Immunomonitoring of minimal residual autoimmunity using highly sensitive flow cytometry in ANCA-associated vasculitis patients treated with rituximab.

Introduction
ANCA-associated vasculitis is a systemic autoimmune disease characterized by
inflammation of the small- to medium-sized blood vessels. RTX, a chimeric CD20
antibody is successfully applied as induction treatment in ANCA-associated
vasculitis (AAV) [1, 2]. Despite successful induction therapy, a subset of
patients relapse while they are being treated with azathioprine, methotrexate
or MMF as maintenance regimen[3-5]. Relapses of AAV can be severe resulting in
organ damage or death[6]. In addition, the adverse effects of these
immunosuppressive therapies have a high morbidity and lead to long-term
complications such as osteoporosis or cardiovascular disease[7]. Therefore,
continuous efforts are directed at the development of better maintenance
regimens for AAV patients but also at early identification of patients at high
risk for relapse.
A recent, pivotal randomized trial (MAINRITSAN study) demonstrated the
superiority of RTX over azathioprine as maintenance treatment in AAV[8]. This
study confirmed previous observations from several uncontrolled cohort studies
that RTX can be used effectively and safely as maintenance treatment in AAV
patients (Table 1)[9-19]. However, in all the published studies RTX is used in
a different regimen, timing and dosing. Roughly, RTX regimens could be
classified into two different regimens, *fixed* or *on-demand* treatment.
*Fixed treatment* applied RTX with repeated dosing at fixed intervals. *On
demand* treatment applied RTX as a (re-)treatment upon clinical signs of a
relapse[16, 18, 19]. The RTX fixed treatment strategy has been widely used in
the recent years (Table 1). This strategy has high rates of sustained remission
of 74%-100 with relapse rates varying from 0%-20% over follow-up periods of
18-84 months versus RTX in non-fixed intervals that has 19-56% of sustained
remission with relapse rates of 32-72% [5]. There is a risk of overtreatment
using the fixed retreatment strategy. On the other hand, a major advantage is
the successful tapering and even discontinuation of glucocorticoids in patients
on a fixed treatment regimen. The RTX *on demand* strategy also seems effective
in uncontrolled cohort studies [16, 18], however, this treatment strategy
inherently holds the risk of serious organ damage and high use of
glucocorticoids because clinical relapse can have acute onset and progression.
As a result, there is increasing tendency to apply RTX retreatment on the basis
of established biomarkers of AAV disease. However, a rise in ANCA titers or B
cell repopulation does not consistently predict a relapse in AAV[5]. In
addition to these different treatment regimens, the approach within each study
to utilize RTX was different. Studies investigating the *fixed treatment*
strategy used very heterogeneous intervals ranging from 4 months[9, 10], 6
months[11-16] to annually [12, 17]. While studies investigating *on demand*
treatment applied RTX as a (re-)treatment upon clinical signs of a relapse[16,
18, 19]. The definition of *relapse*, however, was also heterogeneous: either
based upon the clinician*s discretion or upon specific biomarkers (e.g. ANCA
titer or repopulation of B-cells)[12, 18]. To address these issues, there is
currently a trial ongoing that investigates these two dosing regimens
(ClinicalTrials.gov NCT01731561). Briefly, patients receive retreatment with
RTX based on ANCA titers and CD19+ B cell return or at fixed 6-monthly
intervals. Taken all together, one can appreciate that, although effectiveness
of RTX is well-documented, these studies failed to elucidate the optimal
maintenance strategy for the application of RTX in AAV patients.

Minimal residual autoimmunity after RTX treatment in AAV
As described above, the optimal treatment strategy for RTX in AAV has not been
established. RTX exert its effect through binding of CD20 on the cell membrane
and activating both complement dependent cytotoxicity (CMC) and antibody
dependent cellular cytotoxicity (ADCC) that will deplete CD20+ cells[20].
Previous studies have shown that incomplete B cell depletion after induction
treatment correlated with disease relapses[1, 2]. Also, early repopulation of B
cells correlated with disease relapses[14, 21], especially if the
reconstituting B cells display low levels of the naive subset[19] or high
levels of the switched memory cells[21]. From these studies, we formulated the
concept of minimal residual autoimmunity or MRA (terminology derived from
*minimal residual disease* in the tumor-immunology field). This concept builds
on the hypothesis that the effectiveness of RTX treatment in AAV patients is
associated with successfully depleting autoreactive memory B-cells and plasma
cells. Thus far, studies have been hampered to investigate this hypothesis
because conventional techniques are often not sensitive enough to study small
numbers of B cells. Erroneously, this might lead to the observation that
disease relapse occurred before repopulation of B cells assessed by
conventional flow cytometry [8, 11, 16]. MRA can be assessed by 1) the degree
of B cell depletion after RTX therapy and 2) the timing and pattern of subset
repopulation of B cells after depletion. With respect to the first, incomplete
B cell depletion appears to be more common than appreciated with conventional
techniques [22] and is therefore a direct assessment of the biological efficacy
of RTX treatment. Degree of B-cell depletion is determined by many factors,
such as pharmacological variables (i.e. dosing and regimen) but also
immunological variables (i.e. complement activity, CD20 expression levels and
circulating human anti-chimeric antibodies). Altogether these factors could
lead to incomplete B-cell depletion, i.e. MRA, which can potentially predict
early relapsing AAV. Regarding the timing and pattern of B cell repopulation
after depletion due to RTX, a predominant repopulation of memory B cells (in
contrast to repopulation with naïve or transitional B cells) mirrors MRA and
could be predictive of relapse in AAV patients. This concept was already
demonstrated for RA[22] and SLE patients[23]. Taken together, the detection and
evaluation of MRA could ultimately guide personalized-medicine approach for
RTX-treated AAV patients.
In the present study, we will assess the humoral autoimmune system, including
B-cell and plasma cell subpopulations in AAV patients treated with RTX, using
highly sensitive flow cytometry (HSFC). With this highly sensitive technique,
it is possible to accurately enumerate B cell subsets 50-100 times better than
the conventional techniques by analyzing large numbers of events in combination
with a wide range of cell markers[23]. Therefore, a precise appreciation of the
MRA can be determined.

In this single center cohort study the primary objective is to assess minimal
residual autoimmunity (MRA) in RTX treated AAV patients. The primary objective
is to routinely assess the composition of the memory B-cell and plasma cell
compartment before and after (repeated) RTX treatment in AAV patients through
highly sensitive flow cytometry (HSFC) (EUROFLOW)
Secondary objectives are:
- to routinely assess ANCA titers before and after (repeated) RTX treatment
- to investigate whether MRA is associated with disease flares
- to investigate whether MRA is associated with (a shorter) time to a disease
flares
- to investigate whether the presence or absence of MRA after RTX can guide a
personalized treatment strategy between on-demand and fixed retreatment.

This single-center, non-randomized, prospective cohort study will evaluate MRA
in AAV patients treated with RTX by studying the B-cell and plasma cell
compartment.
AAV patients that are treated with RTX, upon the discretion of the treating
physician, will be included in the study cohort and HSFC analysis will be
performed just before RTX infusion and at predefined timepoints after
treatment.

There is no direct benefit for the subjects participating in this study. Also,
the risks related to the study participation are limited: blood sampling for
experimental studies are combined with standard laboratory assessments for the
routine clinical practice. Consent is requested for 55mL extra blood donation
for the study.