications differed between the two studies. In the Tromsø study cohort, 17 % received intravenous immunoglobulin (IVIG) replacement and 62 % who developed hypogammaglobulinemia discontinued maintenance remission with RTX [8].
To explain differences in outcome, we compared using statistical structural methods the patients' characteristics of the Tromsø study cohort with other published studies of induction and maintenance with RTX in AAV.

Identification of induction and maintenance studies
Two studies from the Northern Norwegian vasculitis register that was approved in 2001 by the Regional Ethical Committee for Medical and Health Research Ethics (REK-V 41/2001) were included [6,8]. Patients (or the patients' parents or guardians in case of children) gave written informed consent in accordance to the declaration of Helsinki at registry inclusion.
All other induction and maintenance studies with RTX in AAV were retrieved either from searching PubMed (www.ncbi.nlm.nih.gov/pubmed) in January 2015 using indexing terms ANCA vasculitis, rituximab maintenance and induction, or from reviewing the reference lists.

Statistical analysis
Principal component analysis (PCA) finds the best linear combinations of important variables at baseline. Number of patients, age, proportion of men, PR3-ANCA status, kidney and lung involvement, Birmingham vasculitis score (BVAS) at baseline, proportion of patients exposed to CYC and type of RTX induction regimen were included in the PCA of induction studies. Number of patients, age, PR3-ANCA status, kidney and lung involvement, exposure to CYC, disease duration prior RTX and follow-up in months were included in the PCA of maintenance studies. Correspondence analysis (CA) of adverse events during RTX maintenance included studies that reported the risks of both severe infections and hypogammaglobulinemia.
Statistical analysis was done with R (R project for statistical computing www.r-project.org).

Induction
Ten studies out of 18 were included in the PCA. Missing data from studies not included in the PCA were PR3-ANCA status [20,21,23], lung involvement [3,22], CYC exposure [13,17] and BVAS [12,20] (Table 1 and Fig. 1). All studies concluded that RTX was effective in inducing remission in AAV patients, even though patients' characteristics and induction regimen were different. The two first PCA dimensions explained 59 % of the variance of the cohorts' characteristics included in the analysis. The RAVE and Cleveland Clinic studies recruited the most patients [4,19]. The two 1g-RTX infusions given one fortnight apart were used more often at induction in the studies from Freiburg and Tromsø [8,24].
Patients in the Tromsø cohort only had GPA, were more often PR3-ANCA positive, had more lung and less kidney involvement, received more frequently CYC and had a higher BVAS at baseline (Fig. 1).

Maintenance
The PCA did not include two studies that reported neither age [31] nor exposure to CYC [29]. Studies were very different in term of patients' characteristics at baseline, however all reported decreased disease activity and relapse rates during RTX maintenance ( Table 2 and Fig. 2). The two first PCA dimensions explained 64 % of the variance of the cohorts' characteristics included in the analysis.
Studies from Freiburg [24] and two other multicentric French studies [5,32] were closely related, as their patients were older at RTX induction, had more kidney and lung involvement and had the shortest follow-up after RTX. Almost all patients were PR3-ANCA positive in the studies from Mayo Clinic and Nottingham [25,28]. Cohorts from Tromsø and Paris were closest to the centroid (point 0) [8,26].
Patients in the Tromsø cohort only had GPA, were younger, more often PR3-ANCA positive and received more frequently CYC prior to RTX (Fig. 2).

Discussion
The Tromsø study cohort only included GPA patients; they were more frequently PR3-ANCA positive and were often exposed to CYC compared with other cohorts. Patients' characteristics of the Tromsø cohort were similar to Freiburg during induction [24] and to an older cohort from Paris during maintenance [26]. The Tromsø study cohort seemed representative of maintenance studies since it was close to the centroid of the PCA.
In AAV, the risks of hypogammaglobulinemia and of severe infection seemed equivalent at 18 % during remission maintenance with RTX. While severe infection was defined alike in all studies, the definition of hypogammaglobulinemia was not standardized and identified patients with different Ig serum levels conferring different risk to receive IVIG and to discontinue RTX. Hypogammaglobulinemia cutoff was lowest in the Boston study: total IgG < 4 g/L [29] vs. IgG < 6 in Cambridge [7], IgG < 7 [24] in Freiburg, total Ig < 6 (corresponding to IgG < 5 g/L) in Tromsø [8] and total Ig < 7 g/L in Paris [30]. Difference in cutoffs explained the difference in results between studies, but hypogammaglobulinemia in the Tromsø study was still more frequent and severe.
Fifty three and 62 % discontinued RTX maintenance due to hypogammaglobulinemia in the Boston and Tromsø cohorts [8,29]. However the risk of hypogammaglobulinemia was four times higher in the Tromsø cohort since the Boston study used a lower cutoff to define hypogammaglobulinemia [29].
PR3-ANCA status and exposure to CYC prior to RTX were similar in the Tromsø and Cambridge cohorts. Nevertheless others factors, such as organ involvement and treatment strategy during remission maintenance, could also explain the differences in severity of hypogammaglobulinemia between both studies. GPA patients in the Tromsø study had more lung and kidney involvement than in Cambridge. They also used concomitant immunosuppressive drugs for a median of two years and continued with pre-emptive RTX maintenance for a median of four years. While in the Cambridge study, patients did not use concomitant immunosuppressive drugs, received RTX remission maintenance during two years and were only re-treated with RTX in case of relapse [7].

Conclusions
In summary, GPA patients' characteristics in the Tromsø cohort were not skewed compared with the other cohorts, apart from IgG levels. In agreement with a previous study on RTX use in autoimmune diseases [33], low IgG level at baseline seemed to contribute the most to hypogammaglobulinemia and its complications during RTX maintenance in AAV.

Ethics approval and consent to participate
Human data originated from the Northern Norwegian vasculitis register that was approved in 2001 by the Regional Ethical Committee for Medical and Health Research Ethics (REK-V 41/2001). Patients (or the patients' parents or guardians in case of children) gave written informed consent in accordance to the declaration of Helsinki at registry inclusion.

Competing interests
The author declares that he has no competing interests.
Author's contribution EB acquired, analyzed and interpreted the data. EB was involved in drafting the manuscript and revising it critically. EB has given formal approval of the version to be published. EB agrees to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The author read and approved the final manuscript.