Association of the FCGR3A -158F/V Gene Polymorphism with the Response to Rituximab Treatment in Spanish Systemic Autoimmune Disease Patients

Introduction

S ystemic autoimmune diseases are a heterogeneous group of diseases with different pathogenesis and clinical manifestations but with common pathogenic mechanisms, including an aberrant B-cell regulation. The crucial role of B cells in autoimmune disorders has evidenced the importance of biological treatments that blockade these cells in refractory patients (De Vita et al., 2002).

Rituximab is a chimeric mouse-human monoclonal immunoglobulin G1 (IgG1) antibody that specifically targets the human B-lymphocyte CD20 surface protein (Pescovitz, 2006), resulting in peripheral B-cell depletion (Taylor and Lindorfer, 2007). Rituximab use has been approved by the FDA for B-cell non-Hodgkin's lymphomas (Grillo-Lopez, 2000), for nonresponders to patients with first-line anti-tumor necrosis factor-α rheumatoid arthritis (RA) (Papagoras et al., 2010), and recently, for vasculitis (Stone et al., 2010; Puechal et al., 2012) or lupus nephritis (Diaz-Lagares et al., 2012; Ezeonyeji and Isenberg, 2012), but there is growing evidence from observational studies and registries of patients that their usefulness can be extended to other autoimmune diseases (Stasi et al., 2001; Looney et al., 2004; Ramos-Casals et al., 2010). Studies have shown several different mechanisms by which rituximab can selectively deplete B cells, such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-mediated cytotoxicity, and direct induction of B-cell apoptosis (Kazkaz and Isenberg, 2004).

Several factors have been associated with a better response to rituximab, for example, in patients with RA, the presence of positive rheumatoid factor (Quartuccio et al., 2009), positive Epstein-Barr virus (Magnusson et al., 2010), low levels of B lymphocyte stimulator (Fabris et al., 2009), and low levels of type I interferons (Thurlings et al., 2010).

The Fc portion of rituximab binds specifically to cell-surface Fc-γ receptors (FCGR), and this may affect certain immune responses such as removal of antigen–antibody complexes from the circulation, ADCC, or phagocytosis. There are three major classes of human FCGR that are encoded by 8 genes (FCGR1A, B, and C; FCGR2A, B, and C; FCGR3A and B), all located on chromosome 1. Three polymorphisms, two in positions 48 and 158 of FCR3A and one at codon 131 of FCGR2A, have been reported to affect receptor affinity for IgG (Vance et al., 1993; de Haas et al., 1996; Wu et al., 1997). Functional studies have described a correlation between the FCGR3A-158 genotype and rituximab efficacy, but no correlation has been found with FCGR2A-131 (Anolik et al., 2003). Moreover, genetic linkage of FCGR-48 and FCGR2A-131 with FCGR3A-158 has been demonstrated and points to the primacy of FCGR3A-158 in predicting rituximab response (Koene et al., 1997; Treon et al., 2005; Hatjiharissi et al., 2007a; Lejeune et al., 2008).

FCGR3A, also known as CD16, is expressed on macrophages, monocytes, and natural killer (NK) cells, all of which are involved in B-cell depletion. The nonsynonymous FCGR3A-158 polymorphism results in either a phenylalanine (F158) or a valine (V158) at this position in the membrane proximal domain of the molecule. The FCGR3A-158V single-nucleotide polymorphism (SNP) exhibits a higher affinity for IgG subtypes than the FCGR3A-158F SNP (Koene et al., 1997; Dall'Ozzo et al., 2004). This SNP has been associated to different autoimmune diseases such as type 1 diabetes, celiac disease, RA (Alizadeh et al., 2007), and systemic lupus erythematosus (SLE) (Koene et al., 1998). In patients with giant-cell arteritis, an association was observed with the FCGR2A-FCGR3A 131R-158F haplotype (Morgan et al., 2006). However, no association between FCGR3A-158 and systemic sclerosis was described (McKinney et al., 2012). Homozygosity for the higher-affinity V allele has also been shown to be associated with susceptibility to antibody-positive RA (Robinson et al., 2010; Thabet et al., 2009).

An important pharmacogenetic association with biological response to rituximab has been shown in this polymorphism. Patients carrying the V/V isoform with non-Hodgkin lymphoma (NHL) and SLE showed a better biological response to rituximab (Anolik et al., 2003; Cartron et al., 2002). Later, two studies conducted in healthy donors determined that this improved response observed in individuals expressing at least one valine at FCGR3A-158 seems to be due to an increased CD16 expression, rituximab binding, and ADCC activity mediated by NK cells (Dall'Ozzo et al., 2004; Hatjiharissi et al., 2007b). In other diseases such as Sjögren's Syndrome (Pers et al., 2007) or chronic lymphocytic leukemia (Farag et al., 2004), this association was not observed, which may indicate that mechanisms of action of rituximab other than ADCC may be more important in these pathologies.

Only one study has examined the influence of FCGR3A-158F/V in the clinical response to rituximab in autoimmunity. This study conducted in patients with RA found that the V allele carriage was significantly associated with a higher response rate (Ruyssen-Witrand et al., 2012).

It is possible however that the relative importance of ADCC as a mechanism for the activity of rituximab may differ between autoimmune disorders. The aim of our work was to investigate the possible involvement of the FCGR3A-158F/V polymorphism in the clinical response to rituximab in Spanish patients with different systemic autoimmune diseases.

Materials and Methods

Results

The response to rituximab was evaluated at month 6 after first infusion, according to the EULAR and ACR criteria (Criteria, 2006; Hellmich et al., 2007; Mosca and Bombardieri, 2007; Ramos-Casals et al., 2010). Eighty of the patients (61%) were considered to be good responders (complete remission of the symptoms and clinical characteristics that recommended the use of the drug); 36 patients (27%) were partial responders (reduction in at least 50% of the disease activity); and sixteen (12%) were classified as nonresponders (reduction in <50% of the disease activity). Of the 132 patients analyzed, 62 (47%) were homozygous for F allele, 48 (36%) were heterozygous FV, and 22 (17%) were homozygous for V allele, similarly to previous studies in SLE in Caucasian populations (Jonsen et al., 2004).

When all patients were pooled, after checking for homogeneity of odds ratios between patients with SLE and the remaining patients with autoimmune diseases by Breslow-Day test (p>0.05), and stratified into two groups according to the response to rituximab, genotypic frequencies in patients presenting total or partial response to rituximab were as follows: 50 patients (43%) were FF; 45 patients (39%) were FV; and 21 patients (18%) were VV, whereas in the subgroup of patients presenting nonresponse to rituximab, 12 (75%) were FF; 3 (19%) were FV; and 1 patient (6%) was VV. In responders, the frequency of V allele carriers (FV+VV) was significantly increased with respect to nonresponders (66 patients=57% vs. 4 patients=25%; p=0.02; OR=3.96, 95% CI 1.10–17.68). A significant association was also found when comparing the FCGR3A-158V allele frequency between responders (87 patients=38%) and nonresponders (5 patients=16%); p=0.01; OR=3.24, 95% CI 1.17–11.13 (Table 2).

In correlation with these results, rituximab was effective in 94% of the patients carrying V allele (66 responders carrying the V allele/70 patients carrying the V allele) and 81% of the homozygous FF patients (50 responders FF homozygotes/62 patients FF homozygotes) (p=0.02) (Table 2).

Finally, we analyzed separately patients with SLE (81/132, 61.4% of the patients) and patients with other autoimmune diseases (51/132, 38.6% of the patients). In both groups, we found a similar trend to those observed in the global analysis. In patients with SLE, 49% of the responders carried the V allele while it was present in 20% of nonresponders (p=0.08, OR=3.89, 95% CI 0.70–39.51). Rituximab was effective in 95% of the V carriers and 82% of the homozygous FF (p=0.08) (Table 3). Likewise, in patients with no SLE, the frequency of the V allele was increased in responder versus nonresponder patients (42% vs. 17%; p=0.08, OR=3.65, 95% CI 0.71–35.81). Rituximab was effective in 94% of the V carriers and 79% of the patients with FF homozygotes (p=0.13) (Table 4).

Discussion

The establishment of pharmacogenetic markers to predict the response to rituximab therapy becomes a pivotal requirement, given the expanding clinical use of this drug in the treatment of several autoimmune diseases.

Rituximab is recognized and bound to the surface of NK cells and macrophages through the FCGR, triggering ADCC immune system mechanism, essential for the activity of rituximab to deplete B cells. FCGR3A is expressed by immune effector cells and shows specific affinity for IgG monoclonal antibodies, such as rituximab. The importance of FCGR3A in the response to rituximab has been shown in studies where mice lacking FCGR3 presented a decrease in the response to this drug (Clynes et al., 2000).

In the present study, we have analyzed the association of the FCGR3A-158F/V polymorphism with the response to rituximab in patients with autoimmune diseases. Genotypic frequencies for this SNP were similar to those described previously for several patients with autoimmune diseases in Caucasian populations (Dijstelbloem et al., 2000; Nieto et al., 2000; Alizadeh et al., 2007). It is remarkable that frequencies were elevated for V carriers in responders, which correlates with the fact that patients carrying the V allele at this position presented a better response to the treatment with the drug than those with homozygous FF genotype.

Functional studies have demonstrated that the 158V allele is correlated with a better biological response to rituximab in autoimmunity. Anolik et al. (2003) showed that in patients with SLE carrying the high-affinity V allele (FV or VV), rituximab was more effective in depleting peripheral B cells than in those homozygous for the low-affinity FF. Recently, the FCGR3A-158F/V SNP has been associated with the clinical response to rituximab in RA. This study conducted in 111 patients found that the V allele carriage was significantly associated with a higher response rate (91% of responder vs. 70%; p=0.006, OR=4.6, 95% CI 1.5–13.6) (Ruyssen-Witrand et al., 2012).

The findings in SLE and RA are in line with our results that showed a better response to rituximab in patients with autoimmune diseases that carried the V allele (FV or VV) than in patients with homozygous FF. Additionally, based on the previous association observed in patients with SLE and on the fact that this was the largest group, we analyzed separately patients with SLE. We found a similar pattern, and patients carrying the V allele showed a better response to rituximab treatment, although it did not reach statistical significance (p=0.08). Finally, we examined the group of patients with no SLE to establish whether this association is shared by different autoimmune disorders. As in the case of patients with SLE, we observed a similar effect, but this association did not reach statistical significance either (p=0.08). This suggests that the influence of the 158F/V polymorphism in the therapeutic response to rituximab is common to various autoimmune diseases; however, the reduced numbers involved in these stratified analysis leads to poor statistical power, and therefore the conclusions are provisional.

It should be noted that copy number variation (CNV) has been shown to be present in the FCGR3A gene (Sebat et al., 2004; Redon et al., 2006; Wong et al., 2007). The presence of common CNVs can cause false SNP genotyping results that can lead to fail the Hardy–Weinberg equilibrium (HWE) and may blur the association of the studied SNPs with disease susceptibility. In our study, the genotypic frequencies were significantly different from those predicted by HWE, but only in the group of patients with SLE. This may be due to existence of an association between the 158F/V polymorphism and this disease (Balding, 2006). In fact, in our cohort of healthy controls (previously published genotypic data), genotype frequencies for this SNP were in the HWE (Alizadeh et al., 2010). Moreover, the frequency of CNV has been reported to vary significantly in different ethnic populations, which can result in contradictory findings, but in this case, frequencies observed in patients were similar to those previously described, and the results reported to date are fairly consistent.

Previous findings showed that patients carrying the V allele in FCGR3A-158F/V increased expression of CD16 in NK cells (Hatjiharissi et al., 2007b). A correlation between the number of cell surface CD16 receptors and the enhancing of the ADCC activity mediated by NK cells was found. These observations would explain the better response to rituximab observed in patients with systemic autoimmune diseases carrying the V allele and would highlight the importance of the ADCC mechanism for clearance of B cells by rituximab in autoimmune diseases.

In summary, our results together with previous findings (Anolik et al., 2003; Cartron et al., 2011; Ruyssen-Witrand et al., 2012) suggest that FCGR3A plays an important role in response to rituximab in patients with systemic autoimmune diseases and support the hypothesis that the 158F/V variant could be used as a potential predictor of those patients who will respond better to treatment with rituximab.