B-cell phenotyping
Rituximab depletes CD20-positive B cells. There has therefore been a focus on enumeration of B-lineage cells in blood and synovium as predictive biomarkers, as well as other markers of B-cell function, such as secreted immunoglobulin and B-cell cytokines. Prior experience using cell-depleting therapies in haematology has demonstrated the value of measuring the extent of B-cell depletion as a biomarker.
In addition to autoantibodies, markers of B-cell activity may also predict better clinical response, such as raised serum IgG, the B-cell cytokine BAFF or the chemokine CCL19 [
94‐
96]. In contrast, in the synovium, higher numbers of CD79a
+ B cells at baseline predict worse clinical response [
97]. In the blood, three studies have used flow cytometry to demonstrate that higher numbers of circulating plasmablasts predict worse clinical response [
98‐
100]. This has been confirmed using a large cohort of patients pooled from randomised trials using a plasmablast gene expression signature based on the combination of IgJ and FCRL5 mRNA expression that predicted non-response to rituximab [
101].
Plasmablasts are a plasma cell precursor differentiated from activated B cells. They are short-lived in the circulation and are CD20 negative, so may act as a biomarker of B-cell activity, especially after depletion of CD20-positive B cells. However, they are not detected in a CD19 lymphocyte gate, requiring specialised flow cytometry protocols for accurate enumeration after rituximab, called high-sensitivity flow cytometry.
Complete depletion of plasmablasts after the first infusion, assessed through high-sensitivity flow cytometry, has been clearly associated with better clinical outcomes, compared with non-complete depletion [
102]. Plasmablast levels may also explain the more variable response to lower dose rituximab: although the rate of complete depletion was lower with lower dose rituximab, patients with lower baseline plasmablasts counts could achieve complete depletion and good EULAR response. Moreover, for patients who failed to deplete, a third extra dose of rituximab increased complete depletion rates and this was associated with better clinical response [
103]. These data provide a basis for modifying therapy. However, studies that used different flow cytometric protocols did not reproduce these findings [
104,
105]. Another study that used high-sensitivity flow cytometry reproduced baseline, but not depletion, results [
100].
Clinical responders have also been found to have lower baseline frequency, more profound suppression and delayed resurgence of memory B cells [
106‐
109]. Also, an increased number of plasma CD95
+ activated B cells and class-switched memory B cells at depletion, and a lower transitional-to-memory B-cell ratio at reconstitution were associated with poor response; class-switched memory B cells accumulated in flaring joints, confirming the pathogenic role of these cells in RA [
110,
111]. Clinical relapse is usually preceded a few months by B-cell compartment repopulation and memory B cells seem to be key players in this process [
107,
112].
Synovial tissue data underline the variable B-cell response to standard-dose rituximab that was demonstrated in blood. Depletion of synovial B cells is more variable. This is less clearly related to treatment response, although these studies have been very modest in size [
97,
105,
113,
114]. In one synovial study, greater local B-cell depletion (assessed through CD19 mRNA expression but not through histology) was seen in ACR50 responders (but not overall responders) compared with non-responders and was coupled with decreased synovial immunoglobulin production [
105,
115].
Greater decrease of synovial plasma cells was reported in good responders (
R
2 = 0.26), correlating with the reduction in serum ACPA levels [
113]. Lymphoid aggregates have been found to decrease after 16 but not 4 weeks. However, baseline synovial plasma cells and lymphoid aggregates did not predict treatment outcome and it is therefore less clear whether the greater normalisation of these changes in clinical responders is a rituximab-specific mechanism of response or just another (generic) reflection of an overall improvement in synovitis [
105,
113]. Interestingly, type I IFN has a key role in promoting the differentiation of plasmablasts and plasma cells from B cells, which may link the negative predictive value of the IFN signature with these blood and synovial findings [
116]. Overall, synovial cellular markers have provided clues to rituximab’s mode of action and RA pathogenesis, but have been limited in associating with clinical outcomes. Large multicentre tissue-based randomised clinical trials (RCTs) further investigating the role of synovium B-cell burden in predicting response to rituximab are ongoing [
117].
Transcriptomic studies
Gene expression studies have emerged, building on evidence from cellular-focused blood and tissue research [
62,
63]. The clearest signal comes from the already mentioned type I IFN signature, negatively associated with response to rituximab at the whole blood and peripheral blood mononuclear cell (PBMC) levels [
35,
36,
118]. Besides baseline expression, the induction of type I IFN response genes 3 months after rituximab treatment was also associated with good clinical response at 6 months [
118]. An important study assessed 68 patients from the SMART study and found a whole-blood transcriptomic signature associated with 6-month response to rituximab, including upregulation of the NF-κB pathway and downregulation of the IFN pathway, which correctly classified treatment response in 92.6% of cases [
119]. This was also confirmed at the tissue level, where patients with a high inflammatory gene score, overexpressing macrophage and T-cell-related genes and under-expressing IFN and remodelling genes, responded better to rituximab [
37]. Also in line with this, another study found responders to have upregulation of synovium immunoglobulin genes and of genes involved in antigen processing and MHC class II presentation [
120].
Investigation into other biomarker candidates is more limited for rituximab response prediction than for TNFi. Some markers have been associated with better response, including: SNPs of the Fc gamma receptor 3A (158 V > F, VV genotype) [
121], BAFF (871C > T, C allele carriage) [
122] and IL-6 (174G > C, CC genotype) [
121] genes; micro-RNA-125b (increased expression) [
123]; and cytokine profile assessed through proteomic analysis (Table
1) [
124].